Title Year Journal Affiliation Abstract Gene Cloning of a cDNA encoding an importin-alpha and down-regulation of the gene by light in rice leaves 1998 Gene Central Research Institute of Electric Power Industry, Chiba, Japan. The import of nuclear proteins into nuclei begins with recognition of nuclear localization signal-harboring proteins and binding to a nuclear pore targeting complex. A cDNA for an importin-alpha protein, a subunit of the complex, was isolated from rice plants. The amino acid sequence deduced from the nucleotide sequence of the cDNA exhibited a high homology to those of importin-alpha proteins from many organisms such as Arabidopsis thaliana, Saccharomyces cerevisiae, human, mouse, Xenopus laevis and Drosophila melanogaster. Down-regulation of the transcription by light was shown in the leaves of light- and dark-grown seedlings by RNA blot analysis. The down-regulation was specific to leaves, whereas no light effect was observed in root tissues or calli, in which higher levels of the transcript were detected. alpha1a Molecular cloning of a novel importin alpha homologue from rice, by which constitutive photomorphogenic 1 (COP1) nuclear localization signal (NLS)-protein is preferentially nuclear imported 2001 J Biol Chem National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan. cjjiang@abr.affrc.go.jp Nuclear import of proteins that contain classical nuclear localization signals (NLS) is initiated by importin alpha, a protein that recognizes and binds to the NLS in the cytoplasm. In this paper, we have cloned a cDNA for a novel importin alpha homologue from rice which is in addition to our previously isolated rice importin alpha1a and alpha2, and we have named it rice importin alpha1b. In vitro binding and nuclear import assays using recombinant importin alpha1b protein demonstrate that rice importin alpha1b functions as a component of the NLS-receptor in plant cells. Analysis of the transcript levels for all three rice importin alpha genes revealed that the genes were not only differentially expressed but that they also responded to dark-adaptation in green leaves. Furthermore, we also show that the COP1 protein bears a bipartite-type NLS and its nuclear import is mediated preferentially by the rice importin alpha1b. These data suggest that each of the different rice importin alpha proteins carry distinct groups of nuclear proteins, such that multiple isoforms of importin alpha contribute to the regulation of plant nuclear protein transport. alpha1a,alpha1b,alpha2 Mutations of genes in synthesis of the carotenoid precursors of ABA lead to pre-harvest sprouting and photo-oxidation in rice 2008 Plant J State Key Laboratory of Plant Genomics and National Centre for Plant Gene Research, Beijing 100101, China. Pre-harvest sprouting (PHS) or vivipary in cereals is an important agronomic trait that results in significant economic loss. A considerable number of mutations that cause PHS have been identified in several species. However, relatively few viviparous mutants in rice (Oryza sativa L.) have been reported. To explore the mechanism of PHS in rice, we carried out an extensive genetic screening and identified 12 PHS mutants (phs). Based on their phenotypes, these phs mutants were classified into three groups. Here we characterize in detail one of these groups, which contains mutations in genes encoding major enzymes of the carotenoid biosynthesis pathway, including phytoene desaturase (OsPDS), zeta-carotene desaturase (OsZDS), carotenoid isomerase (OsCRTISO) and lycopene beta-cyclase (beta-OsLCY), which are essential for the biosynthesis of carotenoid precursors of ABA. As expected, the amount of ABA was reduced in all four phs mutants compared with that in the wild type. Chlorophyll fluorescence analysis revealed the occurrence of photoinhibition in the photosystem and decreased capacity for eliminating excess energy by thermal dissipation. The greatly increased activities of reactive oxygen species (ROS) scavenging enzymes, and reduced photosystem (PS) II core proteins CP43, CP47 and D1 in leaves of the Oscrtiso/phs3-1mutant and OsLCY RNAi transgenic rice indicated that photo-oxidative damage occurred in PS II, consistent with the accumulation of ROS in these plants. These results suggest that the impairment of carotenoid biosynthesis causes photo-oxidation and ABA-deficiency phenotypes, of which the latter is a major factor controlling the PHS trait in rice. beta-OsLCY,OsPDS,OsZDS,OsCRTISO|ZEBRA2 A pair of orthologs of a leucine-rich repeat receptor kinase-like disease resistance gene family regulates rice response to raised temperature 2011 BMC Plant Biol National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. BACKGROUND: Rice Xa3/Xa26 disease-resistance gene encodes a leucine-rich repeat (LRR) receptor kinase-type protein against Xanthomonas oryzae pv. oryzae (Xoo) and belongs to a multigene family. However, the functions of most genes in this family are unknown. RESULTS: Here we report that two orthologs of this family, the NRKe from rice variety Nipponbare and 9RKe from variety 93-11 at the RKe locus, have similar functions although they encode different proteins. This pair of orthologs could not mediate resistance to Xoo, but they were transcriptionally induced by raised temperature. Transcriptional activation of NRKe or 9RKe resulted in the formation of temperature-sensitive lesion mimics, which were spots of dead cells associated with accumulation of superoxides, in different organs of the transgenic plants. These plants were more sensitive to high temperature shock than wild-type controls. Transgenic plants carrying a chimeric protein consisting of the LRR domain of NRKe and the kinase domain of Xa3/Xa26 developed the same lesion mimics as the NRKe-transgenic plants, whereas transgenic plants carrying another chimeric protein consisting of the LRR domain of Xa3/Xa26 and the kinase domain of NRKe were free of lesion mimic. All the transgenic plants carrying a chimeric protein were susceptible to Xoo. CONCLUSION: These results suggest that the RKe locus is involved in rice response to raised temperature. The LRR domain of RKe protein appears to be important to sense increased temperature. The RKe-involved temperature-related pathway and Xa3/Xa26-mediated disease-resistance pathway may partially overlap. RKe,Xa26|Xa3 The ATP-binding cassette transporter OsABCG15 is required for anther development and pollen fertility in rice 2013 J Integr Plant Biol State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. Plant male reproductive development is a complex biological process, but the underlying mechanism is not well understood. Here, we characterized a rice (Oryza sativa L.) male sterile mutant. Based on map-based cloning and sequence analysis, we identified a 1,459-bp deletion in an adenosine triphosphate (ATP)-binding cassette (ABC) transporter gene, OsABCG15, causing abnormal anthers and male sterility. Therefore, we named this mutant osabcg15. Expression analysis showed that OsABCG15 is expressed specifically in developmental anthers from stage 8 (meiosis II stage) to stage 10 (late microspore stage). Two genes CYP704B2 and WDA1, involved in the biosynthesis of very-long-chain fatty acids for the establishment of the anther cuticle and pollen exine, were downregulated in osabcg15 mutant, suggesting that OsABCG15 may play a key function in the processes related to sporopollenin biosynthesis or sporopollenin transfer from tapetal cells to anther locules. Consistently, histological analysis showed that osabcg15 mutants developed obvious abnormality in postmeiotic tapetum degeneration, leading to rapid degredation of young microspores. The results suggest that OsABCG15 plays a critical role in exine formation and pollen development, similar to the homologous gene of AtABCG26 in Arabidopsis. This work is helpful to understand the regulatory network in rice anther development. OsABCG15,CYP704B2,WDA1 ABCG15 encodes an ABC transporter protein, and is essential for post-meiotic anther and pollen exine development in rice 2013 Plant Cell Physiol Rice Research Institute of Sichuan Agricultural University, Chengdu Wenjiang, Sichuan, 611130, PR China. In flowering plants, anther and pollen development is critical for male reproductive success. The anther cuticle and pollen exine play an essential role, and in many cereals, such as rice, orbicules/ubisch bodies are also thought to be important for pollen development. The formation of the anther cuticle, exine and orbicules is associated with the biosynthesis and transport of wax, cutin and sporopollenin components. Recently, progress has been made in understanding the biosynthesis of sporopollenin and cutin components in Arabidopsis and rice, but less is known about the mechanisms by which they are transported to the sites of deposition. Here, we report that the rice ATP-binding cassette (ABC) transporter, ABCG15, is essential for post-meiotic anther and pollen development, and is proposed to play a role in the transport of rice anther cuticle and sporopollenin precursors. ABCG15 is highly expressed in the tapetum at the young microspore stage, and the abcg15 mutant exhibits small, white anthers lacking mature pollen, lipidic cuticle, orbicules and pollen exine. Gas chromatography-mass spectrometry (GC-MS) analysis of the abcg15 anther cuticle revealed significant reductions in a number of wax components and aliphatic cutin monomers. The expression level of genes involved in lipid metabolism in the abcg15 mutant was significantly different from their levels in the wild type, possibly due to perturbations in the homeostasis of anther lipid metabolism. Our study provides new insights for understanding the molecular mechanism of the formation of the anther cuticle, orbicules and pollen wall, as well as the machinery for lipid metabolism in rice anthers. OsABCG15 Overexpression of ACL1 (abaxially curled leaf 1) increased Bulliform cells and induced Abaxial curling of leaf blades in rice 2010 Mol Plant National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Understanding the genetic mechanism underlying rice leaf-shape development is crucial for optimizing rice configuration and achieving high yields; however, little is known about leaf abaxial curling. We isolated a rice transferred DNA (T-DNA) insertion mutant, BY240, which exhibited an abaxial leaf curling phenotype that co-segregated with the inserted T-DNA. The T-DNA was inserted in the promoter of a novel gene, ACL1 (Abaxially Curled Leaf 1), and led to overexpression of this gene in BY240. Overexpression of ACL1 in wild-type rice also resulted in abaxial leaf curling. ACL1 encodes a protein of 116 amino acids with no known conserved functional domains. Overexpression of ACL2, the only homolog of ACL1 in rice, also induced abaxial leaf curling. RT-PCR analysis revealed high expressions of ACLs in leaf sheaths and leaf blades, suggesting a role for these genes in leaf development. In situ hybridization revealed non-tissue-specific expression of the ACLs in the shoot apical meristem, leaf primordium, and young leaf. Histological analysis showed increased number and exaggeration of bulliform cells and expansion of epidermal cells in the leaves of BY240, which caused developmental discoordination of the abaxial and adaxial sides, resulting in abaxially curled leaves. These results revealed an important mechanism in rice leaf development and provided the genetic basis for agricultural improvement. ACL1,ACL2 A Point Mutation of Adh1 Gene is Involved in the Repression of Coleoptile Elongation under Submergence in Rice 2006 Breeding Science Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo In higher plants, alcoholic fermentation is required to supply NAD+ to the glycolytic pathway, which is responsible for ATP synthesis under anaerobic conditions. Matsumura et al. (1995) previously isolated the reduced adh activity (rad) mutant in rice, in which elongation of the coleoptile is repressed under submergence. However, the rad gene had not been characterized. In the present study, we observed that, in the coleoptile, the Adh1 mRNA levels were comparable between the rad mutant and the wild type cultivar Kinmaze, while the amount of ADH1 protein was much lower in the rad mutant than in the wild type. Sequencing showed that G106 of the Adh1 gene of Kinmaze was replaced with A in the rad mutant, resulting in an E36K substitution in the deduced amino acid sequence. A genotyping experiment using F2 plants from a cross of the rad mutant with the indica cultivar Kasalath indicated that the point mutation was involved in the repression of coleoptile elongation. Furthermore, since the reduced ADH activity appeared to cause an ATP deficiency, elongation of the coleoptile was repressed in the submerged rad mutant. Adh1 Molecular characterization of cDNA encoding for adenylate kinase of rice (Oryza sativa L.) 1992 Plant J Institute of Applied Microbiology, University of Tokyo, Japan. Two types of genes (Adk-a, and Adk-b) encoding for adenylate kinase (AK, EC 2.7.4.3.) were isolated from the cDNA library constructed from poly(A)+ RNA of rice (Oryza sativa L.). Two cDNAs were heterogeneous at 5' and 3' ends of non-coding sequences and had possible polyadenylation signals. One of the genes, Adk-a, had 1154 bp sequences encoding 241 amino acid residues, while the other type, Adk-b, contained 1085 bp sequences encoding for 243 amino acid residues. Homology between Adk-a and Adk-b was 73.7% in nucleotide sequences, and 90.8% in amino acid level. Two genes showed about 53% homology to bovine mitochondrial adenylate kinase (AK2) at nucleotide and amino acid levels. Concerning the codon usage of rice AK genes, T was abundant at the third position of a codon in the reading frames. In order to examine the enzyme activity of the protein encoded by the rice cDNA, Adk-a was cloned into an expression vector, pUC119, which was introduced into Escherichia coli strain CV2, a temperature-sensitive mutant of adenylate kinase. We found that the transformant carrying the rice Adk-a gene in the sense orientation recovered cell growth at non-permissive high temperature (42 degrees C) and expressed enzyme activities higher than the untransformed CV2 and the transformant possessing Adk-a cDNA in the antisense orientation. These observations suggest that rice Adk-a codes a biologically active enzyme. Furthermore, sucrose was found to regulate the transcription of AK genes in rice cell cultures. Organ related accumulation of mRNA in whole plants was also found. Adk-a,Adk-b The ANTHER INDEHISCENCE1 gene encoding a single MYB domain protein is involved in anther development in rice 2004 Plant Physiol CSIRO Plant Industry, Canberra, Australian Capital Territories 2601, Australia. Using a two-element iAc/Ds transposon-tagging system, we identified a rice (Oryza sativa L. cv Nipponbare) recessive mutant, anther indehiscence1 (aid1), showing partial to complete spikelet sterility. Spikelets of the aid1 mutant could be classified into three types based on the viability of pollen grains and the extent of anther dehiscence. Type 1 spikelets (approximately 25%) were sterile due to a failure in accumulation of starch in pollen grains. Type 2 spikelets (approximately 55%) had viable pollen grains, but anthers failed to dehisce and/or synchronize with anthesis due to failure in septum degradation and stomium breakage, resulting in sterility. Type 3 spikelets (approximately 20%) had normal fertility. In addition, aid1 mutant plants had fewer tillers and flowered 10 to 15 d later than the wild type. The Ds insertion responsible for the aid1 mutation was mapped within the coding region of the AID1 gene on chromosome 6, which is predicted to encode a novel protein of 426 amino acids with a single MYB domain. The MYB domain of AID1 is closely related to that of the telomere-binding proteins of human, mouse, and Arabidopsis, and of single MYB domain transcriptional regulators in plants such as PcMYB1 and ZmIBP1. AID1 was expressed in both the leaves and panicles of wild-type plants, but not in mutant plants. AID1 Cloning and characterization of AKR4C14, a rice aldo-keto reductase, from Thai Jasmine rice 2012 Protein J Department of Biochemistry, Faculty of Science, Kasetsart University, Pahonyothin Rd, Bangkok, 10903, Thailand. Aldo-keto reductase (AKR) is an enzyme superfamily whose members are involved in the metabolism of aldehydes/ketones. The AKR4 subfamily C (AKR4C) is a group of aldo-keto reductases that are found in plants. Some AKR4C(s) in dicot plants are capable of metabolizing reactive aldehydes whereas, such activities have not been reported for AKR4C(s) from monocot species. In this study, we have screened Indica rice genome for genes with significant homology to dicot AKR4C(s) and identified a cluster of putative AKR4C(s) located on the Indica rice chromosome I. The genes including OsI_04426, OsI_04428 and OsI_04429 were successfully cloned and sequenced by qRT-PCR from leaves of Thai Jasmine rice (KDML105). OsI_04428, later named AKR4C14, was chosen for further studies because it shares highest homology to the dicot AKR4C(s). The bacterially expressed recombinant protein of AKR4C14 was successfully produced as a MBP fusion protein and his-tagged protein. The recombinant AKR4C14 were capable of metabolizing sugars and reactive aldehydes i.e. methylglyoxal, a toxic by-product of the glycolysis pathway, glutaraldehyde, and trans-2-hexenal, a natural reactive 2-alkenal. AKR4C14 was highly expressed in green tissues, i.e. leaf sheets and stems, whereas flowers and roots had a significantly lower level of expression. These findings indicated that monocot AKR4C(s) can metabolize reactive aldehydes like the dicot AKR4C(s) and possibly play a role in detoxification mechanism of reactive aldehydes. AKR4C14|OsI_04428,OsAKR1|OsI_04426,OsI_04429 A novel endosperm transfer cell-containing region-specific gene and its promoter in rice 2011 Plant Mol Biol Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, Japan. The endosperm of cereal grains is an important resource for both food and feed. It contains three major types of tissue: starchy endosperm, the aleurone layer, and transfer cells. To improve grain quality and quantity using molecular methods, control of transgene expression directed by distinct temporal and spatial promoter activity is necessary. To identify aleurone layer-specific and/or transfer cell-specific promoters in rice, microarray analyses were performed, comparing the aleurone layer containing transfer cells and the other reproductive and vegetative tissues. After confirmation by RT-PCR analysis, we identified two putative aleurone layer and/or transfer cell-specific genes, AL1 and AL2. The promoter regions of these genes and beta-glucuronidase (GUS) fusion constructs were stably transformed into rice. The GUS expression patterns indicated that the AL1 promoter was active exclusively in the dorsal aleurone layer adjacent to the main vascular bundle. In rice, transfer cells are differentiated in this region. Therefore, the promoter of the AL1 gene exhibits transfer cell-containing region-specific activity. The AL1 gene encodes a putative anthranilate N-hydroxycinnamoyl/benzoyltransferase. The promoter of this gene will be useful for enhancing uptake of nutrients from the mother cells and protecting filial seeds from pathogen attack. AL1 Molecular characterization of a gene for alanine aminotransferase from rice (Oryza sativa) 1999 Plant Mol Biol Department of Biotechnology, National Institute of Agrobiological Resources, Tsukuba, Ibaraki, Japan. A cDNA clone encoding alanine aminotransferase (AlaAT) has isolated from randomly sequenced clones derived from a cDNA library of maturing rice seeds by comparison to previously identified genes. The deduced amino acid sequence was 88% and 91% homologous to those of the enzymes from barley and broomcorn millet (Panicum miliaceum), respectively. Using this cDNA as a probe, we isolated and sequenced the corresponding genomic clone. Comparison of the sequences of the cDNA and the genomic gene revealed that the coding region of the gene was interrupted by 14 introns 66 to 1547 bp long. Northern and western blotting analyses showed that the gene was expressed at high levels in developing seeds. When the 5'-flanking region between -930 and +85 from the site of initiation of transcription was fused to a reporter gene for beta-glucuronidase (GUS) and then introduced into the rice genome, histochemical staining revealed strong GUS activity in the inner endosperm tissue of developing seeds and weak activity in root tips. Similar tissue-specific expression was also detected by in situ hybridization. These results suggest that AlaAT is involved in nitrogen metabolism during the maturation of rice seed. AlaAT Genetic analysis of rice grain quality 1999 TAG Theoretical and Applied Genetics Institute of Genetics, Chinese Academy of Sciences, Beijing 100101, China The inheritance of grain quality is more complicated than that of other agronomic traits in cereals due to epistasis, maternal and cytoplasmic effects, and the triploid nature of endosperm. In the present study, an established rice DH population derived from anther culture of an indica/japonica hybrid was used for genetic analysis of rice grain quality. A total of five parameters, amylose content (AC), alkali-spreading score (ASS), gel consistency (GC), percentage of grain with a white core (PGWC) and the square of the white core (SWC), were estimated for the DH lines and the parent varieties. For each parent, the value of each parameter was relatively stable in three locations, Beijing, Hangzhou and Chengdu, while the differences between the parents were significant for all five parameters. AC showed a bimodal distribution, and the distribution of ASS was skewed toward the value of JX17, while the other three parameters displayed continuous distributions among the DH lines with partially transgressive segregations. For AC, a minor and a major gene were found on chromosomes 5 and 6 respectively. The major gene, which should be an allele of wx, explained 91.9% of the total variation. For GC, two QTLs were identified on chromosomes 2 and 7 respectively. For ASS, a minor and a major gene were both located on chromosome 6. The major gene should be the same locus as the alkali degeneration gene (alk). Genetic linkage between alk and wx was found in QTL mapping. For PGWC, two QTLs were located on chromosomes 8 and 12. Only a minor QTL was found for SWC on chromosome 3. The results and the molecular markers presented here may be useful in rice breeding for grain quality improvement. ALK|SSIIa OsbZIP58, a basic leucine zipper transcription factor, regulates starch biosynthesis in rice endosperm 2013 J Exp Bot National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, PR China. Starch composition and the amount in endosperm, both of which contribute dramatically to seed yield, cooking quality, and taste in cereals, are determined by a series of complex biochemical reactions. However, the mechanism regulating starch biosynthesis in cereal seeds is not well understood. This study showed that OsbZIP58, a bZIP transcription factor, is a key transcriptional regulator controlling starch synthesis in rice endosperm. OsbZIP58 was expressed mainly in endosperm during active starch synthesis. osbzip58 null mutants displayed abnormal seed morphology with altered starch accumulation in the white belly region and decreased amounts of total starch and amylose. Moreover, osbzip58 had a higher proportion of short chains and a lower proportion of intermediate chains of amylopectin. Furthermore, OsbZIP58 was shown to bind directly to the promoters of six starch-synthesizing genes, OsAGPL3, Wx, OsSSIIa, SBE1, OsBEIIb, and ISA2, and to regulate their expression. These findings indicate that OsbZIP58 functions as a key regulator of starch synthesis in rice seeds and provide new insights into seed quality control. ALK|SSIIa,OsBEIIb,OsISA2,RISBZ1|OsbZIP58,BEI|SBE1,Wx Double repression of soluble starch synthase genes SSIIa and SSIIIa in rice (Oryza sativa L.) uncovers interactive effects on the physicochemical properties of starch 2011 Genome Institute of Crop Science and the National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing 100081, China. Soluble starch synthases (SSs) are major enzymes involved in starch biosynthesis in developing rice (Oryza sativa L.) endosperm. Despite extensive studies of SSs in various plant species including rice, the functional modes of action among multiple SS genes are still not clear. Here, we generated transgenic RNA interference (RNAi) repressed lines for seven of the eight members of the rice SS gene family and studied their effects on starch synthesis and grain formation. Consistent with their expression domains, RNAi repression of genes that encode isozymes SSI, SSIIa, and SSIIIa had strong effects on grain development, whereas no obvious phenotypic changes were observed in transgenic plants with the other SS genes being RNAi repressed, indicating functional redundancies among the genes. To study the potential functional interactions of SS genes, we generated SSIIa/SSIIIa double repression lines whose kernels displayed a chalky kernel appearance and had increased amylose levels, increased pasting temperatures, and decreased viscosities. The double mutation also reduced short (degree of polymerization (DP) 5-6) and long (DP 12-23) amylopectin chain contents in the grain and increased the medium long types (DP 7-11). The nonadditive nature of the double mutation line suggests that SSIIa and SSIIIa interact with each other during starch synthesis. Such interaction may be physical via starch phophorylase as indicated by our pair-wise yeast two-hybrid assays on major starch synthesis enzymes. Collectively, the data showed that SSIIa and SSIIIa play distinctive, but partially overlapping, roles during rice grain starch synthesis. The possibility of extensive redundancy or complementarity among SS isozymes is discussed. ALK|SSIIa,OsSSIIIa|Flo5 Compensation and interaction between RISBZ1 and RPBF during grain filling in rice 2009 Plant J Transgenic Crop Research and Development Centre, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. The rice (Oryza sativa L.) basic leucine Zipper factor RISBZ1 and rice prolamin box binding factor (RPBF) are transcriptional activators of rice seed storage protein (SSP) genes in vivo. To ascertain the functions of these trans-activators in seed development, knock-down (KD) transgenic rice plants were generated in which the accumulation of RISBZ1 and RPBF was reduced in an endosperm-specific manner by co-suppression (KD-RISBZ1 and KD-RPBF). The accumulation of most SSPs changed little between individual KD mutants and wild-type plants, whereas a double KD mutant (KD-RISBZ1/KD-RPBF) resulted in a significant reduction of most SSP gene expression and accumulation. The reduction of both trans-activators also caused a greater reduction in seed starch accumulation than individual KD mutants. Storage lipids were accumulated at reduced levels in KD-RISBZ1 and KD-RISBZ1/KD-RPBF seeds. KD-RPBF and KD-RISBZ1/KD-RPBF seeds exhibited multi-layered aleurone cells. Gene expression of DEFECTIVE KERNEL1 (OsDEK1), CRINKLY4 (OsCR4) and SUPERNUMERARY ALEURONE LAYER 1 (OsSAL1) rice homologues was decreased in the KD mutants, suggesting that these genes are regulated by RISBZ1 and RPBF. These phenotypes suggest that combinatorial interactions between RISBZ1 and RPBF play an essential role during grain filling. The functional redundancy and compensation between RISBZ1 and RPBF possibly account for weak effects on the SSP levels in single KD mutants, and help maintain various processes during seed development in rice. Physical interaction between RISBZ1 and RPBF may ensure that these processes are carried out properly. ALK|SSIIa,GluA,GluD,OsAPL2|osagpl2-3,OsAPS2|OsAGPS2b,OsCR4,ADL1|OsDEK1,RAG1,OsSAL1|RHL,RISBZ1|OsbZIP58,RPBF|OsDof3 Association between nonsynonymous mutations of starch synthase IIa and starch quality in rice (Oryza sativa) 2011 New Phytol Department of Biology, Washington University of St. Louis, Missouri, 63130, USA Present address: Institute of Bioscience and Biotechnology Research, University of Maryland, College Park, Maryland, 20742, USA. guoqinyu@umd.edu Starch quality is one of the most important agronomic traits in Asian rice, Oryza sativa. Starch synthase IIa (SsIIa) is a major candidate gene for starch quality variation. Within SsIIa, three nonsynonymous mutations in exon 8 have been shown to affect enzyme activity when expressed in Escherichia coli. To search for the variation in SsIIa that is responsible for starch quality variation in rice, we sequenced the SsIIa exon 8 region and measured starch quality as starch disintegration in alkali for 289 accessions of cultivated rice and 57 accessions of its wild ancestor, Oryza rufipogon. A general linear model and nested clade analysis were used to identify the associations between the three nonsynonymous single nucleotide polymorphisms (SNPs) and starch quality. Among the three nonsynonymous SNPs, we found strong evidence of association at one nucleotide site ('SNP 3'), corresponding to a Leu/Phe replacement at codon 781. A second SNP, corresponding to a Val/Met replacement at codon 737, could potentially show an association with increased sample sizes. Variation in SsIIa enzyme activity is associated with the cohesiveness of rice grains when cooked, and our findings are consistent with selection for more cohesive grains during the domestication of tropical japonica rice. ALK|SSIIa ALK, the key gene for gelatinization temperature, is a modifier gene for gel consistency in rice 2011 J Integr Plant Biol State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Science, Hangzhou 310006, China. Gelatinization temperature (GT) is an important parameter in evaluating the cooking and eating quality of rice. Indeed, the phenotype, biochemistry and inheritance of GT have been widely studied in recent times. Previous map-based cloning revealed that GT was controlled by ALK gene, which encodes a putative soluble starch synthase II-3. Complementation vector and RNAi vector were constructed and transformed into Nipponbare mediated by Agrobacterium. Phenotypic and molecular analyses of transgenic lines provided direct evidence for ALK as a key gene for GT. Meanwhile, amylose content, gel consistency and pasting properties were also affected in transgenic lines. Two of four nonsynonymous single nucleotide polymorphisms in coding sequence of ALK were identified as essential for GT. Based on the single nucleotide polymorphisms (SNPs), two new sets of SNP markers combined with one cleaved amplified polymorphic sequence marker were developed for application in rice quality breeding. ALK|SSIIa SNP in starch biosynthesis genes associated with nutritional and functional properties of rice 2012 Sci Rep Southern Cross Plant Science, Southern Cross University, Lismore, NSW 2480, Australia. Starch is a major component of human diets. The relative contribution of variation in the genes of starch biosynthesis to the nutritional and functional properties of the rice was evaluated in a rice breeding population. Sequencing 18 genes involved in starch synthesis in a population of 233 rice breeding lines discovered 66 functional SNPs in exonic regions. Five genes, AGPS2b, Isoamylase1, SPHOL, SSIIb and SSIVb showed no polymorphism. Association analysis found 31 of the SNP were associated with differences in pasting and cooking quality properties of the rice lines. Two genes appear to be the major loci controlling traits under human selection in rice, GBSSI (waxy gene) and SSIIa. GBSSI influenced amylose content and retrogradation. Other genes contributing to retrogradation were GPT1, SSI, BEI and SSIIIa. SSIIa explained much of the variation in cooking characteristics. Other genes had relatively small effects. ALK|SSIIa Conservation and implications of eukaryote transcriptional regulatory regions across multiple species 2008 BMC Genomics School of Mathematical Sciences, Peking University, Beijing 100871, PR China Background. Increasing evidence shows that whole genomes of eukaryotes are almost entirely transcribed into both protein coding genes and an enormous number of non-protein-coding RNAs (ncRNAs). Therefore, revealing the underlying regulatory mechanisms of transcripts becomes imperative. However, for a complete understanding of transcriptional regulatory mechanisms, we need to identify the regions in which they are found. We will call these transcriptional regulation regions, or TRRs, which can be considered functional regions containing a cluster of regulatory elements that cooperatively recruit transcriptional factors for binding and then regulating the expression of transcripts. Results. We constructed a hierarchical stochastic language (HSL) model for the identification of core TRRs in yeast based on regulatory cooperation among TRR elements. The HSL model trained based on yeast achieved comparable accuracy in predicting TRRs in other species, e.g., fruit fly, human, and rice, thus demonstrating the conservation of TRRs across species. The HSL model was also used to identify the TRRs of genes, such as p53 or OsALYL1, as well as microRNAs. In addition, the ENCODE regions were examined by HSL, and TRRs were found to pervasively locate in the genomes. Conclusion. Our findings indicate that 1) the HSL model can be used to accurately predict core TRRs of transcripts across species and 2) identified core TRRs by HSL are proper candidates for the further scrutiny of specific regulatory elements and mechanisms. Meanwhile, the regulatory activity taking place in the abundant numbers of ncRNAs might account for the ubiquitous presence of TRRs across the genome. In addition, we also found that the TRRs of protein coding genes and ncRNAs are similar in structure, with the latter being more conserved than the former. OsALYL1 An-1 encodes a basic helix-loop-helix protein that regulates awn development, grain size, and grain number in rice 2013 Plant Cell National Center for Gene Research, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200233, China. Long awns are important for seed dispersal in wild rice (Oryza rufipogon), but are absent in cultivated rice (Oryza sativa). The genetic mechanism involved in loss-of-awn in cultivated rice remains unknown. We report here the molecular cloning of a major quantitative trait locus, An-1, which regulates long awn formation in O. rufipogon. An-1 encodes a basic helix-loop-helix protein, which regulates cell division. The nearly-isogenic line (NIL-An-1) carrying a wild allele An-1 in the genetic background of the awnless indica Guangluai4 produces long awns and longer grains, but significantly fewer grains per panicle compared with Guangluai4. Transgenic studies confirmed that An-1 positively regulates awn elongation, but negatively regulates grain number per panicle. Genetic variations in the An-1 locus were found to be associated with awn loss in cultivated rice. Population genetic analysis of wild and cultivated rice showed a significant reduction in nucleotide diversity of the An-1 locus in rice cultivars, suggesting that the An-1 locus was a major target for artificial selection. Thus, we propose that awn loss was favored and strongly selected by humans, as genetic variations at the An-1 locus that cause awn loss would increase grain numbers and subsequently improve grain yield in cultivated rice. An-1 Retrograde regulation of nuclear gene expression in CW-CMS of rice 2007 Plant Mol Biol Laboratory of Environmental Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. The CW-cytoplasmic male sterility (CMS) line has the cytoplasm of Oryza rufipogon Griff, and mature pollen is morphologically normal under an optical microscope but lacks the ability to germinate; restorer gene Rf17 has been identified as restoring this ability. The difference between nuclear gene expression in mature anthers was compared for the CW-CMS line, [cms-CW] rf17rf17, and a maintainer line with normal cytoplasm of Oryza sativa L., [normal] rf17rf17. Using a 22-k rice oligoarray we detected 58 genes that were up-regulated more than threefold in the CW-CMS line. Expression in other organs was further investigated for 20 genes using RT-PCR. Five genes, including genes for alternative oxidase, were found to be preferentially expressed in [cms-CW] rf17rf17 but not in [normal] rf17rf17 or [cms-CW] Rf17Rf17. Such [cms-CW] rf17rf17-specific gene expression was only observed in mature anthers but not in leaves, stems, or roots, indicating the presence of anther-specific mitochondrial retrograde regulation of nuclear gene expression, and that Rf17 has a role in restoring the ectopic gene expression. We also used a proteomic approach to discover the retrograde regulated proteins and identified six proteins that were accumulated differently. These results reveal organ-specific induced mitochondrial retrograde pathways affecting nuclear gene expression possibly related to CMS. OsAOX1c|AOX1c Unravelling mitochondrial retrograde regulation in the abiotic stress induction of rice ALTERNATIVE OXIDASE 1 genes 2013 Plant Cell Environ Institute of Rice Research, Anhui Academy of Agricultural Science, Hefei 230031, China. Mitochondrial retrograde regulation (MRR) is the transduction of mitochondrial signals to mediate nuclear gene expression. It is not clear whether MRR is a common regulation mechanism in plant abiotic stress response. In this study, we analysed the early abiotic stress response of the rice OsAOX1 genes, and the induction of OsAOX1a and OsAOX1b (OsAOX1a/b) was selected as a working model for the stress-induced MRR studies. We found that the induction mediated by the superoxide ion (O2.(-) )-generating chemical methyl viologen was stronger than that of hydrogen peroxide (H2 O2 ). The addition of reactive oxygen species (ROS) scavengers demonstrated that the stress induction was reduced by eliminating O2.(-) . Furthermore, the stress induction did not rely on chloroplast- or cytosol-derived O2.(-) . Next, we generated transgenic plants overexpressing the superoxide dismutase (SOD) gene at different subcellular locations. The results suggest that only the mitochondrial SOD, OsMSD, attenuated the stress induction of OsAOX1a/b specifically. Therefore, our findings demonstrate that abiotic stress initiates the MRR on OsAOX1a/b and that mitochondrial O2.(-) is involved in the process. OsAOX1c|AOX1c,AOX1a|OsAOX1a,OsAOX1b,OsMSD AOX1c, a novel rice gene for alternative oxidase; comparison with rice AOX1a and AOX1b 2002 Genes Genet Syst Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Japan. A novel gene for alternative oxidase (AOX) was isolated from rice (Oryza sativa L.) and characterized. The deduced amino acid sequence of the novel AOX gene contains features that are conserved among other AOXs. This AOX gene was designated AOX1c based on a phylogenetic analysis of the AOX genes. Northern hybridization analyses revealed that AOX1c and AOX1a/AOX1b transcripts accumulated differently in various rice organs and rice seedlings under low temperature conditions. AOX1c mRNA was mainly present in young leaves under constant light, mature leaves and panicles after heading, but it was not detected in young etiolated leaves and young roots of seedlings or young panicles. On the other hand, the mRNAs of the rice AOX1a and AOX1b genes were mainly present in young roots and mature leaves. Under low temperature conditions, the steady-state mRNA levels of the rice AOX1a and AOX1b genes clearly increased with time but the rice AOX1c gene was apparently not responsive to low temperature. The rice AOX gene family and differences in their regulation are discussed. OsAOX1c|AOX1c The Magnaporthe oryzae effector AvrPiz-t targets the RING E3 ubiquitin ligase APIP6 to suppress pathogen-associated molecular pattern-triggered immunity in rice 2012 Plant Cell Department of Plant Pathology, Ohio State University, Columbus, OH 43210, USA. Although the functions of a few effector proteins produced by bacterial and oomycete plant pathogens have been elucidated in recent years, information for the vast majority of pathogen effectors is still lacking, particularly for those of plant-pathogenic fungi. Here, we show that the avirulence effector AvrPiz-t from the rice blast fungus Magnaporthe oryzae preferentially accumulates in the specialized structure called the biotrophic interfacial complex and is then translocated into rice (Oryza sativa) cells. Ectopic expression of AvrPiz-t in transgenic rice suppresses the flg22- and chitin-induced generation of reactive oxygen species (ROS) and enhances susceptibility to M. oryzae, indicating that AvrPiz-t functions to suppress pathogen-associated molecular pattern (PAMP)-triggered immunity in rice. Interaction assays show that AvrPiz-t suppresses the ubiquitin ligase activity of the rice RING E3 ubiquitin ligase APIP6 and that, in return, APIP6 ubiquitinates AvrPiz-t in vitro. Interestingly, agroinfection assays reveal that AvrPiz-t and AvrPiz-t Interacting Protein 6 (APIP6) are both degraded when coexpressed in Nicotiana benthamiana. Silencing of APIP6 in transgenic rice leads to a significant reduction of flg22-induced ROS generation, suppression of defense-related gene expression, and enhanced susceptibility of rice plants to M. oryzae. Taken together, our results reveal a mechanism in which a fungal effector targets the host ubiquitin proteasome system for the suppression of PAMP-triggered immunity in plants. APIP6 ARAG1, an ABA-responsive DREB gene, plays a role in seed germination and drought tolerance of rice 2010 Ann Bot Research Center for Molecular & Developmental Biology, Key Laboratory of Photosynthesis & Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. BACKGROUND AND AIMS: DREB proteins are involved mainly in plant responses to abiotic stresses such as cold, drought or high salinity as well as ABA signalling. However, the function of most rice DREB genes and the underlying molecular mechanisms controlling these responses remains elusive. In this study, ARAG1, a rice DREB gene, was functionally analysed. METHODS: Antisense and over-expression constructs of ARAG1 were introduced into rice by an Agrobacterium-mediated method. RT-PCR and western blot were used to detect ARAG1 accumulation in transgenics. PEG and ABA were used to test their response to abiotic stresses. KEY RESULTS: ARAG1 was expressed in inflorescences, roots, immature embryos and germinating seeds, but not in coleoptiles, leaves or mature embryos. Drought stress and ABA treatment increased transcript levels of the gene rapidly. ARAG1 knockdown line was hypersensitive to ABA application during seed germination and seedling growth. However, the line over-expressing ARAG1 behaved similarly to wild type in these circumstances. Knockdown of ARAG1 weakened tolerance of the transgenic seedlings to drought stress, while over-expression of it increased the tolerance slightly. In addition, activity of alpha-amylases was enhanced in germinating seeds of the knockdown and over-expression lines. CONCLUSIONS: These results indicate that ARAG1 was involved in the ABA signalling and stress responsive pathways. ARAG1 'Evidence of an auxin signal pathway, microRNA167-ARF8-GH3, and its response to exogenous auxin in cultured rice cells' 2006 Nucleic Acids Res Department of Biological Science and the Basic Science Research Institute, Sungkyunkwan University, Suwon, Korea 440-746. MicroRNA167 (miR167) was shown to cleave auxin responsive factor 8 (ARF8) mRNA in cultured rice cells. MiR167 level was found to be controlled by the presence of auxin in the growth medium. When cells grew in auxin-free medium, miR167 level decreased, resulting in an increase in the level of ARF8 mRNA. Cells growing in the normal growth medium containing auxin showed a reversed trend. It was also shown that expression of OsGH3-2, an rice IAA-conjugating enzyme, was positively regulated by ARF8. Delivery of synthesized miR167 into cells led to decrease of both ARF8 mRNA and OsGH3-2 mRNA. This study provides an evidence in which the exogeneous auxin signal is transduced to OsGH3-2 through miR167 and ARF8 in sequence. This proposed auxin signal transduction pathway, auxin-miR167-ARF8-OsGH3-2, could be, in conjunction with the other microRNA-mediated auxin signals, an important one for responding to exogeneous auxin and for determining the cellular free auxin level which guides appropriate auxin responses. ARF8,OsGH3-2 Identification of a cis-acting element of ART1, a C2H2-type zinc-finger transcription factor for aluminum tolerance in rice 2011 Plant Physiol Institute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki 710-0046, Japan. Rice (Oryza sativa) is one of the most aluminum (Al)-tolerant species among small-grain cereals. Recent identification of a transcription factor AL RESISTANCE TRANSCRIPTION FACTOR1 (ART1) revealed that this high Al tolerance in rice is achieved by multiple genes involved in detoxification of Al at different cellular levels. ART1 is a C2H2-type zinc-finger transcription factor and regulates the expression of 31 genes in the downstream. In this study, we attempted to identify a cis-acting element of ART1. We used the promoter region of SENSITIVE TO AL RHIZOTOXICITY1, an Al tolerance gene in the downstream of ART1. With the help of gel-shift assay, we were able to identify the cis-acting element as GGN(T/g/a/C)V(C/A/g)S(C/G). This element was found in the promoter region of 29 genes among 31 ART1-regulated genes. To confirm this cis-acting element in vivo, we transiently introduced this element one or five times tandemly repeated sequence with 35S minimal promoter and green fluorescent protein reporter together with or without ART1 gene in the tobacco (Nicotiana tabacum) mesophyll protoplasts. The results showed that the expression of green fluorescent protein reporter responded to ART1 expression. Furthermore, the expression increased with repetition of the cis-acting element. Our results indicate that the five nucleotides identified are the target DNA-binding sequence of ART1. ART1 An Al-inducible MATE gene is involved in external detoxification of Al in rice 2011 Plant J Institute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki 710-0046, Japan. A number of plant species, including rice, secretes citrate from roots in response to Al stress. Here we characterized the functions of a gene, OsFRDL4 (Os01g0919100) that belongs to the multidrug and toxic compound extrusion (MATE) family in rice (Oryza sativa). Heterologous expression in Xenopus oocyte showed that the OsFRDL4 protein was able to transport citrate and was activated by Al. The expression level of the OsFRDL4 gene in roots was very low in the absence of Al, but was greatly enhanced by Al after short exposure. Furthermore, the OsFRDL4 expression was regulated by ART1, a C2H2-type zinc finger transcription factor for Al tolerance. Transient expression of OsFRDL4 in onion epidermal cells showed that it localized to the plasma membrane. Immunostaining showed that OsFRDL4 was localized in all cells in the root tip. These expression patterns and cell specificity of localization of OsFRDL4 are different from other MATE members identified previously. Knockout of OsFRDL4 resulted in decreased Al tolerance and decreased citrate secretion compared with the wild-type rice, but did not affect citrate concentration in the xylem sap. Furthermore, there is a positive correlation between OsFRDL4 expression level and the amount of citrate secretion in rice cultivars that are differing in Al tolerance. Taken together, our results show that OsFRDL4 is an Al-induced citrate transporter localized at the plasma membrane of rice root cells and is one of the components of high Al tolerance in rice. ART1,OsFRDL4 A zinc finger transcription factor ART1 regulates multiple genes implicated in aluminum tolerance in rice 2009 Plant Cell Research Institute for Bioresources, Okayama University, Kurashiki 710-0046, Japan. Aluminum (Al) toxicity is the major limiting factor of crop production on acid soils, but some plant species have evolved ways of detoxifying Al. Here, we report a C2H2-type zinc finger transcription factor ART1 (for Al resistance transcription factor 1), which specifically regulates the expression of genes related to Al tolerance in rice (Oryza sativa). ART1 is constitutively expressed in the root, and the expression level is not affected by Al treatment. ART1 is localized in the nucleus of all root cells. A yeast one-hybrid assay showed that ART1 has a transcriptional activation potential and interacts with the promoter region of STAR1, an important factor in rice Al tolerance. Microarray analysis revealed 31 downstream transcripts regulated by ART1, including STAR1 and 2 and a couple of homologs of Al tolerance genes in other plants. Some of these genes were implicated in both internal and external detoxification of Al at different cellular levels. Our findings shed light on comprehensively understanding how plants detoxify aluminum to survive in an acidic environment. ART1,STAR1,STAR2 Plasma membrane-localized transporter for aluminum in rice 2010 Proc Natl Acad Sci U S A Institute for Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan. Aluminum (Al) is the most abundant metal in the Earth's crust, but its trivalent ionic form is highly toxic to all organisms at low concentrations. How Al enters cells has not been elucidated in any organisms. Herein, we report a transporter, Nrat1 (Nramp aluminum transporter 1), specific for trivalent Al ion in rice. Nrat1 belongs to the Nramp (natural resistance-associated macrophage protein) family, but shares a low similarity with other Nramp members. When expressed in yeast, Nrat1 transports trivalent Al ion, but not other divalent ions, such as manganese, iron, and cadmium, or the Al-citrate complex. Nrat1 is localized at the plasma membranes of all cells of root tips except epidermal cells. Knockout of Nrat1 resulted in decreased Al uptake, increased Al binding to cell wall, and enhanced Al sensitivity, but did not affect the tolerance to other metals. Expression of Nrat1 is up-regulated by Al in the roots and regulated by a C2H2 zinc finger transcription factor (ART1). We therefore concluded that Nrat1 is a plasma membrane-localized transporter for trivalent Al, which is required for a prior step of final Al detoxification through sequestration of Al into vacuoles. ART1,Nrat1 Disruption of the rice plastid ribosomal protein s20 leads to chloroplast developmental defects and seedling lethality 2013 G3 (Bethesda) Development Center of Plant Germplasm Resources, College of Life and Environmental Sciences, Shanghai Normal University, Shanghai 200234. Plastid ribosomal proteins (PRPs) are essential for ribosome biogenesis, plastid protein biosynthesis, chloroplast differentiation, and early chloroplast development. This study identifies the first rice PRP mutant, asl1 (albino seedling lethality1), which exhibits an albino lethal phenotype at the seedling stage. This albino phenotype was associated with altered chlorophyll (Chl) content and chloroplast development. Map-based cloning revealed that ASL1 encodes PRP S20 (PRPS20), which localizes to the chloroplast. ASL1 showed tissue-specific expression, as it was highly expressed in plumule and young seedlings but expressed at much lower levels in other tissues. In addition, ASL1 expression was regulated by light. The transcript levels of nuclear genes for Chl biosynthesis and chloroplast development were strongly affected in asl1 mutants; transcripts of some plastid genes for photosynthesis were undetectable. Our findings indicate that nuclear-encoded PRPS20 plays an important role in chloroplast development in rice. ASL1 Mutation of the rice ASL2 gene encoding plastid ribosomal protein L21 causes chloroplast developmental defects and seedling death 2014 Plant Biol (Stuttg) Development Center of Plant Germplasm Resources, College of Life and Environment Sciences, Shanghai Normal University, Shanghai, China. The plastid ribosome proteins (PRPs) play important roles in plastid protein biosynthesis, chloroplast differentiation and early chloroplast development. However, the specialised functions of individual protein components of the chloroplast ribosome in rice (Oryza sativa) remain unresolved. In this paper, we identified a novel rice PRP mutant named asl2 (Albino seedling lethality 2) exhibiting an albino, seedling death phenotype. In asl2 mutants, the alteration of leaf colour was associated with chlorophyll (Chl) content and abnormal chloroplast development. Through map-based cloning and complementation, the mutated ASL2 gene was isolated and found to encode the chloroplast 50S ribosome protein L21 (RPL21c), a component of the chloroplast ribosome large subunit, which was localised in chloroplasts. ASL2 was expressed at a higher level in the plumule and leaves, implying its tissue-specific expression. Additionally, the expression of ASL2 was regulated by light. The transcript levels of the majority of genes for Chl biosynthesis, photosynthesis and chloroplast development were strongly affected in asl2 mutants. Collectively, the absence of functional ASL2 caused chloroplast developmental defects and seedling death. This report establishes the important role of RPL21c in chloroplast development in rice. ASL2 Structure, allelic diversity and selection of Asr genes, candidate for drought tolerance, in Oryza sativa L. and wild relatives 2010 Theor Appl Genet CIRAD, UMR Developpement et Amelioration des Plantes, TA-A 96/03, 34398, Montpellier, France. rphilippe@clermont.inra.fr Asr (ABA, stress, ripening) genes represent a small gene family potentially involved in drought tolerance in several plant species. To analyze their interest for rice breeding for water-limited environments, this gene family was characterized further. Genomic organization of the gene family reveals six members located on four different chromosomes and with the same exon-intron structure. The maintenance of six members of the Asr gene family, which are the result of combination between tandem duplication and whole genome duplication, and their differential regulation under water stress, involves probably some sub-functionalization. The polymorphism of four members was studied in a worldwide collection of 204 accessions of Oryza sativa L. and 14 accessions of wild relatives (O. rufipogon and O. nivara). The nucleotide diversity of the Asr genes was globally low, but contrasted for the different genes, leading to different shapes of haplotype networks. Statistical tests for neutrality were used and compared to their distribution in a set of 111 reference genes spread across the genome, derived from another published study. Asr3 diversity exhibited a pattern concordant with a balancing selection at the species level and with a directional selection in the tropical japonica sub-group. This study provides a thorough description of the organization of the Asr family, and the nucleotide and haplotype diversity of four Asr in Oryza sativa species. Asr3 stood out as the best potential candidate. The polymorphism detected here represents a first step towards an association study between genetic polymorphisms of this gene family and variation in drought tolerance traits. Asr1,Asr2,Asr4,Asr6,ASR5|OsAsr1,Asr3|OsASR3 Involvement of ASR genes in aluminium tolerance mechanisms in rice 2013 Plant Cell Environ Programa de Pos-Graduacao em Genetica e Biologia Molecular Programa de Pos-Graduacao em Biologia Celular e Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil. Among cereal crops, rice is considered the most tolerant to aluminium (Al). However, variability among rice genotypes leads to remarkable differences in the degree of Al tolerance for distinct cultivars. A number of studies have demonstrated that rice plants achieve Al tolerance through an unknown mechanism that is independent of root tip Al exclusion. We have analysed expression changes of the rice ASR gene family as a function of Al treatment. The gene ASR5 was differentially regulated in the Al-tolerant rice ssp. Japonica cv. Nipponbare. However, ASR5 expression did not respond to Al exposure in Indica cv. Taim rice roots, which are highly Al sensitive. Transgenic plants carrying RNAi constructs that targeted the ASR genes were obtained, and increased Al susceptibility was observed in T1 plants. Embryogenic calli of transgenic rice carrying an ASR5-green fluorescent protein fusion revealed that ASR5 was localized in both the nucleus and cytoplasm. Using a proteomic approach to compare non-transformed and ASR-RNAi plants, a total of 41 proteins with contrasting expression patterns were identified. We suggest that the ASR5 protein acts as a transcription factor to regulate the expression of different genes that collectively protect rice cells from Al-induced stress responses. Asr1,Asr2,Asr4,Asr6,ASR5|OsAsr1 Expression of salt-induced 2-Cys peroxiredoxin from Oryza sativa increases stress tolerance and fermentation capacity in genetically engineered yeast Saccharomyces cerevisiae 2013 Appl Microbiol Biotechnol Advanced Bio-resource Research Center, Department of Biology, College of Natural Sciences, Kyungpook National University, #1370 Sankyuk-dong, Buk-gu, Daegu 702-701, Republic of Korea. 92kis@hanmail.net Peroxiredoxins (Prxs), also termed thioredoxin peroxidases (TPXs), are a family of thiol-specific antioxidant enzymes that are critically involved in cell defense and protect cells from oxidative damage. In this study, a putative chloroplastic 2-Cys thioredoxin peroxidase (OsTPX) was identified by proteome analysis from leaf tissue samples of rice (Oryza sativa) seedlings exposed to 0.1 M NaCl for 3 days. To investigate the relationship between the OsTPX gene and the stress response, OsTPX was cloned into the yeast expression vector p426GPD under the control of the glyceraldehyde-3-phosphate dehydrogenase (GPD1) promoter, and the construct was transformed into Saccharomyces cerevisiae cells. OsTPX expression was confirmed by semi-quantitative reverse transcription-polymerase chain reaction and western blot analyses. OsTPX contained two highly conserved cysteine residues (Cys114 and Cys236) and an active site region (FTFVCPT), and it is structurally very similar to human 2-Cys Prx. Heterologous OsTPX expression increased the ability of the transgenic yeast cells to adapt and recover from reactive oxygen species (ROS)-induced oxidative stresses, such as a reduction of cellular hydroperoxide levels in the presence of hydrogen peroxide and menadione, by improving redox homeostasis. OsTPX expression also conferred enhanced tolerance to tert-butylhydroperoxide, heat shock, and high ethanol concentrations. Furthermore, high OsTPX expression improved the fermentation capacity of the yeast during glucose-based batch fermentation at a high temperature (40 degrees C) and at the general cultivation temperature (30 degrees C). The alcohol yield in OsTPX-expressing transgenic yeast increased by approximately 29 % (0.14 g g(-1)) and 21 % (0.12 g g(-1)) during fermentation at 40 and 30 degrees C, respectively, compared to the wild-type yeast. Accordingly, OsTPX-expressing transgenic yeast showed prolonged cell survival during the environmental stresses produced during fermentation. These results suggest that heterologous OsTPX expression increases acquired tolerance to ROS-induced oxidative stress by improving cellular redox homeostasis and improves fermentation capacity due to improved cell survival during fermentation, especially at a high temperature. BAS1|OsTPX A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes 2011 Plant Methods State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, P, R, China. wanghb@mail.sysu.edu.cn. BACKGROUND: Plant protoplasts, a proven physiological and versatile cell system, are widely used in high-throughput analysis and functional characterization of genes. Green protoplasts have been successfully used in investigations of plant signal transduction pathways related to hormones, metabolites and environmental challenges. In rice, protoplasts are commonly prepared from suspension cultured cells or etiolated seedlings, but only a few studies have explored the use of protoplasts from rice green tissue. RESULTS: Here, we report a simplified method for isolating protoplasts from normally cultivated young rice green tissue without the need for unnecessary chemicals and a vacuum device. Transfections of the generated protoplasts with plasmids of a wide range of sizes (4.5-13 kb) and co-transfections with multiple plasmids achieved impressively high efficiencies and allowed evaluations by 1) protein immunoblotting analysis, 2) subcellular localization assays, and 3) protein-protein interaction analysis by bimolecular fluorescence complementation (BiFC) and firefly luciferase complementation (FLC). Importantly, the rice green tissue protoplasts were photosynthetically active and sensitive to the retrograde plastid signaling inducer norflurazon (NF). Transient expression of the GFP-tagged light-related transcription factor OsGLK1 markedly upregulated transcript levels of the endogeneous photosynthetic genes OsLhcb1, OsLhcp, GADPH and RbcS, which were reduced to some extent by NF treatment in the rice green tissue protoplasts. CONCLUSIONS: We show here a simplified and highly efficient transient gene expression system using photosynthetically active rice green tissue protoplasts and its broad applications in protein immunoblot, localization and protein-protein interaction assays. These rice green tissue protoplasts will be particularly useful in studies of light/chloroplast-related processes. BAS1|OsTPX,GADPH,CAB2R The GA octodinucleotide repeat binding factor BBR participates in the transcriptional regulation of the homeobox geneBkn3 2003 The Plant Journal Max-Planck-Institut für Züchtungsforschung, Department of Plant Breeding and Yield Physiology, Carl-von-Linné-Weg 10, 50829 Köln, Germany. In the dominant mutant Hooded (K), the barley gene BKn3 is overexpressed as a result of a duplication of 305 bp in intron IV. When fused to a cauliflower mosaic virus 35S minimal promoter, the 305 bp element activates gene expression in tobacco, as does a 655 bp BKn3 promoter sequence. Both DNA fragments contain a (GA)8 repeat (GA/TC)8. A one-hybrid screen using the 305 bp element as the DNA target led to the cloning of the barley b recombinant (BBR) protein, which binds specifically to the (GA/TC)8 repeat. BBR is nuclear targeted and is a characterized nuclear localization signal (NLS) sequence, a DNA-binding domain extended up to 90 aa at the C-terminus and a putative N-terminal activation domain. The corresponding gene has no introns and is ubiquitously expressed in barley tissues. In co-transfection experiments, BBR activates (GA/TC)8-containing promoters, and its overexpression in tobacco leads to a pronounced leaf shape modification. BBR has properties of a GAGA-binding factor, but the corresponding gene has no sequence homology to Trl and Psq of Drosophila, which encode functionally analogous proteins. In Arabidopsis, (GA/TC)8 repeats occur particularly within 1500 bp upstream of gene start codons included in some homeodomain genes of different classes. The data presented suggest that expression of the barley BKn3 is regulated, at least in part, by the binding of the transcription factor BBR to GA/TC repeats. BBR Rice Brittle culm 6 encodes a dominant-negative form of CesA protein that perturbs cellulose synthesis in secondary cell walls 2011 J Exp Bot Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan. The brittle culm (bc) mutants of Gramineae plants having brittle skeletal structures are valuable materials for studying secondary cell walls. In contrast to other recessive bc mutants, rice Bc6 is a semi-dominant bc mutant with easily breakable plant bodies. In this study, the Bc6 gene was cloned by positional cloning. Bc6 encodes a cellulose synthase catalytic subunit, OsCesA9, and has a missense mutation in its highly conserved region. In culms of the Bc6 mutant, the proportion of cellulose was reduced by 38%, while that of hemicellulose was increased by 34%. Introduction of the semi-dominant Bc6 mutant gene into wild-type rice significantly reduced the percentage of cellulose, causing brittle phenotypes. Transmission electron microscopy analysis revealed that Bc6 mutation reduced the cell wall thickness of sclerenchymal cells in culms. In rice expressing a reporter construct, BC6 promoter activity was detected in the culms, nodes, and flowers, and was localized primarily in xylem tissues. This expression pattern was highly similar to that of BC1, which encodes a COBRA-like protein involved in cellulose synthesis in secondary cell walls in rice. These results indicate that BC6 is a secondary cell wall-specific CesA that plays an important role in proper deposition of cellulose in the secondary cell walls. BC1,Bc6|OsCesA9 BRITTLE CULM1, Which Encodes a COBRA-Like Protein, Affects the Mechanical Properties of Rice Plants 2003 The Plant Cell Online Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Plant mechanical strength is an important agronomic trait. To understand the molecular mechanism that controls the plant mechanical strength of crops, we characterized the classic rice mutant brittle culm1 (bc1) and isolated BC1 using a map-based cloning approach. BC1, which encodes a COBRA-like protein, is expressed mainly in developing sclerenchyma cells and in vascular bundles of rice. In these types of cells, mutations in BC1 cause not only a reduction in cell wall thickness and cellulose content but also an increase in lignin level, suggesting that BC1, a gene that controls the mechanical strength of monocots, plays an important role in the biosynthesis of the cell walls of mechanical tissues. BC1 The carbohydrate-binding module (CBM)-like sequence is crucial for rice CWA1/BC1 function in proper assembly of secondary cell wall materials 2010 Plant Signal Behav Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Japan We recently reported that the cwa1 mutation disturbed the deposition and assembly of secondary cell wall materials in the cortical fiber of rice internodes. Genetic analysis revealed that cwa1 is allelic to bc1, which encodes glycosylphosphatidylinositol (GPI)-anchored COBRA-like protein with the highest homology to Arabidopsis COBRA-like 4 (COBL4) and maize Brittle Stalk 2 (Bk2). Our results suggested that CWA1/BC1 plays a role in assembling secondary cell wall materials at appropriate sites, enabling synthesis of highly ordered secondary cell wall structure with solid and flexible internodes in rice. The N-terminal amino acid sequence of CWA1/BC1, as well as its orthologs (COBL4, Bk2) and other BC1-like proteins in rice, shows weak similarity to a family II carbohydrate-binding module (CBM2) of several bacterial cellulases. To investigate the importance of the CBM-like sequence of CWA1/BC1 in the assembly of secondary cell wall materials, Trp residues in the CBM-like sequence, which is important for carbohydrate binding, were substituted for Val residues and introduced into the cwa1 mutant. CWA1/BC1 with the mutated sequence did not complement the abnormal secondary cell walls seen in the cwa1 mutant, indicating that the CBM-like sequence is essential for the proper function of CWA1/BC1, including assembly of secondary cell wall materials. BC1 Isolation of a novel cell wall architecture mutant of rice with defective Arabidopsis COBL4 ortholog BC1 required for regulated deposition of secondary cell wall components 2010 Planta Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Japan. The plant secondary cell wall is a highly ordered structure composed of various polysaccharides, phenolic components and proteins. Its coordinated regulation of a number of complex metabolic pathways and assembly has not been resolved. To understand the molecular mechanisms that regulate secondary cell wall synthesis, we isolated a novel rice mutant, cell wall architecture1 (cwa1), that exhibits an irregular thickening pattern in the secondary cell wall of sclerenchyma, as well as culm brittleness and reduced cellulose content in mature internodes. Light and transmission electron microscopy revealed that the cwa1 mutant plant has regions of local aggregation in the secondary cell walls of the cortical fibers in its internodes, showing uneven thickness. Ultraviolet microscopic observation indicated that localization of cell wall phenolic components was perturbed and that these components abundantly deposited at the aggregated cell wall regions in sclerenchyma. Therefore, regulation of deposition and assembly of secondary cell wall materials, i.e. phenolic components, appear to be disturbed by mutation of the cwa1 gene. Genetic analysis showed that cwa1 is allelic to brittle culm1 (bc1), which encodes the glycosylphosphatidylinositol-anchored COBRA-like protein specifically in plants. BC1 is known as a regulator that controls the culm mechanical strength and cellulose content in the secondary cell walls of sclerenchyma, but the precise function of BC1 has not been resolved. Our results suggest that CWA1/BC1 has an essential role in assembling cell wall constituents at their appropriate sites, thereby enabling synthesis of solid and flexible internodes in rice. BC1 BC10, a DUF266-containing and Golgi-located type II membrane protein, is required for cell-wall biosynthesis in rice (Oryza sativa L.) 2009 Plant J State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Glycosyltransferases (GTs) are one of the largest enzyme groups required for the synthesis of complex wall polysaccharides and glycoproteins in plants. However, due to the limited number of related mutants that have observable phenotypes, the biological function(s) of most GTs in cell-wall biosynthesis and assembly have remained elusive. We report here the isolation and in-depth characterization of a brittle rice mutant, brittle culm 10 (bc10). bc10 plants show pleiotropic phenotypes, including brittleness of the plant body and retarded growth. The BC10 gene was cloned through a map-based approach, and encodes a Golgi-located type II membrane protein that contains a domain designated as 'domain of unknown function 266' (DUF266) and represents a multiple gene family in rice. BC10 has low sequence similarity with the domain to a core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT), and its in vitro enzymatic activity suggests that it functions as a glycosyltransferase. Monosaccharide analysis of total and fractioned wall residues revealed that bc10 showed impaired cellulose biosynthesis. Immunolocalization and isolation of arabinogalactan proteins (AGPs) in the wild-type and bc10 showed that the level of AGPs in the mutant is significantly affected. BC10 is mainly expressed in the developing sclerenchyma and vascular bundle cells, and its deficiency causes a reduction in the levels of cellulose and AGPs, leading to inferior mechanical properties. BC10 Brittle Culm 12, a dual-targeting kinesin-4 protein, controls cell-cycle progression and wall properties in rice 2010 Plant J State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. Kinesins are encoded by a large gene family involved in many basic processes of plant development. However, the number of functionally identified kinesins in rice is very limited. Here, we report the functional characterization of Brittle Culm12 (BC12), a gene encoding a kinesin-4 protein. bc12 mutants display dwarfism resulting from a significant reduction in cell number and brittleness due to an alteration in cellulose microfibril orientation and wall composition. BC12 is expressed mainly in tissues undergoing cell division and secondary wall thickening. In vitro biochemical analyses verified BC12 as an authentic motor protein. This protein was present in both the nucleus and cytoplasm and associated with microtubule arrays during cell division. Mitotic microtubule array comparison, flow cytometric analysis and expression assays of cyclin-dependent kinase (CDK) complexes in root-tip cells showed that cell-cycle progression is affected in bc12 mutants. BC12 is very probably regulated by CDKA;3 based on yeast two-hybrid and microarray data. Therefore, BC12 functions as a dual-targeting kinesin protein and is implicated in cell-cycle progression, cellulose microfibril deposition and wall composition in the monocot plant rice. BC12 Mutation of rice BC12/GDD1, which encodes a kinesin-like protein that binds to a GA biosynthesis gene promoter, leads to dwarfism with impaired cell elongation 2011 Plant Cell Research Center for Molecular and Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. The kinesins are a family of microtubule-based motor proteins that move directionally along microtubules and are involved in many crucial cellular processes, including cell elongation in plants. Less is known about kinesins directly regulating gene transcription to affect cellular physiological processes. Here, we describe a rice (Oryza sativa) mutant, gibberellin-deficient dwarf1 (gdd1), that has a phenotype of greatly reduced length of root, stems, spikes, and seeds. This reduced length is due to decreased cell elongation and can be rescued by exogenous gibberellic acid (GA(3)) treatment. GDD1 was cloned by a map-based approach, was expressed constitutively, and was found to encode the kinesin-like protein BRITTLE CULM12 (BC12). Microtubule cosedimentation assays revealed that BC12/GDD1 bound to microtubules in an ATP-dependent manner. Whole-genome microarray analysis revealed the expression of ent-kaurene oxidase (KO2), which encodes an enzyme involved in GA biosynthesis, was downregulated in gdd1. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that GDD1 bound to the element ACCAACTTGAA in the KO2 promoter. In addition, GDD1 was shown to have transactivation activity. The level of endogenous GAs was reduced in gdd1, and the reorganization of cortical microtubules was altered. Therefore, BC12/GDD1, a kinesin-like protein with transcription regulation activity, mediates cell elongation by regulating the GA biosynthesis pathway in rice. BC12,D35|OsKOS3|OsKO2 Brittle culm15 encodes a membrane-associated chitinase-like protein required for cellulose biosynthesis in rice 2012 Plant Physiol Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Plant chitinases, a class of glycosyl hydrolases, participate in various aspects of normal plant growth and development, including cell wall metabolism and disease resistance. The rice (Oryza sativa) genome encodes 37 putative chitinases and chitinase-like proteins. However, none of them has been characterized at the genetic level. In this study, we report the isolation of a brittle culm mutant, bc15, and the map-based cloning of the BC15/OsCTL1 (for chitinase-like1) gene affected in the mutant. The gene encodes the rice chitinase-like protein BC15/OsCTL1. Mutation of BC15/OsCTL1 causes reduced cellulose content and mechanical strength without obvious alterations in plant growth. Bioinformatic analyses indicated that BC15/OsCTL1 is a class II chitinase-like protein that is devoid of both an amino-terminal cysteine-rich domain and the chitinase activity motif H-E-T-T but possesses an amino-terminal transmembrane domain. Biochemical assays demonstrated that BC15/OsCTL1 is a Golgi-localized type II membrane protein that lacks classical chitinase activity. Quantitative real-time polymerase chain reaction and beta-glucuronidase activity analyses indicated that BC15/OsCTL1 is ubiquitously expressed. Investigation of the global expression profile of wild-type and bc15 plants, using Illumina RNA sequencing, further suggested a possible mechanism by which BC15/OsCTL1 mediates cellulose biosynthesis and cell wall remodeling. Our findings provide genetic evidence of a role for plant chitinases in cellulose biosynthesis in rice, which appears to differ from their roles as revealed by analysis of Arabidopsis (Arabidopsis thaliana). bc15|OsCTL1 Rice BRITTLE CULM 3 (BC3) encodes a classical dynamin OsDRP2B essential for proper secondary cell wall synthesis 2010 Planta Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan. "Brittle culm" mutants found in Gramineae crops are suitable materials to study the mechanism of secondary cell wall formation. Through positional cloning, we have identified a gene responsible for the brittle culm phenotype in rice, brittle culm 3 (bc3). BC3 encodes a member of the classical dynamin protein family, a family known to function widely in membrane dynamics. The bc3 mutation resulted in reductions of 28-36% in cellulose contents in culms, leaves, and roots, while other cell wall components remained unaffected. Reductions of cell wall thickness and birefringence were observed in both fiber (sclerenchyma) and parenchymal cells, together with blurring of the wall's layered structures. From promoter-GUS analyses, it was suggested that BC3 expression is directly correlated with active secondary cell wall synthesis. These results suggest that BC3 is tightly involved in the synthesis of cellulose and is essential for proper secondary cell wall construction. BC3|OsDRP2B The rice dynamin-related protein DRP2B mediates membrane trafficking, and thereby plays a critical role in secondary cell wall cellulose biosynthesis 2010 Plant J State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Membrane trafficking between the plasma membrane (PM) and intracellular compartments is an important process that regulates the deposition and metabolism of cell wall polysaccharides. Dynamin-related proteins (DRPs), which function in membrane tubulation and vesiculation are closely associated with cell wall biogenesis. However, the molecular mechanisms by which DRPs participate in cell wall formation are poorly understood. Here, we report the functional characterization of Brittle Culm3 (BC3), a gene encoding OsDRP2B. Consistent with the expression of BC3 in mechanical tissues, the bc3 mutation reduces mechanical strength, which results from decreased cellulose content and altered secondary wall structure. OsDRP2B, one of three members of the DRP2 subfamily in rice (Oryza sativa L.), was identified as an authentic membrane-associated dynamin via in vitro biochemical analyses. Subcellular localization of fluorescence-tagged OsDRP2B and several compartment markers in protoplast cells showed that this protein not only lies at the PM and the clathrin-mediated vesicles, but also is targeted to the trans-Golgi network (TGN). An FM4-64 uptake assay in transgenic plants that express green fluorescent protein-tagged OsDRP2B verified its involvement in an endocytic pathway. BC3 mutation and overexpression altered the abundance of cellulose synthase catalytic subunit 4 (OsCESA4) in the PM and in the endomembrane systems. All of these findings lead us to conclude that OsDRP2B participates in the endocytic pathway, probably as well as in post-Golgi membrane trafficking. Mutation of OsDRP2B disturbs the membrane trafficking that is essential for normal cellulose biosynthesis of the secondary cell wall, thereby leading to inferior mechanical properties in rice plants. BC3|OsDRP2B,OsCesA4|Bc7|bc11 Map-based cloning of a novel rice cytochrome P450 gene CYP81A6 that confers resistance to two different classes of herbicides 2006 Plant Mol Biol College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, 310029, Zhejiang, China. Development of hybrid rice has greatly contributed to increased yields during the past three decades. Two bentazon-lethal mutants 8077S and Norin8m are being utilized in developing new hybrid rice systems. When the male sterile lines are developed in such a mutant background, the problem of F1 seed contamination by self-seeds from the sterile lines can be solved by spraying bentazon at seedling stage. We first determined the sensitivity of the mutant plants to bentazon. Both mutants showed symptoms to bentazon starting from 100 mg/l, which was about 60-fold, lower than the sensitivity threshold of their wild-type controls. In addition, both mutants were sensitive to sulfonylurea-type herbicides. The locus for the mutant phenotype is bel for 8077S and bsl for Norin8m. Tests showed that the two loci are allelic to each other. The two genes were cloned by map-based cloning. Interestingly, both mutant alleles had a single-base deletion, which was confirmed by PCR-RFLP. The two loci are renamed bel ( a ) (for bel) and bel ( b ) (for bsl). The wild-type Bel gene encodes a novel cytochrome P450 monooxgenase, named CYP81A6. Analysis of the mutant protein sequence also revealed the reason for bel ( a ) being slightly tolerant than bel ( b ). Introduction of the wild-type Bel gene rescued the bentazon- and sulfonylurea-sensitive phenotype of bel ( a ) mutant. On the other hand, expression of antisense Bel in W6154S induced a mutant phenotype. Based on these results we conclude that the novel cytochrome P450 monooxygenase CYP81A6 encoded by Bel confers resistance to two different classes of herbicides. bel|CYP81A6|bsl A novel rice cytochrome P450 gene, CYP72A31, confers tolerance to acetolactate synthase-inhibiting herbicides in rice and Arabidopsis 2014 Plant Physiol Agrogenomics Research Center, National Institute of Agrobiological Sciences Target-site and non-target-site herbicide tolerance are caused by the prevention of herbicide binding to the target enzyme and the reduction to a non-lethal dose of herbicide reaching the target enzyme, respectively. There is little information on the molecular mechanisms involved in non-target-site herbicide tolerance, although it poses the greater threat in the evolution of herbicide-resistant weeds and could potentially be useful for the production of herbicide-tolerant crops because it is often involved in tolerance to multi-herbicides. Bispyribac sodium (BS) is a herbicide that inhibits the activity of acetolactate synthase (ALS). Rice of the indica variety show BS tolerance while japonica rice varieties are BS sensitive. Map-based cloning and complementation tests revealed that a novel cytochrome P450 monooxygenase, CYP72A31, is involved in BS tolerance. Interestingly, BS tolerance was correlated with CYP72A31 mRNA levels in transgenic plants of rice and Arabidopsis. Moreover, Arabidopsis overexpressing CYP72A31 showed tolerance to bensulfuron-methyl (BSM), which belongs to a different class of ALS-inhibiting herbicides, suggesting that CYP72A31 can metabolize BS and BSM to a compound with reduced phytotoxicity. On the other hand, we showed that the cytochrome P450 monooxygenase CYP81A6, which has been reported to confer BSM tolerance, is barely involved, if at all, in BS tolerance, suggesting that the CYP72A31 enzyme has different herbicide specificities compared to CYP81A6. Thus, the CYP72A31 gene is a potentially useful genetic resource in the field of weed control, herbicide development, and molecular breeding in broad range of crop species. bel|CYP81A6|bsl,CYP72A31 Suppression of a NAC-like transcription factor gene improves boron-toxicity tolerance in rice 2011 Plant Physiol Laboratory of Plant Nutrition, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan. We identified a gene responsible for tolerance to boron (B) toxicity in rice (Oryza sativa), named BORON EXCESS TOLERANT1. Using recombinant inbred lines derived from the B-toxicity-sensitive indica-ecotype cultivar IR36 and the tolerant japonica-ecotype cultivar Nekken 1, the region responsible for tolerance to B toxicity was narrowed to 49 kb on chromosome 4. Eight genes are annotated in this region. The DNA sequence in this region was compared between the B-toxicity-sensitive japonica cultivar Wataribune and the B-toxicity-tolerant japonica cultivar Nipponbare by eco-TILLING analysis and revealed a one-base insertion mutation in the open reading frame sequence of the gene Os04g0477300. The gene encodes a NAC (NAM, ATAF, and CUC)-like transcription factor and the function of the transcript is abolished in B-toxicity-tolerant cultivars. Transgenic plants in which the expression of Os04g0477300 is abolished by RNA interference gain tolerance to B toxicity. BET1 Genetic control of a transition from black to straw-white seed hull in rice domestication 2011 Plant Physiol National Center for Gene Research and Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200233, China. The genetic mechanism involved in a transition from the black-colored seed hull of the ancestral wild rice (Oryza rufipogon and Oryza nivara) to the straw-white seed hull of cultivated rice (Oryza sativa) during grain ripening remains unknown. We report that the black hull of O. rufipogon was controlled by the Black hull4 (Bh4) gene, which was fine-mapped to an 8.8-kb region on rice chromosome 4 using a cross between O. rufipogon W1943 (black hull) and O. sativa indica cv Guangluai 4 (straw-white hull). Bh4 encodes an amino acid transporter. A 22-bp deletion within exon 3 of the bh4 variant disrupted the Bh4 function, leading to the straw-white hull in cultivated rice. Transgenic study indicated that Bh4 could restore the black pigment on hulls in cv Guangluai 4 and Kasalath. Bh4 sequence alignment of all taxa with the outgroup Oryza barthii showed that the wild rice maintained comparable levels of nucleotide diversity that were about 70 times higher than those in the cultivated rice. The results from the maximum likelihood Hudson-Kreitman-Aguade test suggested that the significant reduction in nucleotide diversity in rice cultivars could be caused by artificial selection. We propose that the straw-white hull was selected as an important visual phenotype of nonshattered grains during rice domestication. Bh4 The role of Bh4 in parallel evolution of hull colour in domesticated and weedy rice 2013 J Evol Biol Department of Biology, Washington University, St. Louis, MO, USA. The two independent domestication events in the genus Oryza that led to African and Asian rice offer an extremely useful system for studying the genetic basis of parallel evolution. This system is also characterized by parallel de-domestication events, with two genetically distinct weedy rice biotypes in the US derived from the Asian domesticate. One important trait that has been altered by rice domestication and de-domestication is hull colour. The wild progenitors of the two cultivated rice species have predominantly black-coloured hulls, as does one of the two U.S. weed biotypes; both cultivated species and one of the US weedy biotypes are characterized by straw-coloured hulls. Using Black hull 4 (Bh4) as a hull colour candidate gene, we examined DNA sequence variation at this locus to study the parallel evolution of hull colour variation in the domesticated and weedy rice system. We find that independent Bh4-coding mutations have arisen in African and Asian rice that are correlated with the straw hull phenotype, suggesting that the same gene is responsible for parallel trait evolution. For the U.S. weeds, Bh4 haplotype sequences support current hypotheses on the phylogenetic relationship between the two biotypes and domesticated Asian rice; straw hull weeds are most similar to indica crops, and black hull weeds are most similar to aus crops. Tests for selection indicate that Asian crops and straw hull weeds deviate from neutrality at this gene, suggesting possible selection on Bh4 during both rice domestication and de-domestication. Bh4 Analysis of transcriptional and upstream regulatory sequence activity of two environmental stress-inducible genes, NBS-Str1 and BLEC-Str8, of rice 2012 Transgenic Res Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, 110021, India. Two abiotic stress-inducible upstream regulatory sequences (URSs) from rice have been identified and functionally characterized in rice. NBS-Str1 and BLEC-Str8 genes have been identified, by analysing the transcriptome data of cold, salt and desiccation stress-treated 7-day-old rice (Oryza sativa L. var. IR64) seedling, to be preferentially responsive to desiccation and salt stress, respectively. NBS-Str1 and BLEC-Str8 genes code for putative NBS (nucleotide binding site)-LRR (leucine rich repeat) and beta-lectin domain protein, respectively. NBS-Str1 URS is induced in root tissue, preferentially in vascular bundle, during 3 and 24 h of desiccation stress condition in transgenic 7-day-old rice seedling. In mature transgenic plants, this URS shows induction in root and shoot tissue under desiccation stress as well as under prolonged (1 and 2 day) salt stress. BLEC-Str8 URS shows basal activity under un-stressed condition, however, it is inducible under salt stress condition in both root and leaf tissues in young seedling and mature plants. Activity of BLEC-Str8 URS has been found to be vascular tissue preferential, however, under salt stress condition its activity is also found in the mesophyll tissue. NBS-Str1 and BLEC-Str8 URSs are inducible by heavy metal, copper and manganese. Interestingly, both the URSs have been found to be non responsive to ABA treatment, implying them to be part of ABA-independent abiotic stress response pathway. These URSs could prove useful for expressing a transgene in a stress responsive manner for development of stress tolerant transgenic systems. BLEC-Str8 Identification and mapping of two brown planthopper resistance genes in rice 2001 TAG Theoretical and Applied Genetics College of Life Sciences, Wuhan University, Wuhan 430072, China, CN The brown planthopper (BPH) is one of the most serious insect pests of rice. In this study, we conducted a molecular marker-based genetic analysis of the BPH resistance of ’B5’, a highly resistant line that derived its resistant genes from the wild rice Oryza officinalis. Insect resistance was evaluated using 250 F3 families from a cross between ’B5’ and ’Minghui 63’, based on which the resistance of each F2 plant was inferred. Two bulks were made by mixing, respectively, DNA samples from highly resistant plants and highly susceptible plants selected from the F2 population. The bulks were surveyed for restriction fragment length polymorphism using probes representing all 12 chromosomes at regular intervals. The survey revealed two genomic regions on chromosome 3 and chromosome 4 respectively that contained genes for BPH resistance. The existence of the two loci were further assessed by QTL (quantitative trait locus) analysis, which resolved these two loci to a 14.3-cM interval on chromosome 3 and a 0.4-cM interval on chromosome 4. Comparison of the chromosomal locations and reactions to BPH biotypes indicated that these two genes are different from at least nine of the ten previously identified BPH resistance genes. Both of the genes had large effects on BPH resistance and the two loci acted essentially independent of each other in determining t he resistance. These two genes may be a useful BPH resistance resource for rice breeding programs. Bph14 Identification and characterization of Bph14, a gene conferring resistance to brown planthopper in rice 2009 Proc Natl Acad Sci U S A Key Laboratory of Ministry of Education for Plant Development Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China. Planthoppers are highly destructive pests in crop production worldwide. Brown planthopper (BPH) causes the most serious damage of the rice crop globally among all rice pests. Growing resistant varieties is the most effective and environment-friendly strategy for protecting the crop from BPH. More than 19 BPH-resistance genes have been reported and used to various extents in rice breeding and production. In this study, we cloned Bph14, a gene conferring resistance to BPH at seedling and maturity stages of the rice plant, using a map-base cloning approach. We show that Bph14 encodes a coiled-coil, nucleotide-binding, and leucine-rich repeat (CC-NB-LRR) protein. Sequence comparison indicates that Bph14 carries a unique LRR domain that might function in recognition of the BPH insect invasion and activating the defense response. Bph14 is predominantly expressed in vascular bundles, the site of BPH feeding. Expression of Bph14 activates the salicylic acid signaling pathway and induces callose deposition in phloem cells and trypsin inhibitor production after planthopper infestation, thus reducing the feeding, growth rate, and longevity of the BPH insects. Our work provides insights into the molecular mechanisms of rice defense against insects and facilitates the development of resistant varieties to control this devastating insect. Bph14 The Bphi008a gene interacts with the ethylene pathway and transcriptionally regulates MAPK genes in the response of rice to brown planthopper feeding 2011 Plant Physiol State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan 430072, People's Republic of China. We examined ways in which the Brown planthopper induced008a (Bphi008a; AY256682) gene of rice (Oryza sativa) enhances the plant's resistance to a specialist herbivore, the brown planthopper (BPH; Nilaparvata lugens). Measurement of the expression levels of ethylene synthases and of ethylene emissions showed that BPH feeding rapidly initiated the ethylene signaling pathway and up-regulated Bphi008a transcript levels after 6 to 96 h of feeding. In contrast, blocking ethylene transduction (using 1-methylcyclopropene) reduced Bphi008a transcript levels in wild-type plants fed upon by BPH. In vitro kinase assays showed that Bphi008a can be phosphorylated by rice Mitogen-activated Protein Kinase5 (OsMPK5), and yeast two-hybrid assays demonstrated that the carboxyl-terminal proline-rich region of Bphi008a interacts directly with this kinase. Furthermore, bimolecular fluorescence complementation assays showed that this interaction occurs in the nucleus. Subsequently, we found that Bphi008a up-regulation and down-regulation were accompanied by different changes in transcription levels of OsMPK5, OsMPK12, OsMPK13, and OsMPK17 in transgenic plants. Immunoblot analysis also showed that the OsMPK5 protein level increased in overexpressing plants and decreased in RNA interference plants after BPH feeding. In transgenic lines, changes in the expression levels of several enzymes that are important components of the defenses against the BPH were also observed. Finally, yeast two-hybrid screening results showed that Bphi008a is able to interact with a b-ZIP transcription factor (OsbZIP60) and a RNA polymerase polypeptide (SDRP). Bphi008a,OsACO3,OsACO5|OsACO6,OsACO7,OsACS2,OsACS4,OS-ACS5|OsACS5,OsACS6,OsbZIP60,OsWJUMK1|OsMPK12|OsBWMK1,OsMPK13|OsBIMK2,OsMPK17,OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1,SDRP The Rice brassinosteroid-deficient dwarf2 mutant, defective in the rice homolog of Arabidopsis DIMINUTO/DWARF1, is rescued by the endogenously accumulated alternative bioactive brassinosteroid, dolichosterone 2005 Plant Cell BioScience and Biotechnology Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan. We have identified a rice (Oryza sativa) brassinosteroid (BR)-deficient mutant, BR-deficient dwarf2 (brd2). The brd2 locus contains a single base deletion in the coding region of Dim/dwf1, a homolog of Arabidopsis thaliana DIMINUTO/DWARF1 (DIM/DWF1). Introduction of the wild-type Dim/dwf1 gene into brd2 restored the normal phenotype. Overproduction and repression of Dim/dwf1 resulted in contrasting phenotypes, with repressors mimicking the brd2 phenotype and overproducers having large stature with increased numbers of flowers and seeds. Although brd2 contains low levels of common 6-oxo-type BRs, the severity of the brd2 phenotype is much milder than brd1 mutants and most similar to d2 and d11, which show a semidwarf phenotype at the young seedling stage. Quantitative analysis suggested that in brd2, the 24-methylene BR biosynthesis pathway is activated and the uncommon BR, dolichosterone (DS), is produced. DS enhances the rice lamina joint bending angle, rescues the brd1 dwarf phenotype, and inhibits root elongation, indicating that DS is a bioactive BR in rice. Based on these observations, we discuss an alternative BR biosynthetic pathway that produces DS when Dim/dwf1 is defective. brd2|DIM|DWF1 BRK1, a Bub1-related kinase, is essential for generating proper tension between homologous kinetochores at metaphase I of rice meiosis 2012 Plant Cell State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Bub1 (for budding uninhibited by benzimidazole 1), one of the main spindle checkpoint kinases, acts as a kinetochore scaffold for assembling other checkpoint proteins. Here, we identify a plant Bub1-related kinase 1 (BRK1) in rice (Oryza sativa). The brk1 mutants are sterile due to the precocious separation of sister chromatids at the onset of anaphase I. The centromeric recruitment of SHUGOSHIN1 and phosphorylation of histone H2A at Thr-134 (H2A-pT134) depend on BRK1. Although the homologs can faithfully separate from each other at the end of meiosis I, the uncorrected merotelic attachment of paired sister kinetochores at the early stage of metaphase I in brk1 reduces the tension across homologous kinetochores, causes the metaphase I spindle to be aberrantly shaped, and subsequently affects the synchronicity of homolog separation at the onset of anaphase I. In addition, the phosphorylation of inner centromeric histone H3 at Ser-10 (H3-pS10) during diakinesis depends on BRK1. Therefore, we speculate that BRK1 may be required for normal localization of Aurora kinase before the onset of metaphase I, which is responsible for correcting the merotelic attachment. BRK1 BEAK LIKE SPIKELET1 is Required for Lateral Development of Lemma and Palea in Rice 2012 Plant Molecular Biology Reporter National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, 100081, China Lemma and palea are unique floral structures found only in Poaceae, and are responsible for protecting the inner floral organs and kernels from environmental stresses. However, the mechanism underlying specification of their morphology remains unclear. In this study, we characterized a rice mutant, beak like spikelet1 (bls1), which specifically affects development of the lemma and palea. In bls1 mutant, floral-organ identity and floral-organ patterning are normal, and the defects occur at the stage of the lemma and palea expansion, whereas the other aspects of floral architecture and form are not affected. We isolated BLS1 by positional cloning and found that it encodes a protein with a conserved domain of unknown function. BLS1 is expressed strongly in young inflorescence, specifically the young lemmas and paleas of spikelets. Subcellular localization analysis showed that BLS1 is localized in the nucleus. Expression of the AP1-like and SEP-like floral homeotic genes were not changed in the bls1 mutant. Our study suggested that BLS1 is required for lateral development of the lemma and palea and does not function at stages of floral-organ initiation and patterning. BLS1|BSG1 Beak-shaped grain 1/TRIANGULAR HULL 1, a DUF640 gene, is associated with grain shape, size and weight in rice 2013 Sci China Life Sci National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Grain shape and size both determine grain weight and therefore crop yield. However, the molecular mechanisms controlling grain shape and size are still largely unknown. Here, we isolated a rice mutant, beak-shaped grain1 (bsg1), which produced beak-shaped grains of decreased width, thickness and weight with a loosely interlocked lemma and palea that were unable to close tightly. Starch granules were also irregularly packaged in the bsg1 grains. Consistent with the lemma and palea shapes, the outer parenchyma cell layers of these bsg1 tissues developed fewer cells with decreased size. Map-based cloning revealed that BSG1 encoded a DUF640 domain protein, TRIANGULAR HULL 1, of unknown function. Quantitative PCR and GUS fusion reporter assays showed that BSG1 was expressed mainly in the young panicle and elongating stem. The BSG1 mutation affected the expression of genes potentially involved in the cell cycle and GW2, an important regulator of grain size in rice. Our results suggest that BSG1 determines grain shape and size probably by modifying cell division and expansion in the grain hull. BLS1|BSG1,GW2 Screening for resistance against Pseudomonas syringae in rice-FOX Arabidopsis lines identified a putative receptor-like cytoplasmic kinase gene that confers resistance to major bacterial and fungal pathogens in Arabidopsis and rice 2011 Plant Biotechnol J National Institute of Agrobiological Sciences, Tsukuba, Japan RIKEN, Plant Science Center, Yokohama, Japan. Approximately 20,000 of the rice-FOX Arabidopsis transgenic lines, which overexpress 13,000 rice full-length cDNAs at random in Arabidopsis, were screened for bacterial disease resistance by dip inoculation with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The identities of the overexpressed genes were determined in 72 lines that showed consistent resistance after three independent screens. Pst DC3000 resistance was verified for 19 genes by characterizing other independent Arabidopsis lines for the same genes in the original rice-FOX hunting population or obtained by reintroducing the genes into ecotype Columbia by floral dip transformation. Thirteen lines of these 72 selections were also resistant to the fungal pathogen Colletotrichum higginsianum. Eight genes that conferred resistance to Pst DC3000 in Arabidopsis have been introduced into rice for overexpression, and transformants were evaluated for resistance to the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae. One of the transgenic rice lines was highly resistant to Xanthomonas oryzae pv. oryzae. Interestingly, this line also showed remarkably high resistance to Magnaporthe grisea, the fungal pathogen causing rice blast, which is the most devastating rice disease in many countries. The causal rice gene, encoding a putative receptor-like cytoplasmic kinase, was therefore designated as BROAD-SPECTRUM RESISTANCE 1. Our results demonstrate the utility of the rice-FOX Arabidopsis lines as a tool for the identification of genes involved in plant defence and suggest the presence of a defence mechanism common between monocots and dicots. BSR1 Expression profiling of starch metabolism-related plastidic translocator genes in rice 2006 Planta CREST, Japan Science and Technology Corporation, Omiya, Saitama, Japan. The genes encoding the major putative rice plastidic translocators involved in the carbon flow related to starch metabolism were identified by exhaustive database searches. The genes identified were two for the triose phosphate/phosphate translocator (TPT), five for the glucose 6-phosphate/phosphate translocator (GPT) including putatively non-functional ones, four for the phosphoenolpyruvate/phosphate translocator (PPT), three for the putative ADP-glucose translocator (or Brittle-1 protein, BT1), two for the plastidic nucleotide transport protein (NTT), and one each for the plastidic glucose translocator (pGlcT) and the maltose translocator (MT). The expression patterns of the genes in various photosynthetic and non-photosynthetic organs were examined by quantitative real-time PCR. OsBT1-1 was specifically expressed in the seed and its transcript level tremendously increased at the onset of vigorous starch production in the endosperm, suggesting that the ADP-glucose synthesized in the cytosol is a major precursor for starch biosynthesis in the endosperm amyloplast. In contrast, all of the genes for OsTPT, OsPPT, and OsNTT were mainly expressed in source tissues, suggesting that their proteins play essential roles in the regulation of carbohydrate metabolism in chloroplasts. Substantial expression of the four OsGPT genes and the OspGlcT gene in both source and sink organs suggests that the transport of glucose phosphate and glucose is physiologically important in both photosynthetic and non-photosynthetic tissues. The present study shows that comprehensive analysis of expression patterns of the plastidic translocator genes is a valuable tool for the elucidation of the functions of the translocators in the regulation of starch metabolism in rice. OsBT1-1,OsMT,OsPGLCT,OsBT1-2,OsBT1-3,OsGPT2-3 H3K36 methylation is critical for brassinosteroid-regulated plant growth and development in rice 2012 Plant J State Key Laboratory of Genetic Engineering, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China. Methylation of histone lysine residues plays an essential role in epigenetic regulation of gene expression in eukaryotes. Enzymes involved in establishment of the repressive H3K9 and H3K27 methylation marks have been previously characterized, but the deposition and function of H3K4 and H3K36 methylation remain uncharacterized in rice. Here, we report that rice SDG725 encodes a H3K36 methyltransferase, and its down-regulation causes wide-ranging defects, including dwarfism, shortened internodes, erect leaves and small seeds. These defects resemble the phenotypes previously described for some brassinosteroid-knockdown mutants. Consistently, transcriptome analyses revealed that SDG725 depletion results in down-regulation by more than two-fold of over 1000 genes, including D11, BRI1 and BU1, which are known to be involved in brassinosteroid biosynthesis or signaling pathways. Chromatin immunoprecipitation analyses showed that levels of H3K36me2/3 are reduced in chromatin at some regions of these brassinosteroid-related genes in SDG725 knockdown plants, and that SDG725 protein is able to directly bind to these target genes. Taken together, our data indicate that SDG725-mediated H3K36 methylation modulates brassinosteroid-related gene expression, playing an important role in rice plant growth and development. BU1,D11|CYP724B1,D61|OsBRI1,SDG725 BRASSINOSTEROID UPREGULATED1, encoding a helix-loop-helix protein, is a novel gene involved in brassinosteroid signaling and controls bending of the lamina joint in rice 2009 Plant Physiol Disease Resistance Research Unit, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan. Brassinosteroids (BRs) are involved in many developmental processes and regulate many subsets of downstream genes throughout the plant kingdom. However, little is known about the BR signal transduction and response network in monocots. To identify novel BR-related genes in rice (Oryza sativa), we monitored the transcriptomic response of the brassinosteroid deficient1 (brd1) mutant, with a defective BR biosynthetic gene, to brassinolide treatment. Here, we describe a novel BR-induced rice gene BRASSINOSTEROID UPREGULATED1 (BU1), encoding a helix-loop-helix protein. Rice plants overexpressing BU1 (BU1:OX) showed enhanced bending of the lamina joint, increased grain size, and resistance to brassinazole, an inhibitor of BR biosynthesis. In contrast to BU1:OX, RNAi plants designed to repress both BU1 and its homologs displayed erect leaves. In addition, compared to the wild type, the induction of BU1 by exogenous brassinolide did not require de novo protein synthesis and it was weaker in a BR receptor mutant OsbriI (Oryza sativa brassinosteroid insensitive1, d61) and a rice G protein alpha subunit (RGA1) mutant d1. These results indicate that BU1 protein is a positive regulator of BR response: it controls bending of the lamina joint in rice and it is a novel primary response gene that participates in two BR signaling pathways through OsBRI1 and RGA1. Furthermore, expression analyses showed that BU1 is expressed in several organs including lamina joint, phloem, and epithelial cells in embryos. These results indicate that BU1 may participate in some other unknown processes modulated by BR in rice. BU1,D1|RGA1,D61|OsBRI1 Loss-of-function of a rice brassinosteroid biosynthetic enzyme, C-6 oxidase, prevents the organized arrangement and polar elongation of cells in the leaves and stem 2002 The Plant Journal BioScience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan Molecular genetic and physiological studies on brassinosteroid (BR)-related mutants of dicot plants have revealed that BRs play important roles in normal plant growth and development. However, little is known about the function of BR in monocots (grasses), except for the phenotypic analysis of a rice mutant partially insensitive to BR signaling. To investigate the function of BR in monocots, we identified and characterized BR-deficient mutants of rice, BR-deficient dwarf1 (brd1). The brd1 mutants showed a range of abnormalities in organ development and growth, the most striking of which were defects in the elongation of the stem and leaves. Light microscopic observations revealed that this abnormality was primarily owing to a failure in the organization and polar elongation of the leaf and stem cells. The accumulation profile of BR compounds in the brd1 mutants suggested that these plants may be deficient in the activity of BR C-6 oxidase. Therefore, we cloned a rice gene, OsDWARF, which has a high sequence similarity to the tomato C-6 oxidase gene, DWARF. Introduction of the wild-type OsDWARF gene into brd1 rescued the abnormal phenotype of the mutants. The OsDWARF gene was expressed at a low level in all of the examined tissues, with preferential expression in the leaf sheath, and the expression was negatively regulated by brassinolide treatment. On the basis of these findings, we discuss the biological function of BRs in rice plants. OsDWARF Isolation and characterization of a rice dwarf mutant with a defect in brassinosteroid biosynthesis 2002 Plant Physiol Department of Molecular Genetics, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan. morimasa@nias.affrc.go.jp We have isolated a new recessive dwarf mutant of rice (Oryza sativa L. cv Nipponbare). Under normal growth conditions, the mutant has very short leaf sheaths; has short, curled, and frizzled leaf blades; has few tillers; and is sterile. Longitudinal sections of the leaf sheaths revealed that the cell length along the longitudinal axis is reduced, which explains the short leaf sheaths. Transverse sections of the leaf blades revealed enlargement of the motor cells along the dorsal-ventral axis, which explains the curled and frizzled leaf blades. In addition, the number of crown roots was smaller and the growth of branch roots was weaker than those in the wild-type plant. Because exogenously supplied brassinolide considerably restored the normal phenotypes, we designated the mutant brassinosteroid-dependent 1 (brd1). Further, under darkness, brd1 showed constitutive photomorphogenesis. Quantitative analyses of endogenous sterols and brassinosteroids (BRs) indicated that BR-6-oxidase, a BR biosynthesis enzyme, would be defective. In fact, a 0.2-kb deletion was detected in the genomic region of OsBR6ox (a rice BR-6-oxidase gene) in the brd1 mutant. These results indicate that BRs are involved in many morphological and physiological processes in rice, including the elongation and unrolling of leaves, development of tillers, skotomorphogenesis, root differentiation, and reproductive growth, and that the defect of BR-6-oxidase caused the brd1 phenotype. OsDWARF A CCCH-type zinc finger nucleic acid-binding protein quantitatively confers resistance against rice bacterial blight disease 2012 Plant Physiol National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China. Bacterial blight is a devastating disease of rice (Oryza sativa) caused by Xanthomonas oryzae pv oryzae (Xoo). Zinc finger proteins harboring the motif with three conserved cysteine residues and one histidine residue (CCCH) belong to a large family. Although at least 67 CCCH-type zinc finger protein genes have been identified in the rice genome, their functions are poorly understood. Here, we report that one of the rice CCCH-type zinc finger proteins, C3H12, containing five typical CX(8)-CX(5)-CX(3)-H zinc finger motifs, is involved in the rice-Xoo interaction. Activation of C3H12 partially enhanced resistance to Xoo, accompanied by the accumulation of jasmonic acid (JA) and induced expression of JA signaling genes in rice. In contrast, knockout or suppression of C3H12 resulted in partially increased susceptibility to Xoo, accompanied by decreased levels of JA and expression of JA signaling genes in rice. C3H12 colocalized with a minor disease resistance quantitative trait locus to Xoo, and the enhanced resistance of randomly chosen plants in the quantitative trait locus mapping population correlated with an increased expression level of C3H12. The C3H12 protein localized in the nucleus and possessed nucleic acid-binding activity in vitro. These results suggest that C3H12, as a nucleic acid-binding protein, positively and quantitatively regulates rice resistance to Xoo and that its function is likely associated with the JA-dependent pathway. C3H12 The arbuscular mycorrhizal symbiosis promotes the systemic induction of regulatory defence-related genes in rice leaves and confers resistance to pathogen infection 2012 Mol Plant Pathol Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Parc de Recerca UAB, Edifici CRAG, Campus UAB, Bellaterra (Cerdanyola del Valles), 08193, Barcelona, Spain. Arbuscular mycorrhizal (AM) symbioses are mutualistic associations between soil fungi and most vascular plants. Their association benefits the host plant by improving nutrition, mainly phosphorus nutrition, and by providing increased capability to cope with adverse conditions. In this study, we investigated the transcriptional changes triggered in rice leaves as a result of AM symbiosis, focusing on the relevance of the plant defence response. We showed that root colonization by the AM fungus Glomus intraradices is accompanied by the systemic induction of genes that play a regulatory role in the host defence response, such as OsNPR1, OsAP2, OsEREBP and OsJAmyb. Genes involved in signal transduction processes (OsDUF26 and OsMPK6) and genes that function in calcium-mediated signalling processes (OsCBP, OsCaM and OsCML4) are also up-regulated in leaves of mycorrhizal rice plants in the absence of pathogen infection. In addition, the mycorrhizal rice plants exhibit a stronger induction of defence marker genes [i.e. pathogenesis-related (PR) genes] in their leaves in response to infection by the blast fungus Magnaporthe oryzae. Evidence indicates that mycorrhizal rice plants show enhanced resistance to the rice blast fungus. Overall, these results suggest that the protective effect of the AM symbiosis in rice plants relies on both the systemic activation of defence regulatory genes in the absence of pathogen challenge and the priming for stronger expression of defence effector genes during pathogen infection. The possible mechanisms involved in the mycorrhiza-induced resistance to M. oryzae infection are discussed. OsCaM,OsCBP,OsDUF26 Collapsed abnormal pollen1 gene encoding the Arabinokinase-like protein is involved in pollen development in rice 2013 Plant Physiol Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, Akita 010-0195, Japan. uken@akita-pu.ac.jp We isolated a pollen-defective mutant, collapsed abnormal pollen1 (cap1), from Tos17 insertional mutant lines of rice (Oryza sativa). The cap1 heterozygous plant produced equal numbers of normal and collapsed abnormal grains. The abnormal pollen grains lacked almost all cytoplasmic materials, nuclei, and intine cell walls and did not germinate. Genetic analysis of crosses revealed that the cap1 mutation did not affect female reproduction or vegetative growth. CAP1 encodes a protein consisting of 996 amino acids that showed high similarity to Arabidopsis (Arabidopsis thaliana) l-arabinokinase, which catalyzes the conversion of l-arabinose to l-arabinose 1-phosphate. A wild-type genomic DNA segment containing CAP1 restored mutants to normal pollen grains. During rice pollen development, CAP1 was preferentially expressed in anthers at the bicellular pollen stage, and the effects of the cap1 mutation were mainly detected at this stage. Based on the metabolic pathway of l-arabinose, cap1 pollen phenotype may have been caused by toxic accumulation of l-arabinose or by inhibition of cell wall metabolism due to the lack of UDP-l-arabinose derived from l-arabinose 1-phosphate. The expression pattern of CAP1 was very similar to that of another Arabidopsis homolog that showed 71% amino acid identity with CAP1. Our results suggested that CAP1 and related genes are critical for pollen development in both monocotyledonous and dicotyledonous plants. CAP1,OsARA1 Isolation and characterization of cDNA clones encoding cdc2 homologues from Oryza sativa: a functional homologue and cognate variants 1992 Mol Gen Genet National Institute of Agrobiological Resources, Ibaraki, Japan. Using probes obtained by PCR amplification, we have isolated two cognate rice cDNAs (cdc2Os-1 and cdc2Os-2) encoding structural homologues of the cdc2+/CDC28 (cdc2) protein kinase from a cDNA library prepared from cultured rice cells. Comparison of the deduced amino acid sequences of cdc2Os-1 and cdc2Os-2 showed that they are 83% identical. They are 62% identical to CDC28 of Saccharomyces cerevisiae and much more similar to the yeast and mammalian p34cdc2 kinases than to rice R2, a cdc2-related kinase isolated previously by screening the same rice cDNA library with a different oligonucleotide probe. Southern blot analysis indicated that the three rice clones (cdc2Os-1, cdc2Os-2 and R2) are derived from distinct genes and are each found in a single copy per rice haploid genome. RNA blot analysis revealed that these genes are expressed in proliferating rice cells and in young rice seedlings. cdc2Os-1 could complement a temperature-sensitive yeast mutant of cdc28. However, despite the similarity in structure, both cdc2Os-2 and R2 were unable to complement the same mutant. Thus, the present results demonstrate the presence of structurally related, but functionally distinct cognates of the cdc2 cell cycle kinase in rice. cdc2Os-1,cdc2Os-2,R2 Differential expression of genes for cyclin-dependent protein kinases in rice plants 1999 Plant Physiol Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032, Japan. mumeda@imcbns.iam.u-tokyo.ac.jp Cyclin-dependent protein kinases (CDKs) play key roles in regulating the eukaryotic cell cycle. We have analyzed the expression of four rice (Oryza sativa) CDK genes, cdc2Os1, cdc2Os2, cdc2Os3, and R2, by in situ hybridization of sections of root apices. Transcripts of cdc2Os1, cdc2Os2, and R2 were detected uniformly in the dividing region of the root apex. cdc2Os1 and cdc2Os2 were also expressed in differentiated cells such as those in the sclerenchyma, pericycle, and parenchyma of the central cylinder. By contrast, signals corresponding to transcripts of cdc2Os3 were distributed only in patches in the dividing region. Counterstaining of sections with 4', 6-diamidino-2-phenylindole and double-target in situ hybridization with a probe for histone H4 transcripts revealed that cdc2Os3 transcripts were abundant from the G2 to the M phase, but were less abundant or absent during the S phase. The levels of the Cdc2Os3 protein and its associated histone H1-kinase activity were reduced by treatment of cultured cells with hydroxyurea, which blocks cycling cells at the onset of the S phase. Our results suggest that domains other than the conserved amino acid sequence (the PSTAIRE motif) have important roles in the function of non-PSTAIRE CDKs in distinct cell-cycle phases. cdc2Os-1,cdc2Os-2,CDKB2;1|cdc2Os3,R2 A rice homolog of Cdk7/MO15 phosphorylates both cyclin-dependent protein kinases and the carboxy-terminal domain of RNA polymerase II 1998 The Plant Journal Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan. The activation of cyclin-dependent protein kinases (CDKs) requires phosphorylation of a threonine residue within the T-loop by a CDK-activating kinase (CAK). The R2 protein of rice is very similar to CAKs of animals and fission yeast at the amino acid level but phosphorylation by R2 has not yet been demonstrated. When R2 was overexpressed in a CAK-deficient mutant of budding yeast, it suppressed the temperature sensitivity of the mutation. Immunoprecipitates of rice proteins with the anti-R2 antibody phosphorylated human CDK2, one of the rice CDKs (Cdc2Os1), and the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II of Arabidopsis. Mutational analysis indicated that R2 phosphorylated the threonine residue within the T-loop of CDK2 and Cdc2Os1. R2 was found mainly in two protein complexes which had molecular masses of 190 kDa and 70 kDa, respectively, whilst the CDK- and CTD-kinase activities associated with R2 were identified in a complex of 105 kDa. These results indicate that R2 is closely related to CAKs of animals and fission yeast in terms of its phosphorylation activity and, moreover, that this CAK of rice is distinct from a CAK of the dicotyledonous plant Arabidopsis. cdc2Os-1,R2 Isolation and characterization of a rice cDNA encoding B1-type cyclin-dependent kinase 2006 Plant Biotechnology Institute of Molecular and Cellular Biosciences, The University of Tokyo Cyclin-dependent kinases (CDKs) are central players that control the cell cycle. In plants, A- and B-type CDKs are directly involved in cell cycle regulation. B-type CDK (CDKB) is unique to plants; however, only limited information on this kinase has been accumulated thus far. In the present study, we identified a rice cDNA encoding CDKB1;1 and studied its expression in suspension-cultured cells and plant tissues. We found that this enzyme was expressed in actively dividing cells in suspension cultures and was downregulated by the depletion of sucrose from the medium. In plants, the CDKB1;1 transcripts were highly expressed in the shoot and root apical meristems, but not in mature plant organs. These results suggested that CDKB1 is mainly involved in mitotic cell division during plant development. CDKB1;1 CDKB2 is involved in mitosis and DNA damage response in rice 2012 Plant J Plant Genome Engineering Research Unit, Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan. DNA damage checkpoints delay mitotic cell-cycle progression in response to DNA stress, stalling the cell cycle to allow time for repair. CDKB is a plant-specific cyclin-dependent kinase (CDK) that is required for the G(2)/M transition of the cell cycle. In Arabidopsis, DNA damage leads the degradation of CDKB2, and the subsequent G(2) arrest gives cells time to repair damaged DNA. G(2) arrest also triggers transition from the mitotic cycle to endoreduplication, leading to the presence of polyploid cells in many tissues. In contrast, in rice (Oryza sativa), polyploid cells are found only in the endosperm. It was unclear whether endoreduplication contributes to alleviating DNA damage in rice (Oryza sativa). Here, we show that DNA damage neither down-regulates Orysa;CDKB2;1 nor induces endoreduplication in rice. Furthermore, we found increased levels of Orysa;CDKB2;1 protein upon DNA damage. These results suggest that CDKB2 functions differently in Arabidopsis and rice in response to DNA damage. Arabidopsis may adopt endoreduplication as a survival strategy under genotoxic stress conditions, but rice may enhance DNA repair capacity upon genotoxic stress. In addition, polyploid cells due to endomitosis were present in CDKB2;1 knockdown rice, suggesting an important role for Orysa;CDKB2;1 during mitosis. CDKB2;1|cdc2Os3 An OsCEBiP/OsCERK1-OsRacGEF1-OsRac1 module is an essential early component of chitin-induced rice immunity 2013 Cell Host Microbe Laboratory of Plant Molecular Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. OsCEBiP, a chitin-binding protein, and OsCERK1, a receptor-like kinase, are plasma membrane (PM) proteins that form a receptor complex essential for fungal chitin-driven immune responses in rice. The signaling events immediately following chitin perception are unclear. Investigating the spatiotemporal regulation of the rice small GTPase OsRac1, we find that chitin induces rapid activation of OsRac1 at the PM. Searching for OsRac1 interactors, we identified OsRacGEF1 as a guanine nucleotide exchange factor for OsRac1. OsRacGEF1 interacts with OsCERK1 and is activated when its C-terminal S549 is phosphorylated by the cytoplasmic domain of OsCERK1 in response to chitin. Activated OsRacGEF1 is required for chitin-driven immune responses and resistance to rice blast fungus infection. Further, a protein complex including OsCERK1 and OsRacGEF1 is transported from the endoplasmic reticulum to the PM. Collectively, our results suggest that OsCEBiP, OsCERK1, OsRacGEF1, and OsRac1 function as key components of a "defensome" critically engaged early during chitin-induced immunity. CEBiP Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor 2006 Proc Natl Acad Sci U S A Department of Biochemistry, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan. kaku@isc.meiji.ac.jp Chitin is a major component of fungal cell walls and serves as a molecular pattern for the recognition of potential pathogens in the innate immune systems of both plants and animals. In plants, chitin oligosaccharides have been known to induce various defense responses in a wide range of plant cells including both monocots and dicots. To clarify the molecular machinery involved in the perception and transduction of chitin oligosaccharide elicitor, a high-affinity binding protein for this elicitor was isolated from the plasma membrane of suspension-cultured rice cells. Characterization of the purified protein, CEBiP, as well as the cloning of the corresponding gene revealed that CEBiP is actually a glycoprotein consisting of 328 amino acid residues and glycan chains. CEBiP was predicted to have a short membrane spanning domain at the C terminus. Knockdown of CEBiP gene by RNA interference resulted in the suppression of the elicitor-induced oxidative burst as well as the gene responses, showing that CEBiP plays a key role in the perception and transduction of chitin oligosaccharide elicitor in the rice cells. Structural analysis of CEBiP also indicated the presence of two LysM motifs in the extracellular portion of CEBiP. As the LysM motif has been known to exist in the putative Nod-factor receptor kinases involved in the symbiotic signaling between leguminous plants and rhizobial bacteria, the result indicates the involvement of partially homologous plasma membrane proteins both in defense and symbiotic signaling in plant cells. CEBiP Perception of the chitin oligosaccharides contributes to disease resistance to blast fungus Magnaporthe oryzae in rice 2010 Plant J Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan. Chitin is a component of fungal cell walls, and its fragments act as elicitors in many plants. The plasma membrane glycoprotein CEBiP, which possesses LysM domains, is a receptor for the chitin elicitor (CE) in rice. Here, we report that the perception of CE by CEBiP contributes to disease resistance against the rice blast fungus, Magnaporthe oryzae, and that enhanced responses to CE by engineering CEBiP increase disease tolerance. Knockdown of CEBiP expression allowed increased spread of the infection hyphae. To enhance defense responses to CE, we constructed chimeric genes composed of CEBiP and Xa21, which mediate resistance to rice bacterial leaf blight. The expression of either CRXa1 or CRXa3, each of which contains the whole extracellular portion of CEBiP, the whole intracellular domain of XA21, and the transmembrane domain from either CEBiP or XA21, induced cell death accompanied by an increased production of reactive oxygen and nitrogen species after treatment with CE. Rice plants expressing the chimeric receptor exhibited necrotic lesions in response to CE and became more resistant to M. oryzae. Deletion of the first LysM domain in CRXA1 abolished these cellular responses. These results suggest that CEs are produced and recognized through the LysM domain of CEBiP during the interaction between rice and M. oryzae and imply that engineering pattern recognition receptors represents a new strategy for crop protection against fungal diseases. CEBiP OsLYP4 and OsLYP6 play critical roles in rice defense signal transduction 2013 Plant Signal Behav State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources; School of Life Sciences; Sun Yat-sen University; P.R. China. Plant innate immunity relies on successful detection of trespassing pathogens through recognizing their microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) at the cell surface. We recently reported two rice lysin motif (LysM)-containing proteins, OsLYP4 and OsLYP6, as dual functional PRRs sensing bacterial peptidoglycan (PGN) and fungal chitin. Here we further demonstrated the important roles of OsLYP4 and OsLYP6 in rice defense signaling, as silencing of either LYP impaired the defense marker gene activation induced by either bacterial pathogen Xanthomonas oryzaecola or fungal pathogen Magnaporthe oryzae. Moreover, we found that OsLYP4 and OsLYP6 could form homo- and hetero-dimers, and could interact with CEBiP, suggesting an unexpected complexity of chitin perception in rice. CEBiP,LYP4|OsLYP4,LYP6|OsLYP6 Effector-mediated suppression of chitin-triggered immunity by magnaporthe oryzae is necessary for rice blast disease 2012 Plant Cell School of Biosciences, University of Exeter, Geoffrey Pope Building, Exeter EX4 4QD, United Kingdom. Plants use pattern recognition receptors to defend themselves from microbial pathogens. These receptors recognize pathogen-associated molecular patterns (PAMPs) and activate signaling pathways that lead to immunity. In rice (Oryza sativa), the chitin elicitor binding protein (CEBiP) recognizes chitin oligosaccharides released from the cell walls of fungal pathogens. Here, we show that the rice blast fungus Magnaporthe oryzae overcomes this first line of plant defense by secreting an effector protein, Secreted LysM Protein1 (Slp1), during invasion of new rice cells. We demonstrate that Slp1 accumulates at the interface between the fungal cell wall and the rice plasma membrane, can bind to chitin, and is able to suppress chitin-induced plant immune responses, including generation of reactive oxygen species and plant defense gene expression. Furthermore, we show that Slp1 competes with CEBiP for binding of chitin oligosaccharides. Slp1 is required by M. oryzae for full virulence and exerts a significant effect on tissue invasion and disease lesion expansion. By contrast, gene silencing of CEBiP in rice allows M. oryzae to cause rice blast disease in the absence of Slp1. We propose that Slp1 sequesters chitin oligosaccharides to prevent PAMP-triggered immunity in rice, thereby facilitating rapid spread of the fungus within host tissue. CEBiP CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis 2007 Proc Natl Acad Sci U S A Department of Life Sciences, Faculty of Agriculture, Meiji University, 1-1-1 Higashi-Mita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan. Chitin is a major component of fungal cell walls and serves as a microbe-associated molecular pattern (MAMP) for the detection of various potential pathogens in innate immune systems of both plants and animals. We recently showed that chitin elicitor-binding protein (CEBiP), plasma membrane glycoprotein with LysM motifs, functions as a cell surface receptor for chitin elicitor in rice. The predicted structure of CEBiP does not contain any intracellular domains, suggesting that an additional component(s) is required for signaling through the plasma membrane into the cytoplasm. Here, we identified a receptor-like kinase, designated CERK1, which is essential for chitin elicitor signaling in Arabidopsis. The KO mutants for CERK1 completely lost the ability to respond to the chitin elicitor, including MAPK activation, reactive oxygen species generation, and gene expression. Disease resistance of the KO mutant against an incompatible fungus, Alternaria brassicicola, was partly impaired. Complementation with the WT CERK1 gene showed cerk1 mutations were responsible for the mutant phenotypes. CERK1 is a plasma membrane protein containing three LysM motifs in the extracellular domain and an intracellular Ser/Thr kinase domain with autophosphorylation/myelin basic protein kinase activity, suggesting that CERK1 plays a critical role in fungal MAMP perception in plants. CEBiP Two LysM receptor molecules, CEBiP and OsCERK1, cooperatively regulate chitin elicitor signaling in rice 2010 Plant J Department of Life Sciences, Faculty of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan. Chitin is a major molecular pattern for various fungi, and its fragments, chitin oligosaccharides, are known to induce various defense responses in plant cells. A plasma membrane glycoprotein, CEBiP (chitin elicitor binding protein) and a receptor kinase, CERK1 (chitin elicitor receptor kinase) (also known as LysM-RLK1), were identified as critical components for chitin signaling in rice and Arabidopsis, respectively. However, it is not known whether each plant species requires both of these two types of molecules for chitin signaling, nor the relationships between these molecules in membrane signaling. We report here that rice cells require a LysM receptor-like kinase, OsCERK1, in addition to CEBiP, for chitin signaling. Knockdown of OsCERK1 resulted in marked suppression of the defense responses induced by chitin oligosaccharides, indicating that OsCERK1 is essential for chitin signaling in rice. The results of a yeast two-hybrid assay indicated that both CEBiP and OsCERK1 have the potential to form hetero- or homo-oligomers. Immunoprecipitation using a membrane preparation from rice cells treated with chitin oligosaccharides suggested the ligand-induced formation of a receptor complex containing both CEBiP and OsCERK1. Blue native PAGE and chemical cross-linking experiments also suggested that a major portion of CEBiP exists as homo-oligomers even in the absence of chitin oligosaccharides. CEBiP,OsCERK1 Functional characterization of CEBiP and CERK1 homologs in arabidopsis and rice reveals the presence of different chitin receptor systems in plants 2012 Plant Cell Physiol Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa, 214-8571 Japan. Chitin is a representative microbe-associated molecular pattern (MAMP) molecule for various fungi and induces immune responses in many plant species. It has been clarified that the chitin signaling in rice requires a receptor kinase OsCERK1 and a receptor-like protein (Os)CEBiP, which specifically binds chitin oligosaccharides. On the other hand, Arabidopsis requires a receptor kinase (At)CERK1 for chitin signaling but it is not clear whether the plant also requires a CEBiP-like molecule for chitin perception/signaling. To clarify the similarity/difference of the chitin receptor in these two model plants, we first characterized CEBiP homologs in Arabidopsis. Only one of three CEBiP homologs, AtCEBiP (LYM2), showed a high-affinity binding for chitin oligosaccharides similar to rice CEBiP. AtCEBiP also represented the major chitin-binding protein in the Arabidopsis membrane. However, the single/triple knockout (KO) mutants of Arabidopsis CEBiP homologs and the overexpressor of AtCEBiP showed chitin-induced defense responses similar to wild-type Arabidopsis, indicating that AtCEBiP is biochemically functional as a chitin-binding protein but does not contribute to signaling. Studies of the chitin binding properties of the ectodomains of At/OsCERK1 and the chimeric receptors consisting of ecto/cytosolic domains of these molecules indicated that AtCERK1 is sufficient for chitin perception by itself. CEBiP CFL1, a WW domain protein, regulates cuticle development by modulating the function of HDG1, a class IV homeodomain transcription factor, in rice and Arabidopsis 2011 Plant Cell State Key Laboratory for Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, People's Republic of China. Plants have a chemically heterogeneous lipophilic layer, the cuticle, which protects them from biotic and abiotic stresses. The mechanisms that regulate cuticle development are poorly understood. We identified a rice (Oryza sativa) dominant curly leaf mutant, curly flag leaf1 (cfl1), and cloned CFL1, which encodes a WW domain protein. We overexpressed both rice and Arabidopsis CFL1 in Arabidopsis thaliana; these transgenic plants showed severely impaired cuticle development, similar to that in cfl1 rice. Reduced expression of At CFL1 resulted in reinforcement of cuticle structure. At CFL1 was predominantly expressed in specialized epidermal cells and in regions where dehiscence and abscission occur. Biochemical evidence showed that At CFL1 interacts with HDG1, a class IV homeodomain-leucine zipper transcription factor. Suppression of HDG1 function resulted in similar defective cuticle phenotypes in wild-type Arabidopsis but much alleviated phenotypes in At cfl1-1 mutants. The expression of two cuticle development-associated genes, BDG and FDH, was downregulated in At CFL1 overexpressor and HDG1 suppression plants. HDG1 binds to the cis-element L1 box, which exists in the regulatory regions of BDG and FDH. Our results suggest that rice and Arabidopsis CFL1 negatively regulate cuticle development by affecting the function of HDG1, which regulates the downstream genes BDG and FDH. CFL1 CHIMERIC FLORAL ORGANS1, encoding a monocot-specific MADS box protein, regulates floral organ identity in rice 2012 Plant Physiol Rice Research Institute , Southwest University, Chongqing 400715, China. The control of floral organ identity by homeotic MADS box genes is well established in eudicots. However, grasses have highly specialized outer floral organs, and the identities of the genes that regulate the highly specialized outer floral organs of grasses remain unclear. In this study, we characterized a MIKC-type MADS box gene, CHIMERIC FLORAL ORGANS (CFO1), which plays a key role in the regulation of floral organ identity in rice (Oryza sativa). The cfo1 mutant displayed defective marginal regions of the palea, chimeric floral organs, and ectopic floral organs. Map-based cloning demonstrated that CFO1 encoded the OsMADS32 protein. Phylogenetic analysis revealed that CFO1/OsMADS32 belonged to a monocot-specific clade in the MIKC-type MADS box gene family. The expression domains of CFO1 were mainly restricted to the marginal region of the palea and inner floral organs. The floral organ identity gene DROOPING LEAF (DL) was expressed ectopically in all defective organs of cfo1 flowers. Double mutant analysis revealed that loss of DL function mitigated some of the defects of floral organs in cfo1 flowers. We propose that the CFO1 gene plays a pivotal role in maintaining floral organ identity through negative regulation of DL expression. CFO1|OsMADS32,DL Rice cytokinin GATA transcription Factor1 regulates chloroplast development and plant architecture 2013 Plant Physiol Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1. Chloroplast biogenesis has been well documented in higher plants, yet the complex methods used to regulate chloroplast activity under fluctuating environmental conditions are not well understood. In rice (Oryza sativa), the CYTOKININ-RESPONSIVE GATA TRANSCRIPTION FACTOR1 (Cga1) shows increased expression following light, nitrogen, and cytokinin treatments, while darkness and gibberellin reduce expression. Strong overexpression of Cga1 produces dark green, semidwarf plants with reduced tillering, whereas RNA interference knockdown results in reduced chlorophyll and increased tillering. Coexpression, microarray, and real-time expression analyses demonstrate a correlation between Cga1 expression and the expression of important nucleus-encoded, chloroplast-localized genes. Constitutive Cga1 overexpression increases both chloroplast biogenesis and starch production but also results in delayed senescence and reduced grain filling. Growing the transgenic lines under different nitrogen regimes indicates potential agricultural applications for Cga1, including manipulation of biomass, chlorophyll/chloroplast content, and harvest index. These results indicate a conserved mechanism by which Cga1 regulates chloroplast development in higher plants. Cga1 Chalk5 encodes a vacuolar H+-translocating pyrophosphatase influencing grain chalkiness in rice 2014 Nature Genetics National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China. Grain chalkiness is a highly undesirable quality trait in the marketing and consumption of rice grain. However, the molecular basis of this trait is poorly understood. Here we show that a major quantitative trait locus (QTL), Chalk5, influences grain chalkiness, which also affects head rice yield and many other quality traits. Chalk5 encodes a vacuolar H+-translocating pyrophosphatase (V-PPase) with inorganic pyrophosphate (PPi) hydrolysis and H+-translocation activity. Elevated expression of Chalk5 increases the chalkiness of the endosperm, putatively by disturbing the pH homeostasis of the endomembrane trafficking system in developing seeds, which affects the biogenesis of protein bodies and is coupled with a great increase in small vesicle-like structures, thus forming air spaces among endosperm storage substances and resulting in chalky grain. Our results indicate that two consensus nucleotide polymorphisms in the Chalk5 promoter in rice varieties might partly account for the differences in Chalk5 mRNA levels that contribute to natural variation in grain chalkiness. Chalk5 Combined expression of chitinase and beta-1,3-glucanase genes in indica rice (Oryza sativa L.) enhances resistance against Rhizoctonia solani 2008 Plant Science Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Palkalai Nagar, Madurai 625021, Tamil Nadu, India Agrobacterium-mediated transformation of rice was done using the binary vector pNSP3, harbouring the rice chitinase (chi11) gene under maize ubiquitin promoter and the tobacco beta-1,3-glucanase gene under CaMV 35S promoter in the same T-DNA. Four of the six T0 plants had single copies of complete T-DNAs, while the other two had complex integration patterns. Three of the four single-copy lines showed a 3:1 segregation ratio in the T1 generation. Northern and western blot analyses of T1 plants revealed constitutive expression of chitinase and beta-1,3-glucanase genes. Homozygous T2 plants of the single-copy lines CG20, CG27 and CG53 showed 62-, 9.6- and 11-fold higher chitinase activity over the control plants. beta-1,3-Glucanase activity was 1.1- to 2.5-fold higher in the transgenic plants. Bioassay of homozygous T2 plants of the three single-copy transgenic lines against Rhizoctonia solani revealed a 60% reduction in sheath blight Disease Index in the first week. The Disease Index increased from 61.8 in the first week to 90.6 in the third week in control plants, while it remained low (26.8–34.2) in the transgenic T3 plants in the corresponding period, reflecting the persistence of sheath blight resistance for a longer period. chi11 Mendel's green cotyledon gene encodes a positive regulator of the chlorophyll-degrading pathway 2007 Proc Natl Acad Sci U S A Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. Mutants that retain greenness of leaves during senescence are known as "stay-green" mutants. The most famous stay-green mutant is Mendel's green cotyledon pea, one of the mutants used in determining the law of genetics. Pea plants homozygous for this recessive mutation (known as i at present) retain greenness of the cotyledon during seed maturation and of leaves during senescence. We found tight linkage between the I locus and stay-green gene originally found in rice, SGR. Molecular analysis of three i alleles including one with no SGR expression confirmed that the I gene encodes SGR in pea. Functional analysis of sgr mutants in pea and rice further revealed that leaf functionality is lowered despite a high chlorophyll a (Chl a) and chlorophyll b (Chl b) content in the late stage of senescence, suggesting that SGR is primarily involved in Chl degradation. Consistent with this observation, a wide range of Chl-protein complexes, but not the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit, were shown to be more stable in sgr than wild-type plants. The expression of OsCHL and NYC1, which encode the first enzymes in the degrading pathways of Chl a and Chl b, respectively, was not affected by sgr in rice. The results suggest that SGR might be involved in activation of the Chl-degrading pathway during leaf senescence through translational or posttranslational regulation of Chl-degrading enzymes. OsCHL Rice Chlorina-1 and Chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase, a key enzyme for chlorophyll synthesis and chloroplast development 2006 Plant Mol Biol Department of Plant Science, Seoul National University, Seoul, 151-921, Republic of Korea. Photosynthetic organisms exhibit a green color due to the accumulation of chlorophyll pigments in chloroplasts. Mg-protoporphyrin IX chelatase (Mg-chelatase) comprises three subunits (ChlH, ChlD and ChlI) and catalyzes the insertion of Mg(2+) into protoporphyrin IX, the last common intermediate precursor in both chlorophyll and heme biosyntheses, to produce Mg-protoporphyrin IX (MgProto). Chlorophyll deficiency in higher plants results in chlorina (yellowish-green) phenotype. To date, 10 chlorina (chl) mutants have been isolated in rice, but the corresponding genes have not yet been identified. Rice Chl1 and Chl9 genes were mapped to chromosome 3 and isolated by map-based cloning. A missense mutation occurred in a highly conserved amino acid of ChlD in the chl1 mutant and ChlI in the chl9 mutant. Ultrastructural analyses have revealed that the grana are poorly stacked, resulting in the underdevelopment of chloroplasts. In the seedlings fed with aminolevulinate-dipyridyl in darkness, MgProto levels in the chl1 and chl9 mutants decreased up to 25% and 31% of that in wild-type, respectively, indicating that the Mg-chelatase activity is significantly reduced, causing the eventual decrease in chlorophyll synthesis. Furthermore, Northern blot analysis indicated that the nuclear genes encoding the three subunits of Mg-chelatase and LhcpII in chl1 mutant are expressed about 2-fold higher than those in WT, but are not altered in the chl9 mutant. This result indicates that the ChlD subunit participates in negative feedback regulation of plastid-to-nucleus in the expression of nuclear genes encoding chloroplast proteins, but not the ChlI subunit. Chl1|OsChlD,Chl9|OsChlI CHD3 protein recognizes and regulates methylated histone H3 lysines 4 and 27 over a subset of targets in the rice genome 2012 Proc Natl Acad Sci U S A National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, 430070 Wuhan, China. Histone lysine methylation is an important component of the epigenetic system demarcating transcriptionally active and inactive chromatin domains. It is of primary importance in understanding how different histone lysine methylation marks and a specific combination of them are read and interpreted by chromatin proteins to regulate gene expression. In this paper, we report that the rice CHD3 protein CHR729 that was required for many aspects of plant development can interact with dimethylated histone H3 lysine 4 (H3K4me2, a mark associated with moderately expressed or repressed genes) and with trimethylated histone H3 lysine 27 (H3K27me3, a mark associated with repressed genes), respectively, through the chromodomains and the plant homeodomain (PHD) finger of the protein. A mutation or down-regulation of the gene provoked a decrease of H3K27me3 and H3K4me3 (a mark associated with active genes). Genome-wide analysis revealed that H3K27me3 and H3K4me3, respectively, were lost from about 56 and 23% of marked loci, which correspond mostly to under-expressed or repressed genes. In the mutant, a higher-than-expected proportion of down-regulated genes lost H3K4me3, among which many encode DNA-binding transcription factors. These results suggest that the rice CHD3 protein is a bifunctional chromatin regulator able to recognize and modulate H3K4 and H3K27 methylation over repressed or tissue-specific genes, which may be associated with regulation of a gene transcription program of plant development. CHR729|OsCHD3 Enhanced resistance to blast (Magnaporthe grisea) in transgenic Japonica rice by constitutive expression of rice chitinase 1999 Theor Appl Genet National Institute of Agrobiological Resources, 2-1-2,Kannondai , Tsukuba, 305-8602, Japan, JP. Rice blast is the most devastating plant disease in Japan. Our goal is to create new rice varieties which show enhanced resistance against blast, regardless of the race of blast. By an Agrobacterium-mediated transformation method, we reintroduced a rice class-I chitinase gene, Cht-2 or Cht-3, under the control of the enhanced CaMV 35S promoter and a hygromycin phosphotransferase gene, as a selection marker into the Japonica rice varieties Nipponbare and Koshihikari, which have retained the best popularity over a long period in Japan. In regenerated plants (R(0)), the Cht-2 product was found to accumulate intracellularly whereas the Cht-3 product was found to be targeted extracellularly. The transgenic rice plants which constitutively expressed either chitinase gene showed significantly higher resistance against the rice blast pathogen Magnaporthe grisea races 007.0 and 333. Both high-level expression of the chitinase and blast-resistance were stably inherited by the next generation in several lines. Cht-2,Cht-3 Identification and characterization of two new members of the GRAS gene family in rice responsive to N-acetylchitooligosaccharide elicitor 2003 Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Zi Jin Harbor Campus, Yu Hang Tang Road 388, Hangzhou 310058, China None CIGR1,CIGR2,SLR1|OsGAI A rice virescent-yellow leaf mutant reveals new insights into the role and assembly of plastid caseinolytic protease in higher plants 2013 Plant Physiol National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, People's Republic of China. The plastidic caseinolytic protease (Clp) of higher plants is an evolutionarily conserved protein degradation apparatus composed of a proteolytic core complex (the P and R rings) and a set of accessory proteins (ClpT, ClpC, and ClpS). The role and molecular composition of Clps in higher plants has just begun to be unraveled, mostly from studies with the model dicotyledonous plant Arabidopsis (Arabidopsis thaliana). In this work, we isolated a virescent yellow leaf (vyl) mutant in rice (Oryza sativa), which produces chlorotic leaves throughout the entire growth period. The young chlorotic leaves turn green in later developmental stages, accompanied by alterations in chlorophyll accumulation, chloroplast ultrastructure, and the expression of chloroplast development- and photosynthesis-related genes. Positional cloning revealed that the VYL gene encodes a protein homologous to the Arabidopsis ClpP6 subunit and that it is targeted to the chloroplast. VYL expression is constitutive in most tissues examined but most abundant in leaf sections containing chloroplasts in early stages of development. The mutation in vyl causes premature termination of the predicted gene product and loss of the conserved catalytic triad (serine-histidine-aspartate) and the polypeptide-binding site of VYL. Using a tandem affinity purification approach and mass spectrometry analysis, we identified OsClpP4 as a VYL-associated protein in vivo. In addition, yeast two-hybrid assays demonstrated that VYL directly interacts with OsClpP3 and OsClpP4. Furthermore, we found that OsClpP3 directly interacts with OsClpT, that OsClpP4 directly interacts with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT can homodimerize. Together, our data provide new insights into the function, assembly, and regulation of Clps in higher plants. OsClpP3,CLPP4,OsClpP5,VYL,CLPR4,v-ATP-E COE1, an LRR-RLK responsible for commissural vein pattern formation in rice 2010 Plant J Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. Summary Leaf veins have a complex network pattern. Formation of this vein pattern has been widely studied as a model of tissue pattern formation in plants. To understand the molecular mechanism governing the vascular patterning process, we isolated the rice mutant, commissural vein excessive1 (coe1). The coe1 mutants had short commissural vein (CV) intervals and produced clustered CVs. Application of 1-N-naphthylphthalamic acid and brefeldin A decreased CV intervals, and application of 1-naphthaleneacetic acid increased CV intervals in wild-type rice; however, coe1 mutants were insensitive to these chemicals. COE1 encodes a leucine-rich repeat receptor-like kinase, whose amino acid sequence is similar to that of brassinosteroid-insensitive 1-associated receptor kinase 1 (BAK1), and which is localized at the plasma membrane. Because of the sequence similarity of COE1 to BAK1, we also examined the involvement of brassinosteroids in CV formation. Brassinolide, an active brassinosteroid, decreased the CV intervals of wild-type rice, and brassinazole, an inhibitor of brassinosteroid biosynthesis, increased the CV intervals of wild-type rice, but coe1 mutants showed insensitivity to these chemicals. These results suggest that auxin and brassinosteroids regulate CV intervals in opposite directions, and COE1 may regulate CV intervals downstream of auxin and brassinosteroid signals. COE1,OsBISERK1|OsSERK1|OsBAK1 OsCAD2 is the major CAD gene responsible for monolignol biosynthesis in rice culm 2012 Plant Cell Rep Bioscience and Biotechnology Center, Nagoya University, Chikusa-ku, Nagoya, Aichi, Japan. Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step of monolignol biosynthesis. The rice genome contains 12 CAD-like genes, and whereas the proteins encoded by OsCAD2 and OsCAD7 are known to function in monolignol biosynthesis, the degree to which these enzymes contribute to this process and the involvement of the enzymes encoded by the remaining ten genes is unclear. This paper investigates the role of OsCAD2 and the nine other OsCAD-like proteins in monolignol biosynthesis. Among the OsCAD genes analyzed, OsCAD2, an enzyme belonging to the bona fide CAD phylogenetic group, was the most abundantly expressed gene in the uppermost internode, and was expressed at levels that were more than seven times greater than those of the second most abundantly expressed gene, OsCAD1. Promoter-GUS analysis of OsCAD2 (pCAD::GUS) in the internode, sheath, and roots revealed that GUS expression was strong in tissues that accumulated high levels of lignin. Furthermore, expression always preceded lignin accumulation, showing the tight correlation between OsCAD2 expression and monolignol biosynthesis. Additionally, expression of pCAD::GUS was well synchronized with that of rice caffeic acid 3-O-methyltransferase (OsCOMT::GUS), suggesting that the two enzymes function cooperatively during monolignol biosynthesis. Co-expression network analysis of eight OsCAD genes further revealed that, among the OsCAD genes, expression of OsCAD2 was most tightly associated with the transcription of lignin biosynthesis-related genes. These results suggest that OsCAD2 is largely responsible for monolignol biosynthesis in rice, which is similar to that indicated for the predominant role of other plant bona fide CAD protein to monolignol biosynthesis. COMT,GH2|OsCAD2 Flavonoid 3'-O-methyltransferase from rice: cDNA cloning, characterization and functional expression 2006 Phytochemistry Bio/Molecular Informatics Center, Department of Molecular Biotechnology, Konkuk University, 1 Hwayang-dong, Kwangjin-gu, Seoul 143-701, Republic of Korea. Plant O-methyltransferases (OMTs) are known to be involved in methylation of plant secondary metabolites, especially phenylpropanoid and flavonoid compounds. An OMT, ROMT-9, was cloned and characterized from rice using a reverse transcriptase polymerase chain reaction (RT-PCR). The blast results for ROMT-9 showed a 73% identity with caffeic acid OMTs from maize and Triticum aestivum. ROMT-9 was expressed in Escherichia coli and its recombinant protein was purified using affinity chromatography. It was then tested for its ability to transfer the methyl group of S-adenosyl-l-methionine to the flavonoid substrates, eriodictyol, luteolin, quercetin, and taxifolin, all of which have a 3'-hydroxyl functional group. The reaction products were analyzed using TLC, HPLC, HPLC/MS, and NMR spectroscopy. The NMR analysis showed that ROMT-9 transferred the methyl group specifically to the 3'-hydroxyl group of quercetin, resulting in the formation of its methoxy derivative. Furthermore, ROMT-9 converted flavonoids containing the 3'-hydroxy functional group such as eriodictyol, luteolin, quercetin and taxifolin into the corresponding methoxy derivatives, suggesting that ROMT-9 is an OMT with strict specificity for the 3'-hydroxy group of flavonoids. COMT Proteomic analysis of rice defense response induced by probenazole 2008 Phytochemistry Institute of Plant and Microbial Biology, Academia Sinica, 128, Sec. 2, Academia Road, Taipei 11529, Taiwan. Here, we report the first proteomic analysis of rice defense response induced by probenazole (PBZ), an agricultural chemical that has been widely used to protect rice plants from rice blast and the bacterial blight pathogen. Two-dimensional gel electrophoresis (2-DE) was utilized to identify a total of 40 protein spots including 9 protein spots that are up-regulated by PBZ and 31 abundant protein spots. A total of 11 unique proteins from these 9 spots were identified by LC-MS/MS, and the majority of them were classified and/or possessed orthologs in defense-related functions. Five protein spots with only one protein species identified in each spot appear to be PBZ-regulated proteins. They are a putative glutathione S-transferase GSTU17, a putative phenylalanine ammonia-lyase (PAL, XP_466843), a putative caffeic acid 3-O-methyltransferase (COMT), a putative NADH-ubiquinone oxidoreductase, and a putative glucose-1-phosphate adenyltransferase. However, the other six protein species identified from the remaining four protein spots could not be conclusively described as PBZ-regulated proteins due to either the co-migration of two protein species in one spot or the presence of one protein species in two spots. Through real-time reverse transcription polymerase chain reaction (RT-PCR), it was determined that PAL (XP_466843) is likely regulated at the protein level, whereas GSTU17 and COMT were regulated at the mRNA level after PBZ application. Interestingly, the mRNA transcripts of two PAL paralogs were found to be up-regulated by PBZ. We propose that PAL, COMT, and GSTU17 are likely to confer PBZ-induced disease resistance via such functions as biosynthesis and transport of flavonoid-type phytoalexin and/or lignin biogenesis. COMT Molecular and functional analyses of COPT/Ctr-type copper transporter-like gene family in rice 2011 BMC Plant Biol National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. BACKGROUND: The copper (Cu) transporter (COPT/Ctr) gene family has an important role in the maintenance of Cu homeostasis in different species. The rice COPT-type gene family consists of seven members (COPT1 to COPT7). However, only two, COPT1 and COPT5, have been characterized for their functions in Cu transport. RESULTS: Here we report the molecular and functional characterization of the other five members of the rice COPT gene family (COPT2, COPT3, COPT4, COPT6, and COPT7). All members of the rice COPT family have the conserved features of known COPT/Ctr-type Cu transporter genes. Among the proteins encoded by rice COPTs, COPT2, COPT3, and COPT4 physically interacted with COPT6, respectively, except for the known interaction between COPT1 and COPT5. COPT2, COPT3, or COPT4 cooperating with COPT6 mediated a high-affinity Cu uptake in the yeast Saccharomyces cerevisiae mutant that lacked the functions of ScCtr1 and ScCtr3 for Cu uptake. COPT7 alone could mediate a high-affinity Cu uptake in the yeast mutant. None of the seven COPTs alone or in cooperation could complement the phenotypes of S. cerevisiae mutants that lacked the transporter genes either for iron uptake or for zinc uptake. However, these COPT genes, which showed different tissue-specific expression patterns and Cu level-regulated expression patterns, were also transcriptionally influenced by deficiency of iron, manganese, or zinc. CONCLUSION: These results suggest that COPT2, COPT3, and COPT4 may cooperate with COPT6, respectively, and COPT7 acts alone for Cu transport in different rice tissues. The endogenous concentrations of iron, manganese, or zinc may influence Cu homeostasis by influencing the expression of COPTs in rice. COPT1,COPT5 Expression profiling and bioinformatic analyses of a novel stress-regulated multispanning transmembrane protein family from cereals and Arabidopsis 2003 Plant Physiol Departement des Sciences biologiques, Universite du Quebec a Montreal, Case Postale 8888, succursale Centre-ville, Canada H3C 3P8. Cold acclimation is a multigenic trait that allows hardy plants to develop efficient tolerance mechanisms needed for winter survival. To determine the genetic nature of these mechanisms, several cold-responsive genes of unknown function were identified from cold-acclimated wheat (Triticum aestivum). To identify the putative functions and structural features of these new genes, integrated genomic approaches of data mining, expression profiling, and bioinformatic predictions were used. The analyses revealed that one of these genes is a member of a small family that encodes two distinct groups of multispanning transmembrane proteins. The cold-regulated (COR)413-plasma membrane and COR413-thylakoid membrane groups are potentially targeted to the plasma membrane and thylakoid membrane, respectively. Further sequence analysis of the two groups from different plant species revealed the presence of a highly conserved phosphorylation site and a glycosylphosphatidylinositol-anchoring site at the C-terminal end. No homologous sequences were found in other organisms suggesting that this family is specific to the plant kingdom. Intraspecies and interspecies comparative gene expression profiling shows that the expression of this gene family is correlated with the development of freezing tolerance in cereals and Arabidopsis. In addition, several members of the family are regulated by water stress, light, and abscisic acid. Structure predictions and comparative genome analyses allow us to propose that the cor413 genes encode putative G-protein-coupled receptors. COR413-TM1|Oscor413-tm1,Oscor413-pm1 Identification of cDNA encoding cytochrome c oxidase subunit 5c (COX5c) from rice. Comparison of its expression with nuclear-encoded and mitochondrial-encoded COX genes 1999 Genes Genet Syst Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo Little is presently known about the nuclear-encoded genes for cytochrome c oxidase (COX) in higher plants. In rice, only the nuclear-encoded COX5b gene has been reported. To understand the relationship between the expression of nuclear-encoded and mitochondrial-encoded COX genes in rice, we first characterized a cDNA encoding one of the other nuclear COX genes, COX5c, which encodes 63 amino acids. The deduced amino acid sequence of COX5c from rice was highly homologous to that from sweet potato. Genomic Southern hybridization indicated that the rice COX5c subunit is encoded by a single copy of the COX5c gene. Furthermore, we compared the expression patterns of the nuclear-encoded COX5c and COX5b genes with the expression pattern of the mitochondrial-encoded COX1 gene among several organs by Northern blot analysis. The results suggested that regulatory systems of expression between the nuclear-encoded and the mitochondrial-encoded COX genes are different among different organs in rice. COX5c,coxVb|COX5b Low temperature treatment at the young microspore stage induces protein changes in rice anthers 2006 Molecular & Cellular Proteomics Research Council Centre of Excellence for Integrative Legume Research, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory 0200, Australia. Male reproductive development in rice is very sensitive to various forms of environmental stresses including low temperature. A few days of cold treatment (< 20 degrees C) at the young microspore stage induce severe pollen sterility and thus large grain yield reductions. To investigate this phenomenon, anther proteins at the early stages of microspore development, with or without cold treatment at 12 degrees C, were extracted, separated by two-dimensional gel electrophoresis, and compared. The cold-sensitive cultivar Doongara and the relatively cold-tolerant cultivar HSC55 were used. The abundance of 37 anther proteins was changed more than 2-fold after 1, 2, and 4 days of cold treatment in cv. Doongara. Among them, one protein was newly induced, 32 protein spots were up-regulated, and four protein spots were down-regulated. Of these 37 protein spots, we identified two anther-specific proteins (putative lipid transfer protein and Osg6B) and a calreticulin that were down-regulated and a cystine synthase, a beta-6 subunit of the 20 S proteasome, an H protein of the glycine cleavage system, cytochrome c oxidase subunit VB, an osmotin protein homologue, a putative 6-phospho-gluconolactonase, a putative adenylate kinase, a putative cysteine proteinase inhibitor, ribosomal protein S12E, a caffeoyl-CoA O-methyltransferase, and a monodehydroascorbate reductase that were up-regulated. Identification of these proteins is available upon request. Accumulation of these proteins did not vary greatly after cold treatment in panicles of cv. Doongara or in the anthers of the cv. HSC55. The newly induced protein named Oryza sativa cold-induced anther protein (OsCIA) was identified as an unknown protein. The OsCIA protein was detected in panicles, leaves, and seedling tissues under normal growth conditions. Quantitative real time RT-PCR analysis of OsCIA mRNA expression showed no significant change between low temperature-treated and untreated plants. A possible regulatory role for the newly induced protein is proposed. coxVb|COX5b,OsCIA Enhanced tolerance to chilling stress in OsMYB3R-2 transgenic rice is mediated by alteration in cell cycle and ectopic expression of stress genes 2009 Plant Physiol Research Center for Molecular and Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. MYB transcription factors play central roles in plant responses to abiotic stresses. How stress affects development is poorly understood. Here, we show that OsMYB3R-2 functions in both stress and developmental processes in rice (Oryza sativa). Transgenic plants overexpressing OsMYB3R-2 exhibited enhanced cold tolerance. Cold treatment greatly induced the expression of OsMYB3R-2, which encodes an active transcription factor. We show that OsMYB3R-2 specifically bound to a mitosis-specific activator cis-element, (T/C)C(T/C)AACGG(T/C)(T/C)A, a conserved sequence that was found in promoters of cyclin genes such as OsCycB1;1 and OsKNOLLE2. In addition, overexpression of OsMYB3R-2 in rice led to higher transcript levels of several G2/M phase-specific genes, including OsCycB1;1, OsCycB2;1, OsCycB2;2, and OsCDC20.1, than those in OsMYB3R-2 antisense lines or wild-type plants in response to cold treatment. Flow cytometry analysis revealed an increased cell mitotic index in overexpressed transgenic lines of OsMYB3R-2 after cold treatment. Furthermore, resistance to cold stress in the transgenic plants overexpressing OsCycB1;1 was also enhanced. The level of cellular free proline was increased in the overexpressed rice lines of OsMYB3R-2 and OsCycB1;1 transgenic plants compared with wild-type plants under the cold treatment. These results suggest that OsMYB3R-2 targets OsCycB1;1 and regulates the progress of the cell cycle during chilling stress. OsCPT1, which may be involved in the dehydration-responsive element-binding factor 1A pathway, showed the same transcription pattern in response to cold as did OsCycB1;1 in transgenic rice. Therefore, a cold resistance mechanism in rice could be mediated by regulating the cell cycle, which is controlled by key genes including OsMYB3R-2. CPT1,CycB1;1,OsMYB3R-2 The Rice COLEOPTILE PHOTOTROPISM1 gene encoding an ortholog of Arabidopsis NPH3 is required for phototropism of coleoptiles and lateral translocation of auxin 2005 Plant Cell Botanical Gardens, Graduate School of Science, Osaka City University, Kisaichi, Katano-shi, Osaka 576-0004, Japan. We isolated a mutant, named coleoptile phototropism1 (cpt1), from gamma-ray-mutagenized japonica-type rice (Oryza sativa). This mutant showed no coleoptile phototropism and severely reduced root phototropism after continuous stimulation. A map-based cloning strategy and transgenic complementation test were applied to demonstrate that a NPH3-like gene deleted in the mutant corresponds to CPT1. Phylogenetic analysis of putative CPT1 homologs of rice and related proteins indicated that CPT1 has an orthologous relationship with Arabidopsis thaliana NPH3. These results, along with those for Arabidopsis, demonstrate that NPH3/CPT1 is a key signal transduction component of higher plant phototropism. In an extended study with the cpt1 mutant, it was found that phototropic differential growth is accompanied by a CPT1-independent inhibition of net growth. Kinetic investigation further indicated that a small phototropism occurs in cpt1 coleoptiles. This response, induced only transiently, was thought to be caused by the CPT1-independent growth inhibition. The 3H-indole-3-acetic acid applied to the coleoptile tip was asymmetrically distributed between the two sides of phototropically responding coleoptiles. However, no asymmetry was induced in cpt1 coleoptiles, indicating that lateral translocation of auxin occurs downstream of CPT1. It is concluded that the CPT1-dependent major phototropism of coleoptiles is achieved by lateral auxin translocation and subsequent growth redistribution. CPT1 Central region component1, a novel synaptonemal complex component, is essential for meiotic recombination initiation in rice 2013 Plant Cell State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. In meiosis, homologous recombination entails programmed DNA double-strand break (DSB) formation and synaptonemal complex (SC) assembly coupled with the DSB repair. Although SCs display extensive structural conservation among species, their components identified are poorly conserved at the sequence level. Here, we identified a novel SC component, designated central region component1 (CRC1), in rice (Oryza sativa). CRC1 colocalizes with ZEP1, the rice SC transverse filament protein, to the central region of SCs in a mutually dependent fashion. Consistent with this colocalization, CRC1 interacts with ZEP1 in yeast two-hybrid assays. CRC1 is orthologous to Saccharomyces cerevisiae pachytene checkpoint2 (Pch2) and Mus musculus THYROID receptor-interacting protein13 (TRIP13) and may be a conserved SC component. Additionally, we provide evidence that CRC1 is essential for meiotic DSB formation. CRC1 interacts with homologous pairing aberration in rice meiosis1 (PAIR1) in vitro, suggesting that these proteins act as a complex to promote DSB formation. PAIR2, the rice ortholog of budding yeast homolog pairing1, is required for homologous chromosome pairing. We found that CRC1 is also essential for the recruitment of PAIR2 onto meiotic chromosomes. The roles of CRC1 identified here have not been reported for Pch2 or TRIP13. CRC1,PAIR1,PAIR2,ZEP1 The auxin responsive AP2/ERF transcription factor CROWN ROOTLESS5 is involved in crown root initiation in rice through the induction of OsRR1, a type-A response regulator of cytokinin signaling 2011 Plant J Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi 464-8601, Japan Bioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi 464-8601, Japan. Cytokinin is known to have negative effects on de novo auxin-induced root formation. However, the regulatory mechanisms of root initiation by both cytokinin and auxin are poorly understood. In this study, we characterized a rice mutant, termed crown rootless5 (crl5), which produced fewer crown roots and displayed impaired initiation of crown root primordia. The expression of CRL5, which encodes a member of the large AP2/ERF transcription factor family protein, was observed in the stem region where crown root initiation occurs. Exogenous auxin treatment induced CRL5 expression without de novo protein biosynthesis, which also required the degradation of AUX/IAA proteins. A putative auxin response element in the CRL5 promoter region specifically interacted with a rice ARF, demonstrating that CRL5 may be a direct target of an ARF, similar to CRL1/ADVENTITIOUS ROOTLESS1 (ARL1) that also regulates crown root initiation. A crl1 crl5 double mutant displayed an additive phenotype, indicating that these two genes function in different genetic pathways for crown root initiation. In addition, ProACT:CRL5/WT showed a cytokinin-resistant phenotype for crown root initiation, and also up-regulated the expression of two negative regulators of cytokinin signaling, OsRR1 and OsRR2, which were downregulated in crl5. Transgenic plants that over-expressed OsRR1 under the control of the CRL5 promoter in a crl5 mutant background produced a higher number of crown roots than the crl5 plant. Taken together, these results indicate that auxin-induced CRL5 promotes crown root initiation through repression of cytokinin signaling by positively regulating type-A RR, OsRR1. CRL1|ARL1,Crl5,OsRR1,OsRR2 Constitutive expression of pathogen-inducible OsWRKY31 enhances disease resistance and affects root growth and auxin response in transgenic rice plants 2008 Cell Res State Key Laboratory of Agrobiotechnology, Department of Plant Pathology, China Agricultural University, Yuanmingyuan West Road 2, Beijing 100094, China. WRKY transcription factors have many regulatory roles in response to biotic and abiotic stresses. In this study, we isolated a rice WRKY gene (OsWRKY31) that is induced by the rice blast fungus Magnaporthe grisea and auxin. This gene encodes a polypeptide of 211 amino-acid residues and belongs to a subgroup of the rice WRKY gene family that probably originated after the divergence of monocot and dicot plants. OsWRKY31 was found to be localized to the nucleus of onion epidermis cells to transiently express OsWRKY31-eGFP fusion protein. Analysis of OsWRKY31 and its mutants fused with a Gal4 DNA-binding domain indicated that OsWRKY31 has transactivation activity in yeast. Overexpression of the OsWRKY31 gene was found to enhance resistance against infection with M. grisea, and the transgenic lines exhibited reduced lateral root formation and elongation compared with wild-type and RNAi plants. The lines with overexpression showed constitutive expression of many defense-related genes, such as PBZ1 and OsSci2, as well as early auxin-response genes, such as OsIAA4 and OsCrl1 genes. Furthermore, the plants with overexpression were less sensitive to exogenously supplied IBA, NAA and 2,4-D at high concentrations, suggesting that overexpression of the OsWRKY31 gene might alter the auxin response or transport. These results also suggest that OsWRKY31 might be a common component in the signal transduction pathways of the auxin response and the defense response in rice. CRL1|ARL1,OsIAA4,OsPR10a|PBZ1,OsWRKY31,OsWRKY55|WRKY55a|WRKY55b Studies on sodium bypass flow in lateral rootless mutants lrt1 and lrt2, and crown rootless mutant crl1 of rice (Oryza sativa L.) 2010 Plant Cell Environ Department of Biology and Environmental Science, School of Life Sciences, University of Sussex, Brighton BN19QG, UK. B.Faiyue@sussex.ac.uk An apoplastic pathway, the so-called bypass flow, is important for Na+ uptake in rice (Oryza sativa L.) under saline conditions; however, the precise site of entry is not yet known. We report the results of our test of the hypothesis that bypass flow of Na+ in rice occurs at the site where lateral roots emerge from the main roots. We investigated Na+ uptake and bypass flow in lateral rootless mutants (lrt1, lrt2), a crown rootless mutant (crl1), their wild types (Oochikara, Nipponbare and Taichung 65, respectively) and in seedlings of rice cv. IR36. The results showed that shoot Na+ concentration in lrt1, lrt2 and crl1 was lower (by 20-23%) than that of their wild types. In contrast, the bypass flow quantified using trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS) was significantly increased in the mutants, from an average of 1.1% in the wild types to 3.2% in the mutants. Similarly, bypass flow in shoots of IR36 where the number of lateral and crown roots had been reduced through physical and hormonal manipulations was dramatically increased (from 5.6 to 12.5%) as compared to the controls. The results suggest that the path of bypass flow in rice is not at the sites of lateral root emergence. CRL1|ARL1 ARL1, a LOB-domain protein required for adventitious root formation in rice 2005 Plant J State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310029, China. Adventitious roots constitute the bulk of the fibrous root system in cereals. Compared with the current understanding of shoot development, knowledge of the molecular mechanisms of development of the adventitious roots of cereals is limited. We have isolated and characterized a novel gene controlling the initiation of adventitious root primordia in rice (Oryza sativa L.). The gene, designated Adventitious rootless1 (ARL1), encodes a protein with a LATERAL ORGAN BOUNDARIES (LOB) domain. It is expressed in lateral and adventitious root primordia, tiller primordia, vascular tissues, scutellum, and young pedicels. ARL1 is a nuclear protein and can form homodimers. ARL1 is an auxin- and ethylene-responsive gene, and the expression pattern of ARL1 in roots parallels auxin distribution. Our findings suggest that ARL1 is an auxin-responsive factor involved in auxin-mediated cell dedifferentiation, and that it promotes the initial cell division in the pericycle cells adjacent to the peripheral vascular cylinder in the stem. CRL1|ARL1 Crown rootless1, which is essential for crown root formation in rice, is a target of an AUXIN RESPONSE FACTOR in auxin signaling 2005 Plant Cell Bioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi 464-8601, Japan. Although the importance of auxin in root development is well known, the molecular mechanisms involved are still unknown. We characterized a rice (Oryza sativa) mutant defective in crown root formation, crown rootless1 (crl1). The crl1 mutant showed additional auxin-related abnormal phenotypic traits in the roots, such as decreased lateral root number, auxin insensitivity in lateral root formation, and impaired root gravitropism, whereas no abnormal phenotypic traits were observed in aboveground organs. Expression of Crl1, which encodes a member of the plant-specific ASYMMETRIC LEAVES2/LATERAL ORGAN BOUNDARIES protein family, was localized in tissues where crown and lateral roots are initiated and overlapped with beta-glucuronidase staining controlled by the DR5 promoter. Exogenous auxin treatment induced Crl1 expression without de novo protein biosynthesis, and this induction required the degradation of AUXIN/INDOLE-3-ACETIC ACID proteins. Crl1 contains two putative auxin response elements (AuxREs) in its promoter region. The proximal AuxRE specifically interacted with a rice AUXIN RESPONSE FACTOR (ARF) and acted as a cis-motif for Crl1 expression. We conclude that Crl1 encodes a positive regulator for crown and lateral root formation and that its expression is directly regulated by an ARF in the auxin signaling pathway. CRL1|ARL1 Molecular mechanism of crown root initiation and the different mechanisms between crown root and radicle in rice 2011 Plant Signal Behav Graduate School of Bioagricultural Sciences, Bioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi, Japan. Monocot plants produce numerous adventitious (crown) roots. The plant hormone auxin has positive effects on crown root formation, while cytokinin suppresses it. We have demonstrated that auxin-induced CROWN ROOTLESS5 (CRL5) regulates crown root initiation in rice through the induction of OsRR1, a negative regulator of cytokinin signaling. CRL5 overexpressing calli formed adventitious roots, although CRL5 overexpressing plants did not induce ectopic roots, suggesting that CRL5, which promotes de novo root initiation, might function only in de-differentiated cells. A radicle initiated normally in a crl5 mutant, in spite of the defect in crown root initiation, whereas crown roots, but not a radicle, were produced in a radicleless1 (ral1) mutant. A crl5 ral1 double mutant displayed an additive phenotype, showing that the formation of each root is regulated by different genetic mechanisms in rice. Crl5 Molecular cloning and characterization of calreticulin, a calcium-binding protein involved in the regeneration of rice cultured suspension cells 2000 Eur J Biochem Department of Molecular Biology, National Institute of Agrobiological Resources, Tsukuba, Japan. A full-length cDNA clone encoding a phosphoprotein (pp56) involved in the regeneration of rice (Oryza sativa L.)-cultured suspension cells was isolated by screening a rice cultured suspension cell cDNA library. The 1558-bp cDNA sequence contains an ORF encoding an acidic (pI 4.38) protein of 424 amino acids (47.9 kDa), sharing 70-93% and 50-53% homology with other plant and mammalian calreticulins, respectively. Sequence analysis of the cDNA clone revealed several significant conserved motifs, including a calreticulin family repeat motif in the central domain and two calreticulin family motifs in the N-domain, indicating that this gene is a rice calreticulin (CRO1). The CRO1 gene in long-term rice cultured suspension cells shows constitutive expression in both suspension culture and regeneration media. In contrast, expression of the CRO1 gene in short-term rice cultured suspension cells, which possess regeneration potential, is increased dramatically when these cells are transferred to the regeneration medium. After approximately 2 weeks in the regeneration medium, the expression of the CRO1 gene reverts to constitutive levels. These results demonstrate the presence of calreticulin in rice cultured suspension cells and its developmental regulation during the regeneration of rice cultured suspension cells. CRO1 Cyclic electron flow around photosystem I via chloroplast NAD(P)H dehydrogenase (NDH) complex performs a significant physiological role during photosynthesis and plant growth at low temperature in rice 2011 Plant J Department of Applied Plant Science, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan. wataru.yamori@biochem.tohoku.ac.jp The role of NAD(P)H dehydrogenase (NDH)-dependent cyclic electron flow around photosystem I in photosynthetic regulation and plant growth at several temperatures was examined in rice (Oryza sativa) that is defective in CHLORORESPIRATORY REDUCTION 6 (CRR6), which is required for accumulation of sub-complex A of the chloroplast NDH complex (crr6). NdhK was not detected by Western blot analysis in crr6 mutants, resulting in lack of a transient post-illumination increase in chlorophyll fluorescence, and confirming that crr6 mutants lack NDH activity. When plants were grown at 28 or 35 degrees C, all examined photosynthetic parameters, including the CO(2) assimilation rate and the electron transport rate around photosystems I and II, at each growth temperature at light intensities above growth light (i.e. 800 mumol photons m(-2) sec(-1)), were similar between crr6 mutants and control plants. However, when plants were grown at 20 degrees C, all the examined photosynthetic parameters were significantly lower in crr6 mutants than control plants, and this effect on photosynthesis caused a corresponding reduction in plant biomass. The F(v)/F(m) ratio was only slightly lower in crr6 mutants than in control plants after short-term strong light treatment at 20 degrees C. However, after long-term acclimation to the low temperature, impairment of cyclic electron flow suppressed non-photochemical quenching and promoted reduction of the plastoquinone pool in crr6 mutants. Taken together, our experiments show that NDH-dependent cyclic electron flow plays a significant physiological role in rice during photosynthesis and plant growth at low temperature. CRR6 Calcium-activated (p)ppGpp synthetase in chloroplasts of land plants 2007 J Biol Chem Cell-Free Science and Technology Research Center, Ehime University, Matsuyama, Japan. tozaway@ccr.ehime-u.ac.jp The genetic system of chloroplasts, including the machinery for transcription, translation, and DNA replication, exhibits substantial similarity to that of eubacteria. Chloroplasts are also thought to possess a system for generating guanosine 5'-triphosphate ((p)ppGpp), which triggers the stringent response in eubacteria, with genes encoding chloroplastic (p)ppGpp synthetase having been identified. We now describe the identification and characterization of genes (OsCRSH1, OsCRSH2, and OsCRSH3) for a novel type of (p)ppGpp synthetase in rice. The proteins encoded by these genes contain a putative chloroplast transit peptide at the NH(2) terminus, a central RelA-SpoT-like domain, and two EF-hand motifs at the COOH terminus. The recombinant OsCRSH1 protein was imported into chloroplasts in vitro, and genetic complementation analysis revealed that expression of OsCRSH1 suppressed the phenotype of an Escherichia coli mutant deficient in the RelA and SpoT enzymes. Biochemical analysis showed that the OsCRSH proteins possess (p)ppGpp synthetase activity that is dependent both on Ca(2+) and on the EF-hand motifs. A data base search identified a CRSH homolog in the dicotyledon Arabidopsis thaliana, indicating that such genes are conserved among both monocotyledonous and dicotyledonous land plants. CRSH proteins thus likely function as Ca(2+)-activated (p)ppGpp synthetases in plant chloroplasts, implicating both Ca(2+) and (p)ppGpp signaling in regulation of the genetic system of these organelles. OsCRSH3 Carbon starved anther encodes a MYB domain protein that regulates sugar partitioning required for rice pollen development 2010 Plant Cell School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. In flowering plants, sink tissues rely on transport of carbohydrates from photosynthetic tissues (sources) for nutrition and energy. However, how sugar partitioning in plants is regulated at the molecular level during development remains unknown. We have isolated and characterized a rice (Oryza sativa) mutant, carbon starved anther (csa), that showed increased sugar contents in leaves and stems and reduced levels of sugars and starch in floral organs. In particular, the csa mutant had reduced levels of carbohydrates in later anthers and was male sterile. The csa mutant had reduced accumulation of (14)C-labeled sugars in anther sink tissue. CSA was isolated by map-based cloning and was shown to encode an R2R3 MYB transcription factor that was expressed preferentially in the anther tapetal cells and in the sugar-transporting vascular tissues. In addition, the expression of MST8, encoding a monosaccharide transporter, was greatly reduced in csa anthers. Furthermore, CSA was found to be associated in vivo and in vitro with the promoter of MST8. Our findings suggest that CSA is a key transcriptional regulator for sugar partitioning in rice during male reproductive development. This study also establishes a molecular model system for further elucidation of the genetic control of carbon partitioning in plants. CSA,OsMST8 Mutation in CSA creates a new photoperiod-sensitive genic male sterile line applicable for hybrid rice seed production 2013 Proc Natl Acad Sci U S A State Key Laboratory of Hybrid Rice, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. Rice is a major staple food worldwide. Making hybrid rice has proved to be an effective strategy to significantly increase grain yield. Current hybrid rice technologies rely on male sterile lines and have been used predominantly in indica cultivars. However, intrinsic problems exist in the implementation of these technologies, such as limited germplasms and unpredictable conversions from sterility to fertility in the field. Here, we describe a photoperiod-controlled male sterile line, carbon starved anther (csa), which contains a mutation in an R2R3 MYB transcription regulator of pollen development. This mutation was introduced into indica and japonica rice, and it rendered male sterility under short-day conditions and male fertility under long-day conditions in both lines. Furthermore, F(1) plants of csa and a restorer line JP69 exhibited heterosis (hybrid vigor), suggesting the feasibility of using this mutation to create hybrid rice. The csa-based photoperiod-sensitive male sterile line allows the establishment of a stable two-line hybrid system, which promises to have a significant impact on agriculture. CSA Loss of Cellulose synthase-like F6 function affects mixed-linkage glucan deposition, cell wall mechanical properties, and defense responses in vegetative tissues of rice 2012 Plant Physiol Joint BioEnergy Institute, Emeryville, California 94608, USA. Mixed-linkage glucan (MLG) is a cell wall polysaccharide containing a backbone of unbranched (1,3)- and (1,4)-linked beta-glucosyl residues. Based on its occurrence in plants and chemical characteristics, MLG has primarily been associated with the regulation of cell wall expansion due to its high and transient accumulation in young, expanding tissues. The Cellulose synthase-like F (CslF) subfamily of glycosyltransferases has previously been implicated in mediating the biosynthesis of this polymer. We confirmed that the rice (Oryza sativa) CslF6 gene mediates the biosynthesis of MLG by overexpressing it in Nicotiana benthamiana. Rice cslf6 knockout mutants show a slight decrease in height and stem diameter but otherwise grew normally during vegetative development. However, cslf6 mutants display a drastic decrease in MLG content (97% reduction in coleoptiles and virtually undetectable in other tissues). Immunodetection with an anti-MLG monoclonal antibody revealed that the coleoptiles and leaves retain trace amounts of MLG only in specific cell types such as sclerenchyma fibers. These results correlate with the absence of endogenous MLG synthase activity in mutant seedlings and 4-week-old sheaths. Mutant cell walls are weaker in mature stems but not seedlings, and more brittle in both stems and seedlings, compared to wild type. Mutants also display lesion mimic phenotypes in leaves, which correlates with enhanced defense-related gene expression and enhanced disease resistance. Taken together, our results underline a weaker role of MLG in cell expansion than previously thought, and highlight a structural role for MLG in nonexpanding, mature stem tissues in rice. CslF6 Map-based cloning of the rice cold tolerance gene Ctb1 2010 Plant Science National Agricultural Research Center for Hokkaido Region, Hitsujigaoka 1, Toyohira, Sapporo, 062-8555, Japan Low temperatures during the booting stage reduce rice yields by causing cold-induced male sterility. We previously mapped a quantitative trait locus for cold tolerance, Ctb1, to a 56-kb region containing 7 putative genes. In this study we further mapped Ctb1 to a 17-kb region containing two genes that encode an F-box protein and a ser/thr protein kinase. The F-box protein gene was preferentially expressed in young panicles, while the ser/thr protein kinase gene was expressed in leaves and young panicles. Both genes were cloned from a cold-tolerant variety, Norin-PL8, and introduced into a cold-sensitive variety, Hokkai241, and a cold-sensitive line, BT4-74-8. The cold tolerance of T2 and T3 progenies was assessed by measuring the degree of spikelet fertility in plants treated with cool water irrigation (19 °C, 25 cm) or cool air (12 °C, 4 days). The results indicated that the F-box protein gene confers cold tolerance. Cold tolerance is associated with greater anther length, and the transgenic plants had longer anthers than non-transformed controls. The F-box protein interacts with a subunit of the E3 ubiquitin ligase, Skp1, suggesting that an ubiquitin–proteasome pathway is involved in cold tolerance at the booting stage. Ctb1 Physical mapping and putative candidate gene identification of a quantitative trait locus Ctb1 for cold tolerance at the booting stage of rice 2004 Theor Appl Genet National Agricultural Research Center for Hokkaido Region, Hitsujigaoka 1, Toyohira, Sapporo, Hokkaido, Japan. kjsaito@affrc.go.jp Norin-PL8 is a cold-tolerant variety of rice (Oryza sativa L.) that was developed by introgressing chromosomal segments from a cold-tolerant tropical japonica variety, Silewah, into a template japonica variety, Hokkai241. We previously identified two closely linked quantitative trait loci, Ctb1 and Ctb2, for cold tolerance at the booting stage of Norin-PL8 in the long arm of chromosome 4. We report here the physical mapping of Ctb1 and the identification of the candidate genes. A total of 2,008 segregating individuals were screened for recombination in the Ctb1 region by a PCR-based screening, and a series of near-isogenic lines (NILs) were developed from progenies of recombinants. A comparison of the degrees of cold tolerance of the NILs indicated that Ctb1 is located in the 56-kb region covered by a bacterial artificial chromosome clone, OSJNBa0058 K23, that had been sequenced by the International Rice Genome Sequence Project. We found seven open reading frames (ORFs) in the 56-kb region. Two ORFs encoded receptor-like protein kinases that are possibly involved in signal transduction pathways. Proteins that may be associated with a ubiquitin-proteasome pathway were encoded by three ORFs, two of which encoded F-box proteins and one of which encoded a protein with a BAG domain. The other two ORFs encoded a protein with an OTU domain and an unknown protein. We were also able to show that Ctb1 is likely to be associated with anther length, which is one of major factors in cold tolerance at the booting stage. Ctb1 Identification of two closely linked quantitative trait loci for cold tolerance on chromosome 4 of rice and their association with anther length 2001 TAG Theoretical and Applied Genetics National Agricultural Research Center for Hokkaido Region, Hitsujigaoka 1, Toyohira, Sapporo, Hokkaido 062-8555, Japan Norin-PL8 is a cold-tolerant variety of rice (Oryza sativa L.) that was developed by introgressing chromosomal segments from a cold-tolerant javanica variety, Silewah. We previously detected quantitative trait loci (QTLs) for cold tolerance of Norin-PL8 in the introgressions on chromosomes 3 and 4. We provide fine mapping of the QTLs on chromosome 4 and the association between the QTLs and anther length, which has been reported to be a major component of cold tolerance. Interval mapping using a segregating population derived from an advanced backcross progeny indicated that a QTL for cold tolerance is probably located from the center to the proximal end of the introgression. For fine mapping, we developed a set of near-isogenic lines (NILs) from recombinants in the segregating population. Comparison of cold tolerance between the NILs indicated that either the proximal end or the center of the introgression is necessary for cold tolerance. From these results, we concluded that there are at least two QTLs for cold tolerance, tentatively designated as Ctb-1 and Ctb-2, in the introgression on chromosome 4. The map distance between Ctb-1 and Ctb-2 is estimated to be 4.7–17.2 cM. In order to investigate the mechanism underlying cold tolerance by the QTLs, we compared anther lengths of the NILs. The results indicate that both Ctb-1 and Ctb-2 are associated with anther length. Ctb1 Genome-wide expression analysis of HSP70 family genes in rice and identification of a cytosolic HSP70 gene highly induced under heat stress 2013 Funct Integr Genomics Department of Plant Molecular Systems Biotechnology and Crop Biotech Institute, Kyung Hee University, Yongin 446-701, South Korea. khjung2010@khu.ac.kr The heat shock protein 70 (HSP70) gene family plays a key role in protecting plant cells or tissues from thermal or oxidative stress. Although many studies have elucidated the molecular functions of individual family members, genome-wide analysis of this family is still limited, especially for crop species. Our objective was to integrate various meta-profiling data into the context of a phylogenetic tree, which would enable us to perform fine evaluation of functional dominancy or redundancy within this family. Our data indicated that a loss-of-function mutant of a rice cytosolic HSP70 gene (OsctHSP70-1) did not show a clear defective phenotype in response to high temperature because of the existence of another gene family member that was closely clustered with OsctHSP70-1 and had similar expression patterns. Moreover, the second gene showed much stronger anatomical expression. We indirectly analyzed the function of OsctHSP70-1 by studying GUS activity under the control of the endogenous promoter. We also designed a probable interaction network mediated by OsctHSP70-1 and used co-expression analysis among its components to refine the network, suggesting more probable model to explain the function of OsctHSP70-1. OsctHSP70-1 Thermodynamic basis for the stabilities of three CutA1s from Pyrococcus horikoshii,Thermus thermophilus, and Oryza sativa, with unusually high denaturation temperatures 2008 Biochemistry RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan. In order to elucidate the stabilization mechanism of CutA1 from Pyrococcus horikoshii (PhCutA1) with a denaturation temperature of nearly 150 degrees C, GuHCl denaturation and heat denaturation were examined at neutral and acidic pHs. As a comparison, CutA1 proteins from Thermus thermophilus (TtCutA1) and Oryza sativa (OsCutA1) were also examined, which have lower optimum growth temperatures of 75 and 28 degrees C, respectively, than that (98 degrees C) of P. horikoshii. GuHCl-induced unfolding and refolding curves of the three proteins showed hysteresis effects due to an unusually slow unfolding rate. The midpoints of refolding for PhCutA1, TtCutA1 and OsCutA1 were 5.7 M, 3.3 M, and 2.3 M GuHCl, respectively, at pH 8.0 and 37 degrees C. DSC experiments with TtCutA1 and OsCutA1 showed that the denaturation temperatures were remarkably high, 112.8 and 97.3 degrees C, respectively, at pH 7.0 and that the good heat reversibility was amenable to thermodynamic analyses. At acidic pH, TtCutA1 showed higher stability to both heat and denaturant than PhCutA1. Combined with the data for DSC and denaturant denaturation, the unfolding Gibbs energy of PhCutA1 could be depicted as a function of temperature. It was experimentally revealed that (1) the unusually high stability of PhCutA1 basically originates from a common trimer structure of the three proteins, (2) the stability of PhCutA1 is superior to those of the other two CutA1s over all temperatures above 0 degrees C at neutral pH, due to the decrease in both enthalpy and entropy, and (3) ion pairs of PhCutA1 contribute to the unusually high stability at neutral pH. OsCutA1 The expression of Orysa;CycB1;1 is essential for endosperm formation and causes embryo enlargement in rice 2010 Planta College of Life Sciences, Northeast Forestry University, 150040 Harbin, Heilongjiang, China. The cell cycle is an important process during seed development in plants and its progression is driven by a number of core regulators such as the cyclins. Currently, however, little is known regarding the role of the cyclins in embryo and endosperm development in cereals. In our current study, we show that the knockdown of Orysa;CycB1;1 in rice results in the production of abnormal seeds, which at maturity contain only an enlarged embryo. It was further found that a delayed and abnormal cellularization occurred in the endosperm in these knockdown seeds which eventually became abortive. Moreover, the observed development of the enlarged embryo was also morphologically abnormal and found to be caused by an enlarged cell size rather than an increased cell number. Expression analysis showed that Orysa;CycB1;1 transcripts were localized in the endosperm and embryo. Genome-wide transcriptional profiling further indicated that a large number of genes are responsible for the phenotype of the enlarged embryo. The results of the knockdown of Orysa;CycB1;1 via an endosperm or an embryo-specific promoter also suggest that the enlarged embryo may be correlated to the abortive endosperm. Our results suggest that Orysa;CycB1;1 expression is critical for endosperm formation via the regulation of mitotic division, and that the endosperm plays an important role in maintenance of embryo development in rice. CycB1;1 The novel quantitative trait locus GL3.1 controls rice grain size and yield by regulating Cyclin-T1;3 2012 Cell Res National Key Laboratory of Plant Molecular Genetics and National Center for Plant Gene Research (Shanghai), Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China. Increased crop yields are required to support rapid population growth worldwide. Grain weight is a key component of rice yield, but the underlying molecular mechanisms that control it remain elusive. Here, we report the cloning and characterization of a new quantitative trait locus (QTL) for the control of rice grain length, weight and yield. This locus, GL3.1, encodes a protein phosphatase kelch (PPKL) family - Ser/Thr phosphatase. GL3.1 is a member of the large grain WY3 variety, which is associated with weaker dephosphorylation activity than the small grain FAZ1 variety. GL3.1-WY3 influences protein phosphorylation in the spikelet to accelerate cell division, thereby resulting in longer grains and higher yields. Further studies have shown that GL3.1 directly dephosphorylates its substrate, Cyclin-T1;3, which has only been rarely studied in plants. The downregulation of Cyclin-T1;3 in rice resulted in a shorter grain, which indicates a novel function for Cyclin-T in cell cycle regulation. Our findings suggest a new mechanism for the regulation of grain size and yield that is driven through a novel phosphatase-mediated process that affects the phosphorylation of Cyclin-T1;3 during cell cycle progression, and thus provide new insight into the mechanisms underlying crop seed development. We bred a new variety containing the natural GL3.1 allele that demonstrated increased grain yield, which indicates that GL3.1 is a powerful tool for breeding high-yield crops. Cyclin-T1;3,GL3.1|qGL3-1|qGL3|OsPPKL1 Gibberellin modulates anther development in rice via the transcriptional regulation of GAMYB 2009 Plant Cell Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan. Gibberellins (GAs) play important roles in regulating reproductive development, especially anther development. Our previous studies revealed that the MYB transcriptional factor GAMYB, an important component of GA signaling in cereal aleurone cells, is also important for anther development. Here, we examined the physiological functions of GA during anther development through phenotypic analyses of rice (Oryza sativa) GA-deficient, GA-insensitive, and gamyb mutants. The mutants exhibited common defects in programmed cell death (PCD) of tapetal cells and formation of exine and Ubisch bodies. Microarray analysis using anther RNAs of these mutants revealed that rice GAMYB is involved in almost all instances of GA-regulated gene expression in anthers. Among the GA-regulated genes, we focused on two lipid metabolic genes, a cytochrome P450 hydroxylase CYP703A3 and beta-ketoacyl reductase, both of which might be involved in providing a substrate for exine and Ubisch body. GAMYB specifically interacted with GAMYB binding motifs in the promoter regions in vitro, and mutation of these motifs in promoter-beta-glucuronidase (GUS) transformants caused reduced GUS expression in anthers. Furthermore, a knockout mutant for CYP703A3 showed gamyb-like defects in exine and Ubisch body formation. Together, these results suggest that GA regulates exine formation and the PCD of tapetal cells and that direct activation of CYP703A3 by GAMYB is key to exine formation. CYP703A3,OsMYBGA|OsGAMYB,KAR Cytochrome P450 family member CYP704B2 catalyzes the {omega}-hydroxylation of fatty acids and is required for anther cutin biosynthesis and pollen exine formation in rice 2010 Plant Cell School of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai 200240, China. The anther cuticle and microspore exine act as protective barriers for the male gametophyte and pollen grain, but relatively little is known about the mechanisms underlying the biosynthesis of the monomers of which they are composed. We report here the isolation and characterization of a rice (Oryza sativa) male sterile mutant, cyp704B2, which exhibits a swollen sporophytic tapetal layer, aborted pollen grains without detectable exine, and undeveloped anther cuticle. In addition, chemical composition analysis indicated that cutin monomers were hardly detectable in the cyp704B2 anthers. These defects are caused by a mutation in a cytochrome P450 family gene, CYP704B2. The CYP704B2 transcript is specifically detected in the tapetum and the microspore from stage 8 of anther development to stage 10. Heterologous expression of CYP704B2 in yeast demonstrated that CYP704B2 catalyzes the production of omega -hydroxylated fatty acids with 16 and 18 carbon chains. Our results provide insights into the biosynthesis of the two biopolymers sporopollenin and cutin. Specifically, our study indicates that the omega -hydroxylation pathway of fatty acids relying on this ancient CYP704B family, conserved from moss to angiosperms, is essential for the formation of both cuticle and exine during plant male reproductive and spore development. CYP704B2 CYP714B1 and CYP714B2 encode gibberellin 13-oxidases that reduce gibberellin activity in rice 2013 Proc Natl Acad Sci U S A RIKEN Plant Science Center, Tsurumi, Yokohama, Kanagawa 230-0045, Japan. Bioactive gibberellins (GAs) control many aspects of growth and development in plants. GA(1) has been the most frequently found bioactive GA in various tissues of flowering plants, but the enzymes responsible for GA(1) biosynthesis have not been fully elucidated due to the enzymes catalyzing the 13-hydroxylation step not being identified. Because of the lack of mutants defective in this enzyme, biological significance of GA 13-hydroxylation has been unknown. Here, we report that two cytochrome P450 genes, CYP714B1 and CYP714B2, encode GA 13-oxidase in rice. Transgenic Arabidopsis plants that overexpress CYP714B1 or CYP714B2 show semidwarfism. There was a trend that the levels of 13-OH GAs including GA(1) were increased in these transgenic plants. Functional analysis using yeast or insect cells shows that recombinant CYP714B1 and CYP714B2 proteins can convert GA(12) into GA(53) (13-OH GA(12)) in vitro. Moreover, the levels of 13-OH GAs including GA(1) were decreased, whereas those of 13-H GAs including GA(4) (which is more active than GA(1)) were increased, in the rice cyp714b1 cyp714b2 double mutant. These results indicate that CYP714B1 and CYP714B2 play a predominant role in GA 13-hydroxylation in rice. The double mutant plants appear phenotypically normal until heading, but show elongated uppermost internode at the heading stage. Moreover, CYP714B1 and CYP714B2 expression was up-regulated by exogenous application of bioactive GAs. Our results suggest that GA 13-oxidases play a role in fine-tuning plant growth by decreasing GA bioactivity in rice and that they also participate in GA homeostasis. CYP714B1,CYP714B2 Rice CYP734As function as multisubstrate and multifunctional enzymes in brassinosteroid catabolism 2011 Plant J Institute for Advanced Research, Nagoya University, Nagoya, Aichi 464-8601, Japan. sakamoto@iar.nagoya-u.ac.jp Catabolism of brassinosteroids regulates the endogenous level of bioactive brassinosteroids. In Arabidopsis thaliana, bioactive brassinosteroids such as castasterone (CS) and brassinolide (BL) are inactivated mainly by two cytochrome P450 monooxygenases, CYP734A1/BAS1 and CYP72C1/SOB7/CHI2/SHK1; CYP734A1/BAS1 inactivates CS and BL by means of C-26 hydroxylation. Here, we characterized CYP734A orthologs from Oryza sativa (rice). Overexpression of rice CYP734As in transgenic rice gave typical brassinosteroid-deficient phenotypes. These transformants were deficient in both the bioactive CS and its precursors downstream of the C-22 hydroxylation step. Consistent with this result, recombinant rice CYP734As utilized a range of C-22 hydroxylated brassinosteroid intermediates as substrates. In addition, rice CYP734As can catalyze hydroxylation and the second and third oxidations to produce aldehyde and carboxylate groups at C-26 in vitro. These results indicate that rice CYP734As are multifunctional, multisubstrate enzymes that control the endogenous bioactive brassinosteroid content both by direct inactivation of CS and by the suppression of CS biosynthesis by decreasing the levels of brassinosteroid precursors. CYP734A2,CYP734A4,CYP734A5,CYP734A6 Picking sides: distinct roles for CYP76M6 and CYP76M8 in rice oryzalexin biosynthesis 2013 Biochem J Department of Biochemistry, Iowa State University, Ames, 50011, USA. Natural products biosynthesis often requires the action of multiple CYPs (cytochromes P450), whose ability to introduce oxygen, increasing solubility, is critical for imparting biological activity. In previous investigations of rice diterpenoid biosynthesis, we characterized CYPs that catalyse alternative hydroxylation of ent-sandaracopimaradiene, the precursor to the rice oryzalexin antibiotic phytoalexins. In particular, CYP76M5, CYP76M6 and CYP76M8 were all shown to carry out C-7beta hydroxylation, whereas CYP701A8 catalyses C-3alpha hydroxylation, with oxy groups found at both positions in oryzalexins A-D, suggesting that these may act consecutively in oryzalexin biosynthesis. In the present paper, we report that, although CYP701A8 only poorly reacts with 7beta-hydroxy-ent-sandaracopimaradiene, CYP76M6 and CYP76M8 readily react with 3alpha-hydroxy-ent-sandaracopimaradiene. Notably, their activity yields distinct products, resulting from hydroxylation at C-9beta by CYP76M6 or C-7beta by CYP76M8, on different sides of the core tricyclic ring structure. Thus CYP76M6 and CYP76M8 have distinct non-redundant roles in orzyalexin biosynthesis. Moreover, the resulting 3alpha,7beta- and 3alpha,9beta-diols correspond to oryzalexins D and E respectively. Accordingly, the results of the present study complete the functional identification of the biosynthetic pathway underlying the production of these bioactive phytoalexins. In addition, the altered regiochemistry catalysed by CYP76M6 following C-3alpha hydroxylation has some implications for its active-site configuration, offering further molecular insight. CYP76M6,CYP76M8 CYP76M7 is an ent-cassadiene C11alpha-hydroxylase defining a second multifunctional diterpenoid biosynthetic gene cluster in rice 2009 Plant Cell Department of Biochemistry, Iowa State University, Ames, Iowa 50011, USA. Biosynthetic gene clusters are common in microbial organisms, but rare in plants, raising questions regarding the evolutionary forces that drive their assembly in multicellular eukaryotes. Here, we characterize the biochemical function of a rice (Oryza sativa) cytochrome P450 monooxygenase, CYP76M7, which seems to act in the production of antifungal phytocassanes and defines a second diterpenoid biosynthetic gene cluster in rice. This cluster is uniquely multifunctional, containing enzymatic genes involved in the production of two distinct sets of phytoalexins, the antifungal phytocassanes and antibacterial oryzalides/oryzadiones, with the corresponding genes being subject to distinct transcriptional regulation. The lack of uniform coregulation of the genes within this multifunctional cluster suggests that this was not a primary driving force in its assembly. However, the cluster is dedicated to specialized metabolism, as all genes in the cluster are involved in phytoalexin metabolism. We hypothesize that this dedication to specialized metabolism led to the assembly of the corresponding biosynthetic gene cluster. Consistent with this hypothesis, molecular phylogenetic comparison demonstrates that the two rice diterpenoid biosynthetic gene clusters have undergone independent elaboration to their present-day forms, indicating continued evolutionary pressure for coclustering of enzymatic genes encoding components of related biosynthetic pathways. CYP76M7 Auxin responsiveness of a novel cytochrome p450 in rice coleoptiles 2003 Plant Physiol Institut fur Biologie II, Schanzlestrasse 1, D-79104 Freiburg, Germany. christian.chaban@uni-freiburg.de An early auxin-induced gene was isolated from rice (Oryza sativa L. subsp. japonica cv Nihonmasari) coleoptiles by a fluorescent-labeled differential display screen. The full-length gene contains conserved domains characteristic for the cytochrome p450 superfamily. This gene, designated as CYP87A3, was weakly expressed in dark-grown coleoptiles but was up-regulated rapidly and transiently when coleoptile segments were incubated in 5 microm indole-3-acetic acid. This induction by auxin could not be suppressed by cycloheximide. Depletion of segments from endogenous auxin reduced the amount of CYP87A3 transcripts. The CYP87A3 transcript level was rapidly, although transiently, up-regulated in response to light as well. The observed pattern of gene regulation might indicate a role in the suppression of auxin-induced coleoptile growth. The role of CYP87A3 is discussed with respect to auxin signaling in the regulation of coleoptile growth. CYP87A3 Rice CYP90D2 and CYP90D3 catalyze C-23 hydroxylation of brassinosteroids in vitro 2012 Plant Physiol Biochem Faculty of Bioresources and Environmental Sciences, Ishikawa Prefectural University, Nonoichi, Ishikawa 921 8836, Japan. sakamoto@ishikawa-pu.ac.jp Brassinosteroids are biosynthesized from campesterol via several cytochrome P450 (P450)-catalyzed oxidative reactions. We report the biochemical characterization of two brassinosteroid-biosynthetic P450s from rice: CYP90D2 and CYP90D3. A rice dwarf mutant, ebisu dwarf (d2), which contains a defective copy of CYP90D2, is known to be a brassinosteroid-deficient mutant, and CYP90D2 has been considered to act as a C-3 dehydrogenase. However, in vitro biochemical assays using baculovirus/insect cell-produced proteins revealed that both CYP90D2 and CYP90D3 catalyze C-23 hydroxylation of various 22-hydroxylated brassinosteroids, but with markedly different catalytic efficiencies. Both enzymes preferentially convert (22S,24R)-22-hydroxyergost-4-en-3-one, (22S,24R)-22-hydroxy-5alpha-ergostan-3-one, and 3-epi-6-deoxocathasterone to the corresponding 23-hydroxylated products, but are less active in the conversion of (22S)-22-hydroxycampesterol and 6-deoxocathasterone, in vitro. Consistently, the levels of 23-hydroxylated products of these intermediates, namely, 6-deoxoteasterone, 3-dehydro-6-deoxoteasterone, and 6-deoxotyphasterol were decreased in d2 mutants. These results indicate that CYP90D2 and CYP90D3 can act as brassinosteroid C-23 hydroxylases in rice. CYP90D3,D2|CYP90D2 A rice brassinosteroid-deficient mutant, ebisu dwarf (d2), is caused by a loss of function of a new member of cytochrome P450 2003 Plant Cell BioScience and Biotechnology Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan. We characterized a rice dwarf mutant, ebisu dwarf (d2). It showed the pleiotropic abnormal phenotype similar to that of the rice brassinosteroid (BR)-insensitive mutant, d61. The dwarf phenotype of d2 was rescued by exogenous brassinolide treatment. The accumulation profile of BR intermediates in the d2 mutants confirmed that these plants are deficient in late BR biosynthesis. We cloned the D2 gene by map-based cloning. The D2 gene encoded a novel cytochrome P450 classified in CYP90D that is highly similar to the reported BR synthesis enzymes. Introduction of the wild D2 gene into d2-1 rescued the abnormal phenotype of the mutants. In feeding experiments, 3-dehydro-6-deoxoteasterone, 3-dehydroteasterone, and brassinolide effectively caused the lamina joints of the d2 plants to bend, whereas more upstream compounds did not cause bending. Based on these results, we conclude that D2/CYP90D2 catalyzes the steps from 6-deoxoteasterone to 3-dehydro-6-deoxoteasterone and from teasterone to 3-dehydroteasterone in the late BR biosynthesis pathway. CYP90D3,D2|CYP90D2 CYP93G2 is a flavanone 2-hydroxylase required for C-glycosylflavone biosynthesis in rice 2010 Plant Physiol School of Biological Sciences, The University of Hong Kong, Hong Kong, China. C-Glycosylflavones are ubiquitous in the plant kingdom, and many of them have beneficial effects on human health. They are a special group of flavonoid glycosides in which the sugars are C-linked to the flavone skeleton. It has been long presumed that C-glycosylflavones have a different biosynthetic origin from O-glycosylflavonoids. In rice (Oryza sativa), a C-glucosyltransferase (OsCGT) that accepts 2-hydroxyflavanone substrates and a dehydratase activity that selectively converts C-glucosyl-2-hydroxyflavanones to 6C-glucosylflavones were recently described. In this study, we provide in vitro and in planta evidence that the rice P450 CYP93G2 protein encoded by Os06g01250 is a functional flavanone 2-hydroxylase. CYP93G2 is related to the CYP93B subfamily, which consists of dicot flavone synthase II enzymes. In the presence of NADPH, recombinant CYP93G2 converts naringenin and eriodictyol to the corresponding 2-hydroxyflavanones. In addition, CYP93G2 generates 2-hydroxyflavanones, which are modified by O-glycosylation in transgenic Arabidopsis (Arabidopsis thaliana). Coexpression of CYP93G2 and OsCGT in Arabidopsis resulted in the production of C-glucosyl-2-hydroxyflavanones in the dibenzoylmethane tautomeric form. The same structure was reported previously for the in vitro OsCGT reaction products. Thus, CYP93G2 generates 2-hydroxyflavanone substrates from flavanones for C-glucosylation by OsCGT in planta. Furthermore, knocking down Os06g01250 in rice (O. sativa subsp. japonica 'Zhonghua 11') preferentially depleted the accumulation of C-glycosylapigenin, C-glycosylluteolin, and C-glycosylchrysoeriol but did not affect the levels of tricin, which is frequently present as O-glycosides in cereals. Taken together, our work conclusively assigned CYP93G2 as the first enzyme that channels flavanones to C-glycosylflavone biosynthesis in rice. CYP93G2,OsCGT Oryza sativa cytochrome P450 family member OsCYP96B4 reduces plant height in a transcript dosage dependent manner 2011 PLoS One Rice Functional Genomics Group, Temasek Life Sciences Laboratory, National University of Singapore, Singapore, Singapore. BACKGROUND: Plant cytochromes P450 are involved in a wide range of biosynthetic reactions and play various roles in plant development. However, little is known about the biological functions of the subfamily CYP96 in plants. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report a novel semi-dwarf rice mutant, in which a single copy of transposon dissociator (Ds) was inserted into the gene OsCYP96B4 (Oryza sativa Cytochrome P450 96B4). The mutant exhibits the defects in cell elongation and pollen germination, which can be complemented by the wild type OsCYP96B4 and be rescued by remobilization of the Ds element with the presence of the transposase Activator (Ac). Transgenic plants harboring OsCYP96B4 double-stranded RNA interference construct mimicked the mutant phenotype. The oscyp96b4 mutant phenotype could not be rescued by all the tested phytohormones and it was found that OsCYP96B4 reduced plant height in a transcript dosage dependent manner. Heterologous expression of OsCYP96B4 in Schizosaccharomyces pombe resulted in missegregation and wider cells. Further investigation showed that the mutant exhibited the defects in the metabolism of some lipid molecular species when compared with the wild type. CONCLUSIONS/SIGNIFICANCE: The oscyp96b4 mutant is a novel rice semi-dwarf mutant. Our data suggest that OsCYP96B4 might be involved in lipid metabolism and regulate cell elongation. OsCYP96B4 The rice semi-dwarf mutant sd37, caused by a mutation in CYP96B4, plays an important role in the fine-tuning of plant growth 2014 PLoS One State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China ; National Engineering Research Center for Vegetables, Beijing Academy of Agriculture and Forestry Sciences, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North China), Beijing, China. Plant cytochrome P450 has diverse roles in developmental processes and in the response to environmental cues. Here, we characterized the rice (Oryza sativa L ssp. indica cultivar 3037) semi-dwarf mutant sd37, in which the gene CYP96B4 (Cytochrome P450 96B subfamily) was identified and confirmed as the target by map-based cloning and a complementation test. A point mutation in the SRS2 domain of CYP96B4 resulted in a threonine to lysine substitution in the sd37 mutant. Examination of the subcellular localization of the protein revealed that SD37 was ER-localized protein. And SD37 was predominantly expressed in the shoot apical meristem and developing leaf and root maturation zone but not in the root apical meristem. The sd37 leaves, panicles, and seeds were smaller than those of the wild type. Histological analysis further revealed that a decrease in cell number in the mutant, specifically in the shoots, was the main cause of the dwarf phenotype. Microarray analysis demonstrated that the expression of several cell division-related genes was disturbed in the sd37 mutant. In addition, mutation or strongly overexpression of SD37 results in dwarf plants but moderate overexpression increases plant height. These data suggest that CYP96B4 may be an important regulator of plant growth that affects plant height in rice. OsCYP96B4 Rice carotenoid beta-ring hydroxylase CYP97A4 is involved in lutein biosynthesis 2012 Plant Cell Physiol National Key Laboratory of Plant Molecular Genetics and National Center for Plant Gene Research (Shanghai), Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, PR China. Lutein is the most abundant plant carotenoid and plays essential roles in photosystem assembly and stabilization, as well as protection against photostress. To date, only a few lutein biosynthesis genes have been identified in crop plants. In this study, the rice Cyt P450 gene CYP97A4 encoding a carotenoid beta-ring hydroxylase was shown to be involved in lutein biosynthesis. The results revealed that CYP97A4 was preferentially expressed in leaf compared with spikelet, sheath, stalk and root, and encoded a protein localized at the subcellular level to the chloroplasts. Compared with the wild type, the three allelic mutants of CYP97A4 displayed lutein reductions of 12-24% with substantially increased alpha-carotene, while Chl a/b levels were unaltered. The increased alpha-carotene in the mutants led to greater sensitivity under high light stress. Similarly, reactive oxygen species (ROS) imaging of leaves treated with intense light showed that the mutants generally accumulated greater levels of ROS compared with wild-type plants, which probably caused detrimental effects to the plant photosystem. In conclusion, this study demonstrated the important role of CYP97A4 in alpha-carotene hydroxylation in rice, and knock-out of the gene reduced lutein and increased alpha-carotene, contributing to sensitivity to intense light. CYP97A4 OsTGAP1, a bZIP transcription factor, coordinately regulates the inductive production of diterpenoid phytoalexins in rice 2009 J Biol Chem Biotechnology Research Center, The University of Tokyo, Tokyo 113-8657, Japan. Production of major diterpenoid phytoalexins, momilactones and phytocassanes, is induced in rice upon recognition of pathogenic invasion as plant defense-related compounds. We recently showed that biosynthetic genes for momilactones are clustered on rice chromosome 4 and co-expressed after elicitation, mimicking pathogen attack. Because genes for most metabolic pathways in plants are not organized in gene clusters, examination of the mechanism(s) regulating the expression of such clustered genes is needed. Here, we report a chitin oligosaccharide elicitor-inducible basic leucine zipper transcription factor, OsTGAP1, which is essential for momilactone biosynthesis and regulates the expression of the five genes in the cluster. The knock-out mutant for OsTGAP1 had almost no expression of the five clustered genes (OsCPS4, OsKSL4, CYP99A2, CYP99A3, and OsMAS) or production of momilactones upon elicitor treatment. Inductive expression of OsKSL7 for phytocassane biosynthesis was also largely affected in the ostgap1 mutant, although phytocassane accumulation still occurred. Conversely, OsTGAP1-overexpressing lines exhibited enhanced expression of the clustered genes and hyperaccumulation of momilactones in response to the elicitor. Furthermore, enhanced expression of OsKSL7 and hyperaccumulation of phytocassanes was also observed. We also found that OsTGAP1 overexpression can influence transcriptional up-regulation of OsDXS3 in the methylerythritol phosphate pathway, eventually leading to inductive production of diterpenoid phytoalexins. These results indicate that OsTGAP1 functions as a key regulator of the coordinated transcription of genes involved in inductive diterpenoid phytoalexin production in rice and mainly exerts an essential role on expression of the clustered genes for momilactone biosynthesis. CYP99A2,CYP99A3,MAS,OsCPS4|OsCyc1,OsDTS2|OsKSL4|OsKS4,OsTGAP1|OsbZIP37 CYP99A3: functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice 2011 Plant J Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA. Rice (Oryza sativa) produces momilactone diterpenoids as both phytoalexins and allelochemicals. Strikingly, the rice genome contains a biosynthetic gene cluster for momilactone production, located on rice chromosome 4, which contains two cytochrome P450 (CYP) mono-oxygenases, CYP99A2 and CYP99A3, with undefined roles; although it has been previously shown that RNA interference double knock-down of this pair of closely related CYPs reduced momilactone accumulation. Here we attempted biochemical characterization of CYP99A2 and CYP99A3, which was ultimately achieved by complete gene recoding, enabling functional recombinant expression in bacteria. With these synthetic gene constructs it was possible to demonstrate that while CYP99A2 does not exhibit significant activity with diterpene substrates, CYP99A3 catalyzes consecutive oxidations of the C19 methyl group of the momilactone precursor syn-pimara-7,15-diene to form, sequentially, syn-pimaradien-19-ol, syn-pimaradien-19-al, and syn-pimaradien-19-oic acid. These are presumably intermediates in momilactone biosynthesis, as a C19 carboxylic acid moiety is required for formation of the core 19,6-gamma-lactone ring structure. We further were able to detect syn-pimaradien-19-oic acid in rice plants, which indicates physiological relevance for the observed activity of CYP99A3. In addition, we found that CYP99A3 also oxidized syn-stemod-13(17)-ene at C19 to produce, sequentially, syn-stemoden-19-ol, syn-stemoden-19-al, and syn-stemoden-19-oic acid, albeit with lower catalytic efficiency than with syn-pimaradiene. Although the CYP99A3 syn-stemodene-derived products were not detected in planta, these results nevertheless provide a hint at the currently unknown metabolic fate of this diterpene in rice. Regardless of any wider role, our results strongly indicate that CYP99A3 acts as a multifunctional diterpene oxidase in momilactone biosynthesis. CYP99A2,CYP99A3 Distinct roles of protein disulfide isomerase and P5 sulfhydryl oxidoreductases in multiple pathways for oxidation of structurally diverse storage proteins in rice 2011 Plant Cell Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. In the rice (Oryza sativa) endosperm, storage proteins are synthesized on the rough endoplasmic reticulum (ER), in which prolamins are sorted to protein bodies (PBs) called type-I PB (PB-I). Protein disulfide isomerase (PDI) family oxidoreductase PDIL2;3, an ortholog of human P5, contains a conserved structural disulfide in the redox-inactive thioredoxin-like (TRX) domain and was efficiently targeted to the surface of PB-I in a redox active site-dependent manner, whereas PDIL1;1, an ortholog of human PDI, was localized in the ER lumen. Complementation analyses using PDIL1;1 knockout esp2 mutant indicated that the a and a' TRX domains of PDIL1;1 exhibited similar redox activities and that PDIL2;3 was unable to perform the PDIL1;1 functions. PDIL2;3 knockdown inhibited the accumulation of Cys-rich 10-kD prolamin (crP10) in the core of PB-I. Conversely, crP10 knockdown dispersed PDIL2;3 into the ER lumen. Glutathione S-transferase-PDIL2;3 formed a stable tetramer when it was expressed in Escherichia coli, and the recombinant PDIL2;3 tetramer facilitated alpha-globulin(C79F) mutant protein to form nonnative intermolecular disulfide bonds in vitro. These results indicate that PDIL2;3 and PDIL1;1 are not functionally redundant in sulfhydryl oxidations of structurally diverse storage proteins and play distinct roles in PB development. We discuss PDIL2;3-dependent and PDIL2;3-independent oxidation pathways that sustain disulfide bonds of crP10 in PB-I. CysP13|CpP13|PG5a|Prol-06,CysR10|crP10,PDIL2;3,GLB1 The same nuclear proteins bind to the 5?-flanking regions of genes for the rice seed storage protein: 16 kDa albumin, 13 kDa prolamin and type II glutelin 1996 Plant Mol Biol Department of Applied Biological Sciences, School of Agricultural Sciences, 464-01, Chikusa-ku, Nagoya, Japan Expression of rice seed storage-protein genes is dramatically regulated over a short period of seed maturation. To characterize the expression mechanism of the rice seed storage protein genes, their expression of major storage protein genes (16 kDa albumin, 13 kDa prolamin and type II glutelin) were compared by RNA blot analysis. Their coordinate expression suggests that the transcriptional regulatory machinery is shared among the glutelin, prolamin and albumin-genes. We isolated two novel genomic genes for prolamins (PG5a and PG5b) and obtained the promoter region of the glutelin gene by PCR. The 5'-flanking regions of these three rice seed storage protein genes were found to contain some similar conserved sequences. Nuclear extract partially purified from maturing rice seeds was used for the gel shift assay of the 5' region of the RA gene. We identified two DNA sequences of RA gene which were recognized by independent DNA-binding proteins. The complexes of these DNA sequences and DNA-binding proteins were inhibited by the fragments containing the 5' regions of the prolamin and glutelin genes, suggesting that these three genes share transcription factors. CysP13|CpP13|PG5a|Prol-06,PG5b|Prol-08 Cytokinin activity of cis-zeatin and phenotypic alterations induced by overexpression of putative cis-Zeatin-O-glucosyltransferase in rice 2012 Plant Physiol RIKEN Plant Science Center, Tsurumi, Yokohama 230-0045, Japan. cis-Zeatin (cZ) is generally regarded as a cytokinin with little or no activity, compared with the highly active trans-zeatin (tZ). Although recent studies suggested possible roles for cZ, its physiological significance remains unclear. In our studies with rice (Oryza sativa), cZ inhibited seminal root elongation and up-regulated cytokinin-inducible genes, and its activities were comparable to those of tZ. Tracer experiments showed that exogenously supplied cZ-riboside was mainly converted into cZ derivatives but scarcely into tZ derivatives, indicating that isomerizations of cZ derivatives into tZ derivatives are a minor pathway in rice cytokinin metabolism. We identified three putative cZ-O-glucosyltransferases (cZOGT1, cZOGT2, and cZOGT3) in rice. The cZOGTs preferentially catalyzed O-glucosylation of cZ and cZ-riboside rather than tZ and tZ-riboside in vitro. Transgenic rice lines ectopically overexpressing the cZOGT1 and cZOGT2 genes exhibited short-shoot phenotypes, delay of leaf senescence, and decrease in crown root number, while cZOGT3 overexpressor lines did not show shortened shoots. These results propose that cZ activity has a physiological impact on the growth and development of rice. cZOGT1,cZOGT2,cZOGT3 Isolation and characterization of a dominant dwarf gene, d-h, in rice 2014 PLoS One Department of Plant Science, Research Institute of Agriculture and Life Sciences and Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul National University, Seoul, Korea. Plant height is an important agronomic trait that affects grain yield. Previously, we reported a novel semi-dominant dwarf mutant, HD1, derived from chemical mutagenesis using N-methyl-N-nitrosourea (MNU) on a japonica rice cultivar, Hwacheong. In this study, we cloned the gene responsible for the dwarf mutant using a map-based approach. Fine mapping revealed that the mutant gene was located on the short arm of chromosome 1 in a 48 kb region. Sequencing of the candidate genes and rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) analysis identified the gene, d-h, which encodes a protein of unknown function but whose sequence is conserved in other cereal crops. Real-time (RT)-PCR analysis and promoter activity assays showed that the d-h gene was primarily expressed in the nodes and the panicle. In the HD1 plant, the d-h gene was found to carry a 63-bp deletion in the ORF region that was subsequently confirmed by transgenic experiments to be directly responsible for the gain-of-function phenotype observed in the mutant. Since the mutant plants exhibit a defect in GA response, but not in the GA synthetic pathway, it appears that the d-h gene may be involved in a GA signaling pathway. D-h Rice heterotrimeric G-protein alpha subunit (RGA1): in silico analysis of the gene and promoter and its upregulation under abiotic stress 2013 Plant Physiol Biochem Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India. Heterotrimeric G-protein complexes (Galpha, Gbeta and Ggamma) operate at the apex of diverse signal transduction systems along with their cognate transmembrane G-protein coupled receptors (GPCRs) and appropriate downstream effectors in the plant. Rice Galpha in response to stress has not been well studied. Here, we report the in silico analysis of Galpha subunit from Oryza sativa cv. Indica group Swarna [RGA1(I), accession number HQ634688], its promoter and its transcript upregulation in response to abiotic stresses. Genomic sequence of RGA1(I) contains thirteen exonic and twelve intronic segments. Phylogenetic analysis of RGA1(I) demonstrated high homology with Sorghum and maize and is distantly related to barley and wheat. Promoter sequence analysis of RGA1(I) confirms the presence of stress-related cis-regulatory elements viz. ABA, MeJAE, ARE, GT-1 boxes and LTR suggesting its active and possible independent roles in abiotic stress signalling. Expasy PROSITE database of protein families and domains revealed important motifs, patterns and biologically significant sites in RGA1(I). Three dimensional structure of RGA1(I) protein predicted by I-TASSER server and its stereochemical qualities were validated by PROCHECK and QMEAN server indicating the acceptability of the predicted model. The transcript profiling of RGA1(I) showed upregulation following NaCl, cold and drought stress. Under elevated temperature, its transcript was down regulated. Heavy metal(loid)s stress showed rhythmic and strong upregulation. It showed a rhythmic response in ABA stress. These findings provide a critical evidence for its active role in regulation of abiotic stresses in rice. These findings suggest its possible exploitation in the development of abiotic stress tolerance in crops. D1|RGA1 Molecular cloning and characterization of RGA1 encoding a G protein alpha subunit from rice (Oryza sativa L. IR-36) 1995 Plant Mol Biol Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju, Korea. A cDNA clone, RGA1, was isolated by using a GPA1 cDNA clone of Arabidopsis thaliana G protein alpha subunit as a probe from a rice (Oryza sativa L. IR-36) seedling cDNA library from roots and leaves. Sequence analysis of genomic clone reveals that the RGA1 gene has 14 exons and 13 introns, and encodes a polypeptide of 380 amino acid residues with a calculated molecular weight of 44.5 kDa. The encoded protein exhibits a considerable degree of amino acid sequence similarity to all the other known G protein alpha subunits. A putative TATA sequence (ATATGA), a potential CAAT box sequence (AGCAATAC), and a cis-acting element, CCACGTGG (ABRE), known to be involved in ABA induction are found in the promoter region. The RGA1 protein contains all the consensus regions of G protein alpha subunits except the cysteine residue near the C-terminus for ADP-ribosylation by pertussis toxin. The RGA1 polypeptide expressed in Escherichia coli was, however, ADP-ribosylated by 10 microM [adenylate-32P] NAD and activated cholera toxin. Southern analysis indicates that there are no other genes similar to the RGA1 gene in the rice genome. Northern analysis reveals that the RGA1 mRNA is 1.85 kb long and expressed in vegetative tissues, including leaves and roots, and that its expression is regulated by light. D1|RGA1 Heterotrimeric G protein alpha subunit is involved in rice brassinosteroid response 2006 Cell Res Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Nanxincun 20, Xiangshan, Beijing 100093, China. Heterotrimeric G proteins are known to function as messengers in numerous signal transduction pathways. The null mutation of RGA (rice heterotrimeric G protein alpha subunit), which encodes the alpha subunit of heterotrimeric G protein in rice, causes severe dwarfism and reduced responsiveness to gibberellic acid in rice. However, less is known about heterotrimeric G protein in brassinosteroid (BR) signaling, one of the well-understood phytohormone pathways. In the present study, we used root elongation inhibition assay, lamina inclination assay and coleoptile elongation analysis to demonstrated reduced sensitivity of d1 mutant plants (caused by the null mutation of RGA) to 24-epibrassinolide (24-epiBL), which belongs to brassinosteroids and plays a wide variety of roles in plant growth and development. Moreover, RGA transcript level was decreased in 24-epiBL-treated seedlings in a dose-dependent manner. Our results show that RGA is involved in rice brassinosteroid response, which may be beneficial to elucidate the molecular mechanisms of G protein signaling and provide a novel perspective to understand BR signaling in higher plants. D1|RGA1 Study of novel d1 alleles, defective mutants of the α subunit of heterotrimeric G-protein in rice 2009 Genes Genet Syst Department of Bioscience, Fukui Prefectural University It has been shown that the disruption of the α-subunit gene of heterotorimeric G-proteins (Gα) results in dwarf traits, the erection of leaves and the setting of small seeds in rice. These mutants are called d1. We have studied the expression profiles of the transcripts and translation products of rice Gα in ten alleles of d1 including five additional alleles newly identified. By RT-PCR, the transcripts of the Gα gene were detected in the all d1 alleles. By western blot, the Gα proteins were not detected in the plasma membrane fractions of the d1 alleles with the exception of d1-4. In d1-4, one amino acid change in the GTP-binding box A of the Gα protein was occurred and even in this case the Gα protein was only just detectable in the plasma membrane fraction. Given that the Gα protein did not accumulate in the plasma membrane fraction in d1-8 which has a deletion of just a single amino acid in the Gα protein, it is likely that a proper conformation of the Gα is necessary for accumulation of Gα protein in the plasma membrane. Nine alleles of d1 showed a severer phenotype whilst d1-4 exhibited a mild phenotype with respect to seed size and elongation pattern of internodes. As brassinosteroid signaling was known to be partially impaired in d1s, the sensitivity to 24-epibrassinolide (24-epiBL) was compared among d1 alleles in a T65 genetic background. Only d1-4 showed responses similar to wild type rice. The results show that the d1-4 mutant is a mild allele in terms of the phenotype and mild hyposensitivity to the exogenously applied 24-epiBL. D1|RGA1 Suppression of the rice heterotrimeric G protein beta-subunit gene, RGB1, causes dwarfism and browning of internodes and lamina joint regions 2011 Plant J Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka Kenjyojima, Eiheiji-cho, Yoshida-gun, Fukui 910-1195, Japan. In the present study, we investigated the function of the heterotrimeric G protein beta-subunit (Gbeta) gene (RGB1) in rice. RGB1 knock-down lines were generated in the wild type and d1-5, a mutant deficient for the heterotrimeric G protein alpha-subunit (Galpha) gene (RGA1). Both transgenic lines showed browning of the lamina joint regions and nodes that could be attributed to a reduction of RGB1 function, as the abnormality was not observed in d1-5. The RGB1 knock-down lines generated in d1-5 were shorter, suggesting RGB1 to be a positive regulator of cellular proliferation, in addition to RGA1. The number of sterile seeds also increased in both RGB1 knock-down lines. These results suggest that Gbetagamma and Galpha cooperatively function in cellular proliferation and seed fertility. We discuss the potential predominant role of RGB1 in G protein signaling in rice. D1|RGA1,RGB1 The U-box E3 ubiquitin ligase TUD1 functions with a heterotrimeric G alpha subunit to regulate Brassinosteroid-mediated growth in rice 2013 PLoS Genet State Key Laboratory of Molecular Developmental Biology, Chinese Academy of Sciences and National Center for Plant Gene Research, Beijing, China. Heterotrimeric G proteins are an important group of signaling molecules found in eukaryotes. They function with G-protein-coupled-receptors (GPCRs) to transduce various signals such as steroid hormones in animals. Nevertheless, their functions in plants are not well-defined. Previous studies suggested that the heterotrimeric G protein alpha subunit known as D1/RGA1 in rice is involved in a phytohormone gibberellin-mediated signaling pathway. Evidence also implicates D1 in the action of a second phytohormone Brassinosteroid (BR) and its pathway. However, it is unclear how D1 functions in this pathway, because so far no partner has been identified to act with D1. In this study, we report a D1 genetic interactor Taihu Dwarf1 (TUD1) that encodes a functional U-box E3 ubiquitin ligase. Genetic, phenotypic, and physiological analyses have shown that tud1 is epistatic to d1 and is less sensitive to BR treatment. Histological observations showed that the dwarf phenotype of tud1 is mainly due to decreased cell proliferation and disorganized cell files in aerial organs. Furthermore, we found that D1 directly interacts with TUD1. Taken together, these results demonstrate that D1 and TUD1 act together to mediate a BR-signaling pathway. This supports the idea that a D1-mediated BR signaling pathway occurs in rice to affect plant growth and development. D1|RGA1,TUD1 Function and expression pattern of the alpha subunit of the heterotrimeric G protein in rice 2010 Plant Cell Physiol Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka Kenjyojima, Eiheiji-cho, Yoshida-gun, Fukui, 910-1195 Japan. The d1 mutant, which is deficient for the heterotrimeric G-protein alpha subunit (Galpha) gene of rice, shows dwarfism and sets small round seeds. To determine whether dwarfism in d1 is due to a reduction in cell number or to shortened cell length, the cell number of the leaf sheath, the internode, the root and the lemma was compared between Nipponbare, a wild-type rice and d1-5, a d1 allele derived from Nipponbare. Our results indicate that the cell number was reduced in all organs analyzed in d1-5. In addition, cell enlargement was found in roots and lemma of d1-5, although the organ length in d1-5 was shorter than that of wild-type rice. These results suggest that rice Galpha participates in cell proliferation in rice. Western blot analyses using anti-Galpha antibody and RT-PCR analyses indicate that Galpha is mostly expressed in the developing organs. Galpha promoter activity studies using the GUS reporter gene confirmed that the expression of Galpha was highest in developing organs. We conclude that rice Galpha participates in the regulation of cell number in a developmental stage-dependent manner. D1|RGA1 Heterotrimeric G protein signaling is required for epidermal cell death in rice 2009 Plant Physiol Physiologie und Entwicklungsbiologie der Pflanzen, Botanisches Institut, Universitat Kiel, 24118 Kiel, Germany. In rice (Oryza sativa) adventitious root primordia are formed at the nodes as part of normal development. Upon submergence of rice plants, adventitious roots emerge from the nodes preceded by death of epidermal cells above the root primordia. Cell death is induced by ethylene and mediated by hydrogen peroxide (H(2)O(2)). Pharmacological experiments indicated that epidermal cell death was dependent on signaling through G proteins. Treatment with GTP-gamma-S induced epidermal cell death, whereas GDP-beta-S partially inhibited ethylene-induced cell death. The dwarf1 (d1) mutant of rice has repressed expression of the Galpha subunit RGA1 of heterotrimeric G protein. In d1 plants, cell death in response to ethylene and H(2)O(2) was nearly completely abolished, indicating that signaling through Galpha is essential. Ethylene and H(2)O(2) were previously shown to alter gene expression in epidermal cells that undergo cell death. Transcriptional regulation was not generally affected in the d1 mutant, indicating that altered gene expression is not sufficient to trigger cell death in the absence of Galpha. Analysis of genes encoding proteins related to G protein signaling revealed that four small GTPase genes, two GTPase-activating protein genes, and one GDP dissociation inhibitor gene but not RGA1 were differentially expressed in epidermal cells above adventitious roots, indicating that Galpha activity is regulated posttranscriptionally. D1|RGA1 Rice gibberellin-insensitive dwarf mutant gene Dwarf1 encodes the alpha-subunit of GTP-binding protein 1999 Proc Natl Acad Sci U S A Plant Breeding Laboratory, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan. A rice Dwarf 1 gene was identified by using a map-based cloning strategy. Its recessive mutant allele confers a dwarf phenotype. Linkage analysis revealed that a cDNA encoding the alpha-subunit of GTP-binding protein cosegregated with d1 in 3,185 d1 segregants. Southern hybridization analysis with this cDNA as a probe showed different band patterns in several d1 mutant lines. In at least four independent d1 mutants, no gene transcript was observed by Northern hybridization analysis. Sequencing analysis revealed that an 833-bp deletion had occurred in one of the mutant alleles, which resulted in an inability to express GTP-binding protein. A transgenic d1 mutant with GTP-binding protein gene restored the normal phenotype. We conclude that the rice Dwarf 1 gene encodes GTP-binding protein and that the protein plays an important role in plant growth and development. Because the d1 mutant is classified as gibberellin-insensitive, we suggest that the GTP-binding protein might be associated with gibberellin signal transduction. D1|RGA1 Rice dwarf mutantd1, which is defective in the a subunit of the heterotrimeric G protein, affects gibberellin signal transduction 2000 Proc Natl Acad Sci U S A Bioscience Center and Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, 464-8601, Japan. Previously, we reported that the rice dwarf mutant, d1, is defective in the alpha subunit of the heterotrimeric G protein (Galpha). In the present study, gibberellin (GA) signaling in d1 and the role of the Galpha protein in the GA-signaling pathway were investigated. Compared with the wild type, GA induction of alpha-amylase activity in aleurone cells of d1 was greatly reduced. Relative to the wild type, the GA(3)-treated aleurone layer of d1 had lower expression of Ramy1A, which encodes alpha-amylase, and OsGAMYB, which encodes a GA-inducible transcriptional factor, and no increase in expression of Ca(2 +)-ATPase. However, in the presence of high GA concentrations, alpha-amylase induction occurred even in d1. The GA sensitivity of second leaf sheath elongation in d1 was similar to that of the wild type in terms of dose responsiveness, but the response of internode elongation to GA was much lower in d1. Furthermore, Os20ox expression was up-regulated, and the GA content was elevated in the stunted internodes of d1. All these results suggest that d1 affects a part of the GA-signaling pathway, namely the induction of alpha-amylase in the aleurone layer and internode elongation. In addition, a double mutant between d1 and another GA-signaling mutant, slr, revealed that SLR is epistatic to the D1, supporting that the Galpha protein is involved in GA signaling. However, the data also provide evidence for the presence of an alternative GA-signaling pathway that does not involve the Galpha protein. It is proposed that GA signaling via the Galpha protein may be more sensitive than that of the alternative pathway, as indicated by the low GA responsiveness of this Galpha-independent pathway. D1|RGA1,OsMYBGA|OsGAMYB A metastable DWARF1 epigenetic mutant affecting plant stature in rice 2009 Proc Natl Acad Sci U S A Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan. Epigenetic mutations confer heritable changes in gene expression that are not due to changes in the underlying sequence of the DNA. We identified a spontaneous rice mutant, Epi-d1, that shows a metastable dwarf phenotype. The phenotype is mitotically and meiotically inheritable and corresponds to the metastable epigenetic silencing of the DWARF1 (D1) gene. The silenced state is correlated with repressive histone and DNA methylation marks in the D1 promoter region but is not associated with DNA sequence alterations. Compared with other known epigenetic silenced loci in plants such as paramutable maize alleles and silent Arabidopsis genes, the Epi-d1 silencing phenomenon shows a high level of bidirectional metastable mutability. Epigenetic alleles such as Epi-d1 could thus provide for rapid adaptation under selective conditions. D1|RGA1 Suppression of the heterotrimeric G protein causes abnormal morphology, including dwarfism, in rice 1999 Proc Natl Acad Sci U S A Department of Bioscience, Fukui Prefectural University, Fukui 910-1195, Japan. Transgenic rice containing an antisense cDNA for the alpha subunit of rice heterotrimeric G protein produced little or no mRNA for the subunit and exhibited abnormal morphology, including dwarf traits and the setting of small seeds. In normal rice, the mRNA for the alpha subunit was abundant in the internodes and florets, the tissues closely related to abnormality in the dwarf transformants. The position of the alpha-subunit gene was mapped on rice chromosome 5 by mapping with the restriction fragment length polymorphism. The position was closely linked to the locus of a rice dwarf mutant, Daikoku dwarf (d-1), which is known to exhibit abnormal phenotypes similar to those of the transformants that suppressed the endogenous mRNA for the alpha subunit by antisense technology. Analysis of the cDNAs for the alpha subunits of five alleles of Daikoku dwarf (d-1), ID-1, DK22, DKT-1, DKT-2, and CM1361-1, showed that these dwarf mutants had mutated in the coding region of the alpha-subunit gene. These results show that the G protein functions in the formation of normal internodes and seeds in rice. D1|RGA1 The heterotrimeric G protein alpha subunit acts upstream of the small GTPase Rac in disease resistance of rice 2002 Proc Natl Acad Sci U S A Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan. We used rice dwarf1 (d1) mutants lacking a single-copy Galpha gene and addressed Galpha's role in disease resistance. d1 mutants exhibited a highly reduced hypersensitive response to infection by an avirulent race of rice blast. Activation of PR gene expression in the leaves of the mutants infected with rice blast was delayed for 24 h relative to the wild type. H(2)O(2) production and PR gene expression induced by sphingolipid elicitors (SE) were strongly suppressed in d1 cell cultures. Expression of the constitutively active OsRac1, a small GTPase Rac of rice, in d1 mutants restored SE-dependent defense signaling and resistance to rice blast. Galpha mRNA was induced by an avirulent race of rice blast and SE application on the leaf. These results indicated the role of Galpha in R gene-mediated disease resistance of rice. We have proposed a model for the defense signaling of rice in which the heterotrimeric G protein functions upstream of the small GTPase OsRac1 in the early steps of signaling. D1|RGA1,OsRac1 Erect leaves caused by brassinosteroid deficiency increase biomass production and grain yield in rice 2006 Nat Biotechnol Field Production Science Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Midoricho, Nishi-Tokyo, Tokyo 188-0002, Japan. orchardist@fm.a.u-tokyo.ac.jp New cultivars with very erect leaves, which increase light capture for photosynthesis and nitrogen storage for grain filling, may have increased grain yields. Here we show that the erect leaf phenotype of a rice brassinosteroid-deficient mutant, osdwarf4-1, is associated with enhanced grain yields under conditions of dense planting, even without extra fertilizer. Molecular and biochemical studies reveal that two different cytochrome P450s, CYP90B2/OsDWARF4 and CYP724B1/D11, function redundantly in C-22 hydroxylation, the rate-limiting step of brassinosteroid biosynthesis. Therefore, despite the central role of brassinosteroids in plant growth and development, mutation of OsDWARF4 alone causes only limited defects in brassinosteroid biosynthesis and plant morphology. These results suggest that regulated genetic modulation of brassinosteroid biosynthesis can improve crops without the negative environmental effects of fertilizers. D11|CYP724B1,OsDWARF4|CYP90B2,OsCPD1 A novel cytochrome P450 is implicated in brassinosteroid biosynthesis via the characterization of a rice dwarf mutant, dwarf11, with reduced seed length 2005 Plant Cell Fukui Prefectural University, Faculty of Bioscience, Kenjyojima, Matsuoka-cho, Yoshida-gun, Fukui 910-1195, Japan. We have characterized a rice (Oryza sativa) dwarf mutant, dwarf11 (d11), that bears seeds of reduced length. To understand the mechanism by which seed length is regulated, the D11 gene was isolated by a map-based cloning method. The gene was found to encode a novel cytochrome P450 (CYP724B1), which showed homology to enzymes involved in brassinosteroid (BR) biosynthesis. The dwarf phenotype of d11 mutants was restored by the application of the brassinolide (BL). Compared with wild-type plants, the aberrant D11 mRNA accumulated at higher levels in d11 mutants and was dramatically reduced by treatment with BL, implying that the gene is feedback-regulated by BL. Precise determination of the defective step(s) in BR synthesis in d11 mutants proved intractable because of tissue specificity and the complex control of BR accumulation in plants. However, 6-deoxotyphasterol (6-DeoxoTY) and typhasterol (TY), but not any upstream intermediates before these compounds, effectively restored BR response in d11 mutants in a lamina joint bending assay. Multiple lines of evidence together suggest that the D11/CYP724B1 gene plays a role in BR synthesis and may be involved in the supply of 6-DeoxoTY and TY in the BR biosynthesis network in rice. D11|CYP724B1 RAV-Like1 maintains brassinosteroid homeostasis via the coordinated activation of BRI1 and biosynthetic genes in rice 2010 Plant Cell Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center, Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Korea. Temporal and spatial variation in the levels of and sensitivity to hormones are essential for the development of higher organisms. Traditionally, end-product feedback regulation has been considered as the key mechanism for the achievement of cellular homeostasis. Brassinosteroids (BRs) are plant steroid hormones that are perceived by the cell surface receptor kinase Brassinosteroid Insensitive1. Binding of these hormones to the receptor activates BR signaling and eventually suppresses BR synthesis. This report shows that RAVL1 regulates the expression of the BR receptor. Furthermore, RAVL1 is also required for the expression of the BR biosynthetic genes D2, D11, and BRD1 that are subject to BR negative feedback. Activation by RAVL1 was coordinated via E-box cis-elements in the promoters of the receptor and biosynthetic genes. Also, RAVL1 is necessary for the response of these genes to changes in cellular BR homeostasis. Genetic evidence is presented to strengthen the observation that the primary action of RAVL1 mediates the expression of genes involved in BR signaling and biosynthesis. This study thus describes a regulatory circuit modulating the homeostasis of BR in which RAVL1 ensures the basal activity of both the signaling and the biosynthetic pathways. D11|CYP724B1,D2|CYP90D2,D61|OsBRI1,RAVL1 The multiple contributions of phytochromes to the control of internode elongation in rice 2011 Plant Physiol Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. iwamas@nias.affrc.go.jp Although phyAphyBphyC phytochrome-null mutants in rice (Oryza sativa) have morphological changes and exhibit internode elongation, even as seedlings, it is unknown how phytochromes contribute to the control of internode elongation. A gene for 1-aminocyclopropane-1-carboxylate oxidase (ACO1), which is an ethylene biosynthesis gene contributing to internode elongation, was up-regulated in phyAphyBphyC seedlings. ACO1 expression was controlled mainly by phyA and phyB, and a histochemical analysis showed that ACO1 expression was localized to the basal parts of leaf sheaths of phyAphyBphyC seedlings, similar to mature wild-type plants at the heading stage, when internode elongation was greatly promoted. In addition, the transcription levels of several ethylene- or gibberellin (GA)-related genes were changed in phyAphyBphyC mutants, and measurement of the plant hormone levels indicated low ethylene production and bioactive GA levels in the phyAphyBphyC mutants. We demonstrate that ethylene induced internode elongation and ACO1 expression in phyAphyBphyC seedlings but not in the wild type and that the presence of bioactive GAs was necessary for these effects. These findings indicate that phytochromes contribute to multiple steps in the control of internode elongation, such as the expression of the GA biosynthesis gene OsGA3ox2, ACO1 expression, and the onset of internode elongation. d18|OsGA3ox2,OsACO1,PHYA,PHYB|OsphyB,PHYC Gibberellin is not a regulator of miR156 in rice juvenile-adult phase change 2012 Rice Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, 113-8657, Japan Plant hormone gibberellin (GA) promotes juvenile-adult phase change in higher plants. To confirm the functions of GA in rice, I used dwarf mutant d18-dy. d18-dy is a loss-of-function allele of D18, which encodes GA3ox2. d18-dy mutant exhibited long juvenile phase in morphological traits such as the size of the shoot apical meristem (SAM), shape of leaf blades, presence or absence of midribs and node–internode differentiation in stem. In contrast, expression patterns of juvenile-adult phase change markers miR156 and miR172 were similar between wild type and d18-dy. In addition, d18-dy mutation and GA did not affect expression levels of downstream genes of miR156. GA does not function upstream of miR156 in juvenile-adult phase change. d18|OsGA3ox2 OsDOG, a gibberellin-induced A20/AN1 zinc-finger protein, negatively regulates gibberellin-mediated cell elongation in rice 2011 J Plant Physiol Research Center for Molecular and Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. The A20/AN1 zinc-finger proteins (ZFPs) play pivotal roles in animal immune responses and plant stress responses. From previous gibberellin (GA) microarray data and A20/AN1 ZFP family member association, we chose Oryza sativa dwarf rice with overexpression of gibberellin-induced gene (OsDOG) to examine its function in the GA pathway. OsDOG was induced by gibberellic acid (GA(3)) and repressed by the GA-synthesis inhibitor paclobutrazol. Different transgenic lines with constitutive expression of OsDOG showed dwarf phenotypes due to deficiency of cell elongation. Additional GA(1) and real-time PCR quantitative assay analyses confirmed that the decrease of GA(1) in the overexpression lines resulted from reduced expression of GA3ox2 and enhanced expression of GA2ox1 and GA2ox3. Adding exogenous GA rescued the constitutive expression phenotypes of the transgenic lines. OsDOG has a novel function in regulating GA homeostasis and in negative maintenance of plant cell elongation in rice. d18|OsGA3ox2,GA2OX3,OsGA2ox1,OsDOG|OsSAP11 The rice YABBY1 gene is involved in the feedback regulation of gibberellin metabolism 2007 Plant Physiol National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China. Gibberellin (GA) biosynthesis is regulated by feedback control providing a mechanism for GA homeostasis in plants. However, regulatory elements involved in the feedback control are not known. In this report, we show that a rice (Oryza sativa) YABBY1 (YAB1) gene had a similar expression pattern as key rice GA biosynthetic genes GA3ox2 and GA20ox2. Overexpression of YAB1 in transgenic rice resulted in a semidwarf phenotype that could be fully rescued by applied GA. Quantification of the endogenous GA content revealed increases of GA(20) and decreases of GA(1) levels in the overexpression plants, in which the transcripts of the biosynthetic gene GA3ox2 were decreased. Cosuppression of YAB1 in transgenic plants induced expression of GA3ox2. The repression of GA3ox2 could be obtained upon treatment by dexamethasone of transgenic plants expressing a YAB1-glucocorticoid receptor fusion. Importantly, we show that YAB1 bound to a GA-responsive element within the GA3ox2 promoter. In addition, the expression of YAB1 was deregulated in GA biosynthesis and signaling mutants and could be either transiently induced by GA or repressed by a GA inhibitor. Finally, either overexpression or cosuppression of YAB1 impaired GA-mediated repression of GA3ox2. These data together suggest that YAB1 is involved in the feedback regulation of GA biosynthesis in rice. d18|OsGA3ox2,OsYABBY1|OsYAB1,sd1|GA20ox2 Cloning and functional analysis of two gibberellin 3 beta -hydroxylase genes that are differently expressed during the growth of rice 2001 Proc Natl Acad Sci U S A Bioscience Center, Nagoya University, Chikusa, Nagoya, 464-8601, Japan. We have cloned two gibberellin (GA) 3 beta-hydroxylase genes, OsGA3ox1 and OsGA3ox2, from rice by screening a genomic library with a DNA fragment obtained by PCR using degenerate primers. We have used full-scan GC-MS and Kovats retention indices to show function for the two encoded recombinant fusion proteins. Both proteins show 3 beta-hydroxylase activity for the steps GA(20) to GA(1), GA(5) to GA(3), GA(44) to GA(38), and GA(9) to GA(4). In addition, indirect evidence suggests that the OsGA3ox1 protein also has 2,3-desaturase activity, which catalyzes the steps GA(9) to 2,3-dehydro-GA(9) and GA(20) to GA(5) (2,3-dehydro GA(20)), and 2 beta-hydroxylase activity, which catalyzes the steps GA(1) to GA(8) and GA(4) to GA(34). Molecular and linkage analysis maps the OsGA3ox1 gene to the distal end of the short arm of chromosome 5; the OsGA3ox2 gene maps to the distal end of the short arm of chromosome 1 that corresponds to the D18 locus. The association of the OsGA3ox2 gene with the d18 locus is confirmed by sequence and complementation analysis of three d18 alleles. Complementation of the d18-AD allele with the OxGA3ox2 gene results in transgenic plants with a normal phenotype. Although both genes show transient expression, the highest level for OsGA3ox1 is from unopened flower. The highest level for OsGA3ox2 is from elongating leaves. d18|OsGA3ox2,OsGA3ox1 The rice GERMINATION DEFECTIVE 1, encoding a B3 domain transcriptional repressor, regulates seed germination and seedling development by integrating GA and carbohydrate metabolism 2013 Plant J State Key Laboratory of Plant Genomics and National Center for Plant Gene Research-Beijing, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. It has been shown that seed development is regulated by a network of transcription factors in Arabidopsis including LEC1 (LEAFY COTYLEDON1), L1L (LEC1-like) and the B3 domain factors LEC2, FUS3 (FUSCA3) and ABI3 (ABA-INSENSITIVE3); however, molecular and genetic regulation of seed development in cereals is poorly understood. To understand seed development and seed germination in cereals, a large-scale screen was performed using our T-DNA mutant population, and a mutant germination-defective1 (gd1) was identified. In addition to the severe germination defect, the gd1 mutant also shows a dwarf phenotype and abnormal flower development. Molecular and biochemical analyses revealed that GD1 encodes a B3 domain-containing transcription factor with repression activity. Consistent with the dwarf phenotype of gd1, expression of the gibberelic acid (GA) inactivation gene OsGA2ox3 is increased dramatically, accompanied by reduced expression of GA biosynthetic genes including OsGA20ox1, OsGA20ox2 and OsGA3ox2 in gd1, resulting in a decreased endogenous GA(4) level. Exogenous application of GA not only induced GD1 expression, but also partially rescued the dwarf phenotype of gd1. Furthermore, GD1 binds to the promoter of OsLFL1, a LEC2/FUS3-like gene of rice, via an RY element, leading to significant up-regulation of OsLFL1 and a large subset of seed maturation genes in the gd1 mutant. Plants over-expressing OsLFL1 partly mimic the gd1 mutant. In addition, expression of GD1 was induced under sugar treatment, and the contents of starch and soluble sugar are altered in the gd1 mutant. These data indicate that GD1 participates directly or indirectly in regulating GA and carbohydrate homeostasis, and further regulates rice seed germination and seedling development. d18|OsGA3ox2,GD1,OsGA20ox1,OsLFL1 Proteomic identification of small, copper-responsive proteins in germinating embryos of Oryza sativa 2009 Ann Bot College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China. BACKGROUND AND AIMS: Although copper (Cu) is an essential micronutrient for plants and algae, excess Cu is toxic to most plants and can cause a wide range of deleterious effects. To investigate the response of rice (Oryza sativa) to Cu stress, a proteomic approach was used to analyse Cu stress-induced changes in the expression of low molecular-weight proteins in germinating rice seed embryos. METHODS: Rice seeds were germinated in the presence or absence of 200 microm Cu for 6 d, and embryos, including newly formed shoots and radicles, were isolated. After proteins were extracted from the germinating embryos and separated by two-dimensional PAGE, 16 proteins in the 6- to 25-kDa range were identified using MALDI-TOF mass spectrometry. KEY RESULTS AND CONCLUSIONS: Thirteen of the proteins identified, including metallothionein-like protein, membrane-associated protein-like protein, putative wall-associated protein kinase, pathogenesis-related proteins and the putative small GTP-binding protein Rab2, were up-regulated by Cu stress. Three proteins, a putative small cytochrome P450 (CYP90D2), a putative thioredoxin and a putative GTPase, were down-regulated by Cu stress. As far as is known, this study provides the first proteomic evidence that metallothionein and CYP90D2 are Cu-responsive proteins in plants. These findings may lead to a better understanding of plant molecular responses to toxic metal exposure. D2|CYP90D2 A comprehensive genetic study reveals a crucial role of CYP90D2/D2 in regulating plant architecture in rice (Oryza sativa) 2013 New Phytol State Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing, 210095, China. Brassinosteroids (BRs) are essential regulators of plant architecture. Understanding how BRs control plant height and leaf angle would facilitate development of new plant type varieties by biotechnology. A number of mutants involved in BR biosynthesis have been isolated but many of them lack detailed genetic analysis. Here, we report the isolation and characterization of a severe dwarf mutant, chromosome segment deleted dwarf 1 (csdd1), which was deficient in BR biosynthesis in rice. We isolated the mutant by screening a tissue culture-derived population, cloned the gene by mapping, and confirmed its function by complementary and RNAi experiments, combined with physiological and chemical analysis. We showed that the severe dwarf phenotype was caused by a complete deletion of a cytochrome P450 gene, CYP90D2/D2, which was further confirmed in two independent T-DNA insertion lines in different genetic backgrounds and by RNA interference. Our chemical analysis suggested that CYP90D2/D2 might catalyze C-3 dehydrogenation step in BR biosynthesis. We have demonstrated that the CYP90D2/D2 gene plays a more important role than previously reported. Allelic mutations of CYP90D2/D2 confer varying degrees of dwarfism and leaf angle, thus providing useful information for molecular breeding in grain crop plants. D2|CYP90D2 DWARF10, an RMS1/MAX4/DAD1 ortholog, controls lateral bud outgrowth in rice 2007 Plant J Graduate School of Agriculture and Life Sciences, University of Tokyo, Yayoi, Bunkyo, Tokyo 113-0032, Japan. Plant architecture is mostly determined by shoot branching patterns. Apical dominance is a well-known control mechanism in the development of branching patterns, but little is known regarding its role in monocots such as rice. Here, we show that the concept of apical dominance can be applied to tiller bud outgrowth of rice. In dwarf10 (d10), an enhanced branching mutant of rice, apical dominance can be observed, but the inhibitory effects of the apical meristem was reduced. D10 is a rice ortholog of MAX4/RMS1/DAD1 that encodes a carotenoid cleavage dioxygenase 8 and is supposed to be involved in the synthesis of an unidentified inhibitor of shoot branching. D10 expression predominantly occurs in vascular cells in most organs. Real-time polymerase chain reaction analysis revealed that accumulation of D10 mRNA is induced by exogenous auxin. Moreover, D10 expression is upregulated in six branching mutants, d3, d10, d14, d17, d27 and high tillering dwarf (htd1). No such effects were found for D3 or HTD1, the MAX2 and MAX3 orthologs, respectively, of rice. These findings imply that D10 transcription might be a critical step in the regulation of the branching inhibitor pathway. In addition, we present observations that suggest that FINE CULM1 (FC1), a rice ortholog of teosinte branched 1 (tb1), possibly works independently of the branching inhibitor pathway. D27,D3,HTD1|OsCCD7,HTD2|D88|D14,D10|OsCCD8|OsCCD8b,OsTB1|FC1 DWARF27, an iron-containing protein required for the biosynthesis of strigolactones, regulates rice tiller bud outgrowth 2009 Plant Cell State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Tillering in rice (Oryza sativa) is one of the most important agronomic traits that determine grain yields. Previous studies on rice tillering mutants have shown that the outgrowth of tiller buds in rice is regulated by a carotenoid-derived MAX/RMS/D (more axillary branching) pathway, which may be conserved in higher plants. Strigolactones, a group of terpenoid lactones, have been recently identified as products of the MAX/RMS/D pathway that inhibits axillary bud outgrowth. We report here the molecular genetic characterization of d27, a classic rice mutant exhibiting increased tillers and reduced plant height. D27 encodes a novel iron-containing protein that localizes in chloroplasts and is expressed mainly in vascular cells of shoots and roots. The phenotype of d27 is correlated with enhanced polar auxin transport. The phenotypes of the d27 d10 double mutant are similar to those of d10, a mutant defective in the ortholog of MAX4/RMS1 in rice. In addition, 2'-epi-5-deoxystrigol, an identified strigolactone in root exudates of rice seedlings, was undetectable in d27, and the phenotypes of d27 could be rescued by supplementation with GR24, a synthetic strigolactone analog. Our results demonstrate that D27 is involved in the MAX/RMS/D pathway, in which D27 acts as a new member participating in the biosynthesis of strigolactones. D27 Strigolactone biosynthesis in Medicago truncatula and rice requires the symbiotic GRAS-type transcription factors NSP1 and NSP2 2011 Plant Cell Department of Plant Science, Laboratory of Molecular Biology, Wageningen University, 6708 PB Wageningen, The Netherlands. Legume GRAS (GAI, RGA, SCR)-type transcription factors NODULATION SIGNALING PATHWAY1 (NSP1) and NSP2 are essential for rhizobium Nod factor-induced nodulation. Both proteins are considered to be Nod factor response factors regulating gene expression after symbiotic signaling. However, legume NSP1 and NSP2 can be functionally replaced by nonlegume orthologs, including rice (Oryza sativa) NSP1 and NSP2, indicating that both proteins are functionally conserved in higher plants. Here, we show that NSP1 and NSP2 are indispensable for strigolactone (SL) biosynthesis in the legume Medicago truncatula and in rice. Mutant nsp1 plants do not produce SLs, whereas in M. truncatula, NSP2 is essential for conversion of orobanchol into didehydro-orobanchol, which is the main SL produced by this species. The disturbed SL biosynthesis in nsp1 nsp2 mutant backgrounds correlates with reduced expression of DWARF27, a gene essential for SL biosynthesis. Rice and M. truncatula represent distinct phylogenetic lineages that split approximately 150 million years ago. Therefore, we conclude that regulation of SL biosynthesis by NSP1 and NSP2 is an ancestral function conserved in higher plants. NSP1 and NSP2 are single-copy genes in legumes, which implies that both proteins fulfill dual regulatory functions to control downstream targets after rhizobium-induced signaling as well as SL biosynthesis in nonsymbiotic conditions. D27,OsNSP1,OsNSP2 The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones 2012 Plant Physiol Plant Energy Biology , University of Western Australia, Crawley, Western Australia 6009, Australia. Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. In Arabidopsis (Arabidopsis thaliana), SL biosynthesis requires the sequential action of two carotenoid cleavage dioxygenases, MORE AXILLARY GROWTH3 (MAX3) and MAX4, followed by a cytochrome P450, MAX1. In rice (Oryza sativa), the plastid-localized protein DWARF27 (OsD27) is also necessary for SL biosynthesis, but the equivalent gene in Arabidopsis has not been identified. Here, we use phylogenetic analysis of D27-like sequences from photosynthetic organisms to identify AtD27, the likely Arabidopsis ortholog of OsD27. Using reverse genetics, we show that AtD27 is required for the inhibition of secondary bud outgrowth and that exogenous application of the synthetic SL GR24 can rescue the increased branching phenotype of an Atd27 mutant. Furthermore, we use grafting to demonstrate that AtD27 operates on a nonmobile precursor upstream of MAX1 in the SL biosynthesis pathway. Consistent with the plastid localization of OsD27, we also show that AtD27 possesses a functional plastid transit peptide. We demonstrate that AtD27 transcripts are subject to both local feedback and auxin-dependent signals, albeit to a lesser extent than MAX3 and MAX4, suggesting that early steps in SL biosynthesis are coregulated at the transcriptional level. By identifying an additional component of the canonical SL biosynthesis pathway in Arabidopsis, we provide a new tool to investigate the regulation of shoot branching and other SL-dependent developmental processes. D27 Identification and characterization of HTD2: a novel gene negatively regulating tiller bud outgrowth in rice 2009 Planta State Key Laboratory of Rice Biology, China National Rice Research Institute, 310006 Hangzhou, Zhejiang, China. lwzzju@163.com Tiller number is highly regulated by controlling the formation of tiller bud and its subsequent outgrowth in response to endogenous and environmental signals. Here, we identified a rice mutant htd2 from one of the 15,000 transgenic rice lines, which is characterized by a high tillering and dwarf phenotype. Phenotypic analysis of the mutant showed that the mutation did not affect formation of tiller bud, but promoted the subsequent outgrowth of tiller bud. To isolate the htd2 gene, a map-based cloning strategy was employed and 17 new insertions-deletions (InDels) markers were developed. A high-resolution physical map of the chromosomal region around the htd2 gene was made using the F(2) and F(3) population. Finally, the gene was mapped in 12.8 kb region between marker HT41 and marker HT52 within the BAC clone OSJNBa0009J13. Cloning and sequencing of the target region from the mutant showed that the T-DNA insertion caused a 463 bp deletion between the promoter and first exon of an esterase/lipase/thioesterase family gene in the 12.8 kb region. Furthermore, transgenic rice with reduced expression level of the gene exhibited an enhanced tillering and dwarf phenotype. Accordingly, the esterase/lipase/thioesterase family gene (TIGR locus Os03g10620) was identified as the HTD2 gene. HTD2 transcripts were expressed mainly in leaf. Loss of function of HTD2 resulted in a significantly increased expression of HTD1, D10 and D3, which were involved in the strigolactone biosynthetic pathway. The results suggest that the HTD2 gene could negatively regulate tiller bud outgrowth by the strigolactone pathway. D3,HTD1|OsCCD7,HTD2|D88|D14,D10|OsCCD8|OsCCD8b Rice tillering dwarf mutant dwarf3 has increased leaf longevity during darkness-induced senescence or hydrogen peroxide-induced cell death 2007 Genes Genet Syst Graduate School of Agricultural and Life Sciences, The University of Tokyo Senescence or cell death in plant leaves is known to be inducible by darkness or H(2)O(2). When the Arabidopsis gene MAX2/ORE9 is disrupted, leaf senescence or cell death in response to the above stimuli is delayed. Because the rice (Oryza sativa L.) gene DWARF3 (D3) is orthologous to MAX2/ORE9, we wished to know whether disruption of D3 also results in increased longevity in leaves. We found that darkness-induced senescence or H(2)O(2)-induced cell death in the third leaf [as measured by chlorophyll degradation, membrane ion leakage and expression of senescence-associated genes (SAGs)] in a d3 rice mutant was delayed by 1-3 d compared to that in its reference line Shiokari. Moreover, the mRNA levels of D3, HTD1 and D10, which are orthologs of Arabidopsis MAX2/ORE9, MAX3 and MAX4, respectively, increased during cell death. These results suggest that D3 protein in rice, like MAX2/ORE9 in Arabidopsis, is involved in leaf senescence or cell death. D3,HTD1|OsCCD7,D10|OsCCD8|OsCCD8b Genetic interaction between 2 tillering genes, reduced culm number 1 (rcn1) and tillering dwarf gene d3, in rice 2007 J Hered Department of Crop Science Obihiro University of Agriculture and Veterinary Medicine, Nishi Inada-cho, Obihiro, Hokkaido, Japan. Mutant genes, reduced culm number 1 (rcn1) and bunketsuwaito tillering dwarf (d3), affect tiller number in rice (Oryza sativa L.) in opposite directions. The d3 mutant was reported to increase tiller number and reduce plant stature. Our objective was to compare the phenotype of the d3rcn1 double mutant with each single mutant and parental rice cultivar "Shiokari" and to clarify whether the Rcn1 gene interacted with the D3 gene. We recovered a new rcn1 mutant from Shiokari and developed d3rcn1 double mutant with Shiokari genetic background. A new rcn1 mutant, designated as "S-97-61" exhibited a reduction in tiller number and plant stature to about the same level as the previously reported original rcn1 mutant. Three near-isogenic lines, rcn1 mutant, d3 mutant, and d3rcn1 double mutant, were grown together with the parental Shiokari. The reduction in tillering by the rcn1 mutation was independent of the d3 genotype, and tillering number of d3rcn1 double mutant was between those of the d3 and rcn1 mutants. These results demonstrated that the Rcn1 gene was not involved in the D3-associated pathway in tillering control. D3,Rcn1 The rice HIGH-TILLERING DWARF1 encoding an ortholog of Arabidopsis MAX3 is required for negative regulation of the outgrowth of axillary buds 2006 Plant J State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Rice tillering is an important agronomic trait for grain production. The HIGH-TILLERING DWARF1 (HTD1) gene encodes an ortholog of Arabidopsis MAX3. Complementation analyses for HTD1 confirm that the defect in HTD1 is responsible for both high-tillering and dwarf phenotypes in the htd1 mutant. The rescue of the Arabidopsis max3 mutant phenotype by the introduction of Pro(35S):HTD1 indicates HTD1 is a carotenoid cleavage dioxygenase that has the same function as MAX3 in synthesis of a carotenoid-derived signal molecule. The HTD1 gene is expressed in both shoot and root tissues. By evaluating Pro(HTD1):GUS expression, we found that the HTD1 gene is mainly expressed in vascular bundle tissues throughout the plant. Auxin induction of HTD1 expression suggests that auxin may regulate rice tillering partly through upregulation of HTD1 gene transcription. Restoration of dwarf phenotype after the removal of axillary buds indicates that the dwarfism of the htd1 mutant may be a consequence of excessive tiller production. In addition, the expression of HTD1, D3 and OsCCD8a in the htd1 and d3 mutants suggests a feedback mechanism may exist for the synthesis and perception of the carotenoid-derived signal in rice. Characterization of MAX genes in Arabidopsis, and identification of their orthologs in pea, petunia and rice indicates the existence of a conserved mechanism for shoot-branching regulation in both monocots and dicots. D3,HTD1|OsCCD7,D10|OsCCD8|OsCCD8b DWARF 53 acts as a repressor of strigolactone signalling in rice 2013 Nature State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China Strigolactones (SLs) are a group of newly identified plant hormones that control plant shoot branching. SL signalling requires the hormone-dependent interaction of DWARF 14 (D14), a probable candidate SL receptor, with DWARF 3 (D3), an F-box component of the Skp-Cullin-F-box (SCF) E3 ubiquitin ligase complex. Here we report the characterization of a dominant SL-insensitive rice (Oryza sativa) mutant dwarf 53 (d53) and the cloning of D53, which encodes a substrate of the SCF(D3) ubiquitination complex and functions as a repressor of SL signalling. Treatments with GR24, a synthetic SL analogue, cause D53 degradation via the proteasome in a manner that requires D14 and the SCF(D3) ubiquitin ligase, whereas the dominant form of D53 is resistant to SL-mediated degradation. Moreover, D53 can interact with transcriptional co-repressors known as TOPLESS-RELATED PROTEINS. Our results suggest a model of SL signalling that involves SL-dependent degradation of the D53 repressor mediated by the D14-D3 complex. D3,D53,HTD2|D88|D14 D14-SCF(D3)-dependent degradation of D53 regulates strigolactone signalling 2013 Nature National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, China [2] National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China. Strigolactones (SLs), a newly discovered class of carotenoid-derived phytohormones, are essential for developmental processes that shape plant architecture and interactions with parasitic weeds and symbiotic arbuscular mycorrhizal fungi. Despite the rapid progress in elucidating the SL biosynthetic pathway, the perception and signalling mechanisms of SL remain poorly understood. Here we show that DWARF 53 (D53) acts as a repressor of SL signalling and that SLs induce its degradation. We find that the rice (Oryza sativa) d53 mutant, which produces an exaggerated number of tillers compared to wild-type plants, is caused by a gain-of-function mutation and is insensitive to exogenous SL treatment. The D53 gene product shares predicted features with the class I Clp ATPase proteins and can form a complex with the alpha/beta hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL perception. We demonstrate that, in a D14- and D3-dependent manner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth. Our combined genetic and biochemical data reveal that D53 acts as a repressor of the SL signalling pathway, whose hormone-induced degradation represents a key molecular link between SL perception and responses. D3,D53,HTD2|D88|D14 Suppression of tiller bud activity in tillering dwarf mutants of rice 2005 Plant Cell Physiol Graduate School of Agriculture and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo, 113-8657 Japan. In this study, we analyzed five tillering dwarf mutants that exhibit reduction of plant stature and an increase in tiller numbers. We show that, in the mutants, axillary meristems are normally established but the suppression of tiller bud activity is weakened. The phenotypes of tillering dwarf mutants suggest that they play roles in the control of tiller bud dormancy to suppress bud activity. However, tillering dwarf mutants show the dependence of both node position and planting density on their growth, which implies that the functions of tillering dwarf genes are independent of the developmental and environmental control of bud activity. Map-based cloning of the D3 gene revealed that it encodes an F-box leucine-trich repeat (LRR) protein orthologous to Arabidopsis MAX2/ORE9. This indicates the conservation of mechanisms controlling axillary bud activity between monocots and eudicots. We suggest that tillering dwarf mutants are suitable for the study of bud activity control in rice and believe that future molecular and genetic studies using them may enable significant progress in understanding the control of tillering and shoot branching. D3 DWARF50 (D50), a rice (Oryza sativa L.) gene encoding inositol polyphosphate 5-phosphatase, is required for proper development of intercalary meristem 2012 Plant Cell Environ Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan. ikanna@cc.tuat.ac.jp Rice internodes are vital for supporting high-yield panicles, which are controlled by various factors such as cell division, cell elongation and cell wall biosynthesis. Therefore, formation and regulation of the internode cell-producing intercalary meristem (IM) are important for determining the shape of internodes. To understand the regulation of internode development, we analysed a rice dwarf mutant, dwarf 50 (d50). Previously, we reported that parenchyma cells in the elongated internodes of d50 ectopically deposit cell wall phenolics. In this study, we revealed that D50 encodes putative inositol polyphosphate 5-phosphatase (5PTase), which may be involved in phosphoinositide signalling required for many essential cellular functions, such as cytoskeleton organization, endocytosis and vesicular trafficking in eukaryotes. Analysis of the rice genome revealed 20 putative 5PTases including D50. The d50 mutation induced abnormally oriented cell division, irregular deposition of cell wall pectins and thick actin bundles in the parenchyma cells of the IM, resulting in abnormally organized cell files of the internode parenchyma and dwarf phenotype. Our results suggest that the putative 5PTase, encoded by D50, is essential for IM formation, including the direction of cell division, deposition of cell wall pectins and control of actin organization. D50 Loss-of-function mutations in the rice homeobox gene OSH15 affect the architecture of internodes resulting in dwarf plants 1999 EMBO J Nagoya University, BioScience Center, Chikusa, Nagoya 464-8601, USA. The rice homeobox gene OSH15 (Oryza sativa homeobox) is a member of the knotted1-type homeobox gene family. We report here on the identification and characterization of a loss-of-function mutation in OSH15 from a library of retrotransposon-tagged lines of rice. Based on the phenotype and map position, we have identified three independent deletion alleles of the locus among conventional morphological mutants. All of these recessive mutations, which are considered to be null alleles, exhibit defects in internode elongation. Introduction of a 14 kbp genomic DNA fragment that includes all exons, introns and 5'- and 3'- flanking sequences of OSH15 complemented the defects in internode elongation, confirming that they were caused by the loss-of-function of OSH15. Internodes of the mutants had abnormal-shaped epidermal and hypodermal cells and showed an unusual arrangement of small vascular bundles. These mutations demonstrate a role for OSH15 in the development of rice internodes. This is the first evidence that the knotted1-type homeobox genes have roles other than shoot apical meristem formation and/or maintenance in plant development. D6|OSH15|Oskn3 Overexpression of rice OSH genes induces ectopic shoots on leaf sheaths of transgenic rice plants 2000 Dev Biol BioScience Center, Nagoya University, Chikusa, Nagoya, 464-8601, Japan. Five rice homeobox (OSH) genes were overexpressed under the control of the cauliflower mosaic virus 35S promoter or the rice actin gene promoter in transgenic rice plants. Almost all of the transgenic plants showed abnormal phenotypes, which could be classified into three types according to their severity. Plants with the most severe phenotype formed only green organs, with many shoot apices on their adaxial sides. Plants with an intermediate phenotype formed bladeless leaves with normally developed leaf sheaths. Plants with a mild phenotype formed normal leaf sheaths and blades, but lacked ligules and showed diffusion of the blade-sheath boundary. The leaf structure of this phenotype was similar to that of dominant maize mutants, such as Kn1, Rs1, Lg3, and Lg4. Based on these phenotypes, we suggest that ectopic expression of the rice OSH genes interferes with the development of leaf blades and maintains leaves in less differentiated states. These results are discussed in relation to the leaf maturation schedule hypothesis (M. Freeling et al., 1992, BioEssays 14, 227-236). D6|OSH15|Oskn3,OSH1|Oskn1,OSH43,OSH6,OSH71|Oskn2 Disruption of KNOX gene suppression in leaf by introducing its cDNA in rice 2008 Plant Science Plant Genetics Laboratory, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka-ken 411-8540, Japan KNOX class 1 homeobox genes play a crucial role in formation and/or maintenance of the shoot apical meristem (SAM) in plants, and their SAM-specific expression is essential for normal development of plants. We examined regulation of expression of OSH1, a KNOX gene in rice. A 5′ upstream region of OSH1 had the ability to direct the expression of a reporter gene in leaf in addition to the SAM. Introduction of OSH1 cDNA without a promoter sequence caused ectopic expression of both endogenous OSH1 and introduced OSH1 cDNA itself into the leaf, which resulted in morphological abnormalities in the leaf resembling those of the overexpressors. These results indicate that an extra copy of OSH1 exons with no promoter sequence disrupts suppression of OSH1 in the transgenic leaf. Introduction of cDNAs of two other KNOX genes, OSH15 and OSH71, also showed ectopic expression. These results suggest that the exon sequences of KNOX genes function as one of the major cis-regulatory elements of KNOX gene expression. We present and discuss a possible model which interprets these results. D6|OSH15|Oskn3,OSH1|Oskn1,OSH71|Oskn2 SUI-family genes encode phosphatidylserine synthases and regulate stem development in rice 2013 Planta National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200032, China. In vascular plants, the regulation of stem cell niche determines development of aerial shoot which consists of stems and lateral organs. Intercalary meristem (IM) controls internode elongation in rice and other grasses, however little attention has been paid to the underlying mechanism of stem cell maintenance. Here, we investigated the stem development in rice and showed that the Shortened Uppermost Internode 1 (SUI1) family of genes are pivotal for development of rice stems. We demonstrated that SUI-family genes regulate the development of IM for internode elongation and also the cell expansion of the panicle stem rachis in rice. The SUI-family genes encoded base-exchange types of phosphatidylserine synthases (PSSs), which possessed enzymatic activity in a yeast complementary assay. Overexpression of SUI1 and SUI2 caused outgrowths of internodes during vegetative development, and we showed that expression patterns of Oryza Sativa Homeobox 15 (OSH15) and Histone4 were impaired. Furthermore, genome-wide gene expression analysis revealed that overexpression and RNA knockdown of SUI-family genes affected downstream gene expression related to phospholipid metabolic pathways. Moreover, using Ultra-performance liquid chromatography-quadrupole time of flight-mass spectrometry, we analyzed PS contents in different genetic backgrounds of rice and showed that the quantity of very long chain fatty acids PS is affected by transgene of SUI-family genes. Our study reveals a new mechanism conveyed by the SUI1 pathway and provides evidence to link lipid metabolism with plant stem cell maintenance. D6|OSH15|Oskn3,OsSUI1,SUI2,SUI3 Isolation and characterization of a rice homebox gene, OSH15 1998 Plant Mol Biol Nagoya University, BioScience Center, Chikusa, Japan. In many eukaryotic organisms including plants, homeobox genes are thought to be master regulators that establish the cellular or regional identities and specify the fundamental body plan. We isolated and characterized a cDNA designated OSH15 (Oryza sativa homeobox 15) that encodes a KNOTTED-type homeodomain protein. Transgenic tobacco plants overexpressing the OSH15 cDNA showed a dramatically altered morphological phenotype caused by disturbance of specific aspects of tobacco development, thereby indicating the involvement of OSH15 in plant development. We analyzed the in situ mRNA localization of OSH15 through the whole plant life cycle, comparing the expression pattern with that of another rice homeobox gene, OSH1. In early embryogenesis, both genes were expressed as the same pattern at a region where the shoot apical meristem would develop later. In late embryogenesis, the expression pattern of the two genes became different. Whereas the expression of OSH1 continued within the shoot apical meristem, OSH15 expression within the shoot apical meristem ceased but became observable in a ring shaped pattern at the boundaries of some embryonic organs. This pattern of expression was similar to that observed around vegetative or reproductive shoots, or the floral meristem in mature plants. RNA in situ localization data suggest that OSH15 may play roles in the shoot organization during early embryogenesis and thereafter, OSH15 may be involved in morphogenetic events around the shoot apical meristem. D6|OSH15|Oskn3,OSH1|Oskn1 Characterization of the KNOX class homeobox genes Oskn2 and Oskn3 identified in a collection of cDNA libraries covering the early stages of rice embryogenesis 1999 Plant Mol Biol Institute of Molecular Plant Sciences, Leiden University, The Netherlands. For identification of genes involved in embryogenesis in the model cereal rice, we have constructed a collection of cDNA libraries of well-defined stages of embryo development before, during and after organ differentiation. Here, we focus on the possible role of KNOX (maize Knotted1-like) class homeobox genes in regulation of rice embryogenesis. Three types of KNOX clones were identified in libraries of early zygotic embryos. Two of these, Oskn2 and Oskn3, encode newly described KNOX genes, whereas the third (Oskn1) corresponds to the previously described OSH1 gene. In situ hybridizations showed that during the early stages of embryo development, all three KNOX genes are expressed in the region where the shoot apical meristem (SAM) is organizing, suggesting that these genes are involved in regulating SAM formation. Whereas OSH1 was previously proposed to function also in SAM maintenance, Oskn3 may be involved in patterning organ positions, as its expression was found to mark the boundaries of different embryonic organs following SAM formation. The expression pattern of Oskn2 suggested an additional role in scutellum and epiblast development. Transgenic expression of Oskn2 and Oskn3 in tobacco further supported their involvement in cell fate determination, like previously reported for Knotted1 and OSH1 ectopic expression. Whereas Oskn3 transformants showed the most pronounced phenotypic effects during vegetative development, Oskn2 transformants showed relatively mild alterations in the vegetative phase but a more severely affected flower morphology. The observation that the KNOX genes produce similar though distinct phenotypic reponses in tobacco, indicates that their gene products act on overlapping but different sets of target genes, or that cell-type specific factors determine their precise action. D6|OSH15|Oskn3,OSH1|Oskn1,OSH71|Oskn2 Developmental regulation and downstream effects of the knox class homeobox genes Oskn2 and Oskn3 from rice 2002 Plant Mol Biol Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, The Netherlands. Plant homeobox genes of the class 1 knox (knotted1-like) type are involved in the regulation of shoot apical meristem formation and function. Their expression generally occurs either throughout the meristem or specifically at the lateral organ boundaries. Down-regulation in the organ primordia is tightly controlled and misexpression in leaves leads to a perturbed development. Here, the transcriptional control of two rice knox genes, Oskn2 and Oskn3, was addressed, showing that the promoter sequences of both genes mediate the initial down-regulation during lateral organ formation, but are insufficient to keep expression in lateral organs stably off. Therefore, maintenance of the repressed state requires control elements outside the promoter regions. Ectopic expression of Oskn2 or Oskn3 induced similar defects in panicle branching. internode elongation and leaf patterning. However, small differences suggested that their target gene specificities are not completely overlapping. This was supported by the observation that Oskn3 protein but not Oskn2 could interact with two reported recognition sequences of a KNOX protein from barley. Finally, protein-protein interactions may contribute to the functioning of KNOX proteins, as the ability of Oskn3 and Oskn2 to form heterodimers could be demonstrated. D6|OSH15|Oskn3,OSH71|Oskn2 KNOX homeobox genes are sufficient in maintaining cultured cells in an undifferentiated state in rice 2001 Genesis Plant Genetics Laboratory, National Institute of Genetics, Shizuoka-ken, Japan. We produced transgenic rice calli, which constitutively express each of four KNOX family class 1 homeobox genes of rice, OSH1, OSH16, OSH15, and OSH71, and found that constitutive and ectopic expression of such genes inhibits normal regeneration from transformed calli, which showed continuous growth around their shoot-regenerating stages. Transgenic calli transferred onto regeneration medium began to display green spots, a sign of regeneration, but most of the transformants continued to propagate green spots at given stages. In the normal shoot-regeneration process of calli, expression of endogenous OSH1 was restricted in presumptive shoot-regenerating regions of calli and not observed in other areas. This restricted expression pattern should be required for further differentiation of the regenerating shoots. Thus our present results support the proposed function that KNOX family class 1 homeobox genes play a role in the formation and maintenance of the undetermined meristematic state of cells. D6|OSH15|Oskn3,OSH1|Oskn1,OSH6,OSH71|Oskn2 Regional expression of the rice KN1-type homeobox gene family during embryo, shoot, and flower development 1999 Plant Cell Nagoya University, BioScience Center, Chikusa, Nagoya 464-8601, Japan. We report the isolation, sequence, and pattern of gene expression of members of the KNOTTED1 (KN1)-type class 1 homeobox gene family from rice. Phylogenetic analysis and mapping of the rice genome revealed that all of the rice homeobox genes that we have isolated have one or two direct homologs in maize. Of the homeobox genes that we tested, all exhibited expression in a restricted region of the embryo that defines the position at which the shoot apical meristem (SAM) would eventually develop, prior to visible organ formation. Several distinct spatial and temporal expression patterns were observed for the different genes in this region. After shoot formation, the expression patterns of these homeobox genes were variable in the region of the SAM. These results suggest that the rice KN1-type class 1 homeobox genes function cooperatively to establish the SAM before shoot formation and that after shoot formation, their functions differ. D6|OSH15|Oskn3,OSH1|Oskn1,OSH10,OSH3,OSH43,OSH6,OSH71|Oskn2 Positive autoregulation of a KNOX gene is essential for shoot apical meristem maintenance in rice 2011 Plant Cell Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka, Japan. Self-maintenance of the shoot apical meristem (SAM), from which aerial organs are formed throughout the life cycle, is crucial in plant development. Class I Knotted1-like homeobox (KNOX) genes restrict cell differentiation and play an indispensable role in maintaining the SAM. However, the mechanism that positively regulates their expression is unknown. Here, we show that expression of a rice (Oryza sativa) KNOX gene, Oryza sativa homeobox1 (OSH1), is positively regulated by direct autoregulation. Interestingly, loss-of-function mutants of OSH1 lose the SAM just after germination but can be rescued to grow until reproductive development when they are regenerated from callus. Double mutants of osh1 and d6, a loss-of-function mutant of OSH15, fail to establish the SAM both in embryogenesis and regeneration. Expression analyses in these mutants reveal that KNOX gene expression is positively regulated by the phytohormone cytokinin and by KNOX genes themselves. We demonstrate that OSH1 directly binds to five KNOX loci, including OSH1 and OSH15, through evolutionarily conserved cis-elements and that the positive autoregulation of OSH1 is indispensable for its own expression and SAM maintenance. Thus, the maintenance of the indeterminate state mediated by positive autoregulation of a KNOX gene is an indispensable mechanism of self-maintenance of the SAM. D6|OSH15|Oskn3,OSH1|Oskn1 Functional analysis of the conserved domains of a rice KNOX homeodomain protein, OSH15 2001 Plant Cell BioScience Center, Nagoya University, Chikusa, Nagoya 464-0814, Japan. The rice KNOX protein OSH15 consists of four conserved domains: the MEINOX domain, which can be divided into two subdomains (KNOX1 and KNOX2); the GSE domain; the ELK domain; and the homeodomain (HD). To investigate the function of each domain, we generated 10 truncated proteins with deletions in the conserved domains and four proteins with mutations in the conserved amino acids in the HD. Transgenic analysis suggested that KNOX2 and HD are essential for inducing the abnormal phenotype and that the KNOX1 and ELK domains affect phenotype severity. We also found that both KNOX2 and HD are necessary for homodimerization and that only HD is needed for binding of OSH15 to its target sequence. Transactivation studies suggested that both the KNOX1 and ELK domains play a role in suppressing target gene expression. On the basis of these findings, we propose that overproduced OSH15 probably acts as a dimer and may ectopically suppress the expression of target genes that induce abnormal morphology in transgenic plants. D6|OSH15|Oskn3 OsBLE3, a brassinolide-enhanced gene, is involved in the growth of rice 2006 Phytochemistry National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba 305-8602, Japan. Brassinosteroids (BRs) are a group of plant hormones involved in a wide range of plant growth and developmental processes. To investigate the mechanism of BR action in monocots, a brassinolide (BL) upregulated gene designated OsBLE3 was identified, cloned and characterized in rice. It was mainly expressed in roots and leaf sheaths with levels of expression directly dependent on the dose of BL. In situ hybridization detected OsBLE3 mRNA in the shoot apical meristem, organ primordia and vascular tissue. Furthermore, its expression was enhanced by co-treatment with BL and low concentrations of IAA. These results, and the existence of auxin response elements in the 5'-flanking region of the OsBLE3 gene, indicate that OsBLE3 expression is under control of both BR and auxin. The GUS reporter gene driven by a 2277 bp OsBLE3 putative promoter was mainly expressed in vascular tissues, branch root primordia and was responsive to exogenous BL treatment. OsBLE3 transcript levels were greatly reduced in brd1 plants, a BL deficient mutant, compared to the wild type control. In OsBRI1 antisense transgenic rice and OsBLE3, the BR-insensitive mutant expression of OsBLE3 in response to exogenous BL treatment was significantly lower compared to that in control plants transformed with a vacant vector. Reduced OsBLE3 expression and growth retardation was also observed in OsBLE3 antisense transgenic rice plants. Internode cell length of the OsBLE3 antisense transgenic lines was about 70% of that in the vacant vector transformed control lines. These results suggest that OsBLE3 is involved in cell elongation in rice through dual regulation by BL and IAA. D61|OsBRI1,OsBLE3 Kinase activity of OsBRI1 is essential for brassinosteroids to regulate rice growth and development 2013 Plant Sci National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, China. Brassinosteroids (BRs) are steroid hormones that participate in multiple biological processes. In this paper, we characterized a classic rice mutant Fn189 (dwarf54, d54) showing semi-dwarf stature and erect leaves. The coleoptile elongation and root growth was less affected in Fn189 than wild-type plant by the exogenous application of eBL, the most active form of BRs. Lamina joint inclination assay and morphological analysis in darkness further showed that Fn189 mutant plant was insensitive to exogenous eBL. Through map-based cloning, Fn189 was found to be a novel allelic mutant of the DWARF 61 (D61) gene, which encodes the putative BRs receptor OsBRI1. A single base mutation caused the I834F substitution in the OsBRI1 kinase domain. Consequently, kinase activity of OsBRI1 was found to decrease dramatically. Taken together, the kinase activity of OsBRI1 is essential for brassinosteroids to regulate normal plant growth and development in rice. D61|OsBRI1 Small and round seed 5 gene encodes alpha-tubulin regulating seed cell elongation in rice 2012 Rice (N Y) Faculty of Biotechnology, Fukui Prefectural University, 4-1-1 Kenjojima, Matsuoka, Eiheiji-Town, Fukui 910-1195, Japan. Seed size is an important trait in determinant of rice seed quality and yield. In this study, we report a novel semi-dominant mutant Small and round seed 5 (Srs5) that encodes alpha-tubulin protein. Lemma cell length was reduced in Srs5 compared with that of the wild-type. Mutants defective in the G-protein alpha subunit (d1-1) and brassinosteroid receptor, BRI1 (d61-2) also exhibited short seed phenotypes, the former due to impaired cell numbers and the latter due to impaired cell length. Seeds of the double mutant of Srs5 and d61-2 were smaller than those of Srs5 or d61-2. Furthermore, SRS5 and BRI1 genes were highly expressed in Srs5 and d61-2 mutants. These data indicate that SRS5 independently regulates cell elongation of the brassinosteroid signal transduction pathway. D61|OsBRI1,TID1|SRS5 Loss of function of a rice brassinosteroid insensitive1 homolog prevents internode elongation and bending of the lamina joint 2000 Plant Cell Nagoya University, BioScience Center, Chikusa, Nagoya 464-8601, Japan. Brassinosteroids (BRs) are plant growth-promoting natural products required for plant growth and development. Physiological studies have demonstrated that exogenous BR, alone or in combination with auxin, enhance bending of the lamina joint of rice. However, little is known about the function of endogenous BR in rice or other grass species. We report here the phenotypical and molecular characterization of a rice dwarf mutant, d61, that is less sensitive to BR compared to the wild type. We cloned a rice gene, OsBRI1, with extensive sequence similarity to that of the Arabidopsis BRI gene, which encodes a putative BR receptor kinase. Linkage analysis showed that the OsBRI1 gene is closely linked to the d61 locus. Single nucleotide substitutions found at different sites of the d61 alleles would give rise to amino acid changes in the corresponding polypeptides. Furthermore, introduction of the entire OsBRI1 coding region, including the 5' and 3' flanking sequences, into d61 plants complemented the mutation to display the wild-type phenotype. Transgenic plants carrying the antisense strand of the OsBRI1 transcript showed similar or even more severe phenotypes than those of the d61 mutants. Our results show that OsBRI1 functions in various growth and developmental processes in rice, including (1) internode elongation, by inducing the formation of the intercalary meristem and the longitudinal elongation of internode cells; (2) bending of the lamina joint; and (3) skotomorphogenesis. D61|OsBRI1 The role of OsBRI1 and its homologous genes, OsBRL1 and OsBRL3, in rice 2006 Plant Physiol Bioscience and Biotechnology Center, Nagoya University Chikusa, Nagoya 464-8601, Japan. Since first identifying two alleles of a rice (Oryza sativa) brassinosteroid (BR)-insensitive mutant, d61, that were also defective in an orthologous gene in Arabidopsis (Arabidopsis thaliana) BRASSINOSTEROID INSENSITIVE1 (BRI1), we have isolated eight additional alleles, including null mutations, of the rice BRI1 gene OsBRI1. The most severe mutant, d61-4, exhibited severe dwarfism and twisted leaves, although pattern formation and differentiation were normal. This severe shoot phenotype was caused mainly by a defect in cell elongation and the disturbance of cell division after the determination of cell fate. In contrast to its severe shoot phenotype, the d61-4 mutant had a mild root phenotype. Concomitantly, the accumulation of castasterone, the active BR in rice, was up to 30-fold greater in the shoots, while only 1.5-fold greater in the roots. The homologous genes for OsBRI1, OsBRL1 and OsBRL3, were highly expressed in roots but weakly expressed in shoots, and their expression was higher in d61-4 than in the wild type. Based on these observations, we conclude that OsBRI1 is not essential for pattern formation or organ initiation, but is involved in organ development through controlling cell division and elongation. In addition, OsBRL1 and OsBRL3 are at least partly involved in BR perception in the roots. D61|OsBRI1,OsBRL1,OsBRL3 Morphological alteration caused by brassinosteroid insensitivity increases the biomass and grain production of rice 2006 Plant Physiol Bioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi 464-8601, Japan. The rice (Oryza sativa) dwarf mutant d61 phenotype is caused by loss of function of a rice BRASSINOSTEROID INSENSITIVE1 ortholog, OsBRI1. We have identified nine d61 alleles, the weakest of which, d61-7, confers agronomically important traits such as semidwarf stature and erect leaves. Because erect-leaf habit is considered to increase light capture for photosynthesis, we compared the biomass and grain production of wild-type and d61-7 rice. The biomass of wild type was 38% higher than that of d61-7 at harvest under conventional planting density because of the dwarfism of d61-7. However, the biomass of d61-7 was 35% higher than that of wild type at high planting density. The grain yield of wild type reached a maximum at middensity, but the yield of d61-7 continued to increase with planting density. These results indicate that d61-7 produces biomass more effectively than wild type, and consequently more effectively assimilates the biomass in reproductive organ development at high planting density. However, the small grain size of d61-7 counters any increase in grain yield, leading to the same grain yield as that of wild type even at high density. We therefore produced transgenic rice with partial suppression of endogenous OsBRI1 expression to obtain the erect-leaf phenotype without grain changes. The estimated grain yield of these transformants was about 30% higher than that of wild type at high density. These results demonstrate the feasibility of generating erect-leaf plants by modifying the expression of the brassinosteroid receptor gene in transgenic rice plants. D61|OsBRI1 RPL1, a gene involved in epigenetic processes regulates phenotypic plasticity in rice 2012 Mol Plant National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China. Organisms can adjust their phenotype in response to changing environmental conditions. This phenomenon is termed phenotypic plasticity. Despite its ubiquitous occurrence, there has been very little study on the molecular mechanism of phenotypic plasticity. In this study, we isolated a rice (Oryza sativa L.) mutant, rice plasticity 1 (rpl1), that displayed increased environment-dependent phenotypic variations. RPL1 was expressed in all tissues examined. The protein was localized in the nucleus and its distribution in the nucleus overlapped with heterochromatin. The rpl1 mutation led to an increase in DNA methylation on repetitive sequences and a decrease in overall histone acetylation. In addition, the mutation affected responses of the rice plant to phytohormones such as brassinosteroid, gibberellin, and cytokinin. Analysis of the putative rice brassinosteroid receptor OsBRI1, a key hormone signaling gene, indicated that RPL1 may be involved in the regulation of epigenomic modification of the gene. These data suggest that RPL1 regulated phenotypic plasticity likely through its involvement in epigenetic processes affecting responses of the plant to phytohormones. D61|OsBRI1,RPL1 dad-1, A putative programmed cell death suppressor gene in rice 1997 Plant Cell Physiol Laboratory of Plant Pathology & Genetic Engineering, College of Agriculture, Okayama University, Japan. The human dad-1 cDNA homolog was isolated from rice plants. The amino acid sequence of the predicted protein product is well conserved in both animals and plants. This rice dad-1 homolog can rescue the temperature-sensitive dad-1 mutants of hamster cells from apoptotic death, suggesting that the rice dad-1 homolog also functions as a suppressor for programmed cell death. DAD-1|DAD1 Putrescine differently influences the effect of salt stress on polyamine metabolism and ethylene synthesis in rice cultivars differing in salt resistance 2010 J Exp Bot Groupe de Recherche en Physiologie vegetale (GRPV), Universite catholique de Louvain, 5 (Bte 13) Place Croix du Sud, Louvain-la-Neuve, Belgium. Effects of salt stress on polyamine metabolism and ethylene production were examined in two rice (Oryza sativa L.) cultivars [I Kong Pao (IKP), salt sensitive; and Pokkali, salt resistant] grown for 5 d and 12 d in nutrient solution in the presence or absence of putrescine (1 mM) and 0, 50, and 100 mM NaCl. The salt-sensitive (IKP) and salt-resistant (Pokkali) cultivars differ not only in their mean levels of putrescine, but also in the physiological functions assumed by this molecule in stressed tissues. Salt stress increased the proportion of conjugated putrescine in salt-resistant Pokkali and decreased it in the salt-sensitive IKP, suggesting a possible protective function in response to NaCl. Activities of the enzymes ornithine decarboxylase (ODC; EC 4.1.1.17) and arginine decarboxylase (ADC; EC 4.1.1.19) involved in putrescine synthesis were higher in salt-resistant Pokkali than in salt-sensitive IKP. Both enzymes were involved in the response to salt stress. Salt stress also increased diamine oxidase (DAO; 1.4.3.6) and polyamine oxidase (PAO EC 1.5.3.11) activities in the roots of salt-resistant Pokkali and in the shoots of salt-sensitive IKP. Gene expression followed by reverse transcription-PCR suggested that putrescine could have a post-translational impact on genes coding for ADC (ADCa) and ODC (ODCa and ODCb) but could induce a transcriptional activation of genes coding for PAO (PAOb) mainly in the shoot of salt-stressed plants. The salt-resistant cultivar Pokkali produced higher amounts of ethylene than the salt-sensitive cultivar IKP, and exogenous putrescine increased ethylene synthesis in both cultivars, suggesting no direct antagonism between polyamine and ethylene pathways in rice. DAO,ODCa,ODCb A role for a dioxygenase in auxin metabolism and reproductive development in rice 2013 Dev Cell National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, China. Indole-3-acetic acid (IAA), the natural auxin in plants, regulates many aspects of plant growth and development. Extensive analyses have elucidated the components of auxin biosynthesis, transport, and signaling, but the physiological roles and molecular mechanisms of auxin degradation remain elusive. Here, we demonstrate that the dioxygenase for auxin oxidation (DAO) gene, encoding a putative 2-oxoglutarate-dependent-Fe (II) dioxygenase, is essential for anther dehiscence, pollen fertility, and seed initiation in rice. Rice mutant lines lacking a functional DAO display increased levels of free IAA in anthers and ovaries. Furthermore, exogenous application of IAA or overexpression of the auxin biosynthesis gene OsYUCCA1 phenocopies the dao mutants. We show that recombinant DAO converts the active IAA into biologically inactive 2-oxoindole-3-acetic acid (OxIAA) in vitro. Collectively, these data support a key role of DAO in auxin catabolism and maintenance of auxin homeostasis central to plant reproductive development. DAO,OsYUCCA1 The rice mutant dwarf bamboo shoot 1: a leaky mutant of the NACK-type kinesin-like gene can initiate organ primordia but not organ development 2005 Plant Cell Physiol Bioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi, 464-8601 Japan. sazuka@agr.nagoya-u.ac.jp That plant dwarfism is caused by hormonal defects related to gibberellin and brassinosteroid has been well documented. Other contributing elements, however, have not been elucidated. Here, we report on one of the most severe dwarf mutants of rice, dwarf bamboo shoot 1 (dbs1). Most mutant plants died within 1 month after sowing, but a few (5.2%) survived and grew. Vacuolation enlarged cells in the leaf primordia and seminal root before abortion, which disrupted the organized cell files in these organs. Relative to the severe defects in shoot and root growth, the overall structure of the dbs1 embryo was almost normal. Similarly, initiation and organogenesis of the leaf primordia at the shoot apical meristem and those of the lateral root primordia at the root elongation zone occurred normally. These observations suggest that DBS1 is involved in the growth and development of organs but not in organ initiation or organogenesis. Positional cloning of DBS1 revealed that it encoded a NACK-type kinesin-like protein (OsNACK), homologous to the essential components of a mitogen-activated protein kinase cascade during plant cytokinesis. A BLAST search indicated that DBS1 was the only gene encoding the OsNACK-type protein in the rice genome, and the dbs1 mutant produced only small amounts of the translatable DBS1 mRNA. Thus, we conclude that the dbs1 mutation causes a severe defect in DBS1 function but does not completely shut it down. We discuss the leaky phenotype of dbs1 under the restricted functioning of OsNACK. DBS1|OsNACK DCW11, down-regulated gene 11 in CW-type cytoplasmic male sterile rice, encoding mitochondrial protein phosphatase 2c is related to cytoplasmic male sterility 2008 Plant Cell Physiol Laboratory of Environmental Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. Causes of cytoplasmic male sterility (CMS) in plants have been studied for two decades, and mitochondrial chimeric genes have been predicted to induce CMS. However, it is unclear what happens after CMS-associated proteins accumulate in mitochondria. In our previous study of microarray analysis, we found that 140 genes are aberrantly regulated in anthers of CW-type CMS of rice (Oryza sativa L.). In the present study, we investigated DCW11, one of the down-regulated genes in CW-CMS encoding a protein phosphatase 2C (PP2C). DCW11 mRNA was preferentially expressed in anthers, with the highest expression in mature pollen. As predicted by the N-terminal sequence, DCW11 signal peptide-green fluorescent protein (GFP) fusion protein was localized in mitochondria. Knockdown of DCW11 in wild-type rice by RNA interference caused a major loss of seed-set fertility, without visible defect in pollen development. Since this knockdown phenotype resembled that of CW-CMS, we concluded that the down-regulation of DCW11 is correlated with CW-CMS. This idea was supported by the up-regulation of alternative oxidase 1a (AOX1a), which is known to be regulated by mitochondrial retrograde signaling, in DCW11 knockdown lines. Down-regulation of DCW11 and up-regulation of AOX1a were also observed in two other types of rice CMS. Our result indicates that DCW11 could play a role as a mitochondrial signal transduction mediator in pollen germination. DCW11,AOX1a|OsAOX1a Cytoplasmic male sterility-related protein kinase, OsNek3, is regulated downstream of mitochondrial protein phosphatase 2C, DCW11 2009 Plant Cell Physiol Laboratory of Environmental Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. OsNek3 (Oryza sativa L. NIMA-related kinase) and DCW11 encoding a mitochondrial putative protein phosphatase 2C were found in our previous microarray study as down-regulated genes in the rice CW-CMS line, which lacked pollen germination ability. Further analysis of DCW11 revealed that DCW11 is strongly correlated with CW-CMS occurrence. Here we show the relationship between OsNek3 and DCW11. OsNek3 was preferentially expressed in mature pollen. A knockout mutant with Tos17 inserted into OsNek3 did not show any pollen-defective phenotype. On the other hand, plants overexpressing OsNek3 occasionally produced a peculiar pollen structure in which the outer cell wall of four pollen grains fused together even at the mature pollen stages, which resembled that of quartet mutants in Arabidopsis. OsNek3 was shown to interact with a LIM domain-containing protein, OsPLIM2b, whose expression was strongly specific in mature pollen, suggesting that OsNek3 might play a role in pollen germination. OsNek3 was shown to be down-regulated in DCW11-knockdown lines, whereas osnek3 mutation did not result in DCW11 down-regulation. These results suggest that OsNek3 is downstream of DCW11 in retrograde signaling from the mitochondria to the nucleus and is involved in CW-CMS. DCW11,OsNek3,OsPLIM2b Dwarf and deformed flower 1, encoding an F-box protein, is critical for vegetative and floral development in rice (Oryza sativa L.) 2012 Plant J Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fujian Agriculture & Forestry University, Fuzhou 350002, China. ylduan863@fjau.edu.cn Recent studies have shown that F-box proteins constitute a large family in eukaryotes, and play pivotal roles in regulating various developmental processes in plants. However, their functions in monocots are still obscure. In this study, we characterized a recessive mutant dwarf and deformed flower 1-1 (ddf1-1) in Oryza sativa (rice). The mutant is abnormal in both vegetative and reproductive development, with significant size reduction in all organs except the spikelet. DDF1 controls organ size by regulating both cell division and cell expansion. In the ddf1-1 spikelet, the specification of floral organs in whorls 2 and 3 is altered, with most lodicules and stamens being transformed into glume-like organs and pistil-like organs, respectively, but the specification of lemma/palea and pistil in whorls 1 and 4 is not affected. DDF1 encodes an F-box protein anchored in the nucleolus, and is expressed in almost all vegetative and reproductive tissues. Consistent with the mutant floral phenotype, DDF1 positively regulates B-class genes OsMADS4 and OsMADS16, and negatively regulates pistil specification gene DL. In addition, DDF1 also negatively regulates the Arabidopsis LFY ortholog APO2, implying a functional connection between DDF1 and APO2. Collectively, these results revealed that DDF1, as a newly identified F-box gene, is a crucial genetic factor with pleiotropic functions for both vegetative growth and floral organ specification in rice. These findings provide additional insights into the molecular mechanism controlling monocot vegetative and reproductive development. DDF1,OsMADS16|SPW1,OsMADS4,RFL|APO2 Rice DECUSSATE controls phyllotaxy by affecting the cytokinin signaling pathway 2012 Plant J Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. RIKEN Plant Science Center, Tsurumi, Yokohama 230-0045, Japan. Phyllotaxy is defined as a spatial arrangement of leaves on the stem. The mechanism responsible for this extremely regular pattern is one of the most fascinating enigmas in plant biology. In this study, we identified a gene regulating the phyllotactic pattern in rice. Loss-of-function mutants of the DECUSSATE (DEC) gene displayed a phyllotactic conversion from normal distichous to decussate. The dec mutants had an enlarged SAM with enhanced cell division activity. In contrast to the SAM, the size of the root apical meristem (RAM) in dec was reduced, and cell division activity was suppressed. These phenotypes indicate that DEC has opposite functions in SAM and RAM. Map-based cloning revealed that DEC encodes a plant-specific protein containing a glutamine-rich region and a conserved motif. Although its molecular function is unclear, the conserved domain is shared with fungi and animals. Expression analysis showed that several type-A response regulator genes that act in the cytokinin (CK) signaling pathway were downregulated in the dec mutant. In addition, dec seedlings showed a reduced responsiveness to exogenous CK. Our results suggest that DEC is a factor that controls the phyllotactic pattern by affecting the CK signaling in rice. (c) 2012 The Authors. The Plant Journal (c) 2012 Blackwell Publishing Ltd. DEC The SMALL AND ROUND SEED1 (SRS1/DEP2) gene is involved in the regulation of seed size in rice 2010 Genes Genet Syst Department of Bioscience, Fukui Prefectural University The causal gene of a novel small and round seed mutant 1 (srs1) was identified in rice by map-based cloning and named SMALL AND ROUND SEED 1 (SRS1). The SRS1 gene is identical to the previously identified DENSE AND ERECT PANICLE 2 (DEP2). The SRS1/DEP2 gene encodes a novel protein of 1365 amino acids residues without known functional domains. In the longitudinal direction of the lemma, both cell length and cell number are reduced in srs1-1 compared to the wild type, whereas in the lateral cross section of the lemma, cell length in srs1-1 is greater than that in the wild type, but the cell number in srs1-1 is the same as that in wild type. These results suggest that the small and round seed phenotype of srs1-1 is due to the reduction in both cell length and cell number in the longitudinal direction, and the elongation of the cells in the lateral direction of the lemma. The SRS1 mRNA and proteins are abundant in wild type rice specifically in young organs, namely young leaves, internodes and panicles. Interestingly, the tissues expressing SRS1 are closely related to the tissues that exhibit abnormalities in the srs1 mutants. DEP2|EP2|SRS1 Rice DENSE AND ERECT PANICLE 2 is essential for determining panicle outgrowth and elongation 2010 Cell Res National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. The architecture of the panicle, including grain size and panicle morphology, directly determines grain yield. Panicle erectness, which is selected for achieving ideal plant architecture in the northern part of China, has drawn increasing attention of rice breeders. Here, dense and erect panicle 2 (dep2) mutant, which shows a dense and erect panicle phenotype, was identified. DEP2 encodes a plant-specific protein without any known functional domain. Expression profiling of DEP2 revealed that it is highly expressed in young tissues, with most abundance in young panicles. Morphological and expression analysis indicated that mutation in DEP2 mainly affects the rapid elongation of rachis and primary and secondary branches, but does not impair the initiation or formation of panicle primordia. Further analysis suggests that decrease of panicle length in dep2 is caused by a defect in cell proliferation during the exponential elongation of panicle. Despite a more compact plant type in the dep2 mutant, no significant alteration in grain production was found between wild type and dep2 mutant. Therefore, the study of DEP2 not only strengthens our understanding of the molecular genetic basis of panicle architecture but also has important implications for rice breeding. DEP2|EP2|SRS1 Erect panicle2 encodes a novel protein that regulates panicle erectness in indica rice 2010 Genetics State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Rice (Oryza sativa L.) inflorescence (panicle) architecture is an important agronomic trait for rice breeding. A number of high-yielding japonica rice strains, characterized by an erect panicle (EP) of their architecture, have been released as commercial varieties in China. But no EP-type indica varieties are released so far. Here, we identified two allelic erect-panicle mutants in indica rice, erect panicle2-1 (ep2-1) and erect panicle2-2 (ep2-2), exhibiting the characteristic erect panicle phenotype. Both mutants were derived from spontaneous mutation. We cloned the EP2 gene by way of a map-based cloning strategy, and a transgenic complementation test rescued the phenotype of ep2-1. Anatomical investigations revealed that the ep2 mutants have more vascular bundles and a thicker stem than that of wild-type plants, explaining the panicle erectness phenotype in ep2 mutants. It was shown that EP2 was specifically expressed in the vascular bundles of internodes by GUS staining and RT-PCR. EP2 encodes a novel plant-specific protein, which localizes to the endoplasmic reticulum with unknown biochemical function. In addition, EP2 also regulates other panicle characteristics, such as panicle length and grain size, but grain number per panicle shows little change, indicating that the mutation of the ep2 gene could be applied in EP-type indica rice breeding. DEP2|EP2|SRS1 Fine mapping and candidate gene analysis of dense and erect panicle 3, DEP3, which confers high grain yield in rice (Oryza sativa L.) 2011 Theor Appl Genet Department of Plant Science, Seoul National University, Seoul, 151-921, Korea. Architecture of the rice inflorescence, which is determined mainly by the morphology, number and length of primary and secondary inflorescence branches, is an important agronomical trait. In the current study, we characterized a novel dense and erect panicle (EP) mutant, dep3, derived from the Oryza sativa ssp. japonica cultivar Hwacheong treated with N-methyl-N-nitrosourea. The panicle of the dep3 mutant remained erect from flowering to full maturation, whereas the panicle of the wild type plant began to droop after flowering. The dep3 mutation also regulated other panicle characteristics, including panicle length, grain shape and grain number per panicle. Anatomical observations revealed that the dep3 mutant had more small vascular bundles and a thicker culm than wild type plants, explaining the EP phenotype. Genetic analysis indicated that the phenotype with the dense and EP was controlled by a single recessive gene, termed dep3. The DEP3 gene was identified as the candidate via a map-based cloning approach and was predicted to encode a patatin-like phospholipase A2 (PLA2) superfamily domain-containing protein. The mutant allele gene carried a 408 bp genomic deletion within LOC_Os06g46350, which included the last 47 bp coding region of the third exon and the first 361 bp of the 3'-untranslated region. Taken together, our results indicated that the patatin-like PLA2 might play a significant role in the formation of vascular bundles, and that the dep3 mutant may provide another EP resource for rice breeding programs. DEP3 Over-expression in the nucleotide-binding site-leucine rich repeat gene DEPG1 increases susceptibility to bacterial leaf streak disease in transgenic rice plants 2012 Mol Biol Rep Xiamen Key Laboratory for Plant Genetics, School of Life Sciences, Xiamen University, Xiamen, 361005, People's Republic of China. Bacterial leaf streak of rice (BLS) caused by Xanthomonas oryzae pv. oryzicola (Xoc) is a widely-spread disease in the main rice-producing areas of the world. Investigating the genes that play roles in rice-Xoc interactions helps us to understand the defense signaling pathway in rice. Here we report a differentially expressed protein gene (DEPG1), which regulates susceptibility to BLS. DEPG1 is a nucleotide-binding site (NBS)-leucine rich repeat (LRR) gene, and the deduced protein sequence of DEPG1 has approximately 64% identity with that of the disease resistance gene Pi37. Phylogenetic analysis of DEPG1 and the 18 characterized NBS-LRR genes revealed that DEPG1 is more closely related to Pi37. DEPG1 protein is located to the cytoplasm, which was confirmed by transient expression of DEPG1-GFP (green fluorescent protein) fusion construct in onion epidermal cells. Semi-quantitative PCR assays showed that DEPG1 is widely expressed in rice, and is preferentially expressed in internodes, leaf blades, leaf sheaths and flag leaves. Observation of cross sections of leaves from the transgenic plants with a DEPG1-promoter::glucuronidase (GUS) fusion gene revealed that DEPG1 is also highly expressed in mesophyll tissues where Xoc mainly colonizes. Additionally, Xoc negatively regulates expression of DEPG1 at the early stage of the pathogen infection, and so do the three defense-signal compounds including salicylic acid (SA), methyl jasmonate (MeJA) and 1-aminocyclopropane-1-carboxylic-acid (ACC). Transgenic rice plants overexpressing DEPG1 exhibit enhanced susceptibility to Xoc compared to the wild-type controls. Moreover, enhanced susceptibility to Xoc may be mediated by inhibition of the expression of some SA biosynthesis-related genes and pathogenesis-related genes that may contribute to the disease resistance. Taken together, DEPG1 plays roles in the interactions between rice and BLS pathogen Xoc. DEPG1 Overexpression of OsKTN80a, a katanin P80 ortholog, caused the repressed cell elongation and stalled cell division mediated by microtubule apparatus defects in primary root in Oryza sativa 2014 J Integr Plant Biol State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, 430072, China. Katanin, a microtubule-severing enzyme, consists of two subunits: the catalytic subunit P60, and the regulatory subunit P80. In several species, P80 functions in meiotic spindle organization, the flagella biogenesis, the neuronal development, and the male gamete production. However, the P80 function in higher plants remains elusive. In this study, we found that there are three katanin P80 orthologs (OsKTN80a, OsKTN80b, and OsKTN80c) in Oryza sativa L. Overexpression of OsKTN80a caused the retarded root growth of rice seedlings. Further investigation indicates that the retained root growth was caused by the repressed cell elongation in the elongation zone and the stalled cytokinesis in the division zone in the root tip. The in vivo examination suggests that OsKTN80a acts as a microtubule stabilizer. We prove that OsKTN80a, possibly associated with OsKTN60, is involved in root growth via regulating the cell elongation and division. DGL1|OsKTN60,OsKTN80a,OsKTN80b|OsWD40-111,OsKTN80c|OsWD40-26 Analysis of the rice mutant dwarf and gladius leaf 1. Aberrant katanin-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling 2005 Plant Physiol Bioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi 464-8601, Japan. Molecular genetic studies of plant dwarf mutants have indicated that gibberellin (GA) and brassinosteroid (BR) are two major factors that determine plant height; dwarf mutants that are caused by other defects are relatively rare, especially in monocot species. Here, we report a rice (Oryza sativa) dwarf mutant, dwarf and gladius leaf 1 (dgl1), which exhibits only minimal response to GA and BR. In addition to the dwarf phenotype, dgl1 produces leaves with abnormally rounded tip regions. Positional cloning of DGL1 revealed that it encodes a 60-kD microtubule-severing katanin-like protein. The protein was found to be important in cell elongation and division, based on the observed cell phenotypes. GA biosynthetic genes are up-regulated in dgl1, but the expression of BR biosynthetic genes is not enhanced. The enhanced expression of GA biosynthetic genes in dgl1 is not caused by inappropriate GA signaling because the expression of these genes was repressed by GA3 treatment, and degradation of the rice DELLA protein SLR1 was triggered by GA3 in this mutant. Instead, aberrant microtubule organization caused by the loss of the microtubule-severing function of DGL1 may result in enhanced expression of GA biosynthetic genes in that enhanced expression was also observed in a BR-deficient mutant with aberrant microtubule organization. These results suggest that the function of DGL1 is important for cell and organ elongation in rice, and aberrant DGL1-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling. DGL1|OsKTN60,SLR1|OsGAI DH1, a LOB domain-like protein required for glume formation in rice 2008 Plant Mol Biol Yangzhou University, Yangzhou, Jiangsu, 225009, People's Republic of China. T-DNA tagging is a high throughput strategy for identifying and cloning functional genes in plants. In this study, we screened 4416 lab-created T(1) rice T-DNA tagged lines and identified a mutant, designated dh1 (degenerated hull1), with phenotype of degenerated hull and naked pistils and stamens. Approximately 60% florets on the dh1 panicle defected in forming normal palea and lemma. Instead, they formed degenerative velum-like or filamentous organs accompanying with the lack of lodicules, stamens and pistils at different degree. A 361 bp of genomic sequence flanking the T-DNA isolated using TAIL-PCR (Thermal asymmetric interlaced PCR) co-segregated with the mutation phenotype. Results of blastn and gene prediction revealed the T-DNA inserted into the promoter region of a function-predicted gene at 283 bp upstream of its transcription start site (TSS). The predicted gene encoded a LOB (Lateral Organ Boundaries) domain-like protein. RT-PCR analyses indicated the transcription level of target candidate gene, DH1, decreased significantly in dh1 mutant. RNAi aimed at DH1 in wild type plants could partially result in the mutation phenotype of dh1. DH1 could also rescue the mutation phenotype in the complement experiment. The result of transformation by a fused expression vector, pDH1::GFP, revealed that DH1 had the keen spatial and temporal characteristics of expressing at axillary bud, young panicle and floral organs but not at root, leaf, node and culm, and strongly expressing at young tissues but weakly at mature organs. The dh1 presented severer mutation phenotype under relatively longer daylight than under shorter daylight implied that shorter daylight induced the expression of gene(s) redundant to DH1 in function and partially compensated for the loss-of-function. It is the first time to report the LOB-domain gene participating in the development of floral organs in rice. DH1 Comprehensive expression analysis suggests overlapping and specific roles of rice glutathione S-transferase genes during development and stress responses 2010 BMC Genomics National Institute of Plant Genome Research (NIPGR), Aruna Asaf Ali Marg, New Delhi-110067, India. mjain@nipgr.res.in BACKGROUND: Glutathione S-transferases (GSTs) are the ubiquitous enzymes that play a key role in cellular detoxification. Although several GSTs have been identified and characterized in various plant species, the knowledge about their role in developmental processes and response to various stimuli is still very limited. In this study, we report genome-wide identification, characterization and comprehensive expression analysis of members of GST gene family in crop plant rice, to reveal their function(s). RESULTS: A systematic analysis revealed the presence of at least 79 GST genes in the rice genome. Phylogenetic analysis grouped GST proteins into seven classes. Sequence analysis together with the organization of putative motifs indicated the potential diverse functions of GST gene family members in rice. The tandem gene duplications have contributed a major role in expansion of this gene family. Microarray data analysis revealed tissue-/organ- and developmental stage-specific expression patterns of several rice GST genes. At least 31 GST genes showed response to plant hormones auxin and cytokinin. Furthermore, expression analysis showed the differential expression of quite a large number of GST genes during various abiotic stress (20), arsenate stress (32) and biotic stress (48) conditions. Many of the GST genes were commonly regulated by developmental processes, hormones, abiotic and biotic stresses. CONCLUSION: The transcript profiling suggests overlapping and specific role(s) of GSTs during various stages of development in rice. Further, the study provides evidence for the role of GSTs in mediating crosstalk between various stress and hormone response pathways and represents a very useful resource for functional analysis of selected members of this family in rice. DHAR1|OsDHAR1,OsDHAR2,OsGSTT1,OsTCHQD1 Molecular cloning and characterization of a rice dehydroascorbate reductase 2000 FEBS Lett Department of Biology, Faculty of Science, Shizuoka University, Shizuoka, Japan. Plant dehydroascorbate reductase (DHAR), which re-reduces oxidized ascorbate to maintain an appropriate level of ascorbate, is very important, but no gene or cDNA for plant DHAR has been cloned yet. Here, we describe a cDNA for a rice glutathione-dependent DHAR (designated DHAR1). A recombinant Dhar1p produced in Escherichia coli was functional. The expression sequence tag database suggests that Dhar1p homologs exist in various plants. Furthermore, the rice Dhar1p has a low similarity to rat DHAR, although the rice enzyme has a considerably higher specific activity than the mammalian one. The mRNA level of DHAR1, the protein level of Dhar1p and the DHAR activity in rice seedlings were elevated by high temperature, suggesting the protection role of DHAR at high temperature. DHAR1|OsDHAR1 Genetic interaction of OsMADS3, DROOPING LEAF, and OsMADS13 in specifying rice floral organ identities and meristem determinacy 2011 Plant Physiol School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. Grass plants develop unique floral patterns that determine grain production. However, the molecular mechanism underlying the specification of floral organ identities and meristem determinacy, including the interaction among floral homeotic genes, remains largely unknown in grasses. Here, we report the interactions of rice (Oryza sativa) floral homeotic genes, OsMADS3 (a C-class gene), OsMADS13 (a D-class gene), and DROOPING LEAF (DL), in specifying floral organ identities and floral meristem determinacy. The interaction among these genes was revealed through the analysis of double mutants. osmads13-3 osmads3-4 displayed a loss of floral meristem determinacy and generated abundant carpelloid structures containing severe defective ovules in the flower center, which were not detectable in the single mutant. In addition, in situ hybridization and yeast two-hybrid analyses revealed that OsMADS13 and OsMADS3 did not regulate each other's transcription or interact at the protein level. This indicates that OsMADS3 plays a synergistic role with OsMADS13 in both ovule development and floral meristem termination. Strikingly, osmads3-4 dl-sup6 displayed a severe loss of floral meristem determinacy and produced supernumerary whorls of lodicule-like organs at the forth whorl, suggesting that OsMADS3 and DL synergistically terminate the floral meristem. Furthermore, the defects of osmads13-3 dl-sup6 flowers appeared identical to those of dl-sup6, and the OsMADS13 expression was undetectable in dl-sup6 flowers. These observations suggest that DL and OsMADS13 may function in the same pathway specifying the identity of carpel/ovule and floral meristem. Collectively, we propose a model to illustrate the role of OsMADS3, DL, and OsMADS13 in the specification of flower organ identity and meristem determinacy in rice. DL,OsMADS13,OSMADS3 SUPERWOMAN1 and DROOPING LEAF genes control floral organ identity in rice 2003 Development Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. We analyzed recessive mutants of two homeotic genes in rice, SUPERWOMAN1 (SPW1) and DROOPING LEAF (DL). The homeotic mutation spw1 transforms stamens and lodicules into carpels and palea-like organs, respectively. Two spw1 alleles, spw1-1 and spw1-2, show the same floral phenotype and did not affect vegetative development. We show that SPW1 is a rice APETALA3 homolog, OsMADS16. In contrast, two strong alleles of the dl locus, drooping leaf-superman1 (dl-sup1) and drooping leaf-superman2 (dl-sup2), cause the complete transformation of the gynoecium into stamens. In these strong mutants, many ectopic stamens are formed in the region where the gynoecium is produced in the wild-type flower and they are arranged in a non-whorled, alternate pattern. The intermediate allele dl-1 (T65), results in an increase in the number of stamens and stigmas, and carpels occasionally show staminoid characteristics. In the weakest mutant, dl-2, most of the flowers are normal. All four dl alleles cause midrib-less drooping leaves. The flower of the double mutant, spw1 dl-sup, produces incompletely differentiated organs indefinitely after palea-like organs are produced in the position where lodicules are formed in the wild-type flower. These incompletely differentiated organs are neither stamens nor carpels, but have partial floral identity. Based on genetic and molecular results, we postulate a model of stamen and carpel specification in rice, with DL as a novel gene controlling carpel identity and acting mutually and antagonistically to the class B gene, SPW1. DL,OsMADS16|SPW1 Temporal and spatial regulation of DROOPING LEAF gene expression that promotes midrib formation in rice 2011 Plant J Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8654, Japan. Genes involved in the differentiation and development of tissues and organs are temporally and spatially regulated in plant development. The DROOPING LEAF (DL) gene, a member of the YABBY gene family, promotes midrib formation in the leaf and carpel specification in the flower. Consistent with these functions, DL is initially expressed in the central region of the leaf primordia (presumptive midrib) and in the presumptive carpel primordia in the meristem. To understand the regulatory mechanism underlying DL expression, we tried to identify cis-regulatory regions required for temporal and spatial expression of this gene. We found that the cis region responsible for the presumptive midrib-specific expression in the leaf primordia is located in intron 2. Next, we confined the region to a sequence of about 200bp, which corresponds to a conserved non-coding sequence (CNS) identified by phylogenetic footprinting. In addition, a sequence termed DG1, incorporating a 5' upstream region of about 7.4kb, and introns 1 and 2, was shown to be sufficient to induce DL in the presumptive midrib, and to suppress it in other regions in the leaf primordia. By contrast, the regulatory region required for carpel-specific expression was not included in the DG1 sequence. We modified Oryza sativa (rice) plant architecture by expressing an activated version of DL (DL-VP16) in a precise manner using the DG1 sequence: the resulting transgenic plant produced a midrib in the distal region of the leaf blade, where there is no midrib in wild type, and formed more upright leaves compared with the wild type. DL The YABBY gene DROOPING LEAF regulates carpel specification and midrib development in Oryza sativa 2004 Plant Cell Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan. In this article, we report that carpel specification in the Oryza sativa (rice) flower is regulated by the floral homeotic gene DROOPING LEAF (DL) that is distinct from the well-known ABC genes. Severe loss-of-function mutations of DL cause complete homeotic transformation of carpels into stamens. Molecular cloning reveals that DL is a member of the YABBY gene family and is closely related to the CRABS CLAW (CRC) gene of Arabidopsis thaliana. DL is expressed in the presumptive region (carpel anlagen), where carpel primordia would initiate, and in carpel primordia. These results suggest that carpel specification is regulated by DL in rice flower development. Whereas CRC plays only a partial role in carpel identity, DL may have been recruited to have the more essential function of specifying carpels during the evolution of rice. We also show that DL interacts antagonistically with class B genes and controls floral meristem determinacy. In addition, severe and weak dl alleles fail to form a midrib in the leaf. The phenotypic analysis of dl mutants, together with analyses of the spatial expression patterns and ectopic expression of DL, demonstrate that DL regulates midrib formation by promoting cell proliferation in the central region of the rice leaf. DL Rice MADS6 interacts with the floral homeotic genes SUPERWOMAN1, MADS3, MADS58, MADS13, and DROOPING LEAF in specifying floral organ identities and meristem fate 2011 Plant Cell Institute of Plant Science, State Key Laboratory of Hybrid Rice, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. AGAMOUS-LIKE6 (AGL6) genes play essential roles in flower development, but whether and how they work with floral organ identity genes remain less understood. Here, we describe interactions of the rice (Oryza sativa) AGL6 gene MADS6 with other rice floral homeotic genes in flower development. Genetic analyses revealed that MADS6 specifies the identity of the three inner whorls and floral meristem determinacy redundantly with SUPERWOMAN1/MADS16 (B-gene) or MADS3 (C-gene). MADS6 was shown to define carpel/ovule development and floral determinacy by interacting with MADS13 (D-gene) and control the palea and floral meristem identities together with the YABBY gene DROOPING LEAF. Expression analyses revealed that the transcript levels of six B-, C-, and E-class genes were reduced in mads6-1 at the early flower developmental stage, suggesting that MADS6 is a key regulator of early flower development. Moreover, MADS6 can directly bind to a putative regulatory motif on MADS58 (C-gene), and mads6-1 mads58 displayed phenotypes similar to that of mads6-1. These results suggest that MADS6 is a key player in specifying flower development via interacting with other floral homeotic genes in rice, thus providing new insights into the mechanism by which flower development is controlled. DL,OsMADS13,OsMADS16|SPW1,OSMADS3,OSMADS58,OsMADS6|MFO1 A transposon, Ping, is integrated into intron 4 of the DROOPING LEAF gene of rice, weakly reducing its expression and causing a mild drooping leaf phenotype 2008 Plant Cell Physiol Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657 Japan. The YABBY gene DROOPING LEAF (DL) regulates midrib formation in the leaves and carpel specification in the flowers of rice (Oryza sativa L). We found a new dl allele (dl-5) that caused a mild phenotype in the descendants of a mutable line, IM294. In plants homozygous for this allele, midrib structures were formed but their sizes were reduced. Molecular analysis revealed that a transposon, Ping, was inserted in the fourth intron of DL. Together with mPing and Pong, Ping is a member of a transposon family that was first identified as a group of active transposable elements in rice. Our finding of the Ping insertion in the DL gene is a first indication that Ping is active in planta, and that it can be transposed and integrated in a new locus. Ping seems to be still active because it was excised from intron 4 of DL at a relatively high frequency in rice calli. Real-time PCR analysis and in situ hybridization indicated that DL transcript levels were reduced in dl-5 without alterations in the spatial expression pattern of the DL gene. The reduction of DL expression may be due to inefficient splicing of the large intron caused by Ping insertion. By comparing the expression levels of DL and leaf phenotypes in the dl mutants with different severities, we confirmed our previous hypothesis that DL promotes cell proliferation in the central region of leaf primordia, and that this cell proliferation is critical for midrib formation in the mature leaves. DL The DROOPING LEAF and OsETTIN2 genes promote awn development in rice 2014 Plant J Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657, Japan. The awn is a long needle-like appendage that, in some grass species, is formed on the lemma that encloses floral organs together with the palea. In rice, most wild species and most strains of Oryza sativa ssp. indica generate an awn, whereas most strains of O. sativa ssp. japonica do not. In japonica, the long-awn characteristic appears to have been lost during domestication and breeding programs. Here, we found that the genes DROOPING LEAF (DL) and OsETTIN2 (OsETT2) are involved in awn development in the awned indica strain Kasalath. Genetic analyses and RNA-silencing experiments indicate that DL and OsETT2 act independently in awn formation, and that either gene alone is not sufficient for awn development. Scanning electron microscopy revealed that the top region of the lemma (a putative awn primordium) is larger in an awned floret than in an awnless floret. OsETT2 is expressed in the awn primordium in the awned indica floret, but not in the awnless japonica floret except in the provascular bundle. DL is expressed underneath the primordium at similar levels in both indica and japonica florets, suggesting non-cell-autonomous action. We hypothesize that loss of expression of OsETT2 in the awn primordium is probably associated with the failure of awn formation in japonica strains. DL,OsETT2|OsETTIN2 Identification and localisation of the rice nicotianamine aminotransferase gene OsNAAT1 expression suggests the site of phytosiderophore synthesis in rice 2008 Plant Mol Biol Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Rice plants (Oryza sativa L.) take up iron using iron-chelating compounds known as mugineic acid family phytosiderophores (MAs). In the biosynthetic pathway of MAs, nicotianamine aminotransferase (NAAT) catalyses the key step from nicotianamine to the 3''-keto form. In the present study, we identified six rice NAAT genes (OsNAAT1-6) by screening a cDNA library made from Fe-deficient rice roots and by searching databases. Among the NAAT homologues, OsNAAT1 belongs to a subgroup containing barley functional NAAT (HvNAAT-A and HvNAAT-B) as well as a maize homologue cloned by cDNA library screening (ZmNAAT1). Northern blot and RT-PCR analysis showed that OsNAAT1, but not OsNAAT2-6, was strongly up-regulated by Fe deficiency, both in roots and shoots. The OsNAAT1 protein had NAAT enzyme activity in vitro, confirming that the OsNAAT1 gene encodes functional NAAT. Promoter-GUS analysis revealed that OsNAAT1 was expressed in companion and pericycle cells adjacent to the protoxylem of Fe-sufficient roots. In addition, expression was induced in all cells of Fe-deficient roots, with particularly strong GUS activity evident in the companion and pericycle cells. OsNAAT1 expression was also observed in the companion cells of Fe-sufficient shoots, and was clearly induced in all the cells of Fe-deficient leaves. These expression patterns highly resemble those of OsNAS1, OsNAS2 and OsDMAS1, the genes responsible for MAs biosynthesis for Fe acquisition. These findings strongly suggest that rice synthesizes MAs in whole Fe-deficient roots to acquire Fe from the rhizosphere, and also in phloem cells to maintain metal homeostasis facilitated by MAs-mediated long-distance transport. OsDMAS1,OsNAAT1,OsNAAT2,OsNAAT3,OsNAAT4,OsNAAT5,OsNAAT6 Cloning and characterization of deoxymugineic acid synthase genes from graminaceous plants 2006 J Biol Chem Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657. Graminaceous plants have evolved a unique mechanism to acquire iron through the secretion of a family of small molecules, called mugineic acid family phytosiderophores (MAs). All MAs are synthesized from l-Met, sharing the same pathway from l-Met to 2'-deoxymugineic acid (DMA). DMA is synthesized through the reduction of a 3''-keto intermediate by deoxymugineic acid synthase (DMAS). We have isolated DMAS genes from rice (OsDMAS1), barley (HvDMAS1), wheat (TaD-MAS1), and maize (ZmDMAS1). Their nucleotide sequences indicate that OsDMAS1 encodes a predicted polypeptide of 318 amino acids, whereas the other three orthologs all encode predicted polypeptides of 314 amino acids and are highly homologous (82-97.5%) to each other. The DMAS proteins belong to the aldo-keto reductase superfamily 4 (AKR4) but do not fall within the existing subfamilies of AKR4 and appear to constitute a new subfamily within the AKR4 group. All of the proteins showed DMA synthesis activity in vitro. Their enzymatic activities were highest at pH 8-9, consistent with the hypothesis that DMA is synthesized in subcellular vesicles. Northern blot analysis revealed that the expression of each of the above DMAS genes is up-regulated under iron-deficient conditions in root tissue, and that of the genes OsDMAS1 and TaDMAS1 is up-regulated in shoot tissue. OsDMAS1 promoter-GUS analysis in iron-sufficient roots showed that its expression is restricted to cells participating in long distance transport and that it is highly up-regulated in the entire root under iron-deficient conditions. In shoot tissue, OsDMAS1 promoter drove expression in vascular bundles specifically under iron-deficient conditions. OsDMAS1 A receptor-like protein RMC is involved in regulation of iron acquisition in rice 2013 J Exp Bot State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, PR China. Iron (Fe) is one of the essential mineral elements for plant growth and development. Acquisition of Fe by plants is mediated by a complex network involving Fe mobilization, uptake by root cells, and transport within plants. Here, we evaluated the role of a previously clarified gene encoding a receptor-like protein from rice, OsRMC, in the regulation of Fe acquisition by comparing Fe concentration, biomass, and expression patterns of genes associated with Fe mobilization and transport in wild-type (WT) rice with those in OsRMC overexpression and RNA interference (RNAi) knockdown transgenic rice plants. Expression of OsRMC was upregulated in both shoots and roots upon exposure of WT to Fe-deficient medium. Expression levels of OsRMC were positively correlated with Fe concentration in rice plants under both Fe-sufficient and Fe-deficient conditions such that overexpression and RNAi lines had higher and lower Fe concentration in both roots and shoots than WT plants, respectively. Moreover, overexpression of OsRMC conferred greater accumulation of Fe in mature seeds under Fe-sufficient conditions. OsRMC may also play a role in regulation of Fe deficiency-induced changes in root growth, as evidenced by greater and smaller root systems of OsRMC overexpression lines and RNAi lines than WT under Fe-deficient conditions, respectively. Several Fe deficiency-responsive genes including OsDMAS1, OsNAS1, OsNAS2, OsNAAT1, OsIRT1, OsYSL15, and OsIRO2 were up- and downregulated in OsRMC-overexpressing and RNAi plants compared with WT rice plants. These novel findings highlight an important role of OsRMC played in mediation of Fe acquisition and root growth in rice, particularly under Fe-deficient conditions. OsDMAS1,OsIRO2,OsIRT1,OsNAAT1,OsNAS1,OsNAS2,OsRMC|OsRLK,OsYSL15 Expression of iron-acquisition-related genes in iron-deficient rice is co-ordinately induced by partially conserved iron-deficiency-responsive elements 2005 J Exp Bot Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Rice plants (Oryza sativa L.) utilize the iron chelators known as mugineic acid family phytosiderophores (MAs) to acquire iron from the rhizosphere. Synthesis of MAs and uptake of MA-chelated iron are strongly induced under conditions of iron deficiency. Microarray analysis was used to characterize the expression profile of rice in response to iron deficiency at the genomic level. mRNA extracted from iron-deficient or iron-sufficient rice roots or leaves was hybridized to a rice array containing 8987 cDNA clones. An induction ratio of greater than 2.0 in roots was observed for 57 genes, many of which are involved in iron-uptake mechanisms, including every identified or predicted step in the methionine cycle and the biosynthesis of MAs from methionine. Northern analysis confirmed that the expression of genes encoding every step in the methionine cycle is thoroughly induced by iron deficiency in roots, and almost thoroughly induced in leaves. A promoter search revealed that the iron-deficiency-induced genes related to iron uptake possessed sequences homologous to the iron-deficiency-responsive cis-acting elements IDE1 and IDE2 in their promoter regions, at a higher rate than that showing no induction under Fe deficiency. These results suggest that rice genes involved in iron acquisition are co-ordinately regulated by conserved mechanisms in response to iron deficiency, in which IDE-mediated regulation plays a significant role. OsDMAS1,FDH,OsAPT1|APRT,OsDEP,OsRPI|RPI The expression of iron homeostasis-related genes during rice germination 2007 Plant Mol Biol Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan. To characterize Fe homeostasis during the early stages of seed germination, a microarray analysis was performed. mRNAs extracted from fully mature seeds or seeds harvested 1-3 days after sowing were hybridized to a rice microarray containing approximately 22,000 cDNA oligo probes. Many Fe deficiency-inducible genes were strongly expressed throughout early seed germination. These results suggest that the demand for Fe is extremely high during germination. Under Fe-deficient conditions, rice produces and secretes a metal-cation chelator called deoxymugineic acid (DMA) to acquire Fe from the soil. In addition, DMA and its intermediate nicotianamine (NA) are thought to be involved in long distance Fe transport in rice. Using promoter-beta-glucuronidase (GUS) analysis, we investigated the expression patterns during seed germination of the Fe deficiency-inducible genes OsNAS1, OsNAS2, OsNAS3, OsNAAT1, and OsDMAS1, which encode enzymes that participate in the biosynthesis of DMA, and the transporter genes OsYSL2 and OsIRT1, which are involved in Fe transport. All of these genes were expressed in germinating seeds prior to protrusion of the radicle. These results suggest that DMA and NA are produced and involved in Fe transport during germination. OsDMAS1 Identification of OsbHLH133 as a regulator of iron distribution between roots and shoots in Oryza sativa 2013 Plant Cell Environ State Key Laboratory of Plant Physiology and Biochemistry Joint Research Laboratory in Genomics and Nutriomics, College of Life Sciences, Zhejiang University, Hangzhou, China. Iron (Fe) is an essential micronutrient element for plant growth. Regulation of Fe-deficiency signalling networks is one of the many functions reported for basic helix-loop-helix (bHLH) transcription factors in plants. In the present study, OsbHLH133 was found to be induced by Fe-deficiency conditions in Oryza sativa. Insertional inactivation of OsbHLH133 (bhlh133) resulted in growth retardation, with enhanced Fe concentration seen in shoots, and reduced Fe concentration in roots. Overexpression of OsbHLH133 had the opposite effect, that is resulted in an enhanced Fe concentration in roots and reduced Fe concentration in shoots and also in xylem sap. Microarray analysis showed that some of the genes encoding Fe-related functions were up-regulated under Fe-sufficient conditions, in bhlh133 mutant plants compared to wild-type plants. Significant differential expression of a number of signalling pathways, including calcium signalling, was also seen in bhlh133 plants compared to wild-type plants, independent of Fe conditions. OsDMAS1,OsENA1,OsAPT1|APRT,OsbHLH133,TOM1|OsZIFL4,OsDEP,OsRPI|RPI Deoxymugineic acid increases Zn translocation in Zn-deficient rice plants 2008 Plant Mol Biol Department of Global Agricultural Sciences, Graduate School of Agricultural and Life Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Deoxymugineic acid (DMA) is a member of the mugineic acid family phytosiderophores (MAs), which are natural metal chelators produced by graminaceous plants. Rice secretes DMA in response to Fe deficiency to take up Fe in the form of Fe(III)-MAs complex. In contrast with barley, the roots of which secrete MAs in response to Zn deficiency, the amount of DMA secreted by rice roots was slightly decreased under conditions of low Zn supply. There was a concomitant increase in endogenous DMA in rice shoots, suggesting that DMA plays a role in the translocation of Zn within Zn-deficient rice plants. The expression of OsNAS1 and OsNAS2 was not increased in Zn-deficient roots but that of OsNAS3 was increased in Zn-deficient roots and shoots. The expression of OsNAAT1 was also increased in Zn-deficient roots and dramatically increased in shoots; correspondingly, HPLC analysis was unable to detect nicotianamine in Zn-deficient shoots. The expression of OsDMAS1 was increased in Zn-deficient shoots. Analyses using the positron-emitting tracer imaging system (PETIS) showed that Zn-deficient rice roots absorbed less (62)Zn-DMA than (62)Zn(2+). Importantly, supply of (62)Zn-DMA rather than (62)Zn(2+) increased the translocation of (62)Zn into the leaves of Zn-deficient plants. This was especially evident in the discrimination center (DC). These results suggest that DMA in Zn-deficient rice plants has an important role in the distribution of Zn within the plant rather than in the absorption of Zn from the soil. OsDMAS1 Analysis of anther transcriptomes to identify genes contributing to meiosis and male gametophyte development in rice 2011 BMC Plant Biol Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi, South Campus, New Delhi-110021, India. BACKGROUND: In flowering plants, the anther is the site of male gametophyte development. Two major events in the development of the male germline are meiosis and the asymmetric division in the male gametophyte that gives rise to the vegetative and generative cells, and the following mitotic division in the generative cell that produces two sperm cells. Anther transcriptomes have been analyzed in many plant species at progressive stages of development by using microarray and sequence-by synthesis-technologies to identify genes that regulate anther development. Here we report a comprehensive analysis of rice anther transcriptomes at four distinct stages, focusing on identifying regulatory components that contribute to male meiosis and germline development. Further, these transcriptomes have been compared with the transcriptomes of 10 stages of rice vegetative and seed development to identify genes that express specifically during anther development. RESULTS: Transcriptome profiling of four stages of anther development in rice including pre-meiotic (PMA), meiotic (MA), anthers at single-celled (SCP) and tri-nucleate pollen (TPA) revealed about 22,000 genes expressing in at least one of the anther developmental stages, with the highest number in MA (18,090) and the lowest (15,465) in TPA. Comparison of these transcriptome profiles to an in-house generated microarray-based transcriptomics database comprising of 10 stages/tissues of vegetative as well as reproductive development in rice resulted in the identification of 1,000 genes specifically expressed in anther stages. From this sub-set, 453 genes were specific to TPA, while 78 and 184 genes were expressed specifically in MA and SCP, respectively. The expression pattern of selected genes has been validated using real time PCR and in situ hybridizations. Gene ontology and pathway analysis of stage-specific genes revealed that those encoding transcription factors and components of protein folding, sorting and degradation pathway genes dominated in MA, whereas in TPA, those coding for cell structure and signal transduction components were in abundance. Interestingly, about 50% of the genes with anther-specific expression have not been annotated so far. CONCLUSIONS: Not only have we provided the transcriptome constituents of four landmark stages of anther development in rice but we have also identified genes that express exclusively in these stages. It is likely that many of these candidates may therefore contribute to specific aspects of anther and/or male gametophyte development in rice. In addition, the gene sets that have been produced will assist the plant reproductive community in building a deeper understanding of underlying regulatory networks and in selecting gene candidates for functional validation. DMC1A|DMC1 Filament formation and robust strand exchange activities of the rice DMC1A and DMC1B proteins 2008 Nucleic Acids Res Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Wasedatsurumaki-cho, Shinjuku-ku, Tokyo 162-0041, Japan. The DMC1 protein, a meiosis-specific DNA recombinase, catalyzes strand exchange between homologous chromosomes. In rice, two Dmc1 genes, Dmc1A and Dmc1B, have been reported. Although the Oryza sativa DMC1A protein has been partially characterized, however the biochemical properties of the DMC1B protein have not been defined. In the present study, we expressed the Oryza sativa DMC1A and DMC1B proteins in bacteria and purified them. The purified DMC1A and DMC1B proteins formed helical filaments along single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and promoted robust strand exchange between ssDNA and dsDNA over five thousand base pairs in the presence of RPA, as a co-factor. The DMC1A and DMC1B proteins also promoted strand exchange in the absence of RPA with long DNA substrates containing several thousand base pairs. In contrast, the human DMC1 protein strictly required RPA to promote strand exchange with these long DNA substrates. The strand-exchange activity of the Oryza sativa DMC1A protein was much higher than that of the DMC1B protein. Consistently, the DNA-binding activity of the DMC1A protein was higher than that of the DMC1B protein. These biochemical differences between the DMC1A and DMC1B proteins may provide important insight into their functional differences during meiosis in rice. DMC1A|DMC1 A 5-methylcytosine DNA glycosylase/lyase demethylates the retrotransposon Tos17 and promotes its transposition in rice 2011 Proc Natl Acad Sci U S A Department of Plant Pathology, Ohio State University, Columbus, OH 43210, USA. DNA 5-methylcytosine (5-meC) is an important epigenetic mark for transcriptional gene silencing in many eukaryotes. In Arabidopsis, 5-meC DNA glycosylase/lyases actively remove 5-meC to counteract transcriptional gene silencing in a locus-specific manner, and have been suggested to maintain the expression of transposons. However, it is unclear whether plant DNA demethylases can promote the transposition of transposons. Here we report the functional characterization of the DNA glycosylase/lyase DNG701 in rice. DNG701 encodes a large (1,812 amino acid residues) DNA glycosylase domain protein. Recombinant DNG701 protein showed 5-meC DNA glycosylase and lyase activities in vitro. Knockout or knockdown of DNG701 in rice plants led to DNA hypermethylation and reduced expression of the retrotransposon Tos17. Tos17 showed less transposition in calli derived from dng701 knockout mutant seeds compared with that in wild-type calli. Overexpression of DNG701 in both rice calli and transgenic plants substantially reduced DNA methylation levels of Tos17 and enhanced its expression. The overexpression also led to more frequent transposition of Tos17 in calli. Our results demonstrate that rice DNG701 is a 5-meC DNA glycosylase/lyase responsible for the demethylation of Tos17 and this DNA demethylase plays a critical role in promoting Tos17 transposition in rice calli. DNG701 A leucine-rich repeat receptor-like kinase gene is involved in the specification of outer cell layers in rice roots 2012 Plant J Institute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki 710-0046, Japan. Root outer cell layers of Oryza sativa (rice), which comprise the epidermis, exodermis and sclerenchyma, play an important role in protecting the roots from various stresses in soil, but the molecular mechanisms for the specification of these cell layers are poorly understood. In this work, we report on defective in outer cell layer specification 1 (Docs1), which is involved in the specification of outer cell layers in rice roots. Docs1 was isolated by map-based cloning using a mutant (c68) defective in the outer cell layers of primary roots. It encodes a leucine-rich repeat receptor-like kinase (LRR RLK). Docs1 mRNA was expressed in all tissues including roots, leaf blades and sheaths, and flowers. Immunostaining with an anti-Docs1 antibody showed that Docs1 was localized at the epidermis and exodermis, depending on the root region. Furthermore, Docs1 showed polar localization at the distal side. Subcellular examination showed that Docs1 was localized to the plasma membrane. Comparison of genome-wide transcriptional profiles between the wild-type and the knock-out mutant roots using microarray analysis showed that 61 and 41 genes were up- and downregulated in the mutant, including genes encoding putative transcription factors and genes potentially involved in cell wall metabolism. These results suggest that Docs1 might directly or indirectly regulate multiple genes involved in the proper development of root outer cell layers in rice. Docs1 An AT-hook gene is required for palea formation and floral organ number control in rice 2011 Dev Biol State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. Grasses have highly specialized flowers and their outer floral organ identity remains unclear. In this study, we identified and characterized rice mutants that specifically disrupted the development of palea, one of the outer whorl floral organs. The depressed palea1 (dp1) mutants show a primary defect in the main structure of palea, implying that palea is a fusion between the main structure and marginal tissues on both sides. The sterile lemma at the palea side is occasionally elongated in dp1 mutants. In addition, we found a floral organ number increase in dp1 mutants at low penetration. Both the sterile lemma elongation and the floral organ number increase phenotype are enhanced by the mutation of an independent gene SMALL DEGENERATIVE PALEA1 (SDP1), whose single mutation causes reduced palea size. E function and presumable A function floral homeotic genes were found suppressed in the dp1-2 mutant. We identified the DP1 gene by map-based cloning and found it encodes a nuclear-localized AT-hook DNA binding protein, suggesting a grass-specific role of chromatin architecture modification in flower development. The DP1 enhancer SDP1 was also positional cloned, and was found identical to the recently reported RETARDED PALEA1 (REP1) gene encoding a TCP family transcription factor. We further found that SDP1/REP1 is downstreamly regulated by DP1. DP1,REP1 Rice pollen hybrid incompatibility caused by reciprocal gene loss of duplicated genes 2010 Proc Natl Acad Sci U S A Plant Genetics Laboratory, Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan. Genetic incompatibility is a barrier contributing to species isolation and is caused by genetic interactions. We made a whole genome survey of two-way interacting loci acting within the gametophyte or zygote using independence tests of marker segregations in an F(2) population from an intersubspecific cross between O. sativa subspecies indica and japonica. We detected only one reproducible interaction, and identified paralogous hybrid incompatibility genes, DOPPELGANGER1 (DPL1) and DOPPELGANGER2 (DPL2), by positional cloning. Independent disruptions of DPL1 and DPL2 occurred in indica and japonica, respectively. DPLs encode highly conserved, plant-specific small proteins ( approximately 10 kDa) and are highly expressed in mature anther. Pollen carrying two defective DPL alleles became nonfunctional and did not germinate, suggesting an essential role for DPLs in pollen germination. Although rice has many duplicated genes resulting from ancient whole genome duplication, the origin of this gene duplication was in recent small-scale gene duplication, occurring after Oryza-Brachypodium differentiation. Comparative analyses suggested the geographic and phylogenetic distribution of these two defective alleles, showing that loss-of-function mutations of DPL1 genes emerged multiple times in indica and its wild ancestor, O. rufipogon, and that the DPL2 gene defect is specific to japonica cultivars. DPL1,DPL2 Defective pollen wall is required for anther and microspore development in rice and encodes a fatty acyl carrier protein reductase 2011 Plant Cell Institute of Plant Science, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. Aliphatic alcohols naturally exist in many organisms as important cellular components; however, their roles in extracellular polymer biosynthesis are poorly defined. We report here the isolation and characterization of a rice (Oryza sativa) male-sterile mutant, defective pollen wall (dpw), which displays defective anther development and degenerated pollen grains with an irregular exine. Chemical analysis revealed that dpw anthers had a dramatic reduction in cutin monomers and an altered composition of cuticular wax, as well as soluble fatty acids and alcohols. Using map-based cloning, we identified the DPW gene, which is expressed in both tapetal cells and microspores during anther development. Biochemical analysis of the recombinant DPW enzyme shows that it is a novel fatty acid reductase that produces 1-hexadecanol and exhibits >270-fold higher specificity for palmiltoyl-acyl carrier protein than for C16:0 CoA substrates. DPW was predominantly targeted to plastids mediated by its N-terminal transit peptide. Moreover, we demonstrate that the monocot DPW from rice complements the dicot Arabidopsis thaliana male sterile2 (ms2) mutant and is the probable ortholog of MS2. These data suggest that DPWs participate in a conserved step in primary fatty alcohol synthesis for anther cuticle and pollen sporopollenin biosynthesis in monocots and dicots. DPW Dro1, a major QTL involved in deep rooting of rice under upland field conditions 2011 J Exp Bot National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan. yuga@affrc.go.jp Developing a deep root system is an important strategy for avoiding drought stress in rice. Using the 'basket' method, the ratio of deep rooting (RDR; the proportion of total roots that elongated through the basket bottom) was calculated to evaluate deep rooting. A new major quantitative trait locus (QTL) controlling RDR was detected on chromosome 9 by using 117 recombinant inbred lines (RILs) derived from a cross between the lowland cultivar IR64, with shallow rooting, and the upland cultivar Kinandang Patong (KP), with deep rooting. This QTL explained 66.6% of the total phenotypic variance in RDR in the RILs. A BC(2)F(3) line homozygous for the KP allele of the QTL had an RDR of 40.4%, compared with 2.6% for the homozygous IR64 allele. Fine mapping of this QTL was undertaken using eight BC(2)F(3) recombinant lines. The RDR QTL Dro1 (Deeper rooting 1) was mapped between the markers RM24393 and RM7424, which delimit a 608.4 kb interval in the reference cultivar Nipponbare. To clarify the influence of Dro1 in an upland field, the root distribution in different soil layers was quantified by means of core sampling. A line homozygous for the KP allele of Dro1 (Dro1-KP) and IR64 did not differ in root dry weight in the shallow soil layers (0-25 cm), but root dry weight of Dro1-KP in deep soil layers (25-50 cm) was significantly greater than that of IR64, suggesting that Dro1 plays a crucial role in increased deep rooting under upland field conditions. DRO1 Control of root system architecture by DEEPER ROOTING 1 increases rice yield under drought conditions 2013 Nat Genet National Institute of Agrobiological Sciences, Tsukuba, Japan. yuga@nias.affrc.go.jp The genetic improvement of drought resistance is essential for stable and adequate crop production in drought-prone areas. Here we demonstrate that alteration of root system architecture improves drought avoidance through the cloning and characterization of DEEPER ROOTING 1 (DRO1), a rice quantitative trait locus controlling root growth angle. DRO1 is negatively regulated by auxin and is involved in cell elongation in the root tip that causes asymmetric root growth and downward bending of the root in response to gravity. Higher expression of DRO1 increases the root growth angle, whereby roots grow in a more downward direction. Introducing DRO1 into a shallow-rooting rice cultivar by backcrossing enabled the resulting line to avoid drought by increasing deep rooting, which maintained high yield performance under drought conditions relative to the recipient cultivar. Our experiments suggest that control of root system architecture will contribute to drought avoidance in crops. DRO1 A rice dihydrosphingosine C4 hydroxylase (DSH1) gene, which is abundantly expressed in the stigmas, vascular cells and apical meristem, may be involved in fertility 2007 Plant Cell Physiol Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan. Dihydrosphingosine C4 hydroxylase is a key enzyme in the biosynthesis of phytosphingosine, a major constituent of sphingolipids in plants and yeasts. The rice genome contains five homologue genes for dihydrosphingosine C4 hydroxylase, DSH1-DSH5, whose gene products show high degrees of homology to the yeast counterpart, SUR2. Among them, expression of DSH1, DSH2 and DSH4 was detected, and DSH1 and DSH4 complement the yeast sur2 mutation. The DSH1 gene was specifically and abundantly expressed in vascular bundles and apical meristems. In particular, very strong expression was detected in the stigmas of flowers. Repression of DSH1 expression by the antisense gene or RNA interference (RNAi) resulted in a severe reduction of fertility. In the transformants in which DSH1 expression was suppressed, significantly increased expression of DSH2 was found in leaves but not in pistils, suggesting that there was tissue-specific correlation between DSH1 and DSH2 expression. Our results indicate that the product of DSH1 may be involved in plant viability or reproductive processes, and that the phenotype of sterility is apparently caused by loss of function of DSH1 in the stigma. It is also suggested that there is a complex mechanism controlling the tissue-specific expression of the DSH1 gene. DSH1 A Raf-like MAPKKK gene DSM1 mediates drought resistance through reactive oxygen species scavenging in rice 2010 Plant Physiol National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China. Mitogen-activated protein kinase (MAPK) cascades have been identified in various signaling pathways involved in plant development and stress responses. We identified a drought-hypersensitive mutant (drought-hypersensitive mutant1 [dsm1]) of a putative MAPK kinase kinase (MAPKKK) gene in rice (Oryza sativa). Two allelic dsm1 mutants were more sensitive than wild-type plants to drought stress at both seedling and panicle development stages. The dsm1 mutants lost water more rapidly than wild-type plants under drought stress, which was in agreement with the increased drought-sensitivity phenotype of the mutant plants. DSM1-RNA interference lines were also hypersensitive to drought stress. The predicted DSM1 protein belongs to a B3 subgroup of plant Raf-like MAPKKKs and was localized in the nucleus. By real-time PCR analysis, the DSM1 gene was induced by salt, drought, and abscisic acid, but not by cold. Microarray analysis revealed that two peroxidase (POX) genes, POX22.3 and POX8.1, were sharply down-regulated compared to wild type, suggesting that DSM1 may be involved in reactive oxygen species (ROS) signaling. Peroxidase activity, electrolyte leakage, chlorophyll content, and 3,3'-diaminobenzidine staining revealed that the dsm1 mutant was more sensitive to oxidative stress due to an increase in ROS damage caused by the reduced POX activity. Overexpression of DSM1 in rice increased the tolerance to dehydration stress at the seedling stage. Together, these results suggest that DSM1 might be a novel MAPKKK functioning as an early signaling component in regulating responses to drought stress by regulating scavenging of ROS in rice. DSM1 Characterization of the beta-carotene hydroxylase gene DSM2 conferring drought and oxidative stress resistance by increasing xanthophylls and abscisic acid synthesis in rice 2010 Plant Physiol National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan, China. Drought is a major limiting factor for crop production. To identify critical genes for drought resistance in rice (Oryza sativa), we screened T-DNA mutants and identified a drought-hypersensitive mutant, dsm2. The mutant phenotype was caused by a T-DNA insertion in a gene encoding a putative beta-carotene hydroxylase (BCH). BCH is predicted for the biosynthesis of zeaxanthin, a carotenoid precursor of abscisic acid (ABA). The amounts of zeaxanthin and ABA were significantly reduced in two allelic dsm2 mutants after drought stress compared with the wild type. Under drought stress conditions, the mutant leaves lost water faster than the wild type and the photosynthesis rate, biomass, and grain yield were significantly reduced, whereas malondialdehyde level and stomata aperture were increased in the mutant. The mutant is also hypersensitive to oxidative stresses. The mutant had significantly lower maximal efficiency of photosystem II photochemistry and nonphotochemical quenching capacity than the wild type, indicating photoinhibition in photosystem II and decreased capacity for eliminating excess energy by thermal dissipation. Overexpression of DSM2 in rice resulted in significantly increased resistance to drought and oxidative stresses and increases of the xanthophylls and nonphotochemical quenching. Some stress-related ABA-responsive genes were up-regulated in the overexpression line. DSM2 is a chloroplast protein, and the response of DSM2 to environmental stimuli is distinctive from the other two BCH members in rice. We conclude that the DSM2 gene significantly contributes to control of the xanthophyll cycle and ABA synthesis, both of which play critical roles in the establishment of drought resistance in rice. DSM2 Characterization of an inositol 1,3,4-trisphosphate 5/6-kinase gene that is essential for drought and salt stress responses in rice 2011 Plant Mol Biol National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, 430070 Wuhan, China. Drought and salt stresses are major limiting factors for crop production. To identify critical genes for stress resistance in rice (Oryza sativa L.), we screened T-DNA mutants and identified a drought- and salt-hypersensitive mutant dsm3. The mutant phenotype was caused by a T-DNA insertion in a gene encoding a putative inositol 1,3,4-trisphosphate 5/6-kinase previously named OsITPK2 with unknown function. Under drought stress conditions, the mutant had significantly less accumulation of osmolytes such as proline and soluble sugar and showed significantly reduced root volume, spikelet fertility, biomass, and grain yield; however, malondialdehyde level was increased in the mutant. Interestingly, overexpression of DSM3 (OsITPK2) in rice resulted in drought- and salt-hypersensitive phenotypes and physiological changes similar to those in the mutant. Inositol trisphosphate (IP3) level was decreased in the overexpressors under normal condition and drought stress. A few genes related to osmotic adjustment and reactive oxygen species scavenging were down-regulated in the mutant and overexpression lines. The expression level of DSM3 promoter-driven beta-glucuronidase (GUS) reporter gene in rice was induced by drought, salt and abscisic acid. Protoplast transient expression assay indicated that DSM3 is an endoplasmic reticulum protein. Sequence analysis revealed six putative ITPKs in rice. Transcript level analysis of OsITPK genes revealed that they had different tempo-spatial expression patterns, and the responses of DSM3 to abiotic stresses, including drought, salinity, cold, and high temperature, were distinct from the other five members in rice. These results together suggest that DSM3/OsITPK2 is an important member of the OsITPK family for stress responses, and an optimal expression level is essential for drought and salt tolerance in rice. DSM3|OsITPK2 The SNAC1-targeted gene OsSRO1c modulates stomatal closure and oxidative stress tolerance by regulating hydrogen peroxide in rice 2013 J Exp Bot National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, 430070, China. Abiotic stresses such as drought cause a reduction of plant growth and loss of crop yield. Stomatal aperture controls CO(2) uptake and water loss to the atmosphere, thus playing important roles in both the yield gain and drought tolerance of crops. Here, a rice homologue of SRO (similar to RCD one), termed OsSRO1c, was identified as a direct target gene of SNAC1 (stress-responsive NAC 1) involved in the regulation of stomatal aperture and oxidative response. SNAC1 could bind to the promoter of OsSRO1c and activate the expression of OsSRO1c. OsSRO1c was induced in guard cells by drought stress. The loss-of-function mutant of OsSRO1c showed increased stomatal aperture and sensitivity to drought, and faster water loss compared with the wild-type plant, whereas OsSRO1c overexpression led to decreased stomatal aperture and reduced water loss. Interestingly, OsSRO1c-overexpressing rice showed increased sensitivity to oxidative stress. Expression of DST, a reported zinc finger gene negatively regulating H(2)O(2)-induced stomatal closure, and the activity of H(2)O(2)-scavenging related enzymes were significantly suppressed, and H(2)O(2) in guard cells was accumulated in the overexpression lines. OsSRO1c interacted with various stress-related regulatory and functional proteins, and some of the OsSRO1c-interacting proteins are predicted to be involved in the control of stomatal aperture and oxidative stress tolerance. The results suggest that OsSRO1c has dual roles in drought and oxidative stress tolerance of rice by promoting stomatal closure and H(2)O(2) accumulation through a novel pathway involving regulators SNAC1 and DST. DST,OsSRO1c,OsNAC19|SNAC1|OsNAC9 A previously unknown zinc finger protein, DST, regulates drought and salt tolerance in rice via stomatal aperture control 2009 Genes Dev National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. Abiotic stresses, such as drought and salinity, lead to crop growth damage and a decrease in crop yields. Stomata control CO(2) uptake and optimize water use efficiency, thereby playing crucial roles in abiotic stress tolerance. Hydrogen peroxide (H(2)O(2)) is an important signal molecule that induces stomatal closure. However, the molecular pathway that regulates the H(2)O(2) level in guard cells remains largely unknown. Here, we clone and characterize DST (drought and salt tolerance)-a previously unknown zinc finger transcription factor that negatively regulates stomatal closure by direct modulation of genes related to H(2)O(2) homeostasis-and identify a novel pathway for the signal transduction of DST-mediated H(2)O(2)-induced stomatal closure. Loss of DST function increases stomatal closure and reduces stomatal density, consequently resulting in enhanced drought and salt tolerance in rice. These findings provide an interesting insight into the mechanism of stomata-regulated abiotic stress tolerance, and an important genetic engineering approach for improving abiotic stress tolerance in crops. DST Rice zinc finger protein DST enhances grain production through controlling Gn1a/OsCKX2 expression 2013 Proc Natl Acad Sci U S A State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. The phytohormone cytokinin (CK) positively regulates the activity and function of the shoot apical meristem (SAM), which is a major parameter determining seed production. The rice (Oryza sativa L.) Gn1a/OsCKX2 (Grain number 1a/Cytokinin oxidase 2) gene, which encodes a cytokinin oxidase, has been identified as a major quantitative trait locus contributing to grain number improvement in rice breeding practice. However, the molecular mechanism of how the expression of OsCKX2 is regulated in planta remains elusive. Here, we report that the zinc finger transcription factor DROUGHT AND SALT TOLERANCE (DST) directly regulates OsCKX2 expression in the reproductive meristem. DST-directed expression of OsCKX2 regulates CK accumulation in the SAM and, therefore, controls the number of the reproductive organs. We identify that DST(reg1), a semidominant allele of the DST gene, perturbs DST-directed regulation of OsCKX2 expression and elevates CK levels in the reproductive SAM, leading to increased meristem activity, enhanced panicle branching, and a consequent increase of grain number. Importantly, the DST(reg1) allele provides an approach to pyramid the Gn1a-dependent and Gn1a-independent effects on grain production. Our study reveals that, as a unique regulator of reproductive meristem activity, DST may be explored to facilitate the genetic enhancement of grain production in rice and other small grain cereals. DST,Gn1a|OsCKX2 Association of functional nucleotide polymorphisms at DTH2 with the northward expansion of rice cultivation in Asia 2013 Proc Natl Acad Sci U S A National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, China. Flowering time (i.e., heading date in crops) is an important ecological trait that determines growing seasons and regional adaptability of plants to specific natural environments. Rice (Oryza sativa L.) is a short-day plant that originated in the tropics. Increasing evidence suggests that the northward expansion of cultivated rice was accompanied by human selection of the heading date under noninductive long-day (LD) conditions. We report here the molecular cloning and characterization of DTH2 (for Days to heading on chromosome 2), a minor-effect quantitative trait locus that promotes heading under LD conditions. We show that DTH2 encodes a CONSTANS-like protein that promotes heading by inducing the florigen genes Heading date 3a and RICE FLOWERING LOCUS T 1, and it acts independently of the known floral integrators Heading date 1 and Early heading date 1. Moreover, association analysis and transgenic experiments identified two functional nucleotide polymorphisms in DTH2 that correlated with early heading and increased reproductive fitness under natural LD conditions in northern Asia. Our combined population genetics and network analyses suggest that DTH2 likely represents a target of human selection for adaptation to LD conditions during rice domestication and/or improvement, demonstrating an important role of minor-effect quantitative trait loci in crop adaptation and breeding. DTH2,Hd3a,RFT1 Genetic interactions involved in the inhibition of heading by heading date QTL, Hd2 in rice under long-day conditions 2011 Theor Appl Genet QTL Genomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. Heading date is the one of the most important traits in rice breeding, because it defines where rice can be cultivated and influences the expression of various agronomic traits. To examine the inhibition of heading by Heading date 2 (Hd2), previously detected on the distal end of chromosome 7's long arm by quantitative trait locus (QTL) analysis, we developed backcross inbred lines (BILs) from Koshihikari, a leading Japanese cultivar, and Hayamasari, an extremely early heading cultivar. The BILs were cultivated under natural field conditions in Tsukuba Japan, and under long-day (14.5 h), extremely long-day (18 h), and short-day (10 h) conditions. Combinations of several QTLs near Hd1, Hd2, Ghd7, Hd5, and Hd16 were detected under these four conditions. Analysis of advanced backcross progenies revealed genetic interactions between Hd2 and Hd16 and between Hd2 and Ghd7. In the homozygous Koshihikari genetic background at Hd16, inhibition of heading by the Koshihikari allele at Hd2 was smaller than that with the Hayamasari Hd16 allele. Similarly, in the homozygous Koshihikari genetic background at Ghd7, the difference in heading date caused by different alleles at Hd2 was smaller than in plants homozygous for the Hayamasari Ghd7 allele. Based on these results, we conclude that Hd2 and its genetic interactions play an important role in controlling heading under long-day conditions. In addition, QTLs near Hd2, Hd16, and Ghd7, which are involved in inhibition of heading under long-day conditions, function in the same pathway that controls heading date. Hd5|DTH8|Ghd8|OsHAP3H|LHD1,Ghd7,Hd1,CKI|EL1|Hd16 Roles of the Hd5 gene controlling heading date for adaptation to the northern limits of rice cultivation 2013 Theor Appl Genet Agricultural Research Institute, HOKUREN Federation of Agricultural Cooperatives, Naganuma, Hokkaido, 067-1317, Japan. kfujino@affrc.go.jp During the diversification of cultivated rice after domestication, rice was grown in diverse geographic regions using genetic variations attributed to the combination of alleles in loci for adaptability to various environmental conditions. To elucidate the key gene for adaptation in rice cultivars to the northern limit of rice cultivation, we conducted genetic analyses of heading date using extremely early-heading cultivars. The Hd5 gene controlling heading date (flowering time) generated variations in heading date among cultivars adapted to Hokkaido, where is the northernmost region of Japan and one of the northern limits of rice cultivation in the world. The association of the Hd5 genotype with heading date and genetical analysis clearly showed that the loss-of-function Hd5 has an important role in exhibiting earlier heading among a local population in Hokkaido. Distinct distribution of the loss-of-function Hd5 revealed that this mutation event of the 19-bp deletion occurred in a local landrace Bouzu and that this mutation may have been selected as an early-heading variety in rice breeding programs in Hokkaido in the early 1900s. The loss-of-function Hd5 was then introduced into the rice variety Fanny from France and contributed to its extremely early heading under the presence of functional Ghd7. These results demonstrated that Hd5 plays roles not only in generating early heading in variations of heading date among a local population in Hokkaido, but also in extremely early heading for adaptation to northern limits of rice cultivation. Hd5|DTH8|Ghd8|OsHAP3H|LHD1,Ghd7 Identification, characterization and interaction of HAP family genes in rice 2008 Mol Genet Genomics Genetic Strains Research Center, National Institute of Genetics, Shizuoka-ken, Japan. A HAP complex, which consists of three subunits, namely HAP2 (also called NF-YA or CBF-B), HAP3 (NF-YB/CBF-A) and HAP5 (NF-YC/CBF-C), binds to CCAAT sequences in a promoter to control the expression of target genes. We identified 10 HAP2 genes, 11 HAP3 genes and 7 HAP5 genes in the rice genome. All the three HAP family genes encode a protein with a conserved domain in each family and various non-conserved regions in both length and amino acid sequence. These genes showed various expression patterns depending on genes, and various combinations of overlapped expression of the HAP2, HAP3 and HAP5 genes were observed. Furthermore, protein interaction analyses showed interaction of OsHAP3A, a ubiquitously expressed HAP3 subunit of rice, with specific members of HAP5. These results indicate that the formation of specific complex with various HAP subunits combinations can be achieved by both tissue specific expression of three subunit genes and specific interaction of three subunit proteins. This may suggest that the HAP complexes may control various aspects of rice growth and development through tissue specific expression and complex formation of three subunit members. Hd5|DTH8|Ghd8|OsHAP3H|LHD1,OsHAP3A,OsHAP3B,OsHAP3C,OsHAP3E,OsNF-YB1 LHD1, an allele of DTH8/Ghd8, controls late heading date in common wild rice (Oryza rufipogon) 2012 J Integr Plant Biol State Key Laboratory of Plant Physiology and Biochemistry, National Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China. Flowering at suitable time is very important for plants to adapt to complicated environments and produce their seeds successfully for reproduction. In rice (Oryza rufipogon Griff.) photoperiod regulation is one of the important factors for controlling heading date. Common wild rice, the ancestor of cultivated rice, exhibits a late heading date and a more sensitive photoperiodic response than cultivated rice. Here, through map-based cloning, we identified a major quantitative trait loci (QTL) LHD1 (Late Heading Date 1), an allele of DTH8/Ghd8, which controls the late heading date of wild rice and encodes a putative HAP3/NF-YB/CBF-A subunit of the CCAAT-box-binding transcription factor. Sequence analysis revealed that several variants in the coding region of LHD1 were correlated with a late heading date, and a further complementary study successfully rescued the phenotype. These results suggest that a functional site for LHD1 could be among those variants present in the coding region. We also found that LHD1 could down-regulate the expression of several floral transition activators such as Ehd1, Hd3a and RFT1 under long-day conditions, but not under short-day conditions. This indicates that LHD1 may delay flowering by repressing the expression of Ehd1, Hd3a and RFT1 under long-day conditions. Hd5|DTH8|Ghd8|OsHAP3H|LHD1,Ehd1,Hd3a,RFT1 A major QTL, Ghd8, plays pleiotropic roles in regulating grain productivity, plant height, and heading date in rice 2011 Mol Plant National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Rice yield and heading date are two distinct traits controlled by quantitative trait loci (QTLs). The dissection of molecular mechanisms underlying rice yield traits is important for developing high-yielding rice varieties. Here, we report the cloning and characterization of Ghd8, a major QTL with pleiotropic effects on grain yield, heading date, and plant height. Two sets of near isogenic line populations were developed for the cloning of Ghd8. Ghd8 was narrowed down to a 20-kb region containing two putative genes, of which one encodes the OsHAP3 subunit of a CCAAT-box binding protein (HAP complex); this gene was regarded as the Ghd8 candidate. A complementary test confirmed the identity and pleiotropic effects of the gene; interestingly, the genetic effect of Ghd8 was dependent on its genetic background. By regulating Ehd1, RFT1, and Hd3a, Ghd8 delayed flowering under long-day conditions, but promoted flowering under short-day conditions. Ghd8 up-regulated MOC1, a key gene controlling tillering and branching; this increased the number of tillers, primary and secondary branches, thus producing 50% more grains per plant. The ectopic expression of Ghd8 in Arabidopsis caused early flowering by 10 d-a situation similar to the one observed by its homolog AtHAP3b, when compared to wild-type under long-day conditions; these findings indicate the conserved function of Ghd8 and AtHAP3b in flowering in Arabidopsis. Our results demonstrated the important roles of Ghd8 in rice yield formation and flowering, as well as its opposite functions in flowering between rice and Arabidopsis under long-day conditions. Hd5|DTH8|Ghd8|OsHAP3H|LHD1,Ehd1,Hd3a,MOC1,RFT1 DTH8 suppresses flowering in rice, influencing plant height and yield potential simultaneously 2010 Plant Physiol State Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Provincial Center of Plant Gene Engineering, Nanjing Agricultural University, Nanjing 210095, China. The three most important agronomic traits of rice (Oryza sativa), yield, plant height, and flowering time, are controlled by many quantitative trait loci (QTLs). In this study, a newly identified QTL, DTH8 (QTL for days to heading on chromosome 8), was found to regulate these three traits in rice. Map-based cloning reveals that DTH8 encodes a putative HAP3 subunit of the CCAAT-box-binding transcription factor and the complementary experiment increased significantly days to heading, plant height, and number of grains per panicle in CSSL61 (a chromosome segment substitution line that carries the nonfunctional DTH8 allele) with the Asominori functional DTH8 allele under long-day conditions. DTH8 is expressed in most tissues and its protein is localized to the nucleus exclusively. The quantitative real-time PCR assay revealed that DTH8 could down-regulate the transcriptions of Ehd1 (for Early heading date1) and Hd3a (for Heading date3a; a rice ortholog of FLOWERING LOCUS T) under long-day conditions. Ehd1 and Hd3a can also be down-regulated by the photoperiodic flowering genes Ghd7 and Hd1 (a rice ortholog of CONSTANS). Meanwhile, the transcription of DTH8 has been proved to be independent of Ghd7 and Hd1, and the natural mutation of this gene caused weak photoperiod sensitivity and shorter plant height. Taken together, these data indicate that DTH8 probably plays an important role in the signal network of photoperiodic flowering as a novel suppressor as well as in the regulation of plant height and yield potential. Hd5|DTH8|Ghd8|OsHAP3H|LHD1,Ehd1,Ghd7,Hd1,Hd3a Hd16, a gene for casein kinase I, is involved in the control of rice flowering time by modulating the day-length response 2013 Plant J National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. The alteration of photoperiod sensitivity has let breeders diversify flowering time in Oryza sativa (rice) and develop cultivars adjusted to a range of growing season periods. Map-based cloning revealed that the rice flowering-time quantitative trait locus (QTL) Heading date 16 (Hd16) encodes a casein kinase-I protein. One non-synonymous substitution in Hd16 resulted in decreased photoperiod sensitivity in rice, and this substitution occurred naturally in an old rice cultivar. By using near-isogenic lines with functional or deficient alleles of several rice flowering-time genes, we observed significant digenetic interactions between Hd16 and four other flowering-time genes (Ghd7, Hd1, DTH8 and Hd2). In a near-isogenic line with the weak-photoperiod-sensitivity allele of Hd16, transcription levels of Ehd1, Hd3a, and RFT1 increased under long-day conditions, and transcription levels of Hd3a and RFT1 decreased under short-day conditions. Expression analysis under continuous light and dark conditions showed that Hd16 was not likely to be associated with circadian clock regulation. Biochemical characterization indicated that the functional Hd16 recombinant protein specifically phosphorylated Ghd7. These results demonstrate that Hd16 acts as an inhibitor in the rice flowering pathway by enhancing the photoperiod response as a result of the phosphorylation of Ghd7. Hd5|DTH8|Ghd8|OsHAP3H|LHD1,Ehd1,Ghd7,Hd1,CKI|EL1|Hd16,Hd3a,RFT1 Quantitative trait loci for panicle size, heading date and plant height co-segregating in trait-performance derived near-isogenic lines of rice (Oryza sativa) 2006 Theor Appl Genet National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Near-isogenic lines (NILs) are ideal materials for precise estimation of quantitative trait loci (QTL) effects and map-based gene isolation. With the completion of the rice genome sequence, QTL isolation based on NILs is becoming a routine. In this study, a trait-performance derived NIL strategy was adopted to develop NILs. Two plants were identified within one inbred line of recombinant inbred lines (RILs, F(7) generation), exhibiting a significant difference in panicle size. By marker screening of the whole genome the genetic background of the two plants was estimated to be 98.7% identical. These two plants were selected as parents to produce a near-isogenic F(2) (NIL-F(2)) population, consisting of 125 individuals, in which spikelets per panicle (SPP), grains per panicle (GPP), heading date (HD) and plant height (PH) were recorded. These four traits expressed discontinuous or bimodal distribution in the NIL-F(2) population and followed the expected segregation ratios for a single Mendelian factor by progeny tests. A partial dominant QTL for the four traits was mapped to the same interval flanked by RM310 and RM126 on chromosome 8. The QTL region explained 83.0, 80.2, 94.9 and 93.8% of trait variation of SPP, GPP, HD and PH in the progenies, respectively. Progeny tests also confirmed co-segregation of QTL for the four traits, tall plants consistently flowering late and carrying large panicles. Different NILs development strategies are discussed. Hd5|DTH8|Ghd8|OsHAP3H|LHD1 The rice gene DEFECTIVE TAPETUM AND MEIOCYTES 1 (DTM1) is required for early tapetum development and meiosis 2012 Plant J Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Korea. Tapetum development and meiosis play crucial roles in anther development. Here we identified a rice gene, DEFECTIVE TAPETUM AND MEIOCYTES 1 (DTM1), which controls the early stages of that development. This gene encodes for an endoplasmic reticulum (ER) membrane protein that is present only in cereals. Our T-DNA insertion mutations gave rise to abnormal tapetal formation. Cellular organelles, especially the ER, were underdeveloped, which led to hampered differentiation and degeneration of the tapetum. In addition, the development of pollen mother cells was arrested at the early stages of meiotic prophase I. RNA in-situ hybridization analyses showed that DTM1 transcripts were most abundant in tapetal cells at stages 6 and 7, and moderately in the pollen mother cells and meiocytes. Transcripts of UDT1, which functions in tapetum development during early meiosis, were reduced in dtm1 anthers, as were those of PAIR1, which is involved in chromosome pairing and synapsis during meiosis. However, expression of MSP1 and MEL1, which function in anther wall specification and germ cell division, respectively, was not altered in the dtm1 mutant. Moreover, transcripts of DTM1 were reduced in msp1 mutant anthers, but not in udt1 and pair1 mutants. These results, together with their mutant phenotypes, suggest that DTM1 plays important roles in the ER membrane during early tapetum development, functioning after MSP1 and before UDT1, and also in meiocyte development, after MEL1 and before PAIR1. DTM1,MEL1,OsMSP1,PAIR1,Udt1 Du1, encoding a novel Prp1 protein, regulates starch biosynthesis through affecting the splicing of Wxb pre-mRNAs in rice (Oryza sativa L.) 2007 Plant Mol Biol State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Starch is the major component of cereal grains. In rice, starch properties determine the eating and cooking quality. The dull endosperm of rice grains is a classical morphological and agronomical trait that has long been exploited for breeding and genetics study. To understand the molecular mechanism that regulates the starch biosynthesis in rice grains, we characterized a classic rice mutant dull endosperm1 (du1) and isolated Du1 through a map-based cloning approach. Du1, encoding a member of pre-mRNA processing (Prp1) family, is expressed mainly in panicles. Du1 specifically affects the splicing efficiency of Wx(b) and regulates starch biosynthesis by mediating the expression of starch biosynthesis genes. Analysis of du1wx shows that Du1 acts upstream of Wx(b). These results strongly suggest that Du1 may function as a regulator of the starch biosynthesis by affecting the splicing of Wx(b) and the expression of other genes involved in the rice starch biosynthetic pathways. Du1,Wx Putative megaenzyme DWA1 plays essential roles in drought resistance by regulating stress-induced wax deposition in rice 2013 Proc Natl Acad Sci U S A National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Drought stress is a major limiting factor for crop production. Cuticular wax plays an important role in preventing water loss from drought stress. However, the genetic control of cuticular wax deposition under drought stress conditions has not been characterized. Here, we identified a rice gene Drought-Induced Wax Accumulation 1 (DWA1) encoding a very large protein (2,391 aa in length) containing multiple enzymatic structures, including an oxidoreductase-like domain; a prokaryotic nonribosomal peptide synthetase-like module, including an AMP-binding domain; and an allene oxide synthase-like domain. This previously unreported putative megaenzyme is conserved in vascular plants. A dwa1 KO mutant was highly sensitive to drought stress relative to the WT. DWA1 was preferentially expressed in vascular tissues and epidermal layers and strongly induced by drought stress. The dwa1 mutant was impaired in cuticular wax accumulation under drought stress, which significantly altered the cuticular wax composition of the plant, resulting in increased drought sensitivity. The mutant had reduced levels of very-long-chain fatty acids, and plants overexpressing DWA1 showed elevated levels of very-long-chain fatty acids relative to the WT. The expression of many wax-related genes was significantly suppressed in dwa1 under drought conditions. The AMP-binding domain exhibited in vitro enzymatic activity in activating long-chain fatty acids to form acyl-CoA. Our results suggest that DWA1 controls drought resistance by regulating drought-induced cuticular wax deposition in rice. This finding may have significant implications for improving the drought resistance of crop varieties. DWA1 OsGSR1 is involved in crosstalk between gibberellins and brassinosteroids in rice 2009 Plant J Research Center for Molecular & Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. Gibberellins (GAs) and brassinosteroids (BRs), two growth-promoting phytohormones, regulate many common physiological processes. Their interactions at the molecular level remain unclear. Here, we demonstrate that OsGSR1, a member of the GAST (GA-stimulated transcript) gene family, is induced by GA and repressed by BR. RNA interference (RNAi) transgenic rice plants with reduced OsGSR1 expression show phenotypes similar to plants deficient in BR, including short primary roots, erect leaves and reduced fertility. The OsGSR1 RNAi transgenic rice shows a reduced level of endogenous BR, and the dwarf phenotype could be rescued by the application of brassinolide. The yeast two-hybrid assay revealed that OsGSR1 interacts with DIM/DWF1, an enzyme that catalyzes the conversion from 24-methylenecholesterol to campesterol in BR biosynthesis. These results suggest that OsGSR1 activates BR synthesis by directly regulating a BR biosynthetic enzyme at the post-translational level. Furthermore, OsGSR1 RNAi plants show a reduced sensitivity to GA treatment, an increased expression of the GA biosynthetic gene OsGA20ox2, which is feedback inhibited by GA signaling, and an elevated level of endogenous GA: together, these suggest that OsGSR1 is a positive regulator of GA signaling. These results demonstrate that OsGSR1 plays important roles in both BR and GA pathways, and also mediates an interaction between the two signaling pathways. OsGSR1,sd1|GA20ox2 Dwarf Tiller1, a Wuschel-related homeobox transcription factor, is required for tiller growth in rice 2014 PLoS Genet State Key Laboratory of Hybrid Rice, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China; Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China; Graduate University of the Chinese Academy of Sciences, Beijing, China. Unlike many wild grasses, domesticated rice cultivars have uniform culm height and panicle size among tillers and the main shoot, which is an important trait for grain yield. However, the genetic basis of this trait remains unknown. Here, we report that Dwarf Tiller1 (DWT1) controls the developmental uniformity of the main shoot and tillers in rice (Oryza sativa). Most dwt1 mutant plants develop main shoots with normal height and larger panicles, but dwarf tillers bearing smaller panicles compared with those of the wild type. In addition, dwt1 tillers have shorter internodes with fewer and un-elongated cells compared with the wild type, indicating that DWT1 affects cell division and cell elongation. Map-based cloning revealed that DWT1 encodes a Wuschel-related homeobox (WOX) transcription factor homologous to the Arabidopsis WOX8 and WOX9. The DWT1 gene is highly expressed in young panicles, but undetectable in the internodes, suggesting that DWT1 expression is spatially or temporally separated from its effect on the internode growth. Transcriptomic analysis revealed altered expression of genes involved in cell division and cell elongation, cytokinin/gibberellin homeostasis and signaling in dwt1 shorter internodes. Moreover, the non-elongating internodes of dwt1 are insensitive to exogenous gibberellin (GA) treatment, and some of the slender rice1 (slr1) dwt1 double mutant exhibits defective internodes similar to the dwt1 single mutant, suggesting that the DWT1 activity in the internode elongation is directly or indirectly associated with GA signaling. This study reveals a genetic pathway synchronizing the development of tillers and the main shoot, and a new function of WOX genes in balancing branch growth in rice. DWT1 Fine Mapping and Analysis of DWARF TILLER1 in Controlling Rice Architecture 2013 Journal of Genetics and Genomics Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China Rice is one of the most consumed staple food plants around the world, and its plant architecture is very important to improve the grain yield (Zhang et al., 2008). Plant height, leaf angle, tiller number and angle, and uniformity of panicle layer all can have strong effects on grain yield (Wang and Li, 2008). During the long history of domestication, rice has been selected to develop uniform tiller height architecture that ensures panicle layer uniformity and ease of harvesting (Ma et al., 2009), and is largely determined by the synchronic culm elongation. A large number of dwarf mutants deficient in culm elongation have been isolated and characterized, and gibberellin (GA) and brassinosteroid (BR) have been demonstrated as two major factors in promoting the culm development (Peng et al., 1997; Ikeda et al., 2001; Ashikari et al., 2002; Itoh et al., 2002; Spielmeyer et al., 2002). However, the mechanism of the coordinated elongation between the main shoot and tillers remains unknown. Here, we report a DWARF TILLER1 (DWT1) locus involved in the regulation of the uniform growth of main shoot and tillers in rice. DWT1 EAT1 promotes tapetal cell death by regulating aspartic proteases during male reproductive development in rice 2013 Nat Commun State Key Laboratory of Hybrid Rice, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China. Programmed cell death is essential for the development of multicellular organisms, yet pathways of plant programmed cell death and its regulation remain elusive. Here we report that ETERNAL TAPETUM 1, a basic helix-loop-helix transcription factor conserved in land plants, positively regulates programmed cell death in tapetal cells in rice anthers. eat1 exhibits delayed tapetal cell death and aborted pollen formation. ETERNAL TAPETUM 1 directly regulates the expression of OsAP25 and OsAP37, which encode aspartic proteases that induce programmed cell death in both yeast and plants. Expression and genetic analyses revealed that ETERNAL TAPETUM 1 acts downstream of TAPETUM DEGENERATION RETARDATION, another positive regulator of tapetal programmed cell death, and that ETERNAL TAPETUM 1 can also interact with the TAPETUM DEGENERATION RETARDATION protein. This study demonstrates that ETERNAL TAPETUM 1 promotes aspartic proteases triggering plant programmed cell death, and reveals a dynamic regulatory cascade in male reproductive development in rice. DTD|EAT1,OsAP25,OsAP37,OsAP19 A novel rice bHLH transcription factor, DTD, acts coordinately with TDR in controlling tapetum function and pollen development 2013 Mol Plant State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. None DTD|EAT1 Phytochrome-regulated EBL1 contributes to ACO1 upregulation in rice 2011 Biotechnol Lett Division of Plant Sciences, Photobiology and Photosynthesis Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8602, Japan. iwamas@nias.affrc.go.jp The 1-aminocyclopropane-1-carboxylate oxidase gene (ACO1) was upregulated in rice (Oryza sativa L.) phyAphyBphyC mutants lacking any phytochrome and containing the GCC box element, a binding site for rice ethylene-responsive element binding protein 1 (OsEREBP1), in its promoter region. Since the OsEREBP1-like gene EBL1 (OsEREBP1-LIKE 1) was significantly downregulated in phyAphyBphyC mutants, EBL1 was suspected to repress ACO1 expression in wild-type plants. However, ACO1 was downregulated in EBL1 RNA interference plants, and the total length of these plants was slightly shorter than that of wild-type plants. This study shows that EBL1 is positively regulated by phytochrome B and associated with ACO1 upregulation. EBL1,OsACO1,OsEREBP1 A novel variant of translation elongation factor-1beta: isolation and characterization of the rice gene encoding EF-1beta2 1998 Biochim Biophys Acta Cryobiosystem Research Center, Faculty of Agriculture, Iwate University, Ueda, Morioka, Iwate 020-8550, Japan. A rice gene encoding a novel isoform of translation elongation factor-1beta subunit (termed EF-1beta2) was isolated and characterized. The gene comprises of eight exons, and encodes a 226-amino-acid protein. Expression of EF-1beta2 mRNA is abundant in seeds and cultured cells, but is considerably low in the tissues of the rice seedling. Antiserum raised against an EF-1beta2 synthetic peptide detected a protein with a relative molecular mass of about 32 kDa, indicating the EF-1beta2 gene is actually expressed in rice tissues. EF-1beta2 showed a close similarity to the cognate subunits from plant (beta and beta'). EF-1beta2 A putative lipase gene EXTRA GLUME1 regulates both empty-glume fate and spikelet development in rice 2009 Plant J The State Key Laboratory of Rice Biology, College of Life Sciences, Zhejiang University, Hangzhou, 310029 China. Recent studies have shown that molecular control of inner floral organ identity appears to be largely conserved between monocots and dicots, but little is known regarding the molecular mechanism underlying development of the monocot outer floral organ, a unique floral structure in grasses. In this study, we report the cloning of the rice EXTRA GLUME1 (EG1) gene, a putative lipase gene that specifies empty-glume fate and floral meristem determinacy. In addition to affecting the identity and number of empty glumes, mutations in EG1 caused ectopic floral organs to be formed at each organ whorl or in extra ectopic whorls. Iterative glume-like structures or new floral organ primordia were formed in the presumptive region of the carpel, resulting in an indeterminate floral meristem. EG1 is expressed strongly in inflorescence primordia and weakly in developing floral primordia. We also found that the floral meristem and organ identity gene OsLHS1 showed altered expression with respect to both pattern and levels in the eg1 mutant, and is probably responsible for the pleiotropic floral defects in eg1. As a putative class III lipase that functionally differs from any known plant lipase, EG1 reveals a novel pathway that regulates rice empty-glume fate and spikelet development. EG1,OsMADS1|LHS1|AFO Ehd1, a B-type response regulator in rice, confers short-day promotion of flowering and controls FT-like gene expression independently of Hd1 2004 Genes Dev Plant Breeding Laboratory, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan. kdoi@agr.kyushu-u.ac.jp Two evolutionarily distant plant species, rice (Oryza sativa L.), a short-day (SD) plant, and Arabidopsis thaliana, a long-day plant, share a conserved genetic network controlling photoperiodic flowering. The orthologous floral regulators-rice Heading date 1 (Hd1) and Arabidopsis CONSTANS (CO)-integrate circadian clock and external light signals into mRNA expression of the FLOWERING LOCUS T (FT) group floral inducer. Here, we report that the rice Early heading date 1 (Ehd1) gene, which confers SD promotion of flowering in the absence of a functional allele of Hd1, encodes a B-type response regulator that might not have an ortholog in the Arabidopsis genome. Ehd1 mRNA was induced by 1-wk SD treatment, and Ehd1 may promote flowering by inducing FT-like gene expression only under SD conditions. Microarray analysis further revealed a few MADS box genes downstream of Ehd1. Our results indicate that a novel two-component signaling cascade is integrated into the conserved pathway in the photoperiodic control of flowering in rice. Ehd1 Knockdown of SAMS genes encoding S-adenosyl-l-methionine synthetases causes methylation alterations of DNAs and histones and leads to late flowering in rice 2011 J Plant Physiol Research Center for Molecular and Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. S-Adenosyl-l-methionine synthetase (SAMS) [EC 2.5.1.6] catalyzes to produce SAM (S-adenosyl-l-methionine), a universal methyl group donor in biochemical reactions in cells. However, less is known how SAMS controls plant development. Here, we demonstrate that OsSAMS1, 2 and 3 are essential for histone H3K4me3 and DNA methylation to regulate gene expression related to flowering in Oryza sativa. RNA interference (RNAi) transgenic rice with downregulated transcripts of OsSAMS1, 2 and 3 showed pleiotropic phenotypes, including dwarfism, reduced fertility, delayed germination, as well as late flowering. Delayed germination was largely rescued by application of SAM in the knockdown lines. Knockdown of OsSAMS1, 2 and 3 led to distinguished late flowering and greatly reduced the expression of the flowering key genes, Early heading date 1 (Ehd1), Hd3a and RFT1 (rice FT-like genes). Moreover, the histone H3K4me3 and symmetric DNA methylation at these genes were greatly reduced. Thus, SAM deficiency suppressing DNA and H3K4me3 transmethylations at flowering key genes led to a late-flowering phenotype in rice. This information could help elucidate the mechanism of epigenetic control flowering transition. Ehd1,Hd3a,OsSAMS1,OsSAM2,OsSAM3,RFT1 Heading date gene, dth3 controlled late flowering in O. Glaberrima Steud. by down-regulating Ehd1 2011 Plant Cell Rep National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing, 210095, China. Heading date in rice is an important agronomic trait controlled by several genes. In this study, flowering time of variety Dianjingyou 1 (DJY1) was earlier than a near-isogenic line (named NIL) carried chromosome segment from African rice on chromosome 3S, when grown in both long-day (LD) and short-day (SD) conditions. By analyzing a large F2 population from NIL x DJY1, the locus DTH3 (QTL for days to heading on chromosome 3) controlling early heading date in DJY1 was fine mapped to a 64-kb segment which contained only one annotated gene, a MIKC-type MADS-box protein. We detected a 6-bp deletion and a single base substitution in the C-domain by sequencing DTH3 in DJY1 compared with dth3 in NIL, and overexpression of DTH3 caused early flowering in callus. Quantitative real-time PCR revealed that the transcript level of dth3 in NIL was lower than that DTH3 in DJY1 in both LD and SD conditions. The Early heading date 1 (Ehd1) which promotes the RFT1, was up-regulated by DTH3 in both LD and SD conditions. Based on Indel and dCAPs marker analysis, the dth3 allele was only present in African rice accessions. A phylogenetic analysis based on microsatellite genotyping suggested that African rice had a close genetic relationship to O. rufipogon and O. latifolia, and was similar to japonica cultivars. DTH3 affected flowering time and had no significant effect on the main agronomic traits. Ehd1,Hd1,OsMADS50|OsSOC1|DTH3,RFT1 OsCO3, a CONSTANS-LIKE gene, controls flowering by negatively regulating the expression of FT-like genes under SD conditions in rice 2008 Planta Plant Signaling Network Research Center, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, South Korea. The photoperiod is an important environmental stress that determines flowering time. The CONSTANS (CO) and Heading date 1 (Hd1) genes are known to be central integrators of the photoperiod pathway in Arabidopsis and rice, respectively. Although they are both members of the CONSTANS-LIKE (COL) family and have two B-boxes and a CCT domain, rice also possesses novel COL genes that are not found in Arabidopsis. Here, we demonstrate that a novel COL gene, OsCO3, containing a single B-box and a CCT domain, modulates photoperiodic flowering in rice. The circadian expression pattern of OsCO3 mRNA oscillated in a different phase from Hd1 and was similar to that of OsCO3 pre-mRNA, suggesting that the diurnal expression pattern of OsCO3 transcripts may be regulated at the transcriptional level. Overexpression of OsCO3 specifically caused late flowering under short day (SD) conditions relative to wild-type rice plants. The expression of Hd3a and FTL decreased in these transgenic plants, whereas the expression of Hd1, Early heading date 1 (Ehd1), OsMADS51, and OsMADS50 did not significantly change. Our results suggest that OsCO3 primarily controls flowering time under SD conditions by negatively regulating Hd3a and FTL expression, independent of the SD-promotion pathway. Ehd1,Hd1,Hd3a,OsCO3,OsMADS50|OsSOC1|DTH3,OsMADS51|OsMADS65 Functional characterization of rice OsDof12 2009 Planta State Key Laboratory of Plant Genomics and National Plant Gene Research Centre (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 5 Datun Road, Chaoyang District, 100101, Beijing, China. djli@genetics.ac.cn DNA-binding with one finger (Dof) proteins are a large family of transcription factors involved in a variety of biological processes in plants. In rice, 30 different Dof genes have been identified through genome analysis. Here we report the functional characteristics of a rice Dof gene, OsDof12, which encodes a predicted Dof protein. The nuclear localization of OsDof12 was investigated by the transient expression assays of the OsDof12-GFP fusion protein in onion epidermal cells. Trans-activation assays in a yeast one-hybrid system indicated that OsDof12 had transcriptional activity. RNA expression analyses showed that the expression of OsDof12 was not tissue-specific in general and fluctuated at different development stages in rice. In addition, OsDof12 was strongly inhibited by dark treatments. The transgenic lines overexpressing OsDof12 showed early flowering under long-day (LD) conditions, whereas OsDof12 overexpression had no effect on flowering time under short-day (SD) conditions. In transgenic lines overexpressing OsDof12, the transcription levels of Hd3a and OsMADS14 were up-regulated under LD conditions but not SD conditions, whereas the expression of Hd1, OsMADS51, Ehd1 and OsGI did not change under LD and SD conditions. These results suggested that OsDof12 might regulate flowering by controlling the expression of Hd3a and OsMADS14. Ehd1,Hd1,Hd3a,OsDof12,OsGI,OsMADS14,OsMADS51|OsMADS65 Epistasis among the three major flowering time genes in rice: coordinate changes of photoperiod sensitivity, basic vegetative growth and optimum photoperiod 2007 Euphytica Plant Breeding Laboratory, Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan Flowering time is affected not only by photoperiod sensitivity (PS) but also by basic vegetative growth (BVG) and optimum photoperiod (OP), although their developmental and genetic relationships are not well understood. The present study was carried out in rice to examine to what extent these three developmental components are modified by the three flowering time genes, Se1 (= Hd1), Ef1 and e1 (= m-Ef1), which are known to contribute to flowering time in temperate and tropical regions of rice cultivation. Photoperiodic response curves were estimated under controlled conditions of different growth regimes, using eight near-isogenic lines possessing different combinations of the alleles at the three loci. The results showed that each of the components is greatly affected by the main effect of the genes, temperature and their epistasis, indicating that none of the three genes controls flowering time by altering any single component in PS, BVG or OP. Epistasis was detected more frequently among the three genes than reported before, suggesting that epistasis contributes to flowering time by changing PS, BVG and OP differently. The comparison of the nucleotide sequences suggested that Ef1 is the same as Early heading date 1 (Ehd1). Since the two genes Se1 (= Hd1) and Ef1 (= Ehd1) are known to up-regulate the rice homolog of Arabidopsis FT, it is suggested that the detected epistasis may respond to diverse environments by modulating the CO/FT system conserved in flowering plants. Ehd1,Hd1 Molecular dissection of the roles of phytochrome in photoperiodic flowering in rice 2011 Plant Physiol Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8561, Japan. Phytochromes mediate the photoperiodic control of flowering in rice (Oryza sativa), a short-day plant. Recent molecular genetics studies have revealed a genetic network that enables the critical daylength response of florigen gene expression. Analyses using a rice phytochrome chromophore-deficient mutant, photoperiod sensitivity5, have so far revealed that within this network, phytochromes are required for expression of Grain number, plant height and heading date7 (Ghd7), a floral repressor gene in rice. There are three phytochrome genes in rice, but the roles of each phytochrome family member in daylength response have not previously been defined. Here, we revealed multiple action points for each phytochrome in the critical daylength response of florigen expression by using single and double phytochrome mutant lines of rice. Our results show that either phyA alone or a genetic combination of phyB and phyC can induce Ghd7 mRNA, whereas phyB alone causes some reduction in levels of Ghd7 mRNA. Moreover, phyB and phyA can affect Ghd7 activity and Early heading date1 (a floral inducer) activity in the network, respectively. Therefore, each phytochrome gene of rice has distinct roles, and all of the phytochrome actions coordinately control the critical daylength response of florigen expression in rice. Ehd1,Ghd7,Hd3a,OsCOL4,PHYA,PHYB|OsphyB,PHYC,RFT1 Ectopic expression of OsLFL1 in rice represses Ehd1 by binding on its promoter 2007 Biochem Biophys Res Commun National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China. B3 domain was identified as a novel DNA-binding motif specific to higher plant species. The B3 proteins play important roles in plant development. In the mutant W378, the mutant gene coding OsLFL1, a putative B3 transcription factor gene, was ectopically expressed. In this study, it was found that the flowering promoting gene Ehd1 and its putative downstream genes were all repressed by OsLFL1. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) analyses suggest that OsLFL1 binds to the RY cis-elements (CATGCATG) in the promoter of the Ehd1 gene. Thus, ectopically expressed OsLFL1 might repress Ehd1 via binding directly to the RY cis-elements in its promoter. Ehd1,OsLFL1 A gene network for long-day flowering activates RFT1 encoding a mobile flowering signal in rice 2009 Development Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology (NAIST), Takayama, Ikoma, Japan. Although some genes that encode sensory or regulatory elements for photoperiodic flowering are conserved between the long-day (LD) plant Arabidopsis thaliana and the short-day (SD) plant rice, the gene networks that control rice flowering, and particularly flowering under LD conditions, are not well understood. We show here that RICE FLOWERING LOCUS T 1 (RFT1), the closest homolog to Heading date 3a (Hd3a), is a major floral activator under LD conditions. An RFT1:GFP fusion protein localized in the shoot apical meristem (SAM) under LD conditions, suggesting that RFT1 is a florigen gene in rice. Furthermore, mutants in OsMADS50, a rice ortholog of Arabidopsis SUPPRESOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) did not flower up to 300 days after sowing under LD conditions, indicating that OsMADS50, which acts upstream of RFT1, promotes flowering under LD conditions. We propose that both positive (OsMADS50 and Ehd1) and negative (Hd1, phyB and Ghd7) regulators of RFT1 form a gene network that regulates LD flowering in rice. Among these regulators, Ehd1, a rice-specific floral inducer, integrates multiple pathways to regulate RFT1, leading to flowering under appropriate photoperiod conditions. A rice ortholog of Arabidopsis APETALA1, OsMADS14, was expressed in the floral meristem in wild-type but not in RFT1 RNAi plants, suggesting that OsMADS14 is activated by RFT1 protein in the SAM after the transition to flowering. We have thus exposed a network of genes that regulate LD flowering in rice. Ehd1,Ghd7,Hd1,OsMADS14,OsMADS50|OsSOC1|DTH3,PHYB|OsphyB,RFT1 OsMADS50 and OsMADS56 function antagonistically in regulating long day (LD)-dependent flowering in rice 2009 Plant Cell Environ Department of Life Science and Functional Genomic Center, Pohang University of Science and Technology (POSTECH), Pohang, Korea. In much of the tropics and subtropics, rice (Oryza sativa L.) is grown under long days (LDs). Therefore, LD must play a major role in inducing flowering signal in rice. However, little is known on LD-dependent flowering signal in the species. We previously reported that OsMADS50, which is highly homologous to Arabidopsis SOC1, functions as a positive regulator for flowering. However, its detailed photoperiodic mechanism was not yet elucidated. Here, we report the functional analysis of OsMADS50 and its closely related gene OsMADS56. Knock-out of OsMADS50 caused a late-flowering phenotype only under LD conditions. Overexpression of OsMADS56 (56OX) also resulted in delayed flowering under LD. In the osmads50 mutants and 56OX transgenic plants, transcripts of Ehd1, Hd3a and RFT1 were reduced, although that of OsLFL1 increased. On the other hand, mRNA levels of OsGI, Hd1, OsId1, OsDof12, Ghd7, Hd6 and SE5 were unchanged. These observations imply that OsMADS50 and OsMADS56 function antagonistically through OsLFL1-Ehd1 in regulating LD-dependent flowering. Yeast two-hybrid and co-immunoprecipitation analyses indicated an interaction between those two proteins as well as their formation of homodimers. These results suggest that OsMADS50 and OsMADS56 may form a complex that regulates downstream target genes. Ehd1,Ghd7,Hd1,Hd3a,Hd6|CK2,OsDof12,OsGI,OsLFL1,OsMADS50|OsSOC1|DTH3,OsMADS56,RFT1 Rice Indeterminate 1 (OsId1) is necessary for the expression of Ehd1 (Early heading date 1) regardless of photoperiod 2008 Plant J Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center (PMBBRC), Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Korea. Indeterminate 1 (Id1), a classical flowering gene first reported in 1946, is one of the earliest genes whose expression in leaf tissues affects the floral transition in the shoot meristem. How Id1 is integrated into the flowering process is largely unknown. In this study, we examined the genetic action of the rice (Oryza sativa) ortholog OsId1. In rice, OsId1 is preferentially expressed in young leaves, but the overall expression pattern is broader than that in maize (Zea mays). OsId1 is able to activate transcription in yeast. RNAi mutants show a delay in flowering under both short-day (SD) and long-day (LD) conditions. OsId1 regulates the expression of Ehd1 (Early heading date 1) and its downstream genes, including Hd3a (a rice ortholog of FT) and RFT1 (Rice Flowering Locus T1), under both SD and LD conditions. In rice, the expression of Ehd1 is also controlled by the photoperiodic flowering genes OsGI (a rice ortholog of GI) and OsMADS51. However, the expression of OsId1 is independent of OsGI, OsMADS51, and OsMADS50 (a rice SOC1 ortholog). This study demonstrates that the activation of Ehd1 by OsId1 is required for the promotion of flowering. Ehd1,OsMADS51|OsMADS65,RFT1 OsCOL4 is a constitutive flowering repressor upstream of Ehd1 and downstream of OsphyB 2010 Plant J Department of Life Science, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Korea. Plants recognize environmental factors to determine flowering time. CONSTANS (CO) plays a central role in the photoperiod flowering pathway of Arabidopsis, and CO protein stability is modulated by photoreceptors. In rice, Hd1, an ortholog of CO, acts as a flowering promoter, and phytochromes repress Hd1 expression. Here, we investigated the functioning of OsCOL4, a member of the CONSTANS-like (COL) family in rice. OsCOL4 null mutants flowered early under short or long days. In contrast, OsCOL4 activation-tagging mutants (OsCOL4-D) flowered late in either environment. Transcripts of Ehd1, Hd3a, and RFT1 were increased in the oscol4 mutants, but reduced in the OsCOL4-D mutants. This finding indicates that OsCOL4 is a constitutive repressor functioning upstream of Ehd1. By comparison, levels of Hd1, OsID1, OsMADS50, OsMADS51, and OsMADS56 transcripts were not significantly changed in oscol4 or OsCOL4-D, suggesting that OsCOL4 functions independently from previously reported flowering pathways. In osphyB mutants, OsCOL4 expression was decreased and osphyB oscol4 double mutants flowered at the same time as the osphyB single mutants, indicating OsCOL4 functions downstream of OsphyB. We also present evidence for two independent pathways through which OsPhyB controls flowering time. These pathways are: (i) night break-sensitive, which does not need OsCOL4; and (ii) night break-insensitive, in which OsCOL4 functions between OsphyB and Ehd1. Ehd1,Hd3a,OsCOL4,PHYB|OsphyB,RFT1 Ehd3, encoding a plant homeodomain finger-containing protein, is a critical promoter of rice flowering 2011 Plant J National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. Oryza sativa (rice) flowers in response to photoperiod, and is a facultative short-day (SD) plant. Under SD conditions, flowering is promoted through the activation of FT-like genes (rice florigens) by Heading date 1 (Hd1, a rice CONSTANS homolog) and Early heading date 1 (Ehd1, with no ortholog in the Arabidopsis genome). On the other hand, under long-day (LD) conditions, flowering is delayed by the repressive function of Hd1 on FT-like genes and by downregulation of Ehd1 by the flowering repressor Ghd7 - a unique pathway in rice. We report here that an early heading date 3 (ehd3) mutant flowered later than wild-type plants, particularly under LD conditions, regardless of the Hd1-deficient background. Map-based cloning revealed that Ehd3 encodes a nuclear protein that contains a putative transcriptional regulator with two plant homeodomain (PHD) finger motifs. To identify the role of Ehd3 within the gene regulatory network for rice flowering, we compared the transcript levels of genes related to rice flowering in wild-type plants and ehd3 mutants. Increased transcription of Ghd7 under LD conditions and reduced transcription of downstream Ehd1 and FT-like genes in the ehd3 mutants suggested that Ehd3 normally functions as an LD downregulator of Ghd7 in floral induction. Furthermore, Ehd3 ghd7 plants flowered earlier and show higher Ehd1 transcript levels than ehd3 ghd7 plants, suggesting a Ghd7-independent role of Ehd3 in the upregulation of Ehd1. Our results demonstrate that the PHD-finger gene Ehd3 acts as a promoter in the unique genetic pathway responsible for photoperiodic flowering in rice. Ehd1,Ehd3,Ghd7,Hd1 Footprints of natural and artificial selection for photoperiod pathway genes in Oryza 2012 Plant J Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan. Asian rice, Oryza sativa, consists of two major subspecies, indica and japonica, which are physiologically differentiated and adapted to different latitudes. Genes for photoperiod sensitivity are likely targets of selection along latitude. We examined the footprints of natural and artificial selections for four major genes of the photoperiod pathway, namely PHYTOCHROME B (PhyB), HEADING DATE 1 (Hd1), HEADING DATE 3a (Hd3a), and EARLY HEADING DATE 1 (Ehd1), by investigation of the patterns of nucleotide polymorphisms in cultivated and wild rice. Geographical subdivision between tropical and subtropical O. rufipogon was found for all of the photoperiod genes in plants divided by the Tropic of Cancer (TOC). All of these genes, except for PhyB, were characterized by the existence of clades that split a long time ago and that corresponded to latitudinal subdivisions, and revealed a likely diversifying selection. Ssp. indica showed close affinity to tropical O. rufipogon for all genes, while ssp. japonica, which has a much wider range of distribution, displayed complex patterns of differentiation from O. rufipogon, which reflected various agricultural needs in relation to crop yield. In japonica, all genes, except Hd3a, were genetically differentiated at the TOC, while geographical subdivision occurred at 31 degrees N in Hd3a, probably the result of varying photoperiods. Many other features of the photoperiod genes revealed domestication signatures, which included high linkage disequilibrium (LD) within genes, the occurrence of frequent and recurrent non-functional Hd1 mutants in cultivated rice, crossovers between subtropical and tropical alleles of Hd1, and significant LD between Hd1 and Hd3a in japonica and indica. Ehd1,Hd1,Hd3a,PHYB|OsphyB Ehd4 encodes a novel and Oryza-genus-specific regulator of photoperiodic flowering in rice 2013 PLoS Genet National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing, China. Land plants have evolved increasingly complex regulatory modes of their flowering time (or heading date in crops). Rice (Oryza sativa L.) is a short-day plant that flowers more rapidly in short-day but delays under long-day conditions. Previous studies have shown that the CO-FT module initially identified in long-day plants (Arabidopsis) is evolutionary conserved in short-day plants (Hd1-Hd3a in rice). However, in rice, there is a unique Ehd1-dependent flowering pathway that is Hd1-independent. Here, we report isolation and characterization of a positive regulator of Ehd1, Early heading date 4 (Ehd4). ehd4 mutants showed a never flowering phenotype under natural long-day conditions. Map-based cloning revealed that Ehd4 encodes a novel CCCH-type zinc finger protein, which is localized to the nucleus and is able to bind to nucleic acids in vitro and transactivate transcription in yeast, suggesting that it likely functions as a transcriptional regulator. Ehd4 expression is most active in young leaves with a diurnal expression pattern similar to that of Ehd1 under both short-day and long-day conditions. We show that Ehd4 up-regulates the expression of the "florigen" genes Hd3a and RFT1 through Ehd1, but it acts independently of other known Ehd1 regulators. Strikingly, Ehd4 is highly conserved in the Oryza genus including wild and cultivated rice, but has no homologs in other species, suggesting that Ehd4 is originated along with the diversification of the Oryza genus from the grass family during evolution. We conclude that Ehd4 is a novel Oryza-genus-specific regulator of Ehd1, and it plays an essential role in photoperiodic control of flowering time in rice. Ehd1,Ehd4,Hd3a,RFT1 OsELF3-1, an ortholog of Arabidopsis early flowering 3, regulates rice circadian rhythm and photoperiodic flowering 2012 PLoS One Rice Research Institute, Sichuan Agricultural University, Chengdu, Sichuan, China. Arabidopsis thaliana early flowering 3 (ELF3) as a zeitnehmer (time taker) is responsible for generation of circadian rhythm and regulation of photoperiodic flowering. There are two orthologs (OsELF3-1 and OsELF3-2) of ELF3 in rice (Oryza sativa), but their roles have not yet been fully identified. Here, we performed a functional characterization of OsELF3-1 and revealed it plays a more predominant role than OsELF3-2 in rice heading. Our results suggest OsELF3-1 can affect rice circadian systems via positive regulation of OsLHY expression and negative regulation of OsPRR1, OsPRR37, OsPRR73 and OsPRR95 expression. In addition, OsELF3-1 is involved in blue light signaling by activating early heading date 1 (Ehd1) expression to promote rice flowering under short-day (SD) conditions. Moreover, OsELF3-1 suppresses a flowering repressor grain number, plant height and heading date 7 (Ghd7) to indirectly accelerate flowering under long-day (LD) conditions. Taken together, our results indicate OsELF3-1 is essential for circadian regulation and photoperiodic flowering in rice. Ehd1,Ghd7,Hd17|Ef7|OsELF3-1,OsEF3|OsELF3-2,OsPRR1 A pair of floral regulators sets critical day length for Hd3a florigen expression in rice 2010 Nat Genet Photosynthesis and Photobiology Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan. The critical day length triggering photoperiodic flowering is set as an acute, accurate threshold in many short-day plants, including rice. Here, we show that, unlike the Arabidopsis florigen gene FT, the rice florigen gene Hd3a (Heading date 3a) is toggled by only a 30-min day-length reduction. Hd3a expression is induced by Ehd1 (Early heading date 1) expression when blue light coincides with the morning phase set by OsGIGANTEA(OsGI)-dependent circadian clocks. Ehd1 expression is repressed by both night breaks under short-day conditions and morning light signals under long-day conditions. Ghd7 (Grain number, plant height and heading date 7) was acutely induced when phytochrome signals coincided with a photosensitive phase set differently by distinct photoperiods and this induction repressed Ehd1 the next morning. Thus, two distinct gating mechanisms--of the floral promoter Ehd1 and the floral repressor Ghd7--could enable manipulation of slight differences in day length to control Hd3a transcription with a critical day-length threshold. Ehd1,Ghd7,Hd3a,OsGI Flowering time genes Heading date 1 and Early heading date 1 together control panicle development in rice 2011 Plant Cell Physiol University of Tsukuba, Tsukuba, Japan. Although flowering time is often associated with plant size, little is known about how flowering time genes affect plant architecture. We grew four rice lines having different flowering time genotypes (hd1 ehd1, hd1 Ehd1, Hd1 ehd1 and Hd1 Ehd1) under distinct photoperiod conditions. By using genotype-treatment combinations that resulted in similar flowering times, we were able to compare the effects of flowering time genes on traits related to plant architecture. The results revealed that the combination of Heading-date 1 (Hd1) and Early heading date 1 (Ehd1) can reduce the number of primary branches in a panicle, resulting in smaller spikelet numbers per panicle; this occurs independently of the control of flowering time. In addition, expression of the Hd3a and Rice Flowering-locus T 1 (RFT1) florigen genes was up-regulated in leaves of the Hd1 Ehd1 line at the time of the floral transition. We further revealed that Hd1 and/or Ehd1 caused up-regulation of Terminal Flower 1-like genes and precocious expression of panicle formation-related genes at shoot apical meristems during panicle development. Therefore, two key flowering time genes, Hd1 and Ehd1, can control panicle development in rice; this may affect crop yields in the field through florigen expression in leaf. Ehd1,Hd1,Hd3a,RFT1 OsMADS51 is a short-day flowering promoter that functions upstream of Ehd1, OsMADS14, and Hd3a 2007 Plant Physiol Division of Molecular and Life Sciences and Biotechnology Research Center, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea. Although flowering regulatory mechanisms have been extensively studied in Arabidopsis (Arabidopsis thaliana), those in other species have not been well elucidated. Here, we investigated the role of OsMADS51, a type I MADS-box gene in the short-day (SD) promotion pathway in rice (Oryza sativa). In SDs OsMADS51 null mutants flowered 2 weeks later than normal, whereas in long days loss of OsMADS51 had little effect on flowering. Transcript levels of three flowering regulators-Ehd1, OsMADS14, and Hd3a-were decreased in these mutants, whereas those of OsGI and Hd1 were unchanged. Ectopic expression of OsMADS51 caused flowering to occur about 7 d earlier only in SDs. In ectopic expression lines, transcript levels of Ehd1, OsMADS14, and Hd3a were increased, but those of OsGI and Hd1 remained the same. These results indicate that OsMADS51 is a flowering promoter, particularly in SDs, and that this gene functions upstream of Ehd1, OsMADS14, and Hd3a. To further investigate the relationship with other flowering promoters, we generated transgenic plants in which expression of Ehd1 or OsGI was suppressed. In Ehd1 RNA interference plants, OsMADS51 expression was not affected, supporting our conclusion that the MADS-box gene functions upstream of Ehd1. However, in OsGI antisense plants, the OsMADS51 transcript level was reduced. In addition, the circadian expression pattern for this MADS-box gene was similar to that for OsGI. These results demonstrate that OsMADS51 functions downstream of OsGI. In summary, OsMADS51 is a novel flowering promoter that transmits a SD promotion signal from OsGI to Ehd1. Ehd1,Hd3a,OsGI,OsMADS14,OsMADS51|OsMADS65 Ehd2, a rice ortholog of the maize INDETERMINATE1 gene, promotes flowering by up-regulating Ehd1 2008 Plant Physiol National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. Recent research into the flowering of rice (Oryza sativa) has revealed both unique and conserved genetic pathways in the photoperiodic control of flowering compared with those in Arabidopsis (Arabidopsis thaliana). We discovered an early heading date2 (ehd2) mutant that shows extremely late flowering under both short- and long-day conditions in line with a background deficient in Heading date1 (Hd1), a rice CONSTANS ortholog that belongs to the conserved pathway. This phenotype in the ehd2 mutants suggests that Ehd2 is pivotal for the floral transition in rice. Map-based cloning revealed that Ehd2 encodes a putative transcription factor with zinc finger motifs orthologous to the INDETERMINATE1 (ID1) gene, which promotes flowering in maize (Zea mays). Ehd2 mRNA in rice tissues accumulated most abundantly in developing leaves, but was present at very low levels around the shoot apex and in roots, patterns that are similar to those of ID1. To assign the position of Ehd2 within the flowering pathway of rice, we compared transcript levels of previously isolated flowering-time genes, such as Ehd1, a member of the unique pathway, Hd3a, and Rice FT-like1 (RFT1; rice florigens), between the wild-type plants and the ehd2 mutants. Severely reduced expression of these genes in ehd2 under both short- and long-day conditions suggests that Ehd2 acts as a flowering promoter mainly by up-regulating Ehd1 and by up-regulating the downstream Hd3a and RFT1 genes in the unique genetic network of photoperiodic flowering in rice. Ehd1,Hd1,Hd3a,RFT1,Ehd2|RID1 Analysis of PHOTOPERIOD SENSITIVITY5 sheds light on the role of phytochromes in photoperiodic flowering in rice 2009 Plant Physiol Instituto Valenciano de Investigaciones Agrarias, Carretera Moncada-Naquera Km 7.5, 46113 Moncada, Spain. A great number of plants synchronize flowering with day length. In rice (Oryza sativa), photoperiod is the primary environmental cue that triggers flowering. Here, we show that the s73 mutant, identified in a gamma-irradiated Bahia collection, displays early flowering and photoperiodic insensitivity due to a null mutation in the PHOTOPERIOD SENSITIVITY5 (SE5) gene, which encodes an enzyme implicated in phytochrome chromophore biosynthesis. s73 mutant plants show a number of alterations in the characteristic diurnal expression patterns of master genes involved in photoperiodic control of flowering, resulting in up-regulation of the floral integrator Heading date3a (Hd3a). Early heading date1 (Ehd1), an additional rice floral activator, was also highly expressed in the s73 mutant, suggesting that SE5 represses Ehd1 in wild-type plants. Silencing of Ehd1 in both Bahia and s73 backgrounds indicated that SE5 regulates Ehd1 expression. The data also indicate that SE5 confers photoperiodic sensitivity through regulation of Hd1. These results provide direct evidence that phytochromes inhibit flowering by affecting both Hd1 and Ehd1 flowering pathways. Ehd1,Hd3a,Se5|OsHY1|OsHO1|YGL2 Variations in Hd1 proteins, Hd3a promoters, and Ehd1 expression levels contribute to diversity of flowering time in cultivated rice 2009 Proc Natl Acad Sci U S A Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan. Rice is a facultative short-day plant, and molecular genetic studies have identified the major genes involved in short-day flowering. However, the molecular mechanisms promoting the diversity of flowering time in cultivated rice are not known. We used a core collection of 64 rice cultivars that represent the genetic diversity of 332 accessions from around the world and studied the expression levels and polymorphisms of 6 genes in the short-day flowering pathway. The RNA levels of Heading date 3a (Hd3a), encoding a floral activator, are highly correlated with flowering time, and there is a high degree of polymorphism in the Heading date 1 (Hd1) protein, which is a major regulator of Hd3a expression. Functional and nonfunctional alleles of Hd1 are associated with early and late flowering, respectively, suggesting that Hd1 is a major determinant of variation in flowering time of cultivated rice. We also found that the type of Hd3a promoter and the level of Ehd1 expression contribute to the diversity in flowering time and Hd3a expression level. We evaluated the contributions of these 3 factors by a statistical analysis using a simple linear model, and the results supported our experimental observations. Ehd1,Hd1,Hd3a OsVIL2 functions with PRC2 to induce flowering by repressing OsLFL1 in rice 2013 Plant J Crop Biotech Institute, Kyung Hee University, Yongin, 446-701, Korea. Flowering is exquisitely regulated by both promotive and inhibitory factors. Molecular genetic studies with Arabidopsis have verified several epigenetic repressors that regulate flowering time. However, the roles of chromatin remodeling factors in developmental processes have not been well explored in Oryza sativa (rice). We identified a chromatin remodeling factor OsVIL2 (O. sativa VIN3-LIKE 2) that promotes flowering. OsVIL2 contains a plant homeodomain (PHD) finger, which is a conserved motif of histone binding proteins. Insertion mutations in OsVIL2 caused late flowering under both long and short days. In osvil2 mutants OsLFL1 expression was increased, but that of Ehd1, Hd3a and RFT1 was reduced. We demonstrated that OsVIL2 is bound to native histone H3 in vitro. Chromatin immunoprecipitation analyses showed that OsVIL2 was directly associated with OsLFL1 chromatin. We also observed that H3K27me3 was significantly enriched by OsLFL1 chromatin in the wild type, but that this enrichment was diminished in the osvil2 mutants. These results indicated that OsVIL2 epigenetically represses OsLFL1 expression. We showed that OsVIL2 physically interacts with OsEMF2b, a component of polycomb repression complex 2. As observed from osvil2, a null mutation of OsEMF2b caused late flowering by increasing OsLFL1 expression and decreasing Ehd1 expression. Thus, we conclude that OsVIL2 functions together with PRC2 to induce flowering by repressing OsLFL1. Ehd1,Hd3a,OsEMF2b,OsLFL1,OsVIL2,RFT1 Trithorax group protein Oryza sativa Trithorax1 controls flowering time in rice via interaction with early heading date3 2014 Plant Physiol Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Korea. Trithorax group proteins are chromatin-remodeling factors that activate target gene expression by antagonistically functioning against the Polycomb group. In Arabidopsis (Arabidopsis thaliana), Arabidopsis Trithorax protein1 (ATX1) regulates flowering time and floral organ identity. Here, we observed that suppression of Oryza sativa Trithorax1 (OsTrx1), an ortholog of ATX1, delayed flowering time in rice (Oryza sativa). Because the delay occurred only under long-day conditions, we evaluated the flowering signal pathways that specifically function under long-day conditions. Among them, the OsMADS50 and Heading date1 pathways were not affected by the mutation. However, the Grain number, plant height, and heading date7 (Ghd7) pathway was altered in ostrx1. Transcript levels of OsGI, phytochrome genes, and Early heading date3 (Ehd3), which function upstream of Ghd7, were unchanged in the mutant. Because Trx group proteins form a complex with other proteins to modify the chromatin structure of target genes, we investigated whether OsTrx1 interacts with a previously identified protein that functions upstream of Ghd7. We demonstrated that the plant homeodomain motif of OsTrx1 binds to native histone H3 from the calf thymus and that OsTrx1 binds to Ehd3 through the region between the plant homeodomain and SET domains. Finally, we showed that the SET domain at the C-terminal end of OsTrx1 has histone H3 methyltransferase activity when incubated with oligonucleosomes. Our results suggest that OsTrx1 plays an important role in regulating flowering time in rice by modulating chromatin structure. Ehd3,Ghd7,Hd1,OsGI,OsMADS50|OsSOC1|DTH3,OsTrx1|SDG723 The SUI-homologous translation initiation factor eIF-1 is involved in regulation of ion homeostasis in rice 2008 Plant Biol (Stuttg) Department of Physiology and Biochemistry of Plants, Faculty of Biology, University of Bielefeld, Bielefeld, Germany. Halophytes survive high salinity by using complex adaptive mechanisms. In a search for novel molecular mechanisms involved in salt acclimation, transcript analyses revealed increased expression of a SUI-homologous translation initiation factor eIF-1 in the salt-tolerant grass species Festuca rubra ssp. littoralis but not in rice. Upon analysis of the cell specificity of eIF-1 transcription by in situ polymerase chain reaction (PCR), predominant signals were detected in rice leaf mesophyll. To further examine the role of eIF-1 in salt tolerance, transgenic rice plants were generated that over-express this factor under the control of the CaMV-35S promoter. The eIF-1 over-expressing lines showed improved growth under salt stress that was correlated with maintenance of photosynthetic activity and reduced Na(+) and Cl(-) accumulation in leaves. The transgenic rice lines also activated expression of the vacuolar H(+)-ATPase. In addition, an oxidoreductase that belongs to the aldo/keto reductase family was identified as a gene with modified expression in the eIF-1 over-expressing lines, compared with wild-type rice. Our data suggest that eIF-1 has a central function in salt-stress adaptation in rice by regulating ion accumulation and the intracellular redox status. eIF-1 Inhibition of a basal transcription factor 3-like gene Osj10gBTF3 in rice results in significant plant miniaturization and typical pollen abortion 2012 Plant Cell Physiol Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, PR China. BTF3, which was originally recognized as a basal transcription factor, has been known to be involved in transcription initiation, translational regulation and protein localization in many eukaryotic organisms. However, its function remains largely unknown in plant species. In the present study, we analyzed a BTF3-related sequence in Oryza sativa L. subsp. japonica, which shares the conserved domain of a nascent polypeptide-associated complex with human BTF3, and was referred to as Osj10gBTF3. The expression of Osj10gBTF3 was primarily constitutive and generally modulated by salt, high temperature and exogenous phytohormone stress. The Osj10gBTF3::EGFP (enhanced green fluorescence protein) fusion protein was localized in both the nucleus and cytoplasmic membrane system. Inhibition of Osj10gBTF3 led to significant morphological changes in all detected tissues and organs, with a reduced size of between 25% and 52%. Furthermore, the pollen that developed was completely sterile, which was correlated with the altered expression of two Rf (fertility restorer)-like genes that encode pentatricopeptide repeat-containing proteins OsPPR676 and OsPPR920, translational initiation factors OseIF3e and OseIF3h, and the heat shock protein OsHSP82. These findings were verified through a yeast two-hybrid assay using a Nipponbare callus cDNA library as bait followed by the reverse transcription-PCR analysis of total leaf or anther RNAs. Our demonstration of the important role of Osj10gBTF3 in rice growth and development provides new insights showing that more complex regulatory functions are associated with BTF3 in plants. EIF3E,EIF3H,OsBTF3|Osj10gBTF3,Osj3g2BTF3 Two novel genes rapidly and transiently activated in suspension-cultured rice cells by treatment with N-acetylchitoheptaose, a biotic elicitor for phytoalexin production 1996 Plant Cell Physiol Department of Cell Biology, National Institute of Agrobiological Resources, Japan. By using subtracted probes, two cDNA clones of rice, EL2 and EL3, were isolated as genes responsive within 6 min to N-acetylchitoheptaose, a potent biotic elicitor for phytoalexin biosynthesis. Analyses of the sequence of the cDNAs showed that both of EL2 and EL3 encoded basic proteins with no significant similarities to those of known genes. EL2 Novel plant-specific cyclin-dependent kinase inhibitors induced by biotic and abiotic stresses 2007 J Biol Chem CropDesign N.V., B-9052 Ghent, Belgium. The EL2 gene of rice (Oryza sativa), previously classified as early response gene against the potent biotic elicitor N-acetylchitoheptaose and encoding a short polypeptide with unknown function, was identified as a novel cell cycle regulatory gene related to the recently reported SIAMESE (SIM) gene of Arabidopsis thaliana. Iterative two-hybrid screens, in vitro pull-down assays, and fluorescence resonance energy transfer analyses showed that Orysa; EL2 binds the cyclin-dependent kinase (CDK) CDKA1;1 and D-type cyclins. No interaction was observed with the plant-specific B-type CDKs. The amino acid motif ELERFL was identified to be essential for cyclin, but not for CDK binding. Orysa;EL2 impaired the ability of Orysa; CYCD5;3 to complement a budding yeast (Saccharomyces cerevisiae) triple CLN mutant, whereas recombinant protein inhibited CDK activity in vitro. Moreover, Orysa;EL2 was able to rescue the multicellular trichome phenotype of sim mutants of Arabidopsis, unequivocally demonstrating that Orysa;EL2 operates as a cell cycle inhibitor. Orysa;EL2 mRNA levels were induced by cold, drought, and propionic acid. Our data suggest that Orysa;EL2 encodes a new type of plant CDK inhibitor that links cell cycle progression with biotic and abiotic stress responses. EL2 Isolation and analysis of expression mechanisms of a rice gene, EL5, which shows structural similarity to ATL family from Arabidopsis, in response to N-acetylchitooligosaccharide elicitor 2001 Plant Science Institute of Applied Biochemistry, University of Tsukuba, 305-8072, Tsukuba, Japan Two rice cDNAs, EL5 and RRF1, were isolated and characterized. EL5 was responsive to N-acetylchitooligosaccharide, a biotic elicitor active in suspension-cultured rice cells. The structural specificity of the elicitor required for the expression of EL5 was consistent with other defense reactions observed in the experimental system, indicating that the elicitor signal to EL5 is transmitted through a single class of receptor-mediated recognition events. However, the intracellular signaling pathway to EL5 was distinct from those to other elicitor-responsive genes. Sequence analysis and alignment showed that a genomic sequence stored in rice genome databases in addition to EL5 and RRF1 belongs to the ATL family of RING-H2 finger motif proteins first isolated from Arabidopsis. EL5 EL5, a rice N-acetylchitooligosaccharide elicitor-responsive RING-H2 finger protein, is a ubiquitin ligase which functions in vitro in co-operation with an elicitor-responsive ubiquitin-conjugating enzyme, OsUBC5b 2002 The Plant Journal Institute of Applied Biochemistry, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8572, Japan. EL5, a rice gene responsive to N-acetylchitooligosaccharide elicitor, encodes a RING-H2 finger protein with structural features common to the plant-specific ATL family. We show that the fusion protein of EL5 with maltose binding protein (MBP) was polyubiquitinated by incubation with ubiquitin, ubiquitin-activating enzyme (E1), and the Ubc4/5 subfamily of the ubiquitin-conjugating enzyme (E2). EL5 possesses the activity to catalyse the transfer of ubiquitin to the MBP moiety, and the RING-H2 finger motif of EL5 is necessary for this activity. Thus, we concluded that EL5 represents a ubiquitin ligase (E3). We also show that two rice E2s (OsUBC5a, OsUBC5b) of the Ubc4/5 subfamily function as E2 which catalyses EL5-mediated ubiquitination, and OsUBC5b was induced by elicitor, as well as EL5. These results strongly suggest that EL5 and OsUBC5b have roles in plant defense response through the turnover of protein(s) via the ubiquitin/proteasome system. EL5,OsUBC5b,OsUBC5a RING-H2 type ubiquitin ligase EL5 is involved in root development through the maintenance of cell viability in rice 2007 Plant J Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai, 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. Rice EL5 is an ATL family gene characterized by a transmembrane domain at the N-terminal and a RING-H2 finger domain (RFD), which exhibits ubiquitin ligase (E3) activity. To elucidate the physiological roles of EL5, we analyzed transgenic rice plants overexpressing mutant EL5 proteins that are impaired in E3 activity to various degrees. Plants expressing EL5C153A and EL5W165A, which encode an inactive E3, showed a rootless phenotype accompanied by cell death in root primordia, and those expressing EL5V162A, with moderately impaired E3 activity, formed short crown roots with necrotic lateral roots. The dominant-negative phenotype was specifically observed in root meristems where EL5 is expressed, and not recovered by exogenous auxin. When wild-type EL5 was transcriptionally overexpressed, the EL5 protein was barely detected by Western blotting. Neither treatment with a proteasome inhibitor nor mutation of the sole lysine residue, a potential target of ubiquitination, resulted in increased EL5 accumulation, whereas mutations in the RFD led to increased EL5 accumulation. The stabilized EL5 without the RFD was localized in the plasma membrane. Deletion of the transmembrane domain prevented the EL5 from localizing in the membrane and from exerting an inhibitory effect on root formation. Deletion of the C-terminal region also neutralized the negative effect. We concluded that EL5 plays a major role as a membrane-anchored E3 for the maintenance of cell viability after the initiation of root primordial formation. In addition, we propose that EL5 is an unstable protein, of which degradation is regulated by the RFD in a proteasome-independent manner. EL5 EL5 is involved in root development as an anti-cell death ubiquitin ligase 2008 Plant Signal Behav Division of Plant Sciences; National Institute of Agrobiological Sciences; Tsukuba, Japan. Ubiquitin ligase (E3) plays a central role in substrate recognition during ubiquitination, a post-translational modification of proteins. Rice EL5 is an E3 with a RING-H2 finger domain (RFD) and its transcript is upregulated by a chitin elicitor. The EL5-RFD has been intensively studied and demonstrated to exhibit E3 activity. Its three-dimensional structure was determined for the first time in plant E3, and the amino acid residues required for the interaction with the ubiquitin-conjugating enzyme (E2) were identified. Recent analyses revealed that EL5 plays a crucial role as an E3 in the maintenance of cell viability during root development in rice. In this addendum, we report that the EL5-RFD catalyzes polyubiquitination via the Lys48 residue of ubiquitin. We also discuss the possible role of EL5 as an anti-cell death enzyme. We hypothesize that EL5 might be responsible for mediating the degradation of cytotoxic proteins produced in root cells after the actions of phytohormones. EL5 Overexpression of the trehalose-6-phosphate synthase gene OsTPS1 enhances abiotic stress tolerance in rice 2011 Planta Key Laboratory of Gene Engineering Drug and Biotechnology, College of Life Sciences, Beijing Normal University, Beijing, 100875, People's Republic of China. Trehalose plays an important role in metabolic regulation and abiotic stress tolerance in a variety of organisms. In plants, its biosynthesis is catalyzed by two key enzymes: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). The genome of rice (Oryza sativa) contains 11 OsTPS genes, and only OsTPS1 shows TPS activity. To demonstrate the physiological function of OsTPS1, we introduced it into rice and found that OsTPS1 overexpression improved the tolerance of rice seedling to cold, high salinity and drought treatments without other significant phenotypic changes. In transgenic lines overexpressing OsTPS1, trehalose and proline concentrations were higher than in the wild type and some stress-related genes were up-regulated, including WSI18, RAB16C, HSP70, and ELIP. These results demonstrate that OsTPS1 may enhance the abiotic stress tolerance of plants by increasing the amount of trehalose and proline, and regulating the expression of stress-related genes. Furthermore, we found that overexpression of some Class II TPSs also enhanced plant tolerance of abiotic stress. This work will help to clarify the role of trehalose metabolism in abiotic stress response in higher plants. ELIP,OsTPS1,pwsi18|WSI18,CRP,OsLEA25,OsTRE Cloning of cDNA encoding the rice 22 kDa protein of Photosystem II (PSII-S) and analysis of light-induced expression of the gene 1997 Gene National Institute of Agrobiological Resources, Ibaraki, Japan. Cloning of rice cDNA encoding the chlorophyll-binding 22 kDa protein of Photosystem II (PSII-S) and the light-induced expression of the gene are reported. One of the light-responsive cDNA clones, isolated by screening with a light-specific subtracted cDNA probe, was shown to encode PSII-S of rice. Genomic Southern analysis suggested that the PSII-S gene, psbS, is a single-copy gene in rice. A brief exposure to red light induced a severalfold increase in the steady state level of PSII-S transcripts in etiolated seedlings. The red light effect was reversed by far-red light, suggesting involvement of phytochrome in the PSII-S gene regulation. Prolonged exposure (3 h) to blue light, however, revealed a much stronger effect than red light on the accumulation of PSII-S transcripts in the etiolated seedlings. In dark-adapted green plants, prolonged exposure to blue light induced re-accumulation of transcripts encoding PSII-S, whereas red light had little effect. ELIP,ASCAB9-A|CP26|Lhcb5 Empty pericarp5 encodes a pentatricopeptide repeat protein that is required for mitochondrial RNA editing and seed development in maize 2013 Plant Cell State Key Lab of Agrobiotechnology, Institute of Plant Molecular Biology and Agrobiotechnology, School of Life Science, Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China. In flowering plants, RNA editing is a posttranscriptional mechanism that converts specific cytidines to uridines in both mitochondrial and plastidial transcripts, altering the information encoded by these genes. Here, we report the molecular characterization of the empty pericarp5 (emp5) mutants in maize (Zea mays). Null mutation of Emp5 results in abortion of embryo and endosperm development at early stages. Emp5 encodes a mitochondrion-targeted DYW subgroup pentatricopeptide repeat (PPR) protein. Analysis of the mitochondrial transcripts revealed that loss of the EMP5 function abolishes the C-to-U editing of ribosomal protein L16 at the rpl16-458 site (100% edited in the wild type), decreases the editing at nine sites in NADH dehydrogenase9 (nad9), cytochrome c oxidase3 (cox3), and ribosomal protein S12 (rps12), and surprisingly increases the editing at five sites of ATP synthase F0 subunit a (atp6), apocytochrome b (cob), nad1, and rpl16. Mutant EMP5-4 lacking the E+ and DYW domains still retains the substrate specificity and editing function, only at reduced efficiency. This suggests that the E+ and DYW domains of EMP5 are not essential to the EMP5 editing function but are necessary for efficiency. Analysis of the ortholog in rice (Oryza sativa) indicates that rice EMP5 has a conserved function in C-to-U editing of the rice mitochondrial rpl16-458 site. EMP5 knockdown expression in transgenics resulted in slow growth and defective seeds. These results demonstrate that Emp5 encodes a PPR-DYW protein that is required for the editing of multiple transcripts in mitochondria, and the editing events, particularly the C-to-U editing at the rpl16-458 site, are critical to the mitochondrial functions and, hence, to seed development in maize. EMP5|OsEMP5 Phytosiderophore efflux transporters are crucial for iron acquisition in graminaceous plants 2011 J Biol Chem Department of Global Agricultural Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 Japan. Eukaryotic organisms have developed diverse mechanisms for the acquisition of iron, which is required for their survival. Graminaceous plants use a chelation strategy. They secrete phytosiderophore compounds, which solubilize iron in the soil, and then take up the resulting iron-phytosiderophore complexes. Bacteria and mammals also secrete siderophores to acquire iron. Although phytosiderophore secretion is crucial for plant growth, its molecular mechanism remains unknown. Here, we show that the efflux of deoxymugineic acid, the primary phytosiderophore from rice and barley, involves the TOM1 and HvTOM1 genes, respectively. Xenopus laevis oocytes expressing TOM1 or HvTOM1 released (14)C-labeled deoxymugineic acid but not (14)C-labeled nicotianamine, a structural analog and biosynthetic precursor of deoxymugineic acid, indicating that the TOM1 and HvTOM1 proteins are the phytosiderophore efflux transporters. Under conditions of iron deficiency, rice and barley roots express high levels of TOM1 and HvTOM1, respectively, and the overexpression of these genes increased tolerance to iron deficiency. In rice roots, the efficiency of deoxymugineic acid secretion was enhanced by overexpression of TOM1 and decreased by its repression, providing further evidence that TOM1 encodes the efflux transporter of deoxymugineic acid. We have also identified two genes encoding efflux transporters of nicotianamine, ENA1 and ENA2. Our identification of phytosiderophore efflux transporters has revealed the final piece in the molecular machinery of iron acquisition in graminaceous plants. OsENA1,TOM1|OsZIFL4 ENAC1, a NAC transcription factor, is an early and transient response regulator induced by abiotic stress in rice (Oryza sativa L.) 2012 Mol Biotechnol State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China. The plant-specific NAC (NAM, ATAF, and CUC)-domain proteins play important roles in plant development and stress responses. In this research, a full-length cDNA named ENAC1 (early NAC-domain protein induced by abiotic stress 1) was isolated from rice. ENAC1 possess one NAC domain in the N-terminus. Comparative time-course expression analysis indicated that ENAC1 expression, similar with OsDREB1A, was induced very quickly by various abiotic stresses including salt, drought, cold, and exogenous abscisic acid. However, the induction of ENAC1 by abiotic stress was transient and lasted up to 3 h, whereas that of OsDREB1A maintained longer. The promoter sequence of ENAC1 harbors several cis-elements including ABA response elements, but the well-known dehydration responsive element/C-repeat element is absent. The ENAC1-GFP (green fluorescent protein) fusion protein was localized in the nucleus of rice protoplast cell. Yeast hybrid assays revealed that ENAC1 was a transcription activator and bound to NAC recognition sequence (NACRS). Co-expression analysis suggested that ENAC1 co-expressed with a number of stress-related genes. Taken together, ENAC1 may be an early transcription activator of stress responses and function in the regulation of NACRS-mediated gene expression under abiotic stress. ENAC1,OsDREB1A|OsDREBL Rice SNF2 family helicase ENL1 is essential for syncytial endosperm development 2014 Plant J Bioscience and Biotechnology Center, Nagoya University, Nagoya, 464-8601, Japan. The endosperm of cereal grains represents the most important source of human nutrition. In addition, the endosperm provides many investigatory opportunities for biologists because of the unique processes that occur during its ontogeny, including syncytial development at early stages. Rice endospermless 1 (enl1) develops seeds lacking an endosperm but carrying a functional embryo. The enl1 endosperm produces strikingly enlarged amoeboid nuclei. These abnormal nuclei result from a malfunction in mitotic chromosomal segregation during syncytial endosperm development. The molecular identification of the causal gene revealed that ENL1 encodes an SNF2 helicase family protein that is orthologous to human PICH, which has been implicated in the resolution of persistent DNA catenation during anaphase. ENL1-Venus (enhanced YFP) localizes to the cytoplasm during interphase but moves to the chromosome arms during mitosis. ENL1-Venus is also detected on a thread-like structure that connects separating sister chromosomes. These observations indicate the functional conservation between PICH and ENL1 and confirm the proposed role of PICH. Although ENL1 dysfunction also affects karyokinesis in the root meristem, enl1 plants can grow in a field and set seeds, indicating that its indispensability is tissue-dependent. Notably, despite the wide conservation of ENL1/PICH among eukaryotes, the loss of function of the ENL1 ortholog in Arabidopsis (CHR24) has only marginal effects on endosperm nuclei and results in normal plant development. Our results suggest that ENL1 is endowed with an indispensable role to secure the extremely rapid nuclear cycle during syncytial endosperm development in rice. ENL1 Identification and characterization of the erect-pose panicle gene EP conferring high grain yield in rice (Oryza sativa L.) 2009 Theor Appl Genet Key Laboratory of Crop Physiology, Ecology, Genetics and Breeding, Ministry of Agriculture/Key Laboratory of Northern Japonica Rice Breeding of Liaoning, Shenyang Agricultural University, Shenyang, China. The breeding of japonica varieties with erect-pose panicle (EP) has recently progressed in the northern part of China, because these varieties exhibit a far higher grain yield than the varieties with normal-pose panicle (NP). A genetic analysis using the F(2) population from the cross between Liaojing5, the first japonica EP variety in China, and the Japanese japonica NP variety Toyonishiki revealed that EP is governed by a single dominant gene EP. Based on previous studies, map-based cloning of EP locus was conducted using Liaojing5, Toyonishiki, their F(2) population, and a pair of near-isogenic lines for EP locus (ZF14 and WF14) derived from the cross between the two varieties; consequently, the STS marker H90 was found to completely cosegregate with panicle pose. The H90 is located in the coding sequence AK101247 in the database, and the AK101247 of Liaojing5 has a 12 bp sequence in exon 5 replaced with a 637 bp sequence of its wild type allele. It was therefore considered that the AK101247 encodes the protein of the wild type allele at EP locus, and that the sequence substitution in exon 5 of Liaojing5 is crucial for expression of the EP phenotype. The effects of EP gene on agronomic traits were investigated using two pairs of near-isogenic lines (ZF6 vs. WF6 and ZF14 vs. WF14) derived from the cross between the two varieties. Experimental results showed that EP gene markedly enhanced grain yield, chiefly by increasing number of secondary branches and number of grains on the secondary branch. EP gene also produced a remarkable increase in grain density. EP Evidence for an evolutionary force that prevents epigenetic silencing between tail-to-tail rice genes with a short spacer 2005 Gene Laboratory of Plant Breeding, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. During the course of evolution, the genome should have toned down various types of genomic noise, such as those that cause the unstable expression or gene silencing observed in transgenic organisms. We found a rice genomic segment where two genes, encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) and ribosomal protein small subunit 20 (rps20), are located in a tail-to-tail orientation and separated by only 300 bp of spacer. It is possible that this kind of structure would give rise to unstable expression due to antisense RNA derived from the neighboring gene. We examined this possibility using Northern blot, reverse transcription-polymerase chain reaction (RT-PCR), and 3' RACE analyses, but obtained no evidence for instability or antisense RNAs of these housekeeping genes. Comparison of the sequences in the corresponding regions among related rice species revealed a lower level of genetic divergence of both the 3'-untranslated region (3'-UTRs) than of the other noncoding regions; in particular both of the boundaries between the 3'-UTRs and the spacer were markedly conserved. The conservation of both the terminal regions is most likely the result of purifying selection, implying a functional role for the strict termination of the transcription of these genes to prevent gene-silencing-related events. EPSPs Whole-genome analysis of Oryza sativa reveals similar architecture of two-component signaling machinery with Arabidopsis 2006 Plant Physiol Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India. ashwanip@mail.jnu.ac.in The two-component system (TCS), which works on the principle of histidine-aspartate phosphorelay signaling, is known to play an important role in diverse physiological processes in lower organisms and has recently emerged as an important signaling system in plants. Employing the tools of bioinformatics, we have characterized TCS signaling candidate genes in the genome of Oryza sativa L. subsp. japonica. We present a complete overview of TCS gene families in O. sativa, including gene structures, conserved motifs, chromosome locations, and phylogeny. Our analysis indicates a total of 51 genes encoding 73 putative TCS proteins. Fourteen genes encode 22 putative histidine kinases with a conserved histidine and other typical histidine kinase signature sequences, five phosphotransfer genes encoding seven phosphotransfer proteins, and 32 response regulator genes encoding 44 proteins. The variations seen between gene and protein numbers are assumed to result from alternative splicing. These putative proteins have high homology with TCS members that have been shown experimentally to participate in several important physiological phenomena in plants, such as ethylene and cytokinin signaling and phytochrome-mediated responses to light. We conclude that the overall architecture of the TCS machinery in O. sativa and Arabidopsis thaliana is similar, and our analysis provides insights into the conservation and divergence of this important signaling machinery in higher plants. ERS1,ERS2,ETR3,ETR4,HK1 Nomenclature for two-component signaling elements of rice 2007 Plant Physiol Department of Biological Sciences, Dartmouth College, Hanover, NH 03755 None ERS1,ERS2,ETR3,ETR4,HK1 Differential expression of three genes encoding an ethylene receptor in rice during development, and in response to indole-3-acetic acid and silver ions 2004 J Exp Bot Department of Botany, University of Hong Kong, Pokfulam Road, Hong Kong. Five ethylene receptor genes, OS-ERS1, OS-ERS2, OS-ETR2, OS-ETR3, and OS-ETR4 were isolated and characterized from rice. The genomic structure of OS-ERS1 and OS-ERS2 revealed that the introns within the coding sequences occurred in conserved positions to those of At-ETR1 and At-ERS1, whereas each of the OS-ETR2, OS-ETR3, and OS-ETR4 genes contained 1 intron within its coding region located at a position equivalent to those of At-ERS2, At-ETR2, and At-EIN4. Deduced amino acid sequences of OS-ERS1, OS-ERS2, OS-ETR2, OS-ETR3, and OS-ETR4 showed that they exhibited significant homology to the prokaryotic two-component signal transducer and a wide range of ethylene receptors in a variety of plant species. Northern analysis revealed that the level of OS-ETR2 mRNA was markedly elevated either by the exogenous application of IAA or by ethylene treatment in young etiolated rice seedlings, whereas the OS-ERS1 transcript level was only slightly induced under the same experimental conditions. Pretreatment with silver prevented IAA-induced and ethylene-induced accumulation of both mRNAs (OS-ERS1 and OS-ETR2). However, the abundance of OS-ERS2 mRNA was shown to be down-regulated by both IAA and ethylene treatments, indicating that it was not positively regulated by ethylene. Analysis of the expression of the three ethylene receptor genes in different tissues of rice has unravelled their corresponding tissue-specificity in which OS-ERS1 was constitutively expressed in considerable amounts in all tissues studied, while OS-ERS2 and OS-ETR2 exhibited differential expression patterns in different tissues of rice. Moreover, higher levels of these three mRNAs were commonly observed in anthers when compared with their corresponding levels in other tissues, suggesting the important role played by ethylene involved in the regulation of pollen development in rice. Among the five ethylene receptor genes, the expression levels of both OS-ETR3 and OS-ETR4 were too low to be detected by the northern blot analysis. Results from RT-PCR illustrated that both mRNAs were present in young green rice seedlings and anthers. ERS1,ERS2,ETR3,ETR4 The ethylene receptor ETR2 delays floral transition and affects starch accumulation in rice 2009 Plant Cell Plant Gene Research Center, National Key Lab of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Ethylene regulates multiple aspects of plant growth and development in dicotyledonous plants; however, its roles in monocotyledonous plants are poorly known. Here, we characterized a subfamily II ethylene receptor, ETHYLENE RESPONSE2 (ETR2), in rice (Oryza sativa). The ETR2 receptor with a diverged His kinase domain is a Ser/Thr kinase, but not a His kinase, and can phosphorylate its receiver domain. Mutation of the N box of the kinase domain abolished the kinase activity of ETR2. Overexpression of ETR2 in transgenic rice plants reduced ethylene sensitivity and delayed floral transition. Conversely, RNA interference (RNAi) plants exhibited early flowering and the ETR2 T-DNA insertion mutant etr2 showed enhanced ethylene sensitivity and early flowering. The effective panicles and seed-setting rate were reduced in the ETR2-overexpressing plants, while thousand-seed weight was substantially enhanced in both the ETR2-RNAi plants and the etr2 mutant compared with controls. Starch granules accumulated in the internodes of the ETR2-overexpressing plants, but not in the etr2 mutant. The GIGANTEA and TERMINAL FLOWER1/CENTRORADIALIS homolog (RCN1) that cause delayed flowering were upregulated in ETR2-overexpressing plants but downregulated in the etr2 mutant. Conversely, the alpha-amylase gene RAmy3D was suppressed in ETR2-overexpressing plants but enhanced in the etr2 mutant. Thus, ETR2 may delay flowering and cause starch accumulation in stems by regulating downstream genes. ETR3,ETR4,ETR2|Os-ERL1,Rcn1 Identification of a 98-kb DNA segment containing the rice Eui gene controlling uppermost internode elongation, and construction of a TAC transgene sublibrary 2004 Mol Genet Genomics College of Life Sciences, Zhejiang University, 310029, Hangzhou, China. The recessive 'tall rice' phenotype associated with the mutation eui (elongated upper-most internode) is an important agronomic trait that has been introduced into hybrid rice to eliminate panicle enclosure in all types of male-sterile lines and produce good-quality seeds in high yield and at low cost. Based on our previous Eui mapping data, we conducted fine-structure mapping and positional cloning of the gene using an F2 population comprising more than 5000 individuals derived from a cross of the near-isogenic lines 307T (eui/eui) with the recurrent parent Zhenshan 97 (Eui/Eui). In total 45 CAPS (cleaved amplified polymorphic sequences) markers located within an interval of 14.5 cM were analyzed in the subpopulation of 1298 homozygous recessive plants. The resulting high-resolution map defined a 98-kb interval containing the Eui locus flanked by the markers M0387 and M01, and three markers were found to co-segregate with Eui. In order to facilitate the identification of the Eui gene, we used a transformation-competent artificial chromosome (TAC) vector to construct a set of contiguous TAC clones from the Nipponbare BACs (obtained from the Clemson University Genome Institute; CUGI) spanning this region. These clones can be used to streamline complementation testing. The markers tightly linked to the Eui locus can also be used in breeding male-sterile lines with the elongated uppermost internode. EUI1 Gibberellin homeostasis and plant height control by EUI and a role for gibberellin in root gravity responses in rice 2008 Cell Res National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China. The rice Eui (ELONGATED UPPERMOST INTERNODE) gene encodes a cytochrome P450 monooxygenase that deactivates bioactive gibberellins (GAs). In this study, we investigated controlled expression of the Eui gene and its role in plant development. We found that Eui was differentially induced by exogenous GAs and that the Eui promoter had the highest activity in the vascular bundles. The eui mutant was defective in starch granule development in root caps and Eui overexpression enhanced starch granule generation and gravity responses, revealing a role for GA in root starch granule development and gravity responses. Experiments using embryoless half-seeds revealed that RAmy1A and GAmyb were highly upregulated in eui aleurone cells in the absence of exogenous GA. In addition, the GA biosynthesis genes GA3ox1 and GA20ox2 were downregulated and GA2ox1 was upregulated in eui seedlings. These results indicate that EUI is involved in GA homeostasis, not only in the internodes at the heading stage, but also in the seedling stage, roots and seeds. Disturbing GA homeostasis affected the expression of the GA signaling genes GID1 (GIBBERELLIN INSENSITIVE DWARF 1), GID2 and SLR1. Transgenic RNA interference of the Eui gene effectively increased plant height and improved heading performance. By contrast, the ectopic expression of Eui under the promoters of the rice GA biosynthesis genes GA3ox2 and GA20ox2 significantly reduced plant height. These results demonstrate that a slight increase in Eui expression could dramatically change rice morphology, indicating the practical application of the Eui gene in rice molecular breeding for a high yield potential. EUI1,GF14e|GID2,OsGA2ox1,OsGA3ox1,GID1|OsGID1,SLR1|OsGAI Altered disease development in the eui mutants and Eui overexpressors indicates that gibberellins negatively regulate rice basal disease resistance 2008 Mol Plant National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. Gibberellins (GAs) form a group of important plant tetracyclic diterpenoid hormones that are involved in many aspects of plant growth and development. Emerging evidence implicates that GAs also play roles in stress responses. However, the role of GAs in biotic stress is largely unknown. Here, we report that knockout or overexpression of the Elongated uppermost internode (Eui) gene encoding a GA deactivating enzyme compromises or increases, respectively, disease resistance to bacterial blight (Xanthomonas oryzae pv. oyrzae) and rice blast (Magnaporthe oryzae). Exogenous application of GA(3) and the inhibitor of GA synthesis (uniconazol) could increase disease susceptibility and resistance, respectively, to bacterial blight. Similarly, uniconazol restored disease resistance of the eui mutant and GA(3) decreased disease resistance of the Eui overexpressors to bacterial blight. Therefore, the change of resistance attributes to GA levels. In consistency with this, the GA metabolism genes OsGA20ox2 and OsGA2ox1 were down-regulated during pathogen challenge. We also found that PR1a induction was enhanced but the SA level was decreased in the Eui overexpressor, while the JA level was reduced in the eui mutant. Together, our current study indicates that GAs play a negative role in rice basal disease resistance, with EUI as a positive modulator through regulating the level of bioactive GAs. EUI1,OsGA20ox1 The APETALA-2-Like Transcription Factor OsAP2-39 Controls Key Interactions between Abscisic Acid and Gibberellin in Rice 2010 PLoS Genet Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada The interaction between phytohormones is an important mechanism which controls growth and developmental processes in plants. Deciphering these interactions is a crucial step in helping to develop crops with enhanced yield and resistance to environmental stresses. Controlling the expression level of OsAP2-39 which includes an APETALA 2 (AP2) domain leads to phenotypic changes in rice. Overexpression of OsAP2-39 leads to a reduction in yield by decreasing the biomass and the number of seeds in the transgenic rice lines. Global transcriptome analysis of the OsAP2-39 overexpression transgenic rice revealed the upregulation of a key Abscisic Acid (ABA) biosynthetic gene OsNCED-I which codes for 9-cis-epoxycarotenoid dioxygenase and leads to an increase in the endogenous ABA level. In addition to OsNCED-1, the gene expression analysis revealed the upregulation of a gene that codes for the Elongation of Upper most Internode (EUI) protein, an enzyme that catalyzes 16a, 17-epoxidation of non-13-hydroxylated GAs, which has been shown to deactivate gibberellins (GAs) in rice. The exogenous application of GA restores the wild-type phenotype in the transgenic line and ABA application induces the expression of EUI and suppresses the expression of OsAP2-39 in the wild-type line. These observations clarify the antagonistic relationship between ABA and GA and illustrate a mechanism that leads to homeostasis of these hormones. In vivo and in vitro analysis showed that the expression of both OsNCED-1 and EUI are directly controlled by OsAP2-39. Together, these results reveal a novel mechanism for the control of the ABA/GA balance in rice which is regulated by OsAP2-39 that in turn regulates plant growth and seed production. EUI1,OsAP2-39,OsNCED1 ELONGATED UPPERMOST INTERNODE encodes a cytochrome P450 monooxygenase that epoxidizes gibberellins in a novel deactivation reaction in rice 2006 Plant Cell National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. The recessive tall rice (Oryza sativa) mutant elongated uppermost internode (eui) is morphologically normal until its final internode elongates drastically at the heading stage. The stage-specific developmental effect of the eui mutation has been used in the breeding of hybrid rice to improve the performance of heading in male sterile cultivars. We found that the eui mutant accumulated exceptionally large amounts of biologically active gibberellins (GAs) in the uppermost internode. Map-based cloning revealed that the Eui gene encodes a previously uncharacterized cytochrome P450 monooxygenase, CYP714D1. Using heterologous expression in yeast, we found that EUI catalyzed 16alpha,17-epoxidation of non-13-hydroxylated GAs. Consistent with the tall and dwarfed phenotypes of the eui mutant and Eui-overexpressing transgenic plants, respectively, 16alpha,17-epoxidation reduced the biological activity of GA(4) in rice, demonstrating that EUI functions as a GA-deactivating enzyme. Expression of Eui appeared tightly regulated during plant development, in agreement with the stage-specific eui phenotypes. These results indicate the existence of an unrecognized pathway for GA deactivation by EUI during the growth of wild-type internodes. The identification of Eui as a GA catabolism gene provides additional evidence that the GA metabolism pathway is a useful target for increasing the agronomic value of crops. EUI1 EUI1, encoding a putative cytochrome P450 monooxygenase, regulates internode elongation by modulating gibberellin responses in rice 2006 Plant Cell Physiol National Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, PR China. Elongation of rice internodes is one of the most important agronomic traits, which determines the plant height and underlies the grain yield. It has been shown that the elongation of internodes is under genetic control, and various factors are implicated in the process. Here, we report a detailed characterization of an elongated uppermost internode1 (eui1) mutant, which has been used in hybrid rice breeding. In the eui1-2 mutant, the cell lengths in the uppermost internodes are significantly longer than that of wild type and thus give rise to the elongated uppermost internode. It was found that the level of active gibberellin was elevated in the mutant, whereas its growth in response to gibberellin is similar to that of the wild type, suggesting that the higher level accumulation of gibberellin in the eui1 mutant causes the abnormal elongation of the uppermost internode. Consistently, the expression levels of several genes which encode gibberellin biosynthesis enzymes were altered. We cloned the EUI1 gene, which encodes a putative cytochrome P450 monooxygenase, by map-based cloning and found that EUI1 was weakly expressed in most tissues, but preferentially in young panicles. To confirm its function, transgenic experiments with different constructs of EUI1 were conducted. Overexpression of EUI1 gave rise to the gibberellin-deficient-like phenotypes, which could be partially reversed by supplementation with gibberellin. Furthermore, apart from the alteration of expression levels of the gibberellin biosynthesis genes, accumulation of SLR1 protein was found in the overexpressing transgenic plants, indicating that the expression level of EUI1 is implicated in both gibberellin-mediated SLR1 destruction and a feedback regulation in gibberellin biosynthesis. Therefore, we proposed that EUI1 plays a negative role in gibberellin-mediated regulation of cell elongation in the uppermost internode of rice. EUI1,SLR1|OsGAI Fine mapping and in silico isolation of the EUI1 gene controlling upper internode elongation in rice 2006 Plant Mol Biol Institute of Genetics and Crop Breeding, Fujian Agriculture and Forestry University, 350002, Fuzhou, China. Upper internode elongation in rice is an important agronomic trait. Well-known mutants with an elongated uppermost internode (eui) are important germplasms for developing unsheathed-panicle male-sterile lines in hybrid rice breeding. We finely mapped the eui1 gene and identified its candidate gene using in silico analysis based on previous research work and rice genomic sequence data. The rice eui1 gene was mapped to two overlapping BAC clones, OSJNBa0095J22 and OSJNBb0099O15, between the markers AC40 and AC46, that were 0.64 cM apart and spanned approximately 152 kb. A simple sequence repeat (SSR) marker AC41 that cosegregated with eui1 was located in an intron of a putative cytochrome P450-related gene. In silico analysis suggested that this encoded the cytochrome CYP714D1. Allelic sequencing confirmed that EUI1 corresponded to this P450 gene. A gamma ray-induced eui1 mutant carried a deletion in exon II of the EUI1 gene, and resulted in a frame-shift deletion that produced a truncated polypeptide. We conclude that the EUI1 gene controlling the upper internode elongation in rice is 9,804 bp long, and comprises two exons and one intron. The length of the cDNA is 1,931 bp containing a 1,734 bp ORF, a 110 bp 5'-UTR and a 87 bp 3'-UTR. The ORF encodes an unknown 577 amino acid functional protein, that appears to be a member of the cytochrome P450 family. EUI1 FLEXIBLE CULM 1 encoding a cinnamyl-alcohol dehydrogenase controls culm mechanical strength in rice 2009 Plant Mol Biol National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Culm mechanical strength is an important agronomic trait in crop breeding. To understand the molecular mechanisms that control culm mechanical strength, we identified a flexible culm1 (fc1) mutant by screening a rice T-DNA insertion mutant library. This mutant exhibited an abnormal development phenotype, including late heading time, semi-dwarf habit, and flexible culm. In this study, we cloned the FLEXIBLE CULM1 (FC1) gene in rice using a T-DNA tagging approach. FC1 encodes a cinnamyl-alcohol dehydrogenase and is mainly expressed in the sclerenchyma cells of the secondary cell wall and vascular bundle region. In these types of cells, a deficiency of FC1 in the fc1 mutant caused a reduction in cell wall thickness, as well as a decrease in lignin. Extracts from the first internodes and panicles of the fc1 plants exhibited drastically reduced cinnamyl-alcohol dehydrogenase activity. Further histological and biochemical analyses revealed that the p-hydroxyphenyl and guaiacyl monomers in fc1 cell wall were reduced greatly. Our results indicated that FC1 plays an important role in the biosynthesis of lignin and the control of culm strength in rice. FC1|OsCAD7 FINE CULM1 (FC1) works downstream of strigolactones to inhibit the outgrowth of axillary buds in rice 2010 Plant Cell Physiol Graduate School of Agriculture and Life Sciences, University of Tokyo, Yayoi, Bunkyo, Tokyo, 113-8657 Japan. Recent studies of highly branched mutants of pea, Arabidopsis and rice have demonstrated that strigolactones (SLs) act as hormones that inhibit shoot branching. The identification of genes that work downstream of SLs is required for a better understanding of how SLs control the growth of axillary buds. We found that the increased tillering phenotype of fine culm1 (fc1) mutants of rice is not rescued by the application of 1 microM GR24, a synthetic SL analog. Treatment with a high concentration of GR24 (10 microM) causes suppression of tiller growth in wild-type plants, but is not effective on fc1 mutants, implying that proper FC1 functioning is required for SLs to inhibit bud growth. Overexpression of FC1 partially rescued d3-2 defects in the tiller growth and plant height. An in situ hybridization analysis showed that FC1 mRNA accumulates in axillary buds, the shoot apical meristem, young leaves, vascular tissues and the tips of crown roots. FC1 mRNA expression was not significantly affected by GR24, suggesting that transcriptional induction may not be the mechanism by which SLs affect FC1 functioning. On the other hand, the expression level of FC1 is negatively regulated by cytokinin treatment. We propose that FC1 acts as an integrator of multiple signaling pathways and is essential to the fine-tuning of shoot branching in rice. FC1|OsCAD7,OsTB1|FC1 Functional diversification of CLAVATA3-related CLE proteins in meristem maintenance in rice 2008 Plant Cell Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-8654, Japan. Postembryonic development in plants depends on the activity of the shoot apical meristem (SAM) and root apical meristem (RAM). In Arabidopsis thaliana, CLAVATA signaling negatively regulates the size of the stem cell population in the SAM by repressing WUSCHEL. In other plants, however, studies of factors involved in stem cell maintenance are insufficient. Here, we report that two proteins closely related to CLAVATA3, FLORAL ORGAN NUMBER2 (FON2) and FON2-LIKE CLE PROTEIN1 (FCP1/Os CLE402), have functionally diversified to regulate the different types of meristem in rice (Oryza sativa). Unlike FON2, which regulates the maintenance of flower and inflorescence meristems, FCP1 appears to regulate the maintenance of the vegetative SAM and RAM. Constitutive expression of FCP1 results in consumption of the SAM in the vegetative phase, and application of an FCP1 CLE peptide in vitro disturbs root development by misspecification of cell fates in the RAM. FON1, a putative receptor of FON2, is likely to be unnecessary for these FCP1 functions. Furthermore, we identify a key amino acid residue that discriminates between the actions of FCP1 and FON2. Our results suggest that, although the basic framework of meristem maintenance is conserved in the angiosperms, the functions of the individual factors have diversified during evolution. FCP1|OsCLE402,FON1,FON2|FON4 WUSCHEL-RELATED HOMEOBOX4 is involved in meristem maintenance and is negatively regulated by the CLE gene FCP1 in rice 2013 Plant Cell Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-8654, Japan. The shoot apical meristem is the ultimate source of the cells that constitute the entire aboveground portion of the plant body. In Arabidopsis thaliana, meristem maintenance is regulated by the negative feedback loop of WUSCHEL-CLAVATA (WUS-CLV). Although CLV-like genes, such as FLORAL ORGAN NUMBER1 (FON1) and FON2, have been shown to be involved in maintenance of the reproductive meristems in rice (Oryza sativa), current understanding of meristem maintenance remains insufficient. In this article, we demonstrate that the FON2-LIKE CLE PROTEIN1 (FCP1) and FCP2 genes encoding proteins with similar CLE domains are involved in negative regulation of meristem maintenance in the vegetative phase. In addition, we found that WUSCHEL-RELATED HOMEOBOX4 (WOX4) promotes the undifferentiated state of the meristem in rice and that WOX4 function is associated with cytokinin action. Consistent with similarities in the shoot apical meristem phenotypes caused by overexpression of FCP1 and downregulation of WOX4, expression of WOX4 was negatively regulated by FCP1 (FCP2). Thus, FCP1/2 and WOX4 are likely to be involved in maintenance of the vegetative meristem in rice. FCP1|OsCLE402,FCP2,OsWOX4 A CLE-WOX signalling module regulates root meristem maintenance and vascular tissue development in rice 2013 J Exp Bot School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (ESR)-related (CLE) proteins belong to a small peptide family conserved in plants. Recent studies in Arabidopsis and rice have revealed a key role for CLEs in mediating cell-cell communication and stem cell maintenance during plant development, but how CLE signalling controls root development in the rice remains largely unknown. Here it is shown that exogenous application of a synthetic dodeca-amino acid peptide corresponding to the CLE motif of the rice FON2-LIKE CLE PROTEIN2 (FCP2p) protein or overexpression of FCP2 terminates root apical meristem (RAM) activity and impairs late metaxylem formation. FCP2p treatment suppresses the expression of the rice QUIESCENT-CENTER-SPECIFIC HOMEOBOX (QHB) gene, a putative orthologue of Arabidopsis WUSCHEL (WUS)-RELATED HOMEOBOX 5 (WOX5) gene, in both quiescent centre and late metaxylem cells; whereas inducible overexpression of QHB reduces the sensitivity of rice to FCP2p treatment. These results together suggest that in rice RAM maintenance and late metaxylem development are probably controlled by the mutual regulation between FCP2 and QHB. Moreover, a cross-species peptide treatment experiment in Arabidopsis implies that FCP2 has both evolutionarily conserved and species-specific roles in root development. FCP2 Isolation and characterization of IRO2, a novel iron-regulated bHLH transcription factor in graminaceous plants 2006 J Exp Bot Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1 Yayoi, Tokyo 113-8657, Japan. To clarify the molecular mechanism that regulates iron (Fe) acquisition in graminaceous plants, a time-course analysis of gene expression during Fe deficiency stress was conducted using a rice 22K oligo-DNA microarray. Twenty-one genes for proteins that function in gene regulation were induced by Fe deficiency. Of these genes, a putative basic helix-loop-helix (bHLH) transcription factor gene, named OsIRO2, was strongly expressed in both roots and shoots during Fe deficiency stress. The expression of OsIRO2 was induced exclusively by Fe deficiency, and not by deficiencies in other metals. Expression of the barley HvIRO2 gene, which is a homologue of OsIRO2, was also induced by Fe deficiency. An in silico search revealed that IRO2 is highly conserved among graminaceous plants, which include wheat, sorghum, and maize. The cyclic amplification and selection of targets (CASTing) technique revealed that OsIRO2 bound preferentially to the sequence 5'-ACCACGTGGTTTT-3', and the electrophoretic mobility shift assay revealed 5'-CACGTGG-3' as the core sequence for OsIRO2 binding. Sequences similar to the OsIRO2-binding sequence were found upstream of several genes that are involved in Fe acquisition, such as OsNAS1, OsNAS3, OsIRT1, OsFDH, OsAPT1, and IDS3. The core sequence of the OsIRO2-binding sequence occurred more frequently in the upstream regions of Fe deficiency-inducible genes than in the corresponding regions of non-inducible genes. These results suggest that IRO2 is involved in the regulation of gene expression under Fe-deficient conditions. FDH,OsAPT1|APRT,OsIRO2,OsIRT1,OsNAS1,OsNAS3 Expression profiling of Oryza sativa metal homeostasis genes in different rice cultivars using a cDNA macroarray 2007 Plant Physiol Biochem USDA-ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, 1100 Bates Street, Houston, TX 77030, USA. Rice is an important food crop, but it is a poor source of essential micronutrients such as iron and zinc. In order to improve the metal ion content of rice grains through breeding or biotechnology, more information is needed on the molecular players that help mobilize metals from leaves to developing seeds. To profile several genes simultaneously, a cDNA macroarray was developed using 36 metal-related genes from rice, including ZIPs, NRAMPs, and YSLs (coding for known or potential metal transporters), as well as NAS, FER, FRO, NAAT, FDH, GSTU, and PDR (involved in metal homeostasis). Because flag leaves are the major source of phloem-delivered photoassimilates and remobilized metals for developing seeds, we analyzed the expression of these metal-related genes in flag and non-flag leaves of four different rice cultivars (Cocodrie, Taipei 309, IR58, and IR68144) during the period of mid-grain fill. Genes (24 of 36) exhibited low to non-detectable signals in the macroarray, while 12 genes (OsIRT1, OsZIP1, OsZIP5, OsZIP8, OsYSL5, OsYSL6, OsYSL7, OsYSL8, OsYSL18, OsNRAMP2, OsNRAMP4 and OsNRAMP7) were found to be highly expressed in both flag and non-flag leaves of all four cultivars. Additional expression analysis using semi-quantitative or quantitative PCR provided results that were generally consistent with the macroarray, but semi-quantitative PCR confirmed that OsFDH, OsFER1, OsNAAT, OsNAS1, OsPDR9, OsYSL12, OsYSL13, OsZIP7, and OsZIP10 were also expressed in leaves. This specialized macroarray has provided a short list of potential candidate genes, expressed in leaves, which might contribute to the process of metal transport to distant sinks, such as seeds. FDH,OsZIP1,OsZIP3,OsZIP6 BENT UPPERMOST INTERNODE1 encodes the class II formin FH5 crucial for actin organization and rice development 2011 Plant Cell National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China. The actin cytoskeleton is an important regulator of cell expansion and morphogenesis in plants. However, the molecular mechanisms linking the actin cytoskeleton to these processes remain largely unknown. Here, we report the functional analysis of rice (Oryza sativa) FH5/BENT UPPERMOST INTERNODE1 (BUI1), which encodes a formin-type actin nucleation factor and affects cell expansion and plant morphogenesis in rice. The bui1 mutant displayed pleiotropic phenotypes, including bent uppermost internode, dwarfism, wavy panicle rachis, and enhanced gravitropic response. Cytological observation indicated that the growth defects of bui1 were caused mainly by inhibition of cell expansion. Map-based cloning revealed that BUI1 encodes the class II formin FH5. FH5 contains a phosphatase tensin-like domain at its amino terminus and two highly conserved formin-homology domains, FH1 and FH2. In vitro biochemical analyses indicated that FH5 is capable of nucleating actin assembly from free or profilin-bound monomeric actin. FH5 also interacts with the barbed end of actin filaments and prevents the addition and loss of actin subunits from the same end. Interestingly, the FH2 domain of FH5 could bundle actin filaments directly and stabilize actin filaments in vitro. Consistent with these in vitro biochemical activities of FH5/BUI1, the amount of filamentous actin decreased, and the longitudinal actin cables almost disappeared in bui1 cells. The FH2 or FH1FH2 domains of FH5 could also bind to and bundle microtubules in vitro. Thus, our study identified a rice formin protein that regulates de novo actin nucleation and spatial organization of the actin filaments, which are important for proper cell expansion and rice morphogenesis. FH5|RMD|BUI1 RICE MORPHOLOGY DETERMINANT encodes the type II formin FH5 and regulates rice morphogenesis 2011 Plant Cell School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. Multicellular organisms contain a large number of formins; however, their physiological roles in plants remain poorly understood. Here, we reveal that formin homology 5 (FH5), a type II formin mutated in rice morphology determinant (rmd), plays a crucial role in determining rice (Oryza sativa) morphology. FH5/RMD encodes a formin-like protein consisting of an N-terminal phosphatase tensin (PTEN)-like domain, an FH1 domain, and an FH2 domain. The rmd mutants display a bending growth pattern in seedlings, are stunted as adult plants, and have aberrant inflorescence (panicle) and seed shape. Cytological analysis showed that rmd mutants have severe cell elongation defects and abnormal microtubule and microfilament arrays. FH5/RMD is ubiquitously expressed in rice tissues, and its protein localization to the chloroplast surface is mediated by the PTEN domain. Biochemical assays demonstrated that recombinant FH5 protein can nucleate actin polymerization from monomeric G-actin or actin/profilin complexes, cap the barbed end of actin filaments, and bundle actin filaments in vitro. Moreover, FH5 can directly bind to and bundle microtubules through its FH2 domain in vitro. Our findings suggest that the rice formin protein FH5 plays a critical role in determining plant morphology by regulating actin dynamics and proper spatial organization of microtubules and microfilaments. FH5|RMD|BUI1 Rice actin-binding protein RMD is a key link in the auxin–actin regulatory loop that controls cell growth 2014 Proceedings of the National Academy of Sciences State Key Laboratory of Hybrid Rice, Shanghai Jiao Tong University, Shanghai 20040, China The plant hormone auxin plays a central role in plant growth and development. Auxin transport and signaling depend on actin organization. Despite its functional importance, the mechanistic link between actin filaments (F-actin) and auxin intracellular signaling remains unclear. Here, we report that the actin-organizing protein Rice Morphology Determinant (RMD), a type II formin from rice (Oryza sativa), provides a key link. Mutants lacking RMD display abnormal cell growth and altered configuration of F-actin array direction. The rmd mutants also exhibit an inhibition of auxin-mediated cell elongation, decreased polar auxin transport, altered auxin distribution gradients in root tips, and suppression of plasma membrane localization of auxin transporters O. sativa PIN-FORMED 1b (OsPIN1b) and OsPIN2 in root cells. We demonstrate that RMD is required for endocytosis, exocytosis, and auxin-mediated OsPIN2 recycling to the plasma membrane. Moreover, RMD expression is directly regulated by heterodimerized O. sativa auxin response factor 23 (OsARF23) and OsARF24, providing evidence that auxin modulates the orientation of F-actin arrays through RMD. In support of this regulatory loop, osarf23 and lines with reduced expression of both OsARF23 and OsARF24 display reduced RMD expression, disrupted F-actin organization and cell growth, less sensitivity to auxin response, and altered auxin distribution and OsPIN localization. Our findings establish RMD as a crucial component of the auxin–actin self-organizing regulatory loop from the nucleus to cytoplasm that controls rice cell growth and morphogenesis. FH5|RMD|BUI1,OsARF24,UBQ The rice FISH BONE gene encodes a tryptophan aminotransferase, which affects pleiotropic auxin-related processes 2014 Plant J Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, 113-8657, Japan. Auxin is a fundamental plant hormone and its localization within organs plays pivotal roles in plant growth and development. Analysis of many Arabidopsis mutants that were defective in auxin biosynthesis revealed that the indole-3-pyruvic acid (IPA) pathway, catalyzed by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) families, is the major biosynthetic pathway of indole-3-acetic acid (IAA). In contrast, little information is known about the molecular mechanisms of auxin biosynthesis in rice. In this study, we identified a auxin-related rice mutant, fish bone (fib). FIB encodes an orthologue of TAA genes and loss of FIB function resulted in pleiotropic abnormal phenotypes, such as small leaves with large lamina joint angles, abnormal vascular development, small panicles, abnormal organ identity and defects in root development, together with a reduction in internal IAA levels. Moreover, we found that auxin sensitivity and polar transport activity were altered in the fib mutant. From these results, we suggest that FIB plays a pivotal role in IAA biosynthesis in rice and that auxin biosynthesis, transport and sensitivity are closely interrelated. FIB Genetic analysis of endosperm mutants in rice Oryza sativa L 1991 Theor Appl Genet Division of Plant Breeding, Genetics, and Biochemistry, International Rice Research Institute, P.O. Box 933, Manila, Philippines. Sugary, shrunken, floury, white core, amylose extender and dull mutants induced in japonica varieties were used in this study. The results of an allelic analysis conducted in japonica background indicated that the two sugary mutants 82GF and EM5 are allelic. The two amylose extender mutants 2064 and EM16 are also allelic. The opaque mutant ESD7-3(0) and floury mutants 2047, EM17 and EM28 are allelic as well and have the flo-1 gene. The three white core mutants EM3, EM24 and EM66 were found to be non-allelic. Eleven dull mutants were investigated. Dull mutants 2057, 2083, 2091 and EM15 were found to be allelic to each other. Similarly, dull mutants 2077, 2078 and 2120 have allelic genes. Dull mutants 2035, EM12, EM47, and EM98 are non-allelic to the above loci. Dull genes in EM12, EM15, and EM98 were designated earlier as du-1, du-2 and du-4, respectively.The mutant genes were transferred to indica background by two backcrosses to IR36. Some of the mutant genes were located to respective chromosomes through trisomic analysis using primary trisomics of IR36. In this way the amylose extender gene ae was located to chromosome 2, the flo-1 was located to chromosome 5 and the flo-2 to chromosome 4. Dull genes of EM47, 2120, and 2035 were assigned to chromosomes 6, 9, and 6, respectively. FLO2 A novel factor FLOURY ENDOSPERM2 is involved in regulation of rice grain size and starch quality 2010 Plant Cell Department of Biological Science and Technology, Tokyo University of Science, Noda, Japan. Rice (Oryza sativa) endosperm accumulates a massive amount of storage starch and storage proteins during seed development. However, little is known about the regulatory system involved in the production of storage substances. The rice flo2 mutation resulted in reduced grain size and starch quality. Map-based cloning identified FLOURY ENDOSPERM2 (FLO2), a member of a novel gene family conserved in plants, as the gene responsible for the rice flo2 mutation. FLO2 harbors a tetratricopeptide repeat motif, considered to mediate a protein-protein interactions. FLO2 was abundantly expressed in developing seeds coincident with production of storage starch and protein, as well as in leaves, while abundant expression of its homologs was observed only in leaves. The flo2 mutation decreased expression of genes involved in production of storage starch and storage proteins in the endosperm. Differences between cultivars in their responsiveness of FLO2 expression during high-temperature stress indicated that FLO2 may be involved in heat tolerance during seed development. Overexpression of FLO2 enlarged the size of grains significantly. These results suggest that FLO2 plays a pivotal regulatory role in rice grain size and starch quality by affecting storage substance accumulation in the endosperm. FLO2 FLOURY ENDOSPERM6 encodes a CBM48 domain-containing protein involved in compound granule formation and starch synthesis in rice endosperm 2014 Plant J State Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing, 210095, China. Starch is the most widespread form of energy storage in the plant kingdom. Although many enzymes and related factors have been identified for starch biosynthesis, unknown players remain to be identified, given that it is a complicated and sophisticated process. The endosperm of rice (Oryza sativa) has been used for the study of starch synthesis. Here, we report the cloning and characterization of the FLOURY ENDOSPERM6 (FLO6) gene in rice. In the flo6 mutant, the starch content is decreased and the normal physicochemical features of starch are changed. Significantly, flo6 mutant endosperm cells show obvious defects in compound granule formation. Map-based cloning showed that FLO6 encodes a protein of unknown function. It harbors an N-terminal transit peptide that ensures its correct localization and functions in the plastid, and a C-terminal carbohydrate-binding module 48 (CBM48) domain that binds to starch. Furthermore, FLO6 can interact with isoamylase1 (ISA1) both in vitro and in vivo, whereas ISA1 does not bind to starch directly. We thus propose that FLO6 may act as a starch-binding protein involved in starch synthesis and compound granule formation through a direct interaction with ISA1 in developing rice seeds. Our data provide a novel insight into the role of proteins with the CBM48 domain in plant species. FLO6 The rice FON1 gene controls vegetative and reproductive development by regulating shoot apical meristem size 2006 Mol Cells National Research Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea. Most plant organs develop from meristems. Rice FON1, which is an ortholog of Clv1, regulates stem cell proliferation and organ initiation. The point muta-tions, fon1-1 and fon1-2, disrupt meristem balance, resulting in alteration of floral organ numbers and the architecture of primary rachis branches. In this study, we identified two knockout alleles, fon1-3 and fon1-4, generated by T-DNA and Tos17 insertion, respectively. Unlike the previously isolated point mutants, the null mutants have alterations not only of the reproductive organs but also of vegetative tissues, producing fewer tillers and secondary rachis branches. The mutant plants are semi-dwarfs due to delayed leaf emergence, and leaf senescence is delayed. SEM analysis showed that the shoot apical meristems of fon1-3 mutants are enlarged. These results indicate that FON1 controls vegetative as well as reproductive development by regulating meristem size. FON1 The gene FLORAL ORGAN NUMBER1 regulates floral meristem size in rice and encodes a leucine-rich repeat receptor kinase orthologous to Arabidopsis CLAVATA1 2004 Development Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan. The regulation of floral organ number is closely associated with floral meristem size. Mutations in the gene FLORAL ORGAN NUMBER1 (FON1) cause enlargement of the floral meristem in Oryza sativa (rice), resulting in an increase in the number of all floral organs. Ectopic floral organs develop in the whorl of each organ and/or in the additional whorls that form. Inner floral organs are more severely affected than outer floral organs. Many carpel primordia develop indeterminately, and undifferentiated meristematic tissues remain in the center in almost-mature flowers. Consistent with this result, OSH1, a molecular marker of meristematic indeterminate cells in rice, continues to be expressed in this region. Although floral meristems are strongly affected by the fon1-2 mutation, vegetative and inflorescence meristems are largely normal, even in this strong allele. We isolated the FON1 gene by positional cloning and found that it encodes a leucine-rich repeat receptor-like kinase most similar to CLAVATA1 (CLV1) in Arabidopsis thaliana. This suggests that a pathway similar to the CLV signaling system that regulates meristem maintenance in Arabidopsis is conserved in the grass family. Unlike CLV1, which is predominantly expressed in the L3 layer of the shoot meristem, FON1 is expressed throughout the whole floral meristem, suggesting that small modifications to the CLV signaling pathway may be required to maintain the floral meristem in rice. In addition, FON1 transcripts are detected in all meristems responsible for development of the aerial part of rice, suggesting that genes sharing functional redundancy with FON1 act in the vegetative and inflorescence meristems to mask the effects of the fon1 mutation. FON1,OSH1|Oskn1 The floral organ number4 gene encoding a putative ortholog of Arabidopsis CLAVATA3 regulates apical meristem size in rice 2006 Plant Physiol Shanghai Jiaotong University, Shanghai Institutes for Biological Sciences, Shanghai, China. To understand the molecular mechanism regulating meristem development in the monocot rice (Oryza sativa), we describe here the isolation and characterization of three floral organ number4 (fon4) alleles and the cloning of the FON4 gene. The fon4 mutants showed abnormal enlargement of the embryonic and vegetative shoot apical meristems (SAMs) and the inflorescence and floral meristems. Likely due to enlarged SAMs, fon4 mutants produced thick culms (stems) and increased numbers of both primary rachis branches and floral organs. We identified FON4 using a map-based cloning approach and found it encodes a small putatively secreted protein, which is the putative ortholog of the Arabidopsis (Arabidopsis thaliana) CLAVATA3 (CLV3) gene. FON4 transcripts mainly accumulated in the small group of cells at the apex of the SAMs, whereas the rice ortholog of CLV1 (FON1) is expressed throughout the SAMs, suggesting that the putative FON4 ligand might be sequestered as a possible mechanism for rice meristem regulation. Exogenous application of the peptides FON4p and CLV3p corresponding to the CLV3/ESR-related (CLE) motifs of FON4 and CLV3, respectively, resulted in termination of SAMs in rice, and treatment with CLV3p caused consumption of both rice and Arabidopsis root meristems, suggesting that the CLV pathway in limiting meristem size is conserved in both rice and Arabidopsis. However, exogenous FON4p did not have an obvious effect on limiting both rice and Arabidopsis root meristems, suggesting that the CLE motifs of Arabidopsis CLV3 and FON4 are potentially functionally divergent. FON1,FON2|FON4 FON2 SPARE1 redundantly regulates floral meristem maintenance with FLORAL ORGAN NUMBER2 in rice 2009 PLoS Genet Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan. CLAVATA signaling restricts stem cell identity in the shoot apical meristem (SAM) in Arabidopsis thaliana. In rice (Oryza sativa), FLORAL ORGAN NUMBER2 (FON2), closely related to CLV3, is involved as a signaling molecule in a similar pathway to negatively regulate stem cell proliferation in the floral meristem (FM). Here we show that the FON2 SPARE1 (FOS1) gene encoding a CLE protein functions along with FON2 in maintenance of the FM. In addition, FOS1 appears to be involved in maintenance of the SAM in the vegetative phase, because constitutive expression of FOS1 caused termination of the vegetative SAM. Genetic analysis revealed that FOS1 does not need FON1, the putative receptor of FON2, for its action, suggesting that FOS1 and FON2 may function in meristem maintenance as signaling molecules in independent pathways. Initially, we identified FOS1 as a suppressor that originates from O. sativa indica and suppresses the fon2 mutation in O. sativa japonica. FOS1 function in japonica appears to be compromised by a functional nucleotide polymorphism (FNP) at the putative processing site of the signal peptide. Sequence comparison of FOS1 in about 150 domesticated rice and wild rice species indicates that this FNP is present only in japonica, suggesting that redundant regulation by FOS1 and FON2 is commonplace in species in the Oryza genus. Distribution of the FNP also suggests that this mutation may have occurred during the divergence of japonica from its wild ancestor. Stem cell maintenance may be regulated by at least three negative pathways in rice, and each pathway may contribute differently to this regulation depending on the type of the meristem. This situation contrasts with that in Arabidopsis, where CLV signaling is the major single pathway in all meristems. FON2|FON4,FOS1 Conservation and diversification of meristem maintenance mechanism in Oryza sativa: Function of the FLORAL ORGAN NUMBER2 gene 2006 Plant Cell Physiol Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan. To elucidate the genetic mechanism that regulates meristem maintenance in monocots, here we have examined the function of the gene FLORAL ORGAN NUMBER2 (FON2) in Oryza sativa (rice). Mutations in FON2 cause enlargement of the floral meristem, resulting in an increase in the number of floral organs, although the vegetative and inflorescence meristems are largely normal. Molecular cloning reveals that FON2 encodes a small secreted protein, containing a CLE domain, that is closely related to CLAVATA3 in Arabidopsis thaliana. FON2 transcripts are localized at the apical region in all meristems in the aerial parts of rice plants, showing an expression pattern similar to that of Arabidopsis CLV3. Constitutive expression of FON2 causes a reduction in the number of floral organs and flowers, suggesting that both the flower and inflorescence meristems are reduced in size. This action of FON2 requires the function of FON1, an ortholog of CLV1. Constitutive expression of FON2 also causes premature termination of the shoot apical meristem in Arabidopsis, a phenotype similar to that caused by constitutive expression of CLV3. Together with our previous study of FON1, these results clearly indicate that the FON1-FON2 system in rice corresponds to the CLV signaling system in Arabidopsis and suggest that the negative regulation of stem cell identity by these systems may be principally conserved in a wide range of plants within the Angiosperms. In addition, we propose a model of the genetic regulation of meristem maintenance in rice that includes an alternative pathway independent of FON2-FON1. FON2|FON4 The rice flattened shoot meristem, encoding CAF-1 p150 subunit, is required for meristem maintenance by regulating the cell-cycle period 2008 Dev Biol Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. We isolated flattened shoot meristem (fsm) mutants in rice that showed defective seedling growth and died in the vegetative phase. Since most fsm plants had flat and small shoot apical meristems (SAMs), we suggest that FSM is required for proper SAM maintenance. FSM encodes a putative ortholog of Arabidopsis FASCIATA1 (FAS1) that corresponds to the p150 subunit of chromatin assembly factor-1 (CAF-1). FSM is expressed patchily in tissues with actively dividing cells, suggesting a tight association of FSM with specific cell-cycle phases. Double-target in situ hybridization counterstained with cell-cycle marker genes revealed that FSM is expressed mainly in the G(1) phase. In fsm, expressions of the two marker genes representing S- and G(2)- to M-phases were enhanced in SAM, despite a reduced number of cells in SAM, suggesting that S- and G(2)-phases are prolonged in fsm. In addition, developmental events in fsm leaves took place at the proper time, indicating that the temporal regulation of development occurs independently of the cell-cycle period. In contrast to the fasciated phenotype of Arabidopsis fas1, fsm showed size reduction of SAM. The opposite phenotypes between fsm and fas1 indicate that the SAM maintenance is regulated differently between rice and Arabidopsis. FSM|CAF-1 A novel frameshift mutant allele, fzp-10, affecting the panicle architecture of rice 2011 Euphytica Faculty of Biology-Oriented Science and Technology, Kinki University, 930 Nishimitani, Kinokawa, Wakayama, 649-6493, Japan The rice FRIZZY PANICLE (FZP) locus on chromosome 7, in which an ERF and acidic domain are present, is concerned with the regulation of spikelet meristem identity and the determination of panicle architecture. Many fzp mutants drastically alter panicle morphology with higher-order rachis-branches developing successively instead of the development of floral organs in these mutants. A new mutant showing the same fzp phenotype was induced by γ-ray irradiation of seeds of a rice cultivar “Gimbozu”. Examination of this fzp-like mutant for its allelism to a known allele of fzp-1, nucleotide sequence, and panicle and agronomic characteristics clearly indicated that the allele of this fzp-like mutant is located in FZP. Because there is a previously identified allele called fzp-9, we designated this new allele as fzp-10. fzp-10 has a single nucleotide (cytosine) deletion between the ERF domain and the acidic domain, which results in a frameshift mutation and a premature stop codon, thereby altering the C-terminus of the encoded protein. The degree of higher-order branching in the panicles was significantly reduced in the fzp-10 mutant compared with that of fzp-1. Moreover, the fzp-10 mutant showed highly depressed culm and panicle lengths and panicle number, as well as delayed heading dates compared with its wild type and also with fzp-1. fzp-10 has several new characteristics, its altered nucleotide position and severity of phenotype alteration, and, therefore, could be a new gene resource to examine the function of FZP and the determination of rice panicle architecture. FZP|BFL1 Ds tagging of BRANCHED FLORETLESS 1 (BFL1) that mediates the transition from spikelet to floret meristem in rice (Oryza sativa L) 2003 BMC Plant Biol CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia. qianhao.zhu@csiro.au BACKGROUND: The genetics of spikelet formation, a feature unique to grasses such as rice and maize, is yet to be fully understood, although a number of meristem and organ identity mutants have been isolated and investigated in Arabidopsis and maize. Using a two-element Ac/Ds transposon tagging system we have isolated a rice mutant, designated branched floretless 1 (bfl1) which is defective in the transition from spikelet meristem to floret meristem. RESULTS: The bfl1 mutant shows normal differentiation of the primary rachis-branches leading to initial spikelet meristem (bract-like structure equivalent to rudimentary glumes) formation but fails to develop empty glumes and florets. Instead, axillary meristems in the bract-like structure produce sequential alternate branching, thus resulting in a coral shaped morphology of the branches in the developing panicle. The bfl1 mutant harbours a single Ds insertion in the upstream region of the BFL1 gene on chromosome 7 corresponding to PAC clone P0625E02 (GenBank Acc No. message URL http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=nucleotide&list_uid s=34395191&dopt=GenBank&term=ap004570AP004570). RT-PCR analyses revealed a drastic reduction of BFL1 transcript levels in the bfl1 mutant compared to that in the wild-type. In each of the normal panicle-bearing progeny plants, from occasional revertant seeds of the vegetatively-propagated mutant plant, Ds was shown to be excised from the bfl1 locus. BFL1 contains an EREBP/AP2 domain and is most likely an ortholog of the maize transcription factor gene BRANCHED SILKLESS1 (BD1). CONCLUSIONS: bfl1 is a Ds-tagged rice mutant defective in the transition from spikelet meristem (SM) to floret meristem (FM). BFL1 is most probably a rice ortholog of the maize ERF (EREBP/AP2) transcription factor gene BD1. Based on the similarities in mutant phenotypes bfl1 is likely to be an allele of the previously reported frizzy panicle locus. FZP|BFL1 FRIZZY PANICLE is required to prevent the formation of axillary meristems and to establish floral meristem identity in rice spikelets 2003 Development Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. Inflorescences of grass species have a distinct morphology in which florets are grouped in compact branches called spikelets. Although many studies have shown that the molecular and genetic mechanisms that control floret organ formation are conserved between monocots and dicots, little is known about the genetic pathway leading to spikelet formation. In the frizzy panicle (fzp) mutant of rice, the formation of florets is replaced by sequential rounds of branching. Detailed analyses revealed that several rudimentary glumes are formed in each ectopic branch, indicating that meristems acquire spikelet identity. However, instead of proceeding to floret formation, axillary meristems are formed in the axils of rudimentary glumes and they either arrest or develop into branches of higher order. The fzp mutant phenotype suggests that FZP is required to prevent the formation of axillary meristems within the spikelet meristem and permit the subsequent establishment of floral meristem identity. The FZP gene was isolated by transposon tagging. FZP encodes an ERF transcription factor and is the rice ortholog of the maize BD1 gene. Consistent with observations from phenotypic analyses, FZP expression was found to be restricted to the time of rudimentary glumes differentiation in a half-ring domain at the base of which the rudimentary glume primordium emerged. FZP|BFL1 Two AP2 family genes, supernumerary bract (SNB) and Osindeterminate spikelet 1 (OsIDS1), synergistically control inflorescence architecture and floral meristem establishment in rice 2012 Plant J Department of Plant Molecular Systems Biotechnology, Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Korea. Meristem identity is crucial in determining the inflorescence architecture of grass species. We previously reported that SUPERNUMERARY BRACT (SNB) regulates the transition of spikelet meristems into floral meristems in rice (Oryza sativa). Here we demonstrated that SNB and Oryza sativa INDETERMINATE SPIKELET 1 (OsIDS1) together play important roles in inflorescence architecture and the establishment of floral meristems. In snb osids1 double mutants, the numbers of branches and spikelets within a panicle are significantly decreased, and the transition to a floral meristem is further delayed compared with the snb single mutant. Expression analyses showed that SNB and OsIDS1 are required for spatio-temporal expression of B- and E-function floral organ identity genes in the lodicules. In addition, the AP2 family genes are important for determining the degree of ramification in branch meristems, regulating the spatio-temporal expression of spikelet meristem genes, such as FRIZZY PANICLE (FZP). Furthermore, overexpression of microRNA172 (miR172) causes reductions in SNB and OsIDS1 transcript levels, and phenotypes of the transgenic plants are more severe than for snb osids1. This indicates that additional gene(s) participate in the development of branch and floral meristems. Preferential expression of mature miR172s in the area around the spikelet meristems implies that depletion of the AP2 family genes in those meristems via miR172 is an important step in controlling inflorescence branching and the formation of floral organs. FZP|BFL1,OsIDS1,SNB Morphological and molecular characterization of a new frizzy panicle mutant, "fzp-9(t)", in rice (Oryza sativa L.) 2005 Hereditas Yeongnam Agricultural Research Institute, NICS, RDA, Milyang, Korea. The spikelet identity gene "fzp'' (frizzy panicle) is required for transformation of the floral meristems to inflorescent shoots. In fzp mutants, spikelets are replaced by branches and spikelet meristems produce massive numbers of branch meristems. We have isolated and characterized a new fzp mutant derived from anther culture lines in rice and designated as fzp-9 (t). The fzp-9 (t) mutant showed retarded growth habit and developed fewer tillers than those of the wild- type plant. The primary and secondary rachis branches of fzp-9 (t) appeared to be normal, but higher-order branches formed continuous bract- like structures without developing spikelets. The genetic segregation of fzp-9 (t) showed a good fit to the expected ratio of 3:1. The sequence analysis of fzp-9(t) revealed that there is a single nucleotide base change upstream of the ERF (ethylene-responsive element-binding factor) domain compare to wild-type plant. The mutation point of fzp-9 (t) (W66G) was one of the six amino acids of the ERF domain that contributed to GCC box-specific binding. The premature formation of a stop codon at the beginning of the ERF domain might cause a non-functional product. FZP|BFL1 The homeotic gene long sterile lemma (G1) specifies sterile lemma identity in the rice spikelet 2009 Proc Natl Acad Sci U S A Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-8654, Japan. The mechanism of floral organ specification is principally conserved in angiosperms, as demonstrated by the ABC model. By contrast, mechanisms that regulate the development of organs or structures specific to a group of species remain unclear. Grasses have unique inflorescence units, comprising spikelets and florets. In the genus Oryza (rice), the single spikelet consists of a fertile floret subtended by a lemma and a palea, two sterile lemmas, and rudimentary glumes. Each sterile lemma is a tiny glume-like organ with a smooth surface. Here, we have examined a long sterile lemma1 (g1) mutant, in which the sterile lemma is enlarged like the lemma. Detailed phenotypic analysis reveals that the large sterile lemma in the g1 mutant appears to be caused by homeotic transformation of the sterile lemma into a lemma, suggesting that G1 is involved in the repression of lemma identity to specify the sterile lemma. Gene isolation reveals that G1 is a member of a plant-specific gene family that encodes proteins with a previously uncharacterized domain, named here ALOG (Arabidopsis LSH1 and Oryza G1). G1 mRNA is expressed in sterile lemma primordia throughout their development, and G1 protein is localized in the nucleus. A trans-activation assay using the yeast GAL4 system suggests that G1 is involved in transcriptional regulation. Repression of lemma identity by G1 is consistent with a hypothesis proposed to explain the morphological evolution of rice spikelets. We also show that a wild rice species, Oryza grandiglumis, that forms large sterile lemmas has serious mutations in the G1 gene. G1 Molecular cloning of plant transcripts encoding protein kinase homologs 1989 Proc Natl Acad Sci U S A Plant Biology Laboratory, Salk Institute for Biological Studies, San Diego, CA 92138-9216. Oligonucleotides, corresponding to conserved regions of animal protein-serine/threonine kinases, were used to isolate cDNAs encoding plant homologs in the dicot bean (Phaseolus vulgaris L.) and the monocot rice (Oryzae sativa L.). The C-terminal regions of the deduced polypeptides encoded by the bean (PVPK-1) and rice (G11A) cDNAs, prepared from mRNAs of suspension cultures and leaves, respectively, contain features characteristic of the catalytic domains of eukaryotic protein-serine/threonine kinases, indicating that these cDNAs encode plant protein kinases. The putative catalytic domains are most closely related to cyclic nucleotide-dependent protein kinases and the protein kinase C family, suggesting the plant homologs may likewise transduce extracellular signals. However, outside these domains, PVPK-1 and G11A exhibit no homology either to each other or to regulatory domains of other protein kinases, indicating the plant homologs are modulated by other signals. PVPK-1 corresponds to a 2.4-kb transcript in suspension cultured bean cells. Southern blots of genomic DNA indicate that PVPK-1 and G11A correspond to single copy genes that form part of a family of related plant sequences. G11A Overexpression of a type-A response regulator alters rice morphology and cytokinin metabolism 2007 Plant Cell Physiol RIKEN Plant Science Center, 1-7-22, Suehiro, Tsurumi, Yokohama, 230-0045, Japan. Genome-wide analyses of rice (Oryza sativa L.) cytokinin (CK)-responsive genes using the Affymetrix GeneChip(R) rice genome array were conducted to define the spectrum of genes subject to regulation by CK in monocotyledonous plants. Application of trans-zeatin modulated the expression of a wide variety of genes including those involved in hormone signaling and metabolism, transcriptional regulation, macronutrient transport and protein synthesis. To understand further the function of CK in rice plants, we examined the effects of in planta manipulation of a putative CK signaling factor on morphology, CK metabolism and expression of CK-responsive genes. Overexpression of the CK-inducible type-A response regulator OsRR6 abolished shoot regeneration, suggesting that OsRR6 acts as a negative regulator of CK signaling. Transgenic lines overexpressing OsRR6 (OsRR6-ox) had dwarf phenotypes with poorly developed root systems and panicles. Increased content of trans-zeatin-type CKs in OsRR6-ox lines indicates that homeostatic control of CK levels is regulated by OsRR6 signaling. Expression of genes encoding CK oxidase/dehydrogenase decreased in OsRR6-ox plants, possibly accounting for elevated CK levels in transgenic lines. Expression of a number of stress response genes was also altered in OsRR6-ox plants. GA20OX4,GA2OX3,OsRR3,OsRR5,OsRR6,OsTMK Cryptochrome and phytochrome cooperatively but independently reduce active gibberellin content in rice seedlings under light irradiation 2012 Plant Cell Physiol Photobiology and Photosynthesis Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8602 Japan. fumih@affrc.go.jp In contrast to a wealth of knowledge about the photoregulation of gibberellin metabolism in dicots, that in monocots remains largely unclear. In this study, we found that a blue light signal triggers reduction of active gibberellin content in rice seedlings with simultaneous repression of two gibberellin 20-oxidase genes (OsGA20ox2 and OsGA20ox4) and acute induction of four gibberellin 2-oxidase genes (OsGA2ox4-OsGA2ox7). For further examination of the regulation of these genes, we established a series of cryptochrome-deficient lines through reverse genetic screening from a Tos17 mutant population and construction of knockdown lines based on an RNA interference technique. By using these lines and phytochrome mutants, we elucidated that cryptochrome 1 (cry1), consisting of two species in rice plants (cry1a and cry1b), is indispensable for robust induction of the GA2ox genes. On the other hand, repression of the GA20ox genes is mediated by phytochromes. In addition, we found that the phytochromes also mediate the repression of a gibberellin 3-oxidase gene (OsGA3ox2) in the light. These results imply that, in rice seedlings, phytochromes mediate the repression of gibberellin biosynthesis capacity, while cry1 mediates the induction of gibberellin inactivation capacity. The cry1 action was demonstrated to be dominant in the reduction of active gibberellin content, but, in rice seedlings, the cumulative effects of these independent actions reduced active gibberellin content in the light. This pathway design in which different types of photoreceptors independently but cooperatively regulate active gibberellin content is unique from the viewpoint of dicot research. This redundancy should provide robustness to the response in rice plants. GA20OX4,GA2OX3,OsCRY1a,OsCRY1b,OsCRY2|CRY2,PHYA,PHYB|OsphyB,PHYC Differences and similarities in the photoregulation of gibberellin metabolism between rice and dicots 2013 Plant Signal Behav Functional Plant Research Unit; National Institute of Agrobiological Sciences; Tsukuba, Ibaraki Japan. In rice seedlings, elongation of leaf sheaths is suppressed by light stimuli. The response is mediated by two classes of photoreceptors, phytochromes and cryptochromes. However, it remains unclear how these photoreceptors interact in the process. Our recent study using phytochrome mutants and novel cryptochrome RNAi lines revealed that cryptochromes and phytochromes function cooperatively, but independently to reduce active GA contents in seedlings in visible light. Blue light captured by cryptochrome 1 (cry1a and cry1b) induces robust expression of GA 2-oxidase genes (OsGA2ox4-7). In parallel, phytochrome B with auxiliary action of phytochrome A mediates repression of GA 20-oxidase genes (OsGA20ox2 and OsGA20ox4). The independent effects cumulatively reduce active GA contents, leading to a suppression of leaf sheath elongation. These regulatory mechanisms are distinct from phytochrome B function in dicots. We discuss reasons why the distinct system appeared in rice, and advantages of the rice system in early photomorphogenesis. GA20OX4,OsCRY2|CRY2 An overview of gibberellin metabolism enzyme genes and their related mutants in rice 2004 Plant Physiol Field Production Science Center, University of Tokyo, Nishi-Tokyo, Tokyo 188-0002, Japan. To enhance our understanding of GA metabolism in rice (Oryza sativa), we intensively screened and identified 29 candidate genes encoding the following GA metabolic enzymes using all available rice DNA databases: ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA 20-oxidase (GA20ox), GA 3-oxidase (GA3ox), and GA 2-oxidase (GA2ox). In contrast to the Arabidopsis genome, multiple CPS-like, KS-like, and KO-like genes were identified in the rice genome, most of which are contiguously arranged. We also identified 18 GA-deficient rice mutants at six different loci from rice mutant collections. Based on the mutant and expression analyses, we demonstrated that the enzymes catalyzing the early steps in the GA biosynthetic pathway (i.e. CPS, KS, KO, and KAO) are mainly encoded by single genes, while those for later steps (i.e. GA20ox, GA3ox, and GA2ox) are encoded by gene families. The remaining CPS-like, KS-like, and KO-like genes were likely to be involved in the biosynthesis of diterpene phytoalexins rather than GAs because the expression of two CPS-like and three KS-like genes (OsCPS2, OsCPS4, OsKS4, OsKS7, and OsKS8) were increased by UV irradiation, and four of these genes (OsCPS2, OsCPS4, OsKS4, and OsKS7) were also induced by an elicitor treatment. GA20OX4,GA2OX3,KAO,KS2,OsKS1,OsCPS3 Global gene profiling of laser-captured pollen mother cells indicates molecular pathways and gene subfamilies involved in rice meiosis 2010 Plant Physiol National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Pollen mother cells (PMCs) represent a critical early stage in plant sexual reproduction in which the stage is set for male gamete formation. Understanding the global molecular genetics of this early meiotic stage has so far been limited to whole stamen or floret transcriptome studies, but since PMCs are a discrete population of cells in developmental synchrony, they provide the potential for precise transcriptome analysis and for enhancing our understanding of the transition to meiosis. As a step toward identifying the premeiotic transcriptome, we performed microarray analysis on a homogenous population of rice (Oryza sativa) PMCs isolated by laser microdissection and compared them with those of tricellular pollen and seedling. Known meiotic genes, including OsSPO11-1, PAIR1, PAIR2, PAIR3, OsDMC1, OsMEL1, OsRAD21-4, OsSDS, and ZEP1, all showed preferential expression in PMCs. The Kyoto Encyclopedia of Genes and Genomes pathways significantly enriched in PMC-preferential genes are DNA replication and repair pathways. Our genome-wide survey showed that, in the buildup to meiosis, PMCs accumulate the molecular machinery for meiosis at the mRNA level. We identified 1,158 PMC-preferential genes and suggested candidate genes and pathways involved in meiotic recombination and meiotic cell cycle control. Regarding the developmental context for meiosis, the DEF-like, AGL2-like, and AGL6-like subclades of MADS box transcription factors are PMC-preferentially expressed, the trans-zeatin type of cytokinin might be preferentially synthesized, and the gibberellin signaling pathway is likely active in PMCs. The ubiquitin-mediated proteolysis pathway is enriched in the 127 genes that are expressed in PMCs but not in tricellular pollen or seedling. GA20OX4,GA2OX3,KAO The rice R2R3-MYB transcription factor OsMYB55 is involved in the tolerance to high temperature and modulates amino acid metabolism 2012 PLoS One Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada. Temperatures higher than the optimum negatively affects plant growth and development. Tolerance to high temperature is a complex process that involves several pathways. Understanding this process, especially in crops such as rice, is essential to prepare for predicted climate changes due to global warming. Here, we show that OsMYB55 is induced by high temperature and overexpression of OsMYB55 resulted in improved plant growth under high temperature and decreased the negative effect of high temperature on grain yield. Transcriptome analysis revealed an increase in expression of several genes involved in amino acids metabolism. We demonstrate that OsMYB55 binds to the promoter regions of target genes and directly activates expression of some of those genes including glutamine synthetase (OsGS1;2) glutamine amidotransferase (GAT1) and glutamate decarboxylase 3 (GAD3). OsMYB55 overexpression resulted in an increase in total amino acid content and of the individual amino acids produced by the activation of the above mentioned genes and known for their roles in stress tolerance, namely L-glutamic acid, GABA and arginine especially under high temperature condition. In conclusion, overexpression of OsMYB55 improves rice plant tolerance to high temperature, and this high tolerance is associated with enhanced amino acid metabolism through transcription activation. GAD3,GAT1,GLN1;2|GS1;2,OsMYB55 GIANT EMBRYO encodes CYP78A13, required for proper size balance between embryo and endosperm in rice 2013 Plant J DuPont Agricultural Biotechnology, Experimental Station E353, PO Box 80353, Wilmington, DE 19880, USA. Among angiosperms there is a high degree of variation in embryo/endosperm size in mature seeds. However, little is known about the molecular mechanism underlying size control between these neighboring tissues. Here we report the rice GIANT EMBRYO (GE) gene that is essential for controlling the size balance. The function of GE in each tissue is distinct, controlling cell size in the embryo and cell death in the endosperm. GE, which encodes CYP78A13, is predominantly expressed in the interfacing tissues of the both embryo and endosperm. GE expression is under negative feedback regulation; endogenous GE expression is upregulated in ge mutants. In contrast to the loss-of-function mutant with large embryo and small endosperm, GE overexpression causes a small embryo and enlarged endosperm. A complementation analysis coupled with heterofertilization showed that complementation of ge mutation in either embryo or endosperm failed to restore the wild-type embryo/endosperm ratio. Thus, embryo and endosperm interact in determining embryo/endosperm size balance. Among genes associated with embryo/endosperm size, REDUCED EMBRYO genes, whose loss-of-function causes a phenotype opposite to ge, are revealed to regulate endosperm size upstream of GE. To fully understand the embryo-endosperm size control, the genetic network of the related genes should be elucidated. GE|CYP78A13 Control of rice embryo development, shoot apical meristem maintenance, and grain yield by a novel cytochrome p450 2013 Mol Plant National Key Laboratory of Plant Molecular Genetics and National Center for Plant Gene Research (Shanghai), Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China. Angiosperm seeds usually consist of two major parts: the embryo and the endosperm. However, the molecular mechanism(s) underlying embryo and endosperm development remains largely unknown, particularly in rice, the model cereal. Here, we report the identification and functional characterization of the rice GIANT EMBRYO (GE) gene. Mutation of GE resulted in a large embryo in the seed, which was caused by excessive expansion of scutellum cells. Post-embryonic growth of ge seedling was severely inhibited due to defective shoot apical meristem (SAM) maintenance. Map-based cloning revealed that GE encodes a CYP78A subfamily P450 monooxygenase that is localized to the endoplasmic reticulum. GE is expressed predominantly in the scutellar epithelium, the interface region between embryo and endosperm. Overexpression of GE promoted cell proliferation and enhanced rice plant growth and grain yield, but reduced embryo size, suggesting that GE is critical for coordinating rice embryo and endosperm development. Moreover, transgenic Arabidopsis plants overexpressing AtCYP78A10, a GE homolog, also produced bigger seeds, implying a conserved role for the CYP78A subfamily of P450s in regulating seed development. Taken together, our results indicate that GE plays critical roles in regulating embryo development and SAM maintenance. GE|CYP78A13 The rice 14-3-3 gene family and its involvement in responses to biotic and abiotic stress 2006 DNA Res National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences 300 Fenglin Road, Shanghai 200032, China. 14-3-3 proteins function as major regulators of primary metabolism and cellular signal transduction in plants. However, their involvement in plant defense and stress responses is largely unknown. In order to better address functions of the rice 14-3-3/GF14 proteins in defense and abiotic stress responses, we examined the rice GF14 family that comprises eight numbers. The phylogenetic comparison with the Arabidopsis 14-3-3 family revealed that the majority of rice GF14s might have evolved as an independent branch. At least four rice GF14 genes, GF14b, GF14c, GF14e and Gf14f were differentially regulated in the interactions of rice-Magnaporthe grisea and rice-Xanthomonas oryzae pv. oryzae, and the incompatible interactions stronger induced the genes than the compatible interactions. These GF14 genes were also induced by the defense compounds, benzothiadiazole, methyl jasmonate, ethephon and hydrogen peroxide. Similarly, they were differentially regulated by salinity, drought, wounding and abscisic acid. Tissue-specific analysis and expression of GF14-YFP fusions revealed that the four GF14 isoforms were expressed with tissue specificity and accumulated differentially in the cytoplasm and nucleus. Our current study provides fundamental information for the further investigation of the rice GF14 proteins. GF14b,GF14c,GF14f,GF14e|GID2 14-3-3 proteins act as intracellular receptors for rice Hd3a florigen 2011 Nature Laboratory of Plant Molecular Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. 'Florigen' was proposed 75 years ago to be synthesized in the leaf and transported to the shoot apex, where it induces flowering. Only recently have genetic and biochemical studies established that florigen is encoded by FLOWERING LOCUS T (FT), a gene that is universally conserved in higher plants. Nonetheless, the exact function of florigen during floral induction remains poorly understood and receptors for florigen have not been identified. Here we show that the rice FT homologue Hd3a interacts with 14-3-3 proteins in the apical cells of shoots, yielding a complex that translocates to the nucleus and binds to the Oryza sativa (Os)FD1 transcription factor, a rice homologue of Arabidopsis thaliana FD. The resultant ternary 'florigen activation complex' (FAC) induces transcription of OsMADS15, a homologue of A. thaliana APETALA1 (AP1), which leads to flowering. We have determined the 2.4 A crystal structure of rice FAC, which provides a mechanistic basis for florigen function in flowering. Our results indicate that 14-3-3 proteins act as intracellular receptors for florigen in shoot apical cells, and offer new approaches to manipulate flowering in various crops and trees. GF14c,Hd3a,OsKANADI1|OsKANADI2,OsBIP116b The 14-3-3 protein GF14c acts as a negative regulator of flowering in rice by interacting with the florigen Hd3a 2009 Plant Cell Physiol Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. Hd3a and FT proteins have recently been proposed to act as florigens in rice and Arabidopsis, respectively; however, the molecular mechanisms of their function remain to be determined. In this study, we identified GF14c (a 14-3-3 protein) as an Hd3a-interacting protein in a yeast two-hybrid screen. In vitro and in vivo experiments, using a combination of pull-down assays and bimolecular fluorescence complementation, confirmed the interaction between Hd3a and GF14c. Functional analysis using either GF14c overexpression or knockout transgenic rice plants indicated that this interaction plays a role in the regulation of flowering. GF14c-overexpressing plants exhibited a delay in flowering and the knockout mutants displayed early flowering relative to the wild-type plants under short-day conditions. These results suggest that GF14c acts as a negative regulator of flowering by interacting with Hd3a. Since the 14-3-3 protein has been shown to interact with FT protein in tomato and Arabidopsis, our results in rice provide important findings about FT signaling in plants. GF14c,Hd3a Sugar starvation- and GA-inducible calcium-dependent protein kinase 1 feedback regulates GA biosynthesis and activates a 14-3-3 protein to confer drought tolerance in rice seedlings 2013 Plant Mol Biol Department of Agronomy, National Chiayi University, Chiayi, 600, Taiwan, ROC. slho@mail.ncyu.edu.tw Germination followed by seedling growth constitutes two essential steps in the initiation of a new life cycle in plants, and in cereals, completion of these steps is regulated by sugar starvation and the hormone gibberellin. A calcium-dependent protein kinase 1 gene (OsCDPK1) was identified by differential screening of a cDNA library derived from sucrose-starved rice suspension cells. The expression of OsCDPK1 was found to be specifically activated by sucrose starvation among several stress conditions tested as well as activated transiently during post-germination seedling growth. In gain- and loss-of-function studies performed with transgenic rice overexpressing a constitutively active or RNA interference gene knockdown construct, respectively, OsCDPK1 was found to negatively regulate the expression of enzymes essential for GA biosynthesis. In contrast, OsCDPK1 activated the expression of a 14-3-3 protein, GF14c. Overexpression of either constitutively active OsCDPK1 or GF14c enhanced drought tolerance in transgenic rice seedlings. Hence, our studies demonstrated that OsCDPK1 transduces the post-germination Ca(2+) signal derived from sugar starvation and GA, refines the endogenous GA concentration and prevents drought stress injury, all essential functions to seedling development at the beginning of the life cycle in rice. GF14c,OsCDPK13|OsCDPK11|OsCPK11 A mutation in the rice chalcone isomerase gene causes the golden hull and internode 1 phenotype 2012 Planta State Key Laboratory of Plant Genomics, Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China. The biosynthesis of flavonoids, important secondary plant metabolites, has been investigated extensively, but few mutants of genes in this pathway have been identified in rice (Oryza sativa). The rice gold hull and internode (gh) mutants exhibit a reddish-brown pigmentation in the hull and internode and their phenotype has long been used as a morphological marker trait for breeding and genetic study. Here, we characterized that the gh1 mutant was a mutant of the rice chalcone isomerase gene (OsCHI). The result showed that gh1 had a Dasheng retrotransposon inserted in the 5' UTR of the OsCHI gene, which resulted in the complete loss of OsCHI expression. gh1 exhibited golden pigmentation in hulls and internodes once the panicles were exposed to light. The total flavonoid content in gh1 hulls was increased threefold compared to wild type. Consistent with the gh1 phenotype, OsCHI transcripts were expressed in most tissues of rice and most abundantly in internodes. It was also expressed at high levels in panicles before heading, distributed mainly in lemmas and paleae, but its expression decreased substantially after the panicles emerged from the sheath. OsCHI encodes a protein functionally and structurally conserved to chalcone isomerases in other species. Our findings demonstrated that the OsCHI gene was indispensable for flux of the flavonoid pathway in rice. gh1|OsCHI Structure of the cinnamyl-alcohol dehydrogenase gene family in rice and promoter activity of a member associated with lignification 2005 Planta Agricultural Research Service, Western Regional Research Center, United States Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA. ctobias@pw.usda.gov Analysis of lignification in rice has been facilitated by the availability of the recently completed rice genome sequence, and rice will serve as an important model for understanding the relationship of grass lignin composition to cell wall digestibility. Cinnamyl-alcohol dehydrogenase (CAD) is an enzyme important in lignin biosynthesis. The rice genome contains 12 distinct genes present at nine different loci that encode products with significant similarity to CAD. The rice gene family is diverse with respect to other angiosperm and gymnosperm CAD genes isolated to date and includes one member (OsCAD6) that contains a peroxisomal targeting signal and is substantially diverged relative to other family members. Four closely related family members (OsCAD8A-D) are present at the same locus and represent the product of a localized gene duplication and inversion. Promoter-reporter gene fusions to OsCAD2, an orthologue of the CAD gene present at the bm1 (brown midrib 1) locus of maize, reveal that in rice expression is associated with vascular tissue in aerial parts of the plant and is correlated with the onset of lignification. In root tissue, expression is primarily in the cortical parenchyma adjacent to the exodermis and in vascular tissue. GH2|OsCAD2 GOLD HULL AND INTERNODE2 encodes a primarily multifunctional cinnamyl-alcohol dehydrogenase in rice 2006 Plant Physiol State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, People's Republic of China. Lignin content and composition are two important agronomic traits for the utilization of agricultural residues. Rice (Oryza sativa) gold hull and internode phenotype is a classical morphological marker trait that has long been applied to breeding and genetics study. In this study, we have cloned the GOLD HULL AND INTERNODE2 (GH2) gene in rice using a map-based cloning approach. The result shows that the gh2 mutant is a lignin-deficient mutant, and GH2 encodes a cinnamyl-alcohol dehydrogenase (CAD). Consistent with this finding, extracts from roots, internodes, hulls, and panicles of the gh2 plants exhibited drastically reduced CAD activity and undetectable sinapyl alcohol dehydrogenase activity. When expressed in Escherichia coli, purified recombinant GH2 was found to exhibit strong catalytic ability toward coniferaldehyde and sinapaldehyde, while the mutant protein gh2 completely lost the corresponding CAD and sinapyl alcohol dehydrogenase activities. Further phenotypic analysis of the gh2 mutant plants revealed that the p-hydroxyphenyl, guaiacyl, and sinapyl monomers were reduced in almost the same ratio compared to the wild type. Our results suggest GH2 acts as a primarily multifunctional CAD to synthesize coniferyl and sinapyl alcohol precursors in rice lignin biosynthesis. GH2|OsCAD2 Increased lodging resistance in long-culm, low-lignin gh2 rice for improved feed and bioenergy production 2014 Sci Rep Institute of Agriculture, Graduate School, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan. Lignin modification has been a breeding target for the improvements of forage digestibility and energy yields in forage and bioenergy crops, but decreased lignin levels are often accompanied by reduced lodging resistance. The rice mutant gold hull and internode2 (gh2) has been identified to be lignin deficient. GH2 has been mapped to the short arm of chromosome 2 and encodes cinnamyl-alcohol dehydrogenase (CAD). We developed a long-culm variety, 'Leaf Star', with superior lodging resistance and a gh phenotype similar to one of its parents, 'Chugoku 117'. The gh loci in Leaf Star and Chugoku 117 were localized to the same region of chromosome 2 as the gh2 mutant. Leaf Star had culms with low lignin concentrations due to a natural mutation in OsCAD2 that was not present in Chugoku 117. However, this variety had high culm strength due to its strong, thick culms. Additionally, this variety had a thick layer of cortical fiber tissue with well-developed secondary cell walls. Our results suggest that rice can be improved for forage and bioenergy production by combining superior lodging resistance, which can be obtained by introducing thick and stiff culm traits, with low lignin concentrations, which can be obtained using the gh2 variety. GH2|OsCAD2 A stress-induced rice (Oryza sativa L.) beta-glucosidase represents a new subfamily of glycosyl hydrolase family 5 containing a fascin-like domain 2007 Biochem J School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand. GH5BG, the cDNA for a stress-induced GH5 (glycosyl hydrolase family 5) beta-glucosidase, was cloned from rice (Oryza sativa L.) seedlings. The GH5BG cDNA encodes a 510-amino-acid precursor protein that comprises 19 amino acids of prepeptide and 491 amino acids of mature protein. The protein was predicted to be extracellular. The mature protein is a member of a plant-specific subgroup of the GH5 exoglucanase subfamily that contains two major domains, a beta-1,3-exoglucanase-like domain and a fascin-like domain that is not commonly found in plant enzymes. The GH5BG mRNA is highly expressed in the shoot during germination and in leaf sheaths of mature plants. The GH5BG was up-regulated in response to salt stress, submergence stress, methyl jasmonate and abscisic acid in rice seedlings. A GUS (glucuronidase) reporter tagged at the C-terminus of GH5BG was found to be secreted to the apoplast when expressed in onion (Allium cepa) cells. A thioredoxin fusion protein produced from the GH5BG cDNA in Escherichia coli hydrolysed various pNP (p-nitrophenyl) glycosides, including beta-D-glucoside, alpha-L-arabinoside, beta-D-fucoside, beta-D-galactoside, beta-D-xyloside and beta-D-cellobioside, as well as beta-(1,4)-linked glucose oligosaccharides and beta-(1,3)-linked disaccharide (laminaribiose). The catalytic efficiency (kcat/K(m)) for hydrolysis of beta-(1,4)-linked oligosaccharides by the enzyme remained constant as the DP (degree of polymerization) increased from 3 to 5. This substrate specificity is significantly different from fungal GH5 exoglucanases, such as the exo-beta-(1,3)-glucanase of the yeast Candida albicans, which may correlate with a marked reduction in a loop that makes up the active-site wall in the Candida enzyme. GH5BG Natural variation in Ghd7.1 plays an important role in grain yield and adaptation in rice 2013 Cell Res None None Ghd7.1|Hd2|OsPRR37 Validation and characterization of Ghd7.1, a major quantitative trait locus with pleiotropic effects on spikelets per panicle, plant height, and heading date in rice (Oryza sativa L.) 2013 J Integr Plant Biol National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, 430070, China. A quantitative trait locus (QTL) that affects heading date (HD) and the number of spikelets per panicle (SPP) was previously identified in a small region on chromosome 7 in rice (Oryza sativa L.). In order to further characterize the QTL region, near isogenic lines (NILs) were quickly obtained by self-crossing recombinant inbred line 189, which is heterozygous in the vicinity of the target region. The pleiotropic effects of QTL Ghd7.1 on plant height (PH), SPP, and HD, were validated using an NIL-F2 population. Ghd7.1 explained 50.2%, 45.3%, and 76.9% of phenotypic variation in PH, SPP, and HD, respectively. Ghd7.1 was precisely mapped to a 357-kb region on the basis of analysis of the progeny of the NIL-F2 population. Day-length treatment confirmed that Ghd7.1 is sensitive to photoperiod, with long days delaying heading up to 12.5 d. Identification of panicle initiation and development for the pair of NILs showed that Ghd7.1 elongated the photoperiod-sensitive phase more than 10 d, but did not change the basic vegetative phase and the reproductive growth phase. These findings indicated that Ghd7.1 regulates SPP by controlling the rate of panicle differentiation rather than the duration of panicle development. Ghd7.1|Hd2|OsPRR37 Natural variation in OsPRR37 regulates heading date and contributes to rice cultivation at a wide range of latitudes 2013 Mol Plant Department of Plant Science, Plant Genomics and Breeding Institute, and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea. Heading date and photoperiod sensitivity are fundamental traits that determine rice adaptation to a wide range of geographic environments. By quantitative trait locus (QTL) mapping and candidate gene analysis using whole-genome re-sequencing, we found that Oryza sativa Pseudo-Response Regulator37 (OsPRR37; hereafter PRR37) is responsible for the Early heading7-2 (EH7-2)/Heading date2 (Hd2) QTL which was identified from a cross of late-heading rice 'Milyang23 (M23)' and early-heading rice 'H143'. H143 contains a missense mutation of an invariantly conserved amino acid in the CCT (CONSTANS, CO-like, and TOC1) domain of PRR37 protein. In the world rice collection, different types of nonfunctional PRR37 alleles were found in many European and Asian rice cultivars. Notably, the japonica varieties harboring nonfunctional alleles of both Ghd7/Hd4 and PRR37/Hd2 flower extremely early under natural long-day conditions, and are adapted to the northernmost regions of rice cultivation, up to 53 degrees N latitude. Genetic analysis revealed that the effects of PRR37 and Ghd7 alleles on heading date are additive, and PRR37 down-regulates Hd3a expression to suppress flowering under long-day conditions. Our results demonstrate that natural variations in PRR37/Hd2 and Ghd7/Hd4 have contributed to the expansion of rice cultivation to temperate and cooler regions. Ghd7.1|Hd2|OsPRR37,Ghd7 Evolution and association analysis of Ghd7 in rice 2012 PLoS One National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research Wuhan, Huazhong Agricultural University, Wuhan, China. Plant height, heading date, and yield are the main targets for rice genetic improvement. Ghd7 is a pleiotropic gene that controls the aforementioned traits simultaneously. In this study, a rice germplasm collection of 104 accessions (Oryza sativa) and 3 wild rice varieties (O.rufipogon) was used to analyze the evolution and association of Ghd7 with plant height, heading date, and yield. Among the 104 accessions, 76 single nucleotide polymorphisms (SNPs) and six insertions and deletions were found within a 3932-bp DNA fragment of Ghd7. A higher pairwise pi and theta in the promoter indicated a highly diversified promoter of Ghd7. Sixteen haplotypes and 8 types of Ghd7 protein were detected. SNP changes between haplotypes indicated that Ghd7 evolved from two distinct ancestral gene pools, and independent domestication processes were detected in indica and japonica varietals respectively. In addition to the previously reported premature stop mutation in the first exon of Ghd7, which caused phenotypic changes of multiple traits, we found another functional C/T mutation (SNP S_555) by structure-based association analysis. SNP S_555 is located in the promoter and was related to plant height probably by altering gene expression. Moreover, another seven SNP mutations in complete linkage were found to be associated with the number of spikelets per panicle, regardless of the photoperiod. These associations provide the potential for flexibility of Ghd7 application in rice breeding programs. Ghd7 Natural variation in Ghd7 is an important regulator of heading date and yield potential in rice 2008 Nat Genet National Key Laboratory of Crop Genetic Improvement, National Centre of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Yield potential, plant height and heading date are three classes of traits that determine the productivity of many crop plants. Here we show that the quantitative trait locus (QTL) Ghd7, isolated from an elite rice hybrid and encoding a CCT domain protein, has major effects on an array of traits in rice, including number of grains per panicle, plant height and heading date. Enhanced expression of Ghd7 under long-day conditions delays heading and increases plant height and panicle size. Natural mutants with reduced function enable rice to be cultivated in temperate and cooler regions. Thus, Ghd7 has played crucial roles for increasing productivity and adaptability of rice globally. Ghd7 Natural variation in Hd17, a homolog of Arabidopsis ELF3 that is involved in rice photoperiodic flowering 2012 Plant Cell Physiol National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan. Flowering time of rice depends strongly on photoperiodic responses. We previously identified a quantitative trait locus, Heading date 17 (Hd17), that is associated with a difference in flowering time between Japanese rice (Oryza sativa L.) cultivars. Here, we show that the difference may result from a single nucleotide polymorphism within a putative gene that encodes a homolog of the Arabidopsis EARLY FLOWERING 3 protein, which plays important roles in maintaining circadian rhythms. Our results demonstrate that natural variation in Hd17 may change the transcription level of a flowering repressor, Grain number, plant height and heading date 7 (Ghd7), suggesting that Hd17 is part of rice's photoperiodic flowering pathway. Ghd7,Hd17|Ef7|OsELF3-1 Ghd7 is a central regulator for growth, development, adaptation and responses to biotic and abiotic stresses 2014 Plant physiology National Centre of Plant Gene Research (Wuhan), Huazhong Agricultural University Ghd7 has been regarded as an important regulator of heading date and yield potential in rice. In the study reported in this paper, we investigated new functions of Ghd7 in rice growth, development and environmental response. As a long-day dependent negative regulator of heading date, the degree of phenotypic effect of Ghd7 on heading date and yield traits is quantitatively related to the transcript level, and was also influenced by both environmental conditions and genetic backgrounds. Ghd7 regulates yield traits through modulating panicle branching independent of heading date. Ghd7 also regulates plasticity of tiller branching by mediating the PHYB-OsTB1 pathway as adaption to shade signal. The expression of Ghd7 was strongly repressed by ABA, JA and drought stress while enhanced by low temperature. Over-expression of Ghd7 increased drought sensitivity while knock-down of Ghd7 enhanced drought tolerance. Analysis of expression profiles using gene chip revealed that Ghd7 was involved in regulation of multiple processes, including flowering time, hormone metabolism, biotic and abiotic stresses. This study suggested that Ghd7 functions to link the dynamic environmental inputs with phase transition, architecture regulation and stress response to maximize the reproductive success of the rice plant. Ghd7 Characterization of the molecular mechanism underlying gibberellin perception complex formation in rice 2010 Plant Cell Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan. The DELLA protein SLENDER RICE1 (SLR1) is a repressor of gibberellin (GA) signaling in rice (Oryza sativa), and most of the GA-associated responses are induced upon SLR1 degradation. It is assumed that interaction between GIBBERELLIN INSENSITIVE DWARF1 (GID1) and the N-terminal DELLA/TVHYNP motif of SLR1 triggers F-box protein GID2-mediated SLR1 degradation. We identified a semidominant dwarf mutant, Slr1-d4, which contains a mutation in the region encoding the C-terminal GRAS domain of SLR1 (SLR1(G576V)). The GA-dependent degradation of SLR1(G576V) was reduced in Slr1-d4, and compared with SLR1, SLR1(G576V) showed reduced interaction with GID1 and almost none with GID2 when tested in yeast cells. Surface plasmon resonance of GID1-SLR1 and GID1-SLR1(G576V) interactions revealed that the GRAS domain of SLR1 functions to stabilize the GID1-SLR1 interaction by reducing its dissociation rate and that the G576V substitution in SLR1 diminishes this stability. These results suggest that the stable interaction of GID1-SLR1 through the GRAS domain is essential for the recognition of SLR1 by GID2. We propose that when the DELLA/TVHYNP motif of SLR1 binds with GID1, it enables the GRAS domain of SLR1 to interact with GID1 and that the stable GID1-SLR1 complex is efficiently recognized by GID2. GF14e|GID2,GID1|OsGID1,SLR1|OsGAI GID2, an F-box subunit of the SCF E3 complex, specifically interacts with phosphorylated SLR1 protein and regulates the gibberellin-dependent degradation of SLR1 in rice 2004 The Plant Journal Bioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan. The phytohormone gibberellin (GA) controls growth and development in plants. Previously, we identified a rice F-box protein, gibberellin-insensitive dwarf2 (GID2), which is essential for GA-mediated DELLA protein degradation. In this study, we analyzed the biological and molecular biological properties of GID2. Expression of GID2 preferentially occurred in rice organs actively synthesizing GA. Domain analysis of GID2 revealed that the C-terminal regions were essential for the GID2 function, but not the N-terminal region. Yeast two-hybrid assay and immunoprecipitation experiments demonstrated that GID2 is a component of the SCF complex through an interaction with a rice ASK1 homolog, OsSkp15. Furthermore, an in vitro pull-down assay revealed that GID2 specifically interacted with the phosphorylated Slender Rice 1 (SLR1). Taken these results together, we conclude that the phosphorylated SLR1 is caught by the SCFGID2 complex through an interacting affinity between GID2 and phosphorylated SLR1, triggering the ubiquitin-mediated degradation of SLR1. GF14e|GID2,SLR1|OsGAI Rice 14-3-3 protein (GF14e) negatively affects cell death and disease resistance 2011 Plant J Bioagricultural Sciences and Pest Management and Program in Plant Molecular Biology, Colorado State University, Fort Collins, CO 80523-1177, USA. Plant 14-3-3 proteins regulate important cellular processes, including plant immune responses, through protein-protein interactions with a wide range of target proteins. In rice (Oryza sativa), the GF14e gene, which encodes a 14-3-3 protein, is induced during effector-triggered immunity (ETI) associated with pathogens such as Xanthomonas oryzae pv. oryzae (Xoo). To determine whether the GF14e gene plays a direct role in resistance to disease in rice, we suppressed its expression by RNAi silencing. GF14e suppression was correlated with the appearance of a lesion-mimic (LM) phenotype in the transgenic plants at 3 weeks after sowing. This indicates inappropriate regulation of cell death, a phenotype that is frequently associated with enhanced resistance to pathogens. GF14e-silenced rice plants showed high levels of resistance to a virulent strain of Xoo compared with plants that were not silenced. Enhanced resistance was correlated with GF14e silencing prior to and after development of the LM phenotype, higher basal expression of a defense response peroxidase gene (POX22.3), and accumulation of reactive oxygen species (ROS). In addition, GF14e-silenced plants also exhibit enhanced resistance to the necrotrophic fungal pathogen Rhizoctonia solani. Together, our findings suggest that GF14e negatively affects the induction of plant defense response genes, cell death and broad-spectrum resistance in rice. GF14e|GID2 Release of the repressive activity of rice DELLA protein SLR1 by gibberellin does not require SLR1 degradation in the gid2 mutant 2008 Plant Cell Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan. makoto@nuagr1.agr.nagoya-u.ac.jp The rice (Oryza sativa) DELLA protein SLR1 acts as a repressor of gibberellin (GA) signaling. GA perception by GID1 causes SLR1 protein degradation involving the F-box protein GID2; this triggers GA-associated responses such as shoot elongation and seed germination. In GA-insensitive and GA biosynthesis mutants, SLENDER RICE1 (SLR1) accumulates to high levels, and the severity of dwarfism is usually correlated with the level of SLR1 accumulation. An exception is the GA-insensitive F-box mutant gid2, which shows milder dwarfism than mutants such as gid1 and cps even though it accumulates higher levels of SLR1. The level of SLR1 protein in gid2 was decreased by loss of GID1 function or treatment with a GA biosynthesis inhibitor, and dwarfism was enhanced. Conversely, overproduction of GID1 or treatment with GA(3) increased the SLR1 level in gid2 and reduced dwarfism. These results indicate that derepression of SLR1 repressive activity can be accomplished by GA and GID1 alone and does not require F-box (GID2) function. Evidence for GA signaling without GID2 was also provided by the expression behavior of GA-regulated genes such as GA-20oxidase1, GID1, and SLR1 in the gid2 mutant. Based on these observations, we propose a model for the release of GA suppression that does not require DELLA protein degradation. GF14e|GID2,GID1|OsGID1,SLR1|OsGAI Accumulation of Phosphorylated Repressor for Gibberellin Signaling in an F-box Mutant 2003 Science BioScience Center, Nagoya University, Nagoya 464-8601, Japan. Gibberellin (GA) regulates growth and development in plants. We isolated and characterized a rice GA-insensitive dwarf mutant, gid2. The GID2 gene encodes a putative F-box protein, which interacted with the rice Skp1 homolog in a yeast two-hybrid assay. In gid2, a repressor for GA signaling, SLR1, was highly accumulated in a phosphorylated form and GA increased its concentration, whereas SLR1 was rapidly degraded by GA through ubiquitination in the wild type. We conclude that GID2 is a positive regulator of GA signaling and that regulated degradation of SLR1 is initiated through GA-dependent phosphorylation and finalized by an SCF(GID2)-proteasome pathway. GF14e|GID2 Duplication and independent selection of cell-wall invertase genes GIF1 and OsCIN1 during rice evolution and domestication 2010 BMC Evol Biol National Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. BACKGROUND: Various evolutionary models have been proposed to interpret the fate of paralogous duplicates, which provides substrates on which evolution selection could act. In particular, domestication, as a special selection, has played important role in crop cultivation with divergence of many genes controlling important agronomic traits. Recent studies have indicated that a pair of duplicate genes was often sub-functionalized from their ancestral functions held by the parental genes. We previously demonstrated that the rice cell-wall invertase (CWI) gene GIF1 that plays an important role in the grain-filling process was most likely subjected to domestication selection in the promoter region. Here, we report that GIF1 and another CWI gene OsCIN1 constitute a pair of duplicate genes with differentiated expression and function through independent selection. RESULTS: Through synteny analysis, we show that GIF1 and another cell-wall invertase gene OsCIN1 were paralogues derived from a segmental duplication originated during genome duplication of grasses. Results based on analyses of population genetics and gene phylogenetic tree of 25 cultivars and 25 wild rice sequences demonstrated that OsCIN1 was also artificially selected during rice domestication with a fixed mutation in the coding region, in contrast to GIF1 that was selected in the promoter region. GIF1 and OsCIN1 have evolved into different expression patterns and probable different kinetics parameters of enzymatic activity with the latter displaying less enzymatic activity. Overexpression of GIF1 and OsCIN1 also resulted in different phenotypes, suggesting that OsCIN1 might regulate other unrecognized biological process. CONCLUSION: How gene duplication and divergence contribute to genetic novelty and morphological adaptation has been an interesting issue to geneticists and biologists. Our discovery that the duplicated pair of GIF1 and OsCIN1 has experienced sub-functionalization implies that selection could act independently on each duplicate towards different functional specificity, which provides a vivid example for evolution of genetic novelties in a model crop. Our results also further support the established hypothesis that gene duplication with sub-functionalization could be one solution for genetic adaptive conflict. GIF1|OsCIN2,OsCIN1 Sugar homeostasis mediated by cell wall invertase GRAIN INCOMPLETE FILLING 1 (GIF1) plays a role in pre-existing and induced defence in rice 2014 Mol Plant Pathol College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, 310058, China; National Key Laboratory of Plant Molecular Genetics and National Center of Plant Gene Research, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China. Sugar metabolism and sugar signalling are not only critical for plant growth and development, but are also important for stress responses. However, how sugar homeostasis is involved in plant defence against pathogen attack in the model crop rice remains largely unknown. In this study, we observed that the grains of gif1, a loss-of-function mutant of the cell wall invertase gene GRAIN INCOMPLETE FILLING 1 (GIF1), were hypersusceptible to postharvest fungal pathogens, with decreased levels of sugars and a thinner glume cell wall in comparison with the wild-type. Interestingly, constitutive expression of GIF1 enhanced resistance to both the rice bacterial pathogen Xanthomonas oryzae pv. oryzae and the fungal pathogen Magnaporthe oryzae. The GIF1-overexpressing (GIF1-OE) plants accumulated higher levels of glucose, fructose and sucrose compared with the wild-type plants. More importantly, higher levels of callose were deposited in GIF1-OE plants during pathogen infection. Moreover, the cell wall was much thicker in the infection sites of the GIF1-OE plants when compared with the wild-type plants. We also found that defence-related genes were constitutively activated in the GIF1-OE plants. Taken together, our study reveals that sugar homeostasis mediated by GIF1 plays an important role in constitutive and induced physical and chemical defence. GIF1|OsCIN2 Control of rice grain-filling and yield by a gene with a potential signature of domestication 2008 Nat Genet Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China. Grain-filling, an important trait that contributes greatly to grain weight, is regulated by quantitative trait loci and is associated with crop domestication syndrome. However, the genes and underlying molecular mechanisms controlling crop grain-filling remain elusive. Here we report the isolation and functional analysis of the rice GIF1 (GRAIN INCOMPLETE FILLING 1) gene that encodes a cell-wall invertase required for carbon partitioning during early grain-filling. The cultivated GIF1 gene shows a restricted expression pattern during grain-filling compared to the wild rice allele, probably a result of accumulated mutations in the gene's regulatory sequence through domestication. Fine mapping with introgression lines revealed that the wild rice GIF1 is responsible for grain weight reduction. Ectopic expression of the cultivated GIF1 gene with the 35S or rice Waxy promoter resulted in smaller grains, whereas overexpression of GIF1 driven by its native promoter increased grain production. These findings, together with the domestication signature that we identified by comparing nucleotide diversity of the GIF1 loci between cultivated and wild rice, strongly suggest that GIF1 is a potential domestication gene and that such a domestication-selected gene can be used for further crop improvement. GIF1|OsCIN2 A Kelch motif-containing serine/threonine protein phosphatase determines the large grain QTL trait in rice 2012 J Integr Plant Biol State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China. A thorough understanding of the genetic basis of rice grain traits is critical for the improvement of rice (Oryza sativa L.) varieties. In this study, we generated an F(2) population by crossing the large-grain japonica cultivar CW23 with Peiai 64 (PA64), an elite indica small-grain cultivar. Using QTL analysis, 17 QTLs for five grain traits were detected on four different chromosomes. Eight of the QTLs were newly-identified in this study. In particular, qGL3-1, a newly-identified grain length QTL with the highest LOD value and largest phenotypic variation, was fine-mapped to the 17 kb region of chromosome 3. A serine/threonine protein phosphatase gene encoding a repeat domain containing two Kelch motifs was identified as the unique candidate gene corresponding to this QTL. A comparison of PA64 and CW23 sequences revealed a single nucleotide substitution (C-->A) at position 1092 in exon 10, resulting in replacement of Asp (D) in PA64 with Glu (E) in CW23 for the 364(th) amino acid. This variation is located at the D position of the conserved sequence motif AVLDT of the Kelch repeat. Genetic analysis of a near-isogenic line (NIL) for qGL3-1 revealed that the allele qGL3-1 from CW23 has an additive or partly dominant effect, and is suitable for use in molecular marker-assisted selection. GL3.1|qGL3-1|qGL3|OsPPKL1 Rare allele of OsPPKL1 associated with grain length causes extra-large grain and a significant yield increase in rice 2012 Proc Natl Acad Sci U S A State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China. Grain size and shape are important components determining rice grain yield, and they are controlled by quantitative trait loci (QTLs). Here, we report the cloning and functional characterization of a major grain length QTL, qGL3, which encodes a putative protein phosphatase with Kelch-like repeat domain (OsPPKL1). We found a rare allele qgl3 that leads to a long grain phenotype by an aspartate-to-glutamate transition in a conserved AVLDT motif of the second Kelch domain in OsPPKL1. The rice genome has other two OsPPKL1 homologs, OsPPKL2 and OsPPKL3. Transgenic studies showed that OsPPKL1 and OsPPKL3 function as negative regulators of grain length, whereas OsPPKL2 as a positive regulator. The Kelch domains are essential for the OsPPKL1 biological function. Field trials showed that the application of the qgl3 allele could significantly increase grain yield in both inbred and hybrid rice varieties, due to its favorable effect on grain length, filling, and weight. GL3.1|qGL3-1|qGL3|OsPPKL1,OsPPKL2,OsPPKL3 Influence of different nitrogen inputs on the members of ammonium transporter and glutamine synthetase genes in two rice genotypes having differential responsiveness to nitrogen 2012 Mol Biol Rep Department of Molecular Biology and Genetic Engineering, College of Basic Sciences and Humanities, G. B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand, India. Two aromatic rice genotypes, Pusa Basmati 1 (PB1) and Kalanamak 3119 (KN3119) having 120 and 30 kg/ha optimum nitrogen requirement respectively, to produce optimal yield, were chosen to understand their differential nitrogen responsiveness. Both the genotypes grown under increasing nitrogen inputs showed differences in seed/panicle, 1,000 seed weight, %nitrogen in the biomass and protein content in the seeds. All these parameters in PB1 were found to be in the increasing order in contrast to KN3119 which showed declined response on increasing nitrogen dose exceeding the normal dose indicating that both the genotypes respond differentially to the nitrogen inputs. Gene expression analysis of members of ammonium transporter gene family in flag leaves during active grain filling stage revealed that all the three members of OsAMT3 family genes (OsAMT1;1-3), only one member of OsAMT2 family i.e., OsAMT2;3 and the high affinity OsAMT1;1 were differentially expressed and were affected by different doses of nitrogen. In both the genotypes, both increase and decline in seed protein contents matched with the expressions levels of OsAMT1;1, OsGS1;1 and OsGS1;2 in the flag leaves during grain filling stage indicating that high nitrogen nutrition in KN3119 probably causes the repression of these genes which might be important during grain filling. GLN1;2|GS1;2,OsAMT1;1|OsAMT1.1,OsAMT2;1,OsAMT2;3,OsGS|OsGS1|GS1|OsGS1;1|OsGLN1;1 Biochemical background and compartmentalized functions of cytosolic glutamine synthetase for active ammonium assimilation in rice roots 2004 Plant Cell Physiol RIKEN Plant Science Center, 1-7-22 Suehiro, Tsurumi-ku, Yokohama, 230-0045 Japan. Rice plants in paddy fields prefer to utilize ammonium as a major nitrogen source. Glutamine synthetase (GS) serves for assimilation of ammonium in rice root, and ameliorates the toxic effect of ammonium excess. Among the three isoenzymes of the cytosolic GS1 gene family in rice, OsGLN1;1 and OsGLN1;2 were abundantly expressed in roots. Analysis of the purified enzymes showed that OsGLN1;1 and OsGLN1;2 can be classified into high-affinity subtypes with relatively high V(max) values, as compared with the major high-affinity isoenzyme, GLN1;1, in Arabidopsis. Low-affinity forms of GS1 comparable to those in Arabidopsis (GLN1;2 and GLN1;3) were absent in rice roots. The OsGLN1;1 and OsGLN1;2 transcripts showed reciprocal responses to ammonium supply in the surface cell layers of roots. OsGLN1;1 accumulated in dermatogen, epidermis and exodermis under nitrogen-limited condition. By contrast, OsGLN1;2 was abundantly expressed in the same cell layers under nitrogen-sufficient conditions, replenishing the loss of OsGLN1;1 following ammonium treatment. Within the central cylinder of elongating zone, OsGLN1;1 and OsGLN1;2 were both induced by ammonium, which was distinguishable from the response observed in the surface cell layers. The high-capacity Gln synthetic activities of OsGLN1;1 and OsGLN1;2 facilitate active ammonium assimilation in specific cell types in rice roots. GLN1;2|GS1;2,OsGS1;3|OsGLN1;3,OsGS|OsGS1|GS1|OsGS1;1|OsGLN1;1,OsGS2|lambdaGS31 Severe reduction in growth rate and grain filling of rice mutants lacking OsGS1;1, a cytosolic glutamine synthetase1;1 2005 Plant J Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. Rice (Oryza sativa L.) plants possess three homologous but distinct genes for cytosolic glutamine synthetase (GS1): these are OsGS1;1, OsGS1;2, and OsGS1;3. OsGS1;1 was expressed in all organs tested with higher expression in leaf blades, while OsGS1;2, and OsGS1;3 were expressed mainly in roots and spikelets, respectively. We characterized knockout mutants caused by insertion of endogenous retrotransposon Tos17 into the exon-8 (lines ND8037 and ND9801) or the exon-10 (line NC2327) of OsGS1;1. Mendelian segregation occurred in each progeny. Homozygously inserted mutants showed severe retardation in growth rate and grain filling when grown at normal nitrogen concentrations. Abnormal mRNA for GS1;1 was transcribed, and the GS1 protein and its activity in the leaf blades were barely detectable in these mutants. The glutamine pool in the roots and leaf blades of the mutants was lower than that of the wild type. Re-introduction of OsGS1;1 cDNA under the control of its own promoter into the mutants successfully complemented these phenotypes. Progeny where Tos17 was heterozygously inserted or deleted during segregation showed normal phenotypes. The results indicate that GS1;1 is important for normal growth and grain filling in rice; GS1;2 and GS1;3 were not able to compensate for GS1;1 function. GLN1;2|GS1;2,OsGS1;3|OsGLN1;3,OsGS|OsGS1|GS1|OsGS1;1|OsGLN1;1 Metabolomics data reveal a crucial role of cytosolic glutamine synthetase 1;1 in coordinating metabolic balance in rice 2011 Plant J RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan. Rice plants grown in paddy fields preferentially use ammonium as a source of inorganic nitrogen. Glutamine synthetase (GS) catalyses the conversion of ammonium to glutamine. Of the three genes encoding cytosolic GS in rice, OsGS1;1 is critical for normal growth and grain filling. However, the basis of its physiological function that may alter the rate of nitrogen assimilation and carbon metabolism within the context of metabolic networks remains unclear. To address this issue, we carried out quantitative comparative analyses between the metabolite profiles of a rice mutant lacking OsGS1;1 and its background wild type (WT). The mutant plants exhibited severe retardation of shoot growth in the presence of ammonium compared with the WT. Overaccumulation of free ammonium in the leaf sheath and roots of the mutant indicated the importance of OsGS1;1 for ammonium assimilation in both organs. The metabolite profiles of the mutant line revealed: (i) an imbalance in levels of sugars, amino acids and metabolites in the tricarboxylic acid cycle, and (ii) overaccumulation of secondary metabolites, particularly in the roots under a continuous supply of ammonium. Metabolite-to-metabolite correlation analysis revealed the presence of mutant-specific networks between tryptamine and other primary metabolites in the roots. These results demonstrated a crucial function of OsGS1;1 in coordinating the global metabolic network in rice plants grown using ammonium as the nitrogen source. GLN1;2|GS1;2,OsGDH2,OsGS|OsGS1|GS1|OsGS1;1|OsGLN1;1 Assimilation of ammonium ions and reutilization of nitrogen in rice (Oryza sativa L.) 2007 J Exp Bot Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Sendai 981-8555, Japan. A major source of inorganic nitrogen for rice plants grown in paddy soil is ammonium ions. The ammonium ions are actively taken up by the roots via ammonium transporters and subsequently assimilated into the amide residue of glutamine (Gln) by the reaction of glutamine synthetase (GS) in the roots. The Gln is converted into glutamate (Glu), which is a central amino acid for the synthesis of a number of amino acids, by the reaction of glutamate synthase (GOGAT). Although a small gene family for both GS and GOGAT is present in rice, ammonium-dependent and cell type-specific expression suggest that cytosolic GS1;2 and plastidic NADH-GOGAT1 are responsible for the primary assimilation of ammonium ions in the roots. In the plant top, approximately 80% of the total nitrogen in the panicle is remobilized through the phloem from senescing organs. Since the major form of nitrogen in the phloem sap is Gln, GS in the senescing organs and GOGAT in developing organs are important for nitrogen remobilization and reutilization, respectively. Recent work with a knock-out mutant of rice clearly showed that GS1;1 is responsible for this process. Overexpression studies together with age- and cell type-specific expression strongly suggest that NADH-GOGAT1 is important for the reutilization of transported Gln in developing organs. The overall process of nitrogen utilization within the plant is discussed. GLN1;2|GS1;2 Comparison of ion balance and nitrogen metabolism in old and young leaves of alkali-stressed rice plants 2012 PLoS One Key laboratory of Molecular Epigenetics of MOE, Northeast Normal University, Changchun, Jilin Province, China. BACKGROUND: Alkali stress is an important agricultural contaminant and has complex effects on plant metabolism. The aim of this study was to investigate whether the alkali stress has different effects on the growth, ion balance, and nitrogen metabolism in old and young leaves of rice plants, and to compare functions of both organs in alkali tolerance. METHODOLOGY/PRINCIPAL FINDINGS: The results showed that alkali stress only produced a small effect on the growth of young leaves, whereas strongly damaged old leaves. Rice protected young leaves from ion harm via the large accumulation of Na(+) and Cl(-) in old leaves. The up-regulation of OsHKT1;1, OsAKT1, OsHAK1, OsHAK7, OsHAK10 and OsHAK16 may contribute to the larger accumulation of Na(+) in old leaves under alkali stress. Alkali stress mightily reduced the NO(3)(-) contents in both organs. As old leaf cells have larger vacuole, under alkali stress these scarce NO(3)(-) was principally stored in old leaves. Accordingly, the expression of OsNRT1;1 and OsNRT1;2 in old leaves was up-regulated by alkali stress, revealing that the two genes might contribute to the accumulation of NO(3)(-) in old leaves. NO(3)(-) deficiency in young leaves under alkali stress might induce the reduction in OsNR1 expression and the subsequent lacking of NH(4)(+), which might be main reason for the larger down-regulation of OsFd-GOGAT and OsGS2 in young leaves. CONCLUSIONS/SIGNIFICANCE: Our results strongly indicated that, during adaptation of rice to alkali stress, young and old leaves have distinct mechanisms of ion balance and nitrogen metabolism regulation. We propose that the comparative studies of young and old tissues may be important for abiotic stress tolerance research. GLN1;2|GS1;2,NRT1,OsGDH2,OsGDH3 Reduced expression of glycolate oxidase leads to enhanced disease resistance in rice 2013 PeerJ Department of Plant Pathology , University of California Davis , California , USA. Glycolate oxidase (GLO) is a key enzyme in photorespiration, catalyzing the oxidation of glycolate to glyoxylate. Arabidopsis GLO is required for nonhost defense responses to Pseudomonas syringae and for tobacco Pto/AvrPto-mediated defense responses. We previously described identification of rice GLO1 that interacts with a glutaredoxin protein, which in turn interacts with TGA transcription factors. TGA transcription factors are well known to participate in NPR1/NH1-mediated defense signaling, which is crucial to systemic acquired resistance in plants. Here we demonstrate that reduction of rice GLO1 expression leads to enhanced resistance to Xanthomonas oryzae pv oryzae (Xoo). Constitutive silencing of GLO1 leads to programmed cell death, resulting in a lesion-mimic phenotype and lethality or reduced plant growth and development, consistent with previous reports. Inducible silencing of GLO1, employing a dexamethasone-GVG (Gal4 DNA binding domain-VP16 activation domain-glucocorticoid receptor fusion) inducible system, alleviates these detrimental effects. Silencing of GLO1 results in enhanced resistance to Xoo, increased expression of defense regulators NH1, NH3, and WRKY45, and activation of PR1 expression. GLO1,GLO2,GLO3,GLO4,OsNPR1|NH1,OsWRKY45 Glycolate oxidase isozymes are coordinately controlled by GLO1 and GLO4 in rice 2012 PLoS One State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China. Glycolate oxidase (GLO) is a key enzyme in photorespiratory metabolism. Four putative GLO genes were identified in the rice genome, but how each gene member contributes to GLO activities, particularly to its isozyme profile, is not well understood. In this study, we analyzed how each gene plays a role in isozyme formation and enzymatic activities in both yeast cells and rice tissues. Five GLO isozymes were detected in rice leaves. GLO1 and GLO4 are predominately expressed in rice leaves, while GLO3 and GLO5 are mainly expressed in the root. Enzymatic assays showed that all yeast-expressed GLO members except GLO5 have enzymatic activities. Further analyses suggested that GLO1, GLO3 and GLO4 interacted with each other, but no interactions were observed for GLO5. GLO1/GLO4 co-expressed in yeast exhibited the same isozyme pattern as that from rice leaves. When either GLO1 or GLO4 was silenced, expressions of both genes were simultaneously suppressed and most of the GLO activities were lost, and consistent with this observation, little GLO isozyme protein was detected in the silenced plants. In contrast, no observable effect was detected when GLO3 was suppressed. Comparative analyses between the GLO isoforms expressed in yeast and the isozymes from rice leaves indicated that two of the five isozymes are homo-oligomers composed of either GLO1 or GLO4, and the other three are hetero-oligomers composed of both GLO1 and GLO4. Our current data suggest that GLO isozymes are coordinately controlled by GLO1 and GLO4 in rice, and the existence of GLO isozymes and GLO molecular and compositional complexities implicate potential novel roles for GLO in plants. GLO1,GLO3,GLO4 Glabrous Rice 1, encoding a homeodomain protein, regulates trichome development in rice 2012 Rice (N Y) State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. gsxiong@genetics.ac.cn. BACKGROUND: Glabrous rice, which lacks trichomes on the rice epidermis, is regarded as an important germplasm resource in rice breeding. Trichomes are derived from aerial epidermal cells and used as a model to study the cell fate determination in plant. In Arabidopsis, the molecular mechanisms of trichome development have been well studied. However, little is known about the molecular basis of trichome development in rice. RESULTS: In this study, near isogenic lines harboring the glabrous rice 1 locus were developed. By a map-based approach, we narrowed down the locus to a 21-kb DNA region harboring two genes. One of the genes named Glabrous Rice 1 (GLR1), which is most likely the candidate, encodes a homeodomain protein containing the WOX motif. Constitutive Expression of GLR1 could partially complement the glabrous phenotype of NILglr1. The knock down of GLR1 by RNA interference led to a significant decrease in trichome number on the leaves and glumes of the RNAi transgenic plants. CONCLUSION: GLR1 plays an important role in rice trichome development and will contribute to breeding of glabrous elite rice varieties. GLR1 A rice glutamate receptor-like gene is critical for the division and survival of individual cells in the root apical meristem 2006 Plant Cell State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310029, China. Glu receptors are known to function as Glu-activated ion channels that mediate mostly excitatory neurotransmission in animals. Glu receptor-like genes have also been reported in higher plants, although their function is largely unknown. We have identified a rice (Oryza sativa) Glu receptor-like gene, designated GLR3.1, in which mutation by T-DNA insertion caused a short-root mutant phenotype. Histology and DNA synthesis analyses revealed that the mutant root meristematic activity is distorted and is accompanied by enhanced programmed cell death. Our results supply genetic evidence that a plant Glu receptor-like gene, rice GLR3.1, is essential for the maintenance of cell division and individual cell survival in the root apical meristem at the early seedling stage. GLR3.1 Identification of cis-regulatory elements required for endosperm expression of the rice storage protein glutelin gene GluB-1 1999 Plant Mol Biol Department of Biotechnology, National Institute of Agrobiological Resources, Tsukuba, Ibaraki, Japan. Rice storage protein glutelin genes are coordinately regulated during seed development. A previous 5' deletion analysis using transgenic tobacco revealed that the minimum 5' region necessary for endosperm specificity was within -245 bp of the transcription start site, and included the AACA and GCN4 motifs that are highly conserved in the 5'-flanking regions of all glutelin genes. In this paper, the sequence elements essential for endosperm-specific expression are characterized in stable transgenic tobacco plants by both loss-of-function and gain-of-function experiments using this minimum promoter. Base substitution analysis shows that the proximal AACA motif between -73 and -61, and the GCN4 motif between -165 and -158 act as critical elements. An ACGT motif between -81 and -75, and Skn-I-like elements between -173 and -169 also play important roles in controlling the seed-specific expression. When the distal region between -245 and -145 containing the AACA and the GCN4 motifs or the proximal region between -113 and -46 containing the ACGT and AACA motifs is fused to a truncated promoter (-90 to +9) of the CaMV 35S gene fused to the beta-glucuronidase (GUS) reporter gene, high levels of seed-specific expression are observed in these fusions, thereby indicating that either pair of motifs is sufficient to confer seed expression in these fusions. However, when substituted for by the CaMV 35S core promoter (-46 to +1), seed expression is abolished, suggesting that the sequence between -90 and -46 of the CaMV 35S promoter containing G-box-like motif (as-1 element) is required for such specific expression in addition to AACA and GCN4 motifs. Therefore, we conclude that at least three cis-regulatory elements, the AACA motif, GCN4 motif and ACGT motif, are necessary to mediate endosperm expression of the GluB-1 glutelin gene. GluB-1 The 3'-untranslated region of rice glutelin GluB-1 affects accumulation of heterologous protein in transgenic rice 2009 Biotechnol Lett Transgenic Crop Research and Development Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602, Japan. We compared the effect of the rice storage protein glutelin B-1 (GluB-1) terminator with the nopaline synthase (Nos) terminator on the accumulation of the modified house dust mite allergen mDer f 2 driven by the maize ubiquitin promoter in transgenic rice. Accumulation of mDer f 2 in transgenic seed and leaf using the GluB-1 terminator was greater than when using the Nos terminator construct. The mDer f 2 mRNA containing the GluB-1 3'UTR was processed and polyadenylated at the same sites as the native GluB-1 mRNA in the seeds but diverged in leaves of the transgenic plants. In contrast, the poly(A) sites of mDer f 2 containing Nos 3'UTR were more divergent in both seed and leaf. These results suggest that GluB-1 3'UTR functions as a faithful terminator and that termination at the specific sites may play an important role in mRNA stability and/or translatability, resulting in higher levels of protein accumulation. GluB-1 Functions of the CCCH type zinc finger protein OsGZF1 in regulation of the seed storage protein GluB-1 from rice 2014 Plant Mol Biol Sylvius Laboratory, Institute of Biology (IBL), Leiden University, Sylviusweg 72, 2333 BE, PO Box 9505, 2300 RA, Leiden, The Netherlands. Glutelins are the most abundant storage proteins in rice grain and can make up to 80 % of total protein content. The promoter region of GluB-1, one of the glutelin genes in rice, has been intensively used as a model to understand regulation of seed-storage protein accumulation. In this study, we describe a zinc finger gene of the Cys3His1 (CCCH or C3H) class, named OsGZF1, which was identified in a yeast one-hybrid screening using the core promoter region of GluB-1 as bait and cDNA expression libraries prepared from developing rice panicles and grains as prey. The OsGZF1 protein binds specifically to the bait sequence in yeast and this interaction was confirmed in vitro. OsGZF1 is predominantly expressed in a confined domain surrounding the scutellum of the developing embryo and is localised in the nucleus. Transient expression experiments demonstrated that OsGZF1 can down-regulate a GluB-1-GUS (beta-glucuronidase) reporter and OsGZF1 was also able to significantly reduce activation conferred by RISBZ1 which is a known strong GluB-1 activator. Furthermore, down-regulation of OsGZF1 by an RNAi approach increased grain nitrogen concentration. We propose that OsGZF1 has a function in regulating the GluB-1 promoter and controls accumulation of glutelins during grain development. GluB-1,OsGZF1 Development of Simple Functional Markers for Low Glutelin Content Gene 1 (Lgc1) in Rice (Oryza sativa) 2010 Rice Science Jiangsu High Quality Rice Research and Development Center, Institute of Food Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China Rice with low glutelin content is suitable as functional food for patients affected by kidney failure. Low glutelin-content gene Lgc1 in rice has a 3.5-kb deletion between two highly similar glutelin genes GluB4 and GluB5, which locates on the short arm of chromosome 2. To improve the selection efficiency in low glutelin-content rice breeding, two molecular markers designated as InDel-Lgc1-1 and InDel-Lgc1-2 were developed to detect the low glutelin-content gene Lgc1. A double PCR detection indicated that combined use of the two markers could easily distinguish the genotypes of Lgc1 from different rice varieties. Therefore, as a simple and low-cost technique, the molecular marker could be widely used to identify different varieties with Lgc1 gene and applied in marker-assisted selection of low glutelin-content rice. GluB4,GluB5 Low glutelin content1: A Dominant Mutation That Suppresses the Glutelin Multigene Family via RNA Silencing in Rice 2003 The Plant Cell Online Institute of Radiation Breeding, National Institute of Agrobiological Sciences, Ohmiya-machi, Naka-gun, Ibaraki 319-2293, Japan. Low glutelin content1 (Lgc1) is a dominant mutation that reduces glutelin content in rice grains. Glutelin is a major seed storage protein encoded by a multigene family. RNA gel blot and reverse transcriptase-mediated PCR analyses revealed that Lgc1 acts at the mRNA level in a similarity-dependent manner. In Lgc1 homozygotes, there is a 3.5-kb deletion between two highly similar glutelin genes that forms a tail-to-tail inverted repeat, which might produce a double-stranded RNA molecule, a potent inducer of RNA silencing. The hypothesis that Lgc1 suppresses glutelin expression via RNA silencing is supported by transgenic analysis using this Lgc1 candidate region, by reporter gene analysis, and by the detection of small interfering RNAs. In this context, Lgc1 provides an interesting example of RNA silencing occurring among genes that exhibit various levels of similarity to an RNA-silencing-inducing gene. Possible mechanisms for gene silencing of the glutelin multigene family by Lgc1 are discussed. GluB4,GluB5 Gene-gene interactions between mutants that accumulate abnormally high amounts of proglutelin in rice seed 2010 Breeding Science Faculty of Agriculture, Kyushu University We had previously identified eight mutants, esp2 and g(G)lups1 to 7, which accumulated abnormally high amounts of proglutelin, the major storage protein in rice seeds. Analysis of their seed proteins by SDS-PAGE, their levels of the luminal chaperone BiP and gene-gene interactions indicated that these mutants fell into four classes. The most epistatic class consisted of esp2, which encodes a defective protein disulfide isomerase (PDI). A second class consisting of Glup1, glup2 and glup7 was hypostatic to esp2, and showed abnormally high levels of BiP, suggesting that maturation and export of proglutelins from the ER are inhibited in this class of mutants. The third class containing glup4, Glup5 and glup6 mutations was hypostatic to esp2, Glup1, glup2 and glup7. Since the glup4 allele encodes the small GTPase Rab5a, which participates in the trafficking of proglutelin from Golgi apparatus to the protein storage vacuole (PSV), this third class of mutants is likely affected in this process. Lastly, glup3, which encodes a vacuolar processing enzyme, which proteolytically processes proglutelin into acidic and basic subunits within the PSV, was hypostatic to the other mutants. Overall, these gene relationships are consistent with the sequential intracellular transport and processing of proglutelin and provide novel insights on the trafficking of proglutelin to the PSV. Glup3|OsVPE1,OsRab5a|gpa1|glup4 The vacuolar processing enzyme OsVPE1 is required for efficient glutelin processing in rice 2009 Plant J Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, China. Rice (Oryza sativa L.) accumulates prolamines and glutelins as its major storage proteins. Glutelins are synthesized on rough endoplasmic reticulum as 57-kDa precursors; they are then sorted into protein storage vacuoles where they are processed into acidic and basic subunits. We report a novel rice glutelin mutant, W379, which accumulates higher levels of the 57-kDa glutelin precursor. Genetic analysis revealed that the W379 phenotype is controlled by a single recessive nuclear gene. Using a map-based cloning strategy, we identified this gene, OsVPE1, which is a homolog of the Arabidopsis betaVPE gene. OsVPE1 encodes a 497-amino-acid polypeptide. Nucleotide sequence analysis revealed a missense mutation in W379 that changes Cys269 to Gly. Like the wild-type protein, the mutant protein is sorted into vacuoles; however, the enzymatic activity of the mutant OsVPE1 is almost completely eliminated. Further, we show that OsVPE1 is incorrectly cleaved, resulting in a mature protein that is smaller than the wild-type mature protein. Taken together, these results demonstrate that OsVPE1 is a cysteine protease that plays a crucial role in the maturation of rice glutelins. Further, OsVPE1 Cys269 is a key residue for maintaining the Asn-specific cleavage activity of OsVPE1. Glup3|OsVPE1 Bcl-2 suppresses hydrogen peroxide-induced programmed cell death via OsVPE2 and OsVPE3, but not via OsVPE1 and OsVPE4, in rice 2011 FEBS J State Key Laboratory of Plant Physiology and Biochemistry, Key Laboratory for Cell and Gene Engineering of Zhejiang province, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China. Hydrogen peroxide (H(2)O(2)) is known to be a key player in apoptosis in animals. The components and pathways regulating H(2)O(2)-induced programmed cell death in plants, however, remain largely unknown. In the present study, rice transgenic lines overexpressing Bcl-2, a human apoptotic suppressor, were obtained. These transgenic lines showed increased tolerance to high levels of H(2)O(2), resulting in increased seed germination rates, root elongation, root tip cell viability and chlorophyll retention compared to control lines. In the control lines, treatment with H(2)O(2) resulted in DNA laddering and a clear terminal transferase dUTP nick end labeling signal, which are the hallmarks of programmed cell death. However, this effect was not detected in the Bcl-2-overexpressing transgenic lines. Further investigations indicated that Bcl-2 suppressed H(2)O(2)-induced programmed cell death but did not inhibit stress-elicited reactive oxygen species production in rice. RT-PCR revealed that the expression of the two vacuolar processing enzyme genes (i.e. OsVPE2 and OsVPE3) was dramatically induced by H(2)O(2) in the wild-type line but not in the Bcl-2-overexpressing line. Moreover, treatment with H(2)O(2) resulted in the disruption of the vacuolar membrane in the wild-type line. The expression levels of OsVPE1 and OsVPE4 did not significantly differ between the wild-type line and the transgenic line that was treated or untreated with H(2)O(2). The similar roles of Bcl-2 and OsVPEs during endogenous reactive oxygen species-triggered programmed cell death were also confirmed by NaCl stress in rice. To our knowledge, the present study is the first to demonsatrate that Bcl-2 overexpression inhibits H(2)O(2)-induced programmed cell death and enhances H(2)O(2) tolerance. We propose that Bcl-2 overexpression in rice suppresses the transcriptional activation of OsVPE2 and OsVPE3, but not of OsVPE1 or OsVPE4. Glup3|OsVPE1,OsVPE2,OsVPE4,OsVPE3|REP-2 Vacuolar processing enzyme plays an essential role in the crystalline structure of glutelin in rice seed 2010 Plant Cell Physiol Faculty of Agriculture, Kyushu University, Fukuoka, Japan. kumamaru@agr.kyushu-u.ac.jp To identify the function of genes that regulate the processing of proglutelin, we performed an analysis of glup3 mutants, which accumulates excess amounts of proglutelin and lack the vacuolar processing enzyme (VPE). VPE activity in developing seeds from glup3 lines was reduced remarkably compared with the wild type. DNA sequencing of the VPE gene in glup3 mutants revealed either amino acid substitutions or the appearance of a stop codon within the coding region. Microscopic observations showed that alpha-globulin and proglutelin were distributed homogeneously within glup3 protein storage vacuoles (PSVs), and that glup3 PSVs lacked the crystalline lattice structure typical of wild-type PSVs. This suggests that the processing of proglutelin by VPE in rice is essential for proper PSV structure and compartmentalization of storage proteins. Growth retardation in glup3 seedlings was also observed, indicating that the processing of proglutelin influences early seedling development. These findings indicate that storage of glutelin in its mature form as a crystalline structure in PSVs is required for the rapid use of glutelin as a source of amino acids during early seedling development. In conclusion, VPE plays an important role in the formation of protein crystalline structures in PSVs. Glup3|OsVPE1 A guanine nucleotide exchange factor for Rab5 proteins is essential for intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm 2013 Plant Physiol Kyushu University, Fukuoka 812-8581, Japan. Rice (Oryza sativa) glutelins are synthesized on the endoplasmic reticulum as a precursor, which are then transported via the Golgi to protein storage vacuoles (PSVs), where they are proteolytically processed into acidic and basic subunits. The glutelin precursor mutant6 (glup6) accumulates abnormally large amounts of proglutelin. Map-base cloning studies showed that glup6 was a loss-of-function mutant of guanine nucleotide exchange factor (GEF), which activates Rab GTPase, a key regulator of membrane trafficking. Immunofluorescence studies showed that the transport of proglutelins and alpha-globulins to PSV was disrupted in glup6 endosperm. Secreted granules of glutelin and alpha-globulin were readily observed in young glup6 endosperm, followed by the formation of large dilated paramural bodies (PMBs) containing both proteins as the endosperm matures. The PMBs also contained membrane biomarkers for the Golgi and prevacuolar compartment as well as the cell wall component, beta-glucan. Direct evidence was gathered showing that GLUP6/GEF activated in vitro GLUP4/Rab5 as well as several Arabidopsis (Arabidopsis thaliana) Rab5 isoforms to the GTP-bound form. Therefore, loss-of-function mutations in GEF or Rab5 disrupt the normal transport of proglutelin from the Golgi to PSVs, resulting in the initial extracellular secretion of these proteins followed, in turn, by the formation of PMBs. Overall, our results indicate that GLUP6/GEF is the activator of Rab5 GTPase and that the cycling of GTP- and GDP-bound forms of this regulatory protein is essential for the intracellular transport of proglutelin and alpha-globulin from the Golgi to PSVs and in the maintenance of the general structural organization of the endomembrane system in rice seeds. GLUP6 A loss-of-function mutation of rice DENSE PANICLE 1 causes semi-dwarfness and slightly increased number of spikelets 2011 Breeding Science QTL Genomics Research Center, National Institute of Agrobiological Sciences In cereal breeding, semi-dwarfness and an increased spikelet number are favorable characteristics. We show that the rice DENSE PANICLE 1 (DN1) mutant allele Dn1-1 causes both of these characteristics and that Dn1-1 is a loss-of-function mutation. DN1 is allelic to DENSE AND ERECT PANICLE 1 (DEP1) (=qPE(9-1)). The expression level of OsCKX2 in the shoot apex of Dn1-1 plants is similar to that in the wild type, indicating that OsCKX2 does not contribute to an increased number of spikelets. A comparison of the Dn1-1 and Dn1-3 alleles suggests that the N-terminal region of DN1 contains a coiled-coil domain and a nuclear localization signal that might be responsible for semi-dwarfness. This comparison also revealed that a single transmembrane alpha-helix, a VWFC module, and a four-disulfide core domain can further increase spikelet number. Subcellular localization analysis of the DN1 protein fused with green fluorescent protein (GFP) implies that DN1 is located in the nucleus and cell membrane and that its N-terminal fragment is cleaved. Dn1-1 plants have normal sensitivity to gibberellin, brassinolide, and kinetin, and we observed no genetic epistasis with brassinolide-related mutants, suggesting that DN1 does not function in the signaling pathways of these phytohormones. Gn1a|OsCKX2,DEP1|DN1|qPE9-1|OsDEP1 Mutations in the F-box gene LARGER PANICLE improve the panicle architecture and enhance the grain yield in rice 2011 Plant Biotechnol J State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. Panicle architecture is one of the most important agronomical traits that directly contribute to grain yield in rice (Oryza sativa L.). We report herein an in-depth characterization of two allelic larger panicle (lp) mutants that show significantly increased panicle size as well as improved plant architecture. Morphological analyses reveal that panicles of two mutants produced more inflorescence branches, especially the primary branches, and contained more grains. Moreover, mutant plants also display more lodging resistance than the wild type. The grain yield per plant in mutants is also increased, suggesting that mutant plants have useful potential for high grain yield in rice breeding. Map-based cloning reveals that LARGER PANICLE (LP) encodes a Kelch repeat-containing F-box protein. RNA in situ hybridization studies display that LP expression was enriched in the branch primordial region. Subcellular localization analyses demonstrate that LP is an endoplasmic reticulum (ER) localized protein, suggesting that LP might be involved in ER-associated protein degradation (ERAD). Using yeast two-hybrid assay and bimolecular fluorescence complementation analysis, we confirm that LP is an F-box protein and could interact with rice SKP1-like protein in an F-box domain-dependent manner. Quantitative real-time PCR results show that OsCKX2, which encodes cytokinin oxidase/dehydrogenase, is down-regulated evidently in mutants, implying that LP might be involved in modulating cytokinin level in plant tissues. These results suggest that LP plays an important role in regulating plant architecture, particularly in regulating panicle architecture, thereby representing promising targets for genetic improvement of grain production plants. Gn1a|OsCKX2,EP3|LP Cytokinin oxidase regulates rice grain production 2005 Science Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan. Most agriculturally important traits are regulated by genes known as quantitative trait loci (QTLs) derived from natural allelic variations. We here show that a QTL that increases grain productivity in rice, Gn1a, is a gene for cytokinin oxidase/dehydrogenase (OsCKX2), an enzyme that degrades the phytohormone cytokinin. Reduced expression of OsCKX2 causes cytokinin accumulation in inflorescence meristems and increases the number of reproductive organs, resulting in enhanced grain yield. QTL pyramiding to combine loci for grain number and plant height in the same genetic background generated lines exhibiting both beneficial traits. These results provide a strategy for tailormade crop improvement. Gn1a|OsCKX2 New perspectives on the endo-beta-glucanases of glycosyl hydrolase Family 17 2000 Int J Biol Macromol Section of Molecular and Cellular Biology, University of California, Davis, CA 95616-8535, USA. brthomas@ucdavis.edu Isozymes of glycosyl hydrolase Family 17 hydrolyze 1,3-beta-glucan polysaccharides found in the cell wall matrix of plants and fungi, enabling these plant enzymes to serve diverse roles in plant defense and plant development. Fourteen genes from Family 17 have been characterized in the genome of rice. A sequence dendrogram analysis divided these genes into four subfamilies. The recombinant GNS1 enzyme from subfamily B had 1,3;1,4-beta-glucanase activity, suggesting a role for this isozyme in plant development. Gns1 Characterization of transgenic rice plants over-expressing the stress-inducible beta-glucanase gene Gns1 2003 Plant Mol Biol National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba 305-8602, Japan. ynishi@nias.affrc.go.jp The Gns1 gene of rice (Oryza sativa L. japonica) encodes 1,3;1,4-beta glucanase (EC 3.2.1.73), which hydrolyzes 1,3;1,4-beta-glucosidic linkages on 1,3;1,4-beta-glucan, an important component of cell walls in the Poaceae family. RNA and protein gel blot analyses demonstrated that blast disease or dark treatment induced the expression of the Gns1 gene. To assess the function of the Gns1 gene in disease resistance, we characterized transgenic rice plants constitutively expressing the Gns1 gene. The introduced Gns1 gene was driven by the CaMV 35S promoter and its products were found in the apoplast and accumulated in up to 0.1% of total soluble protein in leaves. Although transgenic plants showed stunted growth and impaired root formation, fertility, germination, and coleoptile elongation appeared unaffected compared to non-transgenic control plants, indicating that Gns1 does not play a crucial role in rice germination and coleoptile elongation. When transgenic plants were inoculated with virulent blast fungus (Magnaporthe grisea), they developed many resistant-type lesions on the inoculated leaf accompanying earlier activation of defense-related genes PR-1 and PBZ1 than in control plants. Transgenic plants spontaneously produced brown specks, similar in appearance to those reported for an initiation type of disease-lesion-mimic mutants, on the third and fourth leaves and occasionally on older leaves without inoculation of pathogens. Expression of the two defense-related genes was drastically increased after the emergence of the lesion-mimic phenotype. Gns1,OsPR10a|PBZ1 N-glycan maturation is crucial for cytokinin-mediated development and cellulose synthesis in Oryza sativa 2013 Plant J Division of Applied Life Science (BK21 Program) and PMBBRC, Gyeongsang National University, 501 Jinju-daero, Jinju, 660-701, Korea. To explore the physiological significance of N-glycan maturation in the plant Golgi apparatus, gnt1, a mutant with loss of N-acetylglucosaminyltransferase I (GnTI) function, was isolated in Oryza sativa. gnt1 exhibited complete inhibition of N-glycan maturation and accumulated high-mannose N-glycans. Phenotypic analyses revealed that gnt1 shows defective post-seedling development and incomplete cell wall biosynthesis, leading to symptoms such as failure in tiller formation, brittle leaves, reduced cell wall thickness, and decreased cellulose content. The developmental defects of gnt1 ultimately resulted in early lethality without transition to the reproductive stage. However, callus induced from gnt1 seeds could be maintained for periods, although it exhibited a low proliferation rate, small size, and hypersensitivity to salt stress. Shoot regeneration and dark-induced leaf senescence assays indicated that the loss of GnTI function results in reduced sensitivity to cytokinin in rice. Reduced expression of A-type O. sativa response regulators that are rapidly induced by cytokinins in gnt1 confirmed that cytokinin signaling is impaired in the mutant. These results strongly support the proposed involvement of N-glycan maturation in transport as well as in the function of membrane proteins that are synthesized via the endomembrane system. GnT1 Expression profiling of rice cultivars differing in their tolerance to long-term drought stress 2009 Plant Mol Biol Max-Planck-Institut fur Molekulare Pflanzenphysiologie, Am Muhlenberg 1, 14476 Potsdam, Germany. Understanding the molecular basis of plant performance under water-limiting conditions will help to breed crop plants with a lower water demand. We investigated the physiological and gene expression response of drought-tolerant (IR57311 and LC-93-4) and drought-sensitive (Nipponbare and Taipei 309) rice (Oryza sativa L.) cultivars to 18 days of drought stress in climate chamber experiments. Drought stressed plants grew significantly slower than the controls. Gene expression profiles were measured in leaf samples with the 20 K NSF oligonucleotide microarray. A linear model was fitted to the data to identify genes that were significantly regulated under drought stress. In all drought stressed cultivars, 245 genes were significantly repressed and 413 genes induced. Genes differing in their expression pattern under drought stress between tolerant and sensitive cultivars were identified by the genotype x environment (G x E) interaction term. More genes were significantly drought regulated in the sensitive than in the tolerant cultivars. Localizing all expressed genes on the rice genome map, we checked which genes with a significant G x E interaction co-localized with published quantitative trait loci regions for drought tolerance. These genes are more likely to be important for drought tolerance in an agricultural environment. To identify the metabolic processes with a significant G x E effect, we adapted the analysis software MapMan for rice. We found a drought stress induced shift toward senescence related degradation processes that was more pronounced in the sensitive than in the tolerant cultivars. In spite of higher growth rates and water use, more photosynthesis related genes were down-regulated in the tolerant than in the sensitive cultivars. gp1 bZIP transcription factor OsbZIP52/RISBZ5: a potential negative regulator of cold and drought stress response in rice 2011 Planta Key Laboratory of Cell Proliferation and Regulation of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, People's Republic of China. OsbZIP52/RISBZ5 is a member of the basic leucine zipper (bZIP) transcription factor (TF) family in rice (Oryza sativa) isolated from rice (Zhonghua11) panicles. Expression of the OsbZIP52 gene was strongly induced by low temperature (4°C) but not by drought, PEG, salt, or ABA. The subcellular localization of OsbZIP52-GFP in onion (Allium cepa) epidermis cells revealed that OsbZIP52 is a nuclear localized protein. A transactivation assay in yeast demonstrated that OsbZIP52 functions as a transcriptional activator and can specifically bind to the G-box promoter motif. In a yeast two-hybrid (Y-2-H) experiment, OsbZIP52 was able to form homodimeric complexes. Rice plants overexpressing OsbZIP52 showed significantly increased sensitivity to cold and drought stress. Real-time PCR analysis revealed that some abiotic stress-related genes, such as OsLEA3, OsTPP1, Rab25, gp1 precursor, beta-gal, LOC_Os05g11910 and LOC_Os05g39250, were down-regulated in OsbZIP52 overexpression lines. These results suggest that OsbZIP52/RISBZ5 could function as a negative regulator in cold and drought stress environments. gp1 GLUTELIN PRECURSOR ACCUMULATION3 encodes a regulator of post-Golgi vesicular traffic essential for vacuolar protein sorting in rice endosperm 2014 Plant Cell State Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, China. In seed plants, a major pathway for sorting of storage proteins to the protein storage vacuole (PSV) depends on the Golgi-derived dense vesicles (DVs). However, the molecular mechanisms regulating the directional trafficking of DVs to PSVs remain largely elusive. Here, we report the functional characterization of the rice (Oryza sativa) glutelin precursor accumulation3 (gpa3) mutant, which exhibits a floury endosperm phenotype and accumulates excess proglutelins in dry seeds. Cytological and immunocytochemistry studies revealed that in the gpa3 mutant, numerous proglutelin-containing DVs are misrouted to the plasma membrane and, via membrane fusion, release their contents into the apoplast to form a new structure named the paramural body. Positional cloning of GPA3 revealed that it encodes a plant-specific kelch-repeat protein that is localized to the trans-Golgi networks, DVs, and PSVs in the developing endosperm. In vitro and in vivo experiments verified that GPA3 directly interacts with the rice Rab5a-guanine exchange factor VPS9a and forms a regulatory complex with Rab5a via VPS9a. Furthermore, our genetic data support the notion that GPA3 acts synergistically with Rab5a and VPS9a to regulate DV-mediated post-Golgi traffic in rice. Our findings provide insights into the molecular mechanisms regulating the plant-specific PSV pathway and expand our knowledge of vesicular trafficking in eukaryotes. GPA3 Rice G-protein coupled receptor (GPCR): In silico analysis and transcription regulation under abiotic stress 2011 Plant Signal Behav International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India. Majority of transmembrane signal transduction in response to diverse external stimuli is mediated by G-protein coupled receptors (GPCRs) and are the principal signal transducers. GPCRs are characterized by seven membrane-spanning domains with an extracellular N-terminus and a cytoplasmic C-terminus which functions along with GTP-binding protein in a highly coordinated fashion. Role of heterotrimeric G-proteins in abiotic stresses has been reported, but the response of GPCR is not yet well characterized. In the present study we report the isolation of one putative GPCR (966 bp) from Indica rice (Oryza sativa cv. Indica group Swarna) and described its transcriptional regulation under abiotic stresses. Amino acid sequence analyses shows the presence of typical heptahelical transmembrane spanning domains with extracellular N-terminus involved in ligand binding and cytoplasm facing C-terminus that binds with heterotrimeric G-protein. Sequence analysis also confirmed the presence of all signature motifs required for functional GPCR. Domain and site prediction shows the presence of myristoylation sites for membrane association, and protein kinase C sites for its desensitization. The transcript levels of rice GPCR was induced following NaCl and ABA treatments. However, in drought condition the expression profile of GPCR up regulated during early exposure which subsequently decreased. On the other hand it seems no significant effect due to cold and heat stress. These findings provide a direct evidence for transcriptional regulation of rice GPCR under abiotic stress conditions. These findings also suggest that GPCR can be exploited for promoting stress tolerance in plants. OsGPCR The rice pentatricopeptide repeat protein RF5 restores fertility in Hong-Lian cytoplasmic male-sterile lines via a complex with the glycine-rich protein GRP162 2012 Plant Cell Key Laboratory of the Ministry of Education for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China. The cytoplasmic male sterility (CMS) phenotype in plants can be reversed by the action of nuclear-encoded fertility restorer (Rf) genes. The molecular mechanism involved in Rf gene-mediated processing of CMS-associated transcripts is unclear, as are the identities of other proteins that may be involved in the CMS-Rf interaction. In this study, we cloned the restorer gene Rf5 for Hong-Lian CMS in rice and studied its fertility restoration mechanism with respect to the processing of the CMS-associated transcript atp6-orfH79. RF5, a pentatricopeptide repeat (PPR) protein, was unable to bind to this CMS-associated transcript; however, a partner protein of RF5 (GRP162, a Gly-rich protein encoding 162 amino acids) was identified to bind to atp6-orfH79. GRP162 was found to physically interact with RF5 and to bind to atp6-orfH79 via an RNA recognition motif. Furthermore, we found that RF5 and GRP162 are both components of a restoration of fertility complex (RFC) that is 400 to 500 kD in size and can cleave CMS-associated transcripts in vitro. Evidence that a PPR protein interacts directly with a Gly-rich protein to form a subunit of the RFC provides a new perspective on the molecular mechanisms underlying fertility restoration. GRP162,OsSUT2|OsSUT2M,Rf1a|Rf5 GS3, a major QTL for grain length and weight and minor QTL for grain width and thickness in rice, encodes a putative transmembrane protein 2006 Theor Appl Genet National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, 430070 Wuhan, China. The GS3 locus located in the pericentromeric region of rice chromosome 3 has been frequently identified as a major QTL for both grain weight (a yield trait) and grain length (a quality trait) in the literature. Near isogenic lines of GS3 were developed by successive crossing and backcrossing Minghui 63 (large grain) with Chuan 7 (small grain), using Minghui 63 as the recurrent parent. Analysis of a random subpopulation of 201 individuals from the BC3F2 progeny confirmed that the GS3 locus explained 80-90% of the variation for grain weight and length in this population. In addition, this locus was resolved as a minor QTL for grain width and thickness. Using 1,384 individuals with recessive phenotype (large grain) from a total of 5,740 BC3F2 plants and 11 molecular markers based on sequence information, GS3 was mapped to a DNA fragment approximately 7.9 kb in length. A full-length cDNA corresponding to the target region was identified, which provided complete sequence information for the GS3 candidate. This gene consists of five exons and encodes 232 amino acids with a putative PEBP-like domain, a transmembrane region, a putative TNFR/NGFR family cysteine-rich domain and a VWFC module. Comparative sequencing analysis identified a nonsense mutation, shared among all the large-grain varieties tested in comparison with the small grain varieties, in the second exon of the putative GS3 gene. This mutation causes a 178-aa truncation in the C-terminus of the predicted protein, suggesting that GS3 may function as a negative regulator for grain size. Cloning of such a gene provided the opportunity for fully characterizing the regulatory mechanism and related processes during grain development. GS3 Evolutionary history of GS3, a gene conferring grain length in rice 2009 Genetics Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan. Unlike maize and wheat, where artificial selection is associated with an almost uniform increase in seed or grain size, domesticated rice exhibits dramatic phenotypic diversity for grain size and shape. Here we clone and characterize GS3, an evolutionarily important gene controlling grain size in rice. We show that GS3 is highly expressed in young panicles in both short- and long-grained varieties but is not expressed in leaves or panicles after flowering, and we use genetic transformation to demonstrate that the dominant allele for short grain complements the long-grain phenotype. An association study revealed that a C to A mutation in the second exon of GS3 (A allele) was associated with enhanced grain length in Oryza sativa but was absent from other Oryza species. Linkage disequilibrium (LD) was elevated and there was a 95.7% reduction in nucleotide diversity (theta(pi)) across the gene in accessions carrying the A allele, suggesting positive selection for long grain. Haplotype analysis traced the origin of the long-grain allele to a Japonica-like ancestor and demonstrated introgression into the Indica gene pool. This study indicates a critical role for GS3 in defining the seed morphologies of modern subpopulations of O. sativa and enhances the potential for genetic manipulation of grain size in rice. GS3 Multiple and independent origins of short seeded alleles of GS3 in rice 2013 Breed Sci Faculty of Agriculture, Graduate School, Kyushu University , 6-10-1, Hakozaki, Higashi, Fukuoka 812-8581, Japan. GRAIN SIZE 3 (GS3) is a cloned gene that is related to seed length. Here we report the discovery of new deletion alleles at the GS3 locus, each of which confer short seed. We selected ten short seeded cultivars from a collection of 282 diverse cultivars. Sequence analysis across the GS3 gene in these ten cultivars identified three novel alleles and a known allele that contain several independent deletion(s) in the fifth exon of GS. These independent deletion variants each resulted in a frameshift mutation that caused a premature stop codon, and they were functionally similar to one another. Each coded for a truncated gene product that behaved as an incomplete dominant allele and conferred a short seeded phenotype. Haplotype analysis of these sequence variants indicated that two of the variants were of japonica origin, and two were from indica. Transformation experiments demonstrated that one of the deletion alleles of GS3 decrease the cell number in the upper epidermis of the glume, resulting in a significant reduction in seed length. The multiple and independent origins of these short seeded alleles indicate that farmers and early breeders imposed artificial selection favoring short seeds. GS3 GS3 participates in stigma exsertion as well as seed length in rice 2011 Breeding Science Plant Breeding Laboratory, Faculty of Agriculture, Graduate School, Kyushu University Stigma exsertion is an important trait that contributes to the improvement of seed production in hybrid rice. We demonstrate that GS3, one of the genes regulating seed length, also regulates stigma length and participates in stigma exsertion in rice. GS3 mRNA is expressed in the basal part of the young stigma, and a nonsense mutation in the second exon of GS3 causes an increase in cell number, resulting in elongation of the stigma. Manipulation of GS3 should contribute to the improvement of hybrid seed-production efficiency. GS3 A causal C-A mutation in the second exon of GS3 highly associated with rice grain length and validated as a functional marker 2009 Theor Appl Genet National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research, Huazhong Agricultural University, 430070, Wuhan, China. Comparative sequencing of GS3, the most important grain length (GL) QTL, has shown that differentiation of rice GL might be principally due to a single nucleotide polymorphism (SNP) between C and A in the second exon. A total of 180 varieties representing a wide range of rice germplasm were used for association analysis between C-A mutation and GL in order to confirm the potential causal mutation. A cleaved amplified polymorphic sequence (CAPS) marker, SF28, was developed based on the C-A polymorphism in the GS3 gene. A total of 142 varieties carried allele C with GL from 6.4 to 8.8 mm, while the remaining 38 varieties carried allele A with GL from 8.8 to 10.7 mm. Twenty-four unlinked SSR markers were selected to genotype 180 varieties for population structure analysis. Population structure was observed when the population was classified to three subpopulations. Average GL of either genotype A or genotype C within japonica among the three subpopulations had no significant difference from that in indica, respectively, although indica rice had longer grains on average than japonica in the 180 varieties. However, genotype C always had longer grain length on average than genotype A among three subpopulations. The mutation could explain 79.1, 66.4 and 34.7% of GL variation in the three subpopulations, respectively. These results clearly confirmed the mutation between C and A was highly associated with GL. The SF28 could be a functional marker for improvement of rice grain length. GS3 Antagonistic actions of HLH/bHLH proteins are involved in grain length and weight in rice 2012 PLoS One Graduate School of Horticulture, Chiba University, Chiba, Japan. Grain size is a major yield component in rice, and partly controlled by the sizes of the lemma and palea. Molecular mechanisms controlling the sizes of these organs largely remain unknown. In this study, we show that an antagonistic pair of basic helix-loop-helix (bHLH) proteins is involved in determining rice grain length by controlling cell length in the lemma/palea. Overexpression of an atypical bHLH, named POSITIVE REGULATOR OF GRAIN LENGTH 1 (PGL1), in lemma/palea increased grain length and weight in transgenic rice. PGL1 is an atypical non-DNA-binding bHLH and assumed to function as an inhibitor of a typical DNA-binding bHLH through heterodimerization. We identified the interaction partner of PGL1 and named it ANTAGONIST OF PGL1 (APG). PGL1 and APG interacted in vivo and localized in the nucleus. As expected, silencing of APG produced the same phenotype as overexpression of PGL1, suggesting antagonistic roles for the two genes. Transcription of two known grain-length-related genes, GS3 and SRS3, was largely unaffected in the PGL1-overexpressing and APG-silenced plants. Observation of the inner epidermal cells of lemma revealed that are caused by increased cell length. PGL1-APG represents a new grain length and weight-controlling pathway in which APG is a negative regulator whose function is inhibited by PGL1. GS3,APG|OsPIL16,PGL1,SRS3 Linking differential domain functions of the GS3 protein to natural variation of grain size in rice 2010 Proc Natl Acad Sci U S A National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Grain yield in many cereal crops is largely determined by grain size. Here we report the genetic and molecular characterization of GS3, a major quantitative trait locus for grain size. It functions as a negative regulator of grain size and organ size. The wild-type isoform is composed of four putative domains: a plant-specific organ size regulation (OSR) domain in the N terminus, a transmembrane domain, a tumor necrosis factor receptor/nerve growth factor receptor (TNFR/NGFR) family cysteine-rich domain, and a von Willebrand factor type C (VWFC) in the C terminus. These domains function differentially in grain size regulation. The OSR domain is both necessary and sufficient for functioning as a negative regulator. The wild-type allele corresponds to medium grain. Loss of function of OSR results in long grain. The C-terminal TNFR/NGFR and VWFC domains show an inhibitory effect on the OSR function; loss-of-function mutations of these domains produced very short grain. This study linked the functional domains of the GS3 protein to natural variation of grain size in rice. GS3 The plant-specific G protein gamma subunit AGG3 influences organ size and shape in Arabidopsis thaliana 2012 New Phytol State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. * Control of organ size and shape by cell proliferation and cell expansion is a fundamental developmental process, but the mechanisms that set the size and shape of determinate organs are largely unknown in plants. * Molecular, genetic, cytological and biochemical approaches were used to characterize the roles of the Arabidopsis thaliana G protein gamma subunit (AGG3) gene in organ growth. * Here, we describe A. thaliana AGG3, which promotes petal growth by increasing the period of cell proliferation. Both the N-terminal region and the C-terminal domains of AGG3 are necessary for the function of AGG3. By contrast, analysis of a series of AGG3 derivatives with deletions in specific domains showed that the deletion of any of these domains cannot completely abolish the function of AGG3. The GFP-AGG3 fusion protein is localized to the plasma membrane. The predicted transmembrane domain plays an important role in the plasma membrane localization of AGG3. Genetic analyses revealed that AGG3 action requires a functional G protein alpha subunit (GPA1) and G protein beta subunit (AGB1). * Our findings demonstrate that AGG3, GPA1 and AGB1 act in the same genetic pathway to influence organ size and shape in A. thaliana. GS3,DEP1|DN1|qPE9-1|OsDEP1 Natural variation in GS5 plays an important role in regulating grain size and yield in rice 2011 Nat Genet National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan, China. Increasing crop yield is one of the most important goals of plant science research. Grain size is a major determinant of grain yield in cereals and is a target trait for both domestication and artificial breeding(1). We showed that the quantitative trait locus (QTL) GS5 in rice controls grain size by regulating grain width, filling and weight. GS5 encodes a putative serine carboxypeptidase and functions as a positive regulator of grain size, such that higher expression of GS5 is correlated with larger grain size. Sequencing of the promoter region in 51 rice accessions from a wide geographic range identified three haplotypes that seem to be associated with grain width. The results suggest that natural variation in GS5 contributes to grain size diversity in rice and may be useful in improving yield in rice and, potentially, other crops(2). GS5 Identification and characterization of dwarf 62, a loss-of-function mutation in DLT/OsGRAS-32 affecting gibberellin metabolism in rice 2010 Planta Department of Agronomy, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, 310029, China. A dwarf mutant, dwarf 62 (d62), was isolated from rice cultivar 93-11 by mutagenesis with gamma-rays. Under normal growth conditions, the mutant had multiple abnormal phenotypes, such as dwarfism, wide and dark-green leaf blades, reduced tiller numbers, late and asynchronous heading, short roots, partial male sterility, etc. Genetic analysis indicated that the abnormal phenotypes were controlled by the recessive mutation of a single nuclear gene. Using molecular markers, the D62 gene was fine mapped in 131-kb region at the short arm of chromosome 6. Positional cloning of D62 gene revealed that it was the same locus as DLT/OsGRAS-32, which encodes a member of the GRAS family. In previous studies, the DLT/OsGRAS-32 is confirmed to play positive roles in brassinosteroid (BR) signaling. Sequence analysis showed that the d62 carried a 2-bp deletion in ORF region of D62 gene which led to a loss-of-function mutation. The function of D62 gene was confirmed by complementation experiment. RT-PCR analysis and promoter activity analysis showed that the D62 gene expressed in all tested tissues including roots, stems, leaves and panicles of rice plant. The d62 mutant exhibited decreased activity of alpha-amylase in endosperm and reduced content of endogenous GA(1). The expression levels of gibberellin (GA) biosynthetic genes including OsCPS1, OsKS1, OsKO1, OsKAO, OsGA20ox2/SD1 and OsGA2ox3 were significantly increased in d62 mutant. Briefly, these results demonstrated that the D62 (DLT/OsGRAS-32) not only participated in the regulation of BR signaling, but also influenced GA metabolism in rice. DLT|OsGRAS-32|D62|GS6,OsCPS|OsCPS1,OsKOS4|OsKO1,OsKS1,sd1|GA20ox2 DWARF AND LOW-TILLERING acts as a direct downstream target of a GSK3/SHAGGY-like kinase to mediate brassinosteroid responses in rice 2012 Plant Cell State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. In Arabidopsis thaliana, the GSK3/SHAGGY-like kinase BRASSINOSTEROID-INSENSITIVE2 (BIN2) plays a critical role in the brassinosteroid (BR) signaling pathway by negatively regulating the activities of bri1-EMS-SUPPRESSOR1/BRASSINAZOLE-RESISTANT1 family transcription factors that regulate the expression of downstream BR-responsive genes. In this study, we analyzed the function of a rice (Oryza sativa) GSK3/SHAGGY-like kinase (GSK2), which is one of the orthologs of BIN2. Overexpression of GSK2 (Go) led to plants with typical BR loss-of-function phenotypes, and suppression of GSK2 resulted in enhanced BR signaling phenotypes. DWARF AND LOW-TILLERING (DLT) is a positive regulator that mediates several BR responses in rice. Suppression of DLT can enhance the phenotypes of BR receptor mutant d61-1, and overexpression of DLT obviously suppressed the BR loss-of-function phenotypes of both d61-1 and Go, suggesting that DLT functions downstream of GSK2 to modulate BR responses. Indeed, GSK2 can interact with DLT and phosphorylate DLT. Moreover, brassinolide treatment can induce the dephosphorylation of DLT, leading to the accumulation of dephosphorylated DLT protein. In GSK2 transgenic plants, the DLT phosphorylation level is dictated by the GSK2 level. These results demonstrate that DLT is a GSK2 substrate, further reinforcing that the BIN2/GSK2 kinase has multiple substrates that carry out various BR responses. DLT|OsGRAS-32|D62|GS6,GSK2,OsGSK4 DWARF AND LOW-TILLERING, a new member of the GRAS family, plays positive roles in brassinosteroid signaling in rice 2009 The Plant Journal State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. Rapid progress has been made regarding the understanding of brassinosteroid (BR) signaling in Arabidopsis. However, little is known about BR signaling in monotyledons. Here, we characterized a rice dwarf and low-tillering (dlt) mutant and cloned the corresponding gene via map-based cloning. DLT encodes a new member of the plant-specific GRAS family. The dwarf phenotype of dlt is similar to BR-deficient or signaling mutants in rice. In addition, both lamina bending and coleoptile elongation assays show that dlt is insensitive or much less responsive to brassinolide (BL), the most active BR, suggesting that DLT is involved in BR signaling. Consistent with this conclusion, the accumulation of transcripts of BR biosynthesis genes in the dlt mutant indicated that DLT is involved in feedback inhibition of BR biosynthesis genes. In addition, transcription of several other BR-regulated genes is altered in the dlt mutant. Finally, consistent with the fact that DLT is also negatively feedback-regulated by BR treatment, a gel mobility shift assay showed that OsBZR1 can bind to the DLT promoter through the BR-response element. Taken together, these studies have enabled us to identify a new signaling component that is involved in several specific BR responses in rice. DLT|OsGRAS-32|D62|GS6,OsBZR1 GS6, a member of the GRAS gene family, negatively regulates grain size in rice 2013 J Integr Plant Biol State Key Laboratory of Plant Physiology and Biochemistry, National Center for Evaluation of Agricultural Wild Plants (Rice), MOE Key Laboratory of Crop Heterosis and Utilization, Beijing 100193, China; Key Laboratory of Crop Genetic Improvement, Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China. Grain size is an important yield-related trait in rice. Intensive artificial selection for grain size during domestication is evidenced by the larger grains of most of today's cultivars compared with their wild relatives. However, the molecular genetic control of rice grain size is still not well characterized. Here, we report the identification and cloning of Grain Size 6 (GS6), which plays an important role in reducing grain size in rice. A premature stop at the +348 position in the coding sequence (CDS) of GS6 increased grain width and weight significantly. Alignment of the CDS regions of GS6 in 90 rice materials revealed three GS6 alleles. Most japonica varieties (95%) harbor the Type I haplotype, and 62.9% of indica varieties harbor the Type II haplotype. Association analysis revealed that the Type I haplotype tends to increase the width and weight of grains more than either of the Type II or Type III haplotypes. Further investigation of genetic diversity and the evolutionary mechanisms of GS6 showed that the GS6 gene was strongly selected in japonica cultivars. In addition, a "ggc" repeat region identified in the region that encodes the GRAS domain of GS6 played an important historic role in the domestication of grain size in rice. Knowledge of the function of GS6 might aid efforts to elucidate the molecular mechanisms that control grain development and evolution in rice plants, and could facilitate the genetic improvement of rice yield. DLT|OsGRAS-32|D62|GS6 Grain setting defect 1, encoding a remorin protein, affects the grain setting in rice through regulating plasmodesmatal conductance 2014 Plant Physiol Institute of Plant Physiology and Ecology, CAS. Effective grain filling is one of the key determinants of grain setting in rice. Grain setting defect 1 (GSD1), which encodes a putative remorin protein, was found to affect grain setting in rice. Investigation of the phenotype of a T-DNA insertion mutant (grain setting defect 1-Dominant, gsd1-D) with enhanced GSD1 expression revealed abnormalities including a reduced grain setting rate, accumulation of carbohydrates in leaves, and lower soluble sugar content in the phloem exudates. GSD1 was found to be specifically expressed in the plasma membrane and plasmodesmata (PD) of phloem companion cells. Experimental evidence suggests the phenotype of the gsd1-D mutant is caused by defects in the grain filling process as a result of the impaired transport of carbohydrates from the photosynthetic site to the phloem. GSD1 functioned in affecting PD conductance by interacting with OsACT1 in association with the PD callose binding protein PDCB1. Together, our results suggest that GSD1 may play a role in regulating photoassimilate translocation through the symplastic pathway to impact grain setting in rice. GSD1,OsACT1 GT-2: a transcription factor with twin autonomous DNA-binding domains of closely related but different target sequence specificity 1992 EMBO J University of California, Berkeley. A triplet of adjacent, highly similar GT motifs in the phyA promoter of rice functions to support maximal expression of this gene. We have obtained a recombinant clone that encodes a full-length nuclear protein, designated GT-2, which binds specifically to these target sequences. This novel protein contains acidic, basic and proline- + glutamine-rich regions, as well as two autonomous DNA-binding domains, one NH2-terminal and the other COOH-terminal, that discriminate with high resolution between the three GT motifs. A duplicated sequence of 75 amino acids, present once in each DNA-binding domain, appears likely to mediate DNA target element recognition. Each copy of this duplicated protein sequence is predicted to form three amphipathic alpha-helices separated from each other by two short loops. The absence of sequence similarity to other known proteins suggests that this predicted structural unit, which we term the trihelix motif, might be representative of a new class of DNA-binding proteins. GT-2 The HMG-I/Y protein PF1 stimulates binding of the transcriptional activator GT-2 to the PHYA gene promoter 1999 The Plant Journal Department of Plant and Microbial Biology, University of California, Berkeley 94720, USA. The DNA-binding proteins PF1 and GT-2 are factors that bind to different functionally defined, positively acting cis-elements in the PHYA genes of oat and rice, respectively. PF1 is an HMG-I/Y protein, with its cognate cis-element being an AT-rich sequence, designated PE1, whereas GT-2 is a transcriptional activator with twin DNA binding domains that recognize a triplet of GT-boxes in a complex motif designated GTE. To further define the DNA-binding activity of PF1 and to explore potential inter-relationships between the two factors, we have performed a series of in vitro DNA-binding experiments with both PE1 and GTE target sites. The data show that, consistent with its membership of the HMG-I/Y protein family, PF1 can bend DNA when bound to PE1. In addition, PF1 can bind promiscuously, with varying affinity, to other AT-containing motifs, including GTE. When co-incubated with GT-2, PF1 enhances the specific DNA-binding activity of GT-2 toward GTE, the first report of such activity for a plant HMG-I/Y protein. This enhancement takes place without demonstrable physical contact between the two proteins, suggesting the possibility of a novel, indirect mechanism of recruitment involving DNA target-site pre-conditioning. The evidence indicates therefore that PF1 and GT-2 do not perform functionally equivalent roles in positively regulating oat and rice PHYA gene expression. However, the data suggest the possibility that PF1 may act as an architectural factor, promiscuously recognizing a spectrum of AT-containing elements in plant promoters, with the general function of catalyzing enhanced binding of conventional cognate transcriptional regulators to these elements via DNA bending. GT-2,PF1,PHYA Transcript profiling of crown rootless1 mutant stem base reveals new elements associated with crown root development in rice 2011 BMC Genomics Universite Montpellier 2, UMR DAP, Place Eugene Bataillon, 34095 Montpellier Cedex 5, France. BACKGROUND: In rice, the major part of the post-embryonic root system is made of stem-derived roots named crown roots (CR). Among the few characterized rice mutants affected in root development, crown rootless1 mutant is unable to initiate crown root primordia. CROWN ROOTLESS1 (CRL1) is induced by auxin and encodes an AS2/LOB-domain transcription factor that acts upstream of the gene regulatory network controlling CR development. RESULTS: To identify genes involved in CR development, we compared global gene expression profile in stem bases of crl1 mutant and wild-type (WT) plants. Our analysis revealed that 250 and 236 genes are down- and up-regulated respectively in the crl1 mutant. Auxin induces CRL1 expression and consequently it is expected that auxin also alters the expression of genes that are early regulated by CRL1. To identify genes under the early control of CRL1, we monitored the expression kinetics of a selected subset of genes, mainly chosen among those exhibiting differential expression, in crl1 and WT following exogenous auxin treatment. This analysis revealed that most of these genes, mainly related to hormone, water and nutrient, development and homeostasis, were likely not regulated directly by CRL1. We hypothesized that the differential expression for these genes observed in the crl1 mutant is likely a consequence of the absence of CR formation. Otherwise, three CRL1-dependent auxin-responsive genes: FSM (FLATENNED SHOOT MERISTEM)/FAS1 (FASCIATA1), GTE4 (GENERAL TRANSCRIPTION FACTOR GROUP E4) and MAP (MICROTUBULE-ASSOCIATED PROTEIN) were identified. FSM/FAS1 and GTE4 are known in rice and Arabidopsis to be involved in the maintenance of root meristem through chromatin remodelling and cell cycle regulation respectively. CONCLUSION: Our data showed that the differential regulation of most genes in crl1 versus WT may be an indirect consequence of CRL1 inactivation resulting from the absence of CR in the crl1 mutant. Nevertheless some genes, FAS1/FSM, GTE4 and MAP, require CRL1 to be induced by auxin suggesting that they are likely directly regulated by CRL1. These genes have a function related to polarized cell growth, cell cycle regulation or chromatin remodelling. This suggests that these genes are controlled by CRL1 and involved in CR initiation in rice. GTE4|OsGTE4,MAP,BTBN18,GIL1,ZOS8-11 A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase 2007 Nat Genet National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Sciences, The Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China. Grain weight is one of the most important components of grain yield and is controlled by quantitative trait loci (QTLs) derived from natural variations in crops. However, the molecular roles of QTLs in the regulation of grain weight have not been fully elucidated. Here, we report the cloning and characterization of GW2, a new QTL that controls rice grain width and weight. Our data show that GW2 encodes a previously unknown RING-type protein with E3 ubiquitin ligase activity, which is known to function in the degradation by the ubiquitin-proteasome pathway. Loss of GW2 function increased cell numbers, resulting in a larger (wider) spikelet hull, and it accelerated the grain milk filling rate, resulting in enhanced grain width, weight and yield. Our results suggest that GW2 negatively regulates cell division by targeting its substrate(s) to proteasomes for regulated proteolysis. The functional characterization of GW2 provides insight into the mechanism of seed development and is a potential tool for improving grain yield in crops. GW2 Isolation and initial characterization of GW5, a major QTL associated with rice grain width and weight 2008 Cell Res National Key Laboratory for Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China. Grain weight is a major determinant of crop grain yield and is controlled by naturally occurring quantitative trait loci (QTLs). We earlier identified a major QTL that controls rice grain width and weight, GW5, which was mapped to a recombination hotspot on rice chromosome 5. To gain a better understanding of how GW5 controls rice grain width, we conducted fine mapping of this locus and uncovered a 1 212-bp deletion associated with the increased grain width in the rice cultivar Asominori, in comparison with the slender grain rice IR24. In addition, genotyping analyses of 46 rice cultivars revealed that this deletion is highly correlated with the grain-width phenotype, suggesting that the GW5 deletion might have been selected during rice domestication. GW5 encodes a novel nuclear protein of 144 amino acids that is localized to the nucleus. Furthermore, we show that GW5 physically interacts with polyubiquitin in a yeast two-hybrid assay. Together, our results suggest that GW5 represents a major QTL underlying rice width and weight, and that it likely acts in the ubiquitin-proteasome pathway to regulate cell division during seed development. This study provides novel insights into the molecular mechanisms controlling rice grain development and suggests that GW5 could serve as a potential tool for high-yield breeding of crops.Cell Research (2008) 18:1199-1209. doi: 10.1038/cr.2008.307; published online 18 November 2008. GW5|qSW5 Quantitative trait loci (QTL) analysis for rice grain width and fine mapping of an identified QTL allele gw-5 in a recombination hotspot region on chromosome 5 2008 Genetics National Key Laboratory for Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China. Rice grain width and shape play a crucial role in determining grain quality and yield. The genetic basis of rice grain width was dissected into six additive quantitative trait loci (QTL) and 11 pairs of epistatic QTL using an F(7) recombinant inbred line (RIL) population derived from a single cross between Asominori (japonica) and IR24 (indica). QTL by environment interactions were evaluated in four environments. Chromosome segment substitution lines (CSSLs) harboring the six additive effect QTL were used to evaluate gene action across eight environments. A major, stable QTL, qGW-5, consistently decreased rice grain width in both the Asominori/IR24 RIL and CSSL populations with the genetic background Asominori. By investigating the distorted segregation of phenotypic values of rice grain width and genotypes of molecular markers in BC(4)F(2) and BC(4)F(3) populations, qGW-5 was dissected into a single recessive gene, gw-5, which controlled both grain width and length-width ratio. gw-5 was narrowed down to a 49.7-kb genomic region with high recombination frequencies on chromosome 5 using 6781 BC(4)F(2) individuals and 10 newly developed simple sequence repeat markers. Our results provide a basis for map-based cloning of the gw-5 gene and for marker-aided gene/QTL pyramiding in rice quality breeding. GW5|qSW5 Deletion in a gene associated with grain size increased yields during rice domestication 2008 Nat Genet Institute of the Society for Techno-Innovation of Agriculture, Forestry, and Fisheries, 446-1 Ippaizuka, Kamiyokoba, Tsukuba, Ibaraki 305-0854, Japan. The domestication of crops involves a complex process of selection in plant evolution and is associated with changes in the DNA regulating agronomically important traits. Here we report the cloning of a newly identified QTL, qSW5 (QTL for seed width on chromosome 5), involved in the determination of grain width in rice. Through fine mapping, complementation testing and association analysis, we found that a deletion in qSW5 resulted in a significant increase in sink size owing to an increase in cell number in the outer glume of the rice flower; this trait might have been selected by ancient humans to increase yield of rice grains. In addition, we mapped two other defective functional nucleotide polymorphisms of rice domestication-related genes with genome-wide RFLP polymorphisms of various rice landraces. These analyses show that the qSW5 deletion had an important historical role in artificial selection, propagation of cultivation and natural crossings in rice domestication, and shed light on how the rice genome was domesticated. GW5|qSW5 Histone acetyltransferases in rice (Oryza sativa L.): phylogenetic analysis, subcellular localization and expression 2012 BMC Plant Biol South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China. BACKGROUND: Histone acetyltransferases (HATs) play an important role in eukaryotic transcription. Eight HATs identified in rice (OsHATs) can be organized into four families, namely the CBP (OsHAC701, OsHAC703, and OsHAC704), TAFII250 (OsHAF701), GNAT (OsHAG702, OsHAG703, and OsHAG704), and MYST (OsHAM701) families. The biological functions of HATs in rice remain unknown, so a comprehensive protein sequence analysis of the HAT families was conducted to investigate their potential functions. In addition, the subcellular localization and expression patterns of the eight OsHATs were analyzed. RESULTS: On the basis of a phylogenetic and domain analysis, monocotyledonous CBP family proteins can be subdivided into two groups, namely Group I and Group II. Similarly, dicotyledonous CBP family proteins can be divided into two groups, namely Group A and Group B. High similarities of protein sequences, conserved domains and three-dimensional models were identified among OsHATs and their homologs in Arabidopsis thaliana and maize. Subcellular localization predictions indicated that all OsHATs might localize in both the nucleus and cytosol. Transient expression in Arabidopsis protoplasts confirmed the nuclear and cytosolic localization of OsHAC701, OsHAG702, and OsHAG704. Real-time quantitative polymerase chain reaction analysis demonstrated that the eight OsHATs were expressed in all tissues examined with significant differences in transcript abundance, and their expression was modulated by abscisic acid and salicylic acid as well as abiotic factors such as salt, cold, and heat stresses. CONCLUSIONS: Both monocotyledonous and dicotyledonous CBP family proteins can be divided into two distinct groups, which suggest the possibility of functional diversification. The high similarities of protein sequences, conserved domains and three-dimensional models among OsHATs and their homologs in Arabidopsis and maize suggested that OsHATs have multiple functions. OsHAC701, OsHAG702, and OsHAG704 were localized in both the nucleus and cytosol in transient expression analyses with Arabidopsis protoplasts. OsHATs were expressed constitutively in rice, and their expression was regulated by exogenous hormones and abiotic stresses, which suggested that OsHATs may play important roles in plant defense responses. OsHAF701,OsHAM701 Expression and in silico structural analysis of a rice (Oryza sativa) hemoglobin 5 2008 Plant Physiol Biochem Laboratorio de Biofisica y Biologia Molecular, Facultad de Ciencias, Universidad Autonoma del Estado de Morelos, Avenida Universidad 1001, Colonia Chamilpa, 62210 Cuernavaca, Morelos, Mexico. This work reports the analysis of an additional hemoglobin (hb) gene copy, hb5, in the genome of rice. The amino acid sequence of Hb5 differs from the previously determined rice Hbs 1-4 in missing 11 residues in helix E. Transcripts of hb5 were found to be ubiquitous in rice organs, and hormone- and stress-response promoters exist upstream of the rice hb5 gene. Furthermore, the modeled structure of Hb5 based on the known crystal structure of rice Hb1 is unusual in that the putative distal His is distant from the heme Fe. This observation suggests that Hb5 binds and releases O(2) easily and thus that it functions as an O(2)-carrier or in some aspects of the O(2) metabolism. Hb5 Identification and linkage mapping of complementary recessive genes causing hybrid breakdown in an intraspecific rice cross 2007 Theor Appl Genet National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. One outcome of hybrid breakdown is poor growth, which we observed as a reduction in the number of panicles per plant and in culm length in an F(2) population derived from a cross between the genetically divergent rice (Oryza sativa L.) cultivars 'Sasanishiki' (japonica) and 'Habataki' (indica). Quantitative trait locus (QTL) analysis of the two traits and two-way ANOVA of the detected QTLs suggested that the poor growth was due mainly to an epistatic interaction between genes at QTLs located on chromosomes 2 and 11. The poor growth was likely to result when a plant was homozygous for the 'Habataki' allele at the QTL on chromosome 2 and homozygous for the 'Sasanishiki' allele at the QTL on chromosome 11. The results suggest that the poor growth found in the F(2) population was due to hybrid breakdown of a set of complementary genes. To test this hypothesis and determine the precise chromosomal location of the genes causing the hybrid breakdown, we performed genetic analyses using a chromosome segment substitution line, in which a part of chromosome 2 from 'Habataki' was substituted into the genetic background of 'Sasanishiki'. The segregation patterns of poor growth in plants suggested that both of the genes underlying the hybrid breakdown were recessive. The gene on chromosome 2, designated hybrid breakdown 2 (hbd2), was mapped between simple sequence repeat markers RM3515 and RM3730. The gene on chromosome 11, hbd3, was mapped between RM5824 and RM1341. hbd2|OsCKI1 Interaction of two recessive genes, hbd2 and hbd3, induces hybrid breakdown in rice 2007 Theor Appl Genet Bioscience and Biotechnology Center, Nagoya University, Nagoya, 464-8601, Japan. Reproductive barriers are important for the maintenance of species identity. We discovered a reproductive barrier via hybrid breakdown among the progeny of a cross between the japonica rice cultivar Koshihikari and the indica rice cultivar Habataki. Genetic analysis indicated that the hybrid breakdown is regulated by the interaction of two recessive genes: hbd2 in Habataki and hbd3 in Koshihikari. Linkage mapping showed that hbd2 is located near the 100 cM region of chromosome 2 in Habataki, whereas hbd3 is located near the 60 cM region of chromosome 11 in Koshihikari. Construction of nearly isogenic lines for hbd2 and Hbd3 (NIL-hbd2 and NIL-Hbd3), as well as a pyramiding line (NIL-hbd2 + Hbd3), confirmed that the hybrid breakdown is induced by the interaction of these two recessive genes. Our results indicate that these genes are novel for the induction of hybrid breakdown in rice. hbd2|OsCKI1 Gain of deleterious function causes an autoimmune response and Bateson-Dobzhansky-Muller incompatibility in rice 2010 Mol Genet Genomics Bioscience and Biotechnology Center, Nagoya University, Nagoya, 464-8601, Japan. Reproductive isolation plays an important role in speciation as it restricts gene flow and accelerates genetic divergence between formerly interbreeding population. In rice, hybrid breakdown is a common reproductive isolation observed in both intra and inter-specific crosses. It is a type of post-zygotic reproductive isolation in which sterility and weakness are manifested in the F(2) and later generations. In this study, the physiological and molecular basis of hybrid breakdown caused by two recessive genes, hbd2 and hbd3, in a cross between japonica variety, Koshihikari, and indica variety, Habataki, were investigated. Fine mapping of hbd2 resulted in the identification of the causal gene as casein kinase I (CKI1). Further analysis revealed that hbd2-CKI1 allele gains its deleterious function that causes the weakness phenotype by a change of one amino acid. As for the other gene, hbd3 was mapped to the NBS-LRR gene cluster region. It is the most common class of R-gene that triggers the immune signal in response to pathogen attack. Expression analysis of pathogen response marker genes suggested that weakness phenotype in this hybrid breakdown can be attributed to an autoimmune response. So far, this is the first evidence linking autoimmune response to post-zygotic isolation in rice. This finding provides a new insight in understanding the molecular and evolutionary mechanisms establishing post-zygotic isolation in plants. hbd2|OsCKI1 Roles of OsCKI1, a rice casein kinase I, in root development and plant hormone sensitivity 2003 The Plant Journal National Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China. Casein kinases are critical in cell division and differentiation across species. A rice cDNA fragment encoding a putative casein kinase I (CKI) was identified via cDNA macroarray under brassinosteroid (BR) treatment, and a 1939-bp full-length cDNA, OsCKI1, was isolated and found to encode a putative 463-aa protein. RT-PCR and Northern blot analysis indicated that OsCKI1 was constitutively expressed in various rice tissues and upregulated by treatments with BR and abscisic acid (ABA). Enzymatic assay of recombinant OsCKI1 proteins expressed in Escherichia coli showed that the protein was capable of phosphorylating casein. The physiological roles of OsCKI1 were studied through antisense transgenic approaches, and homozygous transgenic plants showed abnormal root development, including fewer lateral and adventitious roots, and shortened primary roots as a result of reduced cell elongation. Treatment of wild-type plants with CKI-7, a specific inhibitor of CKI, also confirmed these functions of OsCKI1. Interestingly, in transgenic and CKI-7-treated plants, exogenously supplied IAA could restore normal root development, and measurement of free IAA content in CKI-deficient primary and adventitious roots revealed altered auxin content, indicating that OsCKI1 is involved in auxin metabolism or that it may affect auxin levels. Transgenic plants were less sensitive than control plants to ABA or BR treatment during germination, suggesting that OsCKI1 may be involved in various hormone-signaling pathways. OsCKI1-GFP fusion studies revealed the localization of OsCKI1 to the nucleus, suggesting a possible involvement in regulation of gene expression. In OsCKI1-deficient plants, differential gene expression was investigated using cDNA chip technology, and results indicated that genes related to signal transduction and hormone metabolism were indeed with altered expression. hbd2|OsCKI1 Systematic identification of novel protein domain families associated with nuclear functions 2002 Genome Res European Molecular Biology Laboratory, 69114 Heidelberg, Germany. doerks@embl-heidelberg.de A systematic computational analysis of protein sequences containing known nuclear domains led to the identification of 28 novel domain families. This represents a 26% increase in the starting set of 107 known nuclear domain families used for the analysis. Most of the novel domains are present in all major eukaryotic lineages, but 3 are species specific. For about 500 of the 1200 proteins that contain these new domains, nuclear localization could be inferred, and for 700, additional features could be predicted. For example, we identified a new domain, likely to have a role downstream of the unfolded protein response; a nematode-specific signalling domain; and a widespread domain, likely to be a noncatalytic homolog of ubiquitin-conjugating enzymes. Hd1 An atypical HLH protein OsLF in rice regulates flowering time and interacts with OsPIL13 and OsPIL15 2011 N Biotechnol National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China. In plants, flowering as a crucial developmental event is highly regulated by both genetic programs and environmental signals. Genetic analysis of flowering time mutants is instrumental in dissecting the regulatory pathways of flowering induction. In this study, we isolated the OsLF gene by its association with the T-DNA insertion in the rice late flowering mutant named A654. The OsLF gene encodes an atypical HLH protein composed of 419 amino acids (aa). Overexpression of the OsLF gene in wild type rice recapitulated the late flowering phenotype of A654, indicating that the OsLF gene negatively regulates flowering. Flowering genes downstream of OsPRR1 such as OsGI and Hd1 were down regulated in the A654 mutant. Yeast two hybrid and colocalization assays revealed that OsLF interacts strongly with OsPIL13 and OsPIL15 that are potentially involved in light signaling. In addition, OsPIL13 and OsPIL15 colocalize with OsPRR1, an ortholog of the Arabidopsis APRR1 gene that controls photoperiodic flowering response through clock function. Together, these results suggest that overexpression of OsLF might repress expression of OsGI and Hd1 by competing with OsPRR1 in interacting with OsPIL13 and OsPIL15 and thus induce late flowering. Hd1,OsGI,OsLF,OsPIL13|OsPIL1,OsPIL15,OsPRR1 Identification of quantitative trait loci controlling heading date in rice using a high-density linkage map 1997 TAG Theoretical and Applied Genetics Rice Genome Research Program (RGP), National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305, Japan Quantitative trait locus (QTL) analysis has been carried out to identify genes conferring heading date in rice. One hundred and eighty six F2 plants derived from a cross between a japonica variety, Nipponbare, and an indica variety, Kasalath, were used as a segregating population for QTL mapping and more than 850 markers were employed to identify QTLs. Scan-analysis revealed the existence of two QTLs with large effects, Hd-1 and Hd-2, one in the middle of chromosome 6 and one at the end of chromosome 7, respectively. For both loci, the Kasalath alleles reduced days-to-heading. In addition, three QTLs with minor effects, Hd-3, Hd-4 and Hd-5, were found to be located on chromosomes 6, 7 and 8 based on a secondary scan analysis which was carried out by removing the phenotypic effects of Hd-1 and Hd-2. For the three secondary loci, the Nipponbare alleles reduced days-to-heading. The five QTLs explained 84% of the total phenotypic variation in the F2 population based on a multiple-QTL model. The presence of a digenic interaction between Hd-1 and Hd-2 was clearly suggested. Hd1 Fine mapping of quantitative trait loci Hd-1 , Hd-2 and Hd-3 , controlling heading date of rice, as single Mendelian factors 1998 TAG Theoretical and Applied Genetics Rice Genome Research Program, Institute of Society for Techno-innovation of Agriculture, Forestry and Fisheries (STAFF), Tsukuba, Ibaraki 305, Japan, JP Fine mapping was carried out on three putative QTLs (tentatively designated as Hd-1 to Hd-3) of five such QTLs controlling heading date in rice that had been earlier identified using an F2 population derived from a cross between a japonica variety, ‘Nipponbare’, and an indica variety, ‘Kasalath’, using progeny backcrossed with ‘Nipponbare’ as the recurrent parent. One BC3F2 and two BC3F1 plants, in which the target QTL regions were heterozygous and most other chromosomal regions were homozygous for the ‘Nipponbare’ allele, were selected as the experimental material. Self-pollinated progeny (BC3F2 and BC3F3) of the BC3F1 or BC3F2 showed continuous variation in days to heading. By means of progeny testing based on BC3F3 or BC3F4 lines, we determined the genotypes of each BC3F2 or BC3F3 individual at target QTLs. Their segregation patterns fitted Mendelian inheritance ratios. When the results obtained by RFLP analysis and progeny tests were combined, Hd-1, Hd-2 and Hd-3 were mapped precisely on chromosomes 6, 7 and 6, respectively, of a rice RFLP linkage map. The results demonstrated that QTLs can be treated as Mendelian factors. Moreover, these precise locations were in good agreement with the regions estimated by QTL analysis of the initial F2 population, demonstrating the high reliability of QTL mapping using a high-density linkage map. Hd1,Hd3a Characterization and detection of epistatic interactions of 3 QTLs, Hd1 , Hd2 , and Hd3, controlling heading date in rice using nearly isogenic lines 2000 TAG Theoretical and Applied Genetics Bio-oriented Technology Research Advancement Institution, Omiya, Saitama 331-8537, Japan, JP To characterize quantitative trait loci (QTLs), we used marker-assisted selection (MAS) to develop three nearly isogenic lines (NILs) differing only for the presence of a single, specific QTL (QTL-NILs) –Hd1, Hd2, and Hd3 – for heading date in rice. The three lines contained the chromosomal region of the target QTL from donor variety Kasalath(indica) in the genetic background of var. Nipponbare (japonica). To analyze epistatic interactions in pairs of these QTLs, we also used MAS to develop four combined QTL-NILs with 2 of the 3 QTLs or with all 3. Different daylength treatment testing of the QTL-NILs revealed that the three QTLs control photoperiod sensitivity. Genetic analysis of F2 populations derived from crosses between the three QTL-NILs with a single QTL using molecular markers revealed the existence of epistatic interactions between Hd1 and Hd2, and Hd2 and Hd3. These interactions were also confirmed by the analysis of combined QTL-NILs under different daylength conditions. The existence of an epistatic interaction between Hd1 and Hd3 was also clarified. Based on these results, we suggest that the Kasalath allele of Hd3 does not affect photoperiod sensitivity by itself but that it is involved in enhancement of the expression of the Nipponbare alleles of Hd1 and Hd2. Hd1 Phytochrome B regulates Heading date 1 (Hd1)-mediated expression of rice florigen Hd3a and critical day length in rice 2011 Mol Genet Genomics Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, 630-0192, Japan. Many plants require circadian clock and light information for the photoperiodic control of flowering. In Arabidopsis, a long-day plant (LDP), flowering is triggered by the circadian clock-controlled expression of CONSTANS (CO) and light stabilization of the CO protein to induce FT (FLOWERING LOCUS T). In rice, a short-day plant (SDP), the CO ortholog Heading date 1 (Hd1) regulates FT ortholog Hd3a, but regulation of Hd3a by Hd1 differs from that in Arabidopsis. Here, we report that phytochrome B (phyB)-mediated suppression of Hd3a is a primary cause of long-day suppression of flowering in rice, based on the three complementary discoveries. First, overexpression of Hd1 causes a delay in flowering under SD conditions and this effect requires phyB, suggesting that light modulates Hd1 control of Hd3a transcription. Second, a single extension of day length decreases Hd3a expression proportionately with the length of daylight. Third, Hd1 protein levels in Hd1-overexpressing plants are not altered in the presence of light. These results also suggest that phyB-mediated suppression of Hd3a expression is a component of the molecular mechanism for critical day length in rice. Hd1,Hd3a,PHYB|OsphyB Interactive effects of two heading-time loci, Se1 and Ef1, on pre-flowering developmental phases in rice (Oryza sativa L.) 2002 Euphytica Faculty of Agriculture, Kagoshima University, Kagoshima, 890-0065, Japan The interaction between the Se1 and the Ef1 loci, which chiefly control the photoperiod sensitivity (PS) and the basic vegetative growth (BVG) period of rice (Oryza sativa L.) respectively, was investigated using four tester lines different in genotype for the two heading time loci from each other. The four tester lines were grown under 10, 13, 14, 15, and 16h daylengths to estimate their BVG period and PS. The Taiwanese cultivar Taichung 65 (T65), one of the tester lines, has an extremely long BVG period that has been considered to be conferred by a late heading-time allele ef1 at the Ef1 locus. Experimental results, however, showed that the extremely long BVG of T65 was conferred not by a single effect of ef1 but by a complementary effect of ef1 and Se1-e, a photoperiod insensitivity allele, at the Se1 locus. It was also found that a complementary effect of a PS allele Se1-n at the Se1 locus and ef1 stimulates the PS of rice. Gene analysis for heading time under an optimum daylength (10 h) as well as under natural daylength confirmed the presence of the complementary effect of the two nonallelic genes on BVG, which was found only with homozygosity of both the genes. Based on these results and earlier reports on the Se1 locus, the roles of the Se1 and Ef1 loci on the durations of pre-flowering developmental phases in rice were discussed. Hd1 Heading date 1 (Hd1), an ortholog of Arabidopsis CONSTANS, is a possible target of human selection during domestication to diversify flowering times of cultivated rice 2011 Genes Genet Syst Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology During the domestication of rice (Oryza sativa L.), diversification of flowering time was important in expanding the areas of cultivation. Rice is a facultative short day (SD) plant and requires certain periods of dark to induce flowering. Heading date 1 (Hd1), a regulator of the florigen gene Hd3a, is one of the main factors used to generate diversity in flowering. Loss-of-function alleles of Hd1 are common in cultivated rice and cause the diversity of flowering time. However, it is unclear how these functional nucleotide polymorphisms of Hd1 accumulated in the course of evolution. Nucleotide polymorphisms within Hd1 and Hd3a were analyzed in 38 accessions of ancestral wild rice Oryza rufipogon and compared with those of cultivated rice. In contrast to cultivated rice, no nucleotide changes affecting Hd1 function were found in 38 accessions of wild rice ancestors. No functional changes were found in Hd3a in either cultivated or ancestral rice. A phylogenetic analysis indicated that evolution of the Hd1 alleles may have occurred independently in cultivars descended from various accessions of ancestral rice. The non-functional Hd1 alleles found in cultivated rice may be selected during domestication, because they were not found or very rare in wild ancestral rice. In contrast with Hd3a, which has been highly conserved, Hd1 may have undergone human selection to diversify the flowering times of rice during domestication or the early stage of the cultivation period. Hd1 Functional analyses of the flowering time gene OsMADS50, the putative SUPPRESSOR OF OVEREXPRESSION OF CO 1/AGAMOUS-LIKE 20 (SOC1/AGL20) ortholog in rice 2004 Plant J National Research Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Korea. A late-flowering mutant was isolated from rice T-DNA-tagging lines. T-DNA had been integrated into the K-box region of Oryza sativa MADS50 (OsMADS50), which shares 50.6% amino acid identity with the Arabidopsis MADS-box gene SUPPRESSOR OF OVEREXPRESSION OF CO 1/AGAMOUS-LIKE 20 (SOC1/AGL20). While overexpression of OsMADS50 caused extremely early flowering at the callus stage, OsMADS50 RNAi plants exhibited phenotypes of late flowering and an increase in the number of elongated internodes. This confirmed that the phenotypes observed in the knockout (KO) plants are because of the mutation in OsMADS50. RT-PCR analyses of the OsMADS50 KO and ubiquitin (ubi):OsMADS50 plants showed that OsMADS50 is an upstream regulator of OsMADS1, OsMADS14, OsMADS15, OsMADS18, and Hd (Heading date)3a, but works either parallel with or downstream of Hd1 and O. sativa GIGANTEA (OsGI). These results suggest that OsMADS50 is an important flowering activator that controls various floral regulators in rice. Hd1,Hd3a,OsGI,OsMADS1|LHS1|AFO,OsMADS14,OsMADS15|DEP,OsMADS18,OsMADS50|OsSOC1|DTH3 The effect of the crosstalk between photoperiod and temperature on the heading-date in rice 2009 PLoS One Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, People's Republic of China. Photoperiod and temperature are two important environmental factors that influence the heading-date of rice. Although the influence of the photoperiod on heading has been extensively reported in rice, the molecular mechanism for the temperature control of heading remains unknown. This study reports an early heading mutant derived from tissue culture lines of rice and investigates the heading-date of wild type and mutant in different photoperiod and temperature treatments. The linkage analysis showed that the mutant phenotype cosegregated with the Hd1 locus. Sequencing analysis found that the mutant contained two insertions and several single-base substitutions that caused a dramatic reduction in Hd1mRNA levels compared with wild type. The expression patterns of Hd1 and Hd3a were also analyzed in different photoperiod and temperature conditions, revealing that Hd1 mRNA levels displayed similar expression patterns for different photoperiod and temperature treatments, with high expression levels at night and reduced levels in the daytime. In addition, Hd1 displayed a slightly higher expression level under long-day and low temperature conditions. Hd3a mRNA was present at a very low level under low temperature conditions regardless of the day-length. This result suggests that suppression of Hd3a expression is a principle cause of late heading under low temperature and long-day conditions. Hd1,Hd3a Pleiotropism of the photoperiod-insensitive allele of Hd1 on heading date, plant height and yield traits in rice 2012 PLoS One State Key Laboratory of Rice Biology and Chinese National Center for Rice Improvement, China National Rice Research Institute, Hangzhou, China. Five populations segregated in isogenic backgrounds and three sets of near isogenic lines (NILs) overlapping in a 362.3-kb region covering heading date gene Hd1 were developed from the indica rice cross Zhenshan97 (ZS97)/Milyang 46 (MY46). They were used to analyze the effects of Hd1 on heading date, plant height and yield traits. In a background of the parental mixtures, the photoperiod-sensitive allele derived from ZS97 functioned in promoting and delaying flowering in the natural short-day and long-day conditions, respectively. In the background of ZS97, no response to the photoperiod was observed, whereas the photoperiod-insensitive allele derived from MY46 functioned in delaying flowering, increasing plant height, and enhancing grain productivity. The additive effects estimated in two NIL sets were 6.14 and 6.14 d for heading date, 4.46 and 5.55 cm for plant height, 10.82 and 11.54 for the number of spikelets per panicle, 6.82 and 8.00 for the number of grains per panicle, and 2.16 and 2.23 g for grain yield per plant, which explained 94.1% and 96.3%, 70.5% and 84.8%, 52.4% and 55.2%, 28.9% and 39.2%, and 36.5% and 26.9% of the phenotypic variances, respectively. Since the photoperiod-insensitive allele of Hd1 confers a long vegetative phase, it is a good candidate for breeding rice varieties with high yielding potential for low latitudes. Hd1 Hd1, a Major Photoperiod Sensitivity Quantitative Trait Locus in Rice, Is Closely Related to the Arabidopsis Flowering Time Gene CONSTANS 2000 The Plant Cell Online National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan. A major quantitative trait locus (QTL) controlling response to photoperiod, Hd1, was identified by means of a map-based cloning strategy. High-resolution mapping using 1505 segregants enabled us to define a genomic region of approximately 12 kb as a candidate for Hd1. Further analysis revealed that the Hd1 QTL corresponds to a gene that is a homolog of CONSTANS in Arabidopsis. Sequencing analysis revealed a 43-bp deletion in the first exon of the photoperiod sensitivity 1 (se1) mutant HS66 and a 433-bp insertion in the intron in mutant HS110. Se1 is allelic to the Hd1 QTL, as determined by analysis of two se1 mutants, HS66 and HS110. Genetic complementation analysis proved the function of the candidate gene. The amount of Hd1 mRNA was not greatly affected by a change in length of the photoperiod. We suggest that Hd1 functions in the promotion of heading under short-day conditions and in inhibition under long-day conditions. Hd1 Suppression of the floral activator Hd3a is the principal cause of the night break effect in rice 2005 Plant Cell Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Takayama, Ikoma, Japan. A short exposure to light in the middle of the night causes inhibition of flowering in short-day plants. This phenomenon is called night break (NB) and has been used extensively as a tool to study the photoperiodic control of flowering for many years. However, at the molecular level, very little is known about this phenomenon. In rice (Oryza sativa), 10 min of light exposure in the middle of a 14-h night caused a clear delay in flowering. A single NB strongly suppressed the mRNA of Hd3a, a homolog of Arabidopsis thaliana FLOWERING LOCUS T (FT), whereas the mRNAs of OsGI and Hd1 were not affected. The NB effect on Hd3a mRNA was maximal in the middle of the 14-h night. The phyB mutation abolished the NB effect on flowering and Hd3a mRNA, indicating that the NB effect was mediated by phytochrome B. Because expression of the other FT-like genes was very low and not appreciably affected by NB, our results strongly suggest that the suppression of Hd3a mRNA is the principal cause of the NB effect on flowering in rice. Hd1,Hd3a Identification of dynamin as an interactor of rice GIGANTEA by tandem affinity purification (TAP) 2008 Plant Cell Physiol Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan. GIGANTEA (GI), CONSTANS (CO) and FLOWERING LOCUS T (FT) regulate photoperiodic flowering in Arabidopsis. In rice, OsGI, Hd1 and Hd3a were identified as orthologs of GI, CO and FT, respectively, and are also important regulators of flowering. Although GI has roles in both flowering and the circadian clock, our understanding of its biochemical functions is still limited. In this study, we purified novel OsGI-interacting proteins by using the tandem affinity purification (TAP) method. The TAP method has been used effectively in a number of model species to isolate proteins that interact with proteins of interest. However, in plants, the TAP method has been used in only a few studies, and no novel proteins have previously been isolated by this method. We generated transgenic rice plants and cell cultures expressing a TAP-tagged version of OsGI. After a two-step purification procedure, the interacting proteins were analyzed by mass spectrometry. Seven proteins, including dynamin, were identified as OsGI-interacting proteins. The interaction of OsGI with dynamin was verified by co-immunoprecipitation using a myc-tagged version of OsGI. Moreover, an analysis of Arabidopsis dynamin mutants indicated that although the flowering times of the mutants were not different from those of wild-type plants, an aerial rosette phenotype was observed in the mutants. We also found that OsGI is present in both the nucleus and the cytosol by Western blot analysis and by transient assays. These results indicate that the TAP method is effective for the isolation of novel proteins that interact with target proteins in plants. Hd1,Hd3a,OsGI Breeding strategies for optimum heading date using genotypic information in rice 2009 Molecular Breeding State Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Provincial Center of Plant Gene Engineering, Nanjing Agricultural University, 210095, Nanjing, China Heading date (HD) is a key trait for the adaptation of rice cultivar to a specific growing region. Here, we report conventional and marker-assisted breeding strategies using genetic information related to the determination of HD, where the breeding objectives were to avoid the delayed heading common in indica × japonica hybrids, to increase the efficiency in selecting hybrid rice combinations having a suitable growth duration, and to develop cultivars with target growth duration by quantitative trait locus (QTL) pyramiding. The allelic constitution at the major HD loci was determined for a set of 109 leading Chinese rice cultivars by crossing them with HD tester lines. It was shown that the late heading in indica × japonica hybrids can be overcome by replacing the strong photoperiod-sensitivity allele Se-1 n with Se-1 e . A breeding strategy to enable the selection of hybrid combinations with suitable growth duration was proposed, based on HD genotypic information in rice. Meanwhile, a QTL analysis for HD was conducted over five years based on a recombinant inbred line population, derived from two parents Asominori (japonica) and IR24 (indica). Four QTLs, located on chromosomes 2, 3, 6, and 8, respectively, could be detected in all five years, indicating they were stably expressed QTL. According to this QTL information, and taking Asominori as an example, the HD genotypes for improving the growth duration were designed, and the best breeding selection schemes were determined by use of a genetic breeding simulation tool. Results obtained in this study demonstrate that genetic information related to HD can make a significant contribution to rice breeding. Hd1 SPIN1, a K homology domain protein negatively regulated and ubiquitinated by the E3 ubiquitin ligase SPL11, is involved in flowering time control in rice 2008 Plant Cell Department of Plant Pathology, Plant Molecular Biology and Biotechnology Program, Ohio State University, Columbus, Ohio 43210, USA. The rice (Oryza sativa) E3 ligase SPOTTED LEAF11 (SPL11) negatively regulates programmed cell death and disease resistance. We demonstrate here that SPL11 also regulates flowering via interaction with SPIN1 (for SPL11-interacting protein1), a Signal Transduction and Activation of RNA family member. SPIN1 binds RNA and DNA in vitro and interacts with SPL11 in the nucleus. Spl11 mutants have delayed flowering under long-day conditions. Spin1 overexpression causes late flowering independently of daylength; expression analyses of flowering marker genes in these lines suggested that SPIN1 represses flowering by downregulating the flowering promoter gene Heading date3a (Hd3a) via Hd1-dependent mechanisms in short days and by targeting Hd1-independent factors in long days. Both Spin1 and Spl11 are regulated diurnally in opposing phases. SPL11 negatively regulates Spin1 transcript levels, while SPIN1 also affects Spl11 expression. Moreover, we show that coincidence of high accumulation of Spin1 mRNA with the light in the morning and early evening is needed to repress flowering. SPIN1 is monoubiquitinated by SPL11, suggesting that it is not targeted for degradation. Our data are consistent with a model in which SPIN1 acts as a negative regulator of flowering that itself is negatively regulated by SPL11, possibly via ubiquitination. Hd1,Hd3a,SPIN1,SPL11 Rice early flowering1, a CKI, phosphorylates DELLA protein SLR1 to negatively regulate gibberellin signalling 2010 EMBO J National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, PR China. The plant hormone gibberellin (GA) is crucial for multiple aspects of plant growth and development. To study the relevant regulatory mechanisms, we isolated a rice mutant earlier flowering1, el1, which is deficient in a casein kinase I that has critical roles in both plants and animals. el1 had an enhanced GA response, consistent with the suppression of EL1 expression by exogenous GA(3). Biochemical characterization showed that EL1 specifically phosphorylates the rice DELLA protein SLR1, proving a direct evidence for SLR1 phosphorylation. Overexpression of SLR1 in wild-type plants caused a severe dwarf phenotype, which was significantly suppressed by EL1 deficiency, indicating the negative effect of SLR1 on GA signalling requires the EL1 function. Further studies showed that the phosphorylation of SLR1 is important for maintaining its activity and stability, and mutation of the candidate phosphorylation site of SLR1 results in the altered GA signalling. This study shows EL1 a novel and key regulator of the GA response and provided important clues on casein kinase I activities in GA signalling and plant development. CKI|EL1|Hd16,SLR1|OsGAI Rice homeobox transcription factor HOX1a positively regulates gibberellin responses by directly suppressing EL1 2011 J Integr Plant Biol National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200032, China. Homeobox transcription factors are involved in various aspects of plant development, including maintenance of the biosynthesis and signaling pathways of different hormones. However, few direct targets of homeobox proteins have been identified. We here show that overexpression of rice homeobox gene HOX1a resulted in enhanced gibberellin (GA) response, indicating a positive effect of HOX1a in GA signaling. HOX1a is induced by GA and encodes a homeobox transcription factor with transcription repression activity. In addition, HOX1a suppresses the transcription of early flowering1 (EL1), a negative regulator of GA signaling, and further electrophoretic mobility shift assay and chromatin immunoprecipitation analysis revealed that HOX1a directly bound to the promoter region of EL1 to suppress its expression and stimulate GA signaling. These results demonstrate that HOX1a functions as a positive regulator of GA signaling by suppressing EL1, providing informative hints on the study of GA signaling. CKI|EL1|Hd16,HAZ1|HOX1a Identification of a novel gene ef7 conferring an extremely long basic vegetative growth phase in rice 2009 Theor Appl Genet Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyou, Kyoto, 606-8502, Japan. A late heading-time mutant line, HS276, which was induced by gamma-irradiation of seeds of the japonica rice (Oryza sativa L.) variety Gimbozu, exhibits an extremely long basic vegetative growth phase (BVP). A genetic analysis using the F(2) population from the cross between HS276 and Gimbozu revealed that the late heading of HS276 is governed by a single recessive mutant gene. The subsequent analysis on heading responses of HS276 and Gimbozu to four photoperiods (12, 13, 14, and 15 h) and to the photoperiodic transfer treatment from a short photoperiod to a long photoperiod revealed that the mutant gene confers an extremely long BVP and increases photoperiod sensitivity under long photoperiod (14 and 15 h). The BVP durations of HS276 and Gimbozu were estimated at 30.1 and 16.0 days, respectively; the mutant gene, compared with its wild type allele, elongates the duration of BVP by 14 days. Linkage analysis showed that the mutant gene is located in the 129 kb region between the two INDEL markers, INDELAP0399_6 and INDELAP3487_2, on the distal part of the short arm of chromosome 6. None of the other BVP genes are located in this region; therefore, we declared this a newly detected mutant gene and designated it ef7. A recently established program to breed rice suitable for low latitudes, where short photoperiodic conditions continue throughout the year, aims to develop varieties with extremely long BVPs and weak photoperiod sensitivities; the mutant gene ef7, therefore, will be quite useful in these programs because it confers an extremely long BVP and little enhances photoperiod sensitivity under short photoperiod. Hd17|Ef7|OsELF3-1 OsELF3 is involved in circadian clock regulation for promoting flowering under long-day conditions in rice 2013 Mol Plant National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research-Wuhan, Huazhong Agricultural University, Wuhan 430070, China. Heading date is a critical trait that determines cropping seasons and regional adaptability in rice (Oryza sativa). Research efforts during the last decade have identified some important photoperiod pathway genes that are conserved between Arabidopsis and rice. In this study, we identified a novel gene, Oryza sativa ELF3 (OsELF3), which is a putative homolog of the ELF3 gene in Arabidopsis thaliana. OsELF3 was required for the control of heading date under long-day conditions. Its Tos17-tagging mutants exhibited a delayed heading date phenotype only under long-day, but not short-day, conditions. OsELF3 was highly expressed in leaf blades, and the OsELF3 protein was localized in the nucleolus. An obvious diurnal rhythm of OsELF3 transcript level was observed, with a trough in the early day and a peak in the late night in wild-type plants. However, this expression pattern was disrupted in oself3 mutants. Further investigations showed that the expression of OsGI and Ghd7 was up-regulated in the oself3 mutant, indicating that OsELF3 acts as a negative regulator upstream of OsGI and Ghd7 in the flowering-time control under long-day conditions. The rhythmic expression of circadian clock-related genes, including some OsPRR members, was obviously affected in oself3 mutants. Our results indicated that OsELF3 acts as a floral activator in the long-day photoperiodic pathway via its crosstalk with the circadian clock in rice. Hd17|Ef7|OsELF3-1,OsGI Ef7 encodes an ELF3-like protein and promotes rice flowering by negatively regulating the floral repressor gene Ghd7 under both short- and long-day conditions 2012 Plant Cell Physiol Graduate School of Agriculture, Kyoto University, Kyoto, Japan. Much progress has been made in our understanding of photoperiodic flowering of rice and the mechanisms underlying short-day (SD) promotion and long-day (LD) repression of floral induction. In this study, we identified and characterized the Ef7 gene, one of the rice orthologs of Arabidopsis EARLY FLOWERING 3 (ELF3). The ef7 mutant HS276, which was induced by gamma-irradiation of the japonica rice cultivar 'Gimbozu', flowers late under both SD and LD conditions. Expression analyses of flowering time-related genes demonstrated that Ef7 negatively regulates the expression of Ghd7, which is a repressor of the photoperiodic control of rice flowering, and consequently up-regulates the expression of the downstream Ehd1 and FT-like genes under both SD and LD conditions. Genetic analyses with a non-functional Ghd7 allele provided further evidence that the delayed flowering of ef7 is mediated through the Ghd7 pathway. The analysis of light-induced expression of Ghd7 revealed that the ef7 mutant was more sensitive to red light than the wild-type plant, but the gate of Ghd7 expression was unchanged. Thus, our results show that Ef7 functions as a floral promoter by repressing Ghd7 expression under both SD and LD conditions. Hd17|Ef7|OsELF3-1 Phytochrome dependent quantitative control of Hd3a transcription is the basis of the night break effect in rice flowering 2009 Genes Genet Syst Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology A short exposure to light during relative night (night break; NB) delays flowering in the short day plant rice. NB acts by downregulating Heading date 3a (Hd3a) expression. Because phytochrome B mutants do not respond to NB and their flowering time is not affected even under NB conditions, phyB is required for the suppression of Hd3a expression. The effect of NB is quantitatively controlled by light quality and by either light intensity or duration. However, the molecular mechanisms that regulate these interactions are poorly understood. Here, we examine the roles of phytochromes in the regulation of Hd3a transcription under NB conditions using monochromatic red, far-red and blue light. Red and blue light downregulated Hd3a expression, but far-red light NB did not. The effect of red light NB on Hd3a is dependent on photon fluence and is restored by subsequent far-red light irradiation. Our results suggest that quantitative effect of light on flowering in rice NB is mediated by the regulation of Hd3a transcription by phyB. Hd3a NECK LEAF 1, a GATA type transcription factor, modulates organogenesis by regulating the expression of multiple regulatory genes during reproductive development in rice 2009 Cell Res National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200032, China. In the monocot rice species Oryza sativa L., one of the most striking morphological processes during reproductive development is the concurrence of panicle development with the sequential elongation of upper internodes (UPIs). To elucidate the underlying molecular mechanisms, we cloned the rice gene NECK LEAF 1 (NL1), which when mutated results in delays in flowering time, smaller panicles with overgrown bracts and abnormal UPI elongation patterns. The NL1 gene encodes a GATA-type transcription factor with a single zinc finger domain, and its transcripts are detected predominantly in the bract primordia, which normally degenerate in the wild-type plants. Overexpression of NL1 in transgenic plants often gives rise to severe growth retardation, less vegetative phytomers and smaller leaves, suggesting that NL1 plays an important role in organ differentiation. A novel mutant allele of PLASTOCHRON1 (PLA1), a gene known to play a key role in regulating leaf initiation, was identified in this study. Genetic analysis demonstrated an interaction between nl1 and pla1, with NL1 acting upstream of PLA1. The expression level and spatial pattern of PLA1 were found to be altered in the nl1 mutant. Furthermore, the expression of two regulators of flowering, Hd3a and OsMADS1, was also affected in the nl1 mutant. On the basis of these findings, we propose that NL1 is an intrinsic factor that modulates and coordinates organogenesis through regulating the expression of PLA1 and other regulatory genes during reproductive development in rice. Hd3a,NL1,OsMADS1|LHS1|AFO,PLA1 Hd3a and RFT1 are essential for flowering in rice 2008 Development Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan. RICE FLOWERING LOCUS T 1 (RFT1/FT-L3) is the closest homologue of Heading date 3a (Hd3a), which is thought to encode a mobile flowering signal and promote floral transition under short-day (SD) conditions. RFT1 is located only 11.5 kb from Hd3a on chromosome 6. Although RFT1 RNAi plants flowered normally, double RFT1-Hd3a RNAi plants did not flower up to 300 days after sowing (DAS), indicating that Hd3a and RFT1 are essential for flowering in rice. RFT1 expression was very low in wild-type plants, but there was a marked increase in RFT1 expression by 70 DAS in Hd3a RNAi plants, which flowered 90 DAS. H3K9 acetylation around the transcription initiation site of the RFT1 locus had increased by 70 DAS but not at 35 DAS. In the absence of Hd3a and RFT1 expression, transcription of OsMADS14 and OsMADS15, two rice orthologues of Arabidopsis APETALA1, was strongly reduced, suggesting that they act downstream of Hd3a and RFT1. These results indicate that Hd3a and RFT1 act as floral activators under SD conditions, and that RFT1 expression is partly regulated by chromatin modification. Hd3a,OsMADS14,OsMADS15|DEP,RFT1 RBS1, an RNA Binding Protein, Interacts with SPIN1 and Is Involved in Flowering Time Control in Rice 2014 PLoS One State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China ; College of Agronomy and Biotechnology, China Agricultural University, Beijing, China. The rice U-box/ARM E3 ubiquitin ligase SPL11 negatively regulates programmed cell death (PCD) and disease resistance, and controls flowering time through interacting with the novel RNA/DNA binding KH domain protein SPIN1. Overexpression of Spin1 causes late flowering in transgenic rice under short-day (SD) and long-day (LD) conditions. In this study, we characterized the function of the RNA-binding and SPIN1-interacting 1 (RBS1) protein in flowering time regulation. Rbs1was identified in a yeast-two-hybrid screen using the full-length Spin1 cDNA as a bait and encodes an RNA binding protein with three RNA recognition motifs. The protein binds RNA in vitro and interacts with SPIN1 in the nucleus. Rbs1 overexpression causes delayed flowering under SD and LD conditions in rice. Expression analyses of flowering marker genes show that Rbs1 overexpression represses the expression of Hd3a under SD and LD conditions. Rbs1 is upregulated in both Spin1 overexpression plants and in the spl11 mutant. Interestingly, Spin1 expression is increased but Spl11 expression is repressed in the Rbs1 overexpression plants. Western blot analysis revealed that the SPIN1 protein level is increased in the Rbs1 overexpression plants and that the RBS1 protein level is also up-regulated in the Spin1 overexpression plants. These results suggest that RBS1 is a new negative regulator of flowering time that itself is positively regulated by SPIN1 but negatively regulated by SPL11 in rice. Hd3a,RBS1,SPIN1,SPL11 Inflorescence meristem identity in rice is specified by overlapping functions of three AP1/FUL-like MADS box genes and PAP2, a SEPALLATA MADS box gene 2012 Plant Cell Graduate School of Agriculture and Life Sciences, University of Tokyo, Tokyo, Japan. In plants, the transition to reproductive growth is of particular importance for successful seed production. Transformation of the shoot apical meristem (SAM) to the inflorescence meristem (IM) is the crucial first step in this transition. Using laser microdissection and microarrays, we found that expression of PANICLE PHYTOMER2 (PAP2) and three APETALA1 (AP1)/FRUITFULL (FUL)-like genes (MADS14, MADS15, and MADS18) is induced in the SAM during meristem phase transition in rice (Oryza sativa). PAP2 is a MADS box gene belonging to a grass-specific subclade of the SEPALLATA subfamily. Suppression of these three AP1/FUL-like genes by RNA interference caused a slight delay in reproductive transition. Further depletion of PAP2 function from these triple knockdown plants inhibited the transition of the meristem to the IM. In the quadruple knockdown lines, the meristem continued to generate leaves, rather than becoming an IM. Consequently, multiple shoots were formed instead of an inflorescence. PAP2 physically interacts with MAD14 and MADS15 in vivo. Furthermore, the precocious flowering phenotype caused by the overexpression of Hd3a, a rice florigen gene, was weakened in pap2-1 mutants. Based on these results, we propose that PAP2 and the three AP1/FUL-like genes coordinately act in the meristem to specify the identity of the IM downstream of the florigen signal. Hd3a,OsMADS14,OsMADS15|DEP,OsMADS18,OsMADS34|PAP2 The histone methyltransferase SDG724 mediates H3K36me2/3 deposition at MADS50 and RFT1 and promotes flowering in rice 2012 Plant Cell State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Chromatin modifications affect flowering time in the long-day plant Arabidopsis thaliana, but the role of histone methylation in flowering time regulation of rice (Oryza sativa), a short-day plant, remains to be elucidated. We identified a late-flowering long vegetative phase1 (lvp1) mutant in rice and used map-based cloning to reveal that lvp1 affects the SET domain group protein 724 (SDG724). SDG724 functions as a histone methyltransferase in vitro and contributes to a major fraction of global histone H3 lysine 36 (H3K36) methylation in vivo. Expression analyses of flowering time genes in wild-type and lvp1 mutants revealed that Early heading date1, but not Heading date1, are misregulated in lvp1 mutants. In addition, the double mutant of lvp1 with photoperiod sensitivity5 (se5) flowered later than the se5 single mutant, indicating that lvp1 delays flowering time irrespective of photoperiod. Chromatin immunoprecipitation assays showed that lvp1 had reduced levels of H3K36me2/3 at MADS50 and RFT1. This suggests that the divergent functions of paralogs RFT1 and Hd3a, and of MADS50 and MADS51, are in part due to differential H3K36me2/3 deposition, which also correlates with higher expression levels of MADS50 and RFT1 in flowering promotion in rice. Hd3a,OsMADS50|OsSOC1|DTH3,OsMADS51|OsMADS65,RFT1,SDG724|lvp1,SDG736|OsSET9 Hd3a, a rice ortholog of the Arabidopsis FT gene, promotes transition to flowering downstream of Hd1 under short-day conditions 2002 Plant Cell Physiol Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki, 305-0854 Japan. Heading date 3a (Hd3a) has been detected as a heading-date-related quantitative trait locus in a cross between rice cultivars Nipponbare and Kasalath. A previous study revealed that the Kasalath allele of Hd3a promotes heading under short-day (SD) conditions. High-resolution linkage mapping located the Hd3a locus in a approximately 20-kb genomic region. In this region, we found a candidate gene that shows high similarity to the FLOWERING LOCUS T (FT) gene, which promotes flowering in Arabidopsis: Introduction of the gene caused an early-heading phenotype in rice. The transcript levels of Hd3a were increased under SD conditions. The rice Heading date 1 (Hd1) gene, a homolog of CONSTANS (CO), has been shown to promote heading under SD conditions. By expression analysis, we showed that the amount of Hd3a mRNA is up-regulated by Hd1 under SD conditions, suggesting that Hd3a promotes heading under the control of Hd1. These results indicate that Hd3a encodes a protein closely related to Arabidopsis FT and that the function and regulatory relationship with Hd1 and CO, respectively, of Hd3a and FT are conserved between rice (an SD plant) and Arabidopsis (a long-day plant). Hd3a Genetic dissection of a genomic region for a quantitative trait locus, Hd3, into two loci, Hd3a and Hd3b, controlling heading date in rice 2002 Theor Appl Genet Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305-0854, Japan. The rice photoperiod sensitivity gene Hd3 was originally detected as a heading date-related quantitative trait locus localized on chromosome 6 of rice. High-resolution linkage mapping of Hd3 was performed using a large segregating population derived from advanced backcross progeny between a japonica variety, Nipponbare, and an indica variety, Kasalath. To determine the genotype of Hd3, we employed progeny testing under natural field and short-day conditions. As a result, two tightly linked loci, Hd3a and Hd3b, were identified in the Hd3 region. Nearly-isogenic lines for Hd3a and Hd3b were selected from progeny using marker-assisted selection. The inheritance mode of both Hd3a and Hd3b was found to be additive. Analysis of daylength response in nearly-isogenic lines of Hd3a and Hd3b showed that the Kasalath allele at Hd3a promotes heading under short-day conditions while that at Hd3b causes late heading under long-day and natural field conditions. Hd3a Hd3a Protein Is a Mobile Flowering Signal in Rice 2007 Science Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan. Florigen, the mobile signal that moves from an induced leaf to the shoot apex and causes flowering, has eluded identification since it was first proposed 70 years ago. Understanding the nature of the mobile flowering signal would provide a key insight into the molecular mechanism of floral induction. Recent studies suggest that the Arabidopsis FLOWERING LOCUS T (FT) gene is a candidate for encoding florigen. We show that the protein encoded by Hd3a, a rice ortholog of FT, moves from the leaf to the shoot apical meristem and induces flowering in rice. These results suggest that the Hd3a protein may be the rice florigen. Hd3a Identification of Heading Date Quantitative Trait Locus Hd6 and Characterization of Its Epistatic Interactions With Hd2 in Rice Using Advanced Backcross Progeny 2000 Genetics Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305-0854, Japan A backcrossed population (BC4F2) derived from a cross between a japonica rice variety, Nipponbare, as the recurrent parent and an indica rice variety, Kasalath, as the donor parent showed a long-range variation in days to heading. Quantitative trait loci (QTL) analysis revealed that two QTL, one on chromosome 3, designated Hd6, and another on chromosome 2, designated Hd7, were involved in this variation; and Hd6 was precisely mapped as a single Mendelian factor by using progeny testing (BC4F3). The nearly isogenic line with QTL (QTL-NIL) that carries the chromosomal segment from Kasalath for the Hd6 region in Nipponbare's genetic background was developed by marker-assisted selection. In a day-length treatment test, the QTL-NIL for Hd6 prominently increased days to heading under a 13.5-hr day length compared with the recurrent parent, Nipponbare, suggesting that Hd6 controls photoperiod sensitivity. QTL analysis of the F2 population derived from a cross between the QTL-NILs revealed existence of an epistatic interaction between Hd2, which is one of the photoperiod sensitivity genes detected in a previous analysis, and Hd6. The day-length treatment tests of these QTL-NILs, including the line introgressing both Hd2 and Hd6, also indicated an epistatic interaction for photoperiod sensitivity between them. Hd6|CK2 The role of casein kinase II in flowering time regulation has diversified during evolution 2010 Plant Physiol University of Tsukuba, Tsukuba, Japan 305-8577. Casein kinase II (CK2) is a protein kinase with an evolutionarily conserved function as a circadian clock component in several organisms, including the long-day plant Arabidopsis (Arabidopsis thaliana). The circadian clock component CIRCADIAN CLOCK ASSOCIATED1 (CCA1) is a CK2 target in Arabidopsis, where it influences photoperiodic flowering. In rice (Oryza sativa), a short-day plant, Heading date6 (Hd6) encodes a CK2alpha subunit that delays flowering time under long-day conditions. Here, we demonstrate that control of flowering time in rice by the Hd6 CK2alpha subunit requires a functional Hd1 gene (an Arabidopsis CONSTANS ortholog) and is independent of the circadian clock mechanism. Our findings from overexpressing the dominant-negative CK2 allele in rice support the independence of CK2 function from the circadian clock. This lack of control of the circadian clock by Hd6 CK2alpha might be due to the presence of glutamate in OsLHY (a CCA1 ortholog in rice) instead of the serine at the corresponding CK2 target site in CCA1. However, this glutamate is critical for the control of the OsPRR1 gene (a rice ortholog of the Arabidopsis TOC1/PRR1 gene) by OsLHY for regulation of the circadian clock. We also demonstrated that the other conserved CK2 target sites in OsLHY conferred robust rhythmic expression of OsLHY-LUC under diurnal conditions. These findings imply that the role of CK2 in flowering-time regulation in higher plants has diversified during evolution. Hd6|CK2,OsLHY,OsPRR1 Hd6, a rice quantitative trait locus involved in photoperiod sensitivity, encodes the alpha subunit of protein kinase CK2 2001 Proc Natl Acad Sci U S A Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305-0854, Japan. Hd6 is a quantitative trait locus involved in rice photoperiod sensitivity. It was detected in backcross progeny derived from a cross between the japonica variety Nipponbare and the indica variety Kasalath. To isolate a gene at Hd6, we used a large segregating population for the high-resolution and fine-scale mapping of Hd6 and constructed genomic clone contigs around the Hd6 region. Linkage analysis with P1-derived artificial chromosome clone-derived DNA markers delimited Hd6 to a 26.4-kb genomic region. We identified a gene encoding the alpha subunit of protein kinase CK2 (CK2 alpha) in this region. The Nipponbare allele of CK2 alpha contains a premature stop codon, and the resulting truncated product is undoubtedly nonfunctional. Genetic complementation analysis revealed that the Kasalath allele of CK2 alpha increases days-to-heading. Map-based cloning with advanced backcross progeny enabled us to identify a gene underlying a quantitative trait locus even though it exhibited a relatively small effect on the phenotype. Hd6|CK2 The role of rice HEI10 in the formation of meiotic crossovers 2012 PLoS Genet State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. HEI10 was first described in human as a RING domain-containing protein that regulates cell cycle and cell invasion. Mice HEI10(mei4) mutant displays no obvious defect other than meiotic failure from an absence of chiasmata. In this study, we characterize rice HEI10 by map-based cloning and explore its function during meiotic recombination. In the rice hei10 mutant, chiasma frequency is markedly reduced, and those remaining chiasmata exhibit a random distribution among cells, suggesting possible involvement of HEI10 in the formation of interference-sensitive crossovers (COs). However, mutation of HEI10 does not affect early recombination events and synaptonemal complex (SC) formation. HEI10 protein displays a highly dynamic localization on the meiotic chromosomes. It initially appears as distinct foci and co-localizes with MER3. Thereafter, HEI10 signals elongate along the chromosomes and finally restrict to prominent foci that specially localize to chiasma sites. The linear HEI10 signals always localize on ZEP1 signals, indicating that HEI10 extends along the chromosome in the wake of synapsis. Together our results suggest that HEI10 is the homolog of budding yeast Zip3 and Caenorhabditis elegans ZHP-3, and may specifically promote class I CO formation through modification of various meiotic components. HEI10,MER3|RCK The role of OsMSH5 in crossover formation during rice meiosis 2013 Mol Plant College of Plant Protection, Yunnan Agricultural University, Kunming 650201, China. MSH5, a meiosis-specific member of the MutS-homolog family, is required for normal level of recombination in budding yeast, mice, Caenorhabditis elegans, and Arabidopsis. Here, we report the identification and characterization of its rice homolog, OsMSH5, and demonstrate its function in rice meiosis. Five independent Osmsh5 mutants exhibited normal vegetative growth and severe sterility. The synaptonemal complex is well installed in Osmsh5, while the chiasma frequency is greatly reduced to approximately 10% of that observed in the wild-type, leading to the homologous non-disjunction and complete sterile phenotype. OsMSH5 is predominantly expressed in panicles. Immunofluorescence studies indicate that OsMSH5 chromosomal localization is limited to the early meiotic prophase I. OsMSH5 can be loaded onto meiotic chromosomes in Oszip4, Osmer3, and hei10. However, those ZMM proteins cannot be localized normally in the absence of OsMSH5. Furthermore, the residual chiasmata were shown to be the least frequent among the zmm mutants, including Osmer3, Oszip4, hei10, and Osmsh5. Taken together, we propose that OsMSH5 functions upstream of OsZIP4, OsMER3, and HEI10 in class I crossover formation. HEI10,OsMSH5,MER3|RCK,ZIP4|SPO22 The Rice HGW Gene Encodes a Ubiquitin-Associated (UBA) Domain Protein That Regulates Heading Date and Grain Weight 2012 PLoS One National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China Heading date and grain weight are two determining agronomic traits of crop yield. To date, molecular factors controlling both heading date and grain weight have not been identified. Here we report the isolation of a hemizygous mutation, heading and grain weight (hgw), which delays heading and reduces grain weight in rice. Analysis of hgw mutant phenotypes indicate that the hemizygous hgw mutation decreases latitudinal cell number in the lemma and palea, both composing the spikelet hull that is known to determine the size and shape of brown grain. Molecular cloning and characterization of the HGW gene showed that it encodes a novel plant-specific ubiquitin-associated (UBA) domain protein localized in the cytoplasm and nucleus, and functions as a key upstream regulator to promote expressions of heading date-and grain weight-related genes. Moreover, co-expression analysis in rice and Arabidopsis indicated that HGW and its Arabidopsis homolog are co-expressed with genes encoding various components of ubiquitination machinery, implying a fundamental role for the ubiquitination pathway in heading date and grain weight control. HGW Two types of HKT transporters with different properties of Na+ and K+ transport in Oryza sativa 2001 The Plant Journal Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma-shi, Nara 630-0101, Japan. It is thought that Na+ and K+ homeostasis is crucial for salt-tolerance in plants. To better understand the Na+ and K+ homeostasis in important crop rice (Oryza sativa L.), a cDNA homologous to the wheat HKT1 encoding K+-Na+ symporter was isolated from japonica rice, cv Nipponbare (Ni-OsHKT1). We also isolated two cDNAs homologous to Ni-OsHKT1 from salt-tolerant indica rice, cv Pokkali (Po-OsHKT1, Po-OsHKT2). The predicted amino acid sequence of Ni-OsHKT1 shares 100% identity with Po-OsHKT1 and 91% identity with Po-OsHKT2, and they are 66-67% identical to wheat HKT1. Low-K+ conditions (less than 3 mM) induced the expression of all three OsHKT genes in roots, but mRNA accumulation was inhibited by the presence of 30 mM Na+. We further characterized the ion-transport properties of OsHKT1 and OsHKT2 using an expression system in the heterologous cells, yeast and Xenopus oocytes. OsHKT2 was capable of completely rescuing a K+-uptake deficiency mutation in yeast, whereas OsHKT1 was not under K+-limiting conditions. When OsHKTs were expressed in Na+-sensitive yeast, OsHKT1 rendered the cells more Na+-sensitive than did OsHKT2 in high NaCl conditions. The electrophysiological experiments for OsHKT1 expressed in Xenopus oocytes revealed that external Na+, but not K+, shifted the reversal potential toward depolarization. In contrast, for OsHKT2 either Na+ or K+ in the external solution shifted the reversal potential toward depolarization under the mixed Na+ and K+ containing solutions. These results suggest that two isoforms of HKT transporters, a Na+ transporter (OsHKT1) and a Na+- and K+-coupled transporter (OsHKT2), may act harmoniously in the salt tolerant indica rice. HKT2,OsHKT2;2 Molecular characterization of Hmg2 gene encoding a 3-hydroxy-methylglutaryl-CoA reductase in rice 2001 Mol Cells Division of Biochemistry, National Institute of Agricultural Science and Technology, Rural Development Administration, Suwon, Korea. shha@rda.go.kr Three genes encoding 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR, EC1.1.1.34), which converts HMG-CoA into mevalonate in the early key step of the plant isoprenoid pathway, were isolated by RT-PCR and rice cDNA and genomic library screening. A genomic Southern blot analysis confirmed that HMGR genes are present in three copies in rice. Of the three, the HMGR 2 gene (Hmg2) obtained as a cDNA clone and its genomic clone had 4 exons and 3 introns, and encoded a 576 amino acid peptide containing an open reading frame of 1,728 bp with a calculated Mw. of 61,150. The structure of rice Hmg2 had common features, based on its nucleotide and deduced amino acid sequence homologies, with other plant HMGR genes published to date. Rice Hmg2 transcripts were constitutively detected in all parts of the rice plant, except in lamina and their levels were high particularly in the leaf part of the dark-grown seedlings and mature flowers. Our result showed that mRNA levels of rice Hmg2 were strongly induced in seedlings and influorescence in the early development stage. Rice Hmg2 possibly has a housekeeping role involved in the sterol biosynthesis, among the possible roles of plant HMGR genes that have been suggested in other plants [Weissenborn et al. (1995)]. HMG2 Rice HMGB1 protein recognizes DNA structures and bends DNA efficiently 2003 Archives of Biochemistry and Biophysics Department of Biological Sciences, Faculty of Science, National University of Singapore, 10 Science Drive 4, 117543, Singapore, Singapore. We analyzed the DNA-binding and DNA-bending properties of recombinant HMGB1 proteins based on a rice HMGB1 cDNA. Electrophoretic mobility shift assay demonstrated that rice HMGB1 can bind synthetic four-way junction (4H) DNA and DNA minicircles efficiently but the binding to 4H can be completed out by HMGA and histone H1. Conformational changes were detected by circular dichroism analysis with 4H DNA bound to various concentrations of HMGB1 or its truncated forms. T4 ligase-mediated circularization assays with short DNA fragments of 123 bp showed that the protein is capable of increasing DNA flexibility. The 123-bp DNA formed closed circular monomers efficiently in its presence, similar to that in an earlier study on maize HMG. Additionally, our results show for the first time that the basic N-terminal domain enhances the affinity of the plant HMGB1 protein for 4H DNA, while the acidic C-terminal domain has the converse effects. HMGB1 Cloning and characterization of rice HMGB1 gene 2003 Gene Department of Biological Sciences, National University of Singapore, 10 Science Drive 4, Singapore 117543, Singapore. We isolated a 918 bp long full-length rice HMGB1 cDNA, which has an open reading frame of 471 bp encoding 157 amino acids, with a central domain of high sequence similarity to the HMG-box domain of other plant HMGB1 proteins. RNA gel blot analysis indicated that rice HMGB1 gene is constitutively expressed in various tissues and organs. Southern hybridization and sequence analyses suggested that a single copy of the HMGB1 gene composed of seven exons and six introns exists in rice. We have also cloned a 1755 bp long 5' flanking region of the rice HMGB1 gene, which can be regarded as its promoter. 5' deletion analysis of this promoter indicated that positive cis-elements residing between -1400 and -1115 are important to enhance quantitative expression, whereas negative cis-elements between -1755 and -1400 and between -1115 and -351 inhibit expression. HMGB1 Radial axis differentiation in a globular embryo is marked by HAZ1, a PHD-finger homeobox gene of rice 2004 Gene Plant Genetics Laboratory, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan. Homeobox genes that encode transcription factors play important roles in development and differentiation of both plant and animal systems. From a cDNA library of 3-day after-pollination (DAP) rice embryos we cloned a HAZ1 cDNA that encodes a protein with a PHD-finger domain and a homeodomain. A database search showed that HAZ1 was most similar in its entire amino acid sequence to Zmhox1a (52% identity) and Zmhox1b (50%), PHD-finger family homeodomain proteins of maize. Differing from Zmhox1, overexpression of HAZ1 brought no morphological change either in tobacco or in rice. In situ hybridization showed that HAZ1 was expressed at a higher level in the outer layers of a developing embryo than in the inner parts of the embryo at 3 DAP. At 4 and 5 DAPs, the expression of HAZ1 was concentrated at the ventral part of an embryo. These results indicate that HAZ1 marks outer layer cells of a globular embryo before any morphological differentiation is discerned in it. Radial axis differentiation marked by HAZ1 is then collapsed dynamically along with embryo morphogenesis, and HAZ1 later marks the ventral surface of the embryo. HAZ1|HOX1a A positive feedback loop between HEAT SHOCK PROTEIN101 and HEAT STRESS-ASSOCIATED 32-KD PROTEIN modulates long-term acquired thermotolerance illustrating diverse heat stress responses in rice varieties 2014 Plant Physiol Agricultural Biotechnology Research Center , Academia Sinica, Taipei 11529, Taiwan, Republic of China; Heat stress is an important factor that has a negative impact on rice (Oryza sativa) production. To alleviate this problem, it is necessary to extensively understand the genetic basis of heat tolerance and adaptability to heat stress in rice. Here, we report the molecular mechanism underlying heat acclimation memory that confers long-term acquired thermotolerance (LAT) in this monocot plant. Our results showed that a positive feedback loop formed by two heat-inducible genes, HEAT SHOCK PROTEIN101 (HSP101) and HEAT STRESS-ASSOCIATED 32-KD PROTEIN (HSA32), at the posttranscriptional level prolongs the effect of heat acclimation in rice seedlings. The interplay between HSP101 and HSA32 also affects basal thermotolerance of rice seeds. These findings are similar to those reported for the dicot plant Arabidopsis (Arabidopsis thaliana), suggesting a conserved function in plant heat stress response. Comparison between two rice cultivars, japonica Nipponbare and indica N22 showed opposite performance in basal thermotolerance and LAT assays. 'N22' seedlings have a higher basal thermotolerance level than cv Nipponbare and vice versa at the LAT level, indicating that these two types of thermotolerance can be decoupled. The HSP101 and HSA32 protein levels were substantially higher in cv Nipponbare than in cv N22 after a long recovery following heat acclimation treatment, at least partly explaining the difference in the LAT phenotype. Our results point out the complexity of thermotolerance diversity in rice cultivars, which may need to be taken into consideration when breeding for heat tolerance for different climate scenarios. HSA32,HSP101|OsClpB-cyt|HSP100 Heat-inducible rice hsp82 and hsp70 are not always co-regulated 1994 Planta Laboratorium voor Genetica, Universiteit Gent, K.L. Ledeganckstraat 35, B-9000, Gent, Belgium We have characterized several heat-shock-induced genes in rice (Oryza sativa L.) and compared their expression under a variety of conditions. Three of these genes, which are analogs of the hsp82/90 family, lie within a cloned 18-kilobase (kb) region of the genome. The middle member of this cluster, designated hsp82B, has been fully sequenced. The gene uses a promoter containing six putative heat-shock elements as well as several unusual sequence motifs including a stretch of 11 thymidines alternating with 11 adenosines. The mRNA for this gene reaches its highest relative level of expression within 120 min after plants are shifted to 42 degrees C; no other conditions induce this gene. By contrast, we found that during heat stress the expression of hsp70 correlates well with increases in internal ion concentrations, and can also be induced by excess salt or ethanol at normal growth temperatures. These results appear to indicate that whereas hsp70 is induced by all stresses that lead to protein denaturation-including heat stress-HSP82 mRNA accumulates only upon heat stress. HSP70,hsp82A,hsp82B,hsp82D Mechanisms of plant adaptation/memory in rice seedlings under arsenic and heat stress: expression of heat-shock protein gene HSP70 2010 AoB Plants Centre for Applied Mathematics and Computational Science , Saha Institute of Nuclear Physics , 1/AF Bidhan Nagar , Kolkata- 700064 , India. BACKGROUND AND AIMS: Plants can withstand many abiotic stresses. Stress adaptation through retention of imprints of previous stress exposure has also been described in plants. We have characterized the imprint or memory of adaptive stress responses of rice seedlings to arsenic (As) and heat stress. METHODOLOGY: Two-week-old rice seedlings (both with and without As) were given a 45 degrees C heat shock for 3 h. While under heat shock, the leafy portion of the seedlings was harvested at regular intervals. Subsequently, the seedlings were kept at room temperature for recovery and sampling continued over 3 h. Total RNA and protein were extracted from the leafy portion of the seedlings and complementary DNA (cDNA) was prepared from total RNA. The cDNA was used as a template for the polymerase chain reaction to identify the transcription level of HSP70. Protein extracted from the seedlings was western-blotted. HSP70 and actin (loading control) antibodies were used to recognize the proteins on the same blot. PRINCIPAL RESULTS: Our studies reveal that HSP70, a cellular chaperone gene, is over-expressed at the mRNA and protein levels when rice seedlings are exposed to As and heat. The effect is cumulative and increases with the duration of stress for 3 h. During 3 h recovery from heat stress at ambient temperatures for 3 h, the chaperone remains expressed at higher levels in plants pre-exposed to As. CONCLUSIONS: Our findings demonstrate a retention of the imprint of previous stress exposure, perhaps through sustained activation of the signalling pathways upstream of over-expression of HSP70. Furthermore, stress-induced HSP70 expression was additive/cumulative for continued exposure to similar or different kinds of stress, indicating that a commonality of signal transduction networks is adopted when plants experience more than one stress. HSP70 Characterizations and fine mapping of a mutant gene for high tillering and dwarf in rice (Oryza sativa L.) 2005 Planta National Plant Gene Research Centre (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Science, Beijing, 100101, China. A rice htd-1 mutant, related to tillering and dwarfing, was characterized. We show that the htd-1 mutant increases its tiller number by releasing axillary buds from dormant stage rather than by initiating more axillary buds. The dwarf is caused by averagely reducing each internode and panicle. Based on this dwarfing pattern, the htd-1 mutant could be grouped into dn-type dwarf defined by Takeda (Gamma Field Symp 16:1, 1977). In addition, the dwarfing of the htd-1 mutant was found independent of GA based on the analyses of two GA-mediated processes. Based on the quantitative determination of IAA and ABA and application of the two hormones exogenously to the seedlings, we inferred that the high tillering capacity of the htd-1 mutant should not be attributed to a defect in the synthesis of IAA or ABA. The genetic analysis of the htd-1 mutant indicated that the phenotypes of high tillering and dwarf were controlled by a recessive gene, termed htd1. By map-based cloning, the htd1 gene was fine mapped in a 30-kb DNA region on chromosome 4. Sequencing the target DNA region and comparing the counterpart DNA sequences between the htd-1 mutant and other rice varieties revealed a nucleotide substitution corresponding to an amino acid substitution from prolin to leucine in a predicted rice gene, OsCCD7, the rice orthologous gene of AtMAX3/CCD7. With the evidence of the association between the presence of one amino acid change in OsCCD7 and the abnormal phenotypes of the htd-1 mutant, OsCCD7 was identified as the candidate of the HTD1 gene. HTD1|OsCCD7 Carotenoid oxygenases involved in plant branching catalyse a highly specific conserved apocarotenoid cleavage reaction 2008 Biochem J Albert-Ludwigs University of Freiburg, Faculty of Biology, Institute of Biology II, Schaenzlestrasse 1, D-79104 Freiburg, Germany. Recent studies with the high-tillering mutants in rice (Oryza sativa), the max (more axillary growth) mutants in Arabidopsis thaliana and the rms (ramosus) mutants in pea (Pisum sativum) have indicated the presence of a novel plant hormone that inhibits branching in an auxin-dependent manner. The synthesis of this inhibitor is initiated by the two CCDs [carotenoid-cleaving (di)oxygenases] OsCCD7/OsCCD8b, MAX3/MAX4 and RMS5/RMS1 in rice, Arabidopsis and pea respectively. MAX3 and MAX4 are thought to catalyse the successive cleavage of a carotenoid substrate yielding an apocarotenoid that, possibly after further modification, inhibits the outgrowth of axillary buds. To elucidate the substrate specificity of OsCCD8b, MAX4 and RMS1, we investigated their activities in vitro using naturally accumulated carotenoids and synthetic apocarotenoid substrates, and in vivo using carotenoid-accumulating Escherichia coli strains. The results obtained suggest that these enzymes are highly specific, converting the C27 compounds beta-apo-10'-carotenal and its alcohol into beta-apo-13-carotenone in vitro. Our data suggest that the second cleavage step in the biosynthesis of the plant branching inhibitor is conserved in monocotyledonous and dicotyledonous species. HTD1|OsCCD7,D10|OsCCD8|OsCCD8b Dwarf 88, a novel putative esterase gene affecting architecture of rice plant 2009 Plant Mol Biol National Center for Gene Research/Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200233 Shanghai, China. Rice architecture is an important agronomic trait that affects grain yield. We characterized a tillering dwarf mutant d88 derived from Oryza sativa ssp. japonica cultivar Lansheng treated with EMS. The mutant had excessive shorter tillers and smaller panicles and seeds compared to the wild-type. A reduction in number and size of parenchyma cells around stem marrow cavity as well as a delay in the elongation of parenchyma cells caused slender tillers and dwarfism in the d88 mutant. The D88 gene was isolated via map-based cloning and identified to encode a putative esterase. The gene was expressed in most rice organs, with especially high levels in the vascular tissues. The mutant carried a nucleotide substitution in the first exon of the gene that led to the substitution of arginine for glycine, which presumably disrupted the functionally conserved N-myristoylation domain of the protein. The function of the gene was confirmed by complementation test and antisense analysis. D88, thus, represents a new category of genes that regulates cell growth and organ development and consequently plant architecture. The potential relationship between the tiller formation associated genes and D88 is discussed and future identification of the substrate for D88 may lead to the characterization of new pathways regulating plant development. HTD2|D88|D14 The interaction between OsMADS57 and OsTB1 modulates rice tillering via DWARF14 2013 Nat Commun The Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. Rice tillering is a multigenic trait that influences grain yield, but its regulation molecular module is poorly understood. Here we report that OsMADS57 interacts with OsTB1 (TEOSINTE BRANCHED1) and targets D14 (Dwarf14) to control the outgrowth of axillary buds in rice. An activation-tagged mutant osmads57-1 and OsMADS57-overexpression lines showed increased tillers, whereas OsMADS57 antisense lines had fewer tillers. OsMIR444a-overexpressing lines exhibited suppressed OsMADS57 expression and tillering. Furthermore, osmads57-1 was insensitive to strigolactone treatment to inhibit axillary bud outgrowth, and OsMADS57's function in tillering was dependent on D14. D14 expression was downregulated in osmads57-1, but upregulated in antisense and OsMIR444a-overexpressing lines. OsMADS57 bound to the CArG motif [C(A/T)TTAAAAAG] in the promoter and directly suppressed D14 expression. Interaction of OsMADS57 with OsTB1 reduced OsMADS57 inhibition of D14 transcription. Therefore, OsMIR444a-regulated OsMADS57, together with OsTB1, target D14 to control tillering. This regulation mechanism could have important application in rice molecular breeding programs focused on high grain yield. HTD2|D88|D14,OsMADS57,OsTB1|FC1 d14, a strigolactone-insensitive mutant of rice, shows an accelerated outgrowth of tillers 2009 Plant Cell Physiol Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, Japan. Recent studies using highly branched mutants of pea, Arabidopsis and rice have demonstrated that strigolactones, a group of terpenoid lactones, act as a new hormone class, or its biosynthetic precursors, in inhibiting shoot branching. Here, we provide evidence that DWARF14 (D14) inhibits rice tillering and may act as a new compo-nent of the strigolactone-dependent branching inhibition pathway. The d14 mutant exhibits increased shoot branch-ing with reduced plant height like the previously characterized strigolactone-deficient and -insensitive mutants d10 and d3, respectively. The d10-1 d14-1 double mutant is phenotypically indistinguishable from the d10-1 and d14-1 single mutants, consistent with the idea that D10 and D14 function in the same pathway. However, unlike with d10, the d14 branching phenotype could not be rescued by exogenous strigolactones. In addition, the d14 mutant contained a higher level of 2'-epi-5-deoxystrigol than the wild type. Positional cloning revealed that D14 encodes a protein of the alpha/beta-fold hydrolase superfamily, some members of which play a role in metabolism or signaling of plant hormones. We propose that D14 functions downstream of strigolactone synthesis, as a component of hormone signaling or as an enzyme that participates in the conversion of strigolactones to the bioactive form. HTD2|D88|D14,D10|OsCCD8|OsCCD8b Genetic and physiological analysis of a novel type of interspecific hybrid weakness in rice 2013 Mol Plant National Key Laboratory of Plant Molecular Genetics and National Center for Plant Gene Research Shanghai, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China. Hybrid weakness is an important reproductive barrier that hinders genetic exchange between different species at the post-zygotic stage. However, our understanding of the molecular mechanisms underlying hybrid weakness is limited. In this study, we report discovery of a novel interspecific hybrid weakness in a rice chromosome segment substitution line (CSSL) library derived from a cross between the indica variety Teqing (Oryza sativa) and common wild rice (O. rufipogon). The dominant Hybrid weakness i1 (Hwi1) gene from wild rice is genetically incompatible with Teqing and induced a set of weakness symptoms, including growth suppression, yield decrease, impaired nutrient absorption, and the retardation of crown root initiation. Phytohormone treatment showed that salicylic acid (SA) could restore the height of plants expressing hybrid weakness, while other phytohormones appear to have little effect. Fine mapping indicated that Hwi1 is located in a tandem leucine-rich repeat receptor-like kinase (LRR-RLK) gene cluster. Within the 13.2-kb candidate region on the short arm of chromosome 11, there are two annotated LRR-RLK genes, LOC_Os11g07230 and LOC_Os11g07240. The Teqing allele of LOC_Os11g07230 and the wild rice allele of LOC_Os11g07240 encode predicted functional proteins. Based on the genetic inheritance of hybrid weakness, LOC_Os11g07240 is implicated as the candidate gene for Hwi1. Functional analysis of Hwi1 will expand our knowledge of the regulation of hybrid weakness in rice. Hwi1 Structural, biochemical, and phylogenetic analyses suggest that indole-3-acetic acid methyltransferase is an evolutionarily ancient member of the SABATH family 2008 Plant Physiol Department of Plant Sciences, University of Tennessee, Knoxville, Tennessee 37996, USA. The plant SABATH protein family encompasses a group of related small-molecule methyltransferases (MTs) that catalyze the S-adenosyl-L-methionine-dependent methylation of natural chemicals encompassing widely divergent structures. Indole-3-acetic acid (IAA) methyltransferase (IAMT) is a member of the SABATH family that modulates IAA homeostasis in plant tissues through methylation of IAA's free carboxyl group. The crystal structure of Arabidopsis (Arabidopsis thaliana) IAMT (AtIAMT1) was determined and refined to 2.75 A resolution. The overall tertiary and quaternary structures closely resemble the two-domain bilobed monomer and the dimeric arrangement, respectively, previously observed for the related salicylic acid carboxyl methyltransferase from Clarkia breweri (CbSAMT). To further our understanding of the biological function and evolution of SABATHs, especially of IAMT, we analyzed the SABATH gene family in the rice (Oryza sativa) genome. Forty-one OsSABATH genes were identified. Expression analysis showed that more than one-half of the OsSABATH genes were transcribed in one or multiple organs. The OsSABATH gene most similar to AtIAMT1 is OsSABATH4. Escherichia coli-expressed OsSABATH4 protein displayed the highest level of catalytic activity toward IAA and was therefore named OsIAMT1. OsIAMT1 exhibited kinetic properties similar to AtIAMT1 and poplar IAMT (PtIAMT1). Structural modeling of OsIAMT1 and PtIAMT1 using the experimentally determined structure of AtIAMT1 reported here as a template revealed conserved structural features of IAMTs within the active-site cavity that are divergent from functionally distinct members of the SABATH family, such as CbSAMT. Phylogenetic analysis revealed that IAMTs from Arabidopsis, rice, and poplar (Populus spp.) form a monophyletic group. Thus, structural, biochemical, and phylogenetic evidence supports the hypothesis that IAMT is an evolutionarily ancient member of the SABATH family likely to play a critical role in IAA homeostasis across a wide range of plants. IAMT1 A novel gene IBF1 is required for the inhibition of brown pigment deposition in rice hull furrows 2012 Theor Appl Genet State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. The role of flavonoids as the major red, blue, purple and brown pigments in plants has gained these secondary products a great deal of attention over the years. In this study, we characterized a rice inhibitor for brown furrows1 (ibf1) mutant. In the ibf1 mutant, brown pigments specifically accumulate in hull furrows during seed maturation and reach a maximum level in dry seeds. Higher amounts of total flavonoids and anthocyanin in hull may be responsible for the brown pigmentation of ibf1. The IBF1 gene, which encodes a similar kelch repeat-containing F-box protein, was isolated by map-based cloning approach. Real-time RT-PCR and GUS activity assays revealed that IBF1 specifically expressed in reproductive tissues. GFP-IBF1 fusion protein mainly localized in cytoplasm. The expression of some major structural enzymatic genes involved in flavonoids biosynthesis could be up- or down-regulated to some different extent in ibf1 mutant. Our data suggested that IBF1 as a suppressor could inhibit the brown pigmentation of rice hull furrows. IBF1 The rice transcription factor IDEF1 is essential for the early response to iron deficiency, and induces vegetative expression of late embryogenesis abundant genes 2009 Plant J Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Higher plants maintain iron homeostasis by regulating the expression of iron (Fe)-related genes in accordance with Fe availability. The transcription factor IDEF1 regulates the response to Fe deficiency in Oryza sativa (rice) by recognizing CATGC sequences within the Fe deficiency-responsive cis-acting element IDE1. To investigate the function of IDEF1 in detail, we analyzed the response to Fe deficiency in transgenic rice plants exhibiting induced or repressed IDEF1 expression. Fe-deficiency treatment in hydroponic culture revealed that IDEF1 knock-down plants are susceptible to early-stage Fe deficiency, in contrast to IDEF1-induced plants. Time-course expression analyses using quantitative reverse-transcriptase PCR revealed that the IDEF1 expression level was positively correlated with the level of induction of the Fe utilization-related genes OsIRO2, OsYSL15, OsIRT1, OsYSL2, OsNAS1, OsNAS2, OsNAS3 and OsDMAS1, just after the onset of Fe starvation. However, this overall transactivation mediated by IDEF1 became less evident in subsequent stages. Microarray and in-silico analyses revealed that genes positively regulated by IDEF1, especially at the early stage, exhibit over-representation of CATGC and IDE1-like elements within the proximal promoter regions. These results indicate the existence of early and subsequent responses to Fe deficiency, with the former requiring IDEF1 more specifically. Proximal regions of IDEF1-regulated gene promoters also showed enrichment of RY elements (CATGCA), which regulate gene expression during seed maturation. The expression of several genes encoding late embryogenesis abundant proteins, including Osem, was induced in Fe-deficient roots and/or leaves in an IDEF1-dependent manner, suggesting a possible function of seed maturation-related genes in Fe-deficient vegetative organs. IDEF1,OsIRO2,OsIRT1,OsNAS1,OsNAS2,OsNAS3,OsYSL15,OsYSL2 The rice transcription factor IDEF1 directly binds to iron and other divalent metals for sensing cellular iron status 2012 Plant J Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Iron is essential for most living organisms and its availability often determines survival and proliferation. The Oryza sativa (rice) transcription factor IDEF1 plays a crucial role in regulating iron deficiency-induced genes involved in iron homeostasis. In the present report, we found characteristic histidine-asparagine repeat and proline-rich regions in IDEF1 and its homolog in Hordeum vulgare (barley), HvIDEF1. An immobilized metal ion affinity chromatography assay revealed that IDEF1 and HvIDEF1 bind to various divalent metals, including Fe(2+) and Ni(2+) . Recombinant IDEF1 protein expressed in Escherichia coli contained mainly Fe and Zn. This metal-binding activity of IDEF1 was almost abolished by deletion of the histidine-asparagine and proline-rich regions, but DNA-binding and trans-activation functions were not impaired by the deletion. Transgenic rice plants constitutively overexpressing IDEF1 without these metal-binding domains failed to cause pleiotropic effects conferred by overexpression of full-length IDEF1, including a low germination rate, impaired seedling growth, tolerance to iron deficiency in hydroponic culture, and enhanced expression of various iron deficiency-inducible genes. Impairment of the transcriptional regulation of IDEF1 by deletion of the metal-binding domains occurred primarily at an early stage of iron deficiency. These results suggest that the histidine-asparagine and proline-rich regions in rice IDEF1 directly bind to divalent metals and sense the cellular metal ion balance caused by changes in iron availability. IDEF1 The spatial expression and regulation of transcription factors IDEF1 and IDEF2 2010 Ann Bot Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. BACKGROUND AND AIMS: Under conditions of low iron availability, rice plants induce genes involved in iron uptake and utilization. The iron deficiency-responsive cis-acting element binding factors 1 and 2 (IDEF1 and IDEF2) regulate transcriptional response to iron deficiency in rice roots. Clarification of the functions of IDEF1 and IDEF2 could uncover the gene regulation mechanism. METHODS: Spatial patterns of IDEF1 and IDEF2 expression were analysed by histochemical staining of IDEF1 and IDEF2 promoter-GUS transgenic rice lines. Expression patterns of the target genes of IDEF1 and IDEF2 were analysed using transformants with induced or repressed expression of IDEF1 or IDEF2 grown in iron-rich or in iron-deficient solutions for 1 d. KEY RESULTS: IDEF1 and IDEF2 were highly expressed in the basal parts of the lateral roots and vascular bundles. IDEF1 and IDEF2 expression was dominant in leaf mesophyll and vascular cells, respectively. These expression patterns were similar under both iron-deficient and iron-sufficient conditions. IDEF1 was strongly expressed in pollen, ovaries, the aleurone layer and embryo. IDEF2 was expressed in pollen, ovaries and the dorsal vascular region of the endosperm. During seed germination, IDEF1 and IDEF2 were expressed in the endosperm and embryo. Expression of IDEF1 target genes was regulated in iron-rich roots similar to early iron-deficiency stages. In addition, the expression patterns of IDEF2 target genes were similar between iron-rich conditions and early or subsequent iron deficiency. CONCLUSIONS: IDEF1 and IDEF2 are constitutively expressed during both vegetative and reproductive stages. The spatial expression patterns of IDEF1 and IDEF2 overlap with their target genes in restricted cell types, but not in all cells. The spatial expression patterns and gene regulation of IDEF1 and IDEF2 in roots are generally conserved under conditions of iron sufficiency and deficiency, suggesting complicated interactions with unknown factors for sensing and transmitting iron-deficiency signals. IDEF1,IDEF2 The transcription factor IDEF1 regulates the response to and tolerance of iron deficiency in plants 2007 Proc Natl Acad Sci U S A Laboratory of Plant Biotechnology, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Iron is essential for most living organisms and is often the major limiting nutrient for normal growth. Plants induce iron utilization systems under conditions of low iron availability, but the molecular mechanisms of gene regulation under iron deficiency remain largely unknown. We identified the rice transcription factor IDEF1, which specifically binds the iron deficiency-responsive cis-acting element IDE1. IDEF1 belongs to an uncharacterized branch of the plant-specific transcription factor family ABI3/VP1 and exhibits the sequence recognition property of efficiently binding to the CATGC sequence within IDE1. IDEF1 transcripts are constitutively present in rice roots and leaves. Transgenic tobacco plants expressing IDEF1 under the control of the constitutive cauliflower mosaic virus 35S promoter transactivate IDE1-mediated expression only in iron-deficient roots. Transgenic rice plants expressing an introduced IDEF1 exhibit substantial tolerance to iron deficiency in both hydroponic culture and calcareous soil. IDEF1 overexpression leads to the enhanced expression of the iron deficiency-induced transcription factor gene OsIRO2, suggesting the presence of a sequential gene regulatory network. These findings reveal cis element/trans factor interactions that are functionally linked to the iron deficiency response. Manipulation of IDEF1 also provides another approach for producing crops tolerant of iron deficiency to enhance food and biomass production in calcareous soils. IDEF1 A novel NAC transcription factor, IDEF2, that recognizes the iron deficiency-responsive element 2 regulates the genes involved in iron homeostasis in plants 2008 J Biol Chem Graduate School of Agricultural and Life Sciences, the University of Tokyo, 1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Iron is essential for most living organisms, and thus iron deficiency poses a major abiotic stress in crop production. Plants induce iron utilization systems under conditions of low iron availability, but the molecular mechanisms of gene regulation under iron deficiency remain largely unknown. We identified a novel transcription factor of rice and barley, IDEF2, which specifically binds to the iron deficiency-responsive cis-acting element 2 (IDE2) by yeast one-hybrid screening. IDEF2 belongs to an uncharacterized branch of the NAC transcription factor family and exhibits novel properties of sequence recognition. An electrophoretic mobility shift assay and cyclic amplification and selection of targets experiment revealed that IDEF2 predominantly recognized CA(A/C)G(T/C)(T/C/A)(T/C/A) within IDE2 as the core-binding site. IDEF2 transcripts are constitutively present in rice roots and leaves. Repression of the function of IDEF2 by the RNA interference (RNAi) technique and chimeric repressor gene-silencing technology (CRES-T) caused aberrant iron homeostasis in rice. Several genes up-regulated by iron deficiency, including the Fe(II)-nicotianamine transporter gene OsYSL2, were less induced by iron deficiency in the RNAi rice of IDEF2, suggesting that IDEF2 is involved in the regulation of these genes. Many genes with repressed expression in IDEF2 RNAi rice possessed the IDEF2-binding core sites in their promoters, and the flanking sequences were also highly homologous to IDE2. IDEF2 bound to OsYSL2 promoter region containing the binding core site, suggesting direct regulation of OsYSL2 expression. These results reveal novel cis-element/trans-factor interactions functionally associated with iron homeostasis. IDEF2,OsYSL2 Increased leaf angle1, a Raf-like MAPKKK that interacts with a nuclear protein family, regulates mechanical tissue formation in the Lamina joint of rice 2011 Plant Cell National Key Laboratory of Crop Genetic Improvement and National Center for Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China. Mitogen-activated protein kinase kinase kinases (MAPKKKs), which function at the top level of mitogen-activated protein kinase cascades, are clustered into three groups. However, no Group C Raf-like MAPKKKs have yet been functionally identified. We report here the characterization of a rice (Oryza sativa) mutant, increased leaf angle1 (ila1), resulting from a T-DNA insertion in a Group C MAPKKK gene. The increased leaf angle in ila1 is caused by abnormal vascular bundle formation and cell wall composition in the leaf lamina joint, as distinct from the mechanism observed in brassinosteroid-related mutants. Phosphorylation assays revealed that ILA1 is a functional kinase with Ser/Thr kinase activity. ILA1 is predominantly resident in the nucleus and expressed in the vascular bundles of leaf lamina joints. Yeast two-hybrid screening identified six closely related ILA1 interacting proteins (IIPs) of unknown function. Using representative IIPs, the interaction of ILA1 and IIPs was confirmed in vivo. IIPs were localized in the nucleus and showed transactivation activity. Furthermore, ILA1 could phosphorylate IIP4, indicating that IIPs may be the downstream substrates of ILA1. Microarray analyses of leaf lamina joints provided additional evidence for alterations in mechanical strength in ila1. ILA1 is thus a key factor regulating mechanical tissue formation at the leaf lamina joint. ILA1 Dynamics of brassinosteroid response modulated by negative regulator LIC in rice 2012 PLoS Genet Key Laboratory of Plant Molecular Physiology/Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China. Brassinosteroids (BRs) regulate rice plant architecture, including leaf bending, which affects grain yield. Although BR signaling has been investigated in Arabidopsis thaliana, the components negatively regulating this pathway are less well understood. Here, we demonstrate that Oryza sativa LEAF and TILLER ANGLE INCREASED CONTROLLER (LIC) acts as an antagonistic transcription factor of BRASSINAZOLE-RESISTANT 1 (BZR1) to attenuate the BR signaling pathway. The gain-of-function mutant lic-1 and LIC-overexpressing lines showed erect leaves, similar to BZR1-depleted lines, which indicates the opposite roles of LIC and BZR1 in regulating leaf bending. Quantitative PCR revealed LIC transcription rapidly induced by BR treatment. Image analysis and immunoblotting showed that upon BR treatment LIC proteins translocate from the cytoplasm to the nucleus in a phosphorylation-dependent fashion. Phosphorylation assay in vitro revealed LIC phosphorylated by GSK3-like kinases. For negative feedback, LIC bound to the core element CTCGC in the BZR1 promoter on gel-shift and chromatin immunoprecipitation assay and repressed its transcription on transient transformation assay. LIC directly regulated target genes such as INCREASED LEAF INCLINATION 1 (ILI1) to oppose the action of BZR1. Repression of LIC in ILI1 transcription in protoplasts was partially rescued by BZR1. Phenotypic analysis of the crossed lines depleted in both LIC and BZR1 suggested that BZR1 functionally depends on LIC. Molecular and physiology assays revealed that LIC plays a dominant role at high BR levels, whereas BZR1 is dominant at low levels. Thus, LIC regulates rice leaf bending as an antagonistic transcription factor of BZR1. The phenotypes of lic-1 and LIC-overexpressing lines in erect leaves contribute to ideal plant architecture. Improving this phenotype may be a potential approach to molecular breeding for high yield in rice. ILI1,OsBZR1,OsIBH1|IBH1,LIC|OsLIC1,OsGSK4 Antagonistic HLH/bHLH transcription factors mediate brassinosteroid regulation of cell elongation and plant development in rice and Arabidopsis 2009 Plant Cell Key Laboratory of Photosynthesis and Environmental Molecular Biology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. In rice (Oryza sativa), brassinosteroids (BRs) induce cell elongation at the adaxial side of the lamina joint to promote leaf bending. We identified a rice mutant (ili1-D) showing an increased lamina inclination phenotype similar to that caused by BR treatment. The ili1-D mutant overexpresses an HLH protein homologous to Arabidopsis thaliana Paclobutrazol Resistance1 (PRE1) and the human Inhibitor of DNA binding proteins. Overexpression and RNA interference suppression of ILI1 increase and reduce, respectively, rice laminar inclination, confirming a positive role of ILI1 in leaf bending. ILI1 and PRE1 interact with basic helix-loop-helix (bHLH) protein IBH1 (ILI1 binding bHLH), whose overexpression causes erect leaf in rice and dwarfism in Arabidopsis. Overexpression of ILI1 or PRE1 increases cell elongation and suppresses dwarf phenotypes caused by overexpression of IBH1 in Arabidopsis. Thus, ILI1 and PRE1 may inactivate inhibitory bHLH transcription factors through heterodimerization. BR increases the RNA levels of ILI1 and PRE1 but represses IBH1 through the transcription factor BZR1. The spatial and temporal expression patterns support roles of ILI1 in laminar joint bending and PRE1/At IBH1 in the transition from growth of young organs to growth arrest. These results demonstrate a conserved mechanism of BR regulation of plant development through a pair of antagonizing HLH/bHLH transcription factors that act downstream of BZR1 in Arabidopsis and rice. ILI1,OsIBH1|IBH1 Development of low phytate rice by RNAi mediated seed-specific silencing of inositol 1,3,4,5,6-pentakisphosphate 2-kinase gene (IPK1) 2013 PLoS One Plant Molecular Biology and Biotechnology Laboratory, Department of Botany, University of Calcutta, Kolkata, West Bengal, India. Phytic acid (InsP(6)) is considered to be the major source of phosphorus and inositol phosphates in most cereal grains. However, InsP(6) is not utilized efficiently by monogastric animals due to lack of phytase enzyme. Furthermore, due to its ability to chelate mineral cations, phytic acid is considered to be an antinutrient that renders these minerals unavailable for absorption. In view of these facts, reducing the phytic acid content in cereal grains is a desired goal for the genetic improvement of several crops. In the present study, we report the RNAi-mediated seed-specific silencing (using the Oleosin18 promoter) of the IPK1 gene, which catalyzes the last step of phytic acid biosynthesis in rice. The presence of the transgene cassette in the resulting transgenic plants was confirmed by molecular analysis, indicating the stable integration of the transgene. The subsequent T4 transgenic seeds revealed 3.85-fold down-regulation in IPK1 transcripts, which correlated to a significant reduction in phytate levels and a concomitant increase in the amount of inorganic phosphate (Pi). The low-phytate rice seeds also accumulated 1.8-fold more iron in the endosperm due to the decreased phytic acid levels. No negative effects were observed on seed germination or in any of the agronomic traits examined. The results provide evidence that silencing of IPK1 gene can mediate a substantial reduction in seed phytate levels without hampering the growth and development of transgenic rice plants. IPK1 Rice debranching enzyme isoamylase3 facilitates starch metabolism and affects plastid morphogenesis 2011 Plant Cell Physiol Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Japan. Debranching enzymes, which hydrolyze alpha-1 and 6-glucosidic linkages in alpha-polyglucans, play a dual role in the synthesis and degradation of starch in plants. A transposon-inserted rice mutant of isoamylase3 (isa3) contained an increased amount of starch in the leaf blade at the end of the night, indicating that ISA3 plays a role in the degradation of transitory starch during the night. An epitope-tagged ISA3 expressed in Escherichia coli exhibited hydrolytic activity on beta-limit dextrin and amylopectin. We investigated whether ISA3 plays a role in amyloplast development and starch metabolism in the developing endosperm. ISA3-green fluorescent protein (GFP) fusion protein expressed under the control of the rice ISA3 promoter was targeted to the amyloplast stroma in the endosperm. Overexpression of ISA3 in the sugary1 mutant, which is deficient in ISA1 activity, did not convert water-soluble phytoglycogen to starch granules, indicating that ISA1 and ISA3 are not functionally redundant. Both overexpression and loss of function of ISA3 in the endosperm generated pleomorphic amyloplasts and starch granules. Furthermore, chloroplasts in the leaf blade of isa3 seedlings were large and pleomorphic. These results suggest that ISA3 facilitates starch metabolism and affects morphological characteristics of plastids in rice. ISA3,OsISA1,OsPUL,FtsZ1,FtsZ2-1 Role of the rice transcription factor JAmyb in abiotic stress response 2013 J Plant Res Research Institute for Biological Sciences, Okayama Prefectural Technology Center for Agriculture, Forestry, and Fisheries, 7549-1 Yoshikawa, Kibichuo, Okayama 716-1241, Japan. Plants have developed certain adaptive responses to environmental stresses that cause adverse effects on growth. To identify genes involved in the adaptive mechanisms, we constructed a large population of transgenic Arabidopsis expressing rice full-length cDNAs, and performed gain-of-function screening under high-salinity stress. In this study, we identified a rice R2R3-type MYB transcription factor gene, JAmyb, as a gene whose overexpression causes tolerance to high salinity. JAmyb overexpression in transgenic Arabidopsis improved tolerance to high-salinity stress during seed germination, seedling growth, and root elongation. In rice seedlings, JAmyb expression was induced by high-salinity and high-osmotic stresses and reactive oxygen species (ROS), suggesting that JAmyb is responsible for abiotic stress response. Microarray analysis showed that the overexpression of JAmyb stimulates the expression of several defense-associated genes, some of which have been predicted to be involved in osmotic adjustment, ROS removal, and ion homeostasis. Several transcription factors involved in the jasmonate (JA)-mediated stress response are also regulated by JAmyb. JAmyb has been reported to be associated with disease response. Our observations suggest that JAmyb plays a role in JA-mediated abiotic stress response in addition to biotic stress response in rice. JAmyb Molecular cloning and characterization of a novel Jasmonate inducible pathogenesis-related class 10 protein gene, JIOsPR10, from rice (Oryza sativa L.) seedling leaves 2001 Biochem Biophys Res Commun Department of Molecular Biology, Sejong University, Seoul, 143-747, Korea. A novel rice (Oryza sativa L.) gene, homologous to a sorghum pathogenesis-related class 10 protein gene, was cloned from a cDNA library prepared from 2-week-old jasmonic acid-treated rice seedling leaves, and named as JIOsPR10 (jasmonate inducible). JIOsPR10 encoded a 160-amino-acid polypeptide with a predicted molecular mass of 17,173.23 Da and a pI of 5.84. JIOsPR10 was highly similar (77%) to the sorghum PR10 protein, but showed less than 55% similarity with other identified PR10s at the amino acid level. Genomic Southern analyses indicated the presence of related genes in the rice genome. The JIOsPR10 transcript was not detected in the healthy leaves, and was not induced after cut. Further expression analysis revealed that the signaling components of defense/stress pathways, jasmonate, salicylate, and H(2)O(2) significantly up-regulated the JIOsPR10 mRNA over the cut control, whereas two other stress regulators, ethylene and abscisic acid, failed to induce its expression. Interestingly the protein phosphatase (PP) inhibitors, cantharidin, endothall, and okadaic acid, rapidly and potently up-regulated the JIOsPR10 expression, suggesting involvement of the phosphorylation/dephosphorylation events. Additionally, the inducible expression of the JIOsPR10 gene was influenced by light signal(s). Finally, the blast pathogen (Magnaporthe grisea) also specifically elicited the accumulation of JIOsPR10 mRNA in leaves. Induction of the JIOsPR10 gene expression by signaling molecules, PP inhibitors and pathogen attack, strongly indicate a role for this novel gene in rice self-defense/stress response(s). JIOsPR10|OsPR10 The rice pathogen-related protein 10 (JIOsPR10) is induced by abiotic and biotic stresses and exhibits ribonuclease activity 2008 Plant Cell Rep Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju, Korea. We previously reported that rice blast fungus or jasmonic acid induced the expression of rice pathogenesis-related class 10 (JIOsPR10) proteins (Kim et al. 2003, 2004). However, no further studies have been carried out to examine the expression, localization, and enzymatic activity of this protein in either developmental tissues or in tissues under abiotic stress conditions. In this study, rice JIOsPR10 was examined by Western blot analysis, immunolocalization, and biochemical assays. Western blots revealed that the JIOsPR10 protein was expressed in developmental tissues, including in flower and root. The protein was also expressed under abiotic stresses, such as occurs during senescence and wounding. Using immunohistochemical techniques, we determined that expression of JIOsPR10 was localized to the palea of flower, in the exodermis, and inner part of the endodermis of the root. In senescencing tissues of leaf and coleoptiles, its expression was localized in vascular bundles. The RNase activity using JIOsPR10 recombinant protein was determined and abolished after treatment with DTT in a native in-gel assay. To test this, we created JIOsPR10 mutant proteins containing serine substitutions of amino acids C81S, C83S, or both and examined their RNase activities. The activity of the C83S mutant was decreased in the agarose gel assay compared to the wild type. Taken together, we hypothesize that the JIOsPR10 protein possesses RNase activity that is sensitive to DTT, suggesting the importance of the disulfide bonding between cysteine residues and that it might play a role in constitutive self-defense mechanisms in plants against biotic and abiotic stresses. JIOsPR10|OsPR10 Control of transposon activity by a histone H3K4 demethylase in rice 2013 Proc Natl Acad Sci U S A State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Transposable elements (TEs) are ubiquitously present in plant genomes and often account for significant fractions of the nuclear DNA. For example, roughly 40% of the rice genome consists of TEs, many of which are retrotransposons, including 14% LTR- and approximately 1% non-LTR retrotransposons. Despite their wide distribution and abundance, very few TEs have been found to be transpositional, indicating that TE activities may be tightly controlled by the host genome to minimize the potentially mutagenic effects associated with active transposition. Consistent with this notion, a growing body of evidence suggests that epigenetic silencing pathways such as DNA methylation, RNA interference, and H3K9me2 function collectively to repress TE activity at the transcriptional and posttranscriptional levels. It is not yet clear, however, whether the removal of histone modifications associated with active transcription is also involved in TE silencing. Here, we show that the rice protein JMJ703 is an active H3K4-specific demethylase required for TEs silencing. Impaired JMJ703 activity led to elevated levels of H3K4me3, the misregulation of numerous endogenous genes, and the transpositional reactivation of two families of non-LTR retrotransposons. Interestingly, loss of JMJ703 did not affect TEs (such as Tos17) previously found to be silenced by other epigenetic pathways. These results indicate that the removal of active histone modifications is involved in TE silencing and that different subsets of TEs may be regulated by distinct epigenetic pathways. JMJ703 Jumonji C domain protein JMJ705-mediated removal of histone H3 lysine 27 trimethylation is involved in defense-related gene activation in rice 2013 Plant Cell National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, 430070 Wuhan, China. Histone methylation is an important epigenetic modification in chromatin function, genome activity, and gene regulation. Dimethylated or trimethylated histone H3 lysine 27 (H3K27me2/3) marks silent or repressed genes involved in developmental processes and stress responses in plants. However, the role and the mechanism of the dynamic removal of H3K27me2/3 during gene activation remain unclear. Here, we show that the rice (Oryza sativa) Jumonji C (jmjC) protein gene JMJ705 encodes a histone lysine demethylase that specifically reverses H3K27me2/3. The expression of JMJ705 is induced by stress signals and during pathogen infection. Overexpression of the gene reduces the resting level of H3K27me2/3 resulting in preferential activation of H3K27me3-marked biotic stress-responsive genes and enhances rice resistance to the bacterial blight disease pathogen Xanthomonas oryzae pathovar oryzae. Mutation of the gene reduces plant resistance to the pathogen. Further analysis revealed that JMJ705 is involved in methyl jasmonate-induced dynamic removal of H3K27me3 and gene activation. The results suggest that JMJ705 is a biotic stress-responsive H3K27me2/3 demethylase that may remove H3K27me3 from marked defense-related genes and increase their basal and induced expression during pathogen infection. JMJ705 Rice jmjC domain-containing gene JMJ706 encodes H3K9 demethylase required for floral organ development 2008 Proc Natl Acad Sci U S A National Key Laboratory for Crop Genetic Improvement, Huazhong Agricultural University, 430070 Wuhan, China. Histone lysine methylation is an important epigenetic modification with both activating and repressive roles in gene expression. Jumonji C (jmjC) domain-containing proteins have been shown to reverse histone methylation in nonplant model systems. Here, we show that plant Jumonji C proteins have both conserved and specific features compared with mammalian homologues. In particular, the rice JMJD2 family jmjC gene JMJ706 is shown to encode a heterochromatin-enriched protein. The JMJ706 protein specifically reverses di- and trimethylations of lysine 9 of histone H3 (H3K9) in vitro. Loss-of-function mutations of the gene lead to increased di- and trimethylations of H3K9 and affect the spikelet development, including altered floral morphology and organ number. Gene expression and histone modification analysis indicates that JMJ706 regulates a subset of flower development regulatory genes. Taken together, our data suggest that rice JMJ706 encodes a heterochromatin-associated H3K9 demethylase involved in the regulation of flower development in rice. JMJ706 Crystallographic analysis reveals a unique conformation of the ADP-bound novel rice kinesin K16 2010 Biochem Biophys Res Commun Division of Bioinformatics, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan. Biochemical studies revealed that the novel rice plant-specific kinesin K16 has several unique enzymatic characteristics as compared to conventional kinesins. The ADP-free form of K16 is very stable, whereas the ADP-free form of conventional kinesins is labile. In the present study, the crystal structure of the novel rice kinesin motor domain (K16MD) complexed with Mg-ADP was determined at 2.4 A resolutions. The overall structure of K16MD is similar to that of conventional kinesin motor domains, as expected from the high amino acid sequence similarity (43.2%). However, several unique structures in K16 were observed. The position and length of the L5, L11, and L12 loops, which are key functional regions, were different from those observed in conventional kinesins. Moreover, the neck-linker region of the ADP-bound K16MD showed an ordered conformation at a position quite different from that previously observed in conventional kinesins. These structural differences may reflect the unique enzymatic characteristics of rice kinesin K16. K16 Preparation and characterization of a novel rice plant-specific kinesin 2006 J Biochem Laboratories of Plant and Microbial Genome Control, Graduate School of Science and Technology, Niigata University, Niigata 950-2181. Kinesin is an ATP-driven motor protein that plays important physiological roles in intracellular transport, mitosis and meiosis, control of microtubule dynamics, and signal transduction. The kinesin family is classified into subfamilies. Kinesin species derived from vertebrates have been well characterized. In contrast, plant kinesins have yet to be adequately characterized. In this study, we expressed the motor domain of a novel rice plant-specific kinesin, K16, in Escherichia coli, and then determined its enzymatic characteristics and compared them with those of kinesin 1. Our findings demonstrated that the rice kinesin motor domain has different enzymatic properties from those of well known kinesin 1. K16 Conformational change of the loop L5 in rice kinesin motor domain induced by nucleotide binding 2006 J Biochem Laboratories of Plant and Microbial Genome Control, Graduate School of Science and Technology, Niigata University, Niigata 950-2181. Loop L5 of kinesin is located near the ATPase site, in common with kinesins of various animal species. The rice plant-specific kinesin K16 also has a corresponding loop that is slightly shorter than that of mouse brain kinesin. The present study was designed to monitor conformational changes in loop L5 during ATP hydrolysis. For this purpose, we introduced one reactive cysteine into the L5 of rice kinesin and modified it with fluorescent probes. The cysteine in L5 was labeled with a fluorescent probe 2-(4'(iodoacetamide) anilino-naphthalene-6-sulfonic acid sodium salt) [IAANS]. IAANS was incorporated into L5 at an almost equimolar ratio in the absence of nucleotides. In contrast, the incorporated amount was reduced to 0.62 and 0.32 mol IAANS/mol motor domain in the presence of ATP and ADP, respectively. Upon nucleotide addition, the fluorescent intensity of IAANS incorporated into L5 was significantly reduced to 63% and 51% for ATP and ADP, respectively. These results suggest that L5 of rice kinesin significantly changes its conformation during ATP hydrolysis. K16 Ectopic expression of the K+ channel beta subunits from Puccinellia tenuiflora (KPutB1) and rice (KOB1) alters K+ homeostasis of yeast and Arabidopsis 2011 Mol Biotechnol Asian Natural Environmental Science Center (ANESC), The University of Tokyo, 1-1-1 Midori-cho, Nishitokyo-shi, Tokyo 188-0002, Japan. In this study, we cloned a cDNA for the K+ channel beta subunit from the halophyte Puccinellia tenuiflora and named it KPutB1. KPutB1 was preferentially expressed in the roots and was transiently induced by K+-starvation, salt stress, or the combination of both stresses. By yeast two-hybrid assay, we demonstrated that KPutB1 interacts with PutAKT1, alpha subunit of an AKT1-type K+ channel of P. tenuiflora. The functional relevance of this interaction on K+-nutrition was investigated by co-expression experiments in yeast under various ionic conditions, and K+ channel alpha and beta subunit homologues from rice (OsAKT1 and KOB1, respectively) were included for comparison. Yeast co-expressing PutAKT1 and the beta subunits (KPutB1 and KOB1) had better growth and higher K+-uptake ability than yeast expressing PutAKT1 alone. In contrast, yeast co-expressing the beta subunits (KPutB1 and KOB1) with OsAKT1 had slower growth and lower K+ uptake than yeast expressing OsAKT1 alone. Arabidopsis plants over-expressing the K+ channel beta subunit of P. tenuiflora or rice showed increased shoot K+ content and decreased root Na+ content under control, 75 mM NaCl, and K+-starvation stress conditions. These results suggest that ectopic expression of the K+ channel beta subunit could alter K+ and Na+ homeostasis in plants. KOB1 Molecular cloning and expression characterization of a rice K+ channel beta subunit 1998 Plant Mol Biol Plant Science Department, College of Agriculture and Natural Resources, University of Connecticut, Storrs 06269-4067, USA. K+ channel proteins native to animal membranes have been shown to be composed of two different types of polypeptides: the pore-forming alpha subunit and the beta subunit which may be involved in either modulation of conductance through the channel, or stabilization and surface expression of the channel complex. Several cDNAs encoding animal K+ channel beta subunits have been recently cloned and sequenced. We report the molecular cloning of a rice plant homolog of these animal beta subunits. The rice cDNA (KOB1) described in this report encodes a 36 kDa polypeptide which shares 45% sequence identity with these animal K+ channel beta subunits. and 72% identity with the only other cloned plant (Arabidopsis thaliana) K+ channel beta subunit (KAB1). The KOB1 translation product was demonstrated to form a tight physical association with a plant K+ channel alpha subunit. These results are consistent with the conclusion that the KOB1 cDNA encodes a K+ channel beta subunit. Expression studies indicated that KOB1 protein is more abundant in leaves than in either reproductive structures or roots. Later-developing leaves on a rice plant were found to contain increasing levels of the protein with the flag leaf having the highest titer of KOB1. Leaf sheaths are known to accumulate excess K+ and act as reserve sources of this cation when new growth requires remobilization of K+. Leaf sheaths were found to contain higher levels of KOB1 protein than the blade portions of leaves. It was further determined that when K+ was lost from older leaves of plants grown on K+-deficient fertilizer, the loss of cellular K+ was associated with a decline in both KOB1 mRNA and protein. This finding represents the first demonstration (in either plants or animals) that changes in cellular K+ status may specifically alter expression of a gene encoding a K+ channel subunit. KOB1 Phytochrome B control of total leaf area and stomatal density affects drought tolerance in rice 2012 Plant Mol Biol College of Life Sciences, Shandong Normal University, 250014 Jinan, People's Republic of China. We report that phytochrome B (phyB) mutants exhibit improved drought tolerance compared to wild type (WT) rice (Oryza sativa L. cv. Nipponbare). To understand the underlying mechanism by which phyB regulates drought tolerance, we analyzed root growth and water loss from the leaves of phyB mutants. The root system showed no significant difference between the phyB mutants and WT, suggesting that improved drought tolerance has little relation to root growth. However, phyB mutants exhibited reduced total leaf area per plant, which was probably due to a reduction in the total number of cells per leaf caused by enhanced expression of Orysa;KRP1 and Orysa;KRP4 (encoding inhibitors of cyclin-dependent kinase complex activity) in the phyB mutants. In addition, the developed leaves of phyB mutants displayed larger epidermal cells than WT leaves, resulting in reduced stomatal density. phyB deficiency promoted the expression of both putative ERECTA family genes and EXPANSIN family genes involved in cell expansion in leaves, thus causing greater epidermal cell expansion in the phyB mutants. Reduced stomatal density resulted in reduced transpiration per unit leaf area in the phyB mutants. Considering all these findings, we propose that phyB deficiency causes both reduced total leaf area and reduced transpiration per unit leaf area, which explains the reduced water loss and improved drought tolerance of phyB mutants. KRP1,PHYB|OsphyB The cyclin-dependent kinase inhibitor Orysa;KRP1 plays an important role in seed development of rice 2006 Plant Physiol Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, B-9052 Ghent, Belgium. Kip-related proteins (KRPs) play a major role in the regulation of the plant cell cycle. We report the identification of five putative rice (Oryza sativa) proteins that share characteristic motifs with previously described plant KRPs. To investigate the function of KRPs in rice development, we generated transgenic plants overexpressing the Orysa;KRP1 gene. Phenotypic analysis revealed that overexpressed KRP1 reduced cell production during leaf development. The reduced cell production in the leaf meristem was partly compensated by an increased cell size, demonstrating the existence of a compensatory mechanism in monocot species by which growth rate is less reduced than cell production, through cell expansion. Furthermore, Orysa;KRP1 overexpression dramatically reduced seed filling. Sectioning through the overexpressed KRP1 seeds showed that KRP overproduction disturbed the production of endosperm cells. The decrease in the number of fully formed seeds was accompanied by a drop in the endoreduplication of endosperm cells, pointing toward a role of KRP1 in connecting endocycle with endosperm development. Also, spatial and temporal transcript detection in developing seeds suggests that Orysa;KRP1 plays an important role in the exit from the mitotic cell cycle during rice grain formation. KRP1 Isolation and characterisation of a dwarf rice mutant exhibiting defective gibberellins biosynthesis 2014 Plant Biol (Stuttg) Department of Molecular Biotechnology, Konkuk University, Seoul, Korea. We have isolated a severe dwarf mutant derived from a Ds (Dissociation) insertion mutant rice (Oryza sativa var. japonica c.v. Dongjin). This severe dwarf phenotype, has short and dark green leaves, reduced shoot growth early in the seedling stage, and later severe dwarfism with failure to initiate flowering. When treated with bioactive GA3 , mutants are restored to the normal wild-type phenotype. Reverse transcription PCR analyses of 22 candidate genes related to the gibberellin (GA) biosynthesis pathway revealed that among 22 candidate genes tested, a dwarf mutant transcript was not expressed only in one OsKS2 gene. Genetic analysis revealed that the severe dwarf phenotype was controlled by recessive mutation of a single nuclear gene. The putative OsKS2 gene was a chromosome 4-located ent-kaurene synthase (KS), encoding the enzyme that catalyses an early step of the GA biosynthesis pathway. Sequence analysis revealed that osks2 carried a 1-bp deletion in the ORF region of OsKS2, which led to a loss-of-function mutation. The expression pattern of OsKS2 in wild-type cv Dongjin, showed that it is expressed in all organs, most prominently in the stem and floral organs. Morphological characteristics of the dwarf mutant showed dramatic modifications in internal structure and external morphology. We propose that dwarfism in this mutant is caused by a point mutation in OsKS2, which plays a significant role in growth and development of higher plants. Further investigation on OsKS2 and other OsKS-like proteins is underway and may yield better understanding of the putative role of OsKS in severe dwarf mutants. KS2 cDNA cloning of rice lipoxygenase L-2 and characterization using an active enzyme expressed from the cDNA in Escherichia coli 1992 European Journal of Biochemistry Mitsui Plant Biotechnology Research Institute, Tsukuba, Japan A full-length cDNA of rice lipoxygenase L-2 was cloned from 3-day old seedlings. The identity of the clone was determined by amino acid sequencing of selected peptides of the purified enzyme and immunological characterization of an active enzyme that was produced from the cDNA in Escherichia coli by cultivation at 15°C. The nucleotide sequence showed a strong bias toward G and C in the selection of nucleotides, especially at the third position of the codons(93%G/C). The complete amino acid sequence of the enzyme was deduced from the nucleotide sequence. The molecular mass of the enzyme was calculated to be 96657 Da based on 865 amino acids. The amino acid sequence shares similarity with those of dicot lipoxygenases throughout the enzyme at a level of 50%. A hydropathy profile calculated from the amino acid sequence resembled those of dicot lipoxygenases, suggesting conservation of the secondary structure of these enzymes. The active enzyme, expressed in Escherichia coli, was charaterized for pH dependence of the enzyme activity, intramolecular specificity, heat stability and Km. The enzyme had the same properties as the L-2 enzyme that was isolated from seedlings, but differed from the lipoxygenase L-3 isolated from mature plants. L-2 LAZY1 controls rice shoot gravitropism through regulating polar auxin transport 2007 Cell Res State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Tiller angle of rice (Oryza sativa L.) is an important agronomic trait that contributes to grain production, and has long attracted attentions of breeders for achieving ideal plant architecture to improve grain yield. Although enormous efforts have been made over the past decades to study mutants with extremely spreading or compact tillers, the molecular mechanism underlying the control of tiller angle of cereal crops remains unknown. Here we report the cloning of the LAZY1 (LA1) gene that regulates shoot gravitropism by which the rice tiller angle is controlled. We show that LA1, a novel grass-specific gene, is temporally and spatially expressed, and plays a negative role in polar auxin transport (PAT). Loss-of-function of LA1 enhances PAT greatly and thus alters the endogenous IAA distribution in shoots, leading to the reduced gravitropism, and therefore the tiller-spreading phenotype of rice plants. LA1 Over-expression of OsPIN2 leads to increased tiller numbers, angle and shorter plant height through suppression of OsLAZY1 2012 Plant Biotechnol J State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, China. Crop architecture parameters such as tiller number, angle and plant height are important agronomic traits that have been considered for breeding programmes. Auxin distribution within the plant has long been recognized to alter architecture. The rice (Oryza sativa L.) genome contains 12 putative PIN genes encoding auxin efflux transporters, including four PIN1 and one PIN2 genes. Here, we report that over-expression of OsPIN2 through a transgenic approach in rice (Japonica cv. Nipponbare) led to a shorter plant height, more tillers and a larger tiller angle when compared with wild type (WT). The expression patterns of the auxin reporter DR5::GUS and quantification of auxin distribution showed that OsPIN2 over-expression increased auxin transport from the shoot to the root-shoot junction, resulting in a non-tissue-specific accumulation of more free auxin at the root-shoot junction relative to WT. Over-expression of OsPIN2 enhanced auxin transport from shoots to roots, but did not alter the polar auxin pattern in the roots. Transgenic plants were less sensitive to N-1-naphthylphthalamic acid, an auxin transport inhibitor, than WT in their root growth. OsPIN2-over-expressing plants had suppressed the expression of a gravitropism-related gene OsLazy1 in the shoots, but unaltered expression of OsPIN1b and OsTAC1, which were reported as tiller angle controllers in rice. The data suggest that OsPIN2 has a distinct auxin-dependent regulation pathway together with OsPIN1b and OsTAC1 controlling rice shoot architecture. Altering OsPIN2 expression by genetic transformation can be directly used for modifying rice architecture. LA1,OsPIN2,TAC1 Identification of the gravitropism-related rice gene LAZY1 and elucidation of LAZY1-dependent and -independent gravity signaling pathways 2007 Plant Cell Physiol Botanical Gardens, Graduate School of Science, Osaka City University, Kisaichi, Katano-shi, Osaka, 576-0004 Japan. We identified the gene responsible for three allelic lazy1 mutations of Japonica rice (Oryza sativa L.) by map-based cloning, complementation and RNA interference. Sequence analysis and database searches indicated that the wild-type gene (LAZY1) encodes a novel and unique protein (LAZY1) and that rice has no homologous gene. Two lazy1 mutants were LAZY1 null. Confirming and advancing the previously reported results on lazy1 mutants, we found the following. (i) Gravitropism is impaired, but only partially, in lazy1 coleoptiles. (ii) Circumnutation, observed in dark-grown coleoptiles, is totally absent from lazy1 coleoptiles. (iii) Primary roots of lazy1 mutants show normal gravitropism and circumnutation. (iv) LAZY1 is expressed in a tissue-specific manner in gravity-sensitive shoot tissues (i.e. coleoptiles, leaf sheath pulvini and lamina joints) and is little expressed in roots. (v) The gravitropic response of lazy1 coleoptiles is kinetically separable from that absent from lazy1 coleoptiles. (vi) Gravity-induced lateral translocation of auxin, found in wild-type coleoptiles, does not occur in lazy1 coleoptiles. Based on the genetic and physiological evidence obtained, it is concluded that LAZY1 is specifically involved in shoot gravitropism and that LAZY1-dependent and -independent signaling pathways occur in coleoptiles. It is further concluded that, in coleoptiles, only the LAZY1-dependent gravity signaling involves asymmetric distribution of auxin between the two lateral halves and is required for circumnutation. LA1 Loose Plant Architecture1, an INDETERMINATE DOMAIN protein involved in shoot gravitropism, regulates plant architecture in rice 2013 Plant Physiol State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Tiller angle and leaf angle are two important components of rice (Oryza sativa) plant architecture that play a crucial role in determining grain yield. Here, we report the cloning and characterization of the Loose Plant Architecture1 (LPA1) gene in rice, the functional ortholog of the AtIDD15/SHOOT GRAVITROPISM5 (SGR5) gene in Arabidopsis (Arabidopsis thaliana). LPA1 regulates tiller angle and leaf angle by controlling the adaxial growth of tiller node and lamina joint. LPA1 was also found to affect shoot gravitropism. Expression pattern analysis suggested that LPA1 influences plant architecture by affecting the gravitropism of leaf sheath pulvinus and lamina joint. However, LPA1 only influences gravity perception or signal transduction in coleoptile gravitropism by regulating the sedimentation rate of amyloplasts, distinct from the actions of LAZY1. LPA1 encodes a plant-specific INDETERMINATE DOMAIN protein and defines a novel subfamily of 28 INDETERMINATE DOMAIN proteins with several unique conserved features. LPA1 is localized in the nucleus and functions as an active transcriptional repressor, an activity mainly conferred by a conserved ethylene response factor-associated amphiphilic repression-like motif. Further analysis suggests that LPA1 participates in a complicated transcriptional and protein interaction network and has evolved novel functions distinct from SGR5. This study not only facilitates the understanding of gravitropism mechanisms but also generates a useful genetic material for rice breeding. LA1,LPA1 The LAX1 and FRIZZY PANICLE 2 genes determine the inflorescence architecture of rice by controlling rachis-branch and spikelet development 2001 Dev Biol Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, 630-0101, Japan We have analyzed two mutants that exhibit altered panicle architecture in rice (Oryza sativa L.). In lax1-2, which is a new and stronger allele of the previously reported lax mutant, initiation and/or maintenance of rachis-branches, lateral spikelets, and terminal spikelets was severely prevented. In situ hybridization analysis using OSH1, a rice knotted1 (kn1) ortholog, confirmed the absence of lateral meristems in lax1-2 panicles. These defects indicate that the LAX1 gene is required for the initiation/maintenance of axillary meristems in the rice panicle. In addition to its role in forming lateral meristems, the wild-type LAX1 gene acts as a floral meristem identity gene which specifies the terminal spikelet meristem. A comparison of the defects in lax1-1 and lax1-2 plants suggested that the sensitivities to reduced LAX1 activity were not uniform among different types of meristems. In the fzp2 mutant panicle, the basic branching pattern of the panicle was indistinguishable from that of the wild type; however, specification of both terminal and lateral spikelet meristems was blocked, and sequential rounds of branching occurred at the point where the spikelet meristems are initiated in the wild-type panicle. This resulted in the generation of a panicle composed of excessive ramification of rachis-branches. The lax1-1 fzp2 double mutants exhibited a novel, basically additive, phenotype, which suggests that LAX1 and FZP2 function in genetically independent pathways. LAX1 Two-Step Regulation of LAX PANICLE1 Protein Accumulation in Axillary Meristem Formation in Rice 2009 Plant Cell Graduate School of Agriculture and Life Sciences, University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan. Axillary meristem (AM) formation is an important determinant of plant architecture. In rice (Oryza sativa), LAX PANICLE1 (LAX1) function is required for the generation of AM throughout the plant's lifespan. Here, we show a close relationship between AM initiation and leaf development; specifically, the plastochron 4 (P4) stage of leaf development is crucial for the proliferation of meristematic cells. Coincident with this, LAX1 expression starts in the axils of leaves at P4 stage. LAX1 mRNA accumulates in two to three layers of cells in the boundary region between the initiating AM and the shoot apical meristem. In lax1 mutants, the proliferation of meristematic cells is initiated but fails to progress into the formation of AM. The difference in sites of LAX1 mRNA expression and its action suggests non-cell-autonomous characteristics of LAX1 function. We found that LAX1 protein is trafficked to AM in a stage- and direction-specific manner. Furthermore, we present evidence that LAX1 protein movement is required for the full function of LAX1. Thus, we propose that LAX1 protein accumulates transiently in the initiating AM at P4 stage by a strict regulation of mRNA expression and a subsequent control of protein trafficking. This two-step regulation is crucial to the establishment of the new AM. LAX1 LAX PANICLE2 of rice encodes a novel nuclear protein and regulates the formation of axillary meristems 2011 Plant Cell Crop Development Division, National Agriculture and Food Research Organization Agricultural Research Center, Niigata 943-0193, Japan. Aerial architecture in higher plants is dependent on the activity of the shoot apical meristem (SAM) and axillary meristems (AMs). The SAM produces a main shoot and leaf primordia, while AMs are generated at the axils of leaf primordia and give rise to branches and flowers. Therefore, the formation of AMs is a critical step in the construction of plant architecture. Here, we characterized the rice (Oryza sativa) lax panicle2 (lax2) mutant, which has altered AM formation. LAX2 regulates the branching of the aboveground parts of a rice plant throughout plant development, except for the primary branch in the panicle. The lax2 mutant is similar to lax panicle1 (lax1) in that it lacks an AM in most of the lateral branching of the panicle and has a reduced number of AMs at the vegetative stage. The lax1 lax2 double mutant synergistically enhances the reduced-branching phenotype, indicating the presence of multiple pathways for branching. LAX2 encodes a nuclear protein that contains a plant-specific conserved domain and physically interacts with LAX1. We propose that LAX2 is a novel factor that acts together with LAX1 in rice to regulate the process of AM formation. LAX1,Gnp4|LAX2 LAX and SPA: major regulators of shoot branching in rice 2003 Proc Natl Acad Sci U S A Graduate School of Agriculture and Life Science, University of Tokyo, Yayoi 1-1-1, Bunkyo, Tokyo 113-8657, Japan. The aerial architecture of plants is determined primarily by the pattern of shoot branching. Although shoot apical meristem initiation during embryogenesis has been extensively studied by molecular genetic approaches using Arabidopsis, little is known about the genetic mechanisms controlling axillary meristem initiation, mainly because of the insufficient number of mutants that specifically alter it. We identified the LAX PANICLE (LAX) and SMALL PANICLE (SPA) genes as the main regulators of axillary meristem formation in rice. LAX encodes a basic helix-loop-helix transcription factor and is expressed in the boundary between the shoot apical meristem and the region of new meristem formation. This pattern of LAX expression was repeatedly observed in every axillary meristem, consistent with our observation that LAX is involved in the formation of all types of axillary meristems throughout the ontogeny of a rice plant. Ectopic LAX expression in rice caused pleiotropic effects, including dwarfing, an altered pattern of stem elongation, darker color, bending of the lamina joint, absence of the midribs of leaves, and severe sterility. LAX1 Fine Mapping and Cloning of the Grain Number Per-Panicle Gene (Gnp4) on Chromosome 4 in Rice (Oryza sativa L.) 2011 Agricultural Sciences in China Key Laboratory of Crop Heterosis and Utilization, Ministry of Agriculture/Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing 100193, P.R.China Grain number per-panicle is one of the most important components for rice yield. Spikelets on the primary and secondary branches determine the grain number per-panicle in rice. In this study, we identified a natural mutant, gnp4, lack of lateral spikelet on the secondary branches in the field condition. In addition, the Gnp4 and Lax1-1 double mutant showed dramatically reduced secondary branches and spikelets in panicle at reproductive stage, and tillers at vegetative stage. By map-based cloning approach, and using four F2 segregating populations, the Gnp4 gene was finally mapped to a 10.7-kb region on the long arm of chromosome 4 in rice. In this region, only one gene was predicted, and genomic DNA sequencing of the 10.7-kb region showed no nucleotide differences between the mutant and wild type. Interestingly, we found that the methylation level of several cytosines in the promoter CpG islands region of the predicted gene in gnp4 were different from the wild type. Thus, we propose that the DNA methylation changes at these sites may induce to decrease expression level of Gnp4, consequently, resulting in phenotypic variation. Gnp4|LAX2 Rice leaf inclination2, a VIN3-like protein, regulates leaf angle through modulating cell division of the collar 2010 Cell Res Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China. As an important agronomic trait, inclination of leaves is crucial for crop architecture and grain yields. To understand the molecular mechanism controlling rice leaf angles, one rice leaf inclination2 (lc2, three alleles) mutant was identified and functionally characterized. Compared to wild-type plants, lc2 mutants have enlarged leaf angles due to increased cell division in the adaxial epidermis of lamina joint. The LC2 gene was isolated through positional cloning, and encodes a vernalization insensitive 3-like protein. Complementary expression of LC2 reversed the enlarged leaf angles of lc2 plants, confirming its role in controlling leaf inclination. LC2 is mainly expressed in the lamina joint during leaf development, and particularly, is induced by the phytohormones abscisic acid, gibberellic acid, auxin, and brassinosteroids. LC2 is localized in the nucleus and defects of LC2 result in altered expression of cell division and hormone-responsive genes, indicating an important role of LC2 in regulating leaf inclination and mediating hormone effects. LC2|OsVIL3,OsVIL4 LC2 and OsVIL2 promote rice flowering by photoperoid-induced epigenetic silencing of OsLF 2013 Mol Plant National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese academy of Sciences, Shanghai 200032, PR China. Proper flowering time is essential for plant reproduction. Winter annual Arabidopsis thaliana needs vernalization before flowering, during which AtVILs (VIN3 and VRN5, components of PRC2 complex) mediate the H3K27 tri-methylation at the FLC locus (a floral repressor) to repress the FLC expression and hence to induce flowering. However, how VILs (VIL, VERNALIZATION INSENSITIVE 3-LIKE) function in rice is unknown. Here we demonstrated that rice LC2 (OsVIL3) and OsVIL2 (two OsVILs, possible components of PRC2 complex) promote rice flowering. Our results showed that expressions of LC2 and OsVIL2 are induced by SD (short-day) conditions and both lc2 mutant and OsVIL2-RNAi lines display delayed heading date, consistent with the reduced expression levels of Hd1 and Hd3a. Interestingly, LC2 binds to the promoter region of a floral repressor OsLF and represses the OsLF expression via H3K27 tri-methylation modification. In addition, OsLF directly regulates the Hd1 expression through binding to Hd1 promoter. These results first demonstrated that the putative PRC2 in rice is involved in photoperiod flowering regulation, which is different from that of Arabidopsis, and revealed that LC2 binds the promoter region of target gene, presenting a possible mechanism of the recruitment process of PRC2 complex to its target genes. The studies provide informative clues on the epigenetic control of rice flowering. LC2|OsVIL3,OsLF,OsVIL1,OsVIL2,OsVIL4 Rice LGD1 containing RNA binding activity affects growth and development through alternative promoters 2012 Plant J Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan. Tiller initiation and panicle development are important agronomical traits for grain production in Oryza sativa L. (rice), but their regulatory mechanisms are not yet fully understood. In this study, T-DNA mutant and RNAi transgenic approaches were used to functionally characterize a unique rice gene, LAGGING GROWTH AND DEVELOPMENT 1 (LGD1). The lgd1 mutant showed slow growth, reduced tiller number and plant height, altered panicle architecture and reduced grain yield. The fewer unelongated internodes and cells in lgd1 led to respective reductions in tiller number and to semi-dwarfism. Several independent LGD1-RNAi lines exhibited defective phenotypes similar to those observed in lgd1. Interestingly, LGD1 encodes multiple transcripts with different transcription start sites (TSSs), which were validated by RNA ligase-mediated rapid amplification of 5' and 3' cDNA ends (RLM-RACE). Additionally, GUS assays and a luciferase promoter assay confirmed the promoter activities of LGD1.1 and LGD1.5. LGD1 encoding a von Willebrand factor type A (vWA) domain containing protein is a single gene in rice that is seemingly specific to grasses. GFP-tagged LGD1 isoforms were predominantly detected in the nucleus, and weakly in the cytoplasm. In vitro northwestern analysis showed the RNA-binding activity of the recombinant C-terminal LGD1 protein. Our results demonstrated that LGD1 pleiotropically regulated rice vegetative growth and development through both the distinct spatiotemporal expression patterns of its multiple transcripts and RNA binding activity. Hence, the study of LGD1 will strengthen our understanding of the molecular basis of the multiple transcripts, and their corresponding polypeptides with RNA binding activity, that regulate pleiotropic effects in rice. LGD1 Low-temperature-dependent expression of a rice gene encoding a protein with a leucine-zipper motif 1993 Mol Gen Genet Laboratory of Plant Genetic Engineering, Akita Prefectural College of Agriculture, Japan. We have isolated from rice suspension cells three non-sequence-related cDNAs the expression of which is markedly induced by low, non-freezing temperature. Here we further characterize one of the cDNA clones, lip19. Expression of lip19 is positively regulated by low temperature, but not affected by high (40 degrees C) temperature. Sequencing and primer extension analyses showed that lip19 has a long (552 bp) 5' non-coding sequence followed by a single open reading frame specifying a protein of 148 amino acids. The deduced amino acid sequence of the protein, Lip19, shows at its amino-terminus a conserved basic region followed by a "leucine-zipper" domain. The reported sequence most similar to Lip19 is maize OCSBF-1, which is a bZip-type DNA binding protein. The possibility is suggested that Lip19 is a transcriptional factor that is positively controlled by low temperature. LIP19|OsbZIP38 Genome-wide analysis of basic leucine zipper transcription factor families in Arabidopsis thaliana, Oryza sativa and Populus trichocarpa 2009 Journal of Shanghai University (English Edition) School of Life Sciences, Shanghai University, Shanghai, 200444, P. R. China The basic leucine zipper (bZIP) transcription factors form a large gene family that is important in pathogen defense, light and stress signaling, etc. The Completed whole genome sequences of model plants Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa) and poplar (Populus trichocarpa) constitute a valuable resource for genome-wide analysis and genomic comparative analysis, as they are representatives of the two major evolutionary lineages within the angiosperms: the monocotyledons and the dicotyledons. In this study, bioinformatics analysis identified 74, 89 and 88 bZIP genes respectively in Arabidopsis, rice and poplar. Moreover, a comprehensive overview of this gene family is presented, including the gene structure, phylogeny, chromosome distribution, conserved motifs. As a result, the plant bZIPs were organized into 10 subfamilies on basis of phylogenetic relationship. Gene duplication events during the family evolution history were also investigated. And it was further concluded that chromosomal/segmental duplication might have played a key role in gene expansion of bZIP gene family. LIP19|OsbZIP38,OsABF1|OsABI5|OREB1|OsbZIP10,OsbZIP01,OsbZIP16,OsbZIP23,OsbZIP28|OsbZIP1,OsbZIP39,OsbZIP50|OsbZIP74,OsbZIP52|RISBZ5,OsbZIP60,OsbZIP71,OsbZIP72,OsOBF1|OsbZIP87,OsTGAP1|OsbZIP37,OsZIP-1a|OsbZIP86,OsZIP-2a|OsbZIP80,RF2a|OsbZIP75,RF2b|OsbZIP30 Genomic survey and gene expression analysis of the basic leucine zipper transcription factor family in rice 2008 Plant Physiol Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi 110021, India. The basic leucine (Leu) zipper (bZIP) proteins compose a family of transcriptional regulators present exclusively in eukaryotes. The bZIP proteins characteristically harbor a bZIP domain composed of two structural features: a DNA-binding basic region and the Leu zipper dimerization region. They have been shown to regulate diverse plant-specific phenomena, including seed maturation and germination, floral induction and development, and photomorphogenesis, and are also involved in stress and hormone signaling. We have identified 89 bZIP transcription factor-encoding genes in the rice (Oryza sativa) genome. Their chromosomal distribution and sequence analyses suggest that the bZIP transcription factor family has evolved via gene duplication. The phylogenetic relationship among rice bZIP domains as well as with bZIP domains from other plant bZIP factors suggests that homologous bZIP domains exist in plants. Similar intron/exon structural patterns were observed in the basic and hinge regions of their bZIP domains. Detailed sequence analysis has been done to identify additional conserved motifs outside the bZIP domain and to predict their DNA-binding site specificity as well as dimerization properties, which has helped classify them into different groups and subfamilies, respectively. Expression of bZIP transcription factor-encoding genes has been analyzed by full-length cDNA and expressed sequence tag-based expression profiling. This expression profiling was complemented by microarray analysis. The results indicate specific or coexpression patterns of rice bZIP transcription factors starting from floral transition to various stages of panicle and seed development. bZIP transcription factor-encoding genes in rice also displayed differential expression patterns in rice seedlings in response to abiotic stress and light irradiation. An effort has been made to link the structure and expression pattern of bZIP transcription factor-encoding genes in rice to their function, based on the information obtained from our analyses and earlier known results. This information will be important for functional characterization of bZIP transcription factors in rice. LIP19|OsbZIP38,OsAREB8|OsAREB1|OsbZIP46|OsABF2|ABL1,OSBZ8|OsbZIP05,OsbZIP01,OsbZIP16,OsbZIP23,OsbZIP28|OsbZIP1,OsbZIP39,OsbZIP50|OsbZIP74,OsbZIP52|RISBZ5,OsbZIP60,OsbZIP71,OsbZIP72,OsOBF1|OsbZIP87,OsTGAP1|OsbZIP37,OsZIP-1a|OsbZIP86,OsZIP-2a|OsbZIP80,REB,RF2a|OsbZIP75,RF2b|OsbZIP30,RISBZ1|OsbZIP58,RSG,rTGA2.1|OsbZIP63|OsNIF1,TRAB1|OsbZIP66,RITA1 LIP19, a basic region leucine zipper protein, is a Fos-like molecular switch in the cold signaling of rice plants 2005 Plant Cell Physiol Graduate School of Life Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan. The rice low-temperature-induced lip19 gene encodes a 148-amino-acid basic region/leucine zipper (bZIP) protein, termed LIP19. In this study we characterized LIP19 and showed that it lacks the usual ability of bZIP proteins to homodimerize and to bind DNA, as does the Fos protein in mammals. Using a yeast two-hybrid system, the cDNA clones whose products interact with LIP19 were screened. This search revealed a clone termed OsOBF1 (Oryza sativa OBF1) that encodes a new bZIP protein (OsOBF1). This protein forms a homodimer and binds to the hexamer motif sequence (5'-ACGTCA-3'). The protein-protein interaction in homo- and hetero-combinations between LIP19 and OsOBF1 was confirmed in vitro and in planta. LIP19 and OsOBF1 most likely interact with each other more strongly than OsOBF1 interacts with itself, and the resulting heterodimer binds to the C/G hybrid sequence but not to the hexamer sequence. Whereas the expression patterns of lip19 and OsOBF1 in response to low temperatures were totally opposite, the locations of their expression were almost identical. Based upon the presented data, we propose a model describing the low-temperature signal switching mediated by LIP19 in rice. LIP19|OsbZIP38,OsOBF1|OsbZIP87 Circadian rhythmicity in the expression of a novel light-regulated rice gene 1993 Plant Mol Biol Institute of Plant Biology, University of Zurich, Switzerland. We have identified and analyzed cDNAs corresponding to a single-copy gene from rice, designated lir1, whose expression exhibits dramatic diurnal fluctuations. The cDNAs encode a putative protein of 128 amino acids with no homology to known proteins. Lir1 mRNA accumulates in the light, reaching maximum and minimum steady-state levels at the end of the light and dark period, respectively. The oscillations of lir1 mRNA abundance persist after the plants have been transferred to continuous light or darkness. Plants germinated in the dark have very low levels of lir1 mRNA, whereas plants germinated in continuous light express lir1 at an intermediate but constant level. These results indicate that lir1 expression is controlled by light and a circadian clock. Lir1 Direct control of shoot meristem activity by a cytokinin-activating enzyme 2007 Nature Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8652, Japan. The growth of plants depends on continuous function of the meristems. Shoot meristems are responsible for all the post-embryonic aerial organs, such as leaves, stems and flowers. It has been assumed that the phytohormone cytokinin has a positive role in shoot meristem function. A severe reduction in the size of meristems in a mutant that is defective in all of its cytokinin receptors has provided compelling evidence that cytokinin is required for meristem activity. Here, we report a novel regulation of meristem activity, which is executed by the meristem-specific activation of cytokinins. The LONELY GUY (LOG) gene of rice is required to maintain meristem activity and its loss of function causes premature termination of the shoot meristem. LOG encodes a novel cytokinin-activating enzyme that works in the final step of bioactive cytokinin synthesis. Revising the long-held idea of multistep reactions, LOG directly converts inactive cytokinin nucleotides to the free-base forms, which are biologically active, by its cytokinin-specific phosphoribohydrolase activity. LOG messenger RNA is specifically localized in shoot meristem tips, indicating the activation of cytokinins in a specific developmental domain. We propose the fine-tuning of concentrations and the spatial distribution of bioactive cytokinins by a cytokinin-activating enzyme as a mechanism that regulates meristem activity. LOG LOX genes in blast fungus (Magnaporthe grisea) resistance in rice 2012 Funct Integr Genomics Molecular Biology and Genetic Engineering, G.B. Pant University of Agriculture and Technology, Pantnagar, India. soma.marla@nbpgr.ernet.in Plant Lipoxygenases (LOX) are known to play major role in plant immunity by providing front-line defense against pathogen-induced injury. To verify this, we isolated a full-length OsLOX3 gene and also 12 OsLOX cDNA clones from Oryza sativa indica (cultivar Pusa Basmati 1). We have examined the role played by LOXs in plant development and during attack by blast pathogen Magnaporthe grisea. Gene expression, promoter region analysis, and biochemical and protein structure analysis of isolated OsLOX3 revealed significant homology with LOX super family. Protein sequence comparison of OsLOXs revealed high levels of homology when compared with japonica rice (up to100%) and Arabidopsis (up to 64%). Isolated LOX3 gene and 12 OsLOX cDNAs contained the catalytic LOX domains much required for oxygen binding and synthesis of oxylipins. Amino acid composition, protein secondary structure, and promoter region analysis (with abundance of motifs CGTCA and TGACG) support the role of OsLOX3 gene in providing resistance to diseases in rice plants. OsLOX3 gene expression analysis of root, shoot, flag leaf, and developing and mature seed revealed organ specific patterns during rice plant development and gave evidence to association between tissue location and physiological roles played by individual OsLOXs. Increased defense activity of oxylipins was observed as demonstrated by PCR amplification of OsLOX3 gene and upon inoculation with virulent strains of M. grisea and ectopic application of methyl jasmonate in the injured leaf tissue in adult rice plants. OsLOX3 Map-based cloning of the ERECT PANICLE 3 gene in rice 2009 Theor Appl Genet Department of Plant Science, Plant Genomics and Breeding Institute, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, 151-921, Republic of Korea. Panicle architecture in rice can have a strong influence on yield. Using N-methyl-N-nitrosourea mutagenesis, we isolated an erect panicle mutant, Hep, from Hwasunchalbyeo, a glutinous japonica rice cultivar. Genetic analysis revealed that the erect panicle phenotype was controlled by a single recessive mutation designated erect panicle 3 (ep3). Genetic mapping revealed that the ep3 mutation was located on the short arm of chromosome 2 in a 0.1 cM region delimited by the STS markers STS5803-5 and STS5803-7. The ep3 locus corresponded to 46.8 kb region and contained six candidate genes. Comparison of the DNA sequences of the candidate genes from wild-type and erect panicle plants revealed a single base-pair change in the second exon of LOC_Os02g15950, which is predicted to result in a nonsense mutation. LOC_Os02g15950 encodes a putative F-box protein containing 515 amino acids and is expressed throughout the plant during all growth stages. A line carrying a T-DNA insertion in LOC_ Os02g15950 was obtained and shown to have the same phenotype as the ep3 mutant, thus confirming the identification of LOC_Os02g15950 as the ERECT PANICLE 3 (EP3) gene. The ep3 mutation causes a significant increase in the number of small vascular bundles as well as the thickness of parenchyma in the peduncle, which results in the erect panicle phenotype. EP3|LP The LP2 leucine-rich repeat receptor kinase gene promoter directs organ-specific, light-responsive expression in transgenic rice 2009 Plant Biotechnol J USDA-ARS, Western Regional Research Center, Crop Improvement and Utilization Research Unit, Albany, CA, USA. roger.thilmony@ars.usda.gov Biotechnologists seeking to limit gene expression to nonseed tissues of genetically engineered cereal crops have only a few choices of well characterized organ-specific promoters. We have isolated and characterized the promoter of the rice Leaf Panicle 2 gene (LP2, Os02g40240). The LP2 gene encodes a leucine-rich repeat-receptor kinase-like protein that is strongly expressed in leaves and other photosynthetic tissues. Transgenic rice plants containing an LP2 promoter-GUS::GFP bifunctional reporter gene displayed an organ-specific pattern of expression. This expression corresponded to transcript levels observed on RNA blots of various rice organs and microarray gene expression data. The strongest beta-glucuronidase activity was observed in histochemically stained mesophyll cells, but other green tissues and leaf cell types including epidermal cells also exhibited expression. Low or undetectable levels of LP2 transcript and LP2-mediated reporter gene expression were observed in roots, mature seeds, and reproductive tissues. The LP2 promoter is highly responsive to light and only weak expression was detected in etiolated rice seedlings. The specificity and strength of the LP2 promoter suggests that this promoter will be a useful control element for green tissue-specific expression in rice and potentially other plants. Organ-specific promoters like LP2 will enable precise, localized expression of transgenes in biotechnology-derived crops and limit the potential of unintended impacts on plant physiology and the environment. LP2 Over-expression of the rice LRK1 gene improves quantitative yield components 2009 Plant Biotechnol J State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China. In rice (Oryza sativa L.), the number of panicles, spikelets per panicle and grain weight are important components of grain yield. These characteristics are controlled by quantitative trait loci (QTLs) and are derived from variation inherent in crops. As a result of the complex genetic basis of these traits, only a few genes involved in their control have been cloned and characterized. We have previously map-cloned a gene cluster including eight leucine-rich repeat receptor-like kinase (LRK) genes in Dongxiang wild rice (Oryza rufipogon Griff.), which increased the grain yield by 16%. In the present study, we characterized the LRK1 gene, which was contained in the donor parent (Dongxiang wild rice) genome and absent from the recurrent parent genome (Guichao2, Oryza sativa L. ssp. indica). Our data showed that rice LRK1 is a plasma membrane protein expressed constitutively in leaves, young panicles, roots and culms. The over-expression of rice LRK1 results in increased panicles, spikelets per panicle, weight per grain and enhanced cellular proliferation, leading to a 27.09% increase in total grain yield per plant. The increased number of panicles and spikelets per panicle are associated with increased branch number. Our data suggest that rice LRK1 regulates rice branch number by enhancing cellular proliferation. The functional characterization of rice LRK1 facilitates an understanding of the mechanisms involved in cereal crop yield, and may have utility in improving grain yield in cereal crops. LRK1 Overexpression of rice LRK1 restricts internode elongation by down-regulating OsKO2 2013 Biotechnol Lett State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, China. banmatus@gmail.com Rice (Oryza sativa) has the potential to undergo rapid internodal elongation which determines plant height. Gibberellin is involved in internode elongation. Leucine-rich repeat receptor-like kinases (LRR-RLKs) are the largest subfamily of transmembrane receptor-like kinases in plants. LRR-RLKs play important functions in mediating a variety of cellular processes and regulating responses to environmental signals. LRK1, a PSK receptor homolog, is a member of the LRR-RLK family. In the present study, differences in ectopic expression of LRK1 were consistent with extent of rice internode elongation. Analyses of gene expression demonstrated that LRK1 restricts gibberellin biosynthesis during the internode elongation process by down-regulation of the gibberellin biosynthetic gene coding for ent-kaurene oxidase. LRK1,D35|OsKOS3|OsKO2 The role of the rice aquaporin Lsi1 in arsenite efflux from roots 2010 New Phytol Rothamsted Research, Harpenden, Herts AL5 2JQ, UK. *When supplied with arsenate (As(V)), plant roots extrude a substantial amount of arsenite (As(III)) to the external medium through as yet unidentified pathways. The rice (Oryza sativa) silicon transporter Lsi1 (OsNIP2;1, an aquaporin channel) is the major entry route of arsenite into rice roots. Whether Lsi1 also mediates arsenite efflux was investigated. *Expression of Lsi1 in Xenopus laevis oocytes enhanced arsenite efflux, indicating that Lsi1 facilitates arsenite transport bidirectionally. *Arsenite was the predominant arsenic species in arsenate-exposed rice plants. During 24-h exposure to 5 mum arsenate, rice roots extruded arsenite to the external medium rapidly, accounting for 60-90% of the arsenate uptake. A rice mutant defective in Lsi1 (lsi1) extruded significantly less arsenite than the wild-type rice and, as a result, accumulated more arsenite in the roots. By contrast, Lsi2 mutation had little effect on arsenite efflux to the external medium. *We conclude that Lsi1 plays a role in arsenite efflux in rice roots exposed to arsenate. However, this pathway accounts for only 15-20% of the total efflux, suggesting the existence of other efflux transporters. Lsi1|OsNIP2;1,Lsi2 A silicon transporter in rice 2006 Nature Research Institute for Bioresources, Okayama University, Chuo 2-20-1, Kurashiki 710-0046, Japan. maj@rib.okayama-u.ac.jp Silicon is beneficial to plant growth and helps plants to overcome abiotic and biotic stresses by preventing lodging (falling over) and increasing resistance to pests and diseases, as well as other stresses. Silicon is essential for high and sustainable production of rice, but the molecular mechanism responsible for the uptake of silicon is unknown. Here we describe the Low silicon rice 1 (Lsi1) gene, which controls silicon accumulation in rice, a typical silicon-accumulating plant. This gene belongs to the aquaporin family and is constitutively expressed in the roots. Lsi1 is localized on the plasma membrane of the distal side of both exodermis and endodermis cells, where casparian strips are located. Suppression of Lsi1 expression resulted in reduced silicon uptake. Furthermore, expression of Lsi1 in Xenopus oocytes showed transport activity for silicon only. The identification of a silicon transporter provides both an insight into the silicon uptake system in plants, and a new strategy for producing crops with high resistance to multiple stresses by genetic modification of the root's silicon uptake capacity. Lsi1|OsNIP2;1 An efflux transporter of silicon in rice 2007 Nature Research Institute for Bioresources, Okayama University, Chuo 2-20-1, Kurashiki 710-0046, Japan. maj@rib.okayama-u.ac.jp Silicon is an important nutrient for the optimal growth and sustainable production of rice. Rice accumulates up to 10% silicon in the shoot, and this high accumulation is required to protect the plant from multiple abiotic and biotic stresses. A gene, Lsi1, that encodes a silicon influx transporter has been identified in rice. Here we describe a previously uncharacterized gene, low silicon rice 2 (Lsi2), which has no similarity to Lsi1. This gene is constitutively expressed in the roots. The protein encoded by this gene is localized, like Lsi1, on the plasma membrane of cells in both the exodermis and the endodermis, but in contrast to Lsi1, which is localized on the distal side, Lsi2 is localized on the proximal side of the same cells. Expression of Lsi2 in Xenopus oocytes did not result in influx transport activity for silicon, but preloading of the oocytes with silicon resulted in a release of silicon, indicating that Lsi2 is a silicon efflux transporter. The identification of this silicon transporter revealed a unique mechanism of nutrient transport in plants: having an influx transporter on one side and an efflux transporter on the other side of the cell to permit the effective transcellular transport of the nutrients. Lsi1|OsNIP2;1,Lsi2 Genotypic difference in silicon uptake and expression of silicon transporter genes in rice 2007 Plant Physiol Research Institute for Bioresources, Okayama University, Kurashiki 710-0046, Japan. maj@rib.okayama-u.ac.jp Rice (Oryza sativa) is a highly silicon (Si)-accumulating species that shows genotypic differences in Si accumulation. We investigated the physiological and molecular mechanisms involved in the genotypic difference in Si uptake between the japonica var. Nipponbare and the indica var. Kasalath. Both the Si concentration in the shoot and the Si uptake per root dry weight were higher in Nipponbare than in Kasalath grown in either soil or nutrient solution. The Si uptake by a single root was also higher in Nipponbare than in Kasalath. A kinetics study showed that Nipponbare and Kasalath had a similar K(m) value, whereas the V(max) was higher in Nipponbare. The expression of two Si transporter genes (Low silicon rice 1 [Lsi1] and Lsi2) investigated using real-time reverse transcription polymerase chain reaction revealed higher expression of both genes in Nipponbare than in Kasalath. Immunostaining with Lsi1 and Lsi2 antibodies revealed a similar pattern of subcellular localization of these two Si transporters in both varieties; Lsi1 and Lsi2 were localized at the distal and proximal sides, respectively, of both exodermis and endodermis of the roots. These results revealed that the genotypic difference in the Si accumulation results from the difference in abundance of Si transporters in rice roots. Lsi1|OsNIP2;1,Lsi2 Spatial distribution and temporal variation of the rice silicon transporter Lsi1 2007 Plant Physiol Research Institute for Bioresources, Okayama University, Kurashiki 710-0046, Japan. Rice (Oryza sativa) is a typical silicon (Si) accumulator and requires a large amount of Si for high-yield production. Recently, a gene (Low silicon rice1 [Lsi1]) encoding a Si transporter was identified in rice roots. Here, we characterized Lsi1 in terms of spatial distribution and temporal variation using both physiological and molecular approaches. Results from a multicompartment transport box experiment showed that the major site for Si uptake was located at the basal zone (>10 mm from the root tip) of the roots rather than at the root tips (<10 mm from the root tip). Consistent with the Si uptake pattern, Lsi1 expression and distribution of the Lsi1 protein were found only in the basal zone of roots. In the basal zones of the seminal, crown, and lateral roots, the Lsi1 protein showed a polar localization at the distal side of both the exodermis and endodermis, where the Casparian bands are formed. This indicates that Lsi1 is required for the transport of Si through the cells of the exodermis and endodermis. Expression of Lsi1 displayed a distinct diurnal pattern. Furthermore, expression was transiently enhanced around the heading stage, which coincides with a high Si requirement during this growth stage. Expression was down-regulated by dehydration stress and abscisic acid, suggesting that expression of Lsi1 may be regulated by abscisic acid. Lsi1|OsNIP2;1 The rice aquaporin Lsi1 mediates uptake of methylated arsenic species 2009 Plant Physiol Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, United Kingdom. Pentavalent methylated arsenic (As) species such as monomethylarsonic acid [MMA(V)] and dimethylarsinic acid [DMA(V)] are used as herbicides or pesticides, and can also be synthesized by soil microorganisms or algae through As methylation. The mechanism of MMA(V) and DMA(V) uptake remains unknown. Recent studies have shown that arsenite is taken up by rice (Oryza sativa) roots through two silicon transporters, Lsi1 (the aquaporin NIP2;1) and Lsi2 (an efflux carrier). Here we investigated whether these two transporters also mediate the uptake of MMA(V) and DMA(V). MMA(V) was partly reduced to trivalent MMA(III) in rice roots, but only MMA(V) was translocated to shoots. DMA(V) was stable in plants. The rice lsi1 mutant lost about 80% and 50% of the uptake capacity for MMA(V) and DMA(V), respectively, compared with the wild-type rice, whereas Lsi2 mutation had little effect. The short-term uptake kinetics of MMA(V) can be described by a Michaelis-Menten plus linear model, with the wild type having 3.5-fold higher V(max) than the lsi1 mutant. The uptake kinetics of DMA(V) were linear with the slope being 2.8-fold higher in the wild type than the lsi1 mutant. Heterologous expression of Lsi1 in Xenopus laevis oocytes significantly increased the uptake of MMA(V) but not DMA(V), possibly because of a very limited uptake of the latter. Uptake of MMA(V) and DMA(V) by wild-type rice was increased as the pH of the medium decreased, consistent with an increasing proportion of the undissociated species. The results demonstrate that Lsi1 mediates the uptake of undissociated methylated As in rice roots. Lsi1|OsNIP2;1,Lsi2 Involvement of silicon influx transporter OsNIP2;1 in selenite uptake in rice 2010 Plant Physiol State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, 210008 Nanjing, China. Rice (Oryza sativa) as a staple food, provides a major source of dietary selenium (Se) for humans, which essentially requires Se, however, the molecular mechanism for Se uptake is still poorly understood. Herein, we show evidence that the uptake of selenite, a main bioavailable form of Se in paddy soils, is mediated by a silicon (Si) influx transporter Lsi1 (OsNIP2;1) in rice. Defect of OsNIP2;1 resulted in a significant decrease in the Se concentration of the shoots and xylem sap when selenite was given. However, there was no difference in the Se concentration between the wild-type rice and mutant of OsNIP2;1 when selenate was supplied. A short-term uptake experiment showed that selenite uptake greatly increased with decreasing pH in the external solution. Si as silicic acid did not inhibit the Se uptake from selenite in both rice and yeast (Saccharomyces cerevisiae) at low pHs. Expression of OsNIP2;1 in yeast enhanced the selenite uptake at pH 3.5 and 5.5 but not at pH 7.5. On the other hand, defect of Si efflux transporter Lsi2 did not affect the uptake of Se either from selenite or selenate. Taken together, our results indicate that Si influx transporter OsNIP2;1 is permeable to selenite. Lsi1|OsNIP2;1 Transporters of arsenite in rice and their role in arsenic accumulation in rice grain 2008 Proc Natl Acad Sci U S A Research Institute for Bioresources, Okayama University, Chuo 2-20-1, Kurashiki 710-0046, Japan. maj@rib.okayama-u.ac.jp Arsenic poisoning affects millions of people worldwide. Human arsenic intake from rice consumption can be substantial because rice is particularly efficient in assimilating arsenic from paddy soils, although the mechanism has not been elucidated. Here we report that two different types of transporters mediate transport of arsenite, the predominant form of arsenic in paddy soil, from the external medium to the xylem. Transporters belonging to the NIP subfamily of aquaporins in rice are permeable to arsenite but not to arsenate. Mutation in OsNIP2;1 (Lsi1, a silicon influx transporter) significantly decreases arsenite uptake. Furthermore, in the rice mutants defective in the silicon efflux transporter Lsi2, arsenite transport to the xylem and accumulation in shoots and grain decreased greatly. Mutation in Lsi2 had a much greater impact on arsenic accumulation in shoots and grain in field-grown rice than Lsi1. Arsenite transport in rice roots therefore shares the same highly efficient pathway as silicon, which explains why rice is efficient in arsenic accumulation. Our results provide insight into the uptake mechanism of arsenite in rice and strategies for reducing arsenic accumulation in grain for enhanced food safety. Lsi1|OsNIP2;1,Lsi2 A transporter regulating silicon distribution in rice shoots 2008 Plant Cell Research Institute for Bioresources, Okayama University, Kurashiki 710-0046, Japan. Rice (Oryza sativa) accumulates very high concentrations of silicon (Si) in the shoots, and the deposition of Si as amorphous silica helps plants to overcome biotic and abiotic stresses. Here, we describe a transporter, Lsi6, which is involved in the distribution of Si in the shoots. Lsi6 belongs to the nodulin-26 intrinsic protein III subgroup of aquaporins and is permeable to silicic acid. Lsi6 is expressed in the leaf sheath and leaf blades as well as in the root tips. Cellular localization studies revealed that Lsi6 is found in the xylem parenchyma cells of the leaf sheath and leaf blades. Moreover, Lsi6 showed polar localization at the side facing toward the vessel. Knockdown of Lsi6 did not affect the uptake of Si by the roots but resulted in disordered deposition of silica in the shoots and increased excretion of Si in the guttation fluid. These results indicate that Lsi6 is a transporter responsible for the transport of Si out of the xylem and subsequently affects the distribution of Si in the leaf. Lsi6 A transporter at the node responsible for intervascular transfer of silicon in rice 2009 Plant Cell Research Institute for Bioresources, Okayama University, Kurashiki, Japan. The concentration of essential mineral nutrients in the edible portion of plants such as grains may affect the nutritional value of these foods, while concentrations of toxic minerals in the plant are matter of food safety. Minerals taken up by the roots from soils are normally redirected at plant nodes before they are finally transported into developing seeds. However, the molecular mechanisms involved in this process have not been identified so far. Herein, we report on a transporter (Lsi6) responsible for the redirection of a plant nutrient at the node. Lsi6 is a silicon transporter in rice (Oryza sativa), and its expression in node I below the panicles is greatly enhanced when the panicle is completely emerged. Lsi6 is mainly localized at the xylem transfer cells located at the outer boundary region of the enlarged large vascular bundles in node I. Knockout of Lsi6 decreased Si accumulation in the panicles but increased Si accumulation in the flag leaf. These results suggest that Lsi6 is a transporter involved in intervascular transfer (i.e., transfer of silicon from the large vascular bundles coming from the roots to the diffuse vascular bundles connected to the panicles). These findings will be useful for selectively enhancing the accumulation of essential nutrients and reducing toxic minerals in the edible portion of cereals. Lsi6 Inducibility by pathogen attack and developmental regulation of the rice Ltp1 gene 2002 Plant Mol Biol BIOTROP and CALIM programmes, Cirad, Centre International de Recherches Agronomiques en cooperation pour le Developpement, Montpellier, France. Guiderdoni@cirad.fr Using a genomic clone encoding a rice lipid transfer protein, LTP1, we analysed the activity of the 5' region of the Ltp1 gene in transgenic rice (Oryza sativa L.) during plant development and under pathogen attack. The -1176/+13, -556/+13 and -284/+13 regions of the promoter were fused upstream from the uidA reporter gene and nos 3' polyadenylation signal, resulting in the pdelta1176Gus, pdelta556Gus and pdelta284Gus constructs which were transferred to rice by microprojectile bombardment. Histochemical and fluorometric GUS assays and in situ detection of uidA transcripts in transgenic homozygous lines harbouring the pdelta1176Gus construct demonstrated that the Ltp1 promoter is preferentially active in aerial vegetative and reproductive organs and that both specificity and level of expression are regulated during organ development. In leaf sheath, GUS activity which is initially strictly localized in the epidermis of growing tissue, becomes restricted to the vascular system in mature tissues. In expanded leaf blade, expression of the uidA gene was restricted to the cutting level suggesting inducibility by wounding. Strong activity was detected in lemma and palea, sterile glumes, and immature anther walls and microspores but not in female reproductive organs. No GUS activity was detected during seed embryo maturation whereas the uidA gene was strongly expressed at early stages of somatic embryogenesis in scutellum tissue. The Ltp1 transcripts were found to strongly accumulate in response to inoculation with the fungal agent of the blast disease, Magnaporthe grisea, in two rice cultivars exhibiting compatible or incompatible host-pathogen interactions. Analysis of pdelta1176Gus leaf samples inoculated with the blast fungus demonstrated that the Ltp1 promoter is induced in all cell types of tissues surrounding the lesion and notably in stomata guard cells. In plants harbouring the Ltp1 promoter deletion construct pdelta556Gus, activity was solely detected in the vascular system of mature leaves whereas no uidA gene expression was observed in pdelta284Gus plants. These observations are consistent with the proposed role of LTP1 in strenghtening of structural barriers and organ protection against mechanical disruption and pathogen attack. Ltp1 Evolutionary history of the non-specific lipid transfer proteins 2011 Mol Plant IFM Molecular Genetics, Linkoping University, 581 83 Linkoping, Sweden. The non-specific lipid transfer proteins (nsLTPs) are small, basic proteins characterized by a tunnel-like hydrophobic cavity, capable of transferring various lipid molecules between lipid bilayers. Most nsLTPs are synthesized with an N-terminal signal peptide that localizes the protein to the apoplastic space. The nsLTPs have only been identified in seed plants, where they are encoded by large gene families. We have initiated an analysis of the evolutionary history of the nsLTP family using genomic and EST information from non-seed land plants and green algae to determine: (1) when the nsLTP family arose, (2) how often new nsLTP subfamilies have been created, and (3) how subfamilies differ in their patterns of expansion and loss in different plant lineages. In this study, we searched sequence databases and found that genes and transcripts encoding nsLTPs are abundant in liverworts, mosses, and all other investigated land plants, but not present in any algae. The tertiary structures of representative liverwort and moss nsLTPs were further studied with homology modeling. The results indicate that the nsLTP family has evolved after plants conquered land. Only two of the four major subfamilies of nsLTPs found in flowering plants are present in mosses and liverworts. The additional subfamilies have arisen later, during land plant evolution. In this report, we also introduce a modified nsLTP classification system. LTP2,YY1|OsLTPc2|LTPL45,OsLTPx1 Characterization of a gene encoding an abscisic acid-inducible type-2 lipid transfer protein from rice 1998 FEBS Lett Laboratoire de Physiologie et Biologie Moleculaire Vegetales, UMR 5545 CNRS Universite de Perpignan, France. The cloning and sequence analysis of a novel gene that encodes a type 2 non-specific lipid transfer-like protein (LTP) from rice is reported. Sequence analysis revealed an ORF encoding a protein showing characteristics of the LTP proteins. However, rice LTP2 is more similar to heterologous LTPs than to rice LTP1, supporting the existence of two distinct families of plant LTPs. Ltp2 mRNA is accumulated only in mature seeds. In vegetative tissues, mRNA was only detected after treatment with abscisic acid (ABA), mannitol or NaCl. Transient expression experiments that the 61 nucleotides upstream of the TATA box, containing two ACGT boxes and the motif I, are sufficient for ABA responsiveness of the Ltp gene. LTP2 OsC6, encoding a lipid transfer protein, is required for postmeiotic anther development in rice 2010 Plant Physiol School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, China. Synthesis of lipidic components in anthers, including of the pollen exine, is essential for plant male reproductive development. Plant lipid transfer proteins (LTPs) are small, abundant lipid-binding proteins that have the ability to exchange lipids between membranes in vitro. However, their biological role in male reproductive development remains less understood. Here, we report the crucial role of OsC6 in regulating postmeiotic anther development in rice (Oryza sativa). Found in monocots, OsC6 belongs to a distinct clade from previously identified LTP1 and LTP2 family members found in both dicots and monocots. OsC6 expression is mainly detectable in tapetal cells and weakly in microspores from stage 9 to stage 11 of anther development. Immunological assays indicated that OsC6 is widely distributed in anther tissues such as the tapetal cytoplasm, the extracellular space between the tapetum and middle layer, and the anther locule and anther cuticle. Biochemical assays indicated that recombinant OsC6 has lipid binding activity. Moreover, plants in which OsC6 was silenced had defective development of orbicules (i.e. Ubisch bodies) and pollen exine and had reduced pollen fertility. Furthermore, additional evidence is provided that the expression of OsC6 is positively regulated by a basic helix-loop-helix transcription factor, Tapetum Degeneration Retardation (TDR). Extra granule-like structures were observed on the inner surface of the tdr tapetal layer when the expression of OsC6 was driven by the TDR promoter compared with the tdr mutant. These data suggest that OsC6 plays a crucial role in the development of lipidic orbicules and pollen exine during anther development in rice. LTP2,OSC4,OsC6,TDR,YY1|OsLTPc2|LTPL45 Solution structure of plant nonspecific lipid transfer protein-2 from rice (Oryza sativa) 2002 J Biol Chem Department of Life Sciences, National Tsing Hua University, Taiwan 300, China. The three-dimensional structure of rice nonspecific lipid transfer protein (nsLTP2) has been solved for the first time. The structure of nsLTP2 was obtained using 813 distance constraints, 30 hydrogen bond constraints, and 19 dihedral angle constraints. Fifteen of the 50 random simulated annealing structures satisfied all of the constraints and possessed good nonbonded contacts. The novel three-dimensional fold of rice nsLTP2 contains a triangular hydrophobic cavity formed by three prominent helices. The four disulfide bonds required for stabilization of the nsLTP2 structure show a different pattern of cysteine pairing compared with nsLTP1. The C terminus of the protein is very flexible and forms a cap over the hydrophobic cavity. Molecular modeling studies suggested that the hydrophobic cavity could accommodate large molecules with rigid structures, such as sterols. The positively charged residues on the molecular surface of nsLTP2 are structurally similar to other plant defense proteins. LTP2 Mutation of the light-induced yellow leaf 1 gene, which encodes a geranylgeranyl reductase, affects chlorophyll biosynthesis and light sensitivity in rice 2013 PLoS One Jiangsu Key Laboratory of Crop Genetics and Physiology/Key Laboratory of the Ministry of Education for Plant Functional Genomics, Yangzhou University, Yangzhou, Jiangsu, China. Chlorophylls (Chls) are crucial for capturing light energy for photosynthesis. Although several genes responsible for Chl biosynthesis were characterized in rice (Oryza sativa), the genetic properties of the hydrogenating enzyme involved in the final step of Chl synthesis remain unknown. In this study, we characterized a rice light-induced yellow leaf 1-1 (lyl1-1) mutant that is hypersensitive to high-light and defective in the Chl synthesis. Light-shading experiment suggested that the yellowing of lyl1-1 is light-induced. Map-based cloning of LYL1 revealed that it encodes a geranylgeranyl reductase. The mutation of LYL1 led to the majority of Chl molecules are conjugated with an unsaturated geranylgeraniol side chain. LYL1 is the firstly defined gene involved in the reduction step from Chl-geranylgeranylated (Chl(GG)) and geranylgeranyl pyrophosphate (GGPP) to Chl-phytol (Chl(Phy)) and phytyl pyrophosphate (PPP) in rice. LYL1 can be induced by light and suppressed by darkness which is consistent with its potential biological functions. Additionally, the lyl1-1 mutant suffered from severe photooxidative damage and displayed a drastic reduction in the levels of alpha-tocopherol and photosynthetic proteins. We concluded that LYL1 also plays an important role in response to high-light in rice. LYL1 Overexpression of an F-box protein gene reduces abiotic stress tolerance and promotes root growth in rice 2011 Mol Plant State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. As one of the largest gene families, F-box domain proteins have important roles in regulating various developmental processes and stress responses. In this study, we have investigated a rice F-box domain gene, MAIF1. The MAIF1 protein is mainly localized in the plasma membrane and nucleus. MAIF1 expression is induced rapidly and strongly by abscisic acid (ABA) and abiotic stresses. MAIF1 expression is also induced in root tips by sucrose, independent of its hydrolytic hexose products, glucose and fructose, and the plant hormones auxin and cytokinin. Overexpression of MAIF1 reduces rice ABA sensitivity and abiotic stress tolerance and promotes rice root growth. These results suggest that MAIF1 is involved in multiple signaling pathways in regulating root growth. Growth restraint in plants is an acclimatization strategy against abiotic stress. Our results also suggest that MAIF1 plays the negative role in response to abiotic stress possibly by regulating root growth. MAIF1 Identification of a biosynthetic gene cluster in rice for momilactones 2007 J Biol Chem Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, Japan. Rice diterpenoid phytoalexins such as momilactones and phytocassanes are produced in suspension-cultured rice cells treated with a chitin oligosaccharide elicitor and in rice leaves irradiated with UV light. The common substrate geranylgeranyl diphosphate is converted into diterpene hydrocarbon precursors via a two-step sequential cyclization and then into the bioactive phytoalexins via several oxidation steps. It has been suggested that microsomal cytochrome P-450 monooxygenases (P-450s) are involved in the downstream oxidation of the diterpene hydrocarbons leading to the phytoalexins and that a dehydrogenase is involved in momilactone biosynthesis. However, none of the enzymes involved in the downstream oxidation of the diterpene hydrocarbons have been identified. In this study, we found that a putative dehydrogenase gene (AK103462) and two functionally unknown P-450 genes (CYP99A2 and CYP99A3) form a chitin oligosaccharide elicitor- and UV-inducible gene cluster, together with OsKS4 and OsCyc1, the diterpene cyclase genes involved in momilactone biosynthesis. Functional analysis by heterologous expression in Escherichia coli followed by enzyme assays demonstrated that the AK103462 protein catalyzes the conversion of 3beta-hydroxy-9betaH-pimara-7,15-dien-19,6beta-olide into momilactone A. The double knockdown of CYP99A2 and CYP99A3 specifically suppressed the elicitor-inducible production of momilactones, strongly suggesting that CYP99A2, CYP99A3, or both are involved in momilactone biosynthesis. These results provide strong evidence for the presence on chromosome 4 of a gene cluster involved in momilactone biosynthesis. MAS A germ cell specific gene of the ARGONAUTE family is essential for the progression of premeiotic mitosis and meiosis during sporogenesis in rice 2007 Plant Cell Experimental Farm, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan. knonomur@lab.nig.ac.jp The rice (Oryza sativa) genome contains 18 copies of genes of the ARGONAUTE (AGO) family. Although AGO members play important roles in RNA-mediated silencing during plant development, a family member that is specifically involved in sexual reproduction has not been identified in plants. We identified the rice AGO gene MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1) from the analysis of seed-sterile mutants. In the mel1 mutant, chromosome condensation was arrested at early meiotic stages and irregularly sized, multinucleated, and vacuolated pollen mother cells (PMCs) frequently appeared in developing anthers. In addition, histone H3 lysine-9 dimethylation of pericentromeres was rarely reduced and modification of the nucleolar-organizing region was altered in mel1 mutant PMCs. The mutation also affected female germ cell development. These results indicate that the germ cell-specific rice MEL1 gene regulates the cell division of premeiotic germ cells, the proper modification of meiotic chromosomes, and the faithful progression of meiosis, probably via small RNA-mediated gene silencing, but not the initiation and establishment of germ cells themselves. MEL1 A recurrent general RNA binding domain appended to plant methionyl-tRNA synthetase acts as a cis-acting cofactor for aminoacylation 2000 EMBO J Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 1 Avenue de la Terrasse, 91190 Gif-sur-Yvette, France. The cDNA encoding rice methionyl-tRNA synthetase was isolated. The protein exhibited a C-terminal polypeptide appended to a classical MetRS domain. This supplementary domain is related to endothelial monocyte activating polypeptide II (EMAPII), a cytokine produced in mammals after cleavage of p43, a component of the multisynthetase complex. It is also related to Arc1p and Trbp111, two tRNA binding proteins. We expressed rice MetRS and a derivative with a deletion of its EMAPII-like domain. Band-shift analysis showed that this extra-domain provides MetRS with non-specific tRNA binding properties. The EMAPII-like domain contributed a 10-fold decrease in K:(M) for tRNA in the aminoacylation reaction catalyzed by the native enzyme, as compared with the C-terminally truncated MetRS. Consequently, the EMAPII domain provides MetRS with a better catalytic efficiency at the free tRNA concentration prevailing in vivo. This domain binds the acceptor minihelix of tRNA(Met) and facilitates its aminoacylation. These results suggest that the EMAPII module could be a relic of an ancient tRNA binding domain that was incorporated into primordial synthetases for aminoacylation of RNA minihelices taken as the ancestor of modern tRNA. MetRS|MRS Identification of a rice RNA- and microtubule-binding protein as the multifunctional protein, a peroxisomal enzyme involved in the beta -oxidation of fatty acids 2002 J Biol Chem Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada. The control of subcellular mRNA localization and translation is often mediated by protein factors that are directly or indirectly associated with the cytoskeleton. We report the identification and characterization of a rice seed protein that possesses both RNA and microtubule binding activities. In vitro UV cross-linking assays indicated that this protein binds to all mRNA sequences tested, although there was evidence for preferential binding to RNAs that contained A-C nucleotide sequence motifs. The protein was purified to homogeneity using a two-step procedure, and amino acid sequencing identified it as the multifunctional protein (MFP), a peroxisomal enzyme known to possess a number of activities involved in the beta-oxidation of fatty acids. The recombinant version of this rice MFP binds to RNA in UV cross-linking and gel mobility shift experiments, co-sediments specifically with microtubules, and possesses at least two enzymatic activities involved in peroxisomal fatty acid beta-oxidation. Taken together these data suggest that MFP has an important role in mRNA physiology in the cytoplasm, perhaps in regulating the localization or translation of mRNAs through an interaction with microtubules, in addition to its peroxisomal function. MFP MULTI-FLORET SPIKELET1, which encodes an AP2/ERF protein, determines spikelet meristem fate and sterile lemma identity in rice 2013 Plant Physiol Rice Research Institute, Southwest University, Chongqing 400715, China. The spikelet is a unique inflorescence structure of grass. The molecular mechanism that controls the development of the spikelet remains unclear. In this study, we identified a rice (Oryza sativa) spikelet mutant, multi-floret spikelet1 (mfs1), that showed delayed transformation of spikelet meristems to floral meristems, which resulted in an extra hull-like organ and an elongated rachilla. In addition, the sterile lemma was homeotically converted to the rudimentary glume and the body of the palea was degenerated in mfs1. These results suggest that the MULTI-FLORET SPIKELET1 (MFS1) gene plays an important role in the regulation of spikelet meristem determinacy and floral organ identity. MFS1 belongs to an unknown function clade in the APETALA2/ethylene-responsive factor (AP2/ERF) family. The MFS1-green fluorescent protein fusion protein is localized in the nucleus. MFS1 messenger RNA is expressed in various tissues, especially in the spikelet and floral meristems. Furthermore, our findings suggest that MFS1 positively regulates the expression of LONG STERILE LEMMA and the INDETERMINATE SPIKELET1 (IDS1)-like genes SUPERNUMERARY BRACT and OsIDS1. MFS1 Ethylene-Induced Inhibition of Root Growth Requires Abscisic Acid Function in Rice (Oryza sativa L.) Seedlings 2014 PLoS Genet State Key Lab of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. Ethylene and abscisic acid (ABA) have a complicated interplay in many developmental processes. Their interaction in rice is largely unclear. Here, we characterized a rice ethylene-response mutant mhz4, which exhibited reduced ethylene-response in roots but enhanced ethylene-response in coleoptiles of etiolated seedlings. MHZ4 was identified through map-based cloning and encoded a chloroplast-localized membrane protein homologous to Arabidopsis thaliana (Arabidopsis) ABA4, which is responsible for a branch of ABA biosynthesis. MHZ4 mutation reduced ABA level, but promoted ethylene production. Ethylene induced MHZ4 expression and promoted ABA accumulation in roots. MHZ4 overexpression resulted in enhanced and reduced ethylene response in roots and coleoptiles, respectively. In root, MHZ4-dependent ABA pathway acts at or downstream of ethylene receptors and positively regulates root ethylene response. This ethylene-ABA interaction mode is different from that reported in Arabidopsis, where ethylene-mediated root inhibition is independent of ABA function. In coleoptile, MHZ4-dependent ABA pathway acts at or upstream of OsEIN2 to negatively regulate coleoptile ethylene response, possibly by affecting OsEIN2 expression. At mature stage, mhz4 mutation affects branching and adventitious root formation on stem nodes of higher positions, as well as yield-related traits. Together, our findings reveal a novel mode of interplay between ethylene and ABA in control of rice growth and development. MHZ4 Identification and functional analysis of the MOC1 interacting protein 1 2010 Journal of Genetics and Genomics State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Rice tillering is one of the most important agronomic traits that determine grain yields. Our previous study has demonstrated that the MONOCULM1 (MOC1) gene is a key component that controls the formation of rice tiller buds. To further elucidate the molecular mechanism of MOC1 involved in the regulation of rice tillering, we performed a yeast-two-hybrid screening to identify MOC1 interacting proteins (MIPs). Here we reported that MIP1 interacted with MOC1 both in vitro and in vivo. The overexpression of MIP1 resulted in enhanced tillering and reduced plant height. In-depth characterization of the context of MIP1 and MOC1 would further our understanding of molecular regulatory mechanisms of rice tillering. MIP1,MOC1 Rice-specific mitochondrial iron-regulated gene (MIR) plays an important role in iron homeostasis 2009 Mol Plant Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-Ku, Tokyo 113-8657, Japan. Mitochondria utilize iron (Fe), but the proteins involved in mitochondrial Fe regulation are not characterized in plants. We cloned and characterized a mitochondrial iron-regulated (MIR) gene in rice involved in Fe homeostasis. MIR, when expressed in tobacco BY-2 cells, was localized to the mitochondria. MIR transcripts were greatly increased in response to Fe deficiency in roots and shoot tissue. MIR is not homologous to any known protein, as homologs were not found in the rice or Arabidopsis genome databases, or in the EST database for other organisms. Growth in the MIR T-DNA knockout rice mutant (mir) was significantly impaired compared to wild-type (WT) plants when grown under Fe-deficient or -sufficient conditions. Furthermore, mir plants accumulated more than twice the amount of Fe in shoot and root tissue compared to WT plants when grown under either Fe-sufficient or -deficient conditions. Despite the high accumulation of Fe in roots and shoots, mir plants triggered the expression of Fe-deficiency-inducible genes, indicating that mir may not be able to utilize Fe for physiological functions. These results clearly suggest that MIR is a rice-specific mitochondrial protein, recently evolved, and plays a significant role in Fe homeostasis. MIR The knockdown of OsVIT2 and MIT affects iron localization in rice seed 2013 Rice (N Y) Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. annaoko@mail.ecc.u-tokyo.ac.jp. BACKGROUND: The mechanism of iron (Fe) uptake in plants has been extensively characterized, but little is known about how Fe transport to different subcellular compartments affects Fe localization in rice seed. Here, we discuss the characterization of a rice vacuolar Fe transporter 2 (OsVIT2) T-DNA insertion line (osvit2) and report that the knockdown of OsVIT2 and mitochondrial Fe transporter (MIT) expression affects seed Fe localization. FINDINGS: osvit2 plants accumulated less Fe in their shoots when grown under normal or excess Fe conditions, while the accumulation of Fe was comparable to that in wild-type (WT) plants under Fe-deficient conditions. The accumulation of zinc, copper, and manganese also changed significantly in the shoots of osvit2 plants. The growth of osvit2 plants was also slow compared to that of WT plants. The concentration of Fe increased in osvit2 polished seeds. Previously, we reported that the expression of OsVIT2 was higher in MIT knockdown (mit-2) plants, and in this study, the accumulation of Fe in mit-2 seeds decreased significantly. CONCLUSIONS: These results suggest that vacuolar Fe trafficking is important for plant Fe homeostasis and distribution, especially in plants grown in the presence of excess Fe. Moreover, changes in the expression of OsVIT2 and MIT affect the concentration and localization of metals in brown rice as well as in polished rice seeds. MIT,OsVIT2 The rice mitochondrial iron transporter is essential for plant growth 2011 Nat Commun Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Tokyo 113-8657, Japan. In plants, iron (Fe) is essential for mitochondrial electron transport, heme, and Fe-Sulphur (Fe-S) cluster synthesis; however, plant mitochondrial Fe transporters have not been identified. Here we show, identify and characterize the rice mitochondrial Fe transporter (MIT). Based on a transfer DNA library screen, we identified a rice line showing symptoms of Fe deficiency while accumulating high shoot levels of Fe. Homozygous knockout of MIT in this line resulted in a lethal phenotype. MIT localized to the mitochondria and complemented the growth of Deltamrs3Deltamrs4 yeast defective in mitochondrial Fe transport. The growth of MIT-knockdown (mit-2) plants was also significantly impaired despite abundant Fe accumulation. Further, the decrease in the activity of the mitochondrial and cytosolic Fe-S enzyme, aconitase, indicated that Fe-S cluster synthesis is affected in mit-2 plants. These results indicate that MIT is a mitochondrial Fe transporter essential for rice growth and development. MIT Identification and characterization of the major mitochondrial Fe transporter in rice 2011 Plant Signal Behav Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-Ku, Tokyo, Japan. The uptake, translocation, and compartmentalization of Fe are essential for plant cell function and life cycle. Despite rapid progress in our understanding of Fe homeostasis in plants, Fe transport from the cytoplasm to mitochondria was, until recently, poorly understood. The screening of 3,993 mutant lines for symptoms of Fe deficiency resulted in the identification and characterization of a major mitochondrial Fe transporter (MIT) in rice. MIT was found to localize to mitochondria and to complement the growth of a yeast strain defective in mitochondrial Fe transport. The knock-out of MIT resulted in a lethal phenotype, and in knock-down plants, several agronomic characteristics were compromised, such as plant height, average number of tillers, days to flower, fertility, and yield. Changes in the expression of genes involved in Fe transport suggested a disturbance of cellular Fe transport. Furthermore, the mitochondrial Fe concentration and the activity of the mitochondrial Fe-S enzyme aconitase were significantly reduced compared with wild-type plants. The identification of MIT is a significant advance in the field of plant Fe nutrition and should facilitate the cloning of paralogs from other plant species. MIT Sequence, expression and tissue localization of a gene encoding a makorin RING zinc-finger protein in germinating rice (Oryza sativa L. ssp. Japonica) seeds 2007 Plant Physiol Biochem Laboratory of Molecular Cell Physiology, Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan. thangs@mcb.agr.ehime-u.ac.jp The makorin (MKRN) RING finger protein gene family encodes proteins (makorins) with a characteristic array of zinc-finger motifs and which are present in a wide array of eukaryotes. In the present study, we analyzed the structure and expression of a putative makorin RING finger protein gene in rice (Oryza sativa L. ssp. Japonica cv. Nipponbare). From the analysis of the genomic (AP003543), mRNA (AK120250) and deduced protein (BAD61603) sequences of the putative MKRN gene of rice, obtained from GenBank, we found that it was indeed a bona fide member of the MKRN gene family. The rice MKRN cDNA encoded a protein with four C3H zinc-finger-motifs, one putative Cys-His zinc-finger motif, and one RING zinc-finger motif. The presence of this distinct motif organization and overall amino acid identity clearly indicate that this gene is indeed a true MKRN ortholog. We isolated RNA from embryonic axes of rice seeds at various stages of imbibition and germination and studied the temporal expression profile of MKRN by RT-PCR. This analysis revealed that MKRN transcripts were present at all the time points studied. It was at very low levels in dry seeds, increased slowly during imbibition and germination, and slightly declined in the seedling growth stage. After 6days of germination, an organ-dependent expression pattern of MKRN was observed: highest in roots and moderate in leaves. Similarly to MKRN transcripts, transcripts of cytoskeletal actin and tubulin were also detected in dry embryos, steadily increased during imbibition and germination and leveled off after 24h of germination. We studied the spatial expression profile of MKRN in rice tissues, by using a relatively fast, simple and effective non-radioactive mRNA in situ hybridization (NRISH) technique, which provided the first spatial experimental data that hints at the function of a plant makorin. This analysis revealed that MKRN transcripts were expressed in young plumules, lateral root primordia, leaf primordia, leaves and root tissues at many different stages of germination. The presence of MKRN transcripts in dry seeds, its early induction during germination and its continued spatiotemporal expression during early vegetative growth suggest that MKRN has an important role in germination, leaf and lateral root morphogenesis and overall development in rice. MKRN MKRN expression pattern during embryonic and post-embryonic organogenesis in rice (Oryza sativa L. var. Nipponbare) 2013 Planta Laboratory of Molecular Cell Physiology, Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, 790-8566, Japan. Rice MKRN is a member of the makorin RING finger protein gene (MKRN) family, which encodes a protein with a characteristic array of zinc-finger motifs conserved in various eukaryotes. Using non-radioactive in situ hybridization, we investigated the spatio-temporal gene expression pattern of rice MKRN during embryogenesis, imbibition, seminal and lateral root development of Oryza sativa L. var. Nipponbare. MKRN expression was ubiquitous during early organogenesis in the embryo along the apical-basal and radial axes. The expression of MKRN decreased during embryonic organ elongation and maturation compared to early embryogenesis, but increased again during imbibition. Tissue-specific and position-dependent MKRN expression was found during embryonic and post-embryonic root and shoot development. Meristematic cells ubiquitously expressed MKRN transcripts, while differentiating cells showed a gradual reduction and termination of MKRN expression. Interestingly, during post-germination MKRN expression was prominent and continued in the metabolically active, differentiated companion cells of the phloem. The differential expression pattern was observed both in the differentiating and differentiated cells. Also, MKRN was expressed in the various developmental stages of the lateral root primordia and the cells surrounding them. Expression of MKRN was also observed after periclinal division of the presumptive pericycle founder cells. The MKRN expression pattern during development of various growth stages suggests an important role of makorin RING finger protein gene (MKRN) in embryonic and post-embryonic organogenesis in both apical-basal and radial developmental axes of rice. MKRN OsCERK1 and OsRLCK176 play important roles in peptidoglycan and chitin signaling in rice innate immunity 2014 Plant J State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, 510275, Guangzhou, People's Republic of China. Microbe-associated molecular pattern (MAMP)-triggered immunity plays critical roles in the basal resistance defense response in plants. Chitin and peptidoglycan (PGN) are major molecular patterns for fungi and bacteria, respectively. Two rice (Oryza sativa) lysin motif-containing proteins, OsLYP4 and OsLYP6, function as receptors that sense bacterial PGN and fungal chitin. These membrane receptors, which lack intracellular kinase domains, likely contain another component for transmembrane immune signal transduction. Here, we demonstrate that the rice LysM receptor-like kinase OsCERK1, a key component of the chitin elicitor signaling pathway, also plays an important role in PGN-triggered immunity in rice. Silencing of OsCERK1 suppressed PGN-induced (and chitin-induced) immunity responses, including reactive oxygen species generation, defense gene expression, and callose deposition, indicating that OsCERK1 is essential for both PGN and chitin signaling initiated by OsLYP4 and OsLYP6. OsLYP4 associated with OsLYP6 and the rice chitin receptor CEBiP in the absence of PGN or chitin, and treatment with PGN or chitin led to their disassociation in vivo. OsCERK1 associated with OsLYP4 or OsLYP6 when induced by PGN but it associated with OsLYP4, OsLYP6, or CEBiP under chitin treatment, suggesting the presence of different patterns of ligand-induced heterooligomeric receptor complexes. Furthermore, the receptor-like cytoplasmic kinase OsRLCK176 functions downstream of OsCERK1 in the PGN and chitin signaling pathways, suggesting that these MAMPs share overlapping intracellular signaling components. Therefore, OsCERK1 plays dual roles in PGN and chitin signaling in rice innate immunity and as an adaptor involved in signal transduction at the plasma membrane in conjunction with OsLYP4 and OsLYP6. MLO,OsRLCK176,PAL Methylmalonate-semialdehyde dehydrogenase is induced in auxin-stimulated and zinc-stimulated root formation in rice 2004 Plant Cell Rep Faculty of Agriculture, Miyazaki University, Miyazaki, 889-2192, Japan. Proteins induced in rice by auxin and zinc were determined by proteome analysis. Cultured suspension cells of rice were treated with 2,4-dichlorophenoxyacetic acid and ZnSO4 and then proteins were separated by two-dimensional polyacrylamide gel electrophoresis; seven proteins were found to be induced by auxin and zinc. Of these seven, methylmalonate-semialdehyde dehydrogenase (MMSDH) was elevated by treatment with auxin alone. MMSDH was detected in cultured suspension cells, root and leaf sheath, but not in leaf blades. MMSDH responded to auxin and gibberellin, but did not respond to brassinolide and cytokinin. Furthermore, the amount of MMSDH in slr1, a constitutive gibberellin response mutant, was 2-fold that of wild type. MMSDH mRNA and protein were stimulated in root formation induced by auxin and/or zinc over a 4-week period. These results suggest that MMSDH may be necessary for root formation in rice induced by auxin and/or zinc. MMSDH Control of tillering in rice 2003 Nature Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Tillering in rice (Oryza sativa L.) is an important agronomic trait for grain production, and also a model system for the study of branching in monocotyledonous plants. Rice tiller is a specialized grain-bearing branch that is formed on the unelongated basal internode and grows independently of the mother stem (culm) by means of its own adventitious roots. Rice tillering occurs in a two-stage process: the formation of an axillary bud at each leaf axil and its subsequent outgrowth. Although the morphology and histology and some mutants of rice tillering have been well described, the molecular mechanism of rice tillering remains to be elucidated. Here we report the isolation and characterization of MONOCULM 1 (MOC1), a gene that is important in the control of rice tillering. The moc1 mutant plants have only a main culm without any tillers owing to a defect in the formation of tiller buds. MOC1 encodes a putative GRAS family nuclear protein that is expressed mainly in the axillary buds and functions to initiate axillary buds and to promote their outgrowth. MOC1 Rice APC/C(TE) controls tillering by mediating the degradation of MONOCULM 1 2012 Nat Commun National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China. Rice MONOCULM 1 (MOC1) and its orthologues LS/LAS (lateral suppressor in tomato and Arabidopsis) are key promoting factors of shoot branching and tillering in higher plants. However, the molecular mechanisms regulating MOC1/LS/LAS have remained elusive. Here we show that the rice tiller enhancer (te) mutant displays a drastically increased tiller number. We demonstrate that TE encodes a rice homologue of Cdh1, and that TE acts as an activator of the anaphase promoting complex/cyclosome (APC/C) complex. We show that TE coexpresses with MOC1 in the axil of leaves, where the APC/C(TE) complex mediates the degradation of MOC1 by the ubiquitin-26S proteasome pathway, and consequently downregulates the expression of the meristem identity gene Oryza sativa homeobox 1, thus repressing axillary meristem initiation and formation. We conclude that besides having a conserved role in regulating cell cycle, APC/C(TE) has a unique function in regulating the plant-specific postembryonic shoot branching and tillering, which are major determinants of plant architecture and grain yield. MOC1,OsCCS52A|TAD|TE Degradation of MONOCULM 1 by APC/C(TAD1) regulates rice tillering 2012 Nat Commun State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. A rice tiller is a specialized grain-bearing branch that contributes greatly to grain yield. The MONOCULM 1 (MOC1) gene is the first identified key regulator controlling rice tiller number; however, the underlying mechanism remains to be elucidated. Here we report a novel rice gene, Tillering and Dwarf 1 (TAD1), which encodes a co-activator of the anaphase-promoting complex (APC/C), a multi-subunit E3 ligase. Although the elucidation of co-activators and individual subunits of plant APC/C involved in regulating plant development have emerged recently, the understanding of whether and how this large cell-cycle machinery controls plant development is still very limited. Our study demonstrates that TAD1 interacts with MOC1, forms a complex with OsAPC10 and functions as a co-activator of APC/C to target MOC1 for degradation in a cell-cycle-dependent manner. Our findings uncovered a new mechanism underlying shoot branching and shed light on the understanding of how the cell-cycle machinery regulates plant architecture. MOC1,OsCCS52A|TAD|TE Comparative sequence analysis of MONOCULM1-orthologous regions in 14 Oryza genomes 2009 Proc Natl Acad Sci U S A State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Comparative genomics is a powerful tool to decipher gene and genome evolution. Placing multiple genome comparisons in a phylogenetic context improves the sensitivity of evolutionary inferences. In the genus Oryza, this comparative approach can be used to investigate gene function, genome evolution, domestication, polyploidy, and ecological adaptation. A large genomic region surrounding the MONOCULM1 (MOC1) locus was chosen for study in 14 Oryza species, including 10 diploids and 4 allotetraploids. Sequencing and annotation of 18 bacterial artificial chromosome clones for these species revealed highly conserved gene colinearity and structure in the MOC1 region. Since the Oryza radiation about 14 Mya, differences in transposon amplification appear to be responsible for the different current sizes of the Oryza genomes. In the MOC1 region, transposons were only conserved between genomes of the same type (e.g., AA or BB). In addition to the conserved gene content, several apparent genes have been generated de novo or uniquely retained in the AA lineage. Two different 3-gene segments have been inserted into the MOC1 region of O. coarctata (KK) or O. sativa by unknown mechanism(s). Large and apparently noncoding sequences flanking the MOC1 gene were observed to be under strong purifying selection. The allotetraploids Oryza alta and Oryza minuta were found to be products of recent polyploidization, less than 1.6 and 0.4 Mya, respectively. In allotetraploids, pseudogenization of duplicated genes was common, caused by large deletions, small frame-shifting insertions/deletions, or nonsense mutations. MOC1 Rice monoculm mutation moc2, which inhibits outgrowth of the second tillers, is ascribed to lack of a fructose-1,6-bisphosphatase 2013 Plant Biotechnology Department of Biological Science and Technology, Tokyo University of Science We characterized a rice monoculm mutant moc2, which showed significantly reduced tiller numbers, pale-green leaves, a reduced growth rate, and a consequent dwarf phenotype. The monoculm feature was attributed to a deficiency in the efficient outgrowth of tiller buds, although the moc2 mutant produced tiller buds. Inconsistent change was observed in the expression of genes involved in tiller bud outgrowth, suggesting that the moc2 mutant has a defective function necessary for the tiller bud outgrowth. The gene responsible for the moc2 mutant was mapped to a locus encoding cytosolic fructose-1,6-bisphosphatase 1 (FBP1), in which a Tos17 retrotransposon was inserted in exon 4. Reverse-transcription PCR for the FBP1 gene amplified a shorter transcript from the moc2 mutant than from the wild-type plant. The sequence of the shorter transcript revealed a deletion of exon 4 by abnormal splicing, and the resulting frameshift generated a new translation termination signal. The moc2 mutant showed a very low level of FBPase activity, suggesting that it involves a loss-of-function mutation of FBP1. Cytosolic FBPase is considered a key enzyme in the sucrose biosynthesis pathway. Defective FBPase activity is anticipated to lead a shortage of sucrose supply, which probably causes the inhibition of tiller bud outgrowth in the moc2 mutant. The monoculm phenotype of the moc2 mutant supports the idea that sucrose supply may be an important cue to outgrow tiller buds. MOC2|FBP1 Rice MPR25 encodes a pentatricopeptide repeat protein and is essential for RNA editing of nad5 transcripts in mitochondria 2012 Plant J Laboratory of Environmental Plant Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Sendai 981-8555, Japan. Pentatricopeptide repeat (PPR) proteins are involved in the modification of organelle transcripts. In this study, we investigated the molecular function in rice of the mitochondrial PPR-encoding gene MITOCHONDRIAL PPR25 (MPR25), which belongs to the E subgroup of the PPR family. A Tos17 knockout mutant of MPR25 exhibited growth retardation and pale-green leaves with reduced chlorophyll content during the early stages of plant development. The photosynthetic rate in the mpr25 mutant was significantly decreased, especially under strong light conditions, although the respiration rate did not differ from that of wild-type plants. MPR25 was preferentially expressed in leaves. FLAG-tagged MPR25 accumulated in mitochondria but not in chloroplasts. Direct sequencing revealed that the mpr25 mutant fails to edit a C-U RNA editing site at nucleotide 1580 of nad5, which encodes a subunit of complex I (NADH dehydrogenase) of the respiratory chain in mitochondria. RNA editing of this site is responsible for a change in amino acid from serine to leucine. Recombinant MPR25 directly interacted with the proximal region of the editing site of nad5 transcripts. However, the NADH dehydrogenase activity of complex I was not affected in the mutant. By contrast, genes encoding alternative NADH dehydrogenases and alternative oxidase were up-regulated. The mpr25 mutant may therefore provide new information on the coordinated interaction between mitochondria and chloroplasts. MPR25 Mutation of a rice gene encoding a phenylalanine biosynthetic enzyme results in accumulation of phenylalanine and tryptophan 2008 Plant Cell CREST, Japan Science and Technology Agency, Tokyo 103-0027, Japan. Two distinct biosynthetic pathways for Phe in plants have been proposed: conversion of prephenate to Phe via phenylpyruvate or arogenate. The reactions catalyzed by prephenate dehydratase (PDT) and arogenate dehydratase (ADT) contribute to these respective pathways. The Mtr1 mutant of rice (Oryza sativa) manifests accumulation of Phe, Trp, and several phenylpropanoids, suggesting a link between the synthesis of Phe and Trp. Here, we show that the Mtr1 mutant gene (mtr1-D) encodes a form of rice PDT with a point mutation in the putative allosteric regulatory region of the protein. Transformed callus lines expressing mtr1-D exhibited all the characteristics of Mtr1 callus tissue. Biochemical analysis revealed that rice PDT possesses both PDT and ADT activities, with a preference for arogenate as substrate, suggesting that it functions primarily as an ADT. The wild-type enzyme is feedback regulated by Phe, whereas the mutant enzyme showed a reduced feedback sensitivity, resulting in Phe accumulation. In addition, these observations indicate that rice PDT is critical for regulating the size of the Phe pool in plant cells. Feeding external Phe to wild-type callus tissue and seedlings resulted in Trp accumulation, demonstrating a connection between Phe accumulation and Trp pool size. Mtr1 Expression of an NADP-malic enzyme gene in rice (Oryza sativa. L) is induced by environmental stresses; over-expression of the gene in Arabidopsis confers salt and osmotic stress tolerance 2007 Plant Mol Biol Alkali Soil Natural Environmental Science Center (ASNESC), Stress Molecular Biology Laboratory, Northeast Forestry University, Harbin 150040, P. R. China. NADP-malic enzyme (NADP-ME, EC 1.1.1.40) functions in many different pathways in plants, and has recently been implicated in plant defense such as in responses to wounding and UV-B radiation. In this study, we isolated a complementary DNA (cDNA) clone by using the differential display method and screening of a root cDNA library of rice (Oryza sativa. L) under carbonate (NaHCO3) stress, and identified it as one of the rice NADP-ME genes (we named it NADP-ME2, GenBank accession no. AB053295). The 5' end of NADP-ME2 was obtained by the 5'-RACE method, and the full-length cDNA had a length of 2217 bp encoding 593 amino acids. Expression of NADP-ME2 mRNA in roots was induced by stress from carbonates (NaHCO3 and Na2CO3, NaCl, and environmental pH changes. NADP-ME2 transcripts increased during 72-h exposures to NaHCO3, NaCl, and PEG stresses. Furthermore, NADP-ME activities in leaves and roots of rice seedlings increased by more than 50% in the presence of carbonates (NaHCO3 and Na2CO3), NaCl, and PEG. These results indicate that rice NADP-ME2 responds to salts and osmotic stresses. Transgenic Arabidopsis plants over-expressing NADP-ME2 were obtained through transformation, screening, Northern analysis and in situ NADP-ME activity assay. Transgenic Arabidopsis plants over-expressing NADP-ME2 grew well in 1/2 x MS medium with 100 mM NaCl or 4% mannitol, whereas growth of wild-type (WT) Arabidopsis seedlings was strongly inhibited. In addition, under 125 mM NaCl stress, the root lengths of transgenic lines were about twice as long as those of the WT. These results suggest that NADP-ME2 has a role in enhancing tolerance of plants to salt and osmotic stress. NADP-ME2 Fine mapping of a major QTL for flag leaf width in rice, qFLW4, which might be caused by alternative splicing of NAL1 2012 Plant Cell Rep State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China. Leaf width is an important agricultural trait in rice. QTL mapping in a recombinant inbred line population derived from the cross between the javanica cultivar D50 (narrow-leaved) and the indica cultivar HB277 (wide-leaved) identified five QTLs controlling flag leaf width. Fine mapping of the major QTL qFLW4 narrowed its location to a 74.8 kb interval between the SSR loci RM17483 and RM17486, a region which also contains the gene NAL1 (Narrow leaf 1). There was no difference in the level of NAL1 expression between cvs. D50 and HB277, but an analysis of the NAL1 transcripts showed that while most (if not all) of those produced in cv. D50 were full-length, two-thirds of those in HB277 were non-functional due to either loss or gain of sequence. The inference was that NAL1 is probably synonymous with qFLW4, and that the functional difference between the two alleles was due to alternative splicing. The analysis of expression of other known genes involved in the determination of leaf width provided no evidence of their having any clear functional association with qFLW4/NAL1. NAL1|qFLW4 Mutation of the rice Narrow leaf1 gene, which encodes a novel protein, affects vein patterning and polar auxin transport 2008 Plant Physiol State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. The size and shape of the plant leaf is an important agronomic trait. To understand the molecular mechanism governing plant leaf shape, we characterized a classic rice (Oryza sativa) dwarf mutant named narrow leaf1 (nal1), which exhibits a characteristic phenotype of narrow leaves. In accordance with reduced leaf blade width, leaves of nal1 contain a decreased number of longitudinal veins. Anatomical investigations revealed that the culms of nal1 also show a defective vascular system, in which the number and distribution pattern of vascular bundles are altered. Map-based cloning and genetic complementation analyses demonstrated that Nal1 encodes a plant-specific protein with unknown biochemical function. We provide evidence showing that Nal1 is richly expressed in vascular tissues and that mutation of this gene leads to significantly reduced polar auxin transport capacity. These results indicate that Nal1 affects polar auxin transport as well as the vascular patterns of rice plants and plays an important role in the control of lateral leaf growth. NAL1|qFLW4 NAL1 allele from a rice landrace greatly increases yield in modern indica cultivars 2013 Proc Natl Acad Sci U S A Plant Breeding, Genetics, and Biotechnology, International Rice Research Institute, Metro Manila, Philippines. Increasing crop production is essential for securing the future food supply in developing countries in Asia and Africa as economies and populations grow. However, although the Green Revolution led to increased grain production in the 1960s, no major advances have been made in increasing yield potential in rice since then. In this study, we identified a gene, SPIKELET NUMBER (SPIKE), from a tropical japonica rice landrace that enhances the grain productivity of indica cultivars through pleiotropic effects on plant architecture. Map-based cloning revealed that SPIKE was identical to NARROW LEAF1 (NAL1), which has been reported to control vein pattern in leaf. Phenotypic analyses of a near-isogenic line of a popular indica cultivar, IR64, and overexpressor lines revealed increases in spikelet number, leaf size, root system, and the number of vascular bundles, indicating the enhancement of source size and translocation capacity as well as sink size. The near-isogenic line achieved 13-36% yield increase without any negative effect on grain appearance. Expression analysis revealed that the gene was expressed in all cell types: panicles, leaves, roots, and culms supporting the pleiotropic effects on plant architecture. Furthermore, SPIKE increased grain yield by 18% in the recently released indica cultivar IRRI146, and increased spikelet number in the genetic background of other popular indica cultivars. The use of SPIKE in rice breeding could contribute to food security in indica-growing regions such as South and Southeast Asia. NAL1|qFLW4 The rice narrow leaf2 and narrow leaf3 loci encode WUSCHEL-related homeobox 3A (OsWOX3A) and function in leaf, spikelet, tiller and lateral root development 2013 New Phytol Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-921, Korea. . In order to understand the molecular genetic mechanisms of rice (Oryza sativa) organ development, we studied the narrow leaf2 narrow leaf3 (nal2 nal3; hereafter nal2/3) double mutant, which produces narrow-curly leaves, more tillers, fewer lateral roots, opened spikelets and narrow-thin grains. . We found that narrow-curly leaves resulted mainly from reduced lateral-axis outgrowth with fewer longitudinal veins and more, larger bulliform cells. Opened spikelets, possibly caused by marginal deformity in the lemma, gave rise to narrow-thin grains. . Map-based cloning revealed that NAL2 and NAL3 are paralogs that encode an identical OsWOX3A (OsNS) transcriptional activator, homologous to NARROW SHEATH1 (NS1) and NS2 in maize and PRESSED FLOWER in Arabidopsis. . OsWOX3A is expressed in the vascular tissues of various organs, where nal2/3 mutant phenotypes were displayed. Expression levels of several leaf development-associated genes were altered in nal2/3, and auxin transport-related genes were significantly changed, leading to pin mutant-like phenotypes such as more tillers and fewer lateral roots. OsWOX3A is involved in organ development in rice, lateral-axis outgrowth and vascular patterning in leaves, lemma and palea morphogenesis in spikelets, and development of tillers and lateral roots. NAL3,NAL2|OsWOX3A|WOX3 Two WUSCHEL-related homeobox genes, narrow leaf2 and narrow leaf3, control leaf width in rice 2013 Plant Cell Physiol Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan. Leaf shape is one of the key determinants of plant architecture. Leaf shape also affects the amount of sunlight captured and influences photosynthetic efficiency; thus, it is an important agronomic trait in crop plants. Understanding the molecular mechanisms governing leaf shape is a central issue of plant developmental biology and agrobiotechnology. Here, we characterized the narrow-leaf phenotype of FL90, a linkage tester line of rice (Oryza sativa). Light and scanning electron microscopic analyses of FL90 leaves revealed defects in the development of marginal regions and a reduction in the number of longitudinal veins. The narrow-leaf phenotype of FL90 shows a two-factor recessive inheritance and is caused by the loss of function of two WUSCHEL-related homeobox genes, NAL2 and NAL3 (NAL2/3), which are duplicate genes orthologous to maize NS1 and NS2 and to Arabidopsis PRS. The overexpression of NAL2/3 in transgenic rice plants results in wider leaves containing increased numbers of veins, suggesting that NAL2/3 expression regulates leaf width. Thus, NAL2/3 can be used to modulate leaf shape and improve agronomic yield in crop plants. NAL3,NAL2|OsWOX3A|WOX3 Overexpression of a Rice NPR1 Homolog Leads to Constitutive Activation of Defense Response and Hypersensitivity to Light 2005 Molecular Plant-Microbe Interactions Department of Plant Pathology, University of California, Davis 95616, USA. Arabidopsis NPR1/NIM1 is a key regulator of systemic acquired resistance (SAR), which confers lasting broad-spectrum resistance. Previous reports indicate that rice has a disease-resistance pathway similar to the Arabidopsis SAR pathway. Here we report the isolation and characterization of a rice NPR1 homologue (NH1). Transgenic rice plants overexpressing NH1 (NH1ox) acquire high levels of resistance to Xanthomonas oryzae pv. oryzae. The resistance phenotype is heritable and correlates with the presence of the transgene and reduced bacterial growth. Northern analysis shows that NH1ox rice spontaneously activates defense genes, contrasting with NPR1-overexpressing Arabidopsis, where defense genes are not activated until induction. Wild-type NH1, but not a point mutant corresponding to npr1-1, interacts strongly with the rice transcription factor rTGA2.2 in yeast two-hybrid. Greenhouse-grown NH1ox plants develop lesion-mimic spots on leaves at preflowering stage although no other developmental effects are observed. However, when grown in growth chambers (GCs) under low light, NH1ox plants are dwarfed, indicating elevated sensitivity to light. The GC-grown NH1ox plants show much higher salicylic acid (SA) levels than the wild type, whereas greenhouse-grown NH1ox plants contain lower SA. These results indicate that NH1 may be involved in the regulation of SA in response to environmental changes. NH2 Functional analysis of rice NPR1-like genes reveals that OsNPR1/NH1 is the rice orthologue conferring disease resistance with enhanced herbivore susceptibility 2007 Plant Biotechnol J National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. The key regulator of salicylic acid (SA)-mediated resistance, NPR1, is functionally conserved in diverse plant species, including rice (Oryza sativa L.). Investigation in depth is needed to provide an understanding of NPR1-mediated resistance and a practical strategy for the improvement of disease resistance in the model crop rice. The rice genome contains five NPR1-like genes. In our study, three rice homologous genes, OsNPR1/NH1, OsNPR2/NH2 and OsNPR3, were found to be induced by rice bacterial blight Xanthomonas oryzae pv. oryzae and rice blast Magnaporthe grisea, and the defence molecules benzothiadiazole, methyl jasmonate and ethylene. We confirmed that OsNPR1 is the rice orthologue by complementing the Arabidopsis npr1 mutant. Over-expression of OsNPR1 conferred disease resistance to bacterial blight, but also enhanced herbivore susceptibility in transgenic plants. The OsNPR1-green fluorescent protein (GFP) fusion protein was localized in the cytoplasm and moved into the nucleus after redox change. Mutations in its conserved cysteine residues led to the constitutive localization of OsNPR1(2CA)-GFP in the nucleus and also abolished herbivore hypersensitivity in transgenic rice. Different subcellular localizations of OsNPR1 antagonistically regulated SA- and jasmonic acid (JA)-responsive genes, but not SA and JA levels, indicating that OsNPR1 might mediate antagonistic cross-talk between the SA- and JA-dependent pathways in rice. This study demonstrates that rice has evolved an SA-mediated systemic acquired resistance similar to that in Arabidopsis, and also provides a practical approach for the improvement of disease resistance without the penalty of decreased herbivore resistance in rice. NH2,OsNPR1|NH1 Enhanced disease resistance and hypersensitivity to BTH by introduction of an NH1/OsNPR1 paralog 2011 Plant Biotechnol J Department of Plant Pathology, University of California, Davis, CA, USA. Non-expresser of pathogenesis-related genes 1 (NPR1) is the master regulator of salicylic acid-mediated systemic acquired resistance. Over-expression of Arabidopsis NPR1 and rice NH1 (NPR1 homolog1)/OsNPR1 in rice results in enhanced resistance. While there are four rice NPR1 paralogs in the rice genome, none have been demonstrated to function in disease resistance. To study rice NPR1 paralog 3, we introduced constructs into rice and tested for effects on resistance to infection by Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight. While over-expression of NH3 using the maize ubiquitin-1 promoter failed to enhance resistance, introduction of an extra copy of NH3 driven by its own promoter (nNT-NH3) resulted in clear, enhanced resistance. Progeny analysis confirms that the enhanced resistance phenotype, measured by Xoo-induced lesion length, is associated with the NH3 transgene. Bacterial growth curve analysis indicates that bacterial population levels are reduced 10-fold in nNT-NH3 lines compared to control rice lines. The transgenic plants exhibit higher sensitivity to benzothiadiazole (BTH) and 2,6-dichloroisonicotinic acid (INA) treatment as measured by increased cell death. Expression analysis of pathogenesis-related (PR) genes showed that nNT-NH3 plants display greatly enhanced induction of PR genes only after treatment with BTH. Our study demonstrates an alternative method to employ a regulatory protein to enhance plant defence. This approach avoids using undesirable constitutive, high-level expression and may prove to be more practical for engineering resistance. NH3 A nitrate-inducible GARP family gene encodes an auto-repressible transcriptional repressor in rice 2013 Plant Cell Physiol Biotechnology Research Center, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657 Japan. Nitrogen is the most important macronutrient in plants and its supply induces responses in gene expression, metabolism and developmental processes. However, the molecular mechanisms underlying the nitrogen responses remain poorly understood. Here we show that the supply of nitrate but not ammonium immediately induces the expression of a transcriptional repressor gene in rice, designated NIGT1 (Nitrate-Inducible, GARP-type Transcriptional Repressor 1). The results of DNA-binding site selection experiments and electrophoretic mobility shift assays indicated that NIGT1 binds to DNA containing either of two consensus sequences, GAATC or GAATATTC. In transient reporter assays, NIGT1 was found to repress transcription from the promoters containing the identified NIGT1-binding sequences in vivo. Furthermore, NIGT1 repressed the activity of its own promoter, suggesting an autorepression mechanism. Consistently, nitrate-induced NIGT1 expression was found to be down-regulated after a transient peak during nitrate treatment, and the nitrate-induced expression of NIGT1 decreased in transgenic rice plants in which this gene was constitutively overexpressed. Furthermore, the chlorophyll content that could be a marker of nitrogen utilization was found to be decreased in NIGT1 overexpressors of rice grown with nitrate medium but not with ammonium medium. Thus, we propose NIGT1 as a nitrate-inducible and autorepressible transcriptional repressor that may play a role in the nitrogen response in rice. Taken together with the fact that the NIGT1-binding sites are conserved in promoter sequences of Arabidopsis NIGT1 homologs, our findings imply the presence of a time-dependent complex system for nitrate-responsive transcriptional regulation that is conserved in both monocots and dicots. NIGT1 Semi-dominant mutations in the CC-NB-LRR-type R gene, NLS1, lead to constitutive activation of defense responses in rice 2011 Plant J State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. In this study, we characterized the semi-dominant mutant nls1-1D (necrotic leaf sheath 1) of rice, which displays spontaneous lesions, specifically on leaf sheaths, with a developmental pattern. nls1-1D plants also exhibited constitutively activated defense responses, including extensive cell death, excess hydrogen peroxide and salicylic acid (SA) accumulation, up-regulated expressions of pathogenesis-related genes, and enhanced resistance to bacterial pathogens. Map-based cloning revealed that NLS1 encodes a typical CC-NB-LRR-type protein in rice. The nls1-1D mutation causes a S367N substitution in the non-conserved region close to the GLPL motif of the NB domain. An adjacent S366T substitution was found in another semi-dominant mutant, nls1-2D, which exhibited the same phenotypes as nls1-1D. Combined analyses of wild-type plants transformed with the mutant NLS1 gene (nls1-1D), NLS1 RNAi and over-expression transgenic lines showed that nls1-2D is allelic to nls1-1D, and both mutations may cause constitutive auto-activation of the NLS1 R protein. Further real-time PCR analysis revealed that NLS1 is expressed constitutively in an age-dependent manner. In addition, because the morphology and constitutive defense responses of nls1-1D were not suppressed by blocking SA or NPR1 transcript accumulation, we suggest that NLS1 mediates both SA and NPR1-independent defense signaling pathways in rice. NLS1,OsNPR1|NH1 Genome-wide analysis of the core DNA replication machinery in the higher plants Arabidopsis and rice 2007 Plant Physiol Department of Plant Biology , North Carolina State University, Raleigh, North Carolina 27695, USA. Core DNA replication proteins mediate the initiation, elongation, and Okazaki fragment maturation functions of DNA replication. Although this process is generally conserved in eukaryotes, important differences in the molecular architecture of the DNA replication machine and the function of individual subunits have been reported in various model systems. We have combined genome-wide bioinformatic analyses of Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) with published experimental data to provide a comprehensive view of the core DNA replication machinery in plants. Many components identified in this analysis have not been studied previously in plant systems, including the GINS (go ichi ni san) complex (PSF1, PSF2, PSF3, and SLD5), MCM8, MCM9, MCM10, NOC3, POLA2, POLA3, POLA4, POLD3, POLD4, and RNASEH2. Our results indicate that the core DNA replication machinery from plants is more similar to vertebrates than single-celled yeasts (Saccharomyces cerevisiae), suggesting that animal models may be more relevant to plant systems. However, we also uncovered some important differences between plants and vertebrate machinery. For example, we did not identify geminin or RNASEH1 genes in plants. Our analyses also indicate that plants may be unique among eukaryotes in that they have multiple copies of numerous core DNA replication genes. This finding raises the question of whether specialized functions have evolved in some cases. This analysis establishes that the core DNA replication machinery is highly conserved across plant species and displays many features in common with other eukaryotes and some characteristics that are unique to plants. NOC3,OsORC4,OsORC5,OsORC6 Two short-chain dehydrogenase/reductases, NON-YELLOW COLORING 1 and NYC1-LIKE, are required for chlorophyll b and light-harvesting complex II degradation during senescence in rice 2009 Plant J Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan. Yellowing, which is related to the degradation of chlorophyll and chlorophyll-protein complexes, is a notable phenomenon during leaf senescence. NON-YELLOW COLORING 1 (NYC1) in rice encodes a membrane-localized short-chain dehydrogenase/reductase (SDR) that is thought to represent a chlorophyll b reductase necessary for catalyzing the first step of chlorophyll b degradation. Analysis of the nyc1 mutant, which shows the stay-green phenotype, revealed that chlorophyll b degradation is required for the degradation of light-harvesting complex II and thylakoid grana in leaf senescence. Phylogenetic analysis further revealed the existence of NYC1-LIKE (NOL) as the most closely related protein to NYC1. In the present paper, the nol mutant in rice was also found to show a stay-green phenotype very similar to that of the nyc1 mutant, i.e. the degradation of chlorophyll b was severely inhibited and light-harvesting complex II was selectively retained during senescence, resulting in the retention of thylakoid grana even at a late stage of senescence. The nyc1 nol double mutant did not show prominent enhancement of inhibition of chlorophyll degradation. NOL was localized on the stromal side of the thylakoid membrane despite the lack of a transmembrane domain. Immunoprecipitation analysis revealed that NOL and NYC1 interact physically in vitro. These observations suggest that NOL and NYC1 are co-localized in the thylakoid membrane and act in the form of a complex as a chlorophyll b reductase in rice. NOL,NYC1 Rice NON-YELLOW COLORING1 is involved in light-harvesting complex II and grana degradation during leaf senescence 2007 Plant Cell Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. akusaba@mail.ecc.u-tokyo.ac.jp Chlorophyll degradation is an aspect of leaf senescence, which is an active process to salvage nutrients from old tissues. non-yellow coloring1 (nyc1) is a rice (Oryza sativa) stay-green mutant in which chlorophyll degradation during senescence is impaired. Pigment analysis revealed that degradation of not only chlorophylls but also light-harvesting complex II (LHCII)-bound carotenoids was repressed in nyc1, in which most LHCII isoforms were selectively retained during senescence. Ultrastructural analysis of nyc1 chloroplasts revealed that large and thick grana were present even in the late stage of senescence, suggesting that degradation of LHCII is required for the proper degeneration of thylakoid membranes. Map-based cloning of NYC1 revealed that it encodes a chloroplast-localized short-chain dehydrogenase/reductase (SDR) with three transmembrane domains. The predicted structure of the NYC1 protein and the phenotype of the nyc1 mutant suggest the possibility that NYC1 is a chlorophyll b reductase. Although we were unable to detect the chlorophyll b reductase activity of NYC1, NOL (for NYC1-like), a protein closely related to NYC1 in rice, showed chlorophyll b reductase activity in vitro. We suggest that NYC1 and NOL encode chlorophyll b reductases with divergent functions. Our data collectively suggest that the identified SDR protein NYC1 plays essential roles in the regulation of LHCII and thylakoid membrane degradation during senescence. NOL,NYC1 Rice plastidial N-glycosylated nucleotide pyrophosphatase/phosphodiesterase is transported from the ER-golgi to the chloroplast through the secretory pathway 2006 Plant Cell Laboratory of Plant and Microbial Genome Control, Department of Applied Biological Chemistry, Niigata University, Niigata 950-2181, Japan. A nucleotide pyrophosphatase/phosphodiesterase (NPP) activity that catalyzes the hydrolytic breakdown of ADP-glucose (ADPG) has been shown to occur in the plastidial compartment of both mono- and dicotyledonous plants. To learn more about this enzyme, we purified two NPPs from rice (Oryza sativa) and barley (Hordeum vulgare) seedlings. Both enzymes are glycosylated, since they bind to concanavalin A, stain with periodic acid-Schiff reagent, and are digested by Endo-H. A complete rice NPP cDNA, designated as NPP1, was isolated, characterized, and overexpressed in transgenic plants displaying high ADPG hydrolytic activity. Databank searches revealed that NPP1 belongs to a functionally divergent group of plant nucleotide hydrolases. NPP1 contains numerous N-glycosylation sites and a cleavable hydrophobic signal sequence that does not match with the N-terminal part of the mature protein. Both immunocytochemical analyses and confocal fluorescence microscopy of rice cells expressing NPP1 fused with green fluorescent protein (GFP) revealed that NPP1-GFP occurs in the plastidial compartment. Brefeldin A treatment of NPP1-GFP-expressing cells prevented NPP1-GFP accumulation in the chloroplasts. Endo-H digestibility studies revealed that both NPP1 and NPP1-GFP in the chloroplast are glycosylated. Collectively, these data demonstrate the trafficking of glycosylated proteins from the endoplasmic reticulum-Golgi system to the chloroplast in higher plants. NPP1|OsPAP27b Identification of rice purple acid phosphatases related to phosphate starvation signalling 2011 Plant Biol (Stuttg) State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou, China. Purple acid phosphatases (PAPs) are a family of metallo-phosphoesterases involved in a variety of physiological functions, especially phosphate deficiency adaptations in plants. We identified 26 putative PAP genes by a genome-wide analysis of rice (Oryza sativa), 24 of which have isolated EST sequences in the dbEST database. Amino acid sequence analysis revealed that 25 of these genes possess sets of metal-ligating residues typical of known PAPs. Phylogenetic analysis classified the 26 rice and 29 Arabidopsis PAPs into three main groups and seven subgroups. We detected transcripts of 21 PAP genes in roots or leaves of rice seedlings. The expression levels of ten PAP genes were up-regulated by both phosphate deprivation and over-expression of the transcription factor OsPHR2. These PAP genes all contained one or two OsPHR2 binding elements in their promoter regions, implying that they are directly regulated by OsPHR2. Both acid phosphatase (AP) and surface secretory acid phosphatase (SAP) activity assays showed that the up-regulation of PAPs by Pi starvation, OsPHR2 over-expression, PHO2 knockout or OsSPX1 RNA interference led to an increase in AP and SAP activity in rice roots. This study reveals the potential for developing technologies for crop improvement in phosphorus use efficiency. NPP1|OsPAP27b A novel rice gene, NRR responds to macronutrient deficiency and regulates root growth 2012 Mol Plant State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. To better understand the response of rice to nutrient stress, we have taken a systematic approach to identify rice genes that respond to deficiency of macronutrients and affect rice growth. We report here the expression and biological functions of a previously uncharacterized rice gene that we have named NRR (nutrition response and root growth). NRR is alternatively spliced, producing two 5'-coterminal transcripts, NRRa and NRRb, encoding two proteins of 308 and 223 aa, respectively. Compared to NRRb, NRRa possesses an additional CCT domain at the C-terminus. Expression of NRR in rice seedling roots was significantly influenced by deficiency of macronutrients. Knock-down of expression of NRRa or NRRb by RNA interference resulted in enhanced rice root growth. By contrast, overexpression of NRRa in rice exhibited significantly retarded root growth. These results revealed that both NRRa and NRRb played negative regulatory roles in rice root growth. Our findings suggest that NRRa and NRRb, acting as the key components, modulate the rice root architecture with the availability of macronutrients. NRR A rice transient assay system identifies a novel domain in NRR required for interaction with NH1/OsNPR1 and inhibition of NH1-mediated transcriptional activation 2012 Plant Methods Department of Plant Pathology, University of California, Davis, Davis, CA 95616, USA. pcronald@ucdavis.edu. BACKGROUND: Arabidopsis NPR1 is a master regulator of systemic acquired resistance. NPR1 binds to TGA transcription factors and functions as a transcriptional co-activator. In rice, NH1/OsNPR1 functions to enhance innate immunity. NRR disrupts NH1 function, when over-expressed. RESULTS: We have established a rice transient protoplast assay to demonstrate that NH1 is a transcriptional co-activator and that NRR represses NH1-mediated activation. We identified three NRR homologues (RH1, RH2, and RH3). RH1 and RH3, but not RH2, also effectively repress NH1-mediated transcriptional activation. NRR, RH1, RH2, and RH3 share sequence similarity in a region beyond the previously identified NPR1-interacting domain. This region is required for strong interaction with NH1. A double point mutation, W66A/F70A, in this novel NH1-interacting domain severely reduces interaction with NH1. Mutation W66A/F70A also greatly reduces the ability of NRR to repress NH1-mediated activation. RH2 carries a deviation (amino acids AV) in this region as compared to consensus sequences (amino acids ED) among NRR, RH1, and RH3. A substitution (AV to ED) in RH2 results in strong binding of mutant RH2ED to NH1 and effective repression of NH1-mediated activation. CONCLUSIONS: The protoplast-based transient system can be used to dissect protein domains associated with their functions. Our results demonstrate that the ability of NRR and its homologues to repress NH1-mediated transcriptional activation is tightly correlated with their ability to bind to NH1. Furthermore, a sequence is identified as a novel NH1-interacting domain. Importantly, this novel sequence is widely present in plant species, from cereals to castor bean plants, to poplar trees, to Arabidopsis, indicating its significance in plants. NRR,OsNPR1|NH1,RH1,RH2,RH3 Rice NRR, a negative regulator of disease resistance, interacts with Arabidopsis NPR1 and rice NH1 2005 Plant J Department of Plant Pathology, University of California, Davis, CA 95616, USA. Arabidopsis NPR1/NIM1 is a key regulator of systemic acquired resistance (SAR), which confers lasting broad-spectrum resistance. Over-expression of Arabidopsis NPR1 or the NPR1 homolog 1 (NH1) in rice results in enhanced resistance to the pathogen Xanthomonasoryzae pv. oryzae (Xoo), suggesting the presence of a related defense pathway in rice. We investigated this pathway in rice by identifying proteins that interact with NH1. Here we report the isolation and characterization of a rice cDNA encoding a novel protein, named NRR (for negative regulator of resistance). NRR interacts with NPR1 in the NPR1-interacting domain (NI25) consisting of 25 amino acids. NRR also interacts with NH1; however, NI25 was not sufficient for a strong interaction, indicating a difference between the rice and the Arabidopsis proteins. Silencing of NRR in rice had little effect on resistance to Xoo. When constitutively over-expressed in rice, NRR affected basal resistance, age-related resistance and Xa21-mediated resistance, causing enhanced susceptibility to Xoo. This phenotype was correlated with elevated NRR mRNA and protein levels and increased Xoo growth. Over-expression of NRR suppressed the induction of defense-related genes. NRR:GFP (green fluorescent protein) protein was localized to the nucleus, indicating that NRR may act directly to suppress the activation of defense genes. The fact that NRR compromises Xa21-mediated resistance indicates cross-talk or overlap between NH1- and Xa21-mediated pathways. NRR,OsNPR1|NH1 Suppression of expression of the putative receptor-like kinase gene NRRB enhances resistance to bacterial leaf streak in rice 2014 Mol Biol Rep Xiamen Key Laboratory for Plant Genetics, School of Life Sciences, Xiamen University, Xiamen, 361005, People's Republic of China, heartone@126.com. Bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola (Xoc) is an important disease of rice, which is responsible for the economic losses worldwide. Functional investigation of differentially expressed protein genes (DEPGs) from rice (Oryza sativa L.) upon Xoc infection provides insight into the molecular mechanism of rice-Xoc interactions. Here, we show that one of DEPGs designated NRRB plays a role in rice-Xoc interactions. NRRB, a receptor-like cytoplasmic kinase gene was preferentially expressed in leaf blades and leaf sheaths where the pathogen colonized. Its transcription was depressed by two defense-signal compounds salicylic acid and 1-aminocyclopropane-1-carboxylic-acid, but was activated by wounding and abscisic acid. Additionally, a plenty of cis-elements associated with stress responses were discovered in the promoter region of NRRB. These data suggest that NRRB is involved in stress responses. More importantly, the NRRB-suppressing rice plants exhibited enhanced resistance against BLS, with the markedly shorter average lesion length than that of the wild type. Furthermore, transcription of some salicylic acid synthesis-related and pathogenesis-related genes including PAD4, PR1a and WRKY13 in transgenic plants was activated, implying that enhanced resistance to BLS might be mediated by the activation of the SA signaling pathway. In conclusion, NRRB gene is involved in various stress responses and regulating resistance to BLS, therefore it might be one of useful genes for rice improvement in future. NRRB,OsPR1a,OsWRKY13 Rice OsNAR2.1 interacts with OsNRT2.1, OsNRT2.2 and OsNRT2.3a nitrate transporters to provide uptake over high and low concentration ranges 2011 Plant Cell Environ State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, China. Plants take up both nitrate and ammonium as main nitrogen (N) sources. Although ammonium is the predominant form in anaerobic-flooded paddy soil, it has been proposed that rice and other wetland plants may take up significant amounts of nitrate formed by nitrification of ammonium in the rhizosphere. A two-component system for nitrate transport including NRT2s with a partner protein (NAR2 or NRT3.1) has been identified in Arabidopsis. We report the physiological function of another member of the NAR2 family, OsNAR2.1 in rice (Oryza sativa, ssp. Japonica, cv. Nipponbare). OsNAR2.1 was mainly expressed in roots and induced by nitrate and suppressed by ammonium and some amino acids. Knockdown of OsNAR2.1 by RNA interference synchronously suppressed expression of OsNRT2.1, OsNRT2.2 and OsNRT2.3a in the osnar2.1mutants. Both high- and low-affinity nitrate transports were greatly impaired by OsNAR2.1 knockdown. Yeast two hybridization showed that OsNAR2.1 not only interacted with OsNRT2.1/OsNRT2.2, but also with OsNRT2.3a. Taken together, the data demonstrate that OsNAR2.1 plays a key role in enabling the plant to cope with variable nitrate supply. NRT1,OsNRT2.2,OsNAR2.1,OsNRT2.3a|OsNRT2.3b,OsNAR2.2 Cloning and functional characterization of a constitutively expressed nitrate transporter gene, OsNRT1, from rice 2000 Plant Physiol Department of Life Science, School of Life Science, National Tsing Hua University, 30043, Hsin-Chu, Taiwan. Elucidating how rice (Oryza sativa) takes up nitrate at the molecular level could help improve the low recovery rate (<50%) of nitrogen fertilizer in rice paddies. As a first step toward that goal, we have cloned a nitrate transporter gene from rice called OsNRT1. OsNRT1 is a new member of a growing transporter family called PTR, which consists not only of nitrate transporters from higher plants that are homologs of the Arabidopsis CHL1 (AtNRT1) protein, but also peptide transporters from a wide variety of genera including animals, plants, fungi, and bacteria. However, despite the fact that OsNRT1 shares a higher degree of sequence identity with the two peptide transporters from plants (approximately 50%) than with the nitrate transporters (approximately 40%) of the PTR family, no peptide transport activity was observed when OsNRT1 was expressed in either Xenopus oocytes or yeast. Furthermore, contrasting the dual-affinity nitrate transport activity of CHL1, OsNRT1 displayed only low-affinity nitrate transport activity in Xenopus oocytes, with a K(m) value of approximately 9 mM. Northern-blot and in situ hybridization analysis indicated that OsNRT1 is constitutively expressed in the most external layer of the root, epidermis and root hair. These data strongly indicate that OsNRT1 encodes a constitutive component of a low-affinity nitrate uptake system for rice. NRT1 Knockdown of a rice stelar nitrate transporter alters long-distance translocation but not root influx 2012 Plant Physiol State Key Laboratory of Crop Genetics and Germplasm Enhancement and Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China. Root nitrate uptake is well known to adjust to the plant's nitrogen demand for growth. Long-distance transport and/or root storage pools are thought to provide negative feedback signals regulating root uptake. We have characterized a vascular specific nitrate transporter belonging to the high-affinity Nitrate Transporter2 (NRT2) family, OsNRT2.3a, in rice (Oryza sativa ssp. japonica 'Nipponbare'). Localization analyses using protoplast expression, in planta promoter-beta-glucuronidase assay, and in situ hybridization showed that OsNRT2.3a was located in the plasma membrane and mainly expressed in xylem parenchyma cells of the stele of nitrate-supplied roots. Knockdown expression of OsNRT2.3a by RNA interference (RNAi) had impaired xylem loading of nitrate and decreased plant growth at low (0.5 mm) nitrate supply. In comparison with the wild type, the RNAi lines contained both nitrate and total nitrogen significantly higher in the roots and lower in the shoots. The short-term [(15)N]NO(3)(-) influx (5 min) in entire roots and NO(3)(-) fluxes in root surfaces showed that the knockdown of OsNRT2.3a in comparison with the wild type did not affect nitrate uptake by roots. The RNAi plants showed no significant changes in the expression of some root nitrate transporters (OsNRT2.3b, OsNRT2.4, and OsNAR2.1), but transcripts for nia1 (nitrate reductase) had increased and OsNRT2.1 and OsNRT2.2 had decreased when the plants were supplied with nitrate. Taken together, the data demonstrate that OsNRT2.3a plays a key role in long-distance nitrate transport from root to shoot at low nitrate supply level in rice. OsNRT2.2,OsNRT2.4,OsNRT2.3a|OsNRT2.3b,OsNIA1|OsNia1 Spatial expression and regulation of rice high-affinity nitrate transporters by nitrogen and carbon status 2011 J Exp Bot State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, PR China. ghxu@njau.edu.cn The high affinity nitrate transport system (HATS) plays an important role in rice nitrogen acquisition because, even under flooded anaerobic cultivation when NH(4)(+) dominates, significant nitrification occurs on the root surface. In the rice genome, four NRT2 and two NAR2 genes encoding HATS components have been identified. One gene OsNRT2.3 was mRNA spliced into OsNRT2.3a and OsNRT2.3b and OsNAR2.1 interacts with OsNRT2.1/2.2 and OsNRT2.3a to provide nitrate uptake. Using promoter-GUS reporter plants and semi-quantitative RT-PCR analyses, it was observed that OsNAR2.1 was expressed mainly in the root epidermal cells, differently from the five OsNRT2 genes. OsNAR2.1, OsNRT2.1, OsNRT2.2, and OsNRT2.3a were up-regulated by nitrate and suppressed by NH(4)(+) and high root temperature (37 degrees C). Expression of all these genes was increased by light or external sugar supply. Root transcripts of OsNRT2.3b and OsNRT2.4 were much less abundant and not affected by temperature. Expression of OsNRT2.3b was insensitive to the form of N supply. Expression of OsNRT2.4 responded to changes in auxin supply unlike all the other NRT2 genes. A region from position -311 to -1, relative to the translation start site in the promoter region of OsNAR2.1, was found to contain the cis-element(s) necessary for the nitrate-, but not light- and sugar-dependent activation. However, it was difficult to define a conserved cis-element in the promoters of the nitrate-regulated OsNRT2/OsNAR2 genes. The results imply distinct physiological functions for each OsNRT2 transporter, and differential regulation pathways by N and C status. OsNRT2.2,OsNRT2.4,OsNAR2.1,OsNRT2.3a|OsNRT2.3b,OsNAR2.2 High-affinity nitrate uptake by rice (Oryza sativa) coleoptiles 2011 J Plant Res Graduate School of Environmental Science, University of Shiga Prefecture, 2500 Hassaka-cho, Hikone, Shiga 522-8533, Japan. Nitrate uptake by rice coleoptiles was evaluated using (1)(5)N-nitrate in relation to the expression of high-affinity nitrate uptake-related genes, OsNRT2s (OsNRT2.1-2.4) and OsNAR2s (OsNAR2.1 and 2.2). Apparent nitrate uptake by coleoptiles was about one-sixth of that by hydroponically cultured seedling roots. Real-time RT-PCR analysis revealed that OsNRT2.1, a root-specific key gene of inducible high-affinity transport system for nitrate, was most strongly induced in coleoptiles following nitrate supply initiation, while other OsNRT2s and OsNAR2s showed modest induction. These results suggest that rice coleoptiles may have high-affinity transport systems for nitrate similar to roots, and can be model organs for nutrient uptake by submerged plant shoots. OsNRT2.2,OsNRT2.4,OsNAR2.2 Rice NTRC is a high-efficiency redox system for chloroplast protection against oxidative damage 2006 Plant Cell Instituto de Bioquimica Vegetal y Fotosintesis, Centro de Investigaciones Cientificas Isla de la Cartuja, 41092 Seville, Spain. One of the mechanisms plants have developed for chloroplast protection against oxidative damage involves a 2-Cys peroxiredoxin, which has been proposed to be reduced by ferredoxin and plastid thioredoxins, Trx x and CDSP32, the FTR/Trx pathway. We show that rice (Oryza sativa) chloroplast NADPH THIOREDOXIN REDUCTASE (NTRC), with a thioredoxin domain, uses NADPH to reduce the chloroplast 2-Cys peroxiredoxin BAS1, which then reduces hydrogen peroxide. The presence of both NTR and Trx-like domains in a single polypeptide is absolutely required for the high catalytic efficiency of NTRC. An Arabidopsis thaliana knockout mutant for NTRC shows irregular mesophyll cell shape, abnormal chloroplast structure, and unbalanced BAS1 redox state, resulting in impaired photosynthesis rate under low light. Constitutive expression of wild-type NTRC in mutant transgenic lines rescued this phenotype. Moreover, prolonged darkness followed by light/dark incubation produced an increase in hydrogen peroxide and lipid peroxidation in leaves and accelerated senescence of NTRC-deficient plants. We propose that NTRC constitutes an alternative system for chloroplast protection against oxidative damage, using NADPH as the source of reducing power. Since no light-driven reduced ferredoxin is produced at night, the NTRC-BAS1 pathway may be a key detoxification system during darkness, with NADPH produced by the oxidative pentose phosphate pathway as the source of reducing power. NTRC,CDSP32 Defect in non-yellow coloring 3, an alpha/beta hydrolase-fold family protein, causes a stay-green phenotype during leaf senescence in rice 2009 Plant J Institute of Radiation Breeding, National Institute of Agrobiological Sciences, Hitachi-ohmiya 219-2293, Japan. Chlorophyll degradation is an important phenomenon in the senescence process. It is necessary for the degradation of certain chlorophyll-protein complexes and thylakoid membranes during leaf senescence. Mutants retaining greenness during leaf senescence are known as 'stay-green' mutants. Non-functional type stay-green mutants, which possess defects in chlorophyll degradation, retain greenness but not leaf functionality during senescence. Here, we report a new stay-green mutant in rice, nyc3. nyc3 retained a higher chlorophyll a and chlorophyll b content than the wild-type but showed a decrease in other senescence parameters during dark incubation, suggesting that it is a non-functional stay-green mutant. In addition, a small amount of pheophytin a, a chlorophyll a-derivative without Mg(2+) ions in its tetrapyrrole ring, accumulated in the senescent leaves of nyc3. nyc3 shows a similar but weaker phenotype to stay green (sgr), another non-functional stay-green mutant in rice. The chlorophyll content of nyc3 sgr double mutants at the late stage of leaf senescence was also similar to that of sgr. Linkage analysis revealed that NYC3 is located near the centromere region of chromosome 6. Map-based cloning of genes near the centromere is very difficult because of the low recombination rate; however, we overcame this problem by using ionizing radiation-induced mutant alleles harboring deletions of hundreds of kilobases. Thus, it was revealed that NYC3 encodes a plastid-localizing alpha/beta hydrolase-fold family protein with an esterase/lipase motif. The possible function of NYC3 in the regulation of chlorophyll degradation is discussed. NYC3 NYC4, the rice ortholog of Arabidopsis THF1, is involved in the degradation of chlorophyll - protein complexes during leaf senescence 2013 Plant J Graduate School of Science, Hiroshima University, Higashi-Hiroshima, 739-8526, Japan. Yellowing/chlorophyll breakdown is a prominent phenomenon in leaf senescence, and is associated with the degradation of chlorophyll - protein complexes. From a rice mutant population generated by ionizing radiation, we isolated nyc4-1, a stay-green mutant with a defect in chlorophyll breakdown during leaf senescence. Using gene mapping, nyc4-1 was found to be linked to two chromosomal regions. We extracted Os07g0558500 as a candidate for NYC4 via gene expression microarray analysis, and concluded from further evidence that disruption of the gene by a translocation-related event causes the nyc4 phenotype. Os07g0558500 is thought to be the ortholog of THF1 in Arabidopsis thaliana. The thf1 mutant leaves show variegation in a light intensity-dependent manner. Surprisingly, the Fv /Fm value remained high in nyc4-1 during the dark incubation, suggesting that photosystem II retained its function. Western blot analysis revealed that, in nyc4-1, the PSII core subunits D1 and D2 were significantly retained during leaf senescence in comparison with wild-type and other non-functional stay-green mutants, including sgr-2, a mutant of the key regulator of chlorophyll degradation SGR. The role of NYC4 in degradation of chlorophyll and chlorophyll - protein complexes during leaf senescence is discussed. NYC4 The syncytium-specific expression of the Orysa;KRP3 CDK inhibitor: implication of its involvement in the cell cycle control in the rice (Oryza sativa L.) syncytial endosperm 2010 J Exp Bot United Graduate School of Agricultural Sciences, Iwate University, 3-18-8, Ueda, Morioka, Iwate 020-8550, Japan. During rice (Oryza sativa L.) seed development, the primary endosperm nucleus undergoes a series of divisions without cytokinesis, producing a multinucleate cell, known as a syncytium. After several rounds of rapid nuclear proliferation, the syncytium ceases to undergo mitosis; thereafter, the syncytium is partitioned into individual cells by a specific type of cytokinesis called cellularization. The transition between syncytium and cellularization is important in determining the final seed size and is a model for studying the cell cycle and cytokinesis. The involvement of cyclin-dependent kinase (CDK) inhibitors (CKIs) in cell cycle control was investigated here during the transition between syncytium and cellularization. It was found that one of the rice CKIs, Orysa;KRP3, is strongly expressed in the caryopsis at 2 d after flowering (DAF), and its expression is significantly reduced at 3 DAF. The other CKI transcripts did not show such a shift at 2 DAF. In situ hybridization analysis revealed that Orysa;KRP3 is expressed in multinucleate syncytial endosperm at 2 DAF, but not in cellularized endosperm at 3 DAF. Two-hybrid assays showed that Orysa;KRP3 binds Orysa;CDKA;1, Orysa;CDKA;2, Orysa;CycA1;1, and Orysa;CycD2;2. By contrast, Orysa;CDKB2;1 and Orysa;CycB2;2 do not show binding to Orysa;KRP3. Orysa;KRP3 was able to rescue yeast premature cell division due to the dominant positive expression of mutant rice CDKA;1 indicating that Orysa;KRP3 inhibited rice CDK. These data suggest that Orysa;KRP3 is involved in cell cycle control of syncytial endosperm. KRP3 Characterization of rice anthranilate synthase alpha-subunit genes OASA1 and OASA2. Tryptophan accumulation in transgenic rice expressing a feedback-insensitive mutant of OASA1 2001 Plant Physiol National Agriculture Research Center, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8666, Japan. Anthranilate synthase (AS) is a key enzyme in the synthesis of tryptophan (Trp), indole-3-acetic acid, and indole alkaloids. Two genes, OASA1 and OASA2, encoding AS alpha-subunits were isolated from a monocotyledonous plant, rice (Oryza sativa cv Nipponbare), and were characterized. A phylogenetic tree of AS alpha-subunits from various species revealed a close evolutionary relationship among OASA1 and Arabidopsis ASA2, Ruta graveolens AS alpha 2, and tobacco ASA2, whereas OASA2, Arabidopsis ASA1, and R. graveolens AS alpha 1 were more distantly related. OASA1 is expressed in all tissues tested, but the amount of its mRNA was greater in panicles than in leaves and roots. The abundance of OASA2 transcripts is similar among tissues and greater than that of OASA1 transcripts; furthermore, OASA2 expression was induced by a chitin heptamer, a potent elicitor, suggesting that OASA2 participates in secondary metabolism. Expression of wild-type OASA1 or OASA2 transgenes did not affect the Trp content of rice calli or plants. However, transformed calli and plants expressing a mutated OASA1 gene, OASA1(D323N), that encodes a protein in which aspartate-323 is replaced with asparagine manifested up to 180- and 35-fold increases, respectively, in Trp accumulation. These transgenic calli and plants were resistant to 300 microM 5-methyl-Trp, and AS activity of the calli showed a markedly reduced sensitivity to Trp. These results show that OASA1 is important in the regulation of free Trp concentration, and that mutation of OASA1 to render the encoded protein insensitive to feedback inhibition results in accumulation of Trp at high levels. The OASA1(D323N) transgene may prove useful for the generation of crops with an increased Trp content. OASA1,OASA2 Functional characterization of ObgC in ribosome biogenesis during chloroplast development 2012 Plant J Swine Science and Technology Center, Gyeongnam National University of Science and Technology-GNTECH, Jinju 660-758, Korea. The Spo0B-associated GTP-binding protein (Obg) GTPase, essential for bacterial viability, is also conserved in eukaryotes, but its primary role in eukaryotes remains unknown. Here, our functional characterization of Arabidopsis and rice obgc mutants strongly underlines the evolutionarily conserved role of eukaryotic Obgs in organellar ribosome biogenesis. The mutants exhibited a chlorotic phenotype, caused by retarded chloroplast development. A plastid DNA macroarray revealed a plastid-encoded RNA polymerase (PEP) deficiency in an obgc mutant, caused by incompleteness of the PEP complex, as its western blot exhibited reduced levels of RpoA protein, a component of PEP. Plastid rRNA profiling indicated that plastid rRNA processing is defective in obgc mutants, probably resulting in impaired ribosome biogenesis and, in turn, in reduced levels of RpoA protein. RNA co-immunoprecipitation revealed that ObgC specifically co-precipitates with 23S rRNA in vivo. These findings indicate that ObgC functions primarily in plastid ribosome biogenesis during chloroplast development. Furthermore, complementation analysis can provide new insights into the functional modes of three ObgC domains, including the Obg fold, G domain and OCT. OsObgC1 Characterization of a stress responsive proteinase inhibitor gene with positive effect in improving drought resistance in rice 2007 Planta National Center of Plant Gene Research (Wuhan), National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, China. A full-length cDNA gene, designated Oryza sativa chymotrypsin inhibitor-like 1 (OCPI1), was characterized in rice. The predicted protein of OCPI1 shows very high sequence identity to reported chymotrypsin inhibitors from various plant species. Northern-blot analysis showed that the expression of OCPI1 was strongly induced by dehydration stresses and abscisic acid (ABA). The expression of beta-glucuronidase (GUS) reporter gene under the control of OCPI1 promoter transformed into rice was strongly induced by drought and salt stresses. Interestingly, strong dehydration stress-induced GUS activity was also detected in the transgenic rice containing the reverse sequence of OCPI1 promoter fused to GUS gene, suggesting of a bidirectional transcriptional activity in the OCPI1 promoter. OCPI1 gene was over-expressed in japonica cv. Zhonghua 11 and transgenic plants containing single copy of transgene were tested for drought resistance at reproductive stage. The positive transgenic plants (OCPI1 was over-expressed) had significantly higher grain yield and seed setting rate than the wild type and the negative transgenic control (no over-expression of the transgene) under the severe drought stress conditions, whereas the potential yield of transgenic plants under normal growth conditions was not affected. Chymotrypsin-inhibitor activity assay showed that the crude protein of the positive transgenic plants had stronger inhibitory activity than the negative control. Transgenic plants had less decrease of total proteins than the wild type under drought stress. Taken together, these data indicate that OCPI1 might potentially be useful in the genetic improvement of drought resistance in rice. OCPI1 Rice OGR1 encodes a pentatricopeptide repeat-DYW protein and is essential for RNA editing in mitochondria 2009 Plant J Department of Integrative Bioscience and Biotechnology, National Research Laboratory of Plant Functional Genomics and Functional Genomic Center, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea. RNA editing is the alteration of RNA sequences via insertion, deletion and conversion of nucleotides. In flowering plants, specific cytidine residues of RNA transcribed from organellar genomes are converted into uridines. Approximately 35 editing sites are present in the chloroplasts of higher plants; six pentatricopeptide repeat genes involved in RNA editing have been identified in Arabidopsis. However, although approximately 500 editing sites are found in mitochondrial RNAs of flowering plants, only one gene in Arabidopsis has been reported to be involved in such editing. Here, we identified rice mutants that are defective in seven specific RNA editing sites on five mitochondrial transcripts. Their various phenotypes include delayed seed germination, retarded growth, dwarfism and sterility. Mutant seeds from heterozygous plants are opaque. This mutation, named opaque and growth retardation 1 (ogr1), was generated by T-DNA insertion into a gene that encodes a pentatricopeptide repeat protein containing the DYW motif. The OGR1-sGFP fusion protein is localized to mitochondria. Ectopic expression of OGR1 in the mutant complements the altered phenotypes. We conclude that OGR1 is essential for RNA editing in rice mitochondria and is required for normal growth and development. OGR1 Identification and characterization of cytokinin-signalling gene families in rice 2006 Gene Plant Genetics Laboratory, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka-ken 411-8540, Japan. We identified four histidine kinase (HK) genes of a cytokinin receptor family, two histidine-containing phosphotransmitter (HPt) genes, thirteen A-type response regulator (RR) genes and six B-type RR genes in the rice genome. The HK genes (OHK2, OHK3, OHK4 and OHK5 for Oryza sativa HK), the HPt genes (OHP1 and OHP2 for O. sativa HPt) and the B-type RR genes (ORR1, ORR2, ORR3, ORR4 and ORR6 for O. sativa RR) except one (ORR5) showed expression in various organs. ORR5 was expressed in callus and flower. Three A-type RR genes (OsRR4, OsRR9 and OsRR10 for O. sativa RR) showed cytokinin-induced expression, and three (OsRR8, OsRR12 and OsRR13) showed expression in flower. We also identified two other genes named OHK1 and CHARK (CHASE domain Receptor-like serine/threonine Kinase). OHK1 encodes an HK similar to Arabidopsis CKI1, which is involved in female gametophyte development. CHARK encodes a protein with an extracellular cytokinin-perceiving CHASE domain and a cytoplasmic serine/threonine kinase domain which are connected with a single transmembrane domain. The presence of all four gene families and CHARK in the rice genome suggests that a cytokinin signal is transduced by the phosphotransfer mechanism as is the case in Arabidopsis, and that rice may have an additional novel signalling pathway involving serine/threonine phosphorylation. OHK1,OHK2|OsHk3,OHK3|OsHk5,OHK4,OHK5|OsHk6,OHP1,OHP2,OsRR1,OsRR2,OsRR3,OsRR4,OsRR5,OsRR6,OsRR7,OsRR8,OsRR9 Functional identification of OsHk6 as a homotypic cytokinin receptor in rice with preferential affinity for iP 2012 Plant Cell Physiol Department of Life Sciences and Functional Genomics Center, Pohang University of Science and Technology, Pohang 790-784, Korea. Cytokinins are involved in key developmental processes in rice (Oryza sativa), including the regulation of cell proliferation and grain yield. However, the in vivo action of histidine kinases (OsHks), putative cytokinin receptors, in rice cytokinin signaling remains elusive. This study examined the function and characteristics of OsHk3, 4 and 6 in rice. OsHk6 was highly sensitive to isopentenyladenine (iP) and was capable of restoring cytokinin-dependent ARR6 reporter expression in the ahk2 ahk3 Arabidopsis mutant upon treatment with 1 nM iP. OsHk4 recognized trans-zeatin (tZ) and iP, while OsHk3 scarcely induced cytokinin signaling activity. OsHk4 and OsHk6 mediated the canonical two-component signaling cascade of Arabidopsis to induce phosphorylation of ARR2. OsHk4 and OsHk6 were highly expressed in spikelets, suggesting that tZ and iP might play key roles in grain development. OsHk6 formed a self-interacting homomer in rice protoplasts, although the trans-phosphorylation activity between subunits was much lower than the intra-molecular trans-phosphorylation activity. This indicates that the action mechanism of OsHks is evolutionarily diverged from bacterial histidine kinases. Ectopic expression of OsHk6 in rice calli promoted green pigmentation and subsequent shoot induction, further supporting an OsHk6 in planta function as a cytokinin receptor. From the results of this study, OsHks are homomeric cytokinin receptors with distinctive cytokinin preferences in rice. OHK2|OsHk3,OHK3|OsHk5,OHK4,OHK5|OsHk6 OIP30, a RuvB-like DNA helicase 2, is a potential substrate for the pollen-predominant OsCPK25/26 in rice 2011 Plant Cell Physiol Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan, 40227 ROC. Calcium ions are a well-known essential component for pollen germination and tube elongation. Several calcium-dependent protein kinases (CDPKs) are expressed predominantly in mature pollen grains and play a critical role in pollen. However, none of their interacting proteins or downstream substrates has been identified. Using yeast two-hybrid screening, we isolated OsCPK25/26-interacting protein 30 (OIP30), which is also predominantly expressed in pollen. OIP30 encodes a RuvB-like DNA helicase 2 (RuvBL2) that is well conserved in eukaryotic species from yeast to human. Yeast and Drosophila defective in RuvBL2 are non-viable. The interaction between OsCPK26 and OIP30 was confirmed by far-Western blot and pull-down experiments. OIP30 was phosphorylated in a calcium-dependent manner by OsCPK26 but not OsCPK2, which is highly similar to OsCPK26 in sequence and expression profile. OIP30 unwound partial duplex DNA with a 3' to 5' directionality by ATP hydrolysis. Concurrently, the ATPase activity of OIP30 depended on single-stranded DNA. OsCPK26 phosphorylated OIP30 and enhanced both its helicase and ATPase activity about 3-fold. OIP30 may be the potential downstream substrate for OsCPK25/26 in pollen. This report characterizes a RuvBL in plants and links its activities with its upstream regulator. OIP30,OsCPK25,OsCPK26 Conserved miR164-targeted NAC genes negatively regulate drought resistance in rice 2014 J Exp Bot National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. MicroRNAs constitute a large group of endogenous small RNAs of ~22 nt that emerge as vital regulators, mainly by targeting mRNAs for post-transcriptional repression. Previous studies have revealed that the miR164 family in Arabidopsis is comprised of three members which guide the cleavage of the mRNAs of five NAC genes to modulate developmental processes. However, the functions of the miR164-targeted NAC genes in crops are poorly deciphered. In this study, the conserved features of six miR164-targeted NAC genes (OMTN1-OMTN6) in rice are described, and evidence is provided that four of them confer a negative regulatory role in drought resistance. OMTN proteins have the characteristics of typical NAC transcriptional factors. The miR164 recognition sites of the OMTN genes are highly conserved in rice germplasms. Deletion of the recognition sites impaired the transactivation activity, indicating that the conserved recognition sites play a crucial role in maintaining the function of the OMTN proteins. The OMTN genes were responsive to abiotic stresses, and showed diverse spatio-temporal expression patterns in rice. Overexpression of OMTN2, OMTN3, OMTN4, and OMTN6 in rice led to negative effects on drought resistance at the reproductive stage. The expression of numerous genes related to stress response, development, and metabolism was altered in OMTN2-, OMTN3-, OMTN4-, and OMTN6-overexpressing plants. Most of the up-regulated genes in the OMTN-overexpressing plants were down-regulated by drought stress. The results suggest that the conserved miR164-targeted NAC genes may be negative regulators of drought tolerance in rice, in addition to their reported roles in development. OMTN1,OMTN3,OMTN4,OMTN6 Overexpression of a NAC transcription factor enhances rice drought and salt tolerance 2009 Biochem Biophys Res Commun National Center for Gene Research & Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 500 Caobao Road, Shanghai 200233, China. The plant-specific NAC (NAM, ATAF1/2, CUC2) transcription factors play diverse roles in plant development and stress responses. In this study, a rice NAC gene, ONAC045, was functionally characterized, especially with regard to its role in abiotic stress resistance. Expression analysis revealed that ONAC045 was induced by drought, high salt, and low temperature stresses, and abscisic acid (ABA) treatment in leaves and roots. Transcriptional activation assay in yeast indicated that ONAC045 functioned as a transcriptional activator. Transient expression of GFP-ONAC045 in onion epidermal cells revealed that ONAC045 protein was localized in the nucleus. Transgenic rice plants overexpressing ONAC045 showed enhanced tolerance to drought and salt treatments. Two stress-responsive genes were upregulated in transgenic rice. Together, these results suggest that ONAC045 encodes a novel stress-responsive NAC transcription factor and is potential useful for engineering drought and salt tolerant rice. ONAC045 Tolerance to various environmental stresses conferred by the salt-responsive rice gene ONAC063 in transgenic Arabidopsis 2009 Planta Research Institute for Biological Sciences, Okayama, 7549-1 Yoshikawa, Kibichuo-cho, Okayama 716-1241, Japan. Environmental stresses limit plant growth and crop production worldwide. We attempted to isolate rice genes involved in conferring tolerance to environmental stresses by using a transgenic Arabidopsis population expressing full-length cDNAs of rice. Among these lines, a thermotolerant line, R08946, was detected. The rice cDNA inserted in R08946 encoded a NAC transcription factor, ONAC063. This protein was localized in the nucleus and showed transactivation activity at the C-terminus. ONAC063 expression was not induced by high-temperature but highly induced by high-salinity in rice roots. High-osmotic pressure and reactive oxygen species levels also induced ONAC063 expression. The seeds of ONAC063-expressing transgenic Arabidopsis showed enhanced tolerance to high-salinity and osmotic pressure. Microarray and real-time reverse transcription-polymerase chain reaction analyses showed upregulated expression of some salinity-inducible genes, including the amylase gene AMY1, in ONAC063-expressing transgenic Arabidopsis. Thus, ONAC063 may play an important role in eliciting responses to high-salinity stress. ONAC063 Functions of rice NAC transcriptional factors, ONAC122 and ONAC131, in defense responses against Magnaporthe grisea 2013 Plant Mol Biol National Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, Zhejiang, China. NAC (NAM/ATAF/CUC) transcription factors have important functions in regulating plant growth, development, and abiotic and biotic stress responses. Here, we characterized two rice pathogen-responsive NAC transcription factors, ONAC122 and ONAC131. We determined that these proteins localized to the nucleus when expressed ectopically and had transcriptional activation activities. ONAC122 and ONAC131 expression was induced after infection by Magnaporthe grisea, the causal agent of rice blast disease, and the M. grisea-induced expression of both genes was faster and higher in the incompatible interaction compared with the compatible interaction during early stages of infection. ONAC122 and ONAC131 were also induced by treatment with salicylic acid, methyl jasmonate or 1-aminocyclopropane-1-carboxylic acid (a precursor of ethylene). Silencing ONAC122 or ONAC131 expression using a newly modified Brome mosaic virus (BMV)-based silencing vector resulted in an enhanced susceptibility to M. grisea. Furthermore, expression levels of several other defense- and signaling-related genes (i.e. OsLOX, OsPR1a, OsWRKY45 and OsNH1) were down-regulated in plants silenced for ONAC122 or ONAC131 expression via the BMV-based silencing system. Our results suggest that both ONAC122 and ONAC131 have important roles in rice disease resistance responses through the regulated expression of other defense- and signaling-related genes. ONAC131,ONAC122|OsNAC10,OsNPR1|NH1,OsPR1a,OsWRKY45 Molecular characterization of ONAC300, a novel NAC gene specifically expressed at early stages in various developing tissues of rice 2005 Mol Genet Genomics Genetic Diversity Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. Members of the plant-specific gene family referred to as the NAC family (for NAM-ATAF-CUC-related) are involved in various functions including the regulation of plant development. However, no detailed molecular characterization of any member of the NAC family has yet been reported from monocots. Here, we report such a characterization of ONAC300, a novel NAC-family gene identified using a cDNA cloned from microdissected phloem cells of rice. The predicted ONAC300 protein sequence falls into the NAM subgroup, which also contains the proteins CUC1 and CUC2 from Arabidopsis, CUP from snapdragon, CmNACP from pumpkin and NAM from petunia. High levels of ONAC300 mRNA were detected by in situ hybridization in developing shoot apical meristem (SAM) and in the associated young leaves. The use of an ONAC300:: GUS reporter gene revealed that the ONAC300 promoter was expressed predominantly in developing vascular tissues of the leaves and roots. The construct was also expressed in anther filaments, rachis and carpel styles. RT-PCR analysis further revealed that the levels of ONAC300 transcripts were higher in leaves, roots and culms than in panicles. The observed expression pattern of ONAC300 is quite different from those of the dicot NAC genes previously reported. Thus, ONAC300 is a novel member of the NAC family which is expressed at very early developmental stages in the shoot, root and flower, as well as in the mature phloem of vascular tissues in rice. ONAC300 Fatty acid elongase is required for shoot development in rice 2011 Plant J Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. yukito@bios.tohoku.ac.jp Organisms are covered extracellularly with cuticular waxes that consist of various fatty acids. In higher plants, extracellular waxes act as indispensable barriers to protect the plants from physical and biological stresses such as drought and pathogen attacks. However, the effect of fatty acid composition on plant development under normal growth conditions is not well understood. Here we show that the ONION1 (ONI1) gene, which encodes a fatty acid elongase (beta-ketoacyl CoA synthase) involved in the synthesis of very-long-chain fatty acids, is required for correct fatty acid composition and normal shoot development in rice. oni1 mutants containing a reduced amount of very-long-chain fatty acids produced very small shoots, with an aberrant outermost epidermal cell layer, and ceased to grow soon after germination. These mutants also showed abnormal expression of a KNOX family homeobox gene. ONI1 was specifically expressed in the outermost cell layer of the shoot apical meristem and developing lateral organs. These results show that fatty acid elongase is required for formation of the outermost cell layer, and this layer is indispensable for entire shoot development in rice. ONI1 ONION2 fatty acid elongase is required for shoot development in rice 2013 Plant Cell Physiol Plant Genetics Laboratory, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka-ken, Japan. A plant's surface is covered with epicuticular wax, which protects plants from inappropriate environmental conditions such as drought and pathogen attack. Very-long-chain fatty acids (VLCFAs) are the main component of epicuticular wax on the surface of above-ground organs. Here we show that a fatty acid elongase catalyzing an elongation reaction of VLCFAs is required for shoot development in rice. onion2 (oni2) mutants produced very small shoots in which leaves were fused to each other, and ceased growing after germination. The midrib of oni2 leaf blades was not developed correctly. Molecular cloning showed that ONI2 encodes a fatty acid elongase, which catalyzes the first step of elongation reactions of a carbon chain of VLCFAs, and oni2 had a reduced amount of VLCFAs. Expression analysis showed that ONI2 is specifically expressed in the outermost cell layer of young lateral organs. These results suggest that ONI2 is a layer 1-specific gene required for development of the entire shoot and that VLCFAs play an essential role in normal shoot development in rice. ONI2 Organ fusion and defective shoot development in oni3 mutants of rice 2014 Plant Cell Physiol Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555 Japan. Maintenance of organ separation is one of the essential phenomena for normal plant development. We have identified and analyzed ONION3 (ONI3), which is required for avoiding organ fusions in rice. Loss-of-function mutations of ONI3, which were identified as mutants with ectopic expression of KNOX genes in leaves and morphologically resembling KNOX overexpressors, showed abnormal organ fusions in developing shoots. The mutant seedlings showed fusions between neighboring organs and also within an organ; they stopped growing soon after germination and subsequently died. ONI3 was shown to encode an enzyme that is most similar to Arabidopsis HOTHEAD and is involved in biosynthesis of long-chain fatty acids. Expression analyses showed that ONI3 was specifically expressed in the outermost cell layer in the shoot apex throughout life cycle, and the oni3 mutants had an aberrant outermost cell layer. Our results together with previous studies suggest that long-chain fatty acids are required for avoiding organ fusions and promoting normal shoot development in rice. ONI3 Rice Rab11 is required for JA-mediated defense signaling 2013 Biochem Biophys Res Commun Department of Molecular Biotechnology, Dong-A University, Busan 604-714, South Korea. Rab proteins play an essential role in regulating vesicular transport in eukaryotic cells. Previously, we characterized OsRab11, which in concert with OsGAP1 and OsGDI3 regulates vesicular trafficking from the trans-Golgi network (TGN) to the plasma membrane or vacuole. To further elucidate the physiological function of OsRab11 in plants, we performed yeast two-hybrid screens using OsRab11 as bait. OsOPR8 was isolated and shown to interact with OsRab11. A co-immunoprecipitation assay confirmed this interaction. The green fluorescent protein-OsOPR8 fusion product was targeted to the cytoplasm and peroxisomes of protoplasts from Arabidopsis thaliana. OsOPR8 exhibited NADPH-dependent reduction activity when 2-cyclohexen-1-one (CyHE) and 12-oxo-phytodienoic acid (OPDA) were supplied as possible substrates. Interestingly, NADPH oxidation by OsOPR8 was increased when wild-type OsRab11 or the constitutively active form of OsRab11 (Q78L) were included in the reaction mix, but not when the dominant negative form of OsRab11 (S28N) was included. OsRab11 was expressed broadly in plants and both OsRab11 and OsOPR8 were induced by jasmonic acid (JA) and elicitor treatments. Overexpressed OsRab11 transgenic plants showed resistance to pathogens through induced expression of JA-responsive genes. In conclusion, OsRab11 may be required for JA-mediated defense signaling by activating the reducing activity of OsOPR8. OPR8,OsRab11 Comparative characterization, expression pattern and function analysis of the 12-oxo-phytodienoic acid reductase gene family in rice 2011 Plant Cell Rep State Key Laboratory for Biocontrol and Key Laboratory of Gene Engineering of Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People's Republic of China. The 12-oxo-phytodienoic acid reductases (OPRs) belong to the old yellow enzyme family of flavoenzymes and form multiple subfamilies in angiosperm plants. In our previous study, a comparative genomic analysis showed that five OPR subfamilies (subs. I-V) occur in monocots, and two subfamilies (subs. I and II) in dicots. Here, a comparative study of five OsOPR genes, representing five subfamilies (I-V) in rice, was performed to provide insights into OPR biochemical properties and physiological importance. Comparative analysis of the three-dimensional structure by homology modeling indicated all five OsOPR proteins contained a highly conserved backbone with (alpha/beta)(8)-barrels, while two middle variable regions (MVR i and ii) were also detected and defined. Analysis of enzymatic characteristics revealed that all five OsOPR fusion proteins exhibit distinct substrate specificity. Different catalytic activity was observed using racemic OPDA and trans-2-hexen-1-al as substrates, suggesting OsOPR family genes participate in two main branches of the octadecanoid pathway, including the allene oxide synthase and hydroperoxide lyase pathways which regulate various developmental processes and/or defense responses. The transcript profiles of five OsOPR genes exhibited strong tissue-specific and inducible expression patterns under abiotic stress, hormones and plant wounding treatments. Furthermore, the transcriptions of OsOPR04-1 (OsOPR11) and OsOPR08-1 (OsOPR7), representing subs. I and II, respectively, were observed in all six selected tissues and with all above-stress treatments. This suggests that these two subfamilies play an important role during different developmental stages and in response to stresses; while the expressions of OsOPR06-1 (OsOPR6), OsOPR01-1 (OsOPR10) and OsOPR02-1 (OsOPR8), representing subs. III, IV and V respectively, were strongly up-regulated with abscisic acid (ABA) and indoleacetic acid (IAA) treatments in roots, suggesting these three subfamilies play an important role in responding to hormones especially ABA and IAA signals in roots. OPR8 Identification of the OsOPR7 gene encoding 12-oxophytodienoate reductase involved in the biosynthesis of jasmonic acid in rice 2007 Planta Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Enzyme 12-oxophytodienoate (OPDA) reductase (EC1.3.1.42), which is involved in the biosynthesis of jasmonic acid (JA), catalyses the reduction of 10, 11-double bonds of OPDA to yield 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0). The rice OsOPR1 gene encodes OPDA reductase (OPR) converting (-)-cis-OPDA preferentially, rather than (+)-cis-OPDA, a natural precursor of JA. Here, we provide evidence that an OPR family gene in rice chromosome 8, designated OsOPR7, encodes the enzyme involved in the JA biosynthesis. Recombinant OsOPR7-His protein efficiently catalysed the reduction of both enantiomers of cis-OPDA, similar to the OPR3 protein in Arabidopsis thaliana (L.) Heynh. The expression of OsOPR7 mRNA was induced and reached maximum levels within 0.5 h of mechanical wounding and drought stress, and the endogenous JA level started to increase in accordance with the increase in OsOPR7 expression. The GFP-OsOPR7 fusion protein was detected exclusively in peroxisomes in onion epidermal cells. Furthermore, complementation analysis using an Arabidopsis opr3 mutant indicated that the OsOPR7 gene, but not OsOPR1, was able to complement the phenotypes of male sterility in the mutant caused by JA deficiency, and that JA production in the opr3 mutant was also restored by the expression of the OsOPR7 gene. We conclude that the OsOPR7 gene encodes the enzyme catalysing the reduction of natural (+)-cis-OPDA for the JA biosynthesis in rice. OPR8 OsTZF1, a CCCH-tandem zinc finger protein, confers delayed senescence and stress tolerance in rice by regulating stress-related genes 2013 Plant Physiol Biological Resources and Post-harvest Division, Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki 305-8686, Japan. OsTZF1 is a member of the CCCH-type zinc finger gene family in rice (Oryza sativa). Expression of OsTZF1 was induced by drought, high-salt stress, and hydrogen peroxide. OsTZF1 gene expression was also induced by abscisic acid, methyl jasmonate, and salicylic acid. Histochemical activity of beta-glucuronidase in transgenic rice plants containing the promoter of OsTZF1 fused with beta-glucuronidase was observed in callus, coleoptile, young leaf, and panicle tissues. Upon stress, OsTZF1-green fluorescent protein localization was observed in the cytoplasm and cytoplasmic foci. Transgenic rice plants overexpressing OsTZF1 driven by a maize (Zea mays) ubiquitin promoter (Ubi:OsTZF1-OX [for overexpression]) exhibited delayed seed germination, growth retardation at the seedling stage, and delayed leaf senescence. RNA interference (RNAi) knocked-down plants (OsTZF1-RNAi) showed early seed germination, enhanced seedling growth, and early leaf senescence compared with controls. Ubi:OsTZF1-OX plants showed improved tolerance to high-salt and drought stresses and vice versa for OsTZF1-RNAi plants. Microarray analysis revealed that genes related to stress, reactive oxygen species homeostasis, and metal homeostasis were regulated in the Ubi:OsTZF1-OX plants. RNA-binding assays indicated that OsTZF1 binds to U-rich regions in the 3' untranslated region of messenger RNAs, suggesting that OsTZF1 might be associated with RNA metabolism of stress-responsive genes. OsTZF1 may serve as a useful biotechnological tool for the improvement of stress tolerance in various plants through the control of RNA metabolism of stress-responsive genes. OPT|OsOPT7,OsTZF1,akin-b|akin-beta Characterization of the origin recognition complex (ORC) from a higher plant, rice (Oryza sativa L.) 2005 Gene Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda-shi, Chiba-ken 278-8510, Japan. The origin recognition complex (ORC) protein plays a critical role in DNA replication through binding to sites (origins) where replication commences. The protein is composed of six subunits (ORC1 to 6) in animals and yeasts. Our knowledge of the ORC protein in plants is, however, much less complete. We have performed cDNA cloning and characterization of ORC subunits in rice (Oryza sativa L. cv. Nipponbare) in order to facilitate study of plant DNA replication mechanisms. Our previous report provided a description of a gene, ORC1 (OsORC1), that encodes one of the protein subunits. The present report extends this initial analysis to include the genes that encode four other rice ORC subunits, OsORC2, 3, 4 and 5. Northern hybridization analyses demonstrated the presence of abundant transcripts for all OsORC subunits in shoot apical meristems (SAM) and cultured cells, but not in mature leaves. Interestingly, only OsORC5 showed high levels of expression in organs in which cell proliferation is not active, such as flag leaves, the ears and the non-tip roots. The pattern of expression of OsORC2 also differed from other OsORC subunits. When cell proliferation was temporarily halted for 6-10 days by removal of sucrose from the growth medium, expression of OsORC1, OsORC3, OsORC4 and OsORC5 was substantially reduced. However, the level of expression of OsORC2 remained constant. We suggest from these results that expression of OsORC1, 3, 4 and 5 are correlated with cell proliferation, but the expression of OsORC2 is not. OsORC4,OsORC5 Cloning, expression and immunological characterization of Ory s 1, the major allergen of rice pollen 1995 Gene School of Botany, University of Melbourne, Parkville, Victoria, Australia. We have isolated and characterized a cDNA clone, Ory s 1, encoding a group-1 allergen of rice pollen. The Ory s 1 protein shows significant sequence identity to the major allergen of rye-grass pollen, Lol p 1. RNA gel blot analysis shows that the Ory s 1 gene is expressed in mature anthers, but not in vegetative or other floral tissues tested. Southern blot analysis indicates that this clone represents a member of a small gene family in rice. Western blot analyses of total rice pollen proteins with the group-1 allergen-specific monoclonal 3A2 and IgE antibodies from grass pollen-allergic patients, revealed the presence of cross-reactive antigenic and allergenic epitopes in Ory s 1. Orys1 Agrobacterium-mediated transformation of Australian rice varieties and promoter analysis of major pollen allergen gene, Ory s 1 2011 Plant Cell Rep Plant Molecular Biology and Biotechnology Laboratory, Melbourne School of Land and Environment, The University of Melbourne, Parkville, VIC 3010, Australia. A simple protocol for Agrobacterium-mediated transformation of Australian rice using mature embryos is described. Transgenic plants of two commercial genotypes of Australian rice, Amaroo and Millin, were produced. Transgenic plants were obtained by applying selection pressure to callus and to the regenerated shoots. Exclusion of the selective agent (hygromycin) during plant regeneration was found to be critical for recovery of transgenic plants from these commercial varieties. Transgenic plants were produced after 3 months. The developed system was also used to study spatial and temporal expression of a rice pollen-specific gene, Ory s 1. Expression of pOry s 1::uidA in transgenic rice demonstrated GUS expression in mature pollen, hence indicating potential use of this promoter to direct pollen-specific gene expression. Further Ory s 1 5' deletion study indicated that the pollen-specificity element may reside within -405 bp to the start of the transcription, while the region upstream of -405 contained a cis-acting regulatory element(s) responsible for quantitative expression of this gene. Orys1 Interacting proteins and differences in nuclear transport reveal specific functions for the NAP1 family proteins in plants 2005 Plant Physiol State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai 200433, People's Republic of China. awdong001@yahoo.com.cn Nucleosome assembly protein 1 (NAP1) is conserved from yeast to human and facilitates the in vitro assembly of nucleosomes as a histone chaperone. Inconsistent with their proposed function in the nucleus, however, many NAP1 proteins had been reported to localize in the cytoplasm. We investigated the subcellular localization of tobacco (Nicotiana tabacum) and rice (Oryza sativa) NAP1 family proteins first by identification of interacting partners and by direct examination of the localization of green fluorescent protein-tagged proteins. Through treatment of tobacco cells with leptomycin B and mutagenesis of nuclear export signal, we demonstrated that Nicta;NAP1;1 and Orysa;NAP1;1 shuttle between the cytoplasm and the nucleus. Together with the demonstration that tobacco NAP1 proteins bind histone H2A and H2B, our results support the current model and provide additional evidence that function of NAP1 as histone chaperones appears to be conserved in plants. In addition, we show that tobacco NAP1 proteins interact with tubulin and the mitotic cyclin Nicta;CYCB1;1, suggesting a role for NAP1 in microtubule dynamics. Interestingly, in spite of their high homology with the above NAP1 proteins, the other three tobacco proteins and Orysa;NAP1;2 did not show nucleocytoplasmic shuttling and were localized only in the cytoplasm. Moreover, Orysa;NAP1;3 that lacks a typical nuclear localization signal sequence was localized in both the cytoplasm and the nucleus. Finally, we show that only Orysa;NAP1;3 could be phosphorylated by casein kinase 2alpha in vitro. However, this phosphorylation was not responsible for nuclear import of Orysa;NAP1;3 as being demonstrated through mutagenesis studies. Together, our results provide an important step toward elucidating the molecular mechanism of function of the NAP1 family proteins in plants. Orysa;NAP1;1|OsNAP1_L1,Orysa;NAP1;2|OsNAP1_L2,Orysa;NAP1;3|OsNAP1_L3 Rice Aspartic Proteinase, Oryzasin, Expressed During Seed Ripening and Germination, has a Gene Organization Distinct from Those of Animal and Microbial Aspartic Proteinases 1995 European Journal of Biochemistry Laboratory for Food Science, Atomi Junior College, Tokyo, Japan. The gene organization and nucleotide sequence of an aspartic proteinase (AP) of plant origin were first disclosed by cDNA and genomic DNA cloning of a rice AP (oryzasin). The deduced amino acid sequence of oryzasin 1 was significantly similar to those of other APs (34-85%), with highest similarity (85%) to barley AP (HvAP). Oryzasin 1, as well as HvAP, is distinct from animal and microbial APs in that the plant APs contain a unique 104-amino-acid insertion in the C-terminal region. The oryzasin 1 gene spans approximately 6.6 kbp and is composed of 14 exons and 13 introns. The exon-intron organization of the oryzasin 1 gene is totally different from those of genes for animal and microbial APs such as human cathepsin D, rat renin, bovine chymosin, aspergillopepsin A of Aspergillus awamori, proteinase A of Saccharomyces cerevisiae and rhizopuspepsin of Rhizopus niveus, despite the fact that oryzasin 1 shows overall sequence similarity to these APs. Oryzasin OsAP65, a rice aspartic protease, is essential for male fertility and plays a role in pollen germination and pollen tube growth 2013 J Exp Bot National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Aspartic proteases (APs) comprise a large proteolytic enzyme family widely distributed in animals, microbes, viruses, and plants. The rice genome encodes 96 APs, of which only a few have been functionally characterized. Here, the identification and characterization of a novel AP gene, OsAP65, which plays an indispensable role in pollen tube growth in rice, is reported. The T-DNA insertion line of OsAP65 caused severe segregation distortion. In the progeny derived from an individual heterozygous for the T-DNA insertion, the wild type and T-DNA-carrying heterozygote segregated at a ratio close to 1:1, while homozygotes of disrupted OsAP65 (OsAP65-/-) were not recovered. Reciprocal crosses between heterozygotes and wild-type plants demonstrated that the mutant alleles could not be transmitted through the male gamete. Examination of the anthers from heterozygous plants revealed that the mutant pollen matured normally, but did not germinate or elongate. OsAP65 was expressed in various tissues and the transcript level in heterozygous plants was about half of the amount measured in the wild-type plants. The subcellular localization showed that OsAP65 is a pre-vacuolar compartment (PVC) protein. These results indicated that OsAP65 was essential for rice pollen germination and tube growth. Oryzasin,OsAP65 OVP1, a vacuolar H+-translocating inorganic pyrophosphatase (V-PPase), overexpression improved rice cold tolerance 2011 Plant Physiol Biochem State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100094, China. Vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase, EC 3.6.1.1) is an electrogenic proton pump and has been studied in many plants. Here we report characterization of the OVP1 gene from rice (Oryza sativa L.). Quantitative reverse transcriptase-polymerase chain reaction analysis (RT-qPCR) showed that OVP1 was induced by cold stress. OVP1 overexpression resulted in enhanced cold tolerance in transgenic rice, which was related to an increased integrity of cell membrane, decreased MDA content and accumulation of proline to higher level as compared with wild type rice seedlings. These results indicated that V-PPase was an important element in the survival strategies of plants under cold stress. OVP1 Isolation and characterization of cDNAs encoding vacuolar H+-pyrophosphatase isoforms from rice (Oryza sativa L.) 1996 Plant Mol Biol Molecular Function Laboratory, National Food Research Institute, 2-1-2 Kannondai, 305, Tsukuba, Ibaraki, Japan The vacuolar H(+)-pyrophosphatase (V-PPase) is an electrogenic H+ pump, which was found in the plant vacuolar membrane. Two cDNA clones (OVP1 and OVP2) encoding the V-PPase were isolated from cultured rice (Oryza sativa L.) cells and subsequently sequenced. The sequence analysis has revealed that OVP1 contains 2316 nucleotides of open reading frame (ORF) and 362 nucleotides of the 3'-untranslated region, whereas OVP2 comprises 2304 nucleotides of ORF and 312 nucleotides of the 3'-untranslated region. The nucleotide sequences of ORF of OVP1 and OVP2 are 80.7% identical, and their 5'- and 3'-untranslated regions have 39.4% and 48.4% identity, respectively. The polypeptides encoded by the ORF of OVP1 and OVP2 contain 771 and 767 amino acids, respectively, and the sequences of the OVP proteins are very similar to those of other V-PPases, which are shown to have 85-91% homology. Chromosomal mapping by RFLP techniques demonstrates that OVP1 and OVP2 are isoforms encoded by different genes. Both OVP1 and OVP2 are mapped on the same chromosome (chromosome 6) to a distance of ca.90 cM. Northern analysis indicates that the OVP1 and OVP2 are also expressed in intact rice plants and OVP2 shows higher expression in the calli than the roots and shoots, compared to OVP1. These results show that at least two genes encoding the V-PPases are present in rice genome and their expressions are probably regulated in a different manner. OVP2 Expression of vacuolar H+-pyrophosphatase (OVP3) is under control of an anoxia-inducible promoter in rice 2010 Plant Mol Biol Department of Biological Sciences, Macquarie University, Sydney, NSW, 2109, Australia. Vacuolar H(+)-pyrophosphatase (V-PPase) expression increases in a number of abiotic stresses and is thought to play a role in adaptation to abiotic stresses. This paper reports on the regulation of six V-PPase genes in rice (Oryza sativa L.) coleoptiles under anoxia, using flood tolerant and intolerant cultivars to test our hypothesis. Quantitative PCR analysis showed that one vacuolar H(+)-pyrophosphatase (OVP3) was induced by anoxia, particularly in flood-tolerant rice. Regulation of OVP3 expression under anoxia was investigated by analysing putative OVP promoters. The putative OVP3 promoter contained more previously identified anoxia-inducible motifs than the putative promoters of the other five OVP genes. GUS activity in transgenic rice plants containing the OVP3 promoter region linked to the GUS reporter gene was induced only by anoxia. Salt and cold treatments had little effect on OVP3 promoter-driven GUS expression when compared to the anoxic treatment. OVP3 Os11Gsk gene from a wild rice, Oryza rufipogon improves yield in rice 2012 Funct Integr Genomics Biotechnology Unit, Directorate of Rice Research, Rajendranagar, Hyderabad 500 030, India. Chromosomal segments from wild rice species Oryza rufipogon, introgressed into an elite indica rice restorer line (KMR3) using molecular markers, resulted in significant increase in yield. Here we report the transcriptome analysis of flag leaves and fully emerged young panicles of one of the high yielding introgression lines IL50-7 in comparison to KMR3. A 66-fold upregulated gene Os11Gsk, which showed no transcript in KMR3 was highly expressed in O. rufipogon and IL50-7. A 5-kb genomic region including Os11Gsk and its flanking regions could be PCR amplified only from IL50-7, O. rufipogon, japonica varieties of rice-Nipponbare and Kitaake but not from the indica varieties, KMR3 and Taichung Native-1. Three sister lines of IL50-7 yielding higher than KMR3 showed presence of Os11Gsk, whereas the gene was absent in three other ILs from the same cross having lower yield than KMR3, indicating an association of the presence of Os11Gsk with high yield. Southern analysis showed additional bands in the genomic DNA of O. rufipogon and IL50-7 with Os11Gsk probe. Genomic sequence analysis of ten highly co-expressed differentially regulated genes revealed that two upregulated genes in IL50-7 were derived from O. rufipogon and most of the downregulated genes were either from KMR3 or common to KMR3, IL50-7, and O. rufipogon. Thus, we show that Os11Gsk is a wild rice-derived gene introduced in KMR3 background and increases yield either by regulating expression of functional genes sharing homology with it or by causing epigenetic modifications in the introgression line. Os11Gsk Transcription activator-like (TAL) effectors targeting OsSWEET genes enhance virulence on diverse rice (Oryza sativa) varieties when expressed individually in a TAL effector-deficient strain of Xanthomonas oryzae 2012 New Phytol Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO 80523-1177, USA. Genomes of the rice (Oryza sativa) xylem and mesophyll pathogens Xanthomonas oryzae pv. oryzae (Xoo) and pv. oryzicola (Xoc) encode numerous secreted transcription factors called transcription activator-like (TAL) effectors. In a few studied rice varieties, some of these contribute to virulence by activating corresponding host susceptibility genes. Some activate disease resistance genes. The roles of X. oryzae TAL effectors in diverse rice backgrounds, however, are poorly understood. Xoo TAL effectors that promote infection by activating SWEET sucrose transporter genes were expressed in TAL effector-deficient X. oryzae strain X11-5A, and assessed in 21 rice varieties. Some were also tested in Xoc on variety Nipponbare. Several Xoc TAL effectors were tested in X11-5A on four rice varieties. Xoo TAL effectors enhanced X11-5A virulence on most varieties, but to varying extents depending on the effector and variety. SWEET genes were activated in all tested varieties, but increased virulence did not correlate with activation level. SWEET activators also enhanced Xoc virulence on Nipponbare. Xoc TAL effectors did not alter X11-5A virulence. SWEET-targeting TAL effectors contribute broadly and non-tissue-specifically to virulence in rice, and their function is affected by host differences besides target sequences. Further, the utility of X11-5A for characterizing individual TAL effectors in rice was established. Os11N3,Os8N3|xa13 Rice xa13 recessive resistance to bacterial blight is defeated by induction of the disease susceptibility gene Os-11N3 2010 Plant Cell Department of Plant Pathology, Kansas State University, Manhattan, Kansas 66506, USA. The rice (Oryza sativa) gene xa13 is a recessive resistance allele of Os-8N3, a member of the NODULIN3 (N3) gene family, located on rice chromosome 8. Os-8N3 is a susceptibility (S) gene for Xanthomonas oryzae pv oryzae, the causal agent of bacterial blight, and the recessive allele is defeated by strains of the pathogen producing any one of the type III effectors AvrXa7, PthXo2, or PthXo3, which are all members of the transcription activator-like (TAL) effector family. Both AvrXa7 and PthXo3 induce the expression of a second member of the N3 gene family, here named Os-11N3. Insertional mutagenesis or RNA-mediated silencing of Os-11N3 resulted in plants with loss of susceptibility specifically to strains of X. oryzae pv oryzae dependent on AvrXa7 or PthXo3 for virulence. We further show that AvrXa7 drives expression of Os-11N3 and that AvrXa7 interacts and binds specifically to an effector binding element within the Os-11N3 promoter, lending support to the predictive models for TAL effector binding specificity. The result indicates that variations in the TAL effector repetitive domains are driven by selection to overcome both dominant and recessive forms of resistance to bacterial blight in rice. The finding that Os-8N3 and Os-11N3 encode closely related proteins also provides evidence that N3 proteins have a specific function in facilitating bacterial blight disease. Os11N3,Os8N3|xa13 Structural and enzymatic characterization of Os3BGlu6, a rice beta-glucosidase hydrolyzing hydrophobic glycosides and (1->3)- and (1->2)-linked disaccharides 2009 Plant Physiol School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand. Glycoside hydrolase family 1 (GH1) beta-glucosidases play roles in many processes in plants, such as chemical defense, alkaloid metabolism, hydrolysis of cell wall-derived oligosaccharides, phytohormone regulation, and lignification. However, the functions of most of the 34 GH1 gene products in rice (Oryza sativa) are unknown. Os3BGlu6, a rice beta-glucosidase representing a previously uncharacterized phylogenetic cluster of GH1, was produced in recombinant Escherichia coli. Os3BGlu6 hydrolyzed p-nitrophenyl (pNP)-beta-d-fucoside (k(cat)/K(m) = 67 mm(-1) s(-1)), pNP-beta-d-glucoside (k(cat)/K(m) = 6.2 mm(-1) s(-1)), and pNP-beta-d-galactoside (k(cat)/K(m) = 1.6 mm(-1)s(-1)) efficiently but had little activity toward other pNP glycosides. It also had high activity toward n-octyl-beta-d-glucoside and beta-(1-->3)- and beta-(1-->2)-linked disaccharides and was able to hydrolyze apigenin beta-glucoside and several other natural glycosides. Crystal structures of Os3BGlu6 and its complexes with a covalent intermediate, 2-deoxy-2-fluoroglucoside, and a nonhydrolyzable substrate analog, n-octyl-beta-d-thioglucopyranoside, were solved at 1.83, 1.81, and 1.80 A resolution, respectively. The position of the covalently trapped 2-F-glucosyl residue in the enzyme was similar to that in a 2-F-glucosyl intermediate complex of Os3BGlu7 (rice BGlu1). The side chain of methionine-251 in the mouth of the active site appeared to block the binding of extended beta-(1-->4)-linked oligosaccharides and interact with the hydrophobic aglycone of n-octyl-beta-d-thioglucopyranoside. This correlates with the preference of Os3BGlu6 for short oligosaccharides and hydrophobic glycosides. Os3BGlu6,Os3BGlu7 Rice family GH1 glycoside hydrolases with beta-D-glucosidase and beta-D-mannosidase activities 2009 Arch Biochem Biophys School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand. Plant beta-D-mannosidases and a rice beta-D-glucosidase, Os3BGlu7, with weak beta-D-mannosidase activity, cluster together in phylogenetic analysis. To investigate the relationship between substrate specificity and amino acid sequence similarity in family GH1 glycoside hydrolases, Os3BGlu8 and Os7BGlu26, putative rice beta-D-glucosidases from this cluster, and a beta-D-mannosidase from barley (rHvBII), were expressed in Escherichia coli and characterized. Os3BGlu8, the amino acid sequence and molecular model of which are most similar to Os3BGlu7, hydrolysed 4-nitrophenyl-beta-D-glucopyranoside (4NPGlc) faster than 4-nitrophenyl-beta-D-mannopyranoside (4NPMan), while Os7BGlu26, which is most similar to rHvBII by these criteria, hydrolysed 4NPMan faster than 4NPGlc. All the enzymes hydrolyzed cellooligosaccharides with increased hydrolytic rates as the degree of polymerization increased from 3-6, but only rHvBII hydrolyzed cellobiose with a higher k(cat)/K(m) value than cellotriose. This was primarily due to strong binding of glucosyl residues at the+2 subsite for the rice enzymes, and unfavorable interactions at this subsite with rHvBII. Os3BGlu7,Os3BGlu8,Os7BGlu26 The crystal structure of rice (Oryza sativa L.) Os4BGlu12, an oligosaccharide and tuberonic acid glucoside-hydrolyzing beta-glucosidase with significant thioglucohydrolase activity 2011 Arch Biochem Biophys Institute of Science, Schools of Biochemistry and Chemistry, Suranaree University of Technology, Nakhon Ratchasima, Thailand. Rice Os4BGlu12, a glycoside hydrolase family 1 (GH1) beta-glucosidase, hydrolyzes beta-(1,4)-linked oligosaccharides of 3-6 glucosyl residues and the beta-(1,3)-linked disaccharide laminaribiose, as well as certain glycosides. The crystal structures of apo Os4BGlu12, and its complexes with 2,4-dinitrophenyl-2-deoxyl-2-fluoroglucoside (DNP2FG) and 2-deoxy-2-fluoroglucose (G2F) were solved at 2.50, 2.45 and 2.40A resolution, respectively. The overall structure of rice Os4BGlu12 is typical of GH1 enzymes, but it contains an extra disulfide bridge in the loop B region. The glucose ring of the G2F in the covalent intermediate was found in a (4)C(1) chair conformation, while that of the noncovalently bound DNP2FG had a (1)S(3) skew boat, consistent with hydrolysis via a (4)H(3) half-chair transition state. The position of the catalytic nucleophile (Glu393) in the G2F structure was more similar to that of the Sinapsis alba myrosinase G2F complex than to that in covalent intermediates of other O-glucosidases, such as rice Os3BGlu6 and Os3BGlu7 beta-glucosidases. This correlated with a significant thioglucosidase activity for Os4BGlu12, although with 200- to 1200-fold lower k(cat)/K(m) values for S-glucosides than the comparable O-glucosides, while hydrolysis of S-glucosides was undetectable for Os3BGlu6 and Os3BGlu7. Os4BGlu12 Rice Os4BGlu12 is a wound-induced beta-glucosidase that hydrolyzes cell wall-beta-glucan-derived oligosaccharides and glycosides 2010 Plant Science School of Biochemistry, Institute of Science, Suranaree University of Technology, Muang District, Nakhon Ratchasima 30000, Thailand Rice Os4BGlu12 beta-glucosidase is a family 1 glycoside hydrolase, the transcript levels of which have previously been found to be induced in response to herbivore attack and salinity stress. Here, high levels of Os4bglu12 transcripts were also detected in the shoot during germination, in the leaf sheath and stem of mature rice plants under normal growth conditions. The transcripts of this gene were up-regulated in response to wounding, methyl jasmonate and ethephon in 10-day-old rice seedlings. Os4BGlu12 expressed in recombinant Escherichia coli hydrolyzed beta-(1,3;1,4)-glucooligosaccharides generated by the wounding-induced rice endo-(1,3;1,4)-beta-glucanase OsEGL1, suggesting that both enzymes may act in concert in remodeling of damaged cell wall. Among oligosaccharides tested, Os4BGlu12 hydrolyzed beta-(1,4)-linked glucooligosaccharides with highest catalytic efficiency (kcat/Km = 2.7–4.9 s−1 mM−1) when the degree of polymerization ranged from 3 to 6. It also hydrolyzed the beta-(1,3)-linked disaccharide laminaribiose with high catalytic efficiency (kcat/Km = 4.5 s−1 mM−1). Among the natural glycosides tested, Os4BGlu12 efficiently hydrolyzed deoxycorticosterone 21-glucoside (kcat/Km = 20 s−1 mM−1) and apigenin 7-O-beta-d-glucoside (kcat/Km = 6.7 s−1 mM−1). The amino acid residues predicted to line the active site of Os4BGlu12 are more similar to those of cyanogenic and flavonoid beta-glucosidases than oligosaccharide hydrolases, and it may function in defense, as well as in cell wall-derived oligosaccharide break-down. Os4BGlu12 Analysis of rice glycosyl hydrolase family 1 and expression of Os4bglu12 beta-glucosidase 2006 BMC Plant Biol Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand. opassiri@hotmail.com BACKGROUND: Glycosyl hydrolase family 1 (GH1) beta-glucosidases have been implicated in physiologically important processes in plants, such as response to biotic and abiotic stresses, defense against herbivores, activation of phytohormones, lignification, and cell wall remodeling. Plant GH1 beta-glucosidases are encoded by a multigene family, so we predicted the structures of the genes and the properties of their protein products, and characterized their phylogenetic relationship to other plant GH1 members, their expression and the activity of one of them, to begin to decipher their roles in rice. RESULTS: Forty GH1 genes could be identified in rice databases, including 2 possible endophyte genes, 2 likely pseudogenes, 2 gene fragments, and 34 apparently competent rice glycosidase genes. Phylogenetic analysis revealed that GH1 members with closely related sequences have similar gene structures and are often clustered together on the same chromosome. Most of the genes appear to have been derived from duplications that occurred after the divergence of rice and Arabidopsis thaliana lineages from their common ancestor, and the two plants share only 8 common gene lineages. At least 31 GH1 genes are expressed in a range of organs and stages of rice, based on the cDNA and EST sequences in public databases. The cDNA of the Os4bglu12 gene, which encodes a protein identical at 40 of 44 amino acid residues with the N-terminal sequence of a cell wall-bound enzyme previously purified from germinating rice, was isolated by RT-PCR from rice seedlings. A thioredoxin-Os4bglu12 fusion protein expressed in Escherichia coli efficiently hydrolyzed beta-(1,4)-linked oligosaccharides of 3-6 glucose residues and laminaribiose. CONCLUSION: Careful analysis of the database sequences produced more reliable rice GH1 gene structure and protein product predictions. Since most of these genes diverged after the divergence of the ancestors of rice and Arabidopsis thaliana, only a few of their functions could be implied from those of GH1 enzymes from Arabidopsis and other dicots. This implies that analysis of GH1 enzymes in monocots is necessary to understand their function in the major grain crops. To begin this analysis, Os4bglu12 beta-glucosidase was characterized and found to have high exoglucanase activity, consistent with a role in cell wall metabolism. Os4BGlu12 Expression, purification, crystallization and preliminary X-ray analysis of rice (Oryza sativa L.) Os4BGlu12 beta-glucosidase 2010 Acta Crystallogr Sect F Struct Biol Cryst Commun School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand. Rice (Oryza sativa L.) Os4BGlu12, a glycoside hydrolase family 1 beta-glucosidase (EC 3.2.1.21), was expressed as a fusion protein with an N-terminal thioredoxin/His(6) tag in Escherichia coli strain Origami B (DE3) and purified with subsequent removal of the N-terminal tag. Native Os4BGlu12 and its complex with 2,4-dinitrophenyl-2-deoxy-2-fluoro-beta-D-glucopyranoside (DNP2FG) were crystallized using 19% polyethylene glycol (3350 or 2000, respectively) in 0.1 M Tris-HCl pH 8.5, 0.16 M NaCl at 288 K. Diffraction data sets for the apo and inhibitor-bound forms were collected to 2.50 and 2.45 A resolution, respectively. The space group and the unit-cell parameters of the crystal indicated the presence of two molecules per asymmetric unit, with a solvent content of 50%. The structure of Os4BGlu12 was successfully solved in space group P4(3)2(1)2 by molecular replacement using the white clover cyanogenic beta-glucosidase structure (PDB code 1cbg) as a search model. Os4BGlu12 Functional characterization of evolutionarily divergent 4-coumarate:coenzyme a ligases in rice 2011 Plant Physiol Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. 4-Coumarate:coenzyme A ligase (4CL; EC 6.2.1.12) is a key enzyme in the phenylpropanoid metabolic pathways for monolignol and flavonoid biosynthesis. 4CL has been much studied in dicotyledons, but its function is not completely understood in monocotyledons, which display a different monolignol composition and phenylpropanoid profile. In this study, five members of the 4CL gene family in the rice (Oryza sativa) genome were cloned and analyzed. Biochemical characterization of the 4CL recombinant proteins revealed that the rice 4CL isoforms displayed different substrate specificities and catalytic turnover rates. Among them, Os4CL3 exhibited the highest turnover rate. No apparent tissue-specific expression of the five 4CLs was observed, but significant differences in their expression levels were detected. The rank in order of transcript abundance was Os4CL3 > Os4CL5 > Os4CL1 > Os4CL4 > Os4CL2. Suppression of Os4CL3 expression resulted in significant lignin reduction, shorter plant growth, and other morphological changes. The 4CL-suppressed transgenics also displayed decreased panicle fertility, which may be attributed to abnormal anther development as a result of disrupted lignin synthesis. This study demonstrates that the rice 4CLs exhibit different in vitro catalytic properties from those in dicots and that 4CL-mediated metabolism in vivo may play important roles in regulating a broad range of biological events over the course of rice growth and development. Os4CL1,Os4CL2,Os4CL3,Os4CL4,Os4CL5 Molecular cloning and characterization of rice 6-phosphogluconate dehydrogenase gene that is up-regulated by salt stress 2003 Mol Biol Rep State Key Laboratory of Crop Genetics and Germplasm Enhancement, Rice Research Institute, Nanjing Agricultural University, Nanjing 210095, People's Repulic of China. The pentose phosphate pathway (PPP) is the important metabolism pathway in plant. In the present study, a cDNA encoding one of the key enzymes of PPP, 6-phosphogluconate dehydrogenase( 6PGDH), was isolated from rice and designated as Os6PGDH. The Os6PGDH encoding protein is a cytosolic isoenzyme according to the absence of plastid transit peptide at the N-terminus. The full-length cDNA of 1751 bp encodes 480 amino acids and its putative protein sequence is 94%, 84% and 83% identical to maize, spinach and alfalfa 6PGDHs respectively. Comparison of the cloned mRNA sequence with that of the genomic sequence from the Rice Genome Project showed a simple genomic organization devoid of introns in the translated region of the gene. RT-PCR experiments revealed that Os6PGDH expression was high in inflorescence, low in root and embryos but almost absent in leaves. Furthermore, Os6PGDH was up-regulated in the shoots under salt stress. It is suggested that 6PGDH in plant may play an important role in cell division and salt response. Os6PGDH Rice Os9BGlu31 is a transglucosidase with the capacity to equilibrate phenylpropanoid, flavonoid, and phytohormone glycoconjugates 2013 J Biol Chem Institute of Science, Schools of Biochemistry and Chemistry, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand. Glycosylation is an important mechanism of controlling the reactivities and bioactivities of plant secondary metabolites and phytohormones. Rice (Oryza sativa) Os9BGlu31 is a glycoside hydrolase family GH1 transglycosidase that acts to transfer glucose between phenolic acids, phytohormones, and flavonoids. The highest activity was observed with the donors feruloyl-glucose, 4-coumaroyl-glucose, and sinapoyl-glucose, which are known to serve as donors in acyl and glucosyl transfer reactions in the vacuole, where Os9BGlu31 is localized. The free acids of these compounds also served as the best acceptors, suggesting that Os9BGlu31 may equilibrate the levels of phenolic acids and carboxylated phytohormones and their glucoconjugates. The Os9BGlu31 gene is most highly expressed in senescing flag leaf and developing seed and is induced in rice seedlings in response to drought stress and treatment with phytohormones, including abscisic acid, ethephon, methyljasmonate, 2,4-dichlorophenoxyacetic acid, and kinetin. Although site-directed mutagenesis of Os9BGlu31 indicated a function for the putative catalytic acid/base (Glu(169)), catalytic nucleophile residues (Glu(387)), and His(386), the wild type enzyme displays an unusual lack of inhibition by mechanism-based inhibitors of GH1 beta-glucosidases that utilize a double displacement retaining mechanism. Os9BGlu31 Identification and characterization of a gibberellin-regulated protein, which is ASR5, in the basal region of rice leaf sheaths 2008 Mol Genet Genomics National Institute of Crop Science, Kannondai 2-1-18, Tsukuba, 305-8518, Japan. Gibberellins (GAs) regulate growth and development in higher plants. To identify GA-regulated proteins during rice leaf sheath elongation, a proteomic approach was used. Proteins from the basal region of leaf sheath in rice seedling treated with GA(3) were analyzed by fluorescence two-dimensional difference gel electrophoresis. The levels of abscisic acid-stress-ripening-inducible 5 protein (ASR5), elongation factor-1 beta, translationally controlled tumor protein, fructose-bisphosphate aldolase and a novel protein increased; whereas the level of RuBisCO subunit binding-protein decreased by GA(3) treatment. ASR5 out of these six proteins was significantly regulated by GA(3) at the protein level but not at the mRNA level in the basal region of leaf sheaths. Since this protein is regulated not only by abscisic acid but also by GA(3), these results indicate that ASR5 might be involved in plant growth in addition to stress in the basal regions of leaf sheaths. ASR5|OsAsr1 Ectopic expression of a cold-responsive OsAsr1 cDNA gives enhanced cold tolerance in transgenic rice plants 2009 Mol Cells Department of Life Science, Sogang University, Seoul, Korea. The OsAsrt cDNA clone was isolated from a cDNA library prepared from developing seed coats of rice (Oryza sativa L.). Low-temperature stress increased mRNA levels of OsAsr1 in both vegetative and reproductive organs. In situ analysis showed that OsAsr1 transcript was preferentially accumulated in the leaf mesophyll tissues and parenchyma cells of the palea and lemma. For transgenic rice plants that over-expressed full-length OsAsr1 cDNA in the sense orientation, the Fv/Fm values for photosynthetic efficiency were about 2-fold higher than those of wild type-segregating plants after a 24-h cold treatment. Seedlings exposed to prolonged low temperatures were more tolerant of cold stress, as demonstrated during wilting and regrowth tests. Interestingly, OsAsr1 was highly expressed in transgenic rice plants expressing the C-repeat/dehyhdration responsive element binding factor 1 (CBF1), suggesting the regulation of OsAsr1 by CBF1. Taken together, we suggest that OsAsr1 gene play an important role during temperature stress, and that this gene can be used for generating plants with enhanced cold tolerance. ASR5|OsAsr1 The rice ASR5 protein: a putative role in the response to aluminum photosynthesis disturbance 2012 Plant Signal Behav Programa de Pos-Graduacao em Genetica e Biologia Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil. Under acidic soil conditions, aluminum (Al) becomes available to plants, which must cope with its toxicity by mechanisms involving both internal and external detoxification. Rice is the most Al-tolerant among the crop species, with Al detoxification being managed by both mechanisms. Recently, we focused on ASR (Abscisic acid, Stress and Ripening) gene expression analyses and observed increased ASR5 transcript levels in roots and shoots in response to Al. In addition, ASR5 RNAi knock down plants presented an Al-sensitive phenotype. A proteomic approach showed that ASR5 silencing affected several proteins related to photosynthesis in RNAi rice shoots. Furthermore, an ASR5-GFP fusion in rice protoplasts revealed for the first time a chloroplast localization of this protein. Because it is well known that Al induces photosynthetic dysfunction, here we discuss the hypothesis that ASR5 might be sequestered in the chloroplasts as an inactive transcription factor that could be released to the nucleus in response to Al to regulate genes related to photosynthesis. ASR5|OsAsr1 Abiotic stress responsive rice ASR1 and ASR3 exhibit different tissue-dependent sugar and hormone-sensitivities 2013 Mol Cells Division of Bioscience and Bioinformatics, Myongji University, Seoul, Korea. The expression of the six rice ASR genes is differentially regulated in a tissue-dependent manner according to environmental conditions and reproductive stages. OsASR1 and OsASR3 are the most abundant and are found in most tissues; they are enriched in the leaves and roots, respectively. Coexpression analysis of OsASR1 and OsASR3 and a comparison of the cis-acting elements upstream of OsASR1 and OsASR3 suggested that their expression is regulated in common by abiotic stresses but differently regulated by hormone and sugar signals. The results of quantitative real-time PCR analyses of OsASR1 and OsASR3 expression under various conditions further support this model. The expression of both OsASR1 and OsASR3 was induced by drought stress, which is a major regulator of the expression of all ASR genes in rice. In contrast, ABA is not a common regulator of the expression of these genes. OsASR1 transcription was highly induced by ABA, whereas OsASR3 transcription was strongly induced by GA. In addition, OsASR1 and OsASR3 expression was significantly induced by sucrose and sucrose/glucose treatments, respectively. The induction of gene expression in response to these specific hormone and sugar signals was primarily observed in the major target tissues of these genes (i.e., OsASR1 in leaves and OsASR3 in roots). Our data also showed that the overexpression of either OsASR1 or OsASR3 in transgenic rice plants increased their tolerance to drought and cold stress. Taken together, our results revealed that the transcriptional control of different rice ASR genes exhibit different tissue-dependent sugar and hormone-sensitivities. ASR5|OsAsr1,Asr3|OsASR3 Rice ASR1 protein with reactive oxygen species scavenging and chaperone-like activities enhances acquired tolerance to abiotic stresses in Saccharomyces cerevisiae 2012 Mol Cells Department of Biology, Kyungpook National University, Daegu 702-701, Korea. Abscisic acid stress ripening (ASR1) protein is a small hydrophilic, low molecular weight, and stress-specific plant protein. The gene coding region of ASR1 protein, which is induced under high salinity in rice (Oryza sativa Ilmi), was cloned into a yeast expression vector pVTU260 and transformed into yeast cells. Heterologous expression of ASR1 protein in transgenic yeast cells improved tolerance to abiotic stresses including hydrogen peroxide (H(2)O(2)), high salinity (NaCl), heat shock, menadione, copper sulfate, sulfuric acid, lactic acid, salicylic acid, and also high concentration of ethanol. In particular, the expression of metabolic enzymes (Fba1p, Pgk1p, Eno2p, Tpi1p, and Adh1p), antioxidant enzyme (Ahp1p), molecular chaperone (Ssb1p), and pyrimidine biosynthesis-related enzyme (Ura1p) was up-regulated in the transgenic yeast cells under oxidative stress when compared with wild-type cells. All of these enzymes contribute to an alleviated redox state to H2O2-induced oxidative stress. In the in vitro assay, the purified ASR1 protein was able to scavenge ROS by converting H(2)O(2) to H(2)O. Taken together, these results suggest that the ASR1 protein could function as an effective ROS scavenger and its expression could enhance acquired tolerance of ROS-induced oxidative stress through induction of various cell rescue proteins in yeast cells. ASR5|OsAsr1 Proton pump OsA8 is linked to phosphorus uptake and translocation in rice 2009 J Exp Bot State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, 210095, China. The plasma membrane (PM) proton pump ATPases (H(+)-ATPases) are involved in almost all aspects of biology. They are plant specific and several members of this family are supposed to play a key role in nutrient acquisition. At present, only some members of this gene family in plants have been characterized. However, no nutrient uptake associated H(+)-ATPase gene in rice has been functionally analysed. It is reported here that OsA8, a typical PM H(+)-ATPases gene that was predominantly expressed in roots of rice, is down-regulated by nutrient deficiency. The Osa8 mutant had a relatively smaller size and root to shoot biomass ratio, but higher ATPase activity than its wild-type counterparts under phosphorus (P) starvation conditions. Knockout of OsA8 affected the expression of several OsA genes and the high affinity phosphate transporter, OsPT6, and resulted in a higher P concentration in the roots and a lower amount of P in the shoots. These analyses demonstrate that OsA8 not only influences the uptake of P by roots, but also the translocation of P from the roots to the shoots in rice. OsA8,OsLPT1|OsPht1;6|OsPT6 OsAAP6 functions as an important regulator of grain protein content and nutritional quality in rice 2014 Nature communication National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research (Wuhan), Huazhong Agricultural University, Hongshan District, Wuhan 430070, China NA OsAAP6|qPC1 Identification and functional analysis of a prokaryotic-type aspartate aminotransferase: implications for plant amino acid metabolism 2006 Plant J Departamento de Biologia Molecular y Bioquimica, Instituto Andaluz de Biotecnologia, Unidad Asociada UMA-CSIC, Campus Universitario de Teatinos, Universidad de Malaga, 29071-Malaga, Spain. In this paper, we report the identification of genes from pine (PpAAT), Arabidopsis (AtAAT) and rice (OsAAT) encoding a novel class of aspartate aminotransferase (AAT, EC 2.6.1.1) in plants. The enzyme is unrelated to other eukaryotic AATs from plants and animals but similar to bacterial enzymes. Phylogenetic analysis indicates that this prokaryotic-type AAT is closely related to cyanobacterial enzymes, suggesting it might have an endosymbiotic origin. Interestingly, most of the essential residues involved in the interaction with the substrate and the attachment of pyridoxal phosphate cofactor in the active site of the enzyme were conserved in the deduced polypeptide. The polypeptide is processed in planta to a mature subunit of 45 kDa that is immunologically distinct from the cytosolic, mitochondrial and chloroplastic isoforms of AAT previously characterized in plants. Functional expression of PpAAT sequences in Escherichia coli showed that the processed precursor is assembled into a catalytically active homodimeric holoenzyme that is strictly specific for aspartate. These atypical genes are predominantly expressed in green tissues of pine, Arabidopsis and rice, suggesting a key role of this AAT in nitrogen metabolism associated with photosynthetic activity. Moreover, immunological analyses revealed that the plant prokaryotic-type AAT is a nuclear-encoded chloroplast protein. This implies that two plastidic AAT co-exist in plants: a eukaryotic type previously characterized and the prokaryotic type described here. The respective roles of these two enzymes in plant amino acid metabolism are discussed. OsAAT Phosphorylation of D-allose by hexokinase involved in regulation of OsABF1 expression for growth inhibition in Oryza sativa L 2013 Planta Faculty of Agriculture, Rare Sugar Research Center, and Gene Research Center, Kagawa University, Miki, Kagawa, 761-0795, Japan. We previously reported that a rare sugar D-allose, which is the D-glucose epimer at C3, inhibits the gibberellin-dependent responses such as elongation of the second leaf sheath and induction of alpha-amylase in embryo-less half seeds in rice (Fukumoto et al. 2011). D-Allose suppresses expressions of gibberellin-responsive genes downstream of SLR1 protein in the gibberellin-signaling through hexokinase (HXK)-dependent pathway. In this study, we discovered that D-allose induced expression of ABA-related genes including OsNCED1-3 and OsABA8ox1-3 in rice. Interestingly, D-allose also up-regulated expression of OsABF1, encoding a conserved bZIP transcription factor in ABA signaling, in rice. The D-allose-induced expression of OsABF1 was diminished by a hexokinase inhibitor, D-mannoheptulose (MNH). Consistently, D-allose also inhibited Arabidopsis growth, but failed to trigger growth retardation in the glucose-insensitive2 (gin2) mutant, which is a loss-of-function mutant of the glucose sensor AtHXK1. D-Allose activated AtABI5 expression in transgenic gin2 over-expressing wild-type AtHXK1 but not in gin2 over-expressing the catalytic mutant AtHXK1(S177A), indicating that the D-allose phosphorylation by HXK to D-allose 6-phosphate (A6P) is the first step for the up-regulation of AtABI5 gene expression as well as D-allose-induced growth inhibition. Moreover, overexpression of OsABF1 showed increased sensitivity to D-allose in rice. These findings indicated that the phosphorylation of D-allose at C6 by hexokinase is essential and OsABF1 is involved in the signal transduction for D-allose-induced growth inhibition. OsABA8ox1,OsABA8ox2,OsABA8ox3,OsABF1|OsABI5|OREB1|OsbZIP10,OsNCED1,OsNCED2,OsNCED3 ABA regulates apoplastic sugar transport and is a potential signal for cold-induced pollen sterility in rice 2007 Plant Cell Physiol CSIRO Plant Industry, Canberra, ACT, Australia. Cold temperatures cause pollen sterility and large reductions in grain yield in temperate rice growing regions of the world. Induction of pollen sterility by cold involves a disruption of sugar transport in anthers, caused by the cold-induced repression of the apoplastic sugar transport pathway in the tapetum. Here we demonstrate that the phytohormone ABA is a potential signal for cold-induced pollen sterility (CIPS). Cold treatment of the cold-sensitive cultivar Doongara resulted in increased anther ABA levels. Exogenous ABA treatment at the young microspore stage induced pollen sterility and affected cell wall invertase and monosaccharide transporter gene expression in a way similar to cold treatment. In the cold-tolerant cultivar R31, ABA levels were significantly lower under normal circumstances and remained low after cold treatment. The differences in endogenous ABA levels in Doongara and R31 correlated with differences in expression of the ABA biosynthetic genes encoding zeaxanthin epoxidase (OSZEP1) and 9-cis-epoxycarotenoid dioxygenase (OSNCED2, OSNCED3) in anthers. The expression of three ABA-8-hydroxylase genes (ABA8OX1, 2 and 3) in R31 anthers was higher under control conditions and was regulated differently by cold compared with Doongara. Our results indicate that the cold tolerance phenotype of R31 is correlated with lower endogenous ABA levels and a different regulation of ABA metabolism. OsABA8ox1,OsABA8ox2,OsABA8ox3,OsNCED2,OsNCED3,OsABA1|OSZEP1,OsMST7 Ethylene promotes submergence-induced expression of OsABA8ox1, a gene that encodes ABA 8'-hydroxylase in rice 2007 Plant Cell Physiol Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, Japan. A rapid decrease of the plant hormone ABA under submergence is thought to be a prerequisite for the enhanced elongation of submerged shoots of rice (Oryza sativa L.). Here, we report that the level of phaseic acid (PA), an oxidized form of ABA, increased with decreasing ABA level during submergence. The oxidation of ABA to PA is catalyzed by ABA 8'-hydroxylase, which is possibly encoded by three genes (OsABA8ox1, -2 and -3) in rice. The ABA 8'-hydroxylase activity was confirmed in microsomes from yeast expressing OsABA8ox1. OsABA8ox1-green fluorescent protein (GFP) fusion protein in onion cells was localized to the endoplasmic reticulum. The mRNA level of OsABA8ox1, but not the mRNA levels of other OsABA8ox genes, increased dramatically within 1 h after submergence. On the other hand, the mRNA levels of genes involved in ABA biosynthesis (OsZEP and OsNCEDs) decreased after 1-2 h of submergence. Treatment of aerobic seedlings with ethylene and its precursor, 1-aminocyclopropane-1-carboxylate (ACC), rapidly induced the expression of OsABA8ox1, but the ethylene treatment did not strongly affect the expression of ABA biosynthetic genes. Moreover, pre-treatment with 1-methylcyclopropene (1-MCP), a potent inhibitor of ethylene action, partially suppressed induction of OsABA8ox1 expression under submergence. The ABA level was found to be negatively correlated with OsABA8ox1 expression under ACC or 1-MCP treatment. Together, these results indicate that the rapid decrease in ABA levels in submerged rice shoots is controlled partly by ethylene-induced expression of OsABA8ox1 and partly by ethylene-independent suppression of genes involved in the biosynthesis of ABA. OsABA8ox1,OsABA8ox2,OsABA8ox3,OsNCED1,OsNCED2,OsNCED3 Glucose-induced delay of seed germination in rice is mediated by the suppression of ABA catabolism rather than an enhancement of ABA biosynthesis 2009 Plant Cell Physiol College of Life Science, South China Agricultural University, Guangdong, PR China. Both glucose and ABA play crucial roles in the regulation of seed germination and post-germination development. In Arabidopsis thaliana, up-regulation of ABA biosynthesis is suggested as one of the possible mechanisms mediating the glucose-induced delay in seed germination. Since the endogenous ABA level is controlled by the equilibrium between ABA biosynthesis and catabolism, we investigated how this equilibrium is related to the regulation of seed germination by glucose in rice. When ABA biosynthesis was inhibited by nordihydroguaiaretic acid (NDGA), an inhibitor of the ABA anabolic enzyme 9-cis-epoxycarotenoid dioxygenase (NCED), rice seed germination showed no response. In contrast, inhibition of ABA catabolism by diniconazole significantly arrested seed germination, suggesting that the regulation of ABA catabolism plays a major role. Further experiments indicated that the expression of OsABA8ox3, a key gene in ABA catabolism and encoding ABA 8'-hydroxylase in rice, was significantly increased during the first 6 h of imbibition, which was consistent with the decline of ABA content in the imbibed seeds. Expression of OsABA8ox genes, especially OsABA8ox2 and OsABA8ox3, was sensitively suppressed in the presence of exogenously supplied glucose. In contrast, the expression profiles of OsNCED genes that control the limiting step of ABA biosynthesis showed no significant changes in response to low levels of glucose. Our results demonstrated that the glucose-induced delay of seed germination is a result of the suppression of ABA catabolism rather than any enhancement of ABA biosynthesis during rice seed germination. OsABA8ox1,OsABA8ox2,OsABA8ox3,OsNCED1,OsNCED2,OsNCED3,OsNCED4,OsNCED5 Characterization of an Abc1 kinase family gene OsABC1-2 conferring enhanced tolerance to dark-induced stress in rice 2012 Gene Jiangsu Provincial Key Laboratory of Crop Genetics and Physiology, Key Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou University, Yangzhou 225009, PR China. Leaf senescence is a complex and highly organized process resulting in numerous changes of gene expression and metabolic procedures. However, the exact mechanisms underlying these changes are not well understood. In this study, we reported a rice (Oryza sativa) T-DNA insertion mutant impaired in an Abc1 kinase family gene with a dwarf and pale-green phenotype. The mutant showed reduced pigment content and photosynthetic efficiency and increased superoxide dismutase activity in leaves. The mutated gene, designated OsABC1-2, is expressed primarily in green tissues and/or organs and encodes a protein localized in chloroplast envelope. Expression of the gene was drastically suppressed by dark treatment. Overexpression of the gene in rice enhanced tolerance to prolonged dark-induced stress. Phylogenetic analysis revealed that the plant Abc1 proteins could be divided into three subgroups and OsAbc1-2 resides in a subgroup with potential chloroplast origin. Our results suggest that divergence has occurred among plant Abc1 family and chloroplast Abc1 kinases play potential roles in regulating dark-induced senescence of plants. OsABC1-2 OsABCB14 functions in auxin transport and iron homeostasis in rice (Oryza sativa L.) 2014 Plant J State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, China. Members of the ATP Binding Cassette B/Multidrug-Resistance/P-glyco-protein (ABCB/MDR/PGP) subfamily were shown to function primarily in Oryza sativa (rice) auxin transport; however, none of the rice ABCB transporters have been functionally characterized. Here, we describe that a knock-down of OsABCB14 confers decreased auxin concentrations and polar auxin transport rates, conferring insensitivity to 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetic acid (IAA). OsABCB14 displays enhanced specific auxin influx activity in yeast and protoplasts prepared from rice knock-down alleles. OsABCB14 is localized at the plasma membrane, pointing to an important directionality under physiological conditions. osabcb14 mutants were surprisingly found to be insensitive to iron deficiency treatment (-Fe). Their Fe concentration is higher and upregulation of Fe deficiency-responsive genes is lower in osabcb14 mutants than in wild-type rice (Nipponbare, NIP). Taken together, our results strongly support the role of OsABCB14 as an auxin influx transporter involved in Fe homeostasis. The functional characterization of OsABCB14 provides insights in monocot auxin transport and its relationship to Fe nutrition. OsABCB14 A rice ABC transporter, OsABCC1, reduces arsenic accumulation in the grain 2014 Proc Natl Acad Sci U S A Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang 790-784, Korea; Arsenic (As) is a chronic poison that causes severe skin lesions and cancer. Rice (Oryza sativa L.) is a major dietary source of As; therefore, reducing As accumulation in the rice grain and thereby diminishing the amount of As that enters the food chain is of critical importance. Here, we report that a member of the Oryza sativa C-type ATP-binding cassette (ABC) transporter (OsABCC) family, OsABCC1, is involved in the detoxification and reduction of As in rice grains. We found that OsABCC1 was expressed in many organs, including the roots, leaves, nodes, peduncle, and rachis. Expression was not affected when plants were exposed to low levels of As but was up-regulated in response to high levels of As. In both the basal nodes and upper nodes, which are connected to the panicle, OsABCC1 was localized to the phloem region of vascular bundles. Furthermore, OsABCC1 was localized to the tonoplast and conferred phytochelatin-dependent As resistance in yeast. Knockout of OsABCC1 in rice resulted in decreased tolerance to As, but did not affect cadmium toxicity. At the reproductive growth stage, the As content was higher in the nodes and in other tissues of wild-type rice than in those of OsABCC1 knockout mutants, but was significantly lower in the grain. Taken together, our results indicate that OsABCC1 limits As transport to the grains by sequestering As in the vacuoles of the phloem companion cells of the nodes in rice. OsABCC1|MRP1 RiceABCG43Is Cd Inducible and Confers Cd Tolerance on Yeast 2014 Bioscience, Biotechnology and Biochemistry Department of Applied Biological Chemistry, The University of Tokyo, Tokyo, Japan. An understanding of the cadmium (Cd) transport system in rice can serve as a basis for coping with Cd-related problems in rice and human health. To identify a new gene involved in Cd transport in rice, we screened our yeast library, expressing 140 kinds of rice ATP binding cassette (ABC)-type transporters. From the screening, we found that OsABCG43/PDR5 conferred high Cd tolerance on yeast. The Cd concentration of yeast carrying OsABCG43 was similar to that of the vector control. The OsABCG43 transcript was detected both in shoots and roots. Accumulation of it was elevated by Cd treatment in the roots but not in the shoots. This study indicates that OsABCG43 is a Cd inducible-transporter gene capable of conferring Cd tolerance on yeast. OsABCG43|PDR5 The NAC family transcription factor OsNAP confers abiotic stress response through the ABA pathway 2014 Plant Cell Physiol State Key Laboratory of Genetic Engineering, Institute of Genetics, Institute of Plant Biology, School of Life Science, Fudan University, Shanghai 200433, China. Plants respond to environmental stresses by altering gene expression, and several genes have been found to mediate stress-induced expression, but many additional factors are yet to be identified. OsNAP is a member of the NAC transcription factor family; it is localized in the nucleus, and shows transcriptional activator activity in yeast. Analysis of the OsNAP transcript levels in rice showed that this gene was significantly induced by ABA and abiotic stresses, including high salinity, drought and low temperature. Rice plants overexpressing OsNAP did not show growth retardation, but showed a significantly reduced rate of water loss, enhanced tolerance to high salinity, drought and low temperature at the vegetative stage, and improved yield under drought stress at the flowering stage. Microarray analysis of transgenic plants overexpressing OsNAP revealed that many stress-related genes were up-regulated, including OsPP2C06/OsABI2, OsPP2C09, OsPP2C68 and OsSalT, and some genes coding for stress-related transcription factors (OsDREB1A, OsMYB2, OsAP37 and OsAP59). Our data suggest that OsNAP functions as a transcriptional activator that plays a role in mediating abiotic stress responses in rice. OsABI2|OsPP2C06,OsNAP,OsPP2C09|OsPP15,OsSIPP2C1|OsPP2C68 Characterization of alternative splicing products of bZIP transcription factors OsABI5 2007 Biochem Biophys Res Commun National Centre for Plant Gene Research, Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, South 1-3, Zhongguancun, Beijing 100080, PR China. Alternative splicing allows many gene products to alter their biological functions. A bZIP-type transcription factor, OsABI5, undergoes alternative splicing. Two OsABI5 splicing variants were identified, designated OsABI5-1, and OsABI5-2 and their different expression patterns in tissues were analyzed. Despite a completely identical functional domain, OsABI5-2 could specifically bind to G-box element, but OsABI5-1 could not; the transactivation activity of OsABI5-1 was higher than that of OsABI5-2; the interaction strength of OsABI5-2 and OsVP1 was stronger than that of OsABI5-1 and OsVP1; indicating a different function in the regulation of downstream target genes. Complementation tests and ABA (abscisic acid) hypersensitivity of Arabidopsis transgenic lines revealed the redundant function of OsABI5 splicing variants in ABA signaling. The interaction between OsABI5-1 and OsABI5-2 was also confirmed. These results suggest that OsABI5 variants may have overlapping and distinct functions to fine tune gene expression in ABA signaling as transcription factors together with OsVP1. OsABF1|OsABI5|OREB1|OsbZIP10 Phosphorylation-mediated regulation of a rice ABA responsive element binding factor 2011 Phytochemistry Bio-Crops Development Division, National Academy of Agricultural Sciences, Suwon, Republic of Korea. OREB1 is a rice ABRE binding factor characterized by the presence of multiple highly-conserved phosphorylation domains (C1, C2, C3, and C4) and two kinase recognition motifs, RXXS/T and S/TXXE/D, within different functional domains. An in vitro kinase assay showed that OREB1 is phosphorylated not only by the SnRK2 kinase, but also by other Ser/Thr protein kinases, such as CaMKII, CKII, and SnRK3. Furthermore, the N-terminal phosphorylation domain C1 was found to be differentially phosphorylated by the SnRK2/SnRK3 kinase and by hyperosmotic/cold stress, suggesting that the C1 domain may function in decoding different signals. The phosphorylation-mediated regulation of OREB1 activity was investigated through mutation of the SnRK2 recognition motif RXXS/T within each phosphorylation module. OREB1 contains a crucial nine-amino acid transactivation domain located near the phosphorylation module C1. Deletion of the C1 domain increased OREB1 activity, whereas mutation of Ser 44, Ser 45, and Ser 48 of the C1 domain to aspartates decreased OREB1 activity. In the C2 domain, a double mutation of Ser 118 and Ser 120 to alanines suppressed OREB1 activity. These findings strongly suggest that selective phosphorylation of the C1 or C2 modules may positively or negatively regulate OREB1 transactivation. In addition, mutation of Ser 385 of the C4 domain to alanines completely abolished the interaction between OREB1 and a rice 14-3-3 protein, GF14d, suggesting that SnRK2-mediated phosphorylation may regulate this interaction. These results indicate that phosphorylation domains of OREB1 are not functionally redundant and regulate at least three different functions, including transactivation activity, DNA binding, and protein interactions. The multisite phosphorylation of OREB1 is likely a key for the fine control of its activity and signal integration in the complex stress signaling network of plant cells. OsABF1|OsABI5|OREB1|OsbZIP10 A rice dehydration-inducible SNF1-related protein kinase 2 phosphorylates an abscisic acid responsive element-binding factor and associates with ABA signaling 2007 Plant Mol Biol Cell and Genetics Division, National Institute of Agricultural Biotechnology, Suwon, 441-707, Republic of Korea. By a differential cDNA screening technique, we have isolated a dehydration-inducible gene (designated OSRK1) that encodes a 41.8 kD protein kinase of SnRK2 family from Oryza sativa. The OSRK1 transcript level was undetectable in vegetative tissues, but significantly increased by hyperosmotic stress and Abscisic acid (ABA). To determine its biochemical properties, we expressed and isolated OSRK1 and its mutants as glutathione S-transferase fusion proteins in Escherichia coli. In vitro kinase assay showed that OSRK1 can phosphorylate itself and generic substrates as well. Interestingly, OSRK1 showed strong substrate preference for rice bZIP transcription factors and uncommon cofactor requirement for Mn(2+) over Mg(2+). By deletion of C-terminus 73 amino acids or mutations of Ser-158 and Thr-159 to aspartic acids (Asp) in the activation loop, the activity of OSRK1 was dramatically decreased. OSRK1 can transphosphorylate the inactive deletion protein. A rice family of abscisic acid-responsive element (ABRE) binding factor, OREB1 was phosphorylated in vitro by OSRK1 at multiple sites of different functional domains. MALDI-TOF analysis identified a phosphorylation site at Ser44 of OREB1 and mutation of the residue greatly decreased the substrate specificity for OSRK1. The recognition motif for OSRK1, RQSS is highly similar to the consensus substrate sequence of AMPK/SNF1 kinase family. We further showed that OSRK1 interacts with OREB1 in a yeast two-hybrid system and co-localized to nuclei by transient expression analysis of GFP-fused protein in onion epidermis. Finally, ectopic expression of OSRK1 in transgenic tobacco resulted in a reduced sensitivity to ABA in seed germination and root elongation. These findings suggest that OSRK1 is associated with ABA signaling, possibly through the phosphorylation of ABF family in vivo. The interaction between SnRK2 family kinases and ABF transcription factors may constitute an important part of cross-talk mechanism in the stress signaling networks in plants. OsABF1|OsABI5|OREB1|OsbZIP10,SAPK6|OSRK1 The bZIP transcription factor OsABF1 is an ABA responsive element binding factor that enhances abiotic stress signaling in rice 2010 Plant Mol Biol Department of Bioscience and Biotechnology, The University of Suwon, Hwasung 445-743, Korea. A number of basic leucine zipper (bZIP) transcription factors are known to function in stress signaling in plants but few have thus far been functionally characterized in rice. In our current study in rice, we have newly isolated and characterized the OsABF1 (Oryza sativa ABA responsive element binding factor 1) gene that encodes a bZIP transcription factor. Its expression in seedling shoots and roots was found to be induced by various abiotic stress treatments such as anoxia, salinity, drought, oxidative stress, cold and abscisic acid (ABA). Subcellular localization analysis in maize protoplasts using GFP fusion vectors indicated that OsABF1 is a nuclear protein. In a yeast experiment, OsABF1 was shown to bind to ABA responsive elements (ABREs) and its N-terminal region was necessary to transactivate the downstream reporter gene. The homozygous T-DNA insertional mutants Osabf1-1 and Osabf1-2 were more sensitive in response to drought and salinity treatments than wild type plants. Furthermore, the upregulated expression of some ABA/stress-regulated genes in response to ABA treatment was suppressed in these Osabf1 mutants. Our current results thus suggest that OsABF1 is involved in abiotic stress responses and ABA signaling in rice. OsABF1|OsABI5|OREB1|OsbZIP10,OsABA45 Constitutive activation of transcription factor OsbZIP46 improves drought tolerance in rice 2012 Plant Physiol National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China. OsbZIP46 is one member of the third subfamily of bZIP transcription factors in rice (Oryza sativa). It has high sequence similarity to ABA-responsive element binding factor (ABF/AREB) transcription factors ABI5 and OsbZIP23, two transcriptional activators positively regulating stress tolerance in Arabidopsis (Arabidopsis thaliana) and rice, respectively. Expression of OsbZIP46 was strongly induced by drought, heat, hydrogen peroxide, and abscisic acid (ABA) treatment; however, it was not induced by salt and cold stresses. Overexpression of the native OsbZIP46 gene increased ABA sensitivity but had no positive effect on drought resistance. The activation domain of OsbZIP46 was defined by a series of deletions, and a region (domain D) was identified as having a negative effect on the activation. We produced a constitutive active form of OsbZIP46 (OsbZIP46CA1) with a deletion of domain D. Overexpression of OsbZIP46CA1 in rice significantly increased tolerance to drought and osmotic stresses. Gene chip analysis of the two overexpressors (native OsbZIP46 and the constitutive active form OsbZIP46CA1) revealed that a large number of stress-related genes, many of them predicted to be downstream genes of ABF/AREBs, were activated in the OsbZIP46CA1 overexpressor but not (even down-regulated) in the OsbZIP46 overexpressor. OsbZIP46 can interact with homologs of SnRK2 protein kinases that phosphorylate ABFs in Arabidopsis. These results suggest that OsbZIP46 is a positive regulator of ABA signaling and drought stress tolerance of rice depending on its activation. The stress-related genes activated by OsbZIP46CA1 are largely different from those activated by the other rice ABF/AREB homologs (such as OsbZIP23), further implying the value of OsbZIP46CA1 in genetic engineering of drought tolerance. OsABF1|OsABI5|OREB1|OsbZIP10,OsAREB8|OsAREB1|OsbZIP46|OsABF2|ABL1,OsbZIP23 Rice ABI5-Like1 regulates abscisic acid and auxin responses by affecting the expression of ABRE-containing genes 2011 Plant Physiol Shanghai Institutes for Biological Sciences-University of California, Berkeley, Joint Center on Molecular Life Sciences, National Key Laboratory of Plant Molecular Genetics, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China. Abscisic acid (ABA) regulates plant development and is crucial for plant responses to biotic and abiotic stresses. Studies have identified the key components of ABA signaling in Arabidopsis (Arabidopsis thaliana), some of which regulate ABA responses by the transcriptional regulation of downstream genes. Here, we report the functional identification of rice (Oryza sativa) ABI5-Like1 (ABL1), which is a basic region/leucine zipper motif transcription factor. ABL1 is expressed in various tissues and is induced by the hormones ABA and indole-3-acetic acid and stress conditions including salinity, drought, and osmotic pressure. The ABL1 deficiency mutant, abl1, shows suppressed ABA responses, and ABL1 expression in the Arabidopsis abi5 mutant rescued the ABA sensitivity. The ABL1 protein is localized to the nucleus and can directly bind ABA-responsive elements (ABREs; G-box) in vitro. A gene expression analysis by DNA chip hybridization confirms that a large proportion of down-regulated genes of abl1 are involved in stress responses, consistent with the transcriptional activating effects of ABL1. Further studies indicate that ABL1 regulates the plant stress responses by regulating a series of ABRE-containing WRKY family genes. In addition, the abl1 mutant is hypersensitive to exogenous indole-3-acetic acid, and some ABRE-containing genes related to auxin metabolism or signaling are altered under ABL1 deficiency, suggesting that ABL1 modulates ABA and auxin responses by directly regulating the ABRE-containing genes. OsABF1|OsABI5|OREB1|OsbZIP10,OsAREB8|OsAREB1|OsbZIP46|OsABF2|ABL1 A bZIP transcription factor, OsABI5, is involved in rice fertility and stress tolerance 2008 Plant Mol Biol Key Laboratory of Molecular and Developmental Biology, National Centre for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, P.O. Box 2707, South 1-3, Zhongguancun, Beijing 100080, P.R. China. The phytohormone abscisic acid (ABA) is involved in the adaptive stress response and regulates expression of many stress-responsive genes, including some transcriptional factors. A bZIP transcription factor, OsABI5, was isolated from the panicle of Oryza sativa L. Expression of the OsABI5 gene was induced by abscisic acid (ABA) and high salinity, and down-regulated by drought and cold (4 degrees C) in seedlings. The OsABI5 protein was localized in the nucleus and has trans-activation activity. The N-terminal of the protein is necessary for its activity. OsABI5 could bind to a G-box element and trans-activate reporter gene expression. Complementation analysis revealed that the expression of OsABI5 driven by the 35S promoter could rescue ABA-insensitivity of abi5-1 during seed germination and result in hypersensitivity to ABA. Over-expression of OsABI5 in rice conferred high sensitivity to salt stress. Repression of OsABI5 promoted stress tolerance and resulted in low fertility of rice. These results suggested that OsABI5 could regulate the adaptive stress response and plant fertility of rice as a transcription factor. OsABF1|OsABI5|OREB1|OsbZIP10 A new DEAD-box helicase ATP-binding protein (OsABP) from rice is responsive to abiotic stress 2012 Plant Signal Behav Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India. The DEAD-box RNA helicase family comprise enzymes that participate in every aspect of RNA metabolism, associated with a diverse range of cellular functions including response to abiotic stress. In the present study, we report on the identification of a new DEAD-box helicase ATP-binding protein (OsABP) from rice which is upregulated in response e to multiple abiotic stress treatments including NaCl, dehydration, ABA, blue and red light. It possesses an ORF of 2772 nt, encoding a protein of 923 aa, which contains the DEAD and helicase C-terminal domains, along with the nine conserved motifs specific to DEAD-box helicases. The in silico putative interaction with other proteins showed that OsABP interacts with proteins involved in RNA metabolism, signal transduction or stress response. These results imply that OsABP might perform important functions in the cellular response to specific abiotic stress. OsABP ACO1, a gene for aminocyclopropane-1-carboxylate oxidase: effects on internode elongation at the heading stage in rice 2010 Plant Cell Environ Photobiology and Photosynthesis Research Unit, Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. iwamas@nias.affrc.go.jp Although reports on a gene for 1-amino-cyclopropane-1-carboxylate (ACC) oxidase (ACO1) in rice (Oryza sativa L.) suggest that high levels of its transcript are associated with internode elongation of deep-water rice during submergence, the role of ACO1 in rice development is largely unknown. The tissue-specificity of ACO1 expression indicated that its transcript significantly accumulated in lower parts of elongating internodes at the heading stage. Histochemical analysis and in situ hybridization showed that the ACO1 expression was localized in the basal parts of leaf sheaths immediately above nodes or the lower parts of elongating internodes. To further examine the role of ACO1, ACO1-deficient (aco1) and overexpressing (ACO1-OX) mutants were characterized. The total length of the elongated internodes of aco1 mutants was slightly shorter than that of wild-type plants and that of ACO1-OX mutants was longer. Interestingly, expression of the ACC synthase gene ACS1 and ethylene signalling gene OsEIN2 was up-regulated in the aco1 mutants. This study suggests that the ACO1 has a little effect on internode elongation at the heading stage, and that up-regulation of the ACS1 and OsEIN2 expression may attenuate inhibition of internode elongation. OsACO1,OsACS1,OsEIN2 OsEIL1, a rice homolog of the Arabidopsis EIN3 regulates the ethylene response as a positive component 2006 Plant Mol Biol Institute of Plant Sciences, College of Life Sciences, Zhejiang University, Hangzhou, PR China. The plant gaseous hormone ethylene regulates many aspects of plant growth, development and responses to the environment. ETHYLENE INSENSITIVE3 (EIN3) is a transcription factor involved in the ethylene signal transduction pathway in Arabidopsis. To gain a better understanding of the ethylene signal transduction pathway in rice, six EIN3-like genes (designated OsEIL1-6) were identified. OsEIL1, which showed highest similarity with EIN3, was isolated and functionally characterized. Ectopic expression of OsEIL1 in Arabidopsis can partially complement the ein3-1 mutant. The transgenic rice plants with overexpression of OsEIL1 exhibit short root, coiled primary root and slightly short shoot phenotype and elevated response to exogenous ethylene. OsEBP89, an ethylene responsive element binding protein (EREBP) and OsACO1, an ACC (1-aminocyclopropane-1-carboxylic acid) oxidase gene were enhanced in the OsEIL1 overexpressing transgenic plants. These results indicate that OsEIL1 is involved in ethylene signal transduction pathway and acts as a positive regulator of ethylene response in rice. OsACO1,OsEIL1|EIN3 Submergence enhances expression of a gene encoding 1-aminocyclopropane-1-carboxylate oxidase in deepwater rice 1996 Plant Cell Physiol MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing 48824-1312, USA. Partial submergence greatly stimulates internodal growth in deep water rice (Oryza sativa L.). Previous work has shown that the effect of submergence is, at least in part, mediated by ethylene, which accumulates in the air spaces of submerged internodes. To investigate the expression of the genes encoding ethylene biosynthetic enzymes during accelerated growth of deep water rice, we cloned a 1-aminocyclopropane-1-carboxylate (ACC) oxidase cDNA (OS-ACO1) from internodes of submerged plants and measured the activity of the enzyme in tissue extracts with an improved assay. We found an increase in ACC oxidase mRNA levels and enzyme activity after 4 to 24 h of submergence. Thus, it is likely that ethylene biosynthesis in internodes of deep water rice is controlled, at least in part, at the level of ACC oxidase. OsACO1 OsEIN2 is a positive component in ethylene signaling in rice 2004 Plant and Cell Physiology National Research Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784 Korea. EIN2 is a central signal transducer in the ethylene-signaling pathway, and a unique membrane-anchored protein. By screening a cDNA library, we have isolated a cDNA clone (OsEIN2) that encodes the rice EIN2 homolog. The full-length ORF clone was obtained by reverse transcriptase-polymerase chain reaction. OsEIN2 shares significant amino acid sequence similarity with Arabidopsis EIN2 (57% similarity and 42% identity). Both the numbers and positions of introns and exons in the OsEIN2 and AtEIN2 coding regions are also conserved. To address whether this structural similarity is indicative of functional conservation of the corresponding proteins, we also generated transgenic lines expressing the antisense construct of OsEIN2. Those plants were stunted and shoot elongation was severely inhibited. Their phenotypes were similar to that found with wild-type rice seedlings that were treated with AgNO3, an ethylene signal inhibitor. In the OsEIN2 antisense plants, the expression levels of two ethylene-responsive genes, SC129 and SC255, were decreased compared with the wild types. These results suggest that OsEIN2 is a positive component of the ethylene-signaling pathway in rice, just as AtEIN2 is in Arabidopsis. Our antisense transgenic plants produced approximately 3.5 times more ethylene than the wild-type plants. Expression analysis of rice ACS and ACO genes showed that the transcript levels of OsACS1 and OsACS1 were elevated in the transgenic plants. OsACO1,OsACS1,OsEIN2 EAR motif mutation of rice OsERF3 alters the regulation of ethylene biosynthesis and drought tolerance 2013 Planta Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China. OsERF3 is a transcriptional repressor with an ethylene-responsive element-binding factor-associated amphiphilic repression (EAR) motif (F/LDLNxxP), which transcriptionally represses the ethylene emission and drought tolerance in rice. However, its molecular mechanism to explore repression function remains unknown. Here, we first revealed that the expression of OsERF3 was induced by drought, salt, ACC and ABA treatment. In addition, it showed a higher expression level in the root and sheath than that in the leaf. Then, we generated transgenic rice overexpressing full-length OsERF3 (OE) and its mutation of EAR motif with the A 680/C substitution (mEAR), respectively. The physiological analyses showed that mEAR lines showed better drought tolerance and more ethylene emission compared with those of OE lines and wild type plants. Consistent with our previous research, the expression of ethylene synthesis genes, including ACO2, ACS2, and ACS6 was down-regulated in OE lines. However, the repression of OsERF3 was eliminated in mEAR lines. Specifically, ACS2 was up-regulated in mEAR lines compared with that in OE lines and WT plants, suggesting that the Leu/Ala substitution within the EAR motif resulted in loss of repression of OsERF3. Thus, our data reveal that the EAR motif is required for OsERF3 to transcriptionally regulate the ethylene synthesis and drought tolerance in rice, providing new insight to the roles of ethylene-response factor proteins in regulating ethylene biosynthesis and stress response. OsACO2,OsACS2,OsACS6,AP37|OsERF3 Contribution of ethylene biosynthesis for resistance to blast fungus infection in young rice plants 2006 Plant Physiol National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. The role of ethylene (ET) in resistance to infection with blast fungus (Magnaporthe grisea) in rice (Oryza sativa) is poorly understood. To study it, we quantified ET levels after inoculation, using young rice plants at the four-leaf stage of rice cv Nipponbare (wild type) and its isogenic plant (IL7), which contains the Pi-i resistance gene to blast fungus race 003. Small necrotic lesions by hypersensitive reaction (HR) were formed at 42 to 72 h postinoculation (hpi) in resistant IL7 leaves, and whitish expanding lesions at 96 hpi in susceptible wild-type leaves. Notable was the enhanced ET emission at 48 hpi accompanied by increased 1-aminocyclopropane-1-carboxylic acid (ACC) levels and highly elevated ACC oxidase (ACO) activity in IL7 leaves, whereas only an enhanced ACC increase at 96 hpi in wild-type leaves. Among six ACC synthase (ACS) and seven ACO genes found in the rice genome, OsACS2 was transiently expressed at 48 hpi in IL7 and at 96 hpi in wild type, and OsACO7 was expressed at 48 hpi in IL7. Treatment with an inhibitor for ACS, aminooxyacetic acid, suppressed enhanced ET emission at 48 hpi in IL7, resulting in expanding lesions instead of HR lesions. Exogenously supplied ACC compromised the aminooxyacetic acid-induced breakdown of resistance in IL7, and treatment with 1-methylcyclopropene and silver thiosulfate, inhibitors of ET action, did not suppress resistance. These findings suggest the importance of ET biosynthesis and, consequently, the coproduct, cyanide, for HR-accompanied resistance to blast fungus in young rice plants and the contribution of induced OsACS2 and OsACO7 gene expression to it. OsACO2,OsACO3,OsACO4,OsACO5|OsACO6,OsACO7,OsACS1,OsACS2,OsACS3,OsACS4,OS-ACS5|OsACS5,OsACS6 Hormonal Cross-Talk Between Auxin and Ethylene Differentially Regulates the Expression of Two Members of the 1-Aminocyclopropane-l-Carboxylate Oxidase Gene Family in Rice 2000 Plant Cell Physiol Department of Biology, College of Science, Yonsei University, Seoul, Korea. Two cDNA clones, pOS-ACO2 and pOS-ACO3, encoding 1-aminocyclopropane-1-carboxylate (ACC) oxidase were isolated from rice seedling cDNA library. pOS-ACO3 is a 1,299 bp full-length clone encoding 321 amino acids (Mr=35.9 kDa), while pOS-ACO2 is 1,072 bp long and is a partial cDNA clone encoding 314 amino acids. These two deduced amino acid sequences share 70% identity, and display a high degree of sequence identity (72-92%) with previously isolated pOS-ACO1 of deepwater rice. The chromosomal location studies show that OS-ACO2 is positioned on the long arm of chromosome 9, while OS-ACO3 on the long arm of chromosome 2 of rice genome. A marked increase in the level of OS-ACO2 transcript was observed in IAA-treated etiolated rice seedlings, whereas the OS-ACO3 mRNA was greatly accumulated by ethylene treatment. Results of ethylene inhibitor studies indicated that auxin promotion of the OS-ACO2 transcription was not mediated through the action of auxin-induced ethylene. Thus, it appears that there are two groups of ACC oxidase transcripts in rice plants, either auxin-induced or ethylene-induced. The auxin-induced OS-ACO2 expression was partially inhibited by ethylene, while ethylene induction of OS-ACO3 transcription was completely blocked by auxin. These results indicate that the expression of ACC oxidase genes is regulated by complex hormonal networks in a gene specific manner in rice seedlings. Okadaic acid, a potent inhibitor of protein phosphatase, effectively suppressed the IAA induction of OS-ACO2 expression, suggesting that protein dephosphorylation plays a role in the induction of ACC oxidase by auxin. A scheme of the multiple regulatory pathways for the expression of ACC oxidase gene family by auxin, ethylene and protein phosphatase is presented. OsACO3 Ethylene biosynthesis and signaling in rice 2008 Plant Science Botanisches Institut, Universität Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany Ethylene accumulates to higher levels upon submergence of rice plants where it plays a central role in hypoxia adaptation. In deepwater rice positive feedback regulation of ethylene on the biosynthetic genes 1-aminocyclopropane-1-carboxylic acid synthase (OsACS5), ACC oxidase (OsACO1 and OsACO3) and the Yang cycle OsARD1 gene likely contributes to maintain elevated rates of ethylene synthesis over prolonged periods of time. Ethylene modulates gibberellin and abscisic acid homeostasis thereby promoting internodal growth which keeps part of the foliage above flood waters. Ethylene further induces growth of adventitious roots and programmed cell death to form aerenchyma and to facilitate adventitious root emergence at stem nodes. The ethylene signaling components identified so far in rice include receptors, an EIN2 and an EIN3 ortholog, and CTR1, RTE1, EBF1/2 and EIN5 homologs that are conserved between the dicot Arabidopsis and the monocot rice. Based on the observation that ein2 and eil1 mutants of rice do not display a strong phenotype we conclude that ethylene signaling in rice may be more intricate than in Arabidopsis. In addition, ethylene may have specific signaling outputs in semiaquatic plants such as rice as was shown for Yang cycle regulation. Functional analysis of signaling pathways including the large ethylene response factor (ERF) family may help understand how ethylene co-ordinates adaptive responses to flooding stress in rice. OsACO4 A phosphate starvation-induced acid phosphatase from Oryza sativa: phosphate regulation and transgenic expression 2007 Biotechnol Lett College of Natural Resources and Life Science, Dong-A University, Busan, 604-714, Korea. A phosphate starvation-induced acid phosphatase cDNA was cloned from the rice, Oryza sativa. The cDNA encoding O. sativa acid phosphatase (OsACP1) has 1100 bp with an open reading frame of 274 amino acid residues. The deduced amino acid sequence of OsACP1 cDNA showed 53% identity to tomato acid phosphatase and 46-50% identity to several other plant phosphatases. OsACP1 expression was up-regulated in the rice plant and in cell culture in the absence of phosphate (Pi). The induced expression of OsACP1 was a specific response to Pi starvation, and was not affected by the deprivation of other nutrients. OsACP1 expression was responsive to the level of Pi supply, with transcripts of OsACP1 being abundant in Pi-deprived root. The OsACP1 cDNA was expressed as a 30 kDa polypeptide in baculovirus-infected insect Sf9 cells. In addition, the OsACP1 gene was introduced into Arabidopsis via Agrobacterium-mediated transformation. Functional expression of the OsACP1 gene in the transgenic Arabidopsis lines was confirmed by Northern blot and Western blot analyses, as well as phosphatase activity assays. These results suggest that the OsACP1 gene can be used to develop new transgenic dicotyledonous plants able to adapt to Pi-deficient conditions. OsACP1 A CDC25 homologue from rice functions as an arsenate reductase 2007 New Phytol Department of Soil Environmental Sciences, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, 18 Shuangqing RD, Beijing 100085, People's Republic of China. Enzymatic reduction of arsenate to arsenite is the first step in arsenate metabolism in all organisms studied. The rice genome contains two ACR2-like genes, OsACR2.1 and OsACR2.2, which may be involved in regulating arsenic metabolism in rice. Here, we cloned both OsACR2 genes and expressed them in an Escherichia coli strain in which the arsC gene was deleted and in a yeast (Saccharomyces cerevisiae) strain with a disrupted ACR2 gene. OsACR2.1 complemented the arsenate hypersensitive phenotype of E. coli and yeast. OsACR2.2 showed much less ability to complement. The gene products were purified and demonstrated to reduce arsenate to arsenite in vitro, and both exhibited phosphatase activity. In agreement with the complementation results, OsACR2.1 exhibited higher reductase activity than OsACR2.2. Mutagenesis of cysteine residues in the putative active site HC(X)(5)R motif led to nearly complete loss of both phosphatase and arsenate reductase activities. In planta expression of OsACR2.1 increased dramatically after exposure to arsenate. OsACR2.2 was observed only in roots following arsenate exposure, and its expression was less than OsACR2.1. OsACR2.1,OsACR2.2 ACT domain repeat protein 7, ACR7, interacts with a chaperone HSP18.0-CII in rice nuclei 2006 Plant Cell Physiol Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555 Japan. toshi@biochem.tohoku.ac.jp The regulatory ACT domains serve as amino acid-binding sites in some amino acid metabolic enzymes and transcriptional regulators in bacteria. To elucidate the molecular roles of the glutamine (Gln)-sensing system in nitrogen (N) metabolism in plants, we isolated six genes encoding ACT domain repeat proteins (ACR1, and ACR5-ACR9) from rice (Oryza sativa L.) using genomic information on the primary structure composed of four copies of the domain homologous to those of bacterial Gln sensor GLND. Since expression of ACR7 was the most abundant of the six ACR orthologous genes, we focused on this ACR in the current study. Gene products of ACR7 were most abundant in young developing leaf blades of rice, and ACR7 protein is specifically localized in the nucleus of the parenchyma cells of phloem and xylem in the vascular bundles. A yeast two-hybrid screen identified a small heat stress protein (HSP18.0-CII) as a protein interacting with ACR7. Transient expression analysis of HSP18.0-CII:sGFP in cultured rice cells, followed by co-immunoprecipitation, suggests that the nuclear ACR7 indeed interacted with nucleocytoplasmic HSP18.0-CII in vivo. The potential ability of nuclear protein ACR7 to bind Gln and the possibility of the protein acting as a Gln sensor in rice leaves is discussed. ACR7 Cellular distribution of ACT domain repeat protein 9, a nuclear localizing protein, in rice (Oryza sativa) 2008 Physiol Plant Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. Regulatory ACT domains serve as amino acid-binding sites in certain amino acid metabolic enzymes and transcriptional regulators in bacteria. The ACT domain repeat protein (ACR) family in plants is primarily composed of four copies of the domain homologous to those of the bacteria Gln sensor GLND. In the current study, to evaluate the possible involvement of the protein OsACR9 in the Gln-sensing system related to nitrogen (N) metabolism in rice (Oryza sativa L.), subcellular localization of OsACR9 and its accumulation and cellular distribution in various rice organs were examined by transient expression analysis and immunological methods using a monospecific antibody, respectively. Transient expression analysis of OsACR9 fused with a synthetic green fluorescent protein in cultured rice cells suggested nuclear localization of OsACR9. In rice roots, OsACR9 protein was distributed in epidermis, exodermis, sclerenchyma and vascular parenchyma cells, and its accumulation markedly increased after supply of NH(+)(4). In rice leaf samples, OsACR9 protein was abundant in the vascular parenchyma and mestome-sheath cells of young leaf blades at the early stage of development and in the vascular parenchyma and phloem-companion cells of mature leaf sheaths. OsACR9 protein also showed a high level of accumulation in vascular parenchyma cells of dorsal vascular bundles and aleurone cells in young rice grains at the early stage of ripening. The possibility of the nuclear protein OsACR9 acting as a Gln sensor in rice is subsequently discussed through comparison of its spatiotemporal expression with that of Gln-responsive N-assimilatory genes. OsACR9 Expression characteristics of OS-ACS1 and OS-ACS2, two members of the 1-aminocyclopropane-1-carboxylate synthase gene family in rice (Oryza sativa L. cv. Habiganj Aman II) during partial submergence 1997 Plant Mol Biol Plant Gene Expression Center, 800 Buchanan St., Albany, CA, 94710, USA Deepwater rice can grow in the regions of Southeast Asia that are flooded during the monsoon season because it has several adaptations allowing it to survive under flooded conditions. One such adaptation is the ability for rapid internode elongation upon partial submergence to maintain its foliage above the rising flood water levels. Ethylene is considered to be the trigger of this growth response because deepwater conditions not only trap ethylene in submerged organs, but also enhance the activity of 1-aminocyclopropane-1-carboxylate (ACC) synthase. Herein we have studied the expression characteristics of two members of the five-member multigene family encoding ACC synthase in rice OS-ACS1 and OS-ACS2 and show that partial submergence induces expression of OS-ACS1 and suppresses expression of OS-ACS2. The induction of OS-ACS1 occurs within 12 h of partial submergence and at low oxygen concentrations. The data also suggest that deepwater conditions posttranscriptionally regulate ACC synthase activity. OS-ACS1 gene expression may contribute to longer-term ethylene production, but not to the initial, growth-promoting increase in ethylene synthesis. OsACS1,OsACS2,AP37|OsERF3 The chloroplast-localized phospholipases D alpha4 and alpha5 regulate herbivore-induced direct and indirect defenses in rice 2011 Plant Physiol National Key Laboratory of Rice Biology, Institute of Insect Science, Zhejiang University, Hangzhou 310029, China. The oxylipin pathway is of central importance for plant defensive responses. Yet, the first step of the pathway, the liberation of linolenic acid following induction, is poorly understood. Phospholipases D (PLDs) have been hypothesized to mediate this process, but data from Arabidopsis (Arabidopsis thaliana) regarding the role of PLDs in plant resistance have remained controversial. Here, we cloned two chloroplast-localized PLD genes from rice (Oryza sativa), OsPLDalpha4 and OsPLDalpha5, both of which were up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis, mechanical wounding, and treatment with jasmonic acid (JA). Antisense expression of OsPLDalpha4 and -alpha5 (as-pld), which resulted in a 50% reduction of the expression of the two genes, reduced elicited levels of linolenic acid, JA, green leaf volatiles, and ethylene and attenuated the SSB-induced expression of a mitogen-activated protein kinase (OsMPK3), a lipoxygenase (OsHI-LOX), a hydroperoxide lyase (OsHPL3), as well as a 1-aminocyclopropane-1-carboxylic acid synthase (OsACS2). The impaired oxylipin and ethylene signaling in as-pld plants decreased the levels of herbivore-induced trypsin protease inhibitors and volatiles, improved the performance of SSB and the rice brown planthopper Nilaparvata lugens, and reduced the attractiveness of plants to a larval parasitoid of SSB, Apanteles chilonis. The production of trypsin protease inhibitors in as-pld plants could be partially restored by JA, while the resistance to rice brown planthopper and SSB was restored by green leaf volatile application. Our results show that phospholipases function as important components of herbivore-induced direct and indirect defenses in rice. OsACS2,OsHI-LOX,OsHPL3,OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1,OsPLDalpha4,OsPLDalpha5 OsNPR1 negatively regulates herbivore-induced JA and ethylene signaling and plant resistance to a chewing herbivore in rice 2013 Physiol Plant State Key Laboratory of Rice Biology, Institute of Insect Sciences, Zhejiang University, Hangzhou, 310058, China. NPR1 (a non-expressor of pathogenesis-related genes1) has been reported to play an important role in plant defense by regulating signaling pathways. However, little to nothing is known about its function in herbivore-induced defense in monocot plants. Here, using suppressive substrate hybridization, we identified a NPR1 gene from rice, OsNPR1, and found that its expression levels were upregulated in response to infestation by the rice striped stem borer (SSB) Chilo suppressalis and rice leaf folder (LF) Cnaphalocrocis medinalis, and to mechanical wounding and treatment with jasmonic acid (JA) and salicylic acid (SA). Moreover, mechanical wounding induced the expression of OsNPR1 quickly, whereas herbivore infestation induced the gene more slowly. The antisense expression of OsNPR1 (as-npr1), which reduced the expression of the gene by 50%, increased elicited levels of JA and ethylene (ET) as well as of expression of a lipoxygenase gene OsHI-LOX and an ACC synthase gene OsACS2. The enhanced JA and ET signaling in as-npr1 plants increased the levels of herbivore-induced trypsin proteinase inhibitors (TrypPIs) and volatiles, and reduced the performance of SSB. Our results suggest that OsNPR1 is an early responding gene in herbivore-induced defense and that plants can use it to activate a specific and appropriate defense response against invaders by modulating signaling pathways. OsACS2,OsHI-LOX,OsNPR1|NH1 Tissue localization of a submergence-induced 1-aminocyclopropane-1-carboxylic acid synthase in rice 2002 Plant Physiol Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium. At least two 1-aminocyclopropane-1-carboxylic acid synthase genes (ACS) are implicated in the submergence response of rice (Oryza sativa). Previously, the OS-ACS5 gene has been shown to be induced during short- as well as long-term complete submergence of seedlings and to be controlled by a balance of gibberellin and abscisic acid in both lowland and deepwater rice. This study demonstrates that OS-ACS5 mRNA is localized in specific tissues and cells both during normal development and in response to complete submergence. The temporal and spatial regulation of OS-ACS5 expression is presented by in situ hybridization and histochemical analysis of beta-glucuronidase (GUS) activity in transgenic rice carrying an OS-ACS5-gus fusion. Whole-mount in situ hybridization revealed that in air-grown rice seedlings, OS-ACS5 was expressed at a low level in the shoot apex, meristems, leaf, and adventitious root primordia, and in vascular tissues of nonelongated stems and leaf sheaths. In response to complete submergence, the expression in vascular bundles of young stems and leaf sheaths was strongly induced. The results of histochemical GUS assays were consistent with those found by whole-mount in situ hybridization. Our findings suggest that OS-ACS5 plays a role in vegetative growth of rice under normal conditions and is also recruited for enhanced growth upon complete submergence. The possible implication of OS-ACS5 in root-shoot communication during submergence stress and its putative role in aerenchyma formation upon low-oxygen stress are discussed. OS-ACS5|OsACS5 Identification of a rice actin2 gene regulatory region for high-level expression of transgenes in monocots 2009 Plant Biotechnol J Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA. chengkun.he@hotmail.com We have isolated and characterized the 5' region of the rice actin2 gene (OsAct2), which contains 793 bp of sequence upstream of the OsAct2 transcription initiation site, 58 bp of the first non-coding exon, 1736 bp of the 5' intron and the first 8 bp (non-coding sequence) of the second exon. It was found that the 5' region of OsAct2 is an efficient gene regulatory region for driving the constitutive expression of foreign genes in transgenic rice. In situ histochemical results indicated that OsAct2::GUS (GUS, beta-glucuronidase) gene expression in transgenic rice plants is high in sporophytic and gametophytic tissues. It was demonstrated that a 2.6-kb upstream sequence of the OsAct2 translation initiation codon contains all of the 5' regulatory elements necessary for high-level gus expression in transgenic rice tissues. OsAct2 promoter activity was significantly enhanced by the deletion of a 1590-bp segment from the central region of the first intron. The +96 to +274 region of the intron negatively regulates gus expression in leaves. To identify regulatory elements within the OsAct2 promoter, nested truncations of the promoter region were made and fused to gus. The results showed that the region from -1 to -376 was sufficient for promoter activity. In addition, two OsAct2-based expression vectors for use in monocot transformation were developed to promote the high-level expression of foreign genes. OsAct2|OsActin-2 Expression of rice acyl-CoA oxidase isoenzymes in response to wounding 2007 J Plant Physiol Department of Biological Sciences, Inha University, Incheon 402-751, Republic of Korea. To elucidate the role of acyl-CoA oxidase (ACX; EC 1.3.3.6) in plants, the expression patterns of rice ACXs in response to wounding were characterized. Three isogenes of ACX were identified in the rice genome. The deduced proteins of OsACX1, OsACX2 and OsACX3 consist of 669, 699 and 685 amino acid residues, respectively. The results from reverse transcriptase-PCR indicate that OsACX1 is expressed in leaves, stems, and roots, but was barely detectable in germinating seeds. OsACX2 was expressed predominantly in seeds. Only OsACX1 was upregulated by wounding, both locally and systemically. The expression of OsACX2 and OsACX3 remained unchanged. It is suggested that OsACX2 is involved in providing germinating seeds with sugar and energy, while OsACX1 plays a role in the synthesis of jasmonic acid in response to wounding. OsACX1,OsACX2,OsACX3 Molecular cloning and characterization of an arginine decarboxylase gene up-regulated by chilling stress in rice seedlings 2007 J Plant Physiol Department of Low-Temperature Science, National Agricultural Research Center for Hokkaido Region, 1 Hitsujigaoka, Toyohira-ku, Sapporo, Hokkaido 062-8555, Japan. takiyama@affrc.go.jp We cloned a rice cDNA encoding a putative arginine decarboxylase (ADC) protein, a key enzyme involved with putrescine (Put) biosynthesis in plants. The isolated full-length cDNA (OsADC1) contains an insert consisting of 2451 bp. The longest open reading frame within encodes a putative protein of 702 amino acids, with a calculated molecular mass of 74 kDa and an isoelectric point of 4.9. ClustalW alignment revealed that the deduced OsADC1 protein sequence shares overall 60% and 61% identity at the amino acid level with the Pisum sativum and Glycine max ADC proteins, respectively. Additionally, several OsADC1 regions exhibited striking similarity with these two other plant ADC protein sequences, including motifs characteristic of ADC proteins. Further, RNA gel blot analysis revealed markedly increased OsADC1 mRNA levels in rice seedling leaves subjected to chilling stress. Interestingly, this treatment induced a concomitant increase in free Put levels in these samples, coincident with the observed elevated OsADC1 mRNA levels. To our knowledge, this represents the first direct evidence supporting essentially chilling-specific regulation of a rice ADC gene that also potentially influences Put accumulation, a phenomenon previously noted in cold-stressed rice seedlings. OsADC1 Transcriptional regulation of the rice arginine decarboxylase (Adc1) and S-adenosylmethionine decarboxylase (Samdc) genes by methyl jasmonate 2010 Plant Physiol Biochem Departament de Produccio Vegetal i Ciencia Forestal, ETSEA, Universitat de Lleida, Av. Alcalde Rovira Roure 191, E-25198 Lleida, Spain. We investigated the effect of methyl jasmonate (MeJa) treatment on the expression of two genes in the rice polyamine biosynthesis pathway and on the polyamine content in wild type plants and transgenic rice plants expressing a Datura stramonium (Ds) Adc cDNA, the latter accumulating up to three-fold the normal level of putrescine. Exogenous MeJa transiently inhibited the expression of OsAdc1, OsSamdc and Spermidine synthase (OsSpds) genes in the polyamine biosynthesis pathway, probably through transcriptional repression. There was also a similar negative impact on the DsAdc transgene in transgenic plants, even though a constitutive promoter was used to drive transgene expression. The free putrescine content was reduced significantly in the leaves of both wild type and transgenic plants in response to MeJa, although the magnitude of the effect was greater in wild type plants. We discuss our findings with respect to the previously proposed threshold model of polyamine metabolism in plants subjected to abiotic stress. OsADC1 Comprehensive analysis of differentially expressed rice actin depolymerizing factor gene family and heterologous overexpression of OsADF3 confers Arabidopsis Thaliana drought tolerance 2012 Rice (N Y) Department of Agronomy, National Taiwan University, No, 1, Section 4, Roosevelt Road, Taipei 106, Taiwan, Republic of China. menchi@ntu.edu.tw. BACKGROUND: Actin depolymerizing factors (ADFs) are small actin-binding proteins. Many higher-plant ADFs has been known to involve in plant growth, development and pathogen defense. However, in rice the temporal and spatial expression of OsADF gene family and their relationship with abiotic stresses tolerance is still unknown. RESULTS: Here we reported the first comprehensive gene expression profile analysis of OsADF gene family. The OsADF genes showed distinct and overlapping gene expression patterns at different growth stages, tissues and abiotic stresses. We also demonstrated that both OsADF1 and OsADF3 proteins were localized in the nucleus. OsADF1 and OsADF3 were preferentially expressed in vascular tissues. Under ABA or abiotic stress treatments, OsADF3::GUS activity was enhanced in lateral roots and root tips. Ectopically overexpressed OsADF3 conferred the mannitol- and drought-stress tolerance of transgenic Arabidopsis seedlings by increasing germination rate, primary root length and survival. Several drought-tolerance responsive genes (RD22, ABF4, DREB2A, RD29A, PIP1; 4 and PIP2; 6) were upregulated in transgenic Arabidopsis under drought stress. CONCLUSIONS: These results suggested that OsADF gene family may participate in plant abiotic stresses response or tolerance and would facilitate functional validation of other OsADF genes. OsADF3 The 5'-untranslated region of the Oryza sativa alcohol dehydrogenase gene functions as a translational enhancer in monocotyledonous plant cells 2008 J Biosci Bioeng Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. The 5'-untranslated region (5'-UTR) functions as a translational enhancer in monocotyledonous plant cells is necessary to express a foreign gene efficiently. Here, we show that the 5'-UTR of the rice alcohol dehydrogenase gene contributes to efficient translation in not only dicotyledonous plant cells, but also in monocotyledonous rice cells. OsADH OsTIR1 and OsAFB2 downregulation via OsmiR393 overexpression leads to more tillers, early flowering and less tolerance to salt and drought in rice 2012 PLoS One Key Laboratory of South China Agricultural Plant Genetics and Breeding, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China. The microRNA miR393 has been shown to play a role in plant development and in the stress response by targeting mRNAs that code for the auxin receptors in Arabidopsis. In this study, we verified that two rice auxin receptor gene homologs (OsTIR1 and OsAFB2) could be targeted by OsmiR393 (Os for Oryza sativa). Two new phenotypes (increased tillers and early flowering) and two previously observed phenotypes (reduced tolerance to salt and drought and hyposensitivity to auxin) were observed in the OsmiR393-overexpressing rice plants. The OsmiR393-overexpressing rice demonstrated hyposensitivity to synthetic auxin-analog treatments. These data indicated that the phenotypes of OsmiR393-overexpressing rice may be caused through hyposensitivity to the auxin signal by reduced expression of two auxin receptor genes (OsTIR1 and OsAFB2). The expression of an auxin transporter (OsAUX1) and a tillering inhibitor (OsTB1) were downregulated by overexpression of OsmiR393, which suggested that a gene chain from OsmiR393 to rice tillering may be from OsTIR1 and OsAFB2 to OsAUX1, which affected the transportation of auxin, then to OsTB1, which finally controlled tillering. The positive phenotypes (increased tillers and early flowering) and negative phenotypes (reduced tolerance to salt and hyposensitivity to auxin) of OsmiR393-overexpressing rice present a dilemma for molecular breeding. OsAFB2,OsAUX1,OsTB1|FC1,OsTIR1 Distinctive expression patterns and roles of the miRNA393/TIR1 homolog module in regulating flag leaf inclination and primary and crown root growth in rice (Oryza sativa) 2012 New Phytol Key Laboratory for Cell and Gene Engineering of Zhejiang Province, Institute of Genetics, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China. * MicroRNA (miRNA)-mediated regulation of auxin signaling components plays a critical role in plant development. miRNA expression and functional diversity contribute to the complexity of regulatory networks of miRNA/target modules. * This study functionally characterizes two members of the rice (Oryza sativa) miR393 family and their target genes, OsTIR1 and OsAFB2 (AUXIN SIGNALING F-BOX), the two closest homologs of Arabidopsis TRANSPORT INHIBITOR RESPONSE 1 (TIR1). * We found that the miR393 family members possess distinctive expression patterns, with miR393a expressed mainly in the crown and lateral root primordia, as well as the coleoptile tip, and miR393b expressed in the shoot apical meristem. Transgenic plants overexpressing miR393a/b displayed a severe phenotype with hallmarks of altered auxin signaling, mainly including enlarged flag leaf inclination and altered primary and crown root growth. Furthermore, OsAFB2- and OsTIR1-suppressed lines exhibited increased inclination of flag leaves at the booting stage, resembling miR393-overexpressing plants. Moreover, yeast two-hybrid and bimolecular fluorescence complementation assays showed that OsTIR1 and OsAFB2 interact with OsIAA1. * Expression diversification of miRNA393 implies the potential role of miRNA regulation during species evolution. The conserved mechanisms of the miR393/target module indicate the fundamental importance of the miR393-mediated regulation of auxin signal transduction in rice. OsAFB2,OsIAA1,OsTIR1 Adenosine diphosphate ribosylation factor-GTPase-activating protein stimulates the transport of AUX1 endosome, which relies on actin cytoskeletal organization in rice root development 2011 J Integr Plant Biol Key Laboratory of Photosynthesis and Molecular Environmental Physiology, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China. Polar auxin transport, which depends on polarized subcellular distribution of AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX) influx carriers and PIN-FORMED (PIN) efflux carriers, mediates various processes of plant growth and development. Endosomal recycling of PIN1 is mediated by an adenosine diphosphate (ADP)ribosylation factor (ARF)-GTPase exchange factor protein, GNOM. However, the mediation of auxin influx carrier recycling is poorly understood. Here, we report that overexpression of OsAGAP, an ARF-GTPase-activating protein in rice, stimulates vesicle transport from the plasma membrane to the Golgi apparatus in protoplasts and transgenic plants and induces the accumulation of early endosomes and AUX1. AUX1 endosomes could partially colocalize with FM4-64 labeled early endosome after actin disruption. Furthermore, OsAGAP is involved in actin cytoskeletal organization, and its overexpression tends to reduce the thickness and bundling of actin filaments. Fluorescence recovery after photobleaching analysis revealed exocytosis of the AUX1 recycling endosome was not affected in the OsAGAP overexpression cells, and was only slightly promoted when the actin filaments were completely disrupted by Lat B. Thus, we propose that AUX1 accumulation in the OsAGAP overexpression and actin disrupted cells may be due to the fact that endocytosis of the auxin influx carrier AUX1 early endosome was greatly promoted by actin cytoskeleton disruption. OsAGAP,OsAUX1 OsAGAP, an ARF-GAP from rice, regulates root development mediated by auxin in Arabidopsis 2005 Plant Cell Environ Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, CAS, Beijing, China. Arf (ADP-ribosylation factor) proteins, which mediate vesicular transport, have little or no intrinsic GTPase activity. They rely on the action of GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) for their function. In the present study the OsAGAP gene in rice, which encoded a protein with predicted structure similar to ArfGAP, was identified. The purified OsAGAP-GST fusion protein was able to stimulate the GTPase activity of rice Arf. Furthermore, OsAGAP can rescue the defect of vesicular transport in the yeast gcs1 delta glo3 delta double-mutant cells. Transgenic Arabidopsis with OsAGAP constitutively expression showed reduced apical dominance, shorter primary roots, increasing number of longer adventitious roots. Many of the phenotypes can be phenocopied by treatment of exogenous indoleacetic acid level (IAA) in wild-type plants. Determination of whole-plant IAA level showed that there is a sharp increase of free IAA in OsAGAP transgenic Arabidopsis seedlings. In addition, removal of the 4-day-old shoot apex could inhibit the adventitious root formation in the transgenic seedlings. These results suggest OsAGAP, an ARF-GAP of rice, maybe involved in the mediation of plant root development by regulating auxin level. OsAGAP Over-expression of OsAGAP, an ARF-GAP, interferes with auxin influx, vesicle trafficking and root development 2006 Plant J Key Laboratory of Photosynthesis and Molecular Environmental Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. Development and organogenesis in both dicot and monocot plants are highly dependent on polar auxin transport (PAT), which requires the proper asymmetric localization of both auxin influx and efflux carriers. In the model dicot plant Arabidopsis thaliana, the trafficking and localization of auxin efflux facilitators such as PIN-FORMED1 (PIN1) are mediated by GNOM, a guanine-nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of small GTPases, but molecular regulators of the auxin influx facilitators remain unknown. Here, we show that over-expression of OsAGAP, an ARF-GTPase-activating protein (ARF-GAP) in rice, impaired PAT and interfered with both primary and lateral root development. The lateral root phenotype could be rescued by the membrane-permeable auxin 1-naphthyl acetic acid, but not by indole 3-acetic acid (IAA) or by 2,4-dichloro-phenoxyacetic acid, which require influx facilitators to enter the cells. OsAGAP-over-expressing plants had alterations in vesicle trafficking and localization of the presumptive A. thaliana auxin-influx carrier AUX1, but not in the localization of the auxin efflux facilitators. Together, our data suggest that OsAGAP has a specific role in regulating vesicle trafficking pathways such as the auxin influx pathway, which in turn controls auxin-dependent root growth in plants. OsAGAP Genome-wide identification, organization and phylogenetic analysis of Dicer-like, Argonaute and RNA-dependent RNA Polymerase gene families and their expression analysis during reproductive development and stress in rice 2008 BMC Genomics Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India. kapoors@genomeindia.org BACKGROUND: Important developmental processes in both plants and animals are partly regulated by genes whose expression is modulated at the post-transcriptional level by processes such as RNA interference (RNAi). Dicers, Argonautes and RNA-dependent RNA polymerases (RDR) form the core components that facilitate gene silencing and have been implicated in the initiation and maintenance of the trigger RNA molecules, central to process of RNAi. Investigations in eukaryotes have revealed that these proteins are encoded by variable number of genes with plants showing relatively higher number in each gene family. To date, no systematic expression profiling of these genes in any of the organisms has been reported. RESULTS: In this study, we provide a complete analysis of rice Dicer-like, Argonaute and RDR gene families including gene structure, genomic localization and phylogenetic relatedness among gene family members. We also present microarray-based expression profiling of these genes during 14 stages of reproductive and 5 stages of vegetative development and in response to cold, salt and dehydration stress. We have identified 8 Dicer-like (OsDCLs), 19 Argonaute (OsAGOs) and 5 RNA-dependent RNA polymerase (OsRDRs) genes in rice. Based on phylogeny, each of these genes families have been categorized into four subgroups. Although most of the genes express both in vegetative and reproductive organs, 2 OsDCLs, 14 OsAGOs and 3 OsRDRs were found to express specifically/preferentially during stages of reproductive development. Of these, 2 OsAGOs exhibited preferential up-regulation in seeds. One of the Argonautes (OsAGO2) also showed specific up-regulation in response to cold, salt and dehydration stress. CONCLUSION: This investigation has identified 23 rice genes belonging to DCL, Argonaute and RDR gene families that could potentially be involved in reproductive development-specific gene regulatory mechanisms. These data provide an insight into probable domains of activity of these genes and a basis for further, more detailed investigations aimed at understanding the contribution of individual components of RNA silencing machinery during reproductive phase of plant development. OsAGO1a,OsDCL1,SHO1|OsDCL4,OsDCL2b Rice MicroRNA effector complexes and targets 2009 Plant Cell National Institute of Biological Sciences, Beijing, China. MicroRNAs (miRNAs) are small silencing RNAs with regulatory roles in gene expression. miRNAs interact with Argonaute (AGO) proteins to form effector complexes that cleave target mRNAs or repress translation. Rice (Oryza sativa) encodes four AGO1 homologs (AGO1a, AGO1b, AGO1c, and AGO1d). We used RNA interference (RNAi) to knock down the four AGO1s. The RNAi lines displayed pleiotropic developmental phenotypes and had increased accumulation of miRNA targets. AGO1a, AGO1b, and AGO1c complexes were purified and further characterized. The three AGO1s all have a strong preference for binding small RNAs (sRNAs) with 5' U and have Slicer activity. We cataloged the sRNAs in each AGO1 complex by deep sequencing and found that all three AGO1s predominantly bound known miRNAs. Most of the miRNAs were evenly distributed in the three AGO1 complexes, suggesting a redundant role for the AGO1s. Intriguingly, a subset of miRNAs were specifically incorporated into or excluded from one of the AGO1s, suggesting functional specialization among the AGO1s. Furthermore, we identified rice miRNA targets at a global level. The validated targets include transcription factors that control major stages of development and also genes involved in a variety of physiological processes, indicating a broad regulatory role for miRNAs in rice. OsAGO1a,OsAGO1b,OsAGO1c,OsAGO1d Improved expression, purification and crystallization of a putative N-acetyl-gamma-glutamyl-phosphate reductase from rice (Oryza sativa) 2005 Acta Crystallogr Sect F Struct Biol Cryst Commun National Institute of Agrobiological Sciences, Japan. N-Acetyl-gamma-glutamyl-phosphate reductase (AGPR) catalyzes the third step in an eight-step arginine-biosynthetic pathway that starts with glutamate. This enzyme converts N-acetyl-gamma-glutamyl phosphate to N-acetylglutamate-gamma-semialdehyde by an NADPH-dependent reductive dephosphorylation. AGPR from Oryza sativa (OsAGPR) was expressed in Escherichia coli at 291 K as a soluble fusion protein with an upstream thioredoxin-hexahistidine [Trx-(His)6] extension. OsAGPR(Ala50-Pro366) was purified and crystals were obtained using the sitting-drop vapour-diffusion method at 293 K and diffract X-rays to at least 1.8 A resolution. They belong to the hexagonal space group P6(1), with unit-cell parameters a = 86.11, c = 316.3 A. OsAGPR Identification of a novel plant MAR DNA binding protein localized on chromosomal surfaces 2004 Plant Mol Biol Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka, 565-0871, Japan. We identified a novel nucleoplasm localized protein in Arabidopsis called AT-hook motif nuclear localized protein 1 (AHL1), which was isolated by visual screening of transformants using random GFP::cDNA fusions. AHL1 contains an AT-hook motif and unknown conserved PPC (plants and prokaryotes conserved) domain that includes a hydrophobic region. Approximately 30 paralogues were identified in the Arabidopsis genome. Proteins with PPC-like domains are found in Bacteria, Archaea and the plant kingdom, but in Bacteria and Archaea the PPC containing proteins of do not have an AT-hook motif. Thus, the PPC domain is evolutionary conserved and has a new function such as AT-rich DNA binding. AHL1 was mainly localized in the nucleoplasm, but little in the nucleolus and heterochromatic region, and was concentrated in the boundary region between euchromatin and heterochromatin. Biochemically, AHL1 was also found in the nuclear matrix fraction. In the M phase, AHL1 was localized on the chromosomal surface. The AT-hook motif was essential for matrix attachment region (MAR) binding, and the hydrophobic region of the PPC was indispensable for nuclear localization. Our results suggest that AHL1 is a novel plant MAR binding protein, which is related to the positioning of chromatin fibers in the nucleus by the presence of an AT-hook motif and PPC domain. In addition, AHL1 is located on the surface of chromosomes during mitosis. OsAHL1|AHL1,OsAHL2|AHL2 Rice APOPTOSIS INHIBITOR5 coupled with two DEAD-box adenosine 5'-triphosphate-dependent RNA helicases regulates tapetum degeneration 2011 Plant Cell National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Programmed cell death (PCD) during tapetum degeneration in postmeiotic anthers is critical for the proper development of male gametophytes in flowering plants. Although several genes involved in this process have been identified recently, the molecular mechanism is still poorly understood. Here, we show that knockout of rice (Oryza sativa) APOPTOSIS INHIBITOR5 (API5), which encodes a putative homolog of antiapoptosis protein Api5 in animals, results in delayed degeneration of the tapetum due to inhibition of the tapetal PCD process leading to defects in formation of male gametophytes. Os API5 is a nuclear protein that interacts with two DEAD-box ATP-dependent RNA helicases, API5-INTERACTING PROTEIN1 (AIP1) and AIP2. AIP1 and AIP2 are homologs of yeast (Saccharomyces cerevisiae) Suppressor of Bad Response to Refrigeration1 protein 2 (SUB2p) that have critical roles in transcription elongation and pre-mRNA splicing. Os AIP1 and AIP2 can form dimers and interact directly with the promoter region of CP1, a rice cysteine protease gene. Suppression of Os AIP1/2 leads to down-regulation of CP1, resulting in sterility, which is highly similar to the effects of suppressed expression of Os CP1. Our results uncover a previously unknown pathway for regulating PCD during tapetum degeneration in rice, one that may be conserved among eukaryotic organisms. OsAIP1,OsAIP2,OsAPI5,OsCP1 Oryza sativa actin-interacting protein 1 is required for rice growth by promoting actin turnover 2013 Plant J Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. Rapid actin turnover is essential for numerous actin-based processes. However, how it is precisely regulated remains poorly understood. Actin-interacting protein 1 (AIP1) has been shown to be an important factor by acting coordinately with actin-depolymerizing factor (ADF)/cofilin in promoting actin depolymerization, the rate-limiting factor in actin turnover. However, the molecular mechanism by which AIP1 promotes actin turnover remains largely unknown in plants. Here, we provide a demonstration that AIP1 promotes actin turnover, which is required for optimal growth of rice plants. Specific down-regulation of OsAIP1 increased the level of filamentous actin and reduced actin turnover, whereas over-expression of OsAIP1 induced fragmentation and depolymerization of actin filaments and enhanced actin turnover. In vitro biochemical characterization showed that, although OsAIP1 alone does not affect actin dynamics, it enhances ADF-mediated actin depolymerization. It also caps the filament barbed end in the presence of ADF, but the capping activity is not required for their coordinated action. Real-time visualization of single filament dynamics showed that OsAIP1 enhanced ADF-mediated severing and dissociation of pointed end subunits. Consistent with this, the filament severing frequency and subunit off-rate were enhanced in OsAIP1 over-expressors but decreased in RNAi protoplasts. Importantly, OsAIP1 acts coordinately with ADF and profilin to induce massive net actin depolymerization, indicating that AIP1 plays a major role in the turnover of actin, which is required to optimize F-actin levels in plants. OsAIP1 Alkaline alpha-galactosidase degrades thylakoid membranes in the chloroplast during leaf senescence in rice 2009 New Phytol Department of Life Sciences and Institute of Biodiversity, National Cheng-Kung University, Tainan 700, Taiwan. shanhua@mail.ncku.edu.tw Here, we studied the functional role of a chloroplast alkaline alpha-galactosidase (OsAkalphaGal) in the breakdown of thylakoid membranes during rice (Oryza sativa) leaf senescence. We assayed the enzyme activity of recombinant OsAkalphaGal with different natural substrates and examined the effect of ectopic OsAkalphaGal expression in rice plants. Recombinant OsAkalphaGal showed at least a two-fold greater substrate-binding affinity and a 10-fold greater turnover rate to galactolipid digalactosyl diacylglycerol than the raffinose family of oligosaccharides (verbascose, stachyose, raffinose) and melibiose. The OsAkalphaGal null mutant (osakalphagal) displayed a delayed leaf senescence phenotype. OsAkalphaGal complementation in osakalphagal recovered OsAkalphaGal expression and showed a senescence phenotype similar to that of wild-type plants. Transgenic plants overexpressing OsAkalphaGal (UbiP-OsAkalphaGal) exhibited retarded plant growth and development, and showed a pale-green phenotype coupled with a reduced chlorophyll content to 42% in newly unfolded leaves. UbiP-OsAkalphaGal leaves also showed a 29-fold increase in alkaline alpha-galactosidase activity compared with wild-type leaves. An ultrastructural study of Ubi-OsAkalphaGal chloroplasts in newly unfolded leaves revealed abnormal grana organization. Our findings strongly suggest that OsAkalphaGal is a thylakoid membrane-degrading enzyme involved in the degradation of digalactosyl diacylglycerol during rice leaf senescence. OsAkaGal Overproduction of a rice aldo-keto reductase increases oxidative and heat stress tolerance by malondialdehyde and methylglyoxal detoxification 2011 Plant Mol Biol Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Temesvari krt. 62, 6726, Szeged, Hungary. The accumulation of toxic compounds generated by the interaction between reactive oxygen species and polyunsaturated fatty acids of membrane lipids can significantly damage plant cells. A plethora of enzymes act on these reactive carbonyls, reducing their toxicity. Based on the chromosomal localization and on their homology with other stress-induced aldo-keto reductases (AKRs) we have selected three rice AKR genes. The transcription level of OsAKR1 was greatly induced by abscisic acid and various stress treatments; the other two AKR genes tested were moderately stress-inducible. The OsAKR1 recombinant protein exhibited a high nicotinamide adenine dinucleotide phosphate-dependent catalytic activity to reduce toxic aldehydes including glycolysis-derived methylglyoxal (MG) and lipid peroxidation-originated malondialdehyde (MDA). The function of this enzyme in MG detoxification was demonstrated in vivo in E. coli and in transgenic plants overproducing the OsAKR1 protein. Heterologous synthesis of the OsAKR1 enzyme in transgenic tobacco plants resulted in increased tolerance against oxidative stress generated by methylviologen (MV) and improved resistance to high temperature. In these plants lower levels of MDA were detected both following MV and heat treatment due to the activity of the OsAKR1 enzyme. The transgenic tobaccos also exhibited higher AKR activity and accumulated less MG in their leaves than the wild type plants; both in the presence and absence of heat stress. These results support the positive role of OsAKR1 in abiotic stress-related reactive aldehyde detoxification pathways and its use for improvement of stress tolerance in plants. OsAKR1|OsI_04426 Rice K+ uptake channel OsAKT1 is sensitive to salt stress 2005 Planta Department of Molecular Plant Physiology and Biophysics, University of Wurzburg, Julius-von-Sachs-Platz 2, 97082, Wurzburg, Germany. ifuchs@botanik.uni-wuerzburg.de Potassium ions constitute the most important macronutrients taken up by plants. To unravel the mechanisms of K+ uptake and its sensitivity to salt stress in the model plant rice, we isolated and functionally characterized OsAKT1, a potassium channel homologous to the Arabidopsis root inward rectifier AKT1. OsAKT1 transcripts were predominantly found in the coleoptile and in the roots of young rice seedlings. K+ channel mRNA decreases in response to salt stress, both in the shoot and in the root of 4-day-old rice seedlings. Following expression in HEK293 cells, we were able to characterize OsAKT1 as a voltage-dependent, inward-rectifying K+ channel regulated by extracellular Ca2+ and protons. Patch-clamp studies on rice root protoplasts identified a K+ inward rectifier with similar channel properties as heterologously expressed OsAKT1. In line with the transcriptional downregulation of OsAKT1 in response to salt stress, inward K+ currents were significantly reduced in root protoplasts. Thus, OsAKT1 seems to represent the dominant salt-sensitive K+ uptake channel in rice roots. OsAKT1 The Os-AKT1 Channel Is Critical for K+ Uptake in Rice Roots and Is Modulated by the Rice CBL1-CIPK23 Complex 2014 Plant Cell State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, National Plant Gene Research Centre (Beijing), China Agricultural University, Beijing 100193, China. Potassium (K+) is one of the essential nutrient elements for plant growth and development. Plants absorb K+ ions from the environment via root cell K+ channels and/or transporters. In this study, the Shaker K+ channel Os-AKT1 was characterized for its function in K+ uptake in rice (Oryza sativa) roots, and its regulation by Os-CBL1 (Calcineurin B-Like protein1) and Os-CIPK23 (CBL-Interacting Protein Kinase23) was investigated. As an inward K+ channel, Os-AKT1 could carry out K+ uptake and rescue the low-K+-sensitive phenotype of Arabidopsis thaliana akt1 mutant plants. Rice Os-akt1 mutant plants showed decreased K+ uptake and displayed an obvious low-K+-sensitive phenotype. Disruption of Os-AKT1 significantly reduced the K+ content, which resulted in inhibition of plant growth and development. Similar to the AKT1 regulation in Arabidopsis, Os-CBL1 and Os-CIPK23 were identified as the upstream regulators of Os-AKT1 in rice. The Os-CBL1-Os-CIPK23 complex could enhance Os-AKT1-mediated K+ uptake. A phenotype test confirmed that Os-CIPK23 RNAi lines exhibited similar K+-deficient symptoms as the Os-akt1 mutant under low K+ conditions. These findings demonstrate that Os-AKT1-mediated K+ uptake in rice roots is modulated by the Os-CBL1-Os-CIPK23 complex. OsAKT1,OsCBL1 Induction of mitochondrial aldehyde dehydrogenase by submergence facilitates oxidation of acetaldehyde during re-aeration in rice 2003 FEBS Letters Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 97403-1229, Japan. Post-hypoxic injuries in plants are primarily caused by bursts of reactive oxygen species and acetaldehyde. In agreement with previous studies, we found accumulations of acetaldehyde in rice during re-aeration following submergence. During re-aeration, acetaldehyde-oxidizing aldehyde dehydrogenase (ALDH) activity increased, thereby causing the acetaldehyde content to decrease in rice. Interestingly, re-aerated rice plants showed an intense mitochondrial ALDH2a protein induction, even though ALDH2a mRNA was submergence induced and declined upon re-aeration. This suggests that rice ALDH2a mRNA is accumulated in order to quickly metabolize acetaldehyde that is produced upon re-aeration. ALDH2a|OsALDH2B5 Organ-specific expressions and chromosomal locations of two mitochondrial aldehyde dehydrogenase genes from rice (Oryza sativa L.), ALDH2a and ALDH2b 2003 Gene Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Recent studies have suggested that mitochondrial aldehyde dehydrogenase (aldehyde:NAD(P)(+) oxidoreductase, EC 1.2.1.3) (ALDH2) plays essential roles in pollen development in plants. Rice (Oryza sativa L.) ALDH2 is encoded by at least two ALDH2 genes, one of which (ALDH2a) was previously identified. In this study, to understand the roles of ALDH2 in rice, we isolated and characterized a cDNA clone encoding another rice ALDH2 (ALDH2b). An in vitro ALDH assay indicated that ALDH2b possesses an NAD(+)-linked activity for oxidation of acetaldehyde, glycolaldehyde and propionaldehyde. Northern blot and immunoblot analyses revealed that ALDH2b was constitutively present in all the organs examined, whereas ALDH2a was expressed in leaves of dark-grown seedlings and panicles. By RFLP linkage mapping, the ALDH2a and ALDH2b genes were mapped to the long arm of chromosome 2 and the short arm of chromosome 6, respectively. We suggest that the rice ALDH2a and ALDH2b genes are orthologues of maize mitochondrial ALDH genes, rf2b and rf2a, respectively. ALDH2a|OsALDH2B5,OsALDH2B1|ALDH2b Modeling-dependent protein characterization of the rice aldehyde dehydrogenase (ALDH) superfamily reveals distinct functional and structural features 2010 PLoS One Department of Agronomy, Purdue University, West Lafayette, Indiana, United States of America. skotchon@purdue.edu The completion of the rice genome sequence has made it possible to identify and characterize new genes and to perform comparative genomics studies across taxa. The aldehyde dehydrogenase (ALDH) gene superfamily encoding for NAD(P)(+)-dependent enzymes is found in all major plant and animal taxa. However, the characterization of plant ALDHs has lagged behind their animal- and prokaryotic-ALDH homologs. In plants, ALDHs are involved in abiotic stress tolerance, male sterility restoration, embryo development and seed viability and maturation. However, there is still no structural property-dependent functional characterization of ALDH protein superfamily in plants. In this paper, we identify members of the rice ALDH gene superfamily and use the evolutionary nesting events of retrotransposons and protein-modeling-based structural reconstitution to report the genetic and molecular and structural features of each member of the rice ALDH superfamily in abiotic/biotic stress responses and developmental processes. Our results indicate that rice-ALDHs are the most expanded plant ALDHs ever characterized. This work represents the first report of specific structural features mediating functionality of the whole families of ALDHs in an organism ever characterized. ALDH2a|OsALDH2B5,OsALDH2B1|ALDH2b,OsALDH7|OsALDH7B6|ALDH7B7 Expression of a gene encoding mitochondrial aldehyde dehydrogenase in rice increases under submerged conditions 2000 Plant Physiol Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. anakazo@mail.ecc.u-tokyo.ac.jp It is known that alcoholic fermentation is important for survival of plants under anaerobic conditions. Acetaldehyde, one of the intermediates of alcoholic fermentation, is not only reduced by alcohol dehydrogenase but also can be oxidized by aldehyde dehydrogenase (ALDH). To determine whether ALDH plays a role in anaerobic metabolism in rice (Oryza sativa L. cv Nipponbare), we characterized a cDNA clone encoding mitochondrial ALDH from rice (Aldh2a). Analysis of sub-cellular localization of ALDH2a protein using green fluorescent protein and an in vitro ALDH assay using protein extracts from Escherichia coli cells that overexpressed ALDH2a indicated that ALDH2a functions in the oxidation of acetaldehyde in mitochondria. A Southern-blot analysis indicated that mitochondrial ALDH is encoded by at least two genes in rice. We found that the Aldh2a mRNA was present at high levels in leaves of dark-grown seedlings, mature leaf sheaths, and panicles. It is interesting that expression of the rice Aldh2a gene, unlike the expression of the tobacco (Nicotiana tabacum) Aldh2a gene, was induced in rice seedlings by submergence. Experiments with ruthenium red, which is a blocker of Ca(2+) fluxes in rice as well as maize (Zea mays), suggest that the induction of expression of Adh1 and Pdc1 by low oxygen stress is regulated by elevation of the cytosolic Ca(2+) level. However, the induction of Aldh2a gene expression may not be controlled by the cytosolic Ca(2+) level elevation. A possible involvement of ALDH2a in the submergence tolerance of rice is discussed. ALDH2a|OsALDH2B5 Mutation of OsALDH7 causes a yellow-colored endosperm associated with accumulation of oryzamutaic acid A in rice 2012 Planta State Key Laboratory for Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Weigang 1, Nanjing 210095, China. shenyi1202@gmail.com Aldehyde dehydrogenase proteins consist of a superfamily and the family 7 (ALDH7) is a typical group with highly conserved proteins across species. It catalyzes oxidation of alpha-aminoadipic semialdehyde (AASA) in lysine degradation, participates in protection against hyperosmotic stress, and detoxifies aldehydes in human; however, its function in plants has been much less documented. Here we reported a mutant with yellow-colored endosperm in rice, and showed that the yellow endosperm was caused by mutation of OsALDH7. OsALDH7 is expressed in all tissues detected, with the highest level in mature seeds. We found that oryzamutaic acid A accumulated during late seed development and after a year-long storage in the colored endosperm, whereas it was undetectable in the wild type endosperm. Moreover, lysine degradation was enhanced in yeast over-expressing OsALDH7 and as a result, content of lysine, glutamate and saccharopine was changed, suggesting a role of OsALDH7 in lysine catabolism. OsALDH7|OsALDH7B6|ALDH7B7 Rice aldehyde dehydrogenase7 is needed for seed maturation and viability 2009 Plant Physiol National Research Laboratory, Department of Integrative Bioscience and Biotechnology, and Center for Functional Genomics, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea. Aldehyde dehydrogenases (ALDHs) catalyze the irreversible oxidation of a wide range of reactive aldehydes to their corresponding carboxylic acids. Although the proteins have been studied from various organisms and at different growth stages, their roles in seed development have not been well elucidated. We obtained T-DNA insertional mutants in OsALDH7, which is remarkably inducible by oxidative and abiotic stresses. Interestingly, endosperms from the osaldh7 null mutants accumulated brown pigments during desiccation and storage. Extracts from the mutant seeds showed a maximum absorbance peak at 360 nm, the wavelength that melanoidin absorbs. Under UV light, those extracts also exhibited much stronger fluorescence than the wild type, suggesting that the pigments are melanoidin. These pigments started to accumulate in the late seed developmental stage, the time when OsALDH7 expression began to increase significantly. Purified OsALDH7 protein showed enzyme activities to malondialdehyde, acetaldehyde, and glyceraldehyde. These results suggest that OsALDH7 is involved in removing various aldehydes formed by oxidative stress during seed desiccation. The mutant seeds were more sensitive to our accelerated aging treatment and accumulated more malondialdehyde than the wild type. These data imply that OsALDH7 plays an important role in maintaining seed viability by detoxifying the aldehydes generated by lipid peroxidation. OsALDH7|OsALDH7B6|ALDH7B7 Molecular cloning and differential expression of an aldehyde dehydrogenase gene in rice leaves in response to infection by blast fungus 2007 Biologia None Aldehyde dehydrogenase (ALDH) superfamily is a group of enzymes metabolizing endogenous and exogenous aldehydes. Using differential display RT-PCR and cDNA library screening, a full-length aldehyde dehydrogenase cDNA (ALDH7B7) was isolated from rice leaves infected by incompatible race of blast fungus Magnaporthe grisea. The deduced amino acid sequence consists of 509 amino acid residues and shares 74∼81% identity with those of ALDH7Bs from other plants. ALDH7B7 expression was induced by blast fungus infection, ultraviolet, mechanical wound in rice leaves and was not detected in untreated rice organs. This gene has also been found to be inducible after exogenous phytohormones application, such as salicylic acid, methyl ester of jasmonic acid and abscisic acid. The function of ALDH7B7 in the interaction process between blast fungus and rice is discussed. OsALDH7|OsALDH7B6|ALDH7B7 A novel mutated acetolactate synthase gene conferring specific resistance to pyrimidinyl carboxy herbicides in rice 2007 Plant Mol Biol Laboratory of Environmental Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutumidori-Amamiyamachi, Sendai, Miyagi 981-8555, Japan. Acetolactate synthase (ALS) is the first common enzyme in the biosynthetic pathway of branched-chain amino acids. Mutations of specific amino acids in ALS have been known to confer resistance to ALS-inhibiting herbicides such as sulfonylureas and pyrimidinyl carboxy (PC) herbicides. However, mutations conferring exclusive resistance to PC have not yet been reported to date. We selected PC resistant rice calli, which were derived from anther culture, using one of the PCs, bispyribac-sodium (BS), as a selection agent. Two lines of BS-resistant plants carrying a novel mutation, the 95th Glycine to Alanine (G95A), in ALS were obtained. In vitro ALS activity assay indicated that the recombinant protein of G95A-mutated ALS (ALS-G95A) conferred highly specific resistance to PC herbicides. In order to determine if the ALS-G95A gene could be used as a selection marker for rice transformation, the ALS-G95A gene was connected to ubiquitin promoter and introduced into rice. PC resistant plants containing integrated ALS-G95A gene were obtained after selection with BS as a selection agent. In conclusion, novel G95A mutated ALS gene confers highly specific resistant to PC-herbicides and can be used as a selection marker. OsALS A tonoplast-localized half-size ABC transporter is required for internal detoxification of aluminum in rice 2012 Plant J Institute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki, Japan. Toxic aluminum enters the root cells rapidly, therefore internal detoxification is required. However, the molecular mechanisms underlying this process are poorly understood. Here we functionally characterized a rice gene, Os03g0755100 (OsALS1), that is regulated by ART1, a C2H2-type zinc finger transcription factor. OsALS1 encodes a half-size ABC transporter that is a member of the TAP (transporter associated with antigen processing) sub-group. Expression of OsALS1 was rapidly and specifically induced by Al in the roots, but not by other metals or low pH. OsALS1 was localized at all cells of the roots. Furthermore, OsALS1 is localized to the tonoplast. These expression patterns and cell specificity of localization are different from those of the homologous gene AtALS1 in Arabidopsis. Knockout of OsALS1 in three independent lines resulted in significant increased sensitivity to Al, but did not affect the sensitivity to other metals and low pH. Comparison of Al accumulation patterns between wild-type and osals1 mutants showed that there was no difference in Al levels in the cell sap of root tips between wild-type and the mutants, but the mutants accumulated more Al in the cytosol and nucleus than the wild-type. Expression of OsALS1 in yeast resulted in increased Al sensitivity due to mis-localization. These results indicate that OsALS1 localized at the tonoplast is responsible for sequestration of Al into the vacuoles, which is required for internal detoxification of Al in rice. OsALS1 OsAM1 is required for leptotene-zygotene transition in rice 2011 Cell Res State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. The events occurring at the onset of meiosis have not been fully elucidated. In the present study, OsAM1 was identified in rice (Oryza sativa L.) by map-based cloning. OsAM1, a homolog of Arabidopsis SWI1 and maize AM1, encodes a protein with a coiled-coil domain in its central region. In the Osam1 mutant, pollen mother cells are arrested at leptotene, showing that OsAM1 is required for the leptotene-zygotene transition. Immunocytological analysis revealed that OsAM1 exists as foci in early prophase I meiocytes. Very faint OsREC8 foci persisted in the Osam1 mutant, indicating that OsAM1 is not required for the initial meiotic recruitment of OsREC8. In the absence of OsAM1, many other critical meiotic components, including PAIR2, ZEP1 and OsMER3, could not be correctly installed onto chromosomes. In contrast, in pair2, Osmer3 and zep1 mutants, OsAM1 could be loaded normally, suggesting that OsAM1 plays a fundamental role in building the proper chromosome structure at the beginning of meiosis. OsAM1,OsRad21-4|OsREC8,PAIR2,MER3|RCK,ZEP1 OsSGO1 maintains synaptonemal complex stabilization in addition to protecting centromeric cohesion during rice meiosis 2011 Plant J State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Shugoshin is a conserved protein in eukaryotes that protects the centromeric cohesin of sister chromatids from cleavage by separase during meiosis. In this study, we identify the rice (Oryza sativa, 2n=2x=24) homolog of ZmSGO1 in maize (Zea mays), named OsSGO1. During both mitosis and meiosis, OsSGO1 is recruited from nucleoli onto centromeres at the onset of prophase. In the Tos17-insertional Ossgo1-1 mutant, centromeres of sister chromatids separate precociously from each other from metaphase I, which causes unequal chromosome segregation during meiosis II. Moreover, the release of OsSGO1 from nucleoli is completely blocked in Ossgo1-1, which leads to the absence of OsSGO1 in centromeric regions after the onset of mitosis and meiosis. Furthermore, the timely assembly and maintenance of synaptonemal complexes during early prophase I are affected in Ossgo1 mutants. Finally, we found that the centromeric localization of OsSGO1 depends on OsAM1, not other meiotic proteins such as OsREC8, PAIR2, OsMER3, or ZEP1. OsAM1,OsRad21-4|OsREC8,OsSGO1,PAIR2,MER3|RCK,ZEP1 Diversity and selective sweep in the OsAMT1;1 genomic region of rice 2011 BMC Evol Biol National Key Laboratory of Crop Genetic Improvement, and College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China. BACKGROUND: Ammonium is one of the major forms in which nitrogen is available for plant growth. OsAMT1;1 is a high-affinity ammonium transporter in rice (Oryza sativa L.), responsible for ammonium uptake at low nitrogen concentration. The expression pattern of the gene has been reported. However, variations in its nucleotides and the evolutionary pathway of its descent from wild progenitors are yet to be elucidated. In this study, nucleotide diversity of the gene OsAMT1;1 and the diversity pattern of seven gene fragments spanning a genomic region approximately 150 kb long surrounding the gene were surveyed by sequencing a panel of 216 rice accessions including both cultivated rice and wild relatives. RESULTS: Nucleotide polymorphism (Pi) of OsAMT1;1 was as low as 0.00004 in cultivated rice (Oryza sativa), only 2.3% of that in the common wild rice (O. rufipogon). A single dominant haplotype was fixed at the locus in O. sativa. The test values for neutrality were significantly negative in the entire region stretching 5' upstream and 3' downstream of the gene in all accessions. The value of linkage disequilibrium remained high across a 100 kb genomic region around OsAMT1;1 in O. sativa, but fell rapidly in O. rufipogon on either side of the promoter of OsAMT1;1, demonstrating a strong natural selection within or nearby the ammonium transporter. CONCLUSIONS: The severe reduction in nucleotide variation at OsAMT1;1 in rice was caused by a selective sweep around OsAMT1;1, which may reflect the nitrogen uptake system under strong selection by the paddy soil during the domestication of rice. Purifying selection also occurred before the wild rice diverged into its two subspecies, namely indica and japonica. These findings would provide useful insights into the processes of evolution and domestication of nitrogen uptake genes in rice. OsAMT1;1|OsAMT1.1 Over-expression of the rice OsAMT1-1 gene increases ammonium uptake and content, but impairs growth and development of plants under high ammonium nutrition 2006 Functional Plant Biology Research School of Biological Sciences, The Australian National University, Canberra, ACT 2000, Australia. A transgenic approach was undertaken to investigate the role of a rice ammonium transporter (OsAMT1-1) in ammonium uptake and consequent ammonium assimilation under different nitrogen regimes. Transgenic lines overexpressing OsAMT1-1 were produced by Agrobacterium-mediated transformation of two rice cultivars, Taipei 309 and Jarrah, with an OsAMT1-1 cDNA gene construct driven by the maize ubiquitin promoter. Transcript levels of OsAMT1-1 in both Taipei 309 and Jarrah transgenic lines correlated positively with transgene copy number. Shoot and root biomass of some transgenic lines decreased during seedling and early vegetative stage compared to the wild type, especially when grown under high (2 mm) ammonium nutrition. Transgenic plants, particularly those of cv. Jarrah recovered in the mid-vegetative stage under high ammonium nutrition. Roots of the transgenic plants showed increased ammonium uptake and ammonium content. We conclude that the decreased biomass of the transgenic lines at early stages of growth might be caused by the accumulation of ammonium in the roots owing to the inability of ammonium assimilation to match the greater ammonium uptake. OsAMT1;1|OsAMT1.1 AMT1;1 transgenic rice plants with enhanced NH4(+) permeability show superior growth and higher yield under optimal and suboptimal NH4(+) conditions 2014 J Exp Bot Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1. The major source of nitrogen for rice (Oryza sativa L.) is ammonium (NH4(+)). The NH4(+) uptake of roots is mainly governed by membrane transporters, with OsAMT1;1 being a prominent member of the OsAMT1 gene family that is known to be involved in NH4(+) transport in rice plants. However, little is known about its involvement in NH4(+) uptake in rice roots and subsequent effects on NH4(+) assimilation. This study shows that OsAMT1;1 is a constitutively expressed, nitrogen-responsive gene, and its protein product is localized in the plasma membrane. Its expression level is under the control of circadian rhythm. Transgenic rice lines (L-2 and L-3) overexpressing the OsAMT1;1 gene had the same root structure as the wild type (WT). However, they had 2-fold greater NH4(+) permeability than the WT, whereas OsAMT1;1 gene expression was 20-fold higher than in the WT. Analogous to the expression, transgenic lines had a higher NH4(+) content in the shoots and roots than the WT. Direct NH4(+) fluxes in the xylem showed that the transgenic lines had significantly greater uptake rates than the WT. Higher NH4(+) contents also promoted higher expression levels of genes in the nitrogen assimilation pathway, resulting in greater nitrogen assimilates, chlorophyll, starch, sugars, and grain yield in transgenic lines than in the WT under suboptimal and optimal nitrogen conditions. OsAMT1;1 also enhanced overall plant growth, especially under suboptimal NH4(+) levels. These results suggest that OsAMT1;1 has the potential for improving nitrogen use efficiency, plant growth, and grain yield under both suboptimal and optimal nitrogen fertilizer conditions. OsAMT1;1|OsAMT1.1 Differential expression of three members of the AMT1 gene family encoding putative high-affinity NH4+ transporters in roots of Oryza sativa subspecies indica 2003 Plant Cell Environ Department of Botany, the University of British Columbia, Vancouver, BC, Canada V6T 1Z4, Agriculture and AgriFood Canada, Shelterbelt Centre Indian Head SK, Canada S0G 2K0 and Division of Biology, University of California at San Diego, La Jolla, CA 92093-0116, USA. In order to investigate the molecular basis of high-affinity ammonium absorption by roots of rice plants (Oryza sativa subspecies indica) the expression patterns of three members of the AMT1 family of genes in rice seedling roots in response to altered nitrogen provision and diurnal changes in irradiance were examined. The 13NH4+ influx and transcript levels of OsAMT1.1 in roots decreased several fold within 48 h when plants acclimated to 10 micro m external NH4+ for 3 weeks were transferred to 10 mm NH4+. Likewise when plants acclimated in 10 mm NH4+ were transferred to 10 micro m NH4+, there was an equally rapid up-regulation of OsAMT1.1 and 13NH4+ influx in the roots. Changes in transcript abundance of OsAMT1.2 following these treatments were approximately 50% less than in OsAMT1.1, and changes of OsAMT1.3 expression were even less. By contrast, in response to the diurnal changes of irradiance, root transcript abundance of OsAMT1.3 and 15NH4+ influx increased approximately three-fold late in the photoperiod, whereas OsAMT1.1 and OsAMT1.2 exhibited only modest changes. The present results suggest that high-affinity NH4+ influx is differentially regulated at the transcriptional level through the expression of three members of the OsAMT1 family of genes in roots of rice seedlings in response to changes of N status and daily irradiance. In general, these findings are in agreement with earlier observations in Arabidopsis and tomato. OsAMT1;1|OsAMT1.1,OsAMT1;2|OsAMT1.2,OsAMT1;3|OsAMT1.3 Feedback regulation of the ammonium transporter gene family AMT1 by glutamine in rice 2003 Plant Cell Physiol Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku N10-W8, Sapporo, 060-0810 Japan. The three members of the rice OsAMT1 gene family of ammonium transporters show distinct expression patterns; constitutive and ammonium-promoted expression in shoots and roots for OsAMT1;1; root-specific and ammonium-inducible expression for OsAMT1;2; root-specific and nitrogen-repressible expression for OsAMT1;3 [Sonoda et al. (2003), Plant Cell Physiol. 44: 726]. To clarify the feedback mechanisms, and to identify regulatory factors of the OsAMT1 genes, the accumulation of the three mRNAs and its dependence on endogenous nitrogen compounds (as quantified by capillary electrophoresis) was studied. Ammonium application to roots following a period of nitrogen starvation induced accumulation of OsAMT1;1 and OsAMT1;2 mRNA, but a decrease of OsAMT1;3 mRNA levels. The expression patterns of the three genes showed good correlation (positive in OsAMT1;1 and OsAMT1;2, negative in OsAMT1;3) with the root tissue contents of glutamine but not of ammonium. The ammonium effects on OsAMT1 expression were prevented by methionine sulfoximine, an inhibitor of glutamine synthetase. Moreover, glutamine had the same effect on transcriptional regulation of OsAMT1 genes as ammonium, indicating that glutamine rather than ammonium controls the expression of ammonium transporter genes in rice. These results imply that rice possesses unique mechanisms of adaptation to variable nitrogen sources in the soil. OsAMT1;1|OsAMT1.1,OsAMT1;2|OsAMT1.2,OsAMT1;3|OsAMT1.3 Distinct expression and function of three ammonium transporter genes (OsAMT1;1-1;3) in rice 2003 Plant Cell Physiol Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku N10-W8, Sapporo, 060-0810 Japan. To study the regulation of ammonium uptake into rice roots, three ammonium transporter genes (OsAMT1;1, 1;2 and 1;3; Oryza sativa ammonium transporter) were isolated and examined. OsAMT1s belong to AMT1 family, containing 11 putative transmembrane-spanning domains. Southern blot analysis and screening of the rice genome database confirmed that with OsAMT1;1-1;3 the complete AMT1 family of rice had been isolated. Heterologous expression of OsAMT1s in the yeast Saccharomyces cerevisiae mutant 31019b showed that all three OsAMT1s exhibit ammonium transport activity. Northern blot analysis showed a distinct expression pattern for the three genes; more constitutive expression in shoots and roots for OsAMT1;1, root-specific and ammonium-inducible expression for OsAMT1;2, and root-specific and nitrogen-derepressible expression for OsAMT1;3. In situ mRNA detection revealed that OsAMT1;2 is expressed in the central cylinder and cell surface of root tips. This gene expression analysis revealed a distinct nitrogen-dependent regulation for AMTs in rice, differing from that in tomato or Arabidopsis: OsAMT1;1|OsAMT1.1,OsAMT1;2|OsAMT1.2,OsAMT1;3|OsAMT1.3 Indeterminate domain 10 regulates ammonium-mediated gene expression in rice roots 2013 New Phytol Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, 660-701, South Korea. Indeterminate domain (IDD) genes are a family of plant transcriptional regulators that function in the control of development and metabolism during growth. Here, the function of Oryza sativa indeterminate domain 10 (OsIDD10) has been explored in rice plants. Compared with wild-type roots, idd10 mutant roots are hypersensitive to exogenous ammonium. This work aims to define the action of IDD10 on gene expression involved in ammonium uptake and nitrogen (N) metabolism. The ammonium induction of key ammonium uptake and assimilation genes was examined in the roots of idd10 mutants and IDD10 overexpressors. Molecular studies and transcriptome analysis were performed to identify target genes and IDD10 binding cis-elements. IDD10 activates the transcription of AMT1;2 and GDH2 by binding to a cis-element motif present in the promoter region of AMT1;2 and in the fifth intron of GDH2. IDD10 contributes significantly to the induction of several genes involved in N-linked metabolic and cellular responses, including genes encoding glutamine synthetase 2, nitrite reductases and trehalose-6-phosphate synthase. Furthermore, the possibility that IDD10 might influence the N-mediated feedback regulation of target genes was examined. This study demonstrates that IDD10 is involved in regulatory circuits that determine N-mediated gene expression in plant roots. OsAMT1;2|OsAMT1.2,OsGDH2,OsIDD10 Metabolic regulation of ?-amylase gene expression in transgenic cell cultures of rice (Oryza sativa L.) 1993 Plant Mol Biol Section of Molecular and Cellular Biology, University of California, 95616, Davis, CA, USA Expression of two genes in the α-amylase gene family is controlled by metabolic regulation in rice cultured cells. The levels of RAmy3D and RAmy3E mRNAs in rice cultured cells are inversely related to the concentration of sugar in the culture medium. Other genes in the rice α-amylase gene family have little or no expression in cultured cells; these expression levels are not controlled by metabolic regulation. A RAmy3D promoter/GUS gene fusion was metabolically regulated in the transgenic rice cell line 3DG, just as the endogenous RAmy3D gene is regulated. An assay of GUS enzyme activity in 3DG cells demonstrated that RAmy3D/GUS expression is repressed when sugar is present in the culture medium and induced when sugar is removed from the medium. The 942 bp fragment of the RAmy3D promoter that was linked to the coding region of the GUS reporter gene thus contains all of the regulatory sequences necessary for metabolic regulation of the gene. alphaAmy3|OsAmy3D|RAmy3D Interference with oxidative phosphorylation enhances anoxic expression of rice alpha-amylase genes through abolishing sugar regulation 2010 J Exp Bot Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea. Rice has the unique ability to express alpha-amylase under anoxic conditions, a feature that is critical for successful anaerobic germination and growth. Previously, anaerobic conditions were shown to up-regulate the expression of Amy3 subfamily genes (Amy3B/C, 3D, and 3E) in rice embryos. These genes are known to be feedback regulated by the hydrolytic products of starchy endosperm such as the simple sugar glucose. It was found that oxygen deficiency interferes with the repression of Amy3D gene expression imposed by low concentrations of glucose but not with that imposed by higher amounts. This differential anoxic de-repression depending on sugar concentration suggests the presence of two distinct pathways for sugar regulation of Amy3D gene expression. Anoxic de-repression can be mimicked by treating rice embryos with inhibitors of ATP synthesis during respiration. Other sugar-regulated rice alpha-amylase genes, Amy3B/C and 3E, behave similarly to Amy3D. Treatment with a respiratory inhibitor or anoxia also relieved the sugar repression of the rice CIPK15 gene, a main upstream positive regulator of SnRK1A that is critical for Amy3D expression in response to sugar starvation. SnRK1A accumulation was previously shown to be required for MYBS1 expression, which transactivates Amy3D by binding to a cis-acting element found in the proximal region of all Amy3 subfamily gene promoters (the TA box). Taken together, these results suggest that prevention of oxidative phosphorylation by oxygen deficiency interferes with the sugar repression of Amy3 subfamily gene expression, leading to their enhanced expression in rice embryos during anaerobic germination. alphaAmy3|OsAmy3D|RAmy3D,OsCIPK15,OsMYBS1,SnRK1A Regulatory interplay of the Sub1A and CIPK15 pathways in the regulation of alpha-amylase production in flooded rice plants 2011 Plant Biol (Stuttg) Department of Crop Plant Biology, University of Pisa, Pisa, Italy. Rice (Oryza sativa L.) can successfully germinate and grow even when flooded. Rice varieties possessing the submergence 1A (Sub1A) gene display a distinct flooding-tolerant phenotype, associated with lower carbohydrate consumption and restriction of the fast-elongation phenotype typical of flooding-intolerant rice varieties. Calcineurin B-like interacting protein kinase 15 (CIPK15) was recently indicated as a key regulator of alpha-amylases under oxygen deprivation, linked to both rice germination and flooding tolerance in adult plants. It is still unknown whether the Sub1A- and CIPK15-mediated pathways act as complementary processes for rice survival under O(2) deprivation. In adult plants Sub1A and CIPK15 may perhaps play an antagonistic role in terms of carbohydrate consumption, with Sub1A acting as a starch degradation repressor and CIPK15 as an activator. In this study, we analysed sugar metabolism in the stem of rice plants under water submergence by selecting cultivars with different traits associated with flooding survival. The relation between the Sub1A and the CIPK15 pathways was investigated. The results show that under O(2) deprivation, the CIPK15 pathway is repressed in the tolerant, Sub1A-containing, FR13A variety. CIPK15 is likely to play a role in the up-regulation of Ramy3D in flooding-intolerant rice varieties that display fast elongation under flooding and that do not possess Sub1A. alphaAmy3|OsAmy3D|RAmy3D,OsCIPK15,Sub1A Structural organization and differential expression of rice alpha-amylase genes 1990 Nucleic Acids Res Department of Genetics, University of California, Davis 95616. Rice alpha-amylases are encoded by a multigene family that has previously been classified into 5 hybridization groups. DNA sequence and Southern blot analysis identified three genes (RAmy1A, RAmy1B and RAmy1C) in Group 1 with DNA sequence identity of at least 90%. Hybridization Group 2 is represented by only one gene, RAmy3D, which is identical to a previously characterized cDNA, pOS137. RAmy3D is physically linked to the sole representative of Group 5, RAmy3E. The identity between these two genes is 81.4% in the coding region but less than 50% in the 5' and 3' flanking regions. Northern blot analysis and RNA-PCR were used to detect the expression of alpha-amylase genes in various tissues. Alpha-amylase mRNA was abundant in germinating seeds and callus. Some genes were also expressed at much lower levels in roots, young leaves and immature seeds. RAmy1A and RAmy3E were expressed in all tissues while RAmy3D was expressed in all tissues except the immature seeds. RAmy1B was weakly expressed only in callus. RAmy1A transcript was most abundant in the germinating seeds, while RAmy3D and RAmy3E transcripts were most abundant in callus and immature seeds, respectively. alphaAmy3|OsAmy3D|RAmy3D The SnRK1A protein kinase plays a key role in sugar signaling during germination and seedling growth of rice 2007 Plant Cell Department of Life Sciences, National Central University, Jhongli City, Taoyuan County 320, Taiwan, Republic of China. Sugars repress alpha-amylase expression in germinating embryos and cell cultures of rice (Oryza sativa) through a sugar response complex (SRC) in alpha-amylase gene promoters and its interacting transcription factor MYBS1. The Snf1 protein kinase is required for the derepression of glucose-repressible genes in yeast. In this study, we explored the role of the yeast Snf1 ortholog in rice, SnRK1, in sugar signaling and plant growth. Rice embryo transient expression assays indicated that SnRK1A and SnRK1B act upstream and relieve glucose repression of MYBS1 and alphaAmy3 SRC promoters. Both SnRK1s contain N-terminal kinase domains serving as activators and C-terminal regulatory domains as dominant negative regulators of SRC. The accumulation and activity of SnRK1A was regulated by sugars posttranscriptionally, and SnRK1A relieved glucose repression specifically through the TA box in SRC. A transgenic RNA interference approach indicated that SnRK1A is also necessary for the activation of MYBS1 and alphaAmy3 expression under glucose starvation. Two mutants of SnRK1s, snrk1a and snrk1b, were obtained, and the functions of both SnRK1s were further studied. Our studies demonstrated that SnRK1A is an important intermediate in the sugar signaling cascade, functioning upstream from the interaction between MYBS1 and alphaAmy3 SRC and playing a key role in regulating seed germination and seedling growth in rice. alphaAmy3|OsAmy3D|RAmy3D,OsMYBS1,SnRK1A,SnRK1B The 3' untranslated region of a ricea-amylase gene functions as a sugar-dependent mRNA stability determinant 1998 Proc Natl Acad Sci U S A Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan, Republic of China. In plants, sugar feedback regulation provides a mechanism for control of carbohydrate allocation and utilization among tissues and organs. The sugar repression of alpha-amylase gene expression in rice provides an ideal model for studying the mechanism of sugar feedback regulation. We have shown previously that sugar repression of alpha-amylase gene expression in rice suspension cells involves control of both transcription rate and mRNA stability. The alpha-amylase mRNA is significantly more stable in sucrose-starved cells than in sucrose-provided cells. To elucidate the mechanism of sugar-dependent mRNA turnover, we have examined the effect of alphaAmy3 3' untranslated region (UTR) on mRNA stability by functional analyses in transformed rice suspension cells. We found that the entire alphaAmy3 3' UTR and two of its subdomains can independently mediate sugar-dependent repression of reporter mRNA accumulation. Analysis of reporter mRNA half-lives demonstrated that the entire alphaAmy3 3' UTR and the two subdomains each functioned as a sugar-dependent destabilizing determinant in the turnover of mRNA. Nuclear run-on transcription analysis further confirmed that the alphaAmy3 3' UTR and the two subdomains did not affect the transcription rate of promoter. The identification of sequence elements in the alpha-amylase mRNA that dictate the differential stability has very important implications for the study of sugar-dependent mRNA decay mechanisms. alphaAmy3|OsAmy3D|RAmy3D Signal peptide-dependent targeting of a rice alpha-amylase and cargo proteins to plastids and extracellular compartments of plant cells 2004 Plant Physiol Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, 11529 Taiwan, Republic of China. alpha-Amylases are important enzymes for starch degradation in plants. However, it has been a long-running debate as to whether alpha-amylases are localized in plastids where starch is stored. To study the subcellular localization of alpha-amylases in plant cells, a rice (Oryza sativa) alpha-amylase, alphaAmy3, with or without its own signal peptide (SP) was expressed in transgenic tobacco (Nicotiana tabacum) and analyzed. Loss-of-function analyses revealed that SP was required for targeting of alphaAmy3 to chloroplasts and/or amyloplasts and cell walls and/or extracellular compartments of leaves and suspension cells. SP was also required for in vitro transcribed and/or translated alphaAmy3 to be cotranslationally imported and processed in canine microsomes. alphaAmy3, present in chloroplasts of transgenic tobacco leaves, was processed to a product with Mr similar to alphaAmy3 minus its SP. Amino acid sequence analysis revealed that the SP of chloroplast localized alphaAmy3 was cleaved at a site only one amino acid preceding the predicted cleavage site. Function of the alphaAmy3 SP was further studied by gain-of-function analyses. beta-Glucuronidase (GUS) and green fluorescence protein fused with or without the alphaAmy3 SP was expressed in transgenic tobacco or rice. The alphaAmy3 SP directed translocation of GUS and green fluorescence protein to chloroplasts and/or amyloplasts and cell walls in tobacco leaves and rice suspension cells. The SP of another rice alpha-amylase, alphaAmy8, similarly directed the dual localizations of GUS in transgenic tobacco leaves. This study is the first evidence of SP-dependent dual translocations of proteins to plastids and extracellular compartments, which provides new insights into the role of SP in protein targeting and the pathways of SP-dependent protein translocation in plants. alphaAmy3|OsAmy3D|RAmy3D Interaction between rice MYBGA and the gibberellin response element controls tissue-specific sugar sensitivity of alpha-amylase genes 2006 Plant Cell Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 115, Republic of China. Expression of alpha-amylase genes during cereal grain germination and seedling growth is regulated negatively by sugar in embryos and positively by gibberellin (GA) in endosperm through the sugar response complex (SRC) and the GA response complex (GARC), respectively. We analyzed two alpha-amylase promoters, alphaAmy3 containing only SRC and alphaAmy8 containing overlapped SRC and GARC. alphaAmy3 was sugar-sensitive but GA-nonresponsive in both rice (Oryza sativa) embryos and endosperms, whereas alphaAmy8 was sugar-sensitive in embryos and GA-responsive in endosperms. Mutation of the GA response element (GARE) in the alphaAmy8 promoter impaired its GA response but enhanced sugar sensitivity, and insertion of GARE in the alphaAmy3 promoter rendered it GA-responsive but sugar-insensitive in endosperms. Expression of the GARE-interacting transcription factor MYBGA was induced by GA in endosperms, correlating with the endosperm-specific alphaAmy8 GA response. alphaAmy8 became sugar-sensitive in MYBGA knockout mutant endosperms, suggesting that the MYBGA-GARE interaction overrides the sugar sensitivity of alphaAmy8. In embryos overexpressing MYBGA, alphaAmy8 became sugar-insensitive, indicating that MYBGA affects sugar repression. alpha-Amylase promoters active in endosperms contain GARE, whereas those active in embryos may or may not contain GARE, confirming that the GARE and GA-induced MYBGA interaction prevents sugar feedback repression of endosperm alpha-amylase genes. We demonstrate that the MYBGA-GARE interaction affects sugar feedback control in balanced energy production during seedling growth and provide insight into the control mechanisms of tissue-specific regulation of alpha-amylase expression by sugar and GA signaling interference. alphaAmy3|OsAmy3D|RAmy3D,OsMYBGA|OsGAMYB Sugar sensing and alpha-amylase gene repression in rice embryos 1998 Planta Meijo University, Nagoya, Japan. b42295a@nucc.cc.nagoya-u.ac.jp We used a transient expression system to study the mechanism by which carbohydrates repress a rice (Oryza sativa L.) alpha-amylase (EC 3.2.1.1) gene. Exogenously fed metabolizable carbohydrates are able to elicit repression of the alpha-amylase gene RAmy3D in the rice embryo, and our results indicate that repression is also triggered efficiently by endogenous carbohydrates. Glucose analogs that are taken up by plant cells but not phosphorylated by hexokinase are unable to repress the alpha-amylase gene studied, while 2-deoxyglucose, which is phosphorylable but not further metabolized, down-regulates RAmy3D promoter activity, indicating a role for hexokinase in the sugar-sensing mechanism triggering repression of the RAmy3D gene. We tested two different hexokinase inhibitors, mannoheptulose and glucosamine, but only the latter was able to relieve RAmy3D promoter activity from repression by endogenous carbohydrates. This correlates with the higher ability of glucosamine to inhibit the activity of rice hexokinases in vitro. The glucosamine-mediated relief of RAmy3D promoter activity from repression by endogenous carbohydrates does not correlate with a reduced rate of carbohydrate utilization. alphaAmy3|OsAmy3D|RAmy3D Rice flavonoid pathway genes, OsDfr and OsAns, are induced by dehydration, high salt and ABA, and contain stress responsive promoter elements that interact with the transcription activator, OsC1-MYB 2004 Plant Science Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India Molecular basis of regulation of abiotic stress responses and the flavonoid biosynthesis in rice was investigated. The role of the regulatory gene OsC1-Myb, encoding a MYB class of transcription activators in the stress-induced expression of the structural genes, OsDfr and OsAns, was analyzed. Northern analysis of shoot tissues of rice, Nagina 22, (Oryza sativa L. sub sp. indica) seedlings under dehydration stress or high salt or abscisic acid (ABA) showed a significant enhancement of transcript level and/or transcript stability of OsDfr and OsAns. Enhanced levels of the OsC1-myb transcript were also detected. The expression pattern of these three genes indicates that the stress responsive accumulation of OsDfr and OsAns transcripts is mediated by the transcription factor, OsC1-MYB. The 5′ upstream region of the OsDfr and OsAns genes carry several regulatory domains, which share homology with some of the known stress responsive genes in plants. In addition, several putative myb and myc responsive domains were identified in the promoter region of the genes, OsDfr and OsAns. The recombinant OsC1-MYB protein binds in vitro to the myb responsive elements (MREs) in the OsDfr and OsAns promoters, suggesting that it is a potential transcription activator of stress-induced expression of structural genes of the flavonoid pathway. OsAns,OsDfr Cloning of novel rice allene oxide cyclase (OsAOC): mRNA expression and comparative analysis with allene oxide synthase (OsAOS) gene provides insight into the transcriptional regulation of octadecanoid pathway biosynthetic genes in rice 2003 Plant Science Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), GPO Box 8207, Kathmandu, Nepal None OsAOC Identification of rice Allene Oxide Cyclase mutants and the function of jasmonate for defence against Magnaporthe oryzae 2013 Plant J Botanical Institute, Molecular Cell Biology, Karlsruhe Institute of Technology, Kaiserstrasse 2, 76131, Karlsruhe, Germany. Two photomorphogenic mutants of rice, coleoptile photomorphogenesis 2 (cpm2) and hebiba, were found to be defective in the gene encoding allene oxide cyclase (OsAOC) by map-based cloning and complementation assays. Examination of the enzymatic activity of recombinant GST-OsAOC indicated that OsAOC is a functional enzyme that is involved in the biosynthesis of jasmonic acid and related compounds. The level of jasmonate was extremely low in both mutants, in agreement with the fact that rice has only one gene encoding allene oxide cyclase. Several flower-related mutant phenotypes were observed, including morphological abnormalities of the flower and early flowering. We used these mutants to investigate the function of jasmonate in the defence response to the blast fungus Magnaporthe oryzae. Inoculation assays with fungal spores revealed that both mutants are more susceptible than wild-type to an incompatible strain of M. oryzae, in such a way that hyphal growth was enhanced in mutant tissues. The level of jasmonate isoleucine, a bioactive form of jasmonate, increased in response to blast infection. Furthermore, blast-induced accumulation of phytoalexins, especially that of the flavonoid sakuranetin, was found to be severely impaired in cpm2 and hebiba. Together, the present study demonstrates that, in rice, jasmonate mediates the defence response against blast fungus. OsAOC Phytochrome-mediated transcriptional up-regulation of ALLENE OXIDE SYNTHASE in rice seedlings 2004 Plant Cell Physiol Botanical Gardens, Research School of Science, Osaka City University, Kisaichi, Katano-shi, Osaka, 576-0004 Japan. Allene oxide synthase (AOS) is a key enzyme for the biosynthesis of jasmonic acid (JA). We identified four AOS gene homologs, named OsAOS1-4, in the database of a japonica rice genome and cloned a full-length cDNA of OsAOS1. The analysis of deduced amino acid sequences indicated that only OsAOS1 has a chloroplast transit peptide among all the identified monocot AOSs including OsAOSs. We found that the transcripts of OsAOS1 and OsAOS4 are up-regulated by red and far-red light in seedling shoots. The response in OsAOS1 transcripts occurred rapidly and transiently, while the response in OsAOS4 transcripts was slower and more sustainable; the maximal enhancement was greater in OsAOS1 transcripts than in OsAOS4 transcripts. The transcript of OsAOS1 was also up-regulated transiently in response to wounding, as reported for dicot AOSs. No wound-induced enhancement occurred, however, in OsAOS4 transcripts. Our results also indicated that OsAOS1, responding to both light and wounding, is the most highly expressed of all the OsAOSs in seedling shoots. By using phyA mutants of rice, it was demonstrated that the photoregulation of the AOS transcript level is mediated by phytochrome. It is suggested that this transcriptional photoregulation participates in the phytochrome-mediated inhibition of rice coleoptile growth. OsAOS1,OsAOS2,OsAOS4|OsHPL1,OsAOS3|OsHPL2 OsMPK3 positively regulates the JA signaling pathway and plant resistance to a chewing herbivore in rice 2013 Plant Cell Rep State Key Laboratory of Biocontrol, Institute of Entomology, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China. q.wang302@gmail.com KEY MESSAGE : Silencing OsMPK3 decreased elicited JA levels, which subsequently reduced levels of herbivore-induced trypsin protease inhibitors (TrypPIs) and improved the performance of SSB larvae, but did not influence BPH. Mitogen-activated protein kinases (MPKs) are known to play an important role in plant defense by transferring biotic and abiotic signals into programmed cellular responses. However, their functions in the herbivore-induced defense response in rice remain largely unknown. Here, we identified a MPK3 gene from rice, OsMPK3, and found that its expression levels were up-regulated in response to infestation by the larvae of the striped stem borer (SSB) (Chilo suppressalis), to mechanical wounding and to treatment with jasmonic acid (JA), but not to infestation by the brown planthopper (BPH) Nilaparvata lugens or to treatment with salicylic acid. Moreover, mechanical wounding and SSB infestation induced the expression of OsMPK3 strongly and quickly, whereas JA treatment induced the gene more weakly and slowly. Silencing OsMPK3 (ir-mpk3) reduced the expression of the gene by 50-70 %, decreased elicited levels of JA and diminished the expression of a lipoxygenase gene OsHI-LOX and an allene oxide synthase gene OsAOS1. The reduced JA signaling in ir-mpk3 plants decreased the levels of herbivore-induced trypsin protease inhibitors (TrypPIs) and improved the performance of SSB larvae, but did not influence BPH. Our findings suggest that the gene OsMPK3 responds early in herbivore-induced defense and can be regulated by rice plants to activate a specific and appropriate defense response to different herbivores. OsAOS1,OsHI-LOX,OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1 Constitutive expression of rice WRKY30 gene increases the endogenous jasmonic acid accumulation, PR gene expression and resistance to fungal pathogens in rice 2012 Planta School of Life Sciences, Hunan University of Science and Technology, Taoyuan Rd., Xiangtan, 411201, Hunan, China. WRKY transcription factors are crucial regulatory components of plant responses to pathogen infection. In the present study, we report isolation and functional characterization of the pathogen-responsive rice WRKY30 gene, whose transcripts accumulate rapidly in response to salicylic acid (SA) and jasmonic acid (JA) treatment. Overexpression of WRKY30 in rice enhanced resistance to rice sheath blight fungus Rhizoctonia solani and blast fungus Magnaporthe grisea. The enhanced resistance in the transgenic lines overexpressing WRKY30 was associated with activated expression of JA synthesis-related genes LOX, AOS2 and pathogenesis-related (PR)3 and PR10, and increased endogenous JA accumulation under the challenge of fungal pathogens. WRKY30 was nuclear-localized and had transcriptional activation ability in yeast cells, supporting that it functions as a transcription factor. Together, our findings indicate that JA plays a crucial role in the WRKY30-mediated defense responses to fungal pathogens, and that the rice WRKY30 seems promising as an important candidate gene to improve disease resistance in rice. OsAOS2,OsWRKY30 Inducible overexpression of a rice allene oxide synthase gene increases the endogenous jasmonic acid level, PR gene expression, and host resistance to fungal infection 2006 Mol Plant Microbe Interact Department of Plant Pathology and Program in Cell and Molecular Biology, University of Arkansas, Fayetteville 72071, USA. Many studies in dicotyledonous plants have shown that jasmonates, including jasmonic acid (JA) and methyl jasmonate, are important signal molecules involved in induced resistance to pathogen infection and insect herbivory. However, very little genetic and molecular evidence is available to demonstrate their role in host defense response of rice and other economically important monocot plants. In this study, we have shown that exogenous application of JA was able to activate defense gene expression and local induced resistance in rice seedlings against the rice blast fungus (Magnaporthe grisea). Furthermore, we have characterized a pathogen-inducible rice OsAOS2 gene (which encodes allene oxide synthase, a key enzyme in the JA biosynthetic pathway) and examined the role of endogenous JA in rice defense response through transgenic manipulation of the JA biosynthesis. Sequence analysis indicated that OsAOS2 contains four common domains of the cytochrome P450 enzyme, but does not have the signal peptide for chloroplast targeting. The basal level of OsAOS2 expression is very low in leaves but relatively high in the sheath, culm, and flower of rice plants. Interestingly, the expression of OsAOS2 in rice leaves can be induced significantly upon M. grisea infection. Transgenic rice lines carrying the OsAOS2 transgene under the control of a strong, pathogen-inducible PBZ1 promoter accumulated abundant OsAOS2 transcripts and higher levels of JA, especially after the pathogen infection. These transgenic lines also exhibited enhanced activation of pathogenesis-related (PR) genes such as PR1a, PR3, and PR5 and increased resistance to M. grisea infection. Our results suggest that JA plays a significant role in PR gene induction and blast resistance in rice plants. OsAOS2,OsPR10a|PBZ1,OsPR1a Rice HYDROPEROXIDE LYASES with unique expression patterns generate distinct aldehyde signatures in Arabidopsis 2006 Plant Physiol Section of Plant Biology, University of California, Davis, California 95616, USA. HYDROPEROXIDE LYASE (HPL) genes encode enzymes that catalyze the cleavage of fatty acid hydroperoxides into aldehydes and oxoacids. There are three HPLs in rice (Oryza sativa), designated OsHPL1 through OsHPL3. To explore the possibility of differential functional activities among these genes, we have examined their expression patterns and biochemical properties of their encoded products. Transcript analysis indicates that these genes have distinct patterns and levels of expression. OsHPL1 is ubiquitously expressed, OsHPL2 is expressed in the leaves and leaf sheaths, whereas OsHPL3 is wound inducible and expressed exclusively in leaves. OsHPLs also differ in their substrate preference as determined by in vitro enzyme assays using 9-/13-hydroperoxy linolenic and 9-/13-hydroperoxy linoleic acids as substrates. OsHPL1 and OsHPL2 metabolize 9-/13-hydroperoxides, whereas OsHPL3 metabolizes 13-hydroperoxy linolenic acid exclusively. Sequence alignments of the HPL enzymes have identified signature residues potentially responsible for the substrate specificity/preference of these enzymes. All three OsHPLs are chloroplast localized as determined by chloroplast import assays and green fluorescent protein (GFP) fusion studies. Aldehyde measurements in transgenic Arabidopsis (Arabidopsis thaliana) plants overexpressing individual OsHPL-GFP fusions indicate that all rice HPLs are functional in a heterologous system, and each of them generates a distinct signature of the metabolites. Interestingly, these aldehydes were only detectable in leaves, but not in roots, despite similar levels of OsHPL-GFP proteins in both tissues. Similarly, there were undetectable levels of aldehydes in rice roots, in spite of the presence of OsHPL1 transcripts. Together, these data suggest that additional tissue-specific mechanism(s) beyond transcript and HPL enzyme abundance, regulate the levels of HPL-derived metabolites. OsAOS4|OsHPL1,OsAOS3|OsHPL2,OsHPL3 Transcript levels of tandem-arranged alternative oxidase genes in rice are increased by low temperature 1997 Gene Laboratory of Radiation Genetics, Graduate School of Agricultural and Life Science, The University of Tokyo, Japan. We identified two genes for alternative oxidase (AOX) from rice. One AOX gene (designated AOX1a) is located approx. 1.9 kb downstream of another AOX gene (designated AOX1b). Comparison of the genomic and cDNA sequences of the two AOX genes showed that the AOX1a gene is interrupted by three introns, as are AOX genes of other plants. On the other hand, two introns are inserted in the AOX1b gene. The predicted AOX1a and AOX1b precursor proteins consist of 332 and 335 amino acid residues, respectively. A genomic Southern hybridization analysis indicated that rice has several AOX genes other than the two tandem-arranged AOX genes. Steady-state mRNA levels of both of the genes for AOX1a and AOX1b were increased under low temperature (4 degrees C). However, no difference in the pattern of induction of transcription between the genes for AOX1a and AOX1b was observed. AOX1a|OsAOX1a,OsAOX1b Overexpression of an alternative oxidase gene, OsAOX1a, improves cold tolerance in Oryza sativa L 2013 Genet Mol Res College of Life Sciences, Anhui University, Hefei, China. Low temperature is a major environmental stress in rice cultivating and production. The alternative oxidase 1 (AOX1) gene is potentially important for genetic engineering to increase cold adaptation. However, previous studies related to this effect have mostly focused on the dicot plants Arabidopsis and tobacco, whereas functional research on rice is limited. In this study, we cloned a rice predominant cold-response AOX1 gene, OsAOX1a. Transgenic rice plants with overexpression of OsAOX1a were obtained. We found that OsAOX1a overexpression could strongly enhance the cold growth of seedlings, especially with respect to root extension. However, growth between transgenic and control plants did not differ under normal conditions. Furthermore, the lipid peroxidation and ion leakage rate were determined after cold treatment in transgenic plants. Both factors were reduced by OsAOX1a overexpression, which revealed that OsAOX1a could reduce oxidative damage under cold stress. Taken together, our results suggested that overexpressing OsAOX1a could improve growth performance of rice under cold stress, which might be closely related to the reduction of reactive oxygen species generation and oxidative damage. AOX1a|OsAOX1a,OsAOX1b Transcriptional activation of OsDERF1 in OsERF3 and OsAP2-39 negatively modulates ethylene synthesis and drought tolerance in rice 2011 PLoS One Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China. The phytohormone ethylene is a key signaling molecule that regulates a variety of developmental processes and stress responses in plants. Transcriptional modulation is a pivotal process controlling ethylene synthesis, which further triggers the expression of stress-related genes and plant adaptation to stresses; however, it is unclear how this process is transcriptionally modulated in rice. In the present research, we report the transcriptional regulation of a novel rice ethylene response factor (ERF) in ethylene synthesis and drought tolerance. Through analysis of transcriptional data, one of the drought-responsive ERF genes, OsDERF1, was identified for its activation in response to drought, ethylene and abscisic acid. Transgenic plants overexpressing OsDERF1 (OE) led to reduced tolerance to drought stress in rice at seedling stage, while knockdown of OsDERF1 (RI) expression conferred enhanced tolerance at seedling and tillering stages. This regulation was supported by negative modulation in osmotic adjustment response. To elucidate the molecular basis of drought tolerance, we identified the target genes of OsDERF1 using the Affymetrix GeneChip, including the activation of cluster stress-related negative regulators such as ERF repressors. Biochemical and molecular approaches showed that OsDERF1 at least directly interacted with the GCC box in the promoters of ERF repressors OsERF3 and OsAP2-39. Further investigations showed that OE seedlings had reduced expression (while RI lines showed enhanced expression) of ethylene synthesis genes, thereby resulting in changes in ethylene production. Moreover, overexpression of OsERF3/OsAP2-39 suppressed ethylene synthesis. In addition, application of ACC recovered the drought-sensitive phenotype in the lines overexpressing OsERF3, showing that ethylene production contributed to drought response in rice. Thus our data reveal that a novel ERF transcriptional cascade modulates drought response through controlling the ethylene synthesis, deepening our understanding of the regulation of ERF proteins in ethylene related drought response. OsAP2-39,OsDERF1,AP37|OsERF3 Isolation and characterization of a novel cDNA encoding ERF/AP2-type transcription factor OsAP25 from Oryza sativa L 2007 Biotechnol Lett Department of Horticulture, Nanjing Agricultural University, Weigang, Nanjing, 210095, P.R. China. Using a yeast one-hybrid method, a transcription factor, OsAP25, which interacts specifically with a GCC box was isolated from rice. The OsAP25 protein contained a conserved ethylene-responsive element binding factor (ERF) domain which shared identity with other reported ERF domains. Phylogenetic analysis showed that OsAP25 could be categorized into class III ERF of the previously characterized ERF proteins on an evolutionary relationship. The semi-quantitative RT-PCR analysis revealed that OsAP25 gene was constitutively expressed in leaves, roots, growing points, flower, bolting stage and grain filling stage. In addition, OsAP25 gene was induced by NaCl, cold, drought, abscisic acid and exogenous ethylene. OsAP25 Abnormal endosperm development causes female sterility in rice insertional mutant OsAPC6 2012 Plant Sci Department of Biotechnology, Indian Institute of Technology, Roorkee-247667 India. A T-DNA insertional mutant OsAPC6 of rice, with gibberellic acid insensitivity and reduced height, had up to 45% reduced seed set. The insertion occurred on chromosome 3 of rice in the gene encoding one of the subunits of anaphase promoting complex/Cyclosome APC6. The primary mother cells of the mutant plants had normal meiosis, male gametophyte development and pollen viability. Confocal laser scanning microscopic (CLSM) studies of megagametophyte development showed abnormal mitotic divisions with reduced number or total absence of polar nuclei in about 30-35% megagametophytes of OsAPC6 mutant leading to failure of endosperm and hence embryo and seed development. Abnormal female gametophyte development, high sterility and segregation of tall and gibberellic acid sensitive plants without selectable marker Hpt in the selfed progeny of OsAPC6 mutant plants indicate that the mutant could be maintained in heterozygous condition. The abnormal mitotic divisions during megagametogenesis could be attributed to the inactivation of the APC6/CDC16 of anaphase promoting complex of rice responsible for cell cycle progression during megagametogenesis. Functional validation of the candidate gene through transcriptome profiling and RNAi is in progress. OsAPC6 A candidate gene OsAPC6 of anaphase-promoting complex of rice identified through T-DNA insertion 2010 Funct Integr Genomics Indian Institute of Technology Roorkee, Roorkee, 247 667, Uttarakhand, India. A dwarf mutant (Oryza sativa anaphase-promoting complex 6 (OsAPC6)) of rice cultivar Basmati 370 with 50% reduced plant height as compared to the wild type was isolated by Agrobacterium tumefaciens-mediated transformation using Hm(R) Ds cassette. This mutant was found to be insensitive to exogenous gibberellic acid (GA(3)) application. Homozygous mutant plants showed incomplete penetrance and variable expressivity for plant height and pleiotropic effects including gibberellic acid insensitivity, reduced seed size, panicle length, and female fertility. Single copy insertion of T-DNA and its association with OsAPC6 was confirmed by Southern hybridization, germination on hygromycin, and 3:1 segregation of HPT gene in F(2) from OsAPC6 x Basmati 370 cross. The T-DNA flanking region sequenced through thermal asymmetric interlaced polymerase chain reaction showed a single hit on chromosome 3 of japonica rice cultivar Nipponbare in the second exonic region of a gene which encodes for sixth subunit of anaphase-promoting complex/cyclosome. The candidate gene of 8.6-kb length encodes a 728-amino acid protein containing a conserved tetratricopeptide repeat (TPR) domain and has only a paralog, isopenicillin N-synthase family protein on the same chromosome without the TPR domain. There was no expression of the gene in the mutant while in Basmati 370, it was equal in both roots and shoots. The knockout mutant OsAPC6 interferes with the gibberellic acid signaling pathway leading to reduced height and cell size probably through ubiquitin-mediated proteolysis. Further functional validation of the gene through RNAi is in progress. OsAPC6 Identification of the ADP-glucose pyrophosphorylase isoforms essential for starch synthesis in the leaf and seed endosperm of rice (Oryza sativa L.) 2007 Plant Mol Biol Graduate School of Biotechnology & Plant Metabolism Research Center, Kyung Hee University, Yongin 446-701, Korea. ADP-glucose pyrophosphorylase (AGP) catalyzes the first committed step of starch biosynthesis in higher plants. To identify AGP isoforms essential for this biosynthetic process in sink and source tissues of rice plants, we analyzed the rice AGP gene family which consists of two genes, OsAGPS1 and OsAGPS2, encoding small subunits (SSU) and four genes, OsAGPL1, OsAGPL2, OsAGPL3 and OsAGPL4, encoding large subunits (LSU) of this enzyme heterotetrameric complex. Subcellular localization studies using green fluorescent protein (GFP) fusion constructs indicate that OsAGPS2a, the product of the leaf-preferential transcript of OsAGPS2, and OsAGPS1, OsAGPL1, OsAGPL3, and OsAGPL4 are plastid-targeted isoforms. In contrast, two isoforms, SSU OsAGPS2b which is a product of a seed-specific transcript of OsAGPS2, and LSU OsAGPL2, are localized in the cytosol. Analysis of osagps2 and osagpl2 mutants revealed that a lesion of one of the two cytosolic isoforms, OsAGPL2 and OsAGPS2b, causes a shrunken endosperm due to a remarkable reduction in starch synthesis. In leaves, however, only the osagps2 mutant appears to severely reduce the transitory starch content. Interestingly, the osagps2 mutant was indistinguishable from wild type during vegetative plant growth. Western blot analysis of the osagp mutants and wild type plants demonstrated that OsAGPS2a is an SSU isoform mainly present in leaves, and that OsAGPS2b and OsAGPL2 are the major SSU and LSU isoforms, respectively, in the endosperm. Finally, we propose a spatiotemporal complex model of OsAGP SSU and LSU isoforms in leaves and in developing endosperm of rice plants. OsAPL1|OsAGPL3|OsAGPL3,OsAPL2|osagpl2-3,OsAPL4|OsAGPL4,OsAPS1|OsAGPS1,OsAPS2|OsAGPS2b A mutant of rice lacking the leaf large subunit of ADP-glucose pyrophosphorylase has drastically reduced leaf starch content but grows normally 2007 Functional Plant Biology John Innes Centre, Norwich Research Park, Norfolk NR4 7UH, UK. A mutant of rice was identified with a Tos17 insertion in OsAPL1, a gene encoding a large subunit (LSU) of ADP-glucose pyrophosphorylase (AGPase). The insertion prevents production of a normal transcript from OsAPL1. Characterisation of the mutant (apl1) showed that the LSU encoded by OsAPL1 is required for AGPase activity in rice leaf blades. In mutant leaf blades, the AGPase small subunit protein is not detectable and the AGPase activity and starch content are reduced to <1 and <5% of that in wild type blades, respectively. The mutation also leads to a reduction in starch content in the leaf sheaths but does not significantly affect AGPase activity or starch synthesis in other parts of the plant. The sucrose, glucose and fructose contents of the leaves are not affected by the mutation. Despite the near absence of starch in the leaf blades, apl1 mutant rice plants grow and develop normally under controlled environmental conditions and show no reduction in productivity. OsAPL1|OsAGPL3|OsAGPL3 Expression profiling of genes involved in starch synthesis in sink and source organs of rice 2005 J Exp Bot Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan. A comprehensive analysis of the transcript levels of genes which encode starch-synthesis enzymes is fundamental for the assessment of the function of each enzyme and the regulatory mechanism for starch biosynthesis in source and sink organs. Using quantitative real-time RT-PCR, an examination was made of the expression profiles of 27 rice genes encoding six classes of enzymes, i.e. ADPglucose pyrophosphorylase (AGPase), starch synthase, starch branching enzyme, starch debranching enzyme, starch phosphorylase, and disproportionating enzyme in developing seeds and leaves. The modes of gene expression were tissue- and developmental stage-specific. Four patterns of expression in the seed were identified: group 1 genes, which are expressed very early in grain formation and are presumed to be involved in the construction of fundamental cell machineries, de novo synthesis of glucan primers, and initiation of starch granules; group 2 genes, which are highly expressed throughout endosperm development; group 3 genes, which have transcripts that are low at the onset but which rise steeply at the start of starch synthesis in the endosperm and are thought to play essential roles in endosperm starch synthesis; and group 4 genes, which are expressed scantly, mainly at the onset of grain development, and might be involved in synthesis of starch in the pericarp. The methodology also revealed that the defect in the cytosolic AGPase small subunit2b (AGPS2b) transcription from the AGPS2 gene in endosperm sharply enhanced the expressions of endosperm and leaf plastidial AGPS1, the endosperm cytosolic AGPase large subunit2 (AGPL2), and the leaf plastidial AGPL1. OsAPL1|OsAGPL3|OsAGPL3 Expression profiling of genes related to starch synthesis in rice leaf sheaths during the heading period 2006 Physiologia Plantarum National Agricultural Research Center, Joetsu, Niigata 943-0193, Japan The stems (leaf sheaths and culms) of rice plants (Oryza sativa L.) accumulate high levels of starch before heading, which is subsequently remobilized after heading to provide a carbon source for grain filling. To elucidate the molecular mechanism of starch synthesis in rice leaf sheath, a comprehensive expression analysis was conducted on the gene families encoding starch synthesis–related enzymes, ADP-glucose pyrophosphorylase (EC 2.7.7.27), starch synthase (EC 2.4.1.21) and branching enzyme (EC 2.4.1.18). The changes in the activities of these enzymes in leaf sheaths during the heading period were shown to be accompanied by a coordinated change in the transcript levels of particular members of the corresponding gene families. A similar change before and after heading was also found in the messenger RNA level of GPT2, one of the two genes encoding for a plastidial glucose-6-phosphate/phosphate translocator, suggesting that the capacity of both starch synthesis and its substrate import is under coordinated transcriptional regulation. The members of the gene families predominantly expressed in leaf sheaths are mostly different from those actively expressed in the developing endosperm. The time course for the transcript level of some of these genes appears to correlate to hexose level in the leaf sheaths, suggesting that hexose levels may be a controlling factor in their expression. OsAPL1|OsAGPL3|OsAGPL3 Gene expression of ADP-glucose pyrophosphorylase and starch contents in rice cultured cells are cooperatively regulated by sucrose and ABA 2005 Plant Cell Physiol Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba City, Ibaraki, 305-8572 Japan. Six cDNA clones encoding two small subunits and four large subunits of ADP-glucose pyrophosphorylase (AGPase) were mined from the database of rice full-length cDNAs, cloned and subsequently named: OsAPS1, OsAPS2, OsAPL1, OsAPL2, OsAPL3 and OsAPL4. Expression patterns of the six genes were examined by Northern blot analysis with gene-specific probes. OsAPL3 was predominantly expressed in the middle phases of seed development, and OsAPS1, OsAPL1 and OsAPL2 were expressed later in seed development. OsAPS2 and OsAPL4 were constitutively expressed and these isoforms were coordinated with starch accumulation in the developing rice seed. In order to clarify the effect of sugars and plant hormones on AGPase gene expression more precisely, a rice cell culture system was used. OsAPL3 transcript significantly accumulated in response to increased levels of sucrose and abscisic acid (ABA) concentration in the medium; however, the transcripts of other AGPase genes did not show significant accumulation. Under identical conditions, starch contents in the cultured cells also increased. Interestingly, ABA alone did not affect the gene expression of OsAPL3 and starch content. Collectively, these results indicated that the expression level of OsAPL3 and starch content in the cultured cells were cooperatively controlled by alterations in the concentration of both sucrose and ABA. OsAPL1|OsAGPL3|OsAGPL3,OsAPL2|osagpl2-3,OsAPL3,OsAPL4|OsAGPL4,OsAPS1|OsAGPS1,OsAPS2|OsAGPS2b Phenotypic and Candidate Gene Analysis of a New Floury Endosperm Mutant (osagpl2-3) in Rice 2012 Plant Molecular Biology Reporter Agronomy Department, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, 310058, China A floury endosperm mutant, osagpl2-3, was isolated from the M2 generation of japonica rice cultivar Nipponbare following ethyl methane sulfonate mutagenesis. The osagpl2-3 mutant produced a white-core endosperm compared to the transparent endosperm of the wild type (WT). The results from scanning electron microscope showed that the osagpl2-3 mutant grains comprised of round and loosely packed starch granules, some of which were compounded. The analysis for cooking and nutrition quality traits indicated that the values of gel consistency, gelatinization temperature, and rapid viscosity analysis profile of osagpl2-3 grains were lower than those of the WT. Besides, the protein content, the contents of nine different amino acids, and the thermodynamic parameters of T p and ΔT 1/2 in osagpl2-3 were also different from those of the WT. Genetic analysis revealed that osagpl2-3 mutation was controlled by a single recessive gene. The osagpl2-3 gene was mapped between InDel markers R1M30 and ID1-12 on rice chromosome 1. In the candidate region of the Nipponbare genome, an annotated gene, LOC_Os01g44220 which encodes a large subunit of putative ADP-glucose pyrophosphrylase named OsAPL2 was considered the optimal candidate. Cloning and sequencing of LOC_Os01g44220 in different plants of the osagpl2-3 mutants revealed a single nucleotide mutation (G→A) in the open reading frame region, which led to a substitution of an acidic amino acid Glu (E) by a basic amino acid Lys (K) accordingly. Furthermore, the mutant site is close to the functional domain which interacts with the ADP-Glc. In brief, these results suggested that the osagpl2-3 is a new mutant of OsAPL2. OsAPL2|osagpl2-3 SALT-RESPONSIVE ERF1 Is a Negative Regulator of Grain Filling and Gibberellin-Mediated Seedling Establishment in Rice 2014 Mol Plant Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany Grain quality is an important agricultural trait that is mainly determined by grain size and composition. Here, we characterize the role of the rice transcription factor (TF) SALT-RESPONSIVE ERF1 (SERF1) during grain development. Through genome-wide expression profiling and chromatin immunoprecipitation, we found that SERF1 directly regulates RICE PROLAMIN-BOX BINDING FACTOR (RPBF), a TF that functions as a positive regulator of grain filling. Loss of SERF1 enhances RPBF expression resulting in larger grains with increased starch content, while SERF1 overexpression represses RPBF resulting in smaller grains. Consistently, during grain filling, starch biosynthesis genes such as GRANULE-BOUND STARCH SYNTHASEI (GBSSI), STARCH SYNTHASEI (SSI), SSIIIa, and ADP-GLUCOSE PYROPHOSPHORYLASE LARGE SUBUNIT2 (AGPL2) are up-regulated in SERF1 knockout grains. Moreover, SERF1 is a direct upstream regulator of GBSSI. In addition, SERF1 negatively regulates germination by controlling RPBF expression, which mediates the gibberellic acid (GA)-induced expression of RICE AMYLASE1A (RAmy1A). Loss of SERF1 results in more rapid seedling establishment, while SERF1 overexpression has the opposite effect. Our study reveals that SERF1 represents a negative regulator of grain filling and seedling establishment by timing the expression of RPBF. OsAPL2|osagpl2-3,AmyI-1|RAmy1A,RPBF|OsDof3,SERF1 ABERRANT PANICLE ORGANIZATION 1 temporally regulates meristem identity in rice 2005 Dev Biol Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. We report a recessive mutation of rice, aberrant panicle organization 1 (apo1), which severely affects inflorescence architecture, floral organ identity, and leaf production rate. In the wild-type inflorescence, the main-axis meristem aborts after forming 10-12 primary branch primordia. However, in apo1, the main-axis meristem was converted to a spikelet meristem after producing a small number of branch primordia. In addition, the branch meristems in apo1 became spikelet meristems earlier than in wild type. Therefore, in the inflorescence, the apo1 mutation caused the precocious conversion of the meristem identity. In the apo1 flower, lodicules were increased at the expense of stamens, and carpels were formed indeterminately by the loss of meristem determinacy. Vegetative development is also affected in the apo1. Leaves were formed rapidly throughout the vegetative phase, indicating that APO1 is also involved in temporal regulation of leaf production. These phenotypes suggest that the APO1 plays an important role in the temporal regulation of both vegetative and reproductive development. APO1|OsAPO1|SCM2 A gene controlling the number of primary rachis branches also controls the vascular bundle formation and hence is responsible to increase the harvest index and grain yield in rice 2010 Theor Appl Genet Rice Physiology Research Sub-Team, Hokuriku Research Center, National Agricultural Research Center, National Agriculture and Food Research Organization, 1-2-1, Inada, Joetsu, Niigata, 943-0193, Japan. terao@affrc.go.jp The quantitative trait locus controlling the number of primary rachis branches (PRBs) in rice was identified using backcrossed inbred lines of Sasanishiki/Habataki//Sasanishiki///Sasanishiki. The resultant gene was ABERRANT PANICLE ORGANIZATION 1 (APO1). Habataki-genotype segregated reciprocal recombinant lines for the APO1 locus increased both the number of PRB (12-13%) and the number of grains per panicle (9-12%), which increased the grain yield per plant (5-7%). Further recombination dividing this region revealed that different alleles regulated the number of PRB and the number of grains per panicle. The PRB1 allele, which includes the APO1 open reading frame (ORF) and the proximal promoter region, controlled only the number of PRB but not the number of grains per panicle. In contrast, the HI1 allele, which includes only the distal promoter region, increased the grain yield and harvest index in Habataki-genotype plants, nevertheless, the ORF expressed was Sasanishiki type. It also increased the number of large vascular bundles in the peduncle. APO1 expression occurred not only in developing panicles but also in the developing vascular bundle systems. In addition, Habataki plants displayed increased APO1 expression in comparison to Sasanishiki plants. It suggests that APO1 enhances the formation of vascular bundle systems which, consequently, promote carbohydrate translocation to panicles. The HI1 allele is suggested to regulate the amount of APO1 expression, and thereby control the development of vascular bundle systems. These findings may be useful to improve grain yield as well as quality through the improvement of translocation efficiency. APO1|OsAPO1|SCM2 Rice ABERRANT PANICLE ORGANIZATION 1, encoding an F-box protein, regulates meristem fate 2007 Plant J Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. Inflorescence architecture is one of the most important agronomical traits. Characterization of rice aberrant panicle organization 1 (apo1) mutants revealed that APO1 positively controls spikelet number by suppressing the precocious conversion of inflorescence meristems to spikelet meristems. In addition, APO1 is associated with the regulation of the plastchron, floral organ identity, and floral determinacy. Phenotypic analyses of apo1 and floral homeotic double mutants demonstrate that APO1 positively regulates class-C floral homeotic genes, but not class-B genes. Molecular studies revealed that APO1 encodes an F-box protein, an ortholog of Arabidopsis UNUSUAL FLORAL ORGAN (UFO), which is a positive regulator of class-B genes. Overexpression of APO1 caused an increase in inflorescence branches and spikelets. As the mutant inflorescences and flowers differed considerably between apo1 and ufo, the functions of APO1 and UFO appear to have diverged during evolution. APO1|OsAPO1|SCM2 ABERRANT PANICLE ORGANIZATION 2/RFL, the rice ortholog of Arabidopsis LEAFY, suppresses the transition from inflorescence meristem to floral meristem through interaction with APO1 2012 Plant J Graduate School of Agriculture and Life Sciences, University of Tokyo, Yayoi, Bunkyo, Tokyo 113-0032, Japan. The temporal and spatial control of meristem identity is a key element in plant development. To better understand the molecular mechanisms that regulate inflorescence and flower architecture, we characterized the rice aberrant panicle organization 2 (apo2) mutant which exhibits small panicles with reduced number of primary branches due to the precocious formation of spikelet meristems. The apo2 mutants also display a shortened plastochron in the vegetative phase, late flowering, aberrant floral organ identities and loss of floral meristem determinacy. Map-based cloning revealed that APO2 is identical to previously reported RFL gene, the rice ortholog of the Arabidopsis LEAFY (LFY) gene. Further analysis indicated that APO2/RFL and APO1, the rice ortholog of Arabidopsis UNUSUAL FLORAL ORGANS, act cooperatively to control inflorescence and flower development. The present study revealed functional differences between APO2/RFL and LFY. In particular, APO2/RFL and LFY act oppositely on inflorescence development. Therefore, the genetic mechanisms for controlling inflorescence architecture have evolutionarily diverged between rice (monocots) and Arabidopsis (eudicots). APO1|OsAPO1|SCM2,RFL|APO2 New approach for rice improvement using a pleiotropic QTL gene for lodging resistance and yield 2010 Nat Commun Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan. The use of fertilizer results in tall rice plants that are susceptible to lodging and results in reduced plant yields. In this study, using chromosome segment substitution lines, we identified an effective quantitative trait loci (QTL) for culm strength, which was designated STRONG CULM2 (SCM2). Positional cloning of the gene revealed that SCM2 was identical to ABERRANT PANICLE ORGANIZATION1 (APO1), a gene previously reported to control panicle structure. A near-isogenic line carrying SCM2 showed enhanced culm strength and increased spikelet number because of the pleiotropic effects of the gene. Although SCM2 is a gain-of-function mutant of APO1, it does not have the negative effects reported for APO1 overexpression mutants, such as decreased panicle number and abnormal spikelet morphology. The identification of lodging-resistance genes by QTL analysis combined with positional cloning is a useful approach for improving lodging resistance and overall productivity in rice. APO1|OsAPO1|SCM2 Expression level of ABERRANT PANICLE ORGANIZATION1 determines rice inflorescence form through control of cell proliferation in the meristem 2009 Plant Physiol Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan. Two types of branches, rachis branches (i.e. nonfloral) and spikelets (i.e. floral), are produced during rice (Oryza sativa) inflorescence development. We previously reported that the ABERRANT PANICLE ORGANIZATION1 (APO1) gene, encoding an F-box-containing protein orthologous to Arabidopsis (Arabidopsis thaliana) UNUSUAL FLORAL ORGANS, suppresses precocious conversion of rachis branch meristems to spikelets to ensure generation of certain number of spikelets. Here, we identified four dominant mutants producing an increased number of spikelets and found that they are gain-of-function alleles of APO1. The APO1 expression levels are elevated in all four mutants, suggesting that an increase of APO1 activity caused the delay in the program shift to spikelet formation. In agreement with this result, ectopic overexpression of APO1 accentuated the APO1 gain-of-function phenotypes. In the apo1-D dominant alleles, the inflorescence meristem starts to increase in size more vigorously than the wild type when switching to the reproductive development phase. This alteration in growth rate is opposite to what is observed with the apo1 mutants that have a smaller inflorescence meristem. The difference in meristem size is caused by different rates of cell proliferation. Collectively, these results suggest that the level of APO1 activity regulates the inflorescence form through control of cell proliferation in the meristem. APO1|OsAPO1|SCM2 Mapping QTLs and candidate genes for iron and zinc concentrations in unpolished rice of MadhukarxSwarna RILs 2012 Gene Directorate of Rice Research, Hyderabad, India. Identifying QTLs/genes for iron and zinc in rice grains can help in biofortification programs. 168 F(7) RILs derived from MadhukarxSwarna were used to map QTLs for iron and zinc concentrations in unpolished rice grains. Iron ranged from 0.2 to 224 ppm and zinc ranged from 0.4 to 104ppm. Genome wide mapping using 101 SSRs and 9 gene specific markers showed 5 QTLs on chromosomes 1, 3, 5, 7 and 12 significantly linked to iron, zinc or both. In all, 14 QTLs were identified for these two traits. QTLs for iron were co-located with QTLs for zinc on chromosomes 7 and 12. In all, ten candidate genes known for iron and zinc homeostasis underlie 12 of the 14 QTLs. Another 6 candidate genes were close to QTLs on chromosomes 3, 5 and 7. Thus the high priority candidate genes for high Fe and Zn in seeds are OsYSL1 and OsMTP1 for iron, OsARD2, OsIRT1, OsNAS1, OsNAS2 for zinc and OsNAS3, OsNRAMP1, Heavy metal ion transport and APRT for both iron and zinc together based on our genetic mapping studies as these genes strictly underlie QTLs. Several elite lines with high Fe, high Zn and both were identified. OsAPT1|APRT,OsZIP6 Cloning and characterization of a second form of the rice adenine phosphoribosyl transferase gene (OsAPT2) and its association with TGMS 2006 Plant Mol Biol State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China. A rice gene, OsAPT2, which encodes a putative adenine phosphoribosyl transferase (APRT), was cloned and characterized. Analysis of the cDNA and genomic sequences revealed seven exons and six introns in the OsAPT2. The deduced amino acid sequence of OsAPT2 is highly homologous to those of previously isolated APRTs. RT-PCR analysis indicated that the OsAPT2 transcript in the young panicles of 'Annong S-1' is down-regulated at 29 degrees C, the critical temperature for induction of 'Annong S-1' fertility conversion. Since the panicle is likely the thermo-sensitive organ at the early stages of pollen fertility alternation, the observed heat-induced change in the OsAPT2 expression pattern in young panicles may mediate, at least in part, thermo-sensitive genic male sterility (TGMS) in 'Annong S-1'. An antisense strategy was used to suppress the expression of the OsAPT2 homolog in Arabidopsis, and the obtained homozygous transgenic plants contained lower AMP content, displayed lower pollen germination rates and exhibited some abnormalities in leaf phenotypes and flowering timing. These data suggest that OsAPT2 is likely to be involved in TGMS in the rice line 'Annong S-1'. OsAPT2 Involvement of hydrogen peroxide in heat shock- and cadmium-induced expression of ascorbate peroxidase and glutathione reductase in leaves of rice seedlings 2012 J Plant Physiol Department of Agronomy, National Taiwan University, Taipei, Taiwan, ROC. Hydrogen peroxide (H2O2) is considered a signal molecule inducing cellular stress. Both heat shock (HS) and Cd can increase H2O2 content. We investigated the involvement of H2O2 in HS- and Cd-mediated changes in the expression of ascorbate peroxidase (APX) and glutathione reductase (GR) in leaves of rice seedlings. HS treatment increased the content of H2O2 before it increased activities of APX and GR in rice leaves. Moreover, HS-induced H2O2 production and APX and GR activities could be counteracted by the NADPH oxidase inhibitors dipehenylene iodonium (DPI) and imidazole (IMD). HS-induced OsAPX2 gene expression was associated with HS-induced APX activity but was not regulated by H2O2. Cd-increased H2O2 content and APX and GR activities were lower with than without HS. Cd did not increase the expression of OsAPX and OsGR without HS treatment. Cd increased H2O2 content by Cd before it increased APX and GR activities without HS. Treatment with DPI and IMD effectively inhibited Cd-induced H2O2 production and APX and GR activities. Moreover, the effects of DPI and IMD could be rescued with H2O2 treatment. H2O2 may be involved in the regulation of HS- and Cd-increased APX and GR activities in leaves of rice seedlings. OsAPX1|APXa,OsAPX2|APXb,OsAPx8,OsGR1,OsGR2|RGRC2,OsGR3 Importance of ascorbate peroxidases OsAPX1 and OsAPX2 in the rice pathogen response pathways and growth and reproduction revealed by their transcriptional profiling 2003 Gene Research Laboratory for Agricultural Biotechnology and Biochemistry, GPO Box 8207, Kathmandu, Nepal. None OsAPX1|APXa,OsAPX2|APXb Enhanced chilling tolerance at the booting stage in rice by transgenic overexpression of the ascorbate peroxidase gene, OsAPXa 2011 Plant Cell Rep National Agricultural Research Center for Hokkaido Region, Hitsujigaoka 1, Toyohira, Sapporo 062-8555, Japan, yutaka@affrc.go.jp Low temperatures during the booting stage reduce rice yields by causing cold-induced male sterility. To determine whether antioxidant capacity affects the ability of rice plants to withstand chilling at the booting stage, we produced transgenic rice plants that overexpress OsAPXa and have increased APX activity. The effect of increased APX activity on the levels of H(2)O(2) and lipid peroxidation were determined by measuring H(2)O(2) levels and malondialdehyde (MDA) contents in spikelets during cold treatments at the booting stage. The levels of H(2)O(2) and the MDA content increased by 1.5-fold and twofold, respectively in WT plants subjected to a 12 degrees C treatment for 6 days. In contrast, transgenic lines showed small changes in H(2)O(2) levels and MDA content under cold stress, and H(2)O(2) levels and MDA content were significantly lower than in WT plants. APX activity showed negative correlations with levels of H(2)O(2) and MDA content, which increased during cold treatment. Cold tolerance at the booting stage in transgenic lines and WT plants was evaluated. Spikelet fertility was significantly higher in transgenic lines than in WT plants after a 12 degrees C treatment for 6 days. These results indicate that higher APX activity enhances H(2)O(2)-scavenging capacity and protects spikelets from lipid peroxidation, thereby increasing spikelet fertility under cold stress. OsAPX1|APXa Heat shock-mediated APX gene expression and protection against chilling injury in rice seedlings 2001 J Exp Bot Hokkaido National Agricultural Experiment Station, Hitsujigaoka 1, Toyohira-ku, Sapporo 062-8555, Japan. Rice (Oryza sativa L.) seedlings, when kept at 42 degrees C for 24 h before being kept at 5 degrees C for 7 d, did not develop chilling injury. Chilling resistance was enhanced in parallel with the period of heat-treatment. The level of APX activity was higher in heated seedlings whereas CAT activity was decreased by heat stress. There was no significant difference in SOD activity between heated and unheated seedlings. The elevated activity of APX was sustained after 7 d of chilling. The cytosolic APX gene expression in response to high and low temperature was analysed with an APXa gene probe. APXa mRNA levels increased within 1 h after seedlings were exposed to 42 degrees C. Elevated APXa mRNA levels could also be detected after 24 h of heating. The APXa mRNA level in preheated seedlings was still higher than unheated seedlings under cold stress. The promoter of the APXa gene was cloned from rice genomic DNA by TAIL-PCR, and characterized by DNA sequencing. The promoter had a minimal heat shock factor-binding motif, 5'-nGAAnnTTCn-3', located in the 81 bp upstream of the TATA box. Heat shock induction of the APXa gene could be a possible cause of reduced chilling injury in rice seedlings. OsAPX1|APXa Purification and characterization of two ascorbate peroxidases of rice (Oryza sativa L.) expressed in Escherichia coli 2005 Biotechnol Lett Alkali Soil Natural Environmental Science Center (ASNESC), Stress Molecular Biology Laboratory, Northeast Forestry University, 150040, Harbin, P.R. China. To clarify the diversity and function of isozymes of ascorbate peroxidase (APX) in plants, a method of producing large quantities of these proteins is needed. Here, we describe an Escherichia coli expression system for the rapid and economic expression of two rice APX genes, APXa and APXb (GeneBank accession Nos. D45423 and AB053297, respectively). The two genes were cloned into the pGEX-6p-3 vector to allow expression of APX as a glutathione-S-transferase (GST) fusion protein. The GST-APXa and GST-APXb fusion proteins were purified by affinity chromatography using a glutathione-Sepharose 4B column, with final yields of 40 and 73 mg g(-1) dry cells, respectively. Specific activities were 15 and 20 mM ascorbate min(-1) mg(-1) protein, respectively. The K(m) values for ascorbate were 4 and 1 mM, respectively, and those for H(2)O(2) were 0.3 and 0.7 mM, respectively indicating that the two rice isoenzymes have different properties. OsAPX1|APXa,OsAPX2|APXb Gene knockout study reveals that cytosolic ascorbate peroxidase 2(OsAPX2) plays a critical role in growth and reproduction in rice under drought, salt and cold stresses 2013 PLoS One Biotechnology Research Institute, Chinese Academy of Agricultural Sciences/National Key facility for Gene Resources And Genetic Improvement, Beijing, China. Plant ascorbate peroxidases (APXs), enzymes catalyzing the dismutation of H2O2 into H2O and O2, play an important role in reactive oxygen species homeostasis in plants. The rice genome has eight OsAPXs, but their physiological functions remain to be determined. In this report, we studied the function of OsAPX2 gene using a T-DNA knockout mutant under the treatment of drought, salt and cold stresses. The Osapx2 knockout mutant was isolated by a genetic screening of a rice T-DNA insertion library under 20% PEG-2000 treatment. Loss of function in OsAPX2 affected the growth and development of rice seedlings, resulting in semi-dwarf seedlings, yellow-green leaves, leaf lesion mimic and seed sterility. OsAPX2 expression was developmental- and spatial-regulated, and was induced by drought, salt, and cold stresses. Osapx2 mutants had lower APX activity and were sensitive to abiotic stresses; overexpression of OsAPX2 increased APX activity and enhanced stress tolerance. H2O2 and MDA levels were high in Osapx2 mutants but low in OsAPX2-OX transgenic lines relative to wild-type plants after stress treatments. Taken together, the cytosolic ascorbate peroxidase OsAPX2 plays an important role in rice growth and development by protecting the seedlings from abiotic stresses through scavenging reactive oxygen species. OsAPX2|APXb Genetic transformation and analysis of rice OsAPx2 gene in Medicago sativa 2012 PLoS One Key Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil Field (SAVER), Ministry of Education, Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry University, Harbin, China. The OsAPx2 gene from rice was cloned to produce PBI121::OsAPx2 dual-expression plants, of which expression level would be increasing under stressful conditions. The enzyme ascorbate peroxidase (APX) in the leaves and roots of the plants increased with increasing exposure time to different sodium chloride (NaCl) and hydrogen peroxide (H(2)O(2))concentrations, as indicated by protein gel blot analysis. The increased enzyme yield improved the ability of the plants to resist the stress treatments. The OsAPx2 gene was localized in the cytoplasm of epidermal onion cells as indicated by the instantaneous expression of green fluorescence. An 80% regeneration rate was observed in Medicago sativa L. plants transformed with the OsAPx2 gene using Agrobacterium tumefaciens, as indicated by specific primer PCR. The OsAPx2 gene was expressed at the mRNA level and the individual M. sativa (T#1,T#2,T#5) were obtained through assaying the generation of positive T2 using RNA gel blot analysis. When the seeds of the wild type (WT) and the T2 (T#1,T#5) were incubated in culture containing MS with NaCl for 7 days, the results as shown of following: the root length of transgenic plant was longer than WT plants, the H(2)O(2) content in roots of WT was more than of transgenic plants, the APX activity under stresses increased by 2.89 times compared with the WT, the malondialdehyde (MDA) content of the WT was higher than the transgenic plants, the leaves of the WT turned yellow, but those of the transgenic plants remained green and remained healthy. The chlorophyll content in the WT leaves was less than in the transgenic plants, after soaking in solutions of H(2)O(2), sodium sulfite (Na(2)SO(3)), and sodium bicarbonate (NaHCO(3)). Therefore, the OsAPx2 gene overexpression in transgenic M. sativa improves the removal of H(2)O(2) and the salt-resistance compared with WT plants. A novel strain of M. sativa carrying a salt-resistance gene was obtained. OsAPX2|APXb Expression of ASCORBATE PEROXIDASE 8 in roots of rice (Oryza sativa L.) seedlings in response to NaCl 2007 J Exp Bot Department of Agricultural Chemistry and Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, Republic of China. Reactive oxygen species are thought to play an important role in NaCl stress. Therefore, the expression patterns of the gene family encoding the H(2)O(2)-scavenging enzyme ascorbate peroxidase (APx; EC1.11.1.11) were analysed in roots of etiolated rice (Oryza sativa L.) seedlings in response to NaCl stress. Applying semi-quantitative RT-PCR, the mRNA levels were quantified for two cytosolic (OsAPx1 and OsAPx2), two peroxisomal (OsAPx3 and OsAPx4), and four chloroplastic (OsAPx5, OsAPx6, OsAPx7, and OsAPx8) isoforms identified in the rice genome. NaCl at 150 mM and 200 mM increased the expression of OsAPx8 and the activities of APx, but had no effect on the expression of OsAPx1, OsAPx2, OsAPx3, OsAPx4, OsAPx5, OsAPx6, and OsAPx7 in rice roots. However, NaCl at 300 mM up-regulated OsAPx8 expression, increased APx activity, and down-regulated OsAPx7 expression, but had no effect on the expression of OsAPx1, OsAPx2, OsAPx3, OsAPx4, OsAPx5, and OsAPx6. The accumulation of abscisic acid (ABA) in response to NaCl was observed in rice roots. Exogenously applied ABA also specifically enhanced the expression of OsAPx8 in rice roots. The accumulation of ABA in rice roots in response to NaCl was inhibited by fluridone (Flu), an inhibitor of carotenoid biosynthesis. Flu treatment also suppressed NaCl-enhanced OsAPx8 expression and APx activity. The effect of Flu on the expression of OsAPx8 and increase in APx activity was reversed by the application of ABA. It appears that NaCl-enhanced expression of OsAPx8 in rice roots is mediated through an accumulation of ABA. Evidence is provided to show that Na(+) but not Cl(-) is required for enhancing OsAPx8 expression, APx activity, and ABA accumulation in rice roots treated with NaCl. H(2)O(2) treatment resulted in an enhancement of OsAPx8 induction but no accumulation of ABA. Diphenylene iodonium treatment, which is known to inhibit NaCl-induced accumulation of H(2)O(2) in rice roots, did not suppress OsAPx8 induction and ABA accumulation by NaCl. It appears that H(2)O(2) is not involved in the regulation of NaCl-induced OsAPx8 expression in rice roots. OsAPx8 NaCl-induced expression of ASCORBATE PEROXIDASE 8 in roots of rice (Oryza sativaL.) seedlings is not associated with osmotic component 2008 Plant Signal Behav Department of Agricultural Chemistry and Institute of Biotechnology. Ascorbate peroxidase (APx; EC 1.11.1.11) plays an important role in scavenging the toxic effects of H2O2 in higher plants. Eight types of APx have been described for Oryza sativa: two cytosolic (OsAPx1 and OsAPx2), two putative peroxisomal (OsAPx3 and OsAPx4), and four chloroplastic isoforms (OsAPx5, OsAPx6, OsAPx7, and OsAPx8). We have recently demonstrated that Na+ but not Cl- is required for the NaCl-induced expression of OsAPx8 in rice roots. Evidence is also provided to show that Na+-induced expression of OsAPx8 is mediated through an accumulation of ABA. In addition to its known component of ion toxicity, there is an osmotic effect resulting from salt concentration in the soil. Here we show that ABA level but not OsAPx8 expression was enhanced at a concentration of mannitol iso-osmotic with 150 mM NaCl suggests that NaCl-enhanced OsAPx8 expression is not associated with osmotic component. OsAPx8 Increase in cellulose accumulation and improvement of saccharification by overexpression of arabinofuranosidase in rice 2013 PLoS One Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan. Cellulosic biomass is available for the production of biofuel, with saccharification of the cell wall being a key process. We investigated whether alteration of arabinoxylan, a major hemicellulose in monocots, causes an increase in saccharification efficiency. Arabinoxylans have beta-1,4-D-xylopyranosyl backbones and 1,3- or 1,4-alpha-l-arabinofuranosyl residues linked to O-2 and/or O-3 of xylopyranosyl residues as side chains. Arabinose side chains interrupt the hydrogen bond between arabinoxylan and cellulose and carry an ester-linked feruloyl substituent. Arabinose side chains are the base point for diferuloyl cross-links and lignification. We analyzed rice plants overexpressing arabinofuranosidase (ARAF) to study the role of arabinose residues in the cell wall and their effects on saccharification. Arabinose content in the cell wall of transgenic rice plants overexpressing individual ARAF full-length cDNA (OsARAF1-FOX and OsARAF3-FOX) decreased 25% and 20% compared to the control and the amount of glucose increased by 28.2% and 34.2%, respectively. We studied modifications of cell wall polysaccharides at the cellular level by comparing histochemical cellulose staining patterns and immunolocalization patterns using antibodies raised against alpha-(1,5)-linked l-Ara (LM6) and beta-(1,4)-linked d-Xyl (LM10 and LM11) residues. However, they showed no visible phenotype. Our results suggest that the balance between arabinoxylan and cellulose might maintain the cell wall network. Moreover, ARAF overexpression in rice effectively leads to an increase in cellulose accumulation and saccharification efficiency, which can be used to produce bioethanol. OsARAF1,OsARAF3 Identification and characterization of a novel water-deficit-suppressed gene OsARD encoding an aci-reductone-dioxygenase-like protein in rice 2005 Gene The State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, 310029, PR China. The aci-reductone dioxygenase (ARD) family common to bacteria, plants and animals is involved in the methionine salvage pathway. A water-deficit-suppressed gene, OsARD encoding an aci-reductone-dioxygenase-like protein, was identified from rice (Oryza sativa L.). Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the OsARD expression is regulated by abiotic stresses and phytohormones. OsARD was mainly expressed in roots under flood conditions. It was suppressed by abiotic stresses including water deficit, high salinity and low temperature, and induced by ethylene and gibberellin acid (GA). Our results showed that the genes for S-adenosylmethionine (SAM) synthase and 1-aminocyclopropane-1-carboxylic acid (ACC) synthase were upregulated in RNA-interference (RNAi) transgenic rice plants with a significant reduction of OsARD expression. Furthermore, the expression of two genes for ethylene signal transduction, ETR2 and EIN3, increased in these RNAi transgenic plants, whereas the expression of ERF3 was suppressed. These results suggest that OsARD may play a role in the metabolism of methionine and ethylene in response to abiotic stresses. OsARD|OsARD2,ETR2|Os-ERL1,OsEIL1|EIN3,AP37|OsERF3 The immediate-early ethylene response gene OsARD1 encodes an acireductone dioxygenase involved in recycling of the ethylene precursor S-adenosylmethionine 2005 Plant J Botanisches Institut, Universitat Kiel, Germany. msauter@bot.uni-kiel.de Methylthioadenosine (MTA) is formed as a by-product of ethylene biosynthesis from S-adenosyl-L-methionine (AdoMet). The methionine cycle regenerates AdoMet from MTA. In two independent differential screens for submergence-induced genes and for 1-aminocyclopropane-1-carboxylic acid (ACC)-induced genes from deepwater rice (Oryza sativa L.) we identified an acireductone dioxygenase (ARD). OsARD1 is a metal-binding protein that belongs to the cupin superfamily. Acireductone dioxygenases are unique proteins that can acquire two different activities depending on the metal ion bound. Ectopically expressed apo-OsARD1 preferentially binds Fe(2+) and reconstituted Fe-OsARD1 catalyzed the formation of 2-keto-pentanoate and formate from the model substrate 1,2-dihydroxy-3-ketopent-1-ene and dioxygen, indicating that OsARD1 is capable of catalyzing the penultimate step in the methionine cycle. Two highly homologous ARD genes were identified in rice. OsARD1 mRNA levels showed a rapid, early and transient increase upon submergence and after treatment with ethylene-releasing compounds. The second gene from rice, OsARD2, is constitutively expressed. Accumulation of OsARD1 transcript was observed in the same internodal tissues, i.e. the meristem and elongation zone, which were previously shown to synthesize ethylene. OsARD1 transcripts accumulated in the presence of cycloheximide, an inhibitor of protein synthesis, indicating that OsARD1 is a primary ethylene response gene. Promoter analysis suggests that immediate-early regulation of OsARD1 by ethylene may involve an EIN3-like transcription factor. OsARD1 is induced by low levels of ethylene. We propose that early feedback activation of the methionine cycle by low levels of ethylene ensures the high and continuous rates of ethylene synthesis required for long-term ethylene-mediated submergence adaptation without depleting the tissue of AdoMet. OsARD|OsARD2,OsARD1 OsMTN encodes a 5'-methylthioadenosine nucleosidase that is up-regulated during submergence-induced ethylene synthesis in rice (Oryza sativa L.) 2007 J Exp Bot Botanisches Institut, Universitat Kiel, Am Botanischen Garten 1-9, D-24118 Kiel, Germany. Methylthioadenosine (MTA) is released as a by-product of S-adenosylmethionine (AdoMet)-dependent reactions central to ethylene, polyamine, or phytosiderophore biosynthesis. MTA is hydrolysed by methylthioadenosine nucleosidase (MTN; EC 3.2.2.16) into adenine and methylthioribose which is processed through the methionine (Met) cycle to produce a new molecule of AdoMet. In deepwater rice, submergence enhances ethylene biosynthesis, and ethylene in turn influences the methionine cycle through positive feedback regulation of the acireductone dioxygenase gene OsARD1. In rice, MTN is encoded by a single gene designated OsMTN. Recombinant OsMTN enzyme had a KM for MTA of 2.1 mM and accepted a wide array of 5' substitutions of the substrate. OsMTN also metabolized S-adenosylhomocysteine (AdoHcy) with 15.9% the rate of MTA. OsMTN transcripts and OsMTN-specific activity increased slowly and in parallel upon submergence, indicating that regulation occurred mainly at the transcriptional level. Neither ethylene, MTA, nor Met regulated OsMTN expression. Analysis of steady-state metabolite levels showed that MTN activity was sufficiently high to prevent Met and AdoMet depletion during long-term ethylene biosynthesis. OsARD1,OsMTN OsAREB1, an ABRE-binding protein responding to ABA and glucose, has multiple functions in Arabidopsis 2010 BMB reports Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai, PR China. Expression patterns of OsAREB1 revealed that expression of OsAREB1 gene can be induced by ABA, PEG and heat. Yeast one-hybrid assay demonstrated it can bind to ABA-responsive element (ABRE), which was found in most stress-induced genes. Trdnsgenic Arabidopsis over-expressing OsAREB1 had different responses to ABA and glucose compared to wild-type plants, which suggest OsAREB1 might have a crucial role in these two signaling pathways. Further analysis indicate that OsAREB1 have multiple functions in Arabiclopsis. First, OsAREB1 transgenic plants had higher resistance to drought and heat, and OsAREB1 up-regulated the ABA/stress related gene such as RD29A and RD29B. Second, it delayed plant flowering time by clown-regulating the expression of flowering-related genes, such as FT, SOC1, LFY and AP1. Due to the dates, OsAREB1 may function as a positive regulator in drought/heat stresses response, but a negative regulator in flowering time in Arabiclopsis. [BMB reports 2010; 43(1): 34-39] OsAREB8|OsAREB1|OsbZIP46|OsABF2|ABL1 The ABRE-binding bZIP transcription factor OsABF2 is a positive regulator of abiotic stress and ABA signaling in rice 2010 J Plant Physiol Department of Bioscience and Biotechnology, University of Suwon, San 2-2 Wauri Bongdameup, Hwasung 445-743, Republic of Korea. Abscisic acid (ABA) is an important phytohormone involved in abiotic stress tolerance in plants. The group A bZIP transcription factors play important roles in the ABA signaling pathway in Arabidopsis but little is known about their functions in rice. In our current study, we have isolated and characterized a group A bZIP transcription factor in rice, OsABF2 (Oryza sativa ABA-responsive element binding factor 2). It was found to be expressed in various tissues in rice and induced by different types of abiotic stress treatments such as drought, salinity, cold, oxidative stress, and ABA. Subcellular localization analysis in maize protoplasts using a GFP fusion vector indicated that OsABF2 is a nuclear protein. In yeast experiments, OsABF2 was shown to bind to ABA-responsive elements (ABREs) and its N-terminal region found to be necessary to transactivate a downstream reporter gene. A homozygous T-DNA insertional mutant of OsABF2 is more sensitive to salinity, drought, and oxidative stress compared with wild type plants. In addition, this Osabf2 mutant showed a significantly decreased sensitivity to high levels of ABA at germination and post-germination. Collectively, our present results indicate that OsABF2 functions as a transcriptional regulator that modulates the expression of abiotic stress-responsive genes through an ABA-dependent pathway. OsAREB8|OsAREB1|OsbZIP46|OsABF2|ABL1 Identification of cis-acting promoter elements in cold- and dehydration-induced transcriptional pathways in Arabidopsis, rice, and soybean 2012 DNA Res Biological Resources and Post-harvest Division, Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki 305-8686, Japan. The genomes of three plants, Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and soybean (Glycine max), have been sequenced, and their many genes and promoters have been predicted. In Arabidopsis, cis-acting promoter elements involved in cold- and dehydration-responsive gene expression have been extensively analysed; however, the characteristics of such cis-acting promoter sequences in cold- and dehydration-inducible genes of rice and soybean remain to be clarified. In this study, we performed microarray analyses using the three species, and compared characteristics of identified cold- and dehydration-inducible genes. Transcription profiles of the cold- and dehydration-responsive genes were similar among these three species, showing representative upregulated (dehydrin/LEA) and downregulated (photosynthesis-related) genes. All (4(6) = 4096) hexamer sequences in the promoters of the three species were investigated, revealing the frequency of conserved sequences in cold- and dehydration-inducible promoters. A core sequence of the abscisic acid-responsive element (ABRE) was the most conserved in dehydration-inducible promoters of all three species, suggesting that transcriptional regulation for dehydration-inducible genes is similar among these three species, with the ABRE-dependent transcriptional pathway. In contrast, for cold-inducible promoters, the conserved hexamer sequences were diversified among these three species, suggesting the existence of diverse transcriptional regulatory pathways for cold-inducible genes among the species. OsAREB8|OsAREB1|OsbZIP46|OsABF2|ABL1 Purification, crystallization and preliminary X-ray analysis of OsAREB8 from rice, a member of the AREB/ABF family of bZIP transcription factors, in complex with its cognate DNA 2012 Acta Crystallogr Sect F Struct Biol Cryst Commun Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Tokyo 113-8657, Japan. The AREB/ABF family of bZIP transcription factors play a key role in drought stress response and tolerance during the vegetative stage in plants. To reveal the DNA-recognition mechanism of the AREB/ABF family of proteins, the bZIP domain of OsAREB8, an AREB/ABF-family protein from Oryza sativa, was expressed in Escherichia coli, purified and crystallized with its cognate DNA. Crystals of the OsAREB8-DNA complex were obtained by the sitting-drop vapour-diffusion method at 277 K with a reservoir solution consisting of 50 mM MES pH 6.4, 29% MPD, 2 mM spermidine, 20 mM magnesium acetate and 100 mM sodium chloride. A crystal diffracted X-rays to 3.65 A resolution and belonged to space group C222, with unit-cell parameters a = 155.1, b = 206.7, c = 38.5 A. The crystal contained one OsAREB8-DNA complex in the asymmetric unit. OsAREB8|OsAREB1|OsbZIP46|OsABF2|ABL1 The rice OsDIL gene plays a role in drought tolerance at vegetative and reproductive stages 2013 Plant Mol Biol State Key Laboratory of Genetic Engineering, Institute of Plant Biology, Center for Evolutionary Biology, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China. Drought is one of the critical factors limiting reproductive yields of rice and other crops globally. However, little is known about the molecular mechanism underlying reproductive development under drought stress in rice. To explore the potential gene function for improving rice reproductive development under drought, a drought induced gene, Oryza sativa Drought-Induced LTP (OsDIL) encoding a lipid transfer protein, was identified from our microarray data and selected for further study. OsDIL was primarily expressed in the anther and mainly responsive to abiotic stresses, including drought, cold, NaCl, and stress-related plant hormone abscisic acid (ABA). Compared with wild type, the OsDIL-overexpressing transgenic rice plants were more tolerant to drought stress during vegetative development and showed less severe tapetal defects and fewer defective anther sacs when treated with drought at the reproductive stage. The expression levels of the drought-responsive genes RD22, SODA1, bZIP46 and POD, as well as the ABA synthetic gene ZEP1 were up-regulated in the OsDIL-overexpression lines but the ABA degradation gene ABAOX3 was down-regulated. Moreover, overexpression of OsDIL lessened the down-regulation by drought of anther developmental genes (OsC4, CYP704B2 and OsCP1), providing a mechanism supporting pollen fertility under drought. Overexpression of OsDIL significantly enhanced drought resistance in transgenic rice during reproductive development, while showing no phenotypic changes or yield penalty under normal conditions. Therefore, OsDIL is an excellent candidate gene for genetic improvement of crop yield in adaption to unfavorable environments. OsAREB8|OsAREB1|OsbZIP46|OsABF2|ABL1,OsCP1,OsDIL|OsLTP6,ZEP1 A novel nuclear protein phosphatase 2C negatively regulated by ABL1 is involved in abiotic stress and panicle development in rice 2013 Mol Biotechnol Institute of Food Crops, Jiangsu Academy of Agricultural Sciences, Nanjing, China. Type 2C protein phosphatase plays an important role in the signal transduction of stress response in plants. In this paper, we identified a novel stress-induced type 2C protein phosphatase gene OsSIPP2C1 from rice. OsSIPP2C1 contains a complete open reading frame of 1,074 bp, encoding a protein with 357 amino acids. OsSIPP2C1 expression was up-regulated by high salt, PEG6000 and exogenous ABA, and enhanced in the abl1 mutant under normal, salt, or drought condition. Interestingly, OsSIPP2C1 expression was increased during the early panicle development. Subcellular localization assay using rice protoplast cells indicated that OsSIPP2C1 was predominantly located in the nucleus. Together, it is suggested that a nuclear PP2C protein OsSIPP2C1 negatively regulated by ABL1 is involved in abiotic stress and panicle development in rice. OsAREB8|OsAREB1|OsbZIP46|OsABF2|ABL1,OsSIPP2C1|OsPP2C68 Combinative effects of a bacterial type-III effector and a biocontrol bacterium on rice growth and disease resistance 2006 J Biosci Key Laboratory of Monitoring and Management of Plant Pathogens and Insect Pests, Ministry of Agriculture of China, and Department of Plant Pathology, Nanjing Agricultural University, 1 Weigang, Nanjing, 210095, China. Expression of HpaG(Xoo), a bacterial type-III effector, in transgenic plants induces disease resistance. Resistance also can be elicited by biocontrol bacteria. In both cases, plant growth is often promoted. Here we address whether biocontrol bacteria and HpaG(Xoo) can act together to provide better results in crop improvement. We studied effects of Pseudomonas cepacia on the rice variety R109 and the hpaG(Xoo)-expressing rice line HER1. Compared to R109, HER1 showed increased growth, grain yield, and defense responses toward diseases and salinity stress. Colonization of roots by P. cepacia caused 20% and 13% increase, in contrast to controls, in root growth of R109 and HER1. Growth of leaves and stems also increased in R109 but that of HER1 was inhibited. When P. cepacia colonization was subsequent to plant inoculation with Rhizoctonia solani, a pathogen that causes sheath blight, the disease was less severe than controls in both R109 and HER1; HER1, nevertheless, was more resistant, suggesting that P. cepacia and HpaG(Xoo) cooperate in inducing disease resistance. Several genes that critically regulate growth and defense behaved differentially in HER1 and R109 while responding to P. cepacia. In R109 leaves, the OsARF1 gene, which regulates plant growth, was expressed in consistence with growth promotion by P. cepacia. Inversely, OsARF1 expression was coincident with inhibition in growth of HER1 leaves. In both plants, the expression of OsEXP1, which encodes an expansin protein involved in plant growth,was concomitant with growth promotion in leaves instead of roots,in response to P. cepacia . We also studied OsMAPK, a gene that encodes a mitogen-activated protein kinase and controls defense responses toward salinity and infection by pathogens in rice. In response to P. cepacia, an early expression of OsMAPK was coincident with R109 resistance to the disease, while HER1 expressed the gene similarly whether P. cepacia was present or not. Evidently, P. cepacia and G(Xoo)-gene mediated resistance may act differently in rice growth and resistance. Whereas combinative effects of P. cepacia and HpaG(Xoo) in disease resistance have a great potential in agricultural use, it is interesting to study mechanisms that underlie interactions involving biocontrol bacteria, type-III effectors and pathogens. OsARF1,OsEXP1,OsLP|PR-5 Antisense phenotypes reveal a functional expression of OsARF1, an auxin response factor, in transgenic rice 2009 Curr Issues Mol Biol Rice Biotechnology Lab., Rice Research and Training Center, Sakha, Kafr EL-Sheikh, 33717, Egypt. OsARF1 is the first full-length member of auxin response factor (ARF) gene family to be cloned from monocot plant. Using quantitative RT-PCR this study found that, the transcript abundance of OsARF1 was significantly higher in embryonic tissues than in vegetative tissues. To investigate the effect of OsARF1 on the phenotype of rice, a cDNA fragment of OsARF1 was inserted in inverse orientation to the 35S promoter in vector pBin438 to produce an antisense (AS) construction. The AS-OsARF1 construct was transferred into rice (Oryza sativa L. japonica) calli via Agrobacterium tumefaciens-mediated transformation. Molecular analysis of transgenic plants showed that the functional expression of OsARF1 was inhibited at mRNA level efficiently. The AS-OsARF1 plants showed extremely low growth, poor vigor, short curled leaves and tillered but were sterile. Therefore, the OsARF1 was shown to be essential for growth in vegetative organs and seed development. OsARF1 OsARF1, an auxin response factor from rice, is auxin-regulated and classifies as a primary auxin responsive gene 2002 Plant Mol Biol Institut für Biologie II, Albert-Ludwigs-Universität, Freiburg. We screened for auxin-induced genes with an expression correlated to the auxin-induced growth response from rice coleoptiles by fluorescent differential display. A rice homologue of the auxin response factor (ARF) family of transcriptional regulators, OsARF1, was identified. An OsARF1:GFP fusion protein was localized to the nucleus. Steady-state levels of OsARF1 mRNA correlated positively with auxin-dependent differential growth: gravitropic stimulation enhanced the amount of OsARF1 transcript in the lower, faster-growing flank accompanied by a decrease in the upper flank of gravitropically stimulated rice coleoptiles. Exogenous auxin up-regulated the steady-state level of OsARF1 mRNA within 15-30 min. This up-regulation is independent of de novo protein synthesis. Thus, OsARF1 is the first ARF that classifies as an early auxin-responsive gene. The observed auxin-dependent regulation comprises a new level of regulation in auxin-induced gene expression and is discussed as a possible feedback mechanism in plant growth control. OsARF1 OsARF12, a transcription activator on auxin response gene, regulates root elongation and affects iron accumulation in rice (Oryza sativa) 2012 New Phytol State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China. * Auxin has an important role in maintaining optimal root system architecture (RSA) that can cope with growth reductions of crops caused by water or nutrient shortages. However, the mechanism of controlling RSA remains largely unclear. Here, we found a limiting factor of RSA--OsARF12--an auxin response factor whose knockout led to decreased primary root length in rice (Oryza sativa). * OsARF12 as a transcription activator can facilitate the expression of the auxin response element DR5::GFP, and OsARF12 was inhibited by osa-miRNA167d by transient expression in tobacco and rice callus. * The root elongation zones of osarf12 and osarf12/25, which had lower auxin concentrations, were distinctly shorter than for the wild-type, possibly as a result of decreased expression of auxin synthesis genes OsYUCCAs and auxin efflux carriers OsPINs and OsPGPs. The knockout of OsARF12 also altered the abundance of mitochondrial iron-regulated (OsMIR), iron (Fe)-regulated transporter1 (OsIRT1) and short postembryonic root1 (OsSPR1) in roots of rice, and resulted in lower Fe content. * The data provide evidence for the biological function of OsARF12, which is implicated in regulating root elongation. Our investigation contributes a novel insight for uncovering regulation of RSA and the relationship between auxin response and Fe acquisition. OsARF12,OsARF25,OsIRT1,Osspr1 Auxin response factor (OsARF12), a novel regulator for phosphate homeostasis in rice (Oryza sativa) 2014 New Phytol State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, 310058, China. Phosphorus (P) is crucial nutrient element for crop growth and development. However, the network pathway regulating homeostasis of phosphate (Pi) in crops has many molecular breeding unknowns. Here, we report that an auxin response factor, OsARF12, functions in Pi homeostasis. Measurement of element content, quantitative reverse transcription polymerase chain reaction analysis and acid phosphatases (APases) activity assay showed that the osarf12 mutant and osarf12/25 double mutant with P-intoxicated phenotypes had higher P concentrations, up-regulation of the Pi transporter encoding genes and increased APase activity under Pi-sufficient/-deficient (+Pi/-Pi, 0.32/0 mM NaH2 PO4) conditions. Transcript analysis revealed that Pi-responsive genes--Phosphate starvation (OsIPS)1 and OsIPS2, SYG1/Pho81/XPR1(OsSPX1), Sulfoquinovosyldiacylglycerol 2 (OsSQD2), R2R3 MYB transcription factor (OsMYB2P-1) and Transport Inhibitor Response1 (OsTIR1)--were more abundant in the osarf12 and osarf12/25 mutants under +Pi/-Pi conditions. Knockout of OsARF12 also influenced the transcript abundances of the OsPHR2 gene and its downstream components, such as OsMiR399j, OsPHO2, OsMiR827, OsSPX-MFS1 and OsSPX-MFS2. Results from -Pi/1-naphthylphthalamic acid (NPA) treatments, and auxin reporter DR5::GUS staining suggest that root system alteration and Pi-induced auxin response were at least partially controlled by OsARF12. These findings enrich our understanding of the biological functions of OsARF12, which also acts in regulating Pi homeostasis. OsARF12,OsIPS1,LTN1|OsPHO2,OsSPX-MFS1|OsPSS1,OsSPX-MFS2,OsSPX1,OsTIR1 OsARF16, a transcription factor, is required for auxin and phosphate starvation response in rice (Oryza sativa L.) 2013 Plant Cell Environ State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China. Plant responses to auxin and phosphate (Pi) starvation are closely linked. However, the underlying mechanisms connecting auxin to phosphate starvation (-Pi) responses are largely unclear. Here, we show that OsARF16, an auxin response factor, functions in both auxin and -Pi responses in rice (Oryza sativa L.). The knockout of OsARF16 led to primary roots (PR), lateral roots (LR) and root hair losing sensitivity to auxin and -Pi response. OsARF16 expression and OsARF16::GUS staining in PR and LR of rice Nipponbare (NIP) were induced by indole acetic acid and -Pi treatments. In -Pi conditions, the shoot biomass of osarf16 was slightly reduced, and neither root growth nor iron content was induced, indicating that the knockout of OsARF16 led to loss of response to Pi deficiency in rice. Six phosphate starvation-induced genes (PSIs) were less induced by -Pi in osarf16 and these trends were similar to a knockdown mutant of OsPHR2 or AtPHR1, which was a key regulator under -Pi. These data first reveal the biological function of OsARF16, provide novel evidence of a linkage between auxin and -Pi responses and facilitate the development of new strategies for the efficient utilization of Pi in rice. OsARF16,OsPHR2 OsARG encodes an arginase that plays critical roles in panicle development and grain production in rice 2012 The Plant Journal National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, China. Nitrogen is a crucial nutrient for plant growth and development. Arginine is considered an important amino acid for nitrogen transport and storage, playing a crucial role during plant seedling development. However, little is known about the role of arginine in nitrogen remobilization at the reproductive stage. We isolated a rice mutant with reduced plant height, small panicle and grain size, and low seed-setting rate (10% in nglf-1 compared to 93% in wild-type). Map-based cloning revealed that the mutant was caused by the loss of function of a gene (OsARG) encoding an arginine hydrolysis enzyme, which is consistent with arginine accumulation in the mutant. The phenotype was partially corrected supplying exogenous nitrogen, and fully corrected by a wild type OsARG transgene. Overexpression of OsARG in rice (var. Kitaake) increased grain number per plant under nitrogen-limited conditions. OsARG, ubiquitously expressed in various organs, was more strongly expressed in the developing panicles. The OsARG protein was localized in the mitochondria, consistent with other arginases. Our results suggest that the arginase encoded by OsARG, a key enzyme in Arg catabolism, plays a critical role during panicle development, especially under conditions of insufficient exogenous nitrogen. OsARG is a potential target for crop improvement. OsARG Overexpression of a new rice vacuolar antiporter regulating protein OsARP improves salt tolerance in tobacco 2008 Plant Cell Physiol Laboratory of Plant Biotechnology, The United Graduate School of Agricultural Sciences, Tottori University, Koyama-cho, Minami 4-101, Tottori 680-8553, Japan. We examined the function of the rice (Oryza sativa L.) antiporter-regulating protein OsARP by overexpressing it in tobacco (Nicotiana tabacum L.). In public databases, this protein was annotated as a putative Os02g0465900 protein of rice. The OsARP gene was introduced into tobacco under the control of the cauliflower mosaic virus 35S promoter. The transformants were selected for their ability to grow on medium containing kanamycin. Incorporation of the transgene in the genome of tobacco was confirmed by PCR, and its expression was confirmed by Western blot analysis. Transgenic plants had better growth and vigor than non-transgenic plants under salt stress in vitro. Overexpression of OsARP in transgenic tobacco plants resulted in salt tolerance, and the plants had a higher rate of photosynthesis and effective PSII photon yield when compared with the wild type. The OsARP protein was localized in the tonoplast of rice plants. Transgenic plants accumulated more Na+ in their leaf tissue than did wild-type plants. It is conceivable that the toxic effect of Na+ in the cytosol might be reduced by sequestration into vacuoles. The rate of water loss was higher in the wild type than in transgenic plants under salt stress. Increased vacuolar solute accumulation and water retention could confer salt tolerance in transgenic plants. Tonoplast vesicles isolated from OsARP transgenic plants showed Na+/H+ exchange rates 3-fold higher than those of wild-type plants. These results suggest that OsARP on the tonoplasts plays an important role in compartmentation of Na+ into vacuoles. We suggest that OsARP is a new type of protein participating in Na+ uptake in vacuoles. OsARP|OsCTP The rice nucellin gene ortholog OsAsp1 encodes an active aspartic protease without a plant-specific insert and is strongly expressed in early embryo 2005 Plant Cell Physiol Plant Breeding, Genetics and Biochemistry Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines. dbsbixz@nus.edu.sg The barley nucellin gene was reported to be nucellus specific in its expression and was hypothesized to play a role in the programmed cell death of the nucellus as an aspartic protease. Here we provide direct evidence that the rice ortholog encodes an active aspartic protease, but we prefer the name aspartic protease1 (OsAsp1) to nucellin after a detailed analysis of its expression pattern in rice and barley. Northern blots, RT-PCR and RNA in situ hybridization showed that OsAsp1 is expressed most abundantly in zygotic embryos 1-2 d after fertilization. It is also expressed in pollen, nucellus, ovary wall, shoot and root meristem, coleoptiles of immature seeds, and somatic embryos. A parallel study in barley showed that the barley nucellin gene was expressed not only in the nucellus but also strongly in embryos. Recombinant protein proOsAsp1 expressed in the bacterium Escherichia coli refolded and autolysed at acidic pH 3.5 in vitro, and the mature peptide displayed protease activity. Nucellin has three close homologs in rice on chromosomes 11 and 12 and in Arabidopsis on chromosomes 1 and 4. They lack the plant-specific insert that distinguishes the typical plant aspartic protease from aspartic proteases of other organisms. They constitute a new class of aspartic protease that is present in both monocots and dicots but whose function remains to be explored further. OsAsp1 Isolation and molecular characterization of a Spotted leaf 18 mutant by modified activation-tagging in rice 2007 Plant Mol Biol National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan. morimasa@nias.affrc.go.jp A lesion mimic mutant that we designated Spotted leaf 18 (Spl18) was isolated from 13,000 activation-tagging lines of rice produced by our modified activation-tagging vector and further characterized. Spl18 was dominant and its phenotype was linked to the T-DNA insertion. An ORF was located about 500 bp downstream of the inserted T-DNA, and the deduced protein, designated OsAT1, showed sequence similarity to an acyltransferase whose expression is induced by hypersensitive reaction in tobacco. The transcriptional level of OsAT1 was very low in the WT leaf blade but high in Spl18 leaf blade. In wild-type rice, OsAT1 was transcribed mainly in the young panicle, in the panicle just after heading, and in the leaf sheath. In addition, transcription of the genes for PR protein was upregulated in Spl18, accumulation of phytoalexins (both momilactone A and sakuranetin) was increased, and resistance to blast disease was improved. We then combined OsAT1 genomic DNA downstream of the modified 35S promoter and re-transformed it into rice. Lesion mimic and blast resistance phenotypes were detected in the transgenic lines produced, clearly indicating that overexpression of OsAT1 caused the Spl18 phenotypes. In addition, plants overexpressing OsAT1 showed resistance to bacterial blight. OsAT1 Overexpression of a BAHD acyltransferase, OsAt10, alters rice cell wall hydroxycinnamic acid content and saccharification 2013 Plant Physiol Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma 73019, USA. lbartley@ou.edu Grass cell wall properties influence food, feed, and biofuel feedstock usage efficiency. The glucuronoarabinoxylan of grass cell walls is esterified with the phenylpropanoid-derived hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA). Feruloyl esters undergo oxidative coupling with neighboring phenylpropanoids on glucuronoarabinoxylan and lignin. Examination of rice (Oryza sativa) mutants in a grass-expanded and -diverged clade of BAHD acyl-coenzyme A-utilizing transferases identified four mutants with altered cell wall FA or p-CA contents. Here, we report on the effects of overexpressing one of these genes, OsAt10 (LOC_Os06g39390), in rice. An activation-tagged line, OsAT10-D1, shows a 60% reduction in matrix polysaccharide-bound FA and an approximately 300% increase in p-CA in young leaf tissue but no discernible phenotypic alterations in vegetative development, lignin content, or lignin composition. Two additional independent OsAt10 overexpression lines show similar changes in FA and p-CA content. Cell wall fractionation and liquid chromatography-mass spectrometry experiments isolate the cell wall alterations in the mutant to ester conjugates of a five-carbon sugar with p-CA and FA. These results suggest that OsAT10 is a p-coumaroyl coenzyme A transferase involved in glucuronoarabinoxylan modification. Biomass from OsAT10-D1 exhibits a 20% to 40% increase in saccharification yield depending on the assay. Thus, OsAt10 is an attractive target for improving grass cell wall quality for fuel and animal feed. OsAt10 OsATG10b, an autophagosome component, is needed for cell survival against oxidative stresses in rice 2009 Mol Cells National Research Laboratory of Plant Functional Genomics, POSTECH Biotech Center, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Korea. Autophagy degrades toxic materials and old organelles, and recycles nutrients in eukaryotic cells. Whereas the studies on autophagy have been reported in other eukaryotic cells, its functioning in plants has not been well elucidated. We analyzed the roles of OsATG10 genes, which are autophagy-related. Two rice ATG10 genes - OsATG10a and OsATG10b - share significant sequence homology (about 75%), and were ubiquitously expressed in all organs examined here. GUS assay indicated that OsATG10b was highly expressed in the mesophyll cells and vascular tissue of younger leaves, but its level of expression decreased in older leaves. We identified T-DNA insertional mutants in that gene. Those osatg10b mutants were sensitive to treatments with high salt and methyl viologen (MV). Monodansylcadaverine-staining experiments showed that the number of autophagosomes was significantly decreased in the mutants compared with the WT. Furthermore, the amount of oxidized proteins increased in MV-treated mutant seedlings. These results demonstrate that OsATG10b plays an important role in the survival of rice cells against oxidative stresses. OsATG10b Identification and characterization of two rice autophagy associated genes, OsAtg8 and OsAtg4 2006 Mol Biol Rep Institute of Genetics, State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai, 200433, China. Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components. The molecular machinery responsible for yeast and mammalian autophagy has begun to be elucidated at the cellular level. A genome-wide search revealed significant conservation among autophagy genes in yeast and Arabidopsis. Up till now, however, there is no report about rice autophagy associated genes. Here we cloned OsAtg8 and OsAtg4 from Oryza sativa and detected their expression patterns in various tissues. Immunoblotting analysis showed that carboxyl terminus of OsAtg8 can be cleaved in yeast cell. Mutation analysis revealed that the conserved Gly117 residue of OsAtg8 was essential for its characteristic C-terminal cleavage as similar to that found in mammalian and yeast Atg8. We further proved that OsAtg8 interacted with OsAtg4, and this interaction was not affected by the conserved Gly117 mutation. Our results demonstrate that Atg8 conjugation pathway is conserved in rice and may play important roles in rice autophagy. OsAtg4,OsAtg8 Characterization of a novel rice gene OsATX and modulation of its expression by components of the stress signalling pathways 2002 Physiol Plant Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), GPO Box 8207, Kathmandu, Nepal Japan Science and Technology Corporation (JST), Japan Department of Molecular Biology, College of Natural Science, Sejong University, Seoul 143-747, Korea. In our search to identify gene(s) involved in the rice self-defense responses, we cloned a novel rice (Oryza sativa L. cv. Nipponbare) gene, OsATX, a single copy gene, from the JA treated rice seedling leaves cDNA library. This gene encodes a 69 amino acid polypeptide with a predicted molecular mass of 7649.7 and a pI of 5.6. OsATX was responsive to cutting (wounding by cutting the excised leaf), over its weak constitutive expression in the healthy leaves. The critical signalling molecules, jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), and hydrogen peroxide, together with protein phosphatase inhibitors, effectively up-regulated the OsATX expression with time, over the excised leaf cut control, whereas ethylene had no affect. Furthermore, copper, a heavy metal, also up-regulated OsATX expression. Moreover, induced expression of OsATX mRNA was influenced by light signal(s), and showed a requirement for de novo synthesized protein factors. Additionally, co-application of either JA or ABA with SA drastically suppressed the induced OsATX mRNA level. Finally, the blast pathogen, Magnaporthe grisea, triggered OsATX mRNA accumulation. These results strongly suggest a function/role(s) for OsATX in defense/stress responses in rice. OsATX OsBADH1 is possibly involved in acetaldehyde oxidation in rice plant peroxisomes 2009 FEBS Lett Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan. Although rice (Oryza sativa L.) produces little glycine betaine (GB), it has two betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8) gene homologs (OsBADH1 and OsBADH2). We found that OsBADH1 catalyzes the oxidation of acetaldehyde efficiently, while the activity of OsBADH2 is extremely low. The accumulation of OsBADH1 mRNA decreases following submergence treatment, but quickly recovers after re-aeration. We confirmed that OsBADH1 localizes in peroxisomes. In this paper, a possible physiological function of OsBADH1 in the oxidation of acetaldehyde produced by catalase in rice plant peroxisomes is discussed. BAD1|OsBADH1,OsBADH2|fgr Dissecting substrate specificity of two rice BADH isoforms: Enzyme kinetics, docking and molecular dynamics simulation studies 2012 Biochimie Departmant of Biochemistry, Faculty of Science, Kasetsart University, Chatuchak, Bangkok 10900, Thailand. Fragrance rice (Oryza sativa) contains two isoforms of BADH, named OsBADH1 and OsBADH2. OsBADH1 is implicated in acetaldehyde oxidation in rice plant peroxisomes, while the non-functional OsBADH2 is believed to be involved in the accumulation of 2-acetyl-1-pyrroline, the major compound of aroma in fragrance rice. In the present study, site-directed mutagenesis, molecular docking and molecular dynamics simulation studies were used to investigate the substrate specificity towards Bet-ald and GAB-ald. Consistent with our previous study, kinetics data indicated that the enzymes catalyze the oxidation of GAB-ald more efficiently than Bet-ald and the OsBADH1 W172F and OsBADH2 W170F mutants displayed a higher catalytic efficiency towards GAB-ald. Molecular docking analysis and molecular dynamics simulations for the first time provided models for aldehyde substrate-bound complexes of OsBADHs. The amino acid residues, E262, L263, C296 and W461 of OsBADH1 and E260, L261, C294 and W459 of OsBADH2 located within 5 A of the OsBADH active site mainly interacted with GAB-ald forming strong hydrogen bonds in both OsBADH isoforms. Residues W163, N164, Q294, C296 and F397 of OsBADH1-Bet-ald and Y163, M167, W170, E260, S295 and C453 of OsBADH2-Bet-ald formed the main interaction sites while E260 showed an interaction energy of -14.21 kcal/mol. Unconserved A290 in OsBADH1 and W288 in OsBADH2 appeared to be important for substrate recognition similar to that observed in PsAMADHs. Overall, the results here help to explain how two homologous rice BADHs recognize the aldehyde substrate differently, a key property to their biological role. BAD1|OsBADH1,OsBADH2|fgr Genetic manipulation of Japonica rice using the OsBADH1 gene from Indica rice to improve salinity tolerance 2010 Plant Cell, Tissue and Organ Culture (PCTOC) Laboratory of Plant Cell Technology, Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo City, Chiba, 271-8510, Japan Betaine aldehyde dehydrogenase (BADH) is a major oxidative enzyme that converts betaine aldehyde to glycine betaine (GB), an osmoprotectant compound in plants. Japonica rice (salt-sensitive) was genetically engineered to enhance salt tolerance by introducing the OsBADH1 gene from Indica rice (salt-tolerant), which is a GB accumulator. We produced transgenic rice plants overexpressing the modified OsBADH1 gene under the control of the maize ubiquitin promoter. The transgenic rice showed increased OsBADH1 gene expression and OsBADH1 enzyme production, resulting in the accumulation of GB. It also exhibited enhanced salt tolerance in immature and mature transgenic rice seedlings. The adverse effect of salt stress on seed germination, the growth of immature and mature seedlings, water status, and photosynthetic pigments was alleviated in transgenic seedlings. BAD1|OsBADH1 Expression of OsBADH1 gene in Indica rice (Oryza sativaL.) in correlation with salt, plasmolysis, temperature and light stresses 2011 Plant Omics Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand The relationship between environmental factors, salt tolerant and the expression of betaine aldehyde dehydrogenase (BADH) gene, salt stress related gene, was investigated in Indica rice. The expression was observed in various rice cultivars as well as under different environmental conditions. Northern blot analysis revealed that salt-tolerant in each rice cultivar is correlated to the expression level of OsBADH1 mRNA. The expression studies showed that OsBADH1 can be induced by a variety of environmental factors such as salinity, drought, cold, heat, light intensity and CO2 concentration. The results demonstrated that the OsBADH1 mRNA expression was up-regulated by salinity, drought, cold and high light intensity but down-regulated by CO2 enrichment and heat stress. The primary response of OsBADH1 gene expression was induced within 24 h after salinity, cold or drought stress treatment. Moreover, these results suggest that the expression of OsBADH1 gene in response to salt stress could be magnified under high light conditions. Interestingly, the effect of salt stress on the expression of OsBADH1 gene was alleviated by CO2 enrichment. This report showed that BADH1 gene not only plays role in the response of indica rice to salt stress but also to plasmolysis, temperature and light stresses. Therefore, BADH1 gene might involve in multifunctional mechanisms in response to environmental stresses of indica rice. BAD1|OsBADH1 Inactivation of an aminoaldehyde dehydrogenase is responsible for fragrance in rice 2008 Plant Mol Biol Centre for Plant Conservation Genetics, Southern Cross University, Military Road, Lismore, NSW 2480, Australia. Rice (Oryza sativa) has two betaine aldehyde dehydrogenase homologs, BAD1 and BAD2, encoded on chromosome four and chromosome eight respectively. BAD2 is responsible for the characteristic aroma of fragrant rice. Complementary DNA clones of both BAD1 and BAD2 were isolated and expressed in E. coli. BAD2 had optimum activity at pH 10, little to no affinity towards N-acetyl-gamma-aminobutyraldehyde (NAGABald) with a Km of approximately 10 mM and moderate affinity towards gamma-guanidinobutyraldehyde (GGBald) and betaine aldehyde (bet-ald) with Km values of approximately 260 microM and 63 microM respectively. A lower Km of approximately 9 microM was observed with gamma-aminobutyraldehyde (GABald), suggesting BAD2 has a higher affinity towards this substate in vivo. The enzyme encoded on chromosome four, BAD1, had optimum activity at pH 9.5, showed little to no affinity towards bet-ald with a Km of 3 mM and had moderate affinity towards GGBald, NAGABald and GABald with Km values of approximately 545, 420 and 497 microM respectively. BAD1 had a half life roughly double that of BAD2. We discuss the implications of these findings on the pathway of fragrance generation in Basmati and Jasmine rice and the potential of rice to accumulate the osmoprotectant glycine betaine. BAD1|OsBADH1,OsBADH2|fgr Expression of Indica rice OsBADH1 gene under salinity stress in transgenic tobacco 2009 Plant Biotechnology Reports Laboratory of Plant Cell Technology, Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo City, Chiba, 271-8510, Japan Glycine betaine has been reported as an osmoprotectant compound conferring tolerance to salinity and osmotic stresses in plants. We previously found that the expression of betaine aldehyde dehydrogenase 1 gene (OsBADH1), encoding a key enzyme for glycine betaine biosynthesis pathway, showed close correlation with salt tolerance of rice. In this study, the expression of the OsBADH1 gene in transgenic tobacco was investigated in response to salt stress using a transgenic approach. Transgenic tobacco plants expressing the OsBADH1 gene were generated under the control of a promoter from the maize ubiquitin gene. Three homozygous lines of T2 progenies with single transgene insert were chosen for gene expression analysis. RT-PCR and western blot analysis results indicated that the OsBADH1 gene was effectively expressed in transgenic tobacco leading to the accumulation of glycine betaine. Transgenic lines demonstrated normal seed germination and morphology, and normal growth rates of seedlings under salt stress conditions. These results suggest that the OsBADH1 gene could be an excellent candidate for producing plants with osmotic stress tolerance. BAD1|OsBADH1 Aroma in rice: genetic analysis of a quantitative trait 1996 Theor Appl Genet ORSTOM-LRGAPT, BP 5045 34032, Montpellier Cedex, 1, France. A new approach was developed which succeeded in tagging for the first time a major gene and two QTLs controlling grain aroma in rice. It involved a combination of two techniques, quantification of volatile compounds in the cooking water by gas chromatography, and molecular marker mapping. Four types of molecular marker were used (RFLPs, RAPDs, STSs, isozymes). Evaluation and mapping were performed on a doubled haploid line population which (1) conferred a precise character evaluation by enabling the analysis of large quantities of grains per genotype and (2) made possible the comparison of gas chromatography results and sensitive tests. The population size (135 lines) provided a good mapping precision. Several markers on chromosome 8 were found to be closely linked to a major gene controlling the presence of 2-acetyl-1-pyrroline (AcPy), the main compound of rice aroma. Moreover, our results showed that AcPy concentration in plants is regulated by at least two chromosomal regions. Estimations of recombination fractions on chromosome 8 were corrected for strong segregation distortion. This study confirms that AcPy is the major component of aroma. Use of the markers linked to AcPy major gene and QTLs for marker-assisted selection by successive backcrosses may be envisaged. OsBADH2|fgr Haplotype variation at Badh2, the gene determining fragrance in rice 2012 Genomics State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China. Fragrance is an important component of end-use quality in rice. A set of 516 fragrant rice accessions were genotyped and over 80% of them carried the badh2.7 allele. A subset of 144 mostly fragrant accessions, including nine of Oryza rufipogon, was then subjected to a detailed diversity and haplotype analysis. The level of linkage disequilibrium in the Badh2 region was higher among the fragrant accessions. Re-sequencing in the Badh2 region showed that badh2.7, badh2.2 and badh2.4-5 all arose in the japonica genepool, and spread later into the indica genepool as a result of deliberate crossing. However, loss-of-function alleles of Badh2 are also found in the indica genepools, and then transferred into japonica. Evidence for three new possible FNPs was obtained from the Badh2 sequence of 62 fragrant accessions. Based on these data, we have elaborated a model for the evolution of Badh2 and its participation in the rice domestication process. OsBADH2|fgr RFLP tagging of a gene for aroma in rice 1992 Theor Appl Genet Department of Plant Breeding and Biometry, 252 Emerson Hall, Cornell University, 14853, Ithaca, NY, USA. We report here the identification of a DNA marker closely linked to a gene for aroma in rice. The DNA marker was identified by testing 126 mapped rice genomic, cDNA, and oat cDNA, clones as hybridization probes against Southern blots, consisting of DNA from a pair of nearly isogenic lines (NILs) with or without the aroma gene. Chromosomal segments introgressed from the donor genome were distinguished by RFLPs between the NILs. Linkage association of the clone with the gene was verified using an F3 segregating for aroma. Cosegregation of the scented phenotype and donor-derived allele indicated the presence of linkage between the DNA marker and the gene. RFLP analysis showed that the gene is linked to a single-copy DNA clone, RG28, on chromosome 8, at a distance of 4.5 cM. The availability of a linked DNA marker may facilitate early selection for the aroma gene in rice breeding programs. OsBADH2|fgr Characterization of the major fragance gene from an aromatic japonica rice and analysis of its diversity in Asian cultivated rice 2008 Theor Appl Genet UMR 5096, IRD-CNRS-Universite de Perpignan, Laboratoire Genome et Developpement des Plantes, Perpignan, France. In Asian cultivated rice (Oryza sativa L.), aroma is one of the most valuable traits in grain quality and 2-ACP is the main volatile compound contributing to the characteristic popcorn-like odour of aromatic rices. Although the major locus for grain fragrance (frg gene) has been described recently in Basmati rice, this gene has not been characterised in true japonica varieties and molecular information available on the genetic diversity and evolutionary origin of this gene among the different varieties is still limited. Here we report on characterisation of the frg gene in the Azucena variety, one of the few aromatic japonica cultivars. We used a RIL population from a cross between Azucena and IR64, a non-aromatic indica, the reference genomic sequence of Nipponbare (japonica) and 93-11 (indica) as well as an Azucena BAC library, to identify the major fragance gene in Azucena. We thus identified a betaine aldehyde dehydrogenase gene, badh2, as the candidate locus responsible for aroma, which presented exactly the same mutation as that identified in Basmati and Jasmine-like rices. Comparative genomic analyses showed very high sequence conservation between Azucena and Nipponbare BADH2, and a MITE was identified in the promotor region of the BADH2 allele in 93-11. The badh2 mutation and MITE were surveyed in a representative rice collection, including traditional aromatic and non-aromatic rice varieties, and strongly suggested a monophylogenetic origin of this badh2 mutation in Asian cultivated rices. Altogether these new data are discussed here in the light of current hypotheses on the origin of rice genetic diversity. OsBADH2|fgr RNAi-directed downregulation of OsBADH2 results in aroma (2-acetyl-1-pyrroline) production in rice (Oryza sativa L.) 2008 BMC Plant Biol Ministry of Education Key Laboratory for Bio-resource and Eco-environment, College of Life Science, Sichuan University, Chengdu 610064, PR China. niu_xiangli@163.com BACKGROUND: Aromatic rice is popular worldwide because of its characteristic fragrance. Genetic studies and physical fine mapping reveal that a candidate gene (fgr/OsBADH2) homologous to betaine aldehyde dehydrogenase is responsible for aroma metabolism in fragrant rice varieties, but the direct evidence demonstrating the functions of OsBADH2 is lacking. To elucidate the physiological roles of OsBADH2, sequencing approach and RNA interference (RNAi) technique were employed to analyze allelic variation and functions of OsBADH2 gene in aroma production. Semi-quantitative, real-time reverse transcription-polymerase chain reaction (RT-PCR), as well as gas chromatography-mass spectrometry (GC-MS) were conducted to determine the expression levels of OsBADH2 and the fragrant compound in wild type and transgenic OsBADH2-RNAi repression lines, respectively. RESULTS: The results showed that multiple mutations identical to fgr allele occur in the 13 fragrant rice accessions across China; OsBADH2 is expressed constitutively, with less expression abundance in mature roots; the disrupted OsBADH2 by RNA interference leads to significantly increased 2-acetyl-1-pyrroline production. CONCLUSION: We have found that the altered expression levels of OsBADH2 gene influence aroma accumulation, and the prevalent aromatic allele probably has a single evolutionary origin. OsBADH2|fgr Purification, crystallization and preliminary X-ray analysis of recombinant betaine aldehyde dehydrogenase 2 (OsBADH2), a protein involved in jasmine aroma, from Thai fragrant rice (Oryza sativa L.) 2011 Acta Crystallogr Sect F Struct Biol Cryst Commun Synchrotron Light Research Institute (Public Organization), 111 University Avenue, Muang District, Nakhon Ratchasima 30000, Thailand. buabarn@slri.or.th Fragrant rice (Oryza sativa L.) betaine aldehyde dehydrogenase 2 (OsBADH2) is a key enzyme in the synthesis of fragrance aroma compounds. The extremely low activity of OsBADH2 in catalyzing the oxidation of acetaldehyde is believed to be crucial for the accumulation of the volatile compound 2-acetyl-1-pyrroline (2AP) in many scented plants, including fragrant rice. Recombinant fragrant rice OsBADH2 was expressed in Escherichia coli as an N-terminal hexahistidine fusion protein, purified using Ni Sepharose affinity chromatography and crystallized using the microbatch method. Initial crystals were obtained within 24 h using 0.1 M Tris pH 8.5 with 30%(w/v) PEG 4000 and 0.2 M magnesium chloride as the precipitating agent at 291 K. Crystal quality was improved when the enzyme was cocrystallized with NAD(+). Improved crystals were grown in 0.1 M HEPES pH 7.4, 24%(w/v) PEG 4000 and 0.2 M ammonium chloride and diffracted to beyond 2.95 A resolution after being cooled in a stream of N(2) immediately prior to X-ray diffraction experiments. The crystals belonged to space group C222(1), with unit-cell parameters a = 66.03, b = 183.94, c = 172.28 A. An initial molecular-replacement solution has been obtained and refinement is in progress. OsBADH2|fgr The gene for fragrance in rice 2005 Plant Biotechnol J Centre for Plant Conservation Genetics, Southern Cross University, Lismore, NSW 2480, Australia. The flavour or fragrance of basmati and jasmine rice is associated with the presence of 2-acetyl-1-pyrroline. A recessive gene (fgr) on chromosome 8 of rice has been linked to this important trait. Here, we show that a gene with homology to the gene that encodes betaine aldehyde dehydrogenase (BAD) has significant polymorphisms in the coding region of fragrant genotypes relative to non-fragrant genotypes. The accumulation of 2-acetyl-1-pyrroline in fragrant rice genotypes may be explained by the presence of mutations resulting in a loss of function of the fgr gene product. The allele in fragrant genotypes has a mutation introducing a stop codon upstream of key amino acid sequences conserved in other BADs. The fgr gene corresponds to the gene encoding BAD2 in rice, while BAD1 is encoded by a gene on chromosome 4. BAD has been linked to stress tolerance in plants. However, the apparent loss of function of BAD2 does not seem to limit the growth of fragrant rice genotypes. Fragrance in domesticated rice has apparently originated from a common ancestor and may have evolved in a genetically isolated population, or may be the outcome of a separate domestication event. This is an example of effective human selection for a recessive trait during domestication. OsBADH2|fgr Badh2, encoding betaine aldehyde dehydrogenase, inhibits the biosynthesis of 2-acetyl-1-pyrroline, a major component in rice fragrance 2008 Plant Cell National Maize Improvement Center of China, China Agricultural University, Beijing 100193, People's Republic of China. In rice (Oryza sativa), the presence of a dominant Badh2 allele encoding betaine aldehyde dehydrogenase (BADH2) inhibits the synthesis of 2-acetyl-1-pyrroline (2AP), a potent flavor component in rice fragrance. By contrast, its two recessive alleles, badh2-E2 and badh2-E7, induce 2AP formation. Badh2 was found to be transcribed in all tissues tested except for roots, and the transcript was detected at higher abundance in young, healthy leaves than in other tissues. Multiple Badh2 transcript lengths were detected, and the complete, full-length Badh2 transcript was much less abundant than partial Badh2 transcripts. 2AP levels were significantly reduced in cauliflower mosaic virus 35S-driven transgenic lines expressing the complete, but not the partial, Badh2 coding sequences. In accordance, the intact, full-length BADH2 protein (503 residues) appeared exclusively in nonfragrant transgenic lines and rice varieties. These results indicate that the full-length BADH2 protein encoded by Badh2 renders rice nonfragrant by inhibiting 2AP biosynthesis. The BADH2 enzyme was predicted to contain three domains: NAD binding, substrate binding, and oligomerization domains. BADH2 was distributed throughout the cytoplasm, where it is predicted to catalyze the oxidization of betaine aldehyde, 4-aminobutyraldehyde (AB-ald), and 3-aminopropionaldehyde. The presence of null badh2 alleles resulted in AB-ald accumulation and enhanced 2AP biosynthesis. In summary, these data support the hypothesis that BADH2 inhibits 2AP biosynthesis by exhausting AB-ald, a presumed 2AP precursor. OsBADH2|fgr The origin and evolution of fragrance in rice (Oryza sativa L.) 2009 Proc Natl Acad Sci U S A Department of Plant Breeding and Genetics, Cornell University, Ithaca, NY 14853, USA. Fragrance in the grain is one of the most highly valued grain quality traits in rice, yet the origin and evolution of the betaine aldehyde dehydrogenase gene (BADH2) underlying this trait remains unclear. In this study, we identify eight putatively nonfunctional alleles of the BADH2 gene and show that these alleles have distinct geographic and genetic origins. Despite multiple origins of the fragrance trait, a single allele, badh2.1, is the predominant allele in virtually all fragrant rice varieties today, including the widely recognized Basmati and Jasmine types. Haplotype analysis allowed us to establish a single origin of the badh2.1 allele within the Japonica varietal group and demonstrate the introgression of this allele from Japonica to Indica. Basmati-like accessions were nearly identical to the ancestral Japonica haplotype across a 5.3-Mb region flanking BADH2 regardless of their fragrance phenotype, demonstrating a close evolutionary relationship between Basmati varieties and the Japonica gene pool. These results clarify the relationships among fragrant rice varieties and challenge the traditional assumption that the fragrance trait arose in the Indica varietal group. OsBADH2|fgr Biochemical and enzymatic study of rice BADH wild-type and mutants: an insight into fragrance in rice 2011 Protein J Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok, Thailand. Betaine aldehyde dehydrogenase 2 (BADH2) is believed to be involved in the accumulation of 2-acetyl-1-pyrroline (2AP), one of the major aromatic compounds in fragrant rice. The enzyme can oxidize omega-aminoaldehydes to the corresponding omega-amino acids. This study was carried out to investigate the function of wild-type BADHs and four BADH2 mutants: BADH2_Y420, containing a Y420 insertion similar to BADH2.8 in Myanmar fragrance rice, BADH2_C294A, BADH2_E260A and BADH2_N162A, consisting of a single catalytic-residue mutation. Our results showed that the BADH2_Y420 mutant exhibited less catalytic efficiency towards gamma-aminobutyraldehyde but greater efficiency towards betaine aldehyde than wild-type. We hypothesized that this point mutation may account for the accumulation of gamma-aminobutyraldehyde/Delta(1)-pyrroline prior to conversion to 2AP, generating fragrance in Myanmar rice. In addition, the three catalytic-residue mutants confirmed that residues C294, E260 and N162 were involved in the catalytic activity of BADH2 similar to those of other BADHs. OsBADH2|fgr Rice RING protein OsBBI1 with E3 ligase activity confers broad-spectrum resistance against Magnaporthe oryzae by modifying the cell wall defence 2011 Cell Res National Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310029, China. Emerging evidence suggests that E3 ligases play critical roles in diverse biological processes, including innate immune responses in plants. However, the mechanism of the E3 ligase involvement in plant innate immunity is unclear. We report that a rice gene, OsBBI1, encoding a RING finger protein with E3 ligase activity, mediates broad-spectrum disease resistance. The expression of OsBBI1 was induced by rice blast fungus Magnaporthe oryzae, as well as chemical inducers, benzothiadiazole and salicylic acid. Biochemical analysis revealed that OsBBI1 protein possesses E3 ubiquitin ligase activity in vitro. Genetic analysis revealed that the loss of OsBBI1 function in a Tos17-insertion line increased susceptibility, while the overexpression of OsBBI1 in transgenic plants conferred enhanced resistance to multiple races of M. oryzae. This indicates that OsBBI1 modulates broad-spectrum resistance against the blast fungus. The OsBBI1-overexpressing plants showed higher levels of H(2)O(2) accumulation in cells and higher levels of phenolic compounds and cross-linking of proteins in cell walls at infection sites by M. oryzae compared with wild-type (WT) plants. The cell walls were thicker in the OsBBI1-overexpressing plants and thinner in the mutant plants than in the WT plants. Our results suggest that OsBBI1 modulates broad-spectrum resistance to blast fungus by modifying cell wall defence responses. The functional characterization of OsBBI1 provides insight into the E3 ligase-mediated innate immunity, and a practical tool for constructing broad-spectrum resistance against the most destructive disease in rice. OsBBI1 Characterization of a rice (Oryza sativa L.) Bowman–Birk proteinase inhibitor: tightly light regulated induction in response to cut, jasmonic acid, ethylene and protein phosphatase 2A inhibitors 2001 Gene United Graduate School, Tokyo University of Agriculture and Technology, Tokyo, Japan. The Bowman-Birk (BB) family of proteinase inhibitors (PI), initially reported from legume seeds, and thereafter also from wounded alfalfa and maize leaves appear to be regulated in similar ways as the extensively characterized PI I and PI II family from dicots. Here, we report a first characterization of the expression profiles of a rice (Oryza sativa L. cv. Nipponbare) BBPI gene, OsBBPI, which is part of a multigene family as demonstrated by genomic Southern hybridization. OsBBPI was found to be rapidly induced in rice seedling leaf in response to cut, exogenous jasmonic acid (JA), and two potent protein phosphatase 2A (PP2A) inhibitors, cantharidin (CN) and endothall (EN), in a light/dark-, time- and dose-dependent manner; this induction was completely inhibited by cycloheximide (CHX), indicating a requirement for de novo protein synthesis in its induction. Surprisingly, dark strongly up regulated cut-, JA-, CN-, and EN-induced OsBBPI expression, with the strongest enhancement observed with JA. A simultaneous application of a serine/threonine protein kinase inhibitor staurosporine (ST) did not affect significantly the JA-, CN-, and EN-induced OsBBPI transcript. Besides JA, it was found that the ethylene generator ethephon (ET) also had an enhancing effect on OsBBPI transcript, suggesting a direct effect of ethylene on OsBBPI expression. However, a simultaneous application of salicylic acid (SA) and abscisic acid (ABA), with JA, respectively, completely blocked OsBBPI gene expression, whereas kinetin (KN) was only partially effective. To the best of our knowledge, complete inhibition of JA-induced OsBBPI expression by SA is the first report in monocots, and with ABA in plants. Taken together, these results suggest that among the phytohormones tested here, JA and ethylene play important role(s) in regulating OsBBPI expression, with an intimate interaction with light signals. Finally, that the induced OsBBPI expression follows a kinase-signaling cascade is implied by the use of PP2A inhibitors. OsBBPI OsBC1L4 encodes a COBRA-like protein that affects cellulose synthesis in rice 2011 Plant Mol Biol National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, 430070, Wuhan, China. Plant morphogenesis is highly dependent on the regulation of cell division and expansion. The organization of the cellulose microfibrils in the cell wall is a key determinant of cell expansion. Previously, a dwarf mutant with fewer tillers, Osbc1l4 (Oryza sativa brittle culm 1 like 4), was identified by screening a rice T-DNA insertion mutant library. It is reported here that OsBC1L4 encodes a COBRA-like protein that exhibits typical structural features of a glycosylphosphatidylinositol-anchor protein. The T-DNA insertion in OsBC1L4 results in abnormal cell expansion. A decrease in cellulose content but the increase in pectin and starch contents was identified in Osbc1l4 mutants by measuring the content of wall components. OsBC1L4 was expressed in all tissues/organs examined, with a low level in leaves. OsBC1L4 protein is mainly located in the cell wall and plasma membrane. Correlation analysis indicated that the expression of OsBC1L4 was highly correlated to that of several primary wall-forming cellulose synthase genes (CESAs). Moreover, the expression level of several cellulose-related genes is increased in Osbc1l4 mutants, which suggests that a feedback mechanism may exist during cellulose synthesis. OsBC1L4 An SNP downstream of the OsBEIIb gene is significantly associated with amylose content and viscosity properties in rice (Oryza sativa L.) 2012 Journal of Cereal Science Department of Plant Resources, College of Industrial Sciences, Kongju National University, Yesan 340-802, Republic of Korea Allele mining in starch synthesis-related genes (SSRGs) has facilitated the discovery of desired natural sequence variations for eating quality in rice. This study investigated the sequence variations from 10 SSRGs, and further evaluated their relationship with the amylose content (AC) and rapid viscosity analysis profiles in a global collection of rice accessions by association mapping (AM). In total, 83 sequence variations were found in 10 sequenced amplicons, including 73 single nucleotide polymorphisms (SNPs), eight insertion-deletions (InDels) and two polymorphic simple sequence repeats (SSRs). Four subpopulations were identified by population structure analysis based on 170 genome-wide SSR genotypes. AM revealed 11 significant associations between three phenotypic indices and three sequence variations. One SNP with a g/c transversion at the 63rd nucleotide downstream of the OsBEIIb gene termination codon on rice chromosome 2 was significantly associated with multiple trait indices in both the general linear and mixed linear models (GLM and MLM), including the final viscosity (p < 0.001, R2 = 23.87%) in both 2009 and 2010, and AC (p < 0.01, R2 = 11.25%) and trough viscosity (p < 0.01, R2 = 20.43) in 2010. This study provides a new perspective of allele mining for breeding strategies based on marker-assisted selection. OsBEIIb The structure of starch can be manipulated by changing the expression levels of starch branching enzyme IIb in rice endosperm 2004 Plant Biotechnol J National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki 305-8602, Japan. When the starch branching enzyme IIb (BEIIb) gene was introduced into a BEIIb-defective mutant, the resulting transgenic rice plants showed a wide range of BEIIb activity and the fine structure of their amylopectins showed considerable variation despite having the two other BE isoforms, BEI and BEIIa, in their endosperm at the same levels as in the wild-type. The properties of the starch granules, such as their gelatinization behaviour, morphology and X-ray diffraction pattern, also changed dramatically depending on the level of BEIIb activity, even when this was either slightly lower or higher than that of the wild-type. The over-expression of BEIIb resulted in the accumulation of excessive branched, water-soluble polysaccharides instead of amylopectin. These results imply that the manipulation of BEIIb activity is an effective strategy for the generation of novel starches for use in foodstuffs and industrial applications. OsBEIIb Molecular characterization of beta-galactosidases from germinating rice (Oryza sativa) 2007 Plant Science School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand Staining of germinating rice with X-Gal and immunohistochemical localization indicated that beta-d-galactosidase (EC 3.2.1.23) is localized to the embryo, aleurone layer and surrounding tissues, and in the radicle and shoot after germination. The genes encoding two isoforms of beta-galactosidase expressed in germinating rice and panicle, designated OsBGal1 and OsBGal2, were cloned and sequenced. The full-length cDNA sequence of OsBGal1 encodes 843 amino acids, including a signal peptide of 25 residues. The related OsBGal2 clone from panicle at flowering stage encodes 715 amino acid residues, including a signal peptide of 20 residues. The two genes were found to be relatively highly expressed in seedling roots and shoots and in leaf sheath in 15–30-day-old plants, which contained major immunoreactive proteins of approximately 90 kDa for OsBGal1 protein and 55 kDa for OsBGal2 in extracts of these tissues. Both were also expressed at low levels in flowers and immature seeds, but only the OsBGal2 transcript was found in mature seeds. The predicted mature proteins of the rice beta-galactosidases were expressed as thioredoxin fusion proteins in E. coli strain OrigamiB (DE3) to produce beta-galactosidase activities not found in control extracts. Purified OsBGal1 fusion protein hydrolyzed and transglycosylated p-nitrophenyl (pNP) beta-d-galactopyranoside and hydrolyzed pNP beta-d-fucopyranoside. Galactose was released by this enzyme from beta-(1 → 3)-, beta-(1 → 4)- and beta-(1 → 6)-linked di- and trisaccharides of galactose and rice root and rice coleoptile alcohol-insoluble residue, but it did not hydrolyze larger [3H]oligo-(1 → 4)-galactans (penta-, octa- and undecasaccharides), lactose, a galactosylated xyloglucan oligosaccharide (XLLG), total rice hemicellulosic polysaccharides or pectin. OsBGal1,OsBGal2 A rice transcription factor OsbHLH1 is involved in cold stress response 2003 Theor Appl Genet Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 100101 Beijing, PR China. Cold stress adversely affects plant growth and crop production. Some plants express a series of cold-responsive genes during cold acclimation to reduce the damage of cold stress. Among them, transcription factors play important roles in enhancing plant cold tolerance. A bHLH-type gene OsbHLH1 was isolated from rice. The predicted OsbHLH1 protein has a putative nuclear-localization signal and a putative DNA binding-domain bHLH-ZIP. The genomic sequence of the OsbHLH1 gene is unique in rice genome and has four introns. The transcription of the OsbHLH1 gene was specifically induced in roots of rice seedlings by cold but not by NaCl, PEG and ABA treatments. The OsbHLH1 protein was located in the nucleus of plant cells and had the ability to activate the transcription of the reporter gene in yeast. In addition, OsbHLH1 had the ability to dimerize. These results indicate that the OsbHLH1 may function as a transcription factor in a cold signal-transduction pathway. OsbHLH1 OsbHLH148, a basic helix-loop-helix protein, interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice 2011 Plant J Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea. Jasmonates play important roles in development, stress responses and defense in plants. Here, we report the results of a study using a functional genomics approach that identified a rice basic helix-loop-helix domain gene, OsbHLH148, that conferred drought tolerance as a component of the jasmonate signaling module in rice. OsbHLH148 transcript levels were rapidly increased by treatment with methyl jasmonate (MeJA) or abscisic acid, and abiotic stresses including dehydration, high salinity, low temperature and wounding. Transgenic over-expression of OsbHLH148 in rice confers plant tolerance to drought stress. Expression profiling followed by DNA microarray and RNA gel-blot analyses of transgenic versus wild-type rice identified genes that are up-regulated by OsbHLH148 over-expression. These include OsDREB and OsJAZ genes that are involved in stress responses and the jasmonate signaling pathway, respectively. OsJAZ1, a rice ZIM domain protein, interacted with OsbHLH148 in yeast two-hybrid and pull-down assays, but it interacted with the putative OsCOI1 only in the presence of coronatine. Furthermore, the OsJAZ1 protein was degraded by rice and Arabidopsis extracts in the presence of coronatine, and its degradation was inhibited by MG132, a 26S proteasome inhibitor, suggesting 26S proteasome-mediated degradation of OsJAZ1 via the SCF(OsCOI1) complex. The transcription level of OsJAZ1 increased upon exposure of rice to MeJA. These results show that OsJAZ1 could act as a transcriptional regulator of the OsbHLH148-related jasmonate signaling pathway leading to drought tolerance. Thus, our study suggests that OsbHLH148 acts on an initial response of jasmonate-regulated gene expression toward drought tolerance, constituting the OsbHLH148-OsJAZ-OsCOI1 signaling module in rice. OsbHLH148,OsCOI1|OsCOI1a,OsJAZ1|OsTIFY3|EG2,UspA Overexpression of Bax inhibitor suppresses the fungal elicitor-induced cell death in rice (Oryza sativaL.) cells 2003 The Plant Journal Iwate Biotechnology Research Center, Kitakami, Iwate 024-0003, Japan. Treatment of suspension-cultured cells of rice (Oryza sativa L.) with cell wall extract of rice blast fungus (Magnaporthe grisea) elicits a rapid generation of H2O2, alkalinization of culture medium, and eventual cell death. To elucidate genes involved in these processes, we exploited SAGE (Serial Analysis of Gene Expression) technique for the molecular analysis of cell death in suspension-cultured cells treated with the elicitor. Among the downregulated genes in the elicitor-treated cells, a BI-1 gene coding for Bax inhibitor was identified. Transgenic rice cells overexpressing Arabidopsis BI-1 gene showed sustainable cell survival when challenged with M. grisea elicitor. Thus, the plant Bax inhibitor plays a functional role in regulating cell death in the rice cell culture system. OsBI-1 OsBi1, a rice gene, encodes a novel protein with a CBS-like domain and its expression is induced in responses to herbivore feeding 2004 Plant Science Key Laboratory of Ministry of Education for Plant Development Biology, Wuhan University College of Life Sciences, Wuhan 420072, PR China Plant–herbivore interactions are complex. Plant genes respond differentially to insects of different feeding modes. In this paper, we describe the cloning and characterization of OsBi1, a rice (Oryza sativa L.) gene significantly induced by feeding of the sucking herbivore brown planthopper (Nilaparvata lugens Stål.). The OsBi1 gene contains an open reading frame that encodes a polypeptide of 283 amino acid residues with a predicted molecular weight of 36.2 kDa. Homology search in GenBank databases showed that the deduced OSBI1 protein was homologous to the inosine monophosphate dehydrogenase (IMPDH) protein in the CBS domain. Genomic Southern analysis indicated that the OsBi1 gene was present as a single copy sequence in the rice genome. Restriction fragment length polymorphism mapping assigned the OsBi1 gene to the long arm of chromosome 2. Increase in the level of OsBi1 expression was detected in rice plants shortly after brown planthopper feeding. OsBi1 gene expression was up-regulated in response to ethephone and water deficit. In situ hybridization indicated that transcripts of the gene accumulated specifically around the vascular bundle of stem. Evidences suggest that OsBi1 is a BPH-inducible gene that is implicated in the resistance of rice plants to brown planthopper. OsBi1 Overexpression of a rice diacylglycerol kinase gene OsBIDK1 enhances disease resistance in transgenic tobacco 2008 Mol Cells State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University at Huajiachi Campus, Hangzhou, Zhejiang 310029, People's Republic of China. A rice diacylglycerol kinase (DGK) gene, OsBIDK1, which encodes a 499-amino acid protein, was cloned and characterized. OsBIDK1 contains a conserved DGK domain, consisting of a diacylglycerol kinase catalytic subdomain and a diacylglycerol kinase accessory subdomain. Expression of OsBIDK1 in rice seedlings was induced by treatment with benzothiadiazole (BTH), a chemical activator of the plant defense response, and by infection with Magnaporthe grisea, causal agent of blast disease. In BTH-treated rice seedlings, expression of OsBIDK1 was induced earlier and at a higher level than in water-treated control seedlings after inoculation with M. grisea. Transgenic tobacco plants that constitutively express the OsBIDK1 gene were generated and disease resistance assays showed that overexpression of OsBIDK1 in transgenic tobacco plants resulted in enhanced resistance against infection by tobacco mosaic virus and Phytophthora parasitica var. nicotianae. These results suggest that OsBIDK1 may play a role in disease resistance responses. OsBIDK1 Molecular characterization of four rice genes encoding ethylene-responsive transcriptional factors and their expressions in response to biotic and abiotic stress 2006 J Plant Physiol Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang 310029, PR China. We isolated and identified four rice genes, OsBIERF1 to OsBIERF4 (Oryza sativa benzothiadiazole (BTH)-induced ethylene responsive transcriptional factors (ERF)) and analyzed their expressions in rice disease resistance response and under various abiotic stress conditions. The OsBIERF1-4 proteins contain conserved ERF domains, but are categorized into different classes of the previously characterized ERF proteins based on their structural organizations. OsBIERF3 and OsBIERF2 belong to Classes I and II, respectively; while OsBIRERF1 and OsBIERF4 are members of Class IV. OsBIERF3 could bind specifically to the GCC box sequence and was targeted to nucleus when transiently expressed in onion epidermis cells. Expression of OsBIERF1, OsBIERF3 and OsBIERF4 was induced by treatments with BTH and salicylic acid, chemical inducers capable of inducing disease resistance response in rice. In the BTH-treated rice seedlings, expression of OsBIERF1, OsBIERF3 and OsBIERF4 was further induced by infection with Magnaporthe grisea, the rice blast fungus, as compared with those in water-treated seedlings. OsBIERF1 and OsBIERF3 were activated in an incompatible interaction but not in compatible interaction between rice and M. grisea. Moreover, OsBIERF1, OsBIERF3 and OsBIERF4 were also up-regulated by salt, cold, drought and wounding. These results suggest that OsBIERF proteins may participate in different signaling pathways that mediate disease resistance response and stress responses to abiotic factors. OsBIERF1,OsBIERF2,AP59|OsBIERF3,OsBIERF4 Overexpression of the rice EREBP-like gene OsBIERF3 enhances disease resistance and salt tolerance in transgenic tobacco 2005 Physiological and Molecular Plant Pathology Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang 310029, People's Republic of China The rice OsBIERF3 encodes a protein belonging to the ethylene-responsive element binding protein (EREBP) transcription factor family and was shown to be induced by treatments with chemical inducers of disease resistance response, infection with the blast fungus, Magnaporthe grisea, and by treatments with some abiotic stress conditions, e.g. salt and cold. To better understand the function of OsBIERF3 in disease resistance response and abiotic tolerance, we overexpressed the OsBIERF3 gene in tobacco by Agrobacterium-mediated leaf disc transformation with a construct containing the OsBIERF3 ORF under control of the cauliflower mosaic virus 35S promoter. Disease resistance assays showed that the transgenic tobacco plants enhanced disease resistance against infection by tomato mosaic virus and the bacterial wild fire pathogen, Pseudomonas syringae pv. tabaci. Elevated level of defense-related PR-1a gene expression was also detected in the transgenic tobacco plants. Moreover, the OsBIERF3-overexpressing transgenic tobacco plants also showed increased tolerance to salt stress. Our results suggest that OsBIERF3 may play important roles in disease resistance response and salt tolerance. AP59|OsBIERF3,OsPR1a Overexpression of the Transcription Factor AP37 in Rice Improves Grain Yield under Drought Conditions 2009 Plant Physiol School of Biotechnology and Environmental Engineering, Myongji University, Yongin 449-728, Korea. Transcription factors with an APETELA2 (AP2) domain have been implicated in various cellular processes involved in plant development and stress responses. Of the 139 AP2 genes predicted in rice (Oryza sativa), we identified 42 genes in our current study that are induced by one or more stress conditions, including drought, high salinity, low temperature, and abscisic acid. Phylogenic analysis of these 42 stress-inducible AP2 genes revealed the presence of six subgroups (I-VI) with distinct signature motifs. Two genes, AP37 and AP59, representing subgroups I and II, respectively, were functionally characterized. Both genes were found to be induced upon 2 h of exposure to drought and high-salinity conditions but to differ in their expression profile upon exposure to low temperature and abscisic acid. The overexpression of AP37 and AP59 in rice under the control of the constitutive promoter OsCc1 increased the tolerance to drought and high salinity at the vegetative stage. Increased tolerance to low temperatures was observed only in OsCc1:AP37 plants. More importantly, the OsCc1:AP37 plants showed significantly enhanced drought tolerance in the field, which increased grain yield by 16% to 57% over controls under severe drought conditions, yet exhibited no significant difference under normal growth conditions. In contrast, grain yield in OsCc1:AP59 plants in the field was reduced by 23% to 43% compared with controls under both normal and drought stress conditions. Microarray experiments identified 10 and 38 genes that are up-regulated by AP37 and AP59, respectively, in addition to 37 genes that are commonly induced by both factors. Our results suggest that the AP37 gene has the potential to improve drought tolerance in rice without causing undesirable growth phenotypes. AP59|OsBIERF3,AP37|OsERF3 Up-regulation of OsBIHD1, a rice gene encoding BELL homeodomain transcriptional factor, in disease resistance responses 2005 Plant Biol (Stuttg) Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang, PR China. In the present study, we cloned and identified a full-length cDNA of a rice gene, OsBIHD1, encoding a homeodomain type transcriptional factor. OsBIHD1 is predicted to encode a 642 amino acid protein and the deduced protein sequence of OsBIHD1 contains all conserved domains, a homeodomain, a BELL domain, a SKY box, and a VSLTLGL box, which are characteristics of the BELL type homedomain proteins. The recombinant OsBIHD1 protein expressed in Escherichia coli bound to the TGTCA motif that is the characteristic cis-element DNA sequence of the homeodomain transcriptional factors. Subcellular localization analysis revealed that the OsBIHD1 protein localized in the nucleus of the plant cells. The OsBIHD1 gene was mapped to chromosome 3 of the rice genome and is a single-copy gene with four exons and three introns. Northern blot analysis showed that expression of OsBIHD1 was activated upon treatment with benzothiadiazole (BTH), which is capable of inducing disease resistance. Expression of OsBIHD1 was also up-regulated rapidly during the first 6 h after inoculation with Magnaporthe grisea in BTH-treated rice seedlings and during the incompatible interaction between M. grisea and a resistant genotype. These results suggest that OsBIHD1 is a BELL type of homeodomain transcription factor present in the nucleus, whose induction is associated with resistance response in rice. OsBIHD1 Overexpression in transgenic tobacco reveals different roles for the rice homeodomain gene OsBIHD1 in biotic and abiotic stress responses 2005 J Exp Bot Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang 310029, PR China. The rice OsBIHD1 gene encodes a transcriptional factor belonging to the homeodomain class. It had previously been shown to be activated by treatment with benzothiadiazole, a chemical inducer of disease resistance, and in an incompatible interaction between rice and the blast fungus. To allow a better understanding of the function of OsBIHD1 in plant disease resistance response, the OsBIHD1 gene in tobacco was overexpressed by Agrobacterium-mediated leaf disc transformation with a construct containing the OsBIHD1 ORF under control of the 35S promoter. Overexpression of the rice OsBIHD1 gene in some of the transgenic tobacco lines led to some morphological abnormalities in the top buds and roots. The transgenic tobacco plants showed an elevated level of defence-related PR-1 gene expression and enhanced disease resistance against infection by tomato mosaic virus, tobacco mosaic virus, and Phytophthora parasitica var. nicotianae. However, the transgenic tobacco plants overexpressing OsBIHD1 showed enhanced sensitivity to salt and oxidative stress as compared with the wild-type plants. The results suggested that the OsBIHD1 protein may be positively involved in activating expression of the defence-related genes in disease resistance responses, and is also important in rice development and abiotic stress tolerance. OsBIHD1 Expression of OsBiP4 and OsBiP5 is highly correlated with the endoplasmic reticulum stress response in rice 2012 Planta Functional Transgenic Crops Research Unit, Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602, Japan. Binding protein (BiP) is a chaperone protein involved in the folding of secretory proteins in the ER lumen. OsBiP1 is constitutively expressed in various tissues, whereas the expression of OsBiP4 and OsBiP5 (OsBiP4&5) is not detected in any tissue under normal conditions. However, expression of OsBiP4&5 was highly and specifically activated under ER stress conditions induced by DTT treatment, OsBiP1 knockdown, OsBiP1 overexpression, OsIRE1 overexpression, or various exogenous recombinant proteins in transgenic rice. In contrast, OsBiP4&5 did not accumulate in OsIRE1 knockdown transgenic rice even after DTT treatment. When the subcellular localization of OsBiP4&5 was investigated in seed endosperm cells under the ER stress condition, OsBiP4&5 were localized to the ER, but did not participate in ER-derived protein body (PB-I) formation in a different manner to OsBiP1. These results indicate that OsBiP4&5 levels were positively correlated with stress levels in the ER. Taken together, these results suggest that OsBiP4&5 are ER stress-related BiP proteins that are regulated by OsIRE1/OsbZIP50 pathway and that they may have a distinct function from that of OsBiP1 in rice. OsBip1,OsBiP4,OsBiP5,IRE1|OsIRE1,OsBiP2|Bip2,OsBiP3|BiP3 Signal transduction by IRE1-mediated splicing of bZIP50 and other stress sensors in the endoplasmic reticulum stress response of rice 2012 Plant J Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. The endoplasmic reticulum (ER) stress sensor IRE1 transduces signals by inducing the unconventional splicing of mRNAs encoding key transcription factors: HAC1 in yeast and XBP1 in animals. However, no HAC1 or XBP1 homologues have been found in plants, and until recently the substrate for plant IRE1 has remained unknown. This study demonstrates that the Oryza sativa (rice) OsbZIP50 transcription factor, an orthologue of Arabidopsis AtbZIP60, is regulated by IRE1-mediated splicing of its RNA. Despite the presence of a transcriptional activation domain, OsbZIP50 protein is not translocated into the nucleus efficiently in the absence of OsbZIP50 mRNA splicing. Unconventional splicing of OsbZIP50 mRNA causes a frame shift, which results in the appearance of a nuclear localization signal in the newly translated OsbZIP50. OsbZIP50 mRNA is spliced in a similar manner to HAC1 and XBP1 mRNAs; however, this splicing has very different effects on the translation products, a finding that shows the diversity of IRE1-related transcription factors in eukaryotes. In addition, the expression of OsbZIP50 is affected by ER stress sensor proteins OsIRE1, OsbZIP39 and OsbZIP60. ER stress-related genes differ with respect to their dependency on OsbZIP50 for their expression. The findings of this study improve our understanding of the molecular mechanisms underlying the plant ER stress response. OsBip1,OsBiP4,OsBiP5,OsbZIP39,OsbZIP50|OsbZIP74,OsbZIP60,OsEro1,IRE1|OsIRE1,NEF,OsBiP2|Bip2,OsBiP3|BiP3 Expression of ER quality control-related genes in response to changes in BiP1 levels in developing rice endosperm 2011 Plant J Transgenic Crop Research and Development Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. Binding protein (BiP) is the key chaperone involved in folding of secretory proteins such as seed storage proteins in the ER lumen. To obtain functional information about BiP1, a gene that is predominantly expressed during rice seed maturation, we generated several transgenic rice plants in which various levels of BiP1 protein accumulated in an endosperm-specific manner. Severe suppression (BiP1 KD) or significant over-expression (BiP1 OEmax) of BiP1 not only altered seed phenotype and the intracellular structure of endosperm cells, but also reduced seed storage protein content, starch accumulation and grain weight. Microarray and RT-PCR analyses indicated that expression of many chaperone and co-chaperone genes was induced in transgenic plants, with more prominent expression in the BiP1 KD line than in the BiP1 OEmax line. Transcriptional induction of most chaperones was observed in calli treated with dithiothreitol or tunicamycin, treatments that trigger ER stress, indicating that induction of the chaperone genes in transgenic rice was caused by an ER stress response. In transient assays using rice protoplasts, the ortholog (Os06g0622700) of the AtbZIP60 transcription factor was shown to be involved in activation of some chaperone genes. Slight increases in the BiP1 level compared with wild-type, accompanied by increased levels of calnexin and protein disulfide isomerase-like proteins, resulted in significant enhancement of seed storage protein content, without any change in intracellular structure or seed phenotype. Judicious modification of BiP1 levels in transgenic rice can provide suitable conditions for the production of secretory proteins by alleviating ER stress. OsBip1,CRT,GLB1,NEF,Stt3a Overexpression of the endoplasmic reticulum chaperone BiP3 regulates XA21-mediated innate immunity in rice 2010 PLoS One Department of Plant Pathology, University of California Davis, Davis, California, United States of America. Recognition of pathogen-associated molecular patterns by pattern recognition receptors (PRRs) activates the innate immune response. Although PRR-mediated signaling events are critical to the survival of plants and animals, secretion and localization of PRRs have not yet been clearly elucidated. Here we report the in vivo interaction of the endoplasmic reticulum (ER) chaperone BiP3 with the rice XA21 PRR, which confers resistance to the Gram negative bacterium, Xanthomonas oryzae pv. oryzae (Xoo). We show that XA21 is glycosylated and is primarily localized to the ER and also to the plasma membrane (PM). In BiP3-overexpressing rice plants, XA21-mediated immunity is compromised, XA21 stability is significantly decreased, and XA21 proteolytic cleavage is inhibited. BiP3 overexpression does not affect the general rice defense response, cell death or brassinolide-induced responses. These results indicate that BiP3 regulates XA21 protein stability and processing and that this regulation is critical for resistance to Xoo. OsBip1,xa21 Functional analysis reveals pleiotropic effects of rice RING-H2 finger protein gene OsBIRF1 on regulation of growth and defense responses against abiotic and biotic stresses 2008 Plant Mol Biol State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Huajiachi Campus, Hangzhou, Zhejiang, People's Republic of China. RING finger proteins comprise a large family and play key roles in regulating growth/developmental processes, hormone signaling and responses to biotic and abiotic stresses in plants. A rice gene, OsBIRF1, encoding a putative RING-H2 finger protein, was cloned and identified. OsBIRF1 encodes a 396 amino acid protein belonging to the ATL family characterized by a conserved RING-H2 finger domain (C-X2-C-X15-C-X1-H-X2-H-X2-C-X10-C-X2-C), a transmembrane domain at the N-terminal, a basic amino acid rich region and a characteristic GLD region. Expression of OsBIRF1 was up-regulated in rice seedlings after treatment with benzothaidiazole, salicylic acid, l-aminocyclopropane-1-carboxylic acid and jasmonic acid, and was induced differentially in incompatible but not compatible interactions between rice and Magnaporthe grisea, the causal agent of blast disease. Transgenic tobacco plants that constitutively express OsBIRF1 exhibit enhanced disease resistance against tobacco mosaic virus and Pseudomonas syringae pv. tabaci and elevated expression levels of defense-related genes, e.g. PR-1, PR-2, PR-3 and PR-5. The OsBIRF1-overexpressing transgenic tobacco plants show increased oxidative stress tolerance to exogenous treatment with methyl viologen and H2O2, and up-regulate expression of oxidative stress-related genes. Reduced ABA sensitivity in root elongation and increased drought tolerance in seed germination were also observed in OsBIRF1 transgenic tobacco plants. Furthermore, the transgenic tobacco plants show longer roots and higher plant heights as compared with the wild-type plants, suggesting that overexpression of OsBIRF1 promote plant growth. These results demonstrate that OsBIRF1 has pleiotropic effects on growth and defense response against multiple abiotic and biotic stresses. OsBIRF1 OsBIRH1, a DEAD-box RNA helicase with functions in modulating defence responses against pathogen infection and oxidative stress 2008 J Exp Bot State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University-Huajiachi Campus, Hangzhou, Zhejiang 310029, People's Republic of China. DEAD-box proteins comprise a large protein family with members from all kingdoms and play important roles in all types of processes in RNA metabolism. In this study, a rice gene OsBIRH1, which encodes a DEAD-box RNA helicase protein, was cloned and characterized. The predicted OsBIRH1 protein contains a DEAD domain and all conserved motifs that are common characteristics of DEAD-box RNA helicases. Recombinant OsBIRH1 protein purified from Escherichia coli was shown to have both RNA-dependent ATPase and ATP-dependent RNA helicase activities in vitro. Expression of OsBIRH1 was activated in rice seedling leaves after treatment with defence-related signal chemicals, for example benzothiadiazole, salicylic acid, l-aminocyclopropane-1-carboxylic acid, and jasmonic acid, and was also up-regulated in an incompatible interaction between a resistant rice genotype and the blast fungus, Magnaporthe grisea. Transgenic Arabidopsis plants that overexpress the OsBIRH1 gene were generated. Disease resistance phenotype assays revealed that the OsBIRH1-overexpressing transgenic plants showed an enhanced disease resistance against Alternaria brassicicola and Pseudomonas syringae pv. tomato DC3000. Meanwhile, defence-related genes, for example PR-1, PR-2, PR-5, and PDF1.2, showed an up-regulated expression in the transgenic plants. Moreover, the OsBIRH1 transgenic Arabidopsis plants also showed increased tolerance to oxidative stress and elevated expression levels of oxidative defence genes, AtApx1, AtApx2, and AtFSD1. The results suggest that OsBIRH1 encodes a functional DEAD-box RNA helicase and plays important roles in defence responses against biotic and abiotic stresses. OsBIRH1 OsBISAMT1, a gene encoding S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase, is differentially expressed in rice defense responses 2006 Mol Biol Rep Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang, 310029, People's Republic of China. We isolated and identified a full-length cDNA, OsBISAMT1 [Oryza sativa L. benzothiadiazole (BTH)-induced SAMT 1], which encodes a putative S-adenosyl-L: -methionine:salicylic acid carboxyl methyltransferase (SAMT) from rice. OsBISAMT1 contains an ORE of 1128 bp, which predicts to encode a 375 aa protein. The OsBISAMT1 protein sequence shows a high level of identity to known plant SAMTs and contains a conserved characteristic methyltransferase domain. OsBISAMT1 is a member of a small gene family in the rice genome. Expression of OsBISAMT1 in rice leaves was induced by treatments with benzothiadiazole and salicylic acid, which are capable of inducing rice disease resistance. OsBISAMT1 was also up-regulated in both incompatible and compatible interactions between rice and the blast fungus, Magnaporthe grsiea, but the induced expression of OsBISAMT1 was greater and more rapid in the incompatible interaction than that in the compatible one. Moreover, mechanical wounding also activated OsBISAMT1 expression. The results suggest that OsBISAMT1 may be involved in disease resistance responses as well as in wound response in rice. OsBISAMT1 A rice serine carboxypeptidase-like gene OsBISCPL1 is involved in regulation of defense responses against biotic and oxidative stress 2008 Gene State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Huajiachi Campus, Hangzhou, Zhejiang 310029, People's Republic of China. Serine carboxypeptidase-like proteins (SCPLs) comprise a large family of protein hydrolyzing enzymes that play roles in multiple cellular processes. During the course of study aimed at elucidating the molecular basis of induced immunity in rice, a gene, OsBISCPL1, encoding a putative SCPL, was isolated and identified. OsBISCPL1 contains a conserved peptidase S10 domain, serine active site and a signal peptide at N-terminus. OsBISCPL1 is expressed ubiquitously in rice, including roots, stems, leaves and spikes. Expression of OsBISCPL1 in leaves was significantly up-regulated after treatments with benzothiadiazole, salicylic acid, jasmonic acid and 1-amino cyclopropane-1-carboxylic acid, and also up-regulated in incompatible interactions between rice and the blast fungus, Magnaporthe grisea. Transgenic Arabidopsis plants with constitutive expression of OsBISCPL1 were generated and disease resistance assays indicated that the OsBISCPL1-overexpressing plants showed an enhanced disease resistance against Pseudomonas syringae pv. tomato and Alternaria brassicicola. Expression levels of defense-related genes, e.g. PR1, PR2, PR5 and PDF1.2, were constitutively up-regulated in transgenic plants as compared with those in wild-type plants. Furthermore, the OsBISCPL1-overexpressing plants also showed an increased tolerance to oxidative stress and up-regulated expression of oxidative stress-related genes. The results suggest that the OsBISCPL1 may be involved in regulation of defense responses against pathogen infection and oxidative stress. OsBISCPL1 Molecular cloning and characterization of a novel brassinolide enhanced gene OsBLE1 in Oryza sativa seedlings 2004 Plant Physiol Biochem Department of Molecular Genetics, National Institute of Agrobiological Sciences, 2-1-2, Kannondai, Tsukuba, 305-8602, Japan. Brassinosteroids (BRs) are plant steroids essential for normal growth and development. To gain insight into the molecular mechanism by which BRs regulate the growth and development of plants, it is necessary to identify and analyze more genes that are regulated by BRs. A novel brassinolide (BL)-enhanced gene designated OsBLE1, which was originally identified by using rice (Oryza sativa L.) cDNA microarray, was cloned and characterized. Its cDNA is 598 bp long, encoding a predicted polypeptide with 81 amino acid residues. Northern blots analysis revealed that OsBLE1 expression began to increase at 6 h and reached its maximum at 12 h after BL treatment. OsBLE1 expression was most responsive to BL in lamina joint in rice seedlings; besides, IAA and GA3 also enhanced its expression. OsBLE1 expressed mainly in active tissues such as vascular bundles and root primordial. Transgenic rice expressing antisense OsBLE1 exhibits various degrees of repressed growth. Results suggest that OsBLE1 might be involved in BL-regulated growth processes in rice seedlings. OsBLE1 A novel brassinolide-enhanced gene identified by cDNA microarray is involved in the growth of rice 2003 Plant Mol Biol National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan. Brassinosteroids (BRs) are growth-promoting natural substances required for normal plant growth and development. To understand the molecular mechanism of BR action, a cDNA microarray containing 1265 rice genes was analyzed for expression differences in rice lamina joint treated with brassinolide (BL). A novel BL-enhanced gene, designated OsBLE2, was identified and cloned. The full-length cDNA is 3243 bp long, encoding a predicted polypeptide of 761 amino acid residues and nine possible transmembrane regions. OsBLE2 expression was most responsive to BL in the lamina joint and leaf sheath in rice seedlings. Besides, auxin and gibberellins also increased its expression. OsBLE2 expressed more, as revealed by in situ hybridization, in vascular bundles and root primordia, where the cells are actively undergoing division, elongation, and differentiation. Transgenic rice expressing antisense OsBLE2 exhibits various degrees of repressed growth. BL could not enhance its expression in transgenic rice expressing antisense BRI1, a BR receptor, indicating that BR signaling to the enhanced expression of OsBLE2 is through BRI1. BL effect in the d1 mutant rice was much weaker than that in its wild-type control, indicating that heterotrimeric G protein may be a component of BRs signaling. These results suggest that OsBLE2 is involved in BL-regulated growth and development processes in rice. OsBLE2 Rice LHS1/OsMADS1 controls floret meristem specification by coordinated regulation of transcription factors and hormone signaling pathways 2013 Plant Physiol Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India. SEPALLATA (SEP) MADS box transcription factors mediate floral development in association with other regulators. Mutants in five rice (Oryza sativa) SEP genes suggest both redundant and unique functions in panicle branching and floret development. leafy hull sterile1/OsMADS1, from a grass-specific subgroup of LOFSEP genes, is required for specifying a single floret on the spikelet meristem and for floret organ development, but its downstream mechanisms are unknown. Here, key pathways and directly modulated targets of OsMADS1 were deduced from expression analysis after its knockdown and induction in developing florets and by studying its chromatin occupancy at downstream genes. The negative regulation of OsMADS34, another LOFSEP gene, and activation of OsMADS55, a SHORT VEGETATIVE PHASE-like floret meristem identity gene, show its role in facilitating the spikelet-to-floret meristem transition. Direct regulation of other transcription factor genes like OsHB4 (a class III homeodomain Leu zipper member), OsBLH1 (a BEL1-like homeodomain member), OsKANADI2, OsKANADI4, and OsETTIN2 show its role in meristem maintenance, determinacy, and lateral organ development. We found that the OsMADS1 targets OsETTIN1 and OsETTIN2 redundantly ensure carpel differentiation. The multiple effects of OsMADS1 in promoting auxin transport, signaling, and auxin-dependent expression and its direct repression of three cytokinin A-type response regulators show its role in balancing meristem growth, lateral organ differentiation, and determinacy. Overall, we show that OsMADS1 integrates transcriptional and signaling pathways to promote rice floret specification and development. OsBLH1,OsETT2|OsETTIN2,OsETTIN1|OsARF15,OSHB4,OsKANADI1|OsKANADI2,OsKANADI4,OsMADS1|LHS1|AFO Identification of transcription factors involved in rice secondary cell wall formation 2013 Plant Cell Physiol Bioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi, Japan. Using co-expression network analysis, we identified 123 transcription factors (TFs) as candidate secondary cell wall regulators in rice. To validate whether these TFs are associated with secondary cell wall formation, six TF genes belonging to the MYB, NAC or homeodomain-containing TF families were overexpressed or downregulated in rice. With the exception of OsMYB58/63-RNAi plants, all transgenic plants showed phenotypes possibly related to secondary cell wall alteration, such as dwarfism, narrow and dark green leaves, and also altered rice cinnamyl alcohol dehydrogenase 2 (OsCAD2) gene expression and lignin content. These results suggest that many of the 123 candidate secondary cell wall-regulating TFs are likely to function in secondary cell wall formation in rice. Further analyses were performed for the OsMYB55/61 and OsBLH6 TFs, the former being a TF in which the Arabidopsis ortholog is known to participate in lignin biosynthesis (AtMYB61) and the latter being one for which no previous involvement in cell wall formation has been reported even in Arabidopsis (BLH6). OsMYB55/61 and OsBLH6-GFP fusion proteins localized to the nucleus of onion epidermal cells. Moreover, expression of a reporter gene driven by the OsCAD2 promoter was enhanced in rice calli when OsMYB55/61 or OsBLH6 was transiently expressed, demonstrating that they function in secondary cell wall formation. These results show the validity of identifying potential secondary cell wall TFs in rice by the use of rice co-expression network analysis. OsBLH6 Cell-type specificity of the expression of Os BOR1, a rice efflux boron transporter gene, is regulated in response to boron availability for efficient boron uptake and xylem loading 2007 Plant Cell Biotechnology Research Center, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. We describe a boron (B) transporter, Os BOR1, in rice (Oryza sativa). Os BOR1 is a plasma membrane-localized efflux transporter of B and is required for normal growth of rice plants under conditions of limited B supply (referred to as -B). Disruption of Os BOR1 reduced B uptake and xylem loading of B. The accumulation of Os BOR1 transcripts was higher in roots than that in shoots and was not affected by B deprivation; however, Os BOR1 was detected in the roots of wild-type plants under -B conditions, but not under normal conditions, suggesting regulation of protein accumulation in response to B nutrition. Interestingly, tissue specificity of Os BOR1 expression is affected by B treatment. Transgenic rice plants containing an Os BOR1 promoter-beta-glucuronidase (GUS) fusion construct grown with a normal B supply showed the strongest GUS activity in the steles, whereas after 3 d of -B treatment, GUS activity was elevated in the exodermis. After 6 d of -B treatment, GUS activity was again strong in the stele. Our results demonstrate that Os BOR1 is required both for efficient B uptake and for xylem loading of B. Possible roles of the temporal changes in tissue-specific patterns of Os BOR1 expression in response to B condition are discussed. OsBOR1 Roles of pollen-specific boron efflux transporter, OsBOR4, in the rice fertilization process 2013 Plant Cell Physiol Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, 113-8657 Japan. Arabidopsis thaliana BOR1 was the first boron (B) transporter identified in living systems. There are four AtBOR1-like genes, OsBOR1, 2, 3 and 4, present in the rice genome. We characterized the activity, expression and physiological function of OsBOR4. OsBOR4 is an active efflux transporter of B. Quantitative PCR analysis and OsBOR4 promoter-green fluorescent protein (GFP) fusion revealed that OsBOR4 was both highly and specifically expressed in pollen. We obtained five Tos17 insertion mutants of osbor4. The pollen grains were viable and development of floral organs was normal in the homozygous osbor4 mutants. We observed that in all Tos17 insertion lines tested, the frequency of osbor4 homozygous plants was lower than expected in the progeny of self-fertilized heterozygous plants. These results establish that OsBOR4 is essential for normal reproductive processes. Pollen from osbor4 homozygous plants elongated fewer tubes on wild-type stigmas, and tube elongation of mutant pollen was less efficient compared with the wild-type pollen, suggesting reduced competence of osbor4 mutant pollen. The reduced competence of mutant pollen was further supported by the crosses of independent Tos17-inserted alleles of OsBOR4. Our results suggest that OsBOR4, a boron efflux transporter, is required for normal pollen germination and/or tube elongation. OsBOR4 OsBP-73, a rice gene, encodes a novel DNA-binding protein with a SAP-like domain and its genetic interference by double-stranded RNA inhibits rice growth 2003 Plant Mol Biol National Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China. The SAP domain is a recently defined DNA binding domain that forms a helix-extended-helix structure. SAP proteins have been implicated in nuclear architecture and/or RNA metabolism. In this paper, we describe the cloning and characterization of a rice gene, OsBP-73, encoding a 375 amino acid protein with a SAP-like domain. We identified the binding sequence of OsBP-73 by gel retardation assays and southwestern blotting. Northern blot analysis demonstrated that OsBP-73 is weakly expressed in root, leaf and immature seed. OsBP-73 gene expression was also examined by histochemical studies of transgenic rice plants carrying an OsBP-73 5'/GUS reporter gene. The reporter gene is mainly expressed in the tissues with high cell division activities, such as root tip, stem node, panicle and immature seed. Genetic interference of OsBP-73 gene expression by double-stranded RNA strikingly inhibits the whole plant growth but does not affect the passage from the juvenile to adult phase. These results suggest that OsBP-73 may play an important role in the regulation of cell proliferation. OsBP-73 Molecular tagging of the Bph1 locus for resistance to brown planthopper (Nilaparvata lugens Stal) through representational difference analysis 2008 Mol Genet Genomics Rice Division, Yeongnam Agricultural Research Institute, National Institute of Crop Science, RDA, Milyang, South Korea. During brown planthopper (BPH) feeding on rice plants, we employed a modified representational difference analysis (RDA) method to detect rare transcripts among those differentially expressed in SNBC61, a BPH resistant near-isogenic line (NIL) carrying the Bph1 resistance gene. This identified 3 RDA clones: OsBphi237, OsBphi252 and OsBphi262. DNA gel-blot analysis revealed that the loci of the RDA clones in SNBC61 corresponded to the alleles of the BPH resistant donor Samgangbyeo. Expression analysis indicated that the RDA genes were up-regulated in SNBC61 during BPH feeding. Interestingly, analysis of 64 SNBC NILs, derived from backcrosses of Samgangbyeo with a BPH susceptible Nagdongbyeo, using a cleaved amplified polymorphic sequence (CAPS) marker indicated that OsBphi252, which encodes a putative lipoxygenase (LOX), co-segregates with BPH resistance. Our results suggest that OsBphi252 is tightly linked to Bph1, and may be useful in marker-assisted selection (MAS) for resistance to BPH. OsBphi262 A putative leucine-rich repeat receptor kinase, OsBRR1, is involved in rice blast resistance 2009 Planta Biotechnology Research Institute, National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, 100081, Beijing, China. penghao78@yahoo.com.cn Leucine-rich repeat receptor-like kinases (LRR-RLKs) comprise the largest subfamily of transmembrane receptor-like kinases in plants, and they regulate a wide variety of developmental and defense-related processes. In this study, RNA interference (RNAi) strategy was used to specifically knockdown 59 individual rice genes encoding putative LRR-RLKs, and a novel rice blast resistance-related gene (designated as OsBRR1) was identified by screening T(0) RNAi population using a weakly virulent isolate of Magnaporthe oryzae, Ken 54-04. Wild-type plants (Oryza sativa L. cv. 'Nipponbare') showed intermediate resistance to Ken 54-04, while OsBRR1 suppression plants were susceptible to Ken 54-04. Furthermore, OsBRR1-overexpressing plants exhibited enhanced resistance to some virulent isolates (97-27-2, 99-31-1 and zhong 10-8-14). OsBRR1 expression was low in leaves and undetectable in roots under normal growth conditions, while its transcript was significantly induced in leaves infected with the blast fungus (Ken 54-04) and was moderately affected by ABA, JA and SA treatment. Overexpression or RNAi suppression of OsBRR1 did not cause visible developmental changes in rice plants. These results indicate that OsBRR1 is involved in rice resistance responses to blast fungus and mediates resistance to rice blast. OsBRR1 Systematic identification and expression analysis of BREVIS RADIX-like homologous genes in rice 2010 Plant Science National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China BREVIS RADIX is a novel plant-specific gene family with highly conserved protein sequences; the key member of this family, BRX, controls cell proliferation and root elongation in Arabidopsis. In this study, six members of rice BRX-like gene family containing the conserved BRX domain were identified in japonica rice. Phytogenetic analysis suggested that BRX-like genes may be evolved before the divergence of vascular plant and bryophytes species. Four rice BRX-like genes were predominantly (OsBRXL1, OsBRXL2, and OsBRXL4) or specifically (OsBRXL3) expressed in young panicle. Except one member (OsBRXL6) that was not expressed in all the tissues and organs investigated, the other five genes were differentially responsive to major stresses including drought, salt, and cold, suggesting that the BRX-like family may also function in abiotic stress responses. OsBRXL1 and OsBRXL4 were significantly up-regulated by brassinosteroid and auxin, respectively. To further test the function of BRX-like genes in rice, the OsBRXL4 was overexpressed in rice Zhonghua 11. The OsBRXL4-overexpression plant showed significantly longer root and increased sensitivity to auxin than wild type, suggesting that OsBRXL4 may be involved in primary root growth through the auxin signaling pathway. OsBRXL1,OsBRXL2,OsBRXL3,OsBRXL4,OsBRXL5,OsBRXL6 Overexpression of stress-inducible OsBURP16, the beta subunit of polygalacturonase 1, decreases pectin content and cell adhesion and increases abiotic stress sensitivity in rice 2014 Plant Cell Environ Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China; Graduate University of the Chinese Academy of Sciences, Beijing, 100049, China. Polygalacturonase (PG), one of the hydrolases responsible for cell wall pectin degradation, is involved in organ consenescence and biotic stress in plants. PG1 is composed of a catalytic subunit, PG2, and a non-catalytic PG1beta subunit. OsBURP16 belongs to the PG1beta-like subfamily of BURP-family genes and encodes one putative PG1beta subunit precursor in rice (Oryza sativa L.). Transcription of OsBURP16 is induced by cold, salinity and drought stresses, as well as by abscisic acid (ABA) treatment. Analysis of plant survival rates, relative ion leakage rates, accumulation levels of H2 O2 and water loss rates of leaves showed that overexpression of OsBURP16 enhanced sensitivity to cold, salinity and drought stresses compared with controls. Young leaves of Ubi::OsBURP16 transgenic plants showed reduced cell adhesion and increased cuticular transpiration rate. Mechanical strength measurement of Ubi::OsBURP16 plants showed that reduced force was required to break leaves as compared with wild type. Transgenic rice showed enhanced PG activity and reduced pectin content. All these results suggested that overexpression of OsBURP16 caused pectin degradation and affected cell wall integrity as well as transpiration rate, which decreased tolerance to abiotic stresses. OsBURP16 Trans-acting factor designated OSBZ8 interacts with both typical abscisic acid responsive elements as well as abscisic acid responsive element-like sequences in the vegetative tissues of indica rice cultivars 2008 Plant Cell Rep Department of Botany, Bose Institute, Kolkata, 700009, West Bengal, India. Specific cis-acting elements, identified in the stress-regulated promoters, can respond to the changes in the levels of abscisic acid. Most of our previous works were done with ACGT-containing typical abscisic acid responsive elements (ABREs) but not with non-ACGT, GC-rich sequences also present in such promoters. The current communication shows a comparative analysis performed on the binding of rice nuclear proteins, together with the purified transcription factor OSBZ8, to the cis-elements in the promoters of Rab16A (Motif I/Motif II), Osem (Motif A-1/Motif B) and Em (4X ABRE/2X ABRC). Our data show that the extent of binding of nuclear protein from salt-tolerant rice to both typical ABREs and non-ACGT, ABRE-like sequences such as Motif IIa, is much higher than that from salt-sensitive rice and occurs constitutively, i.e., even with the protein from unstressed plants. The complex formation is low and inducible only by salt in the salt-sensitive variety. While Motif I bind to a single 38 kDa protein, Motif IIa bind to two polypeptides of 38 and 29 kDa. We also show here that the activation and binding of OSBZ8 to the upstream regions of salt-inducible genes depends on its phosphorylated state. The novelty of our work is that it shows rice OSBZ8 as the prime factor interacting with both typical ABRE(s) and ABRE-like sequences. To our knowledge, this is also the first report for the detection and identification of Motif IIa (non-ACGT, coupling element-like)-binding factor(s) from rice and their expression pattern in different rice cultivars. OSBZ8|OsbZIP05 Spermidine-mediated in vitro phosphorylation of transcriptional regulator OSBZ8 by SNF1-type serine/threonine protein kinase SAPK4 homolog in indica rice 2012 Acta Physiologiae Plantarum Division of Plant Biology, Bose Institute (Main Campus), 93/1 A.P.C Road, Kolkata, 700009, India Plants respond to abiotic stresses such as salinity, extreme temperature and drought by the activation of complex intracellular signaling cascades that regulate acclimatory biochemical and physiological changes. Protein kinases are major signal transduction factors that play a central role in mediating acclimation to environmental changes in eukaryotic organisms. It is well known that changes in abiotic conditions such as the concentration of ions, temperature and humidity lead to modulation of polyamine contents in plants. However, little is known about the relevant part these polyamines play in abiotic stress responses. Here, we address a specific role of spermidine during high salt stress by studying its interaction with OSPDK, a sucrose nonfermenting 1-related protein kinase2 (SnRK2)-type serine/threonine protein kinase SAPK4 homolog in indica rice. In this report, we demonstrate that spermidine mediates in vitro phosphorylation of OSBZ8, a bZIP class of ABRE-binding transcription factor, by OSPDK. Our results give a first-hand indication of the pivotal role played by polyamines in abiotic stress cell signaling in plants. OSBZ8|OsbZIP05,SAPK4|OSPDK An ABRE-binding factor, OSBZ8, is highly expressed in salt tolerant cultivars than in salt sensitive cultivars of indica rice 2006 BMC Plant Biol Department of Botany, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Kolkata 700 009, India. mkakali@yahoo.com BACKGROUND: The bZIP class Abscisic acid Responsive Element (ABRE)-binding factor, OSBZ8 (38.5 kD) has been considered to regulate ABA-mediated transcription in the suspension cultured cells of japonica rice. Still, nothing is known about the expression of OSBZ8 at protein level in vegetative tissue of salt sensitive and salt tolerant rice plants. In our previous study, Electrophoretic Mobility Shift Assay (EMSA) of [32P]ABRE-DNA and nuclear extracts prepared from the lamina of Pokkali rice plants has detected the presence of an ABRE-binding factor. Northern analysis has also detected salinity stress induced accumulation of transcripts for bZIP class of factor. Therefore, OSBZ8 was considered to play an important role in the regulation of transcription in the vegetative tissue of rice. The aim of this study is to find out whether OSBZ8 has any role in regulating the NaCl-stress induced gene expression in vegetative tissue and whether the expression of OSBZ8 factor directly correlates with the stress tolerance of different varieties of indica type rice. RESULTS: Northern analysis of total RNA from roots and lamina of salt-sensitive M-I-48 and salt-tolerant Nonabokra, when probed with the N-terminal unique region of OSBZ8 (OSBZ8p, without the highly conserved basic region), a transcript of 1.3 kb hybridized and its level was much higher in tolerant cultivar. EMSA with Em1a, the strongest ABA Responsive Element till reported from the upstream of EmBP1, and the nuclear extracts from laminar tissue of untreated and salt-treated seedlings of three salt sensitive, one moderately sensitive and two salt tolerant indica rice cultivars showed specific binding of nuclear factor to ABRE element. Intensity of binding was low and inducible in salt sensitive rice cultivars while high and constitutive in salt tolerant cultivars. EMSA with 300 bp 5'upstream region of Rab16A gene, a well known salt stress and ABA-inducible gene of rice, showed formation of two complexes, again very weak in salt sensitive and strong in salt tolerant rice cultivar. CONCLUSION: The bZIP factor OSBZ8 was found to be present in the ABRE-DNA: protein complex as shown by the supershift of the complex by the purified antiserum raised against OSBZ8p. Treatment of the seedlings with NaCl was found to enhance the complex formation, suggesting the regulation of OSBZ8 gene at both transcriptional and post-translational steps. Comparative EMSA with different varieties of rice suggests a positive correlation with the expression pattern of OSBZ8 and salt tolerance in rice cultivars. OSBZ8|OsbZIP05 A rice bZIP protein, designated OSBZ8, is rapidly induced by abscisic acid 1996 The Plant Journal Center for Molecular Biology and Genetics, Mie University, Tsu, Japan. A cDNA that encoded a bZIP protein, designated OSBZ8, was isolated from a rice embryo cDNA library by use of degenerate oligonucleotide probes that corresponded to the amino acid sequences conserved among the basic regions of plant G-box-binding factor-type bZIP proteins (GBF). OSBZ8 was shown to have structural features typical of the GBF-type bZIP proteins and to bind to G-box and G-box-like sequences that include ABA-responsive elements (ABREs) which have been functionally identified in the promoters of ABA-inducible genes, such as Em, Osem and Rab16. The accumulation of OSBZ8 mRNA was induced by treatment with ABA of imbibed mature rice embryos, of young plant tissues and of suspension-cultured cells. The accumulation of OSBZ8 mRNA in response to ABA preceded that of Osem and Rab16A mRNAs and was not inhibited by an inhibitor of protein synthesis, cycloheximide. By contrast, the induction of Osem and Rab16A was partially inhibited and almost completely inhibited, respectively, by cycloheximide. These results strongly suggest that OSBZ8 might be involved in the regulation of transcription by ABA in rice. OSBZ8|OsbZIP05 Identification of a novel rice bZIP-type transcription factor gene,OsbZIP1, involved in response to infection ofMagnaporthe grisea 2012 Plant Molecular Biology Reporter State Key Laboratory of Agrobiotechnology and Department of Plant Pathology, China Agricultural University, 100094, Beijing, China The basic leucine zipper (bZIP) proteins belong to a large family of transcriptional factors, some of which are thought to be involved in plant responses to biotic and abiotic stimuli. In this work, we cloned a novel bZIP gene from rice, designatedOsbZIP1, which is inducible in response to infection withMagnaporthe grisea. Sequence analysis showed that the deduced protein had a potential nuclear localization signal and 2 possible acidic activation domains. Results of transient expression experiments demonstrated that OsbZIP1 is localized in the nucleus. The ability of OsbZIP1 to activate the transcription of the reporter gene was also determined in yeast. Northern blotting analysis showed thatOsbZIP1 was constitutively expressed in the roots and strongly induced in rice leaves in response to infection with both the compatible race MS220 and incompatible race 131 ofM. grisea. Furthermore, OsbZIP1 expression could be rapidly induced in leaves treated with salicylic acid, jasmonic acid, and abscisic acid. These results indicate that the OsbZIP1 may function as a transcription factor and may play a role in salicylic acid-dependent signal transduction pathway for defense of rice against pathogens. OsbZIP01,OsbZIP28|OsbZIP1 Basic leucine zipper transcription factor OsbZIP16 positively regulates drought resistance in rice 2012 Plant Sci College of Life Science, Hunan Normal University, Changsha 410081, China. Abiotic stress has been shown to limit the growth, development, and productivity of crops. Here, we characterized the function of a rice bZIP transcription factor OsbZIP16 in drought stress. Expression of OsbZIP16 was dramatically induced under drought conditions. Transient expression and transactivation assays demonstrated that OsbZIP16 was localized in the nucleus and had transactivation activity. At both the seedling and tillering stages, transgenic rice plants overexpressing OsbZIP16 exhibited significantly improved drought resistance, which was positively correlated with the observed expression levels of OsbZIP16. Representative downstream drought-inducible genes were observed to have significantly higher expression levels in transgenic rice plants than in the wild type plants under drought conditions. OsbZIP16 was shown to be induced by exogenous ABA treatment, while overexpression of OsbZIP16 was observed to make transgenic plants more sensitive to ABA than wild type plants were. Transcriptome analysis identified a number of differentially expressed genes between wild type plants and plants overexpressing OsbZIP16, many of which are involved in stress response according to their gene ontologies. Overall, our findings suggest that OsbZIP16 positively regulates drought resistance in rice. OsbZIP16 Characterization of OsbZIP23 as a key player of the basic leucine zipper transcription factor family for conferring abscisic acid sensitivity and salinity and drought tolerance in rice 2008 Plant Physiol National Center of Plant Gene Research , National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China. OsbZIP23 is a member of the basic leucine zipper (bZIP) transcription factor family in rice (Oryza sativa). Expression of OsbZIP23 is strongly induced by a wide spectrum of stresses, including drought, salt, abscisic acid (ABA), and polyethylene glycol treatments, while other stress-responsive genes of this family are slightly induced only by one or two of the stresses. Transactivation assay in yeast demonstrated that OsbZIP23 functions as a transcriptional activator, and the sequences at the N terminus (amino acids 1-59) and a region close to the C terminus (amino acids 210-240) are required for the transactivation activity. Transient expression of OsbZIP23-green fluorescent protein in onion (Allium cepa) cells revealed a nuclear localization of the protein. Transgenic rice overexpressing OsbZIP23 showed significantly improved tolerance to drought and high-salinity stresses and sensitivity to ABA. On the other hand, a null mutant of this gene showed significantly decreased sensitivity to a high concentration of ABA and decreased tolerance to high-salinity and drought stress, and this phenotype can be complemented by transforming the OsbZIP23 back into the mutant. GeneChip and real-time polymerase chain reaction analyses revealed that hundreds of genes were up- or down-regulated in the rice plants overexpressing OsbZIP23. More than half of these genes have been annotated or evidenced for their diverse functions in stress response or tolerance. In addition, more than 30 genes that are possible OsbZIP23-specific target genes were identified based on the comparison of the expression profiles in the overexpressor and the mutant of OsbZIP23. Collectively, these results indicate that OsbZIP23 functions as a transcriptional regulator that can regulate the expression of a wide spectrum of stress-related genes in response to abiotic stresses through an ABA-dependent regulation pathway. We propose that OsbZIP23 is a major player of the bZIP family in rice for conferring ABA-dependent drought and salinity tolerance and has high potential usefulness in genetic improvement of stress tolerance. OsbZIP23 Overexpression of a new stress-repressive gene OsDSR2 encoding a protein with a DUF966 domain increases salt and simulated drought stress sensitivities and reduces ABA sensitivity in rice 2014 Plant Cell Rep Xiamen Key Laboratory for Plant Genetics, School of Life Sciences, Xiamen University, Xiamen, 361005, China. Domain of Unknown Function 966 (DUF966) gene family was found in the protein family database, which consisted of seven genes in rice. The proteins encoded by these genes contained one or two highly conserved DUF966 domains. The available data of public microarray databases implied that these genes might play crucial roles in plant response to abiotic stresses. In this study, a member of the DUF966 gene family, DUF966-stress repressive gene 2 in Oryza sativa (OsDSR2, Loc_Os01g62200), was cloned and its role in rice responding to salt and simulated drought stresses was functionally characterized. OsDSR2 was expressed mainly in nodes of stems and leaf blades from rice. Expression profile analysis of adversity showed that OsDSR2 had different transcriptional responses to salt, drought, cold, heat and oxidative (H2O2) stresses, as well as abscisic acid (ABA), methyl jasmonate, salicylic acid, gibberellin acid and auxin treatments. Transient expression demonstrated that OsDSR2 was localized in the membrane and nucleus. Overexpression of OsDSR2 could increase salt and simulated drought (polyethyleneglycol)-stress sensitivities in rice by downregulating the expression of ABA- and stress-responsive genes including OsNCED4, SNAC1, OsbZIP23, P5CS, Oslea3 and rab16C. Furthermore, OsDSR2-overexpressing plants showed reduced ABA sensitivity during the post-germination stage. These results suggested that OsDSR2 negatively regulated rice response to salt and simulated drought stresses as well as ABA signaling, which provided some useful data for understanding the functional roles of DUF966 family genes in abiotic stress responses in plants. OsbZIP23,OsDSR2,OsLEA3|OsLEA3-1,OsNCED4,OsP5CS|OsP5CS1,OsNAC19|SNAC1|OsNAC9 A rice transmembrane bZIP transcription factor, OsbZIP39, regulates the endoplasmic reticulum stress response 2012 Plant Cell Physiol Functional Transgenic Crops Research Unit, Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan. The endoplasmic reticulum (ER) responds to the accumulation of unfolded proteins in its lumen (ER stress) by activating intracellular signal transduction pathways. These pathways are known as the ER stress response or the unfolded protein response. In this study, three rice basic leucine zipper (bZIP) transcription factors (OsbZIP39, OsbZIP50 and OsbZIP60) containing putative transmembrane domains (TMDs) in their C-terminal regions were identified as candidates of the ER stress sensor transducer. One of these proteins, OsbZIP39, was characterized in this study. OsbZIP39 was shown to associate with microsomes as a membrane-integrated protein using the subcellular fractionation method. When the full length and a truncated form of OsbZIP39 without the TMD (OsbZIP39DeltaC) was fused to green fluorescent protein (GFP) and transfected into rice protoplasts, the proteins were identified in the cytoplasm and nucleus, respectively. This suggests that OsbZIP39 may be converted into a soluble truncated form by proteolytic cleavage and subsequently translocated to the nucleus. Expression of OsbZIP39DeltaC clearly activated the binding protein 1 (BiP1) promoter in a rice protoplast transient assay. Overexpression of OsbZIP39DeltaC in stable transgenic rice also led to the up-regulation of several ER stress response genes including BiP1 and OsbZIP50 in the absence of ER stress. However, in the OsbZIP39DeltaC-overexpressing line, OsbZIP50 mRNA did not undergo IRE1 (inositol-requiring protein 1)-mediated cytoplasmic splicing that is required for its activation. These data indicate that OsbZIP39 may be directly involved in the regulation of several ER stress response genes. OsbZIP39 Cis-element of the rice PDIL2-3 promoter is responsible for inducing the endoplasmic reticulum stress response 2014 J Biosci Bioeng Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. A protein disulfide isomerase (PDI) family oxidoreductase, PDIL2-3, is involved in endoplasmic reticulum (ER) stress responses in rice. We identified a critical cis-element required for induction of the ER stress response. The activation of PDIL2-3 in response to ER stress strongly depends on the IRE1-OsbZIP50 signaling pathway. OsbZIP50|OsbZIP74,IRE1|OsIRE1,PDIL2;3 Conservation of IRE1-regulated bZIP74 mRNA unconventional splicing in rice (Oryza sativa L.) involved in ER stress responses 2012 Mol Plant State Key Laboratory of Genetic Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, 200433, China. Protein folding in the endoplasmic reticulum (ER) is a fundamental process in plant cells that is vulnerable to many environmental stresses. When unfolded or misfolded proteins accumulate in the ER, the well-conserved unfolded protein response (UPR) is initiated to mitigate the ER stress by enhancing the protein folding capability and/or accelerating the ER-associated protein degradation. Here, we report the conservation of the activation mechanism of OsbZIP74 (also known as OsbZIP50), an important ER stress regulator in monocot plant rice (Oryza sativa L.). Under normal conditions, OsbZIP74 mRNA encodes a basic leucine-zipper transcription factor with a putative transmembrane domain. When treating with ER stress-inducing agents such as tunicamycin and DTT, the conserved double stem-loop structures of OsbZIP74 mRNA are spliced out. Thereafter, the resulting new OsbZIP74 mRNA produces the nucleus-localized form of OsbZIP74 protein, eliminating the hydrophobic region. The activated form of OsbZIP74 has transcriptional activation activity in both yeast cells and Arabidopsis leaf protoplasts. The induction of OsbZIP74 splicing is much suppressed in the OsIRE1 knock-down rice plants, indicating the involvement of OsIRE1 in OsbZIP74 splicing. We also demonstrate that the unconventional splicing of OsbZIP74 mRNA is associated with heat stress and salicylic acid, which is an important plant hormone in systemic acquired resistance against pathogen or parasite. OsbZIP50|OsbZIP74,IRE1|OsIRE1 Multiple roles of the ER stress sensor IRE1 demonstrated by gene targeting in rice 2012 Sci Rep Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences , Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. The endoplasmic reticulum (ER) stress sensor, IRE1, contains a kinase domain and a ribonuclease domain. Ribonuclease mediates the unconventional splicing of mRNA encoding the transcription factor AtbZIP60 in Arabidopsis, or OsbZIP50 in rice, and thereby transduces signals from stressed ER. Here, we demonstrate the additional roles of plant IRE1 using genetically modified rice plants. Using a gene targeting system based on homologous recombination, genomic IRE1 was replaced with two types of missense alleles, leading to a defect in the kinase or ribonuclease activity of IRE1. Genetic analysis of these alleles demonstrated that the kinase activity of IRE1 plays a vital role independent of ribonuclease activity. Furthermore, the existence of ribonuclease substrates other than OsbZIP50 mRNA is demonstrated for the first time. This study provides new insights into higher plant signalling using a gene targeting approach. OsbZIP50|OsbZIP74,IRE1|OsIRE1 bZIP transcription factor OsbZIP52/RISBZ5: a potential negative regulator of cold and drought stress response in rice 2012 Planta Key Laboratory of Cell Proliferation and Regulation of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, People's Republic of China. OsbZIP52/RISBZ5 is a member of the basic leucine zipper (bZIP) transcription factor (TF) family in rice (Oryza sativa) isolated from rice (Zhonghua11) panicles. Expression of the OsbZIP52 gene was strongly induced by low temperature (4 degrees C) but not by drought, PEG, salt, or ABA. The subcellular localization of OsbZIP52-GFP in onion (Allium cepa) epidermis cells revealed that OsbZIP52 is a nuclear localized protein. A transactivation assay in yeast demonstrated that OsbZIP52 functions as a transcriptional activator and can specifically bind to the G-box promoter motif. In a yeast two-hybrid (Y-2-H) experiment, OsbZIP52 was able to form homodimeric complexes. Rice plants overexpressing OsbZIP52 showed significantly increased sensitivity to cold and drought stress. Real-time PCR analysis revealed that some abiotic stress-related genes, such as OsLEA3, OsTPP1, Rab25, gp1 precursor, beta-gal, LOC_Os05g11910 and LOC_Os05g39250, were down-regulated in OsbZIP52 overexpression lines. These results suggest that OsbZIP52/RISBZ5 could function as a negative regulator in cold and drought stress environments. OsbZIP52|RISBZ5,OsLEA3|OsLEA3-1,OsTPP1 Analysis of ER stress in developing rice endosperm accumulating beta-amyloid peptide 2010 Plant Biotechnol J Transgenic Crop Research and Development Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan. The common neurodegenerative disorder known as Alzheimer's disease is characterized by cerebral neuritic plaques of amyloid beta (Abeta) peptide. Plaque formation is related to the highly aggregative property of this peptide, because it polymerizes to form insoluble plaques or fibrils causing neurotoxicity. Here, we expressed Abeta peptide as a new causing agent to endoplasmic reticulum (ER) stress to study ER stress occurred in plant. When the dimer of Abeta(1-42) peptide was expressed in maturing seed under the control of the 2.3-kb glutelin GluB-1 promoter containing its signal peptide, a maximum of about 8 mug peptide per grain accumulated and was deposited at the periphery of distorted ER-derived PB-I protein bodies. Synthesis of Abeta peptide in the ER lumen severely inhibited the synthesis and deposition of seed storage proteins, resulting in the generation of many small and abnormally appearing PB bodies. This ultrastructural change was accounted for by ER stress leading to the accumulation of aggregated Abeta peptide in the ER lumen and a coordinated increase in ER-resident molecular chaperones such as BiPs and PDIs in Abeta-expressing plants. Microarray analysis also confirmed that expression of several BiPs, PDIs and OsbZIP60 containing putative transmembrane domains was affected by the ER stress response. Abeta-expressing transgenic rice kernels exhibited an opaque and shrunken phenotype. When grain phenotype and expression levels were compared among transgenic rice grains expressing several different recombinant peptides, such detrimental effects on grain phenotype were correlated with the expressed peptide causing ER stress rather than expression levels. OsbZIP60 OsbZIP71, a bZIP transcription factor, confers salinity and drought tolerance in rice 2014 Plant Mol Biol State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China. The bZIP transcription factor (TF) family plays an important role in the abscisic acid (ABA) signaling pathway of abiotic stress in plants. We here report the cloning and characterization of OsbZIP71, which encodes a rice bZIP TF. Functional analysis showed that OsbZIP71 is a nuclear-localized protein that specifically binds to the G-box motif, but has no transcriptional activity both in yeast and rice protoplasts. In yeast two-hybrid assays, OsbZIP71 can form both homodimers and heterodimers with Group C members of the bZIP gene family. Expression of OsbZIP71 was strongly induced by drought, polyethylene glycol (PEG), and ABA treatments, but repressed by salt treatment. OsbZIP71 overexpressing (p35S::OsbZIP71) rice significantly improved tolerance to drought, salt and PEG osmotic stresses. In contrast, RNAi knockdown transgenic lines were much more sensitive to salt, PEG osmotic stresses, and also ABA treatment. Inducible expression (RD29A::OsbZIP71) lines were significantly improved their tolerance to PEG osmotic stresses, but hypersensitivity to salt, and insensitivity to ABA. Real-time PCR analysis revealed that the abiotic stress-related genes, OsVHA-B, OsNHX1, COR413-TM1, and OsMyb4, were up-regulated in overexpressing lines, while these same genes were down-regulated in RNAi lines. Chromatin immunoprecipitation analysis confirmed that OsbZIP71 directly binds the promoters of OsNHX1 and COR413-TM1 in vivo. These results suggest that OsbZIP71 may play an important role in ABA-mediated drought and salt tolerance in rice. OsbZIP71,Osmyb4,OsNHX1 Identification of OsbZIP72 as a positive regulator of ABA response and drought tolerance in rice 2009 Planta Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. Abscisic Acid (ABA) is an important phytohormone involved in abiotic stress resistance in plants. A group of bZIP transcription factors play important roles in the ABA signaling pathway in Arabidopsis. However, little is known about the function of their orthologs in rice, where they may hold a great potential for developing drought resistant food crops. In this study, our phylogenetic analysis showed that this group of bZIPs was evolutionarily conserved between Arabidopsis and rice, which implies that they may share similar functions. We demonstrated with quantitative RT-PCR that the expressions of most of these OsbZIPs were significantly induced by ABA, ACC, and abiotic stresses. OsbZIP72, a member of this group, was proved to be an ABRE binding factor in rice using the yeast hybrid systems. We showed that it could bind to ABRE and transactivate the downstream reporter genes in yeast, and the transactivity was depending on its N-terminal region. Transgenic rice overexpressing OsbZIP72 showed a hypersensitivity to ABA, elevated levels of expression of ABA response gene such as LEAs, and an enhanced ability of drought tolerance. These results suggest that OsbZIP72 plays a positive role in drought resistance through ABA signaling, and is potential useful for engineering drought tolerant rice. OsbZIP72 Functions of OsBZR1 and 14-3-3 proteins in brassinosteroid signaling in rice 2007 Proc Natl Acad Sci U S A Key Laboratory of Photosynthesis and Environmental Molecular Biology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. Brassinosteroids (BR) are essential growth hormones found throughout the plant kingdom. BR bind to the receptor kinase BRI1 on the cell surface to activate a signal transduction pathway that regulates nuclear gene expression and plant growth. To understand the downstream BR signaling mechanism in rice, we studied the function of OsBZR1 using reverse genetic approaches and identified OsBZR1-interacting proteins. Suppressing OsBZR1 expression by RNAi resulted in dwarfism, erect leaves, reduced BR sensitivity, and altered BR-responsive gene expression in transgenic rice plants, demonstrating an essential role of OsBZR1 in BR responses in rice. Moreover, a yeast two-hybrid screen identified 14-3-3 proteins as OsBZR1-interacting proteins. Mutation of a putative 14-3-3-binding site of OsBZR1 abolished its interaction with the 14-3-3 proteins in yeast and in vivo. Such mutant OsBZR1 proteins suppressed the phenotypes of the Arabidopsis bri1-5 mutant and showed an increased nuclear distribution compared with the wild-type protein, suggesting that 14-3-3 proteins directly inhibit OsBZR1 function at least in part by reducing its nuclear localization. These results demonstrate a conserved function of OsBZR1 and an important role of 14-3-3 proteins in brassinosteroid signal transduction in rice. OsBZR1 OsCIPK31, a CBL-interacting protein kinase is involved in germination and seedling growth under abiotic stress conditions in rice plants 2010 Mol Cells Division of Applied Life Science (Brain Korea 21 Program), Plant Molecular Biology and Biotechnology Research Center, Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Korea. Calcineurin B-like protein-interacting protein kinases (CIPKs) are a group of typical Ser/Thr protein kinases that mediate calcium signals. Extensive studies using Arabidopsis plants have demonstrated that many calcium signatures that activate CIPKs originate from abiotic stresses. However, there are few reports on the functional demonstration of CIPKs in other plants, especially in grasses. In this study, we used a loss-of-function mutation to characterize the function of the rice CIPK gene OsCIPK31. Exposure to high concentrations of NaCl or mannitol effected a rapid and transient enhancement of OsCIPK31 expression. These findings were observed only in the light. However, longer exposure to most stresses resulted in downregulation of OsCIPK31 expression in both the presence and absence of light. To determine the physiological roles of OsCIPK31 in rice plants, the sensitivity of oscipk31::Ds, which is a transposon Ds insertion mutant, to abiotic stresses was examined during germination and seedling stages. oscipk31::Ds mutants exhibited hypersensitive phenotypes to ABA, salt, mannitol, and glucose. Compared with wild-type rice plants, mutants exhibited retarded germination and slow seedling growth. In addition, oscipk31::Ds seedlings exhibited enhanced expression of several stress-responsive genes after exposure to these abiotic stresses. However, the expression of ABA metabolic genes and the endogenous levels of ABA were not altered significantly in the oscipk31::Ds mutant. This study demonstrated that rice plants use OsCIPK31 to modulate responses to abiotic stresses during the seed germination and seedling stages and to modulate the expression of stress-responsive genes. OsCK1|OsCIPK31|CIPK03 Calcium sensors and their interacting protein kinases: genomics of the Arabidopsis and rice CBL-CIPK signaling networks 2004 Plant Physiol Abteilung Pflanzenphysiologie, Universitat Rostock, Albert-Einstein-Strasse 3, 18051 Rostock, Germany. Calcium signals mediate a multitude of plant responses to external stimuli and regulate a wide range of physiological processes. Calcium-binding proteins, like calcineurin B-like (CBL) proteins, represent important relays in plant calcium signaling. These proteins form a complex network with their target kinases being the CBL-interacting protein kinases (CIPKs). Here, we present a comparative genomics analysis of the full complement of CBLs and CIPKs in Arabidopsis and rice (Oryza sativa). We confirm the expression and transcript composition of the 10 CBLs and 25 CIPKs encoded in the Arabidopsis genome. Our identification of 10 CBLs and 30 CIPKs from rice indicates a similar complexity of this signaling network in both species. An analysis of the genomic evolution suggests that the extant number of gene family members largely results from segmental duplications. A phylogenetic comparison of protein sequences and intron positions indicates an early diversification of separate branches within both gene families. These branches may represent proteins with different functions. Protein interaction analyses and expression studies of closely related family members suggest that even recently duplicated representatives may fulfill different functions. This work provides a basis for a defined further functional dissection of this important plant-specific signaling system. OsCK1|OsCIPK31|CIPK03,PK4|OsPK4|OsCIPK19 Characterization of stress-responsive CIPK genes in rice for stress tolerance improvement 2007 Plant Physiol National Center of Plant Gene Research, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China. Plants respond to adverse environments by initiating a series of signaling processes that often involves diverse protein kinases, including calcineurin B-like protein-interacting protein kinases (CIPKs). In this study, putative CIPK genes (OsCIPK01-OsCIPK30) in the rice (Oryza sativa) genome were surveyed for their transcriptional responses to various abiotic stresses. The results showed that 20 OsCIPK genes were differentially induced by at least one of the stresses, including drought, salinity, cold, polyethylene glycol, and abscisic acid treatment. Most of the genes induced by drought or salt stress were also induced by abscisic acid treatment but not by cold. A few CIPK genes containing none of the reported stress-responsive cis-elements in their promoter regions were also induced by multiple stresses. To prove that some of these stress-responsive OsCIPK genes are potentially useful for stress-tolerance improvement, three CIPK genes (OsCIPK03, OsCIPK12, and OsCIPK15) were overexpressed in japonica rice 'Zhonghua 11'. Transgenic plants overexpressing the transgenes OsCIPK03, OsCIPK12, and OsCIPK15 showed significantly improved tolerance to cold, drought, and salt stress, respectively. Under cold and drought stresses, OsCIPK03- and OsCIPK12-overexpressing transgenic plants accumulated significantly higher contents of proline and soluble sugars than the wild type. Putative proline synthetase and transporter genes had significantly higher expression level in the transgenic plants than in the wild type. The differentially induced expression of OsCIPK genes by different stresses and the examples of improved stress tolerance of the OsCIPK transgenic rice suggest that rice CIPK genes have diverse roles in different stress responses and some of them may possess potential usefulness in stress-tolerance improvement of rice. OsCK1|OsCIPK31|CIPK03,OsCIPK12,OsCIPK15,PK4|OsPK4|OsCIPK19 Isolation and characterization of a novel rice Ca2+-regulated protein kinase gene involved in responses to diverse signals including cold, light, cytokinins, sugars and salts 2003 Plant Mol Biol Department of Molecular Physiology, National Institute of Agricultural Biotechnology, Suweon 441-707, Korea. We have isolated a cold-inducible gene (designated OsCK1) from Oryza sativa by a differential cDNA screening technique. Sequence analysis indicated that the open reading frame of the OsCK1 gene consists of 1350 bp encoding 449 amino acid residues, which is very similar to a family of calcineurin B-like protein (CBL)-interacting protein kinases (CIPKs) or salt overly sensitive 2 (SOS2)-like protein kinases (PKS) in Arabidopsis. CIPKs/PKS are a group of Ser/Thr protein kinases associated with the AtCBL/SOS3-like calcium-binding proteins (SCaBP). OsCK1 actually interacts with AtCBL3 through the C-terminal region in a yeast two-hybrid system, suggesting that OsCK1 is probably a rice orthologue of one of the CIPK/PKS members. Expression of the OsCK1 gene was detected mainly in the shoots and highly inducible by diverse signals such as cold, light, salt, sugar and cytokinins. In addition, calcium increased the OsCK1 transcript level, whereas a calcium ionophore, A23187, partially abolished stimulus-induced expressions. OsCK1 phosphorylated itself and a generic substrate, myelin basic protein, in the preference of Mn2+. Deletion of the C-terminal region from OsCK1 significantly decreased autophosphorylation activity without affecting the ability for substrate phosphorylation. These findings suggest that the CBL/CIPK or SCaBP/PKS signaling pathways recently found in Arabidopsis may also exist in rice and function in cold response in which calcium signal serves as a second messenger. OsCK1|OsCIPK31|CIPK03 Antiquity and function of CASTOR and POLLUX, the twin ion channel-encoding genes key to the evolution of root symbioses in plants 2009 Plant Physiol Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky 40546, USA. Root symbioses with arbuscular mycorrhizal fungi and rhizobial bacteria share a common signaling pathway in legumes. Among the common symbiosis genes are CASTOR and POLLUX, the twin homologous genes in Lotus japonicus that encode putative ion channel proteins. Here, we show that the orthologs of CASTOR and POLLUX are ubiquitously present and highly conserved in both legumes and nonlegumes. Using rice (Oryza sativa) as a study system, we employ reverse genetic tools (knockout mutants and RNA interference) to demonstrate that Os-CASTOR and Os-POLLUX are indispensable for mycorrhizal symbiosis in rice. Furthermore, a cross-species complementation test indicates that Os-POLLUX can restore nodulation, but not rhizobial infection, to a Medicago truncatula dmi1 mutant. Os-CASTOR,Os-POLLUX Allelic diversification at the C (OsC1) locus of wild and cultivated rice: nucleotide changes associated with phenotypes 2004 Genetics Plant Breeding Laboratory, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. Divergent phenotypes are often detected in domesticated plants despite the existence of invariant phenotypes in their wild forms. One such example in rice is the occurrence of varying degrees of apiculus coloration due to anthocyanin pigmentation, which was previously reported to be caused by a series of alleles at the C locus. The present study reveals, on the basis of comparison of its maps, that the C gene appears to be the rice homolog (OsC1) of maize C1, which belongs to the group of R2R3-Myb factors. Two different types of deletions causing a frameshift were detected in the third exon, and both of the deleted nucleotides corresponded to the positions of putative base-contacting residues, suggesting that the Indica and Japonica types carry loss-of-function mutations with independent origins. In addition, replacement substitutions were frequently detected in OsC1 of strains carrying the previously defined C alleles. Molecular population analysis revealed that 17 haplotypes were found in 39 wild and cultivated rices, and the haplotypes of most cultivated forms could be classified into one of three distinct groups, with few shared haplotypes among taxa, including Indica and Japonica types. The genealogy of the OsC1 gene suggests that allelic diversification causing phenotypic change might have resulted from mutations in the coding region rather than from recombination between preexisting alleles. The McDonald and Kreitman test revealed that the changes in amino acids might be associated with selective forces acting on the lineage of group A whose haplotypes were carried by most Asian cultivated forms. The results regarding a significant implication for genetic diversity in landraces of rice are also discussed. OsC1 Tapetum degeneration retardation is critical for aliphatic metabolism and gene regulation during rice pollen development 2008 Mol Plant Shanghai Jiao Tong University, Shanghai Institutes for Biological Sciences, China. As a complex wall system in flowering plants, the pollen outer wall mainly contains aliphatic sporopollenin; however, the mechanism for synthesizing these lipidic precursors during pollen development remains less well understood. Here, we report on the function of the rice tapetum-expressing TDR (Tapetum Degeneration Retardation) gene in aliphatic metabolism and its regulatory role during rice pollen development. The observations of transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses suggested that pollen wall formation was significantly altered in the tdr mutant. The contents of aliphatic compositions of anther were greatly changed in the tdr mutant revealed by GC-MS (gas chromatography-mass spectrometry) testing, particularly less accumulated in fatty acids, primary alcohols, alkanes and alkenes, and an abnormal increase in secondary alcohols with carbon lengths from C29 to C35 in tdr. Microarray data revealed that a group of genes putatively involved in lipid transport and metabolism were significantly altered in the tdr mutant, indicating the critical role of TDR in the formation of the pollen wall. Also, a wide range of genes (236 in total-154 up-regulated and 82 down-regulated) exhibited statistically significant expressional differences between wild-type and tdr. In addition to its function in promoting tapetum PCD, TDR possibly plays crucial regulatory roles in several basic biological processes during rice pollen development. OSC4,OsPKS1|YY2,TDR,OsACS,OsCER1,OsCHS,RAFTIN1 Isolation of genes abundantly expressed in rice anthers at the microspore stage 1992 Plant Mol Biol Institute for Cell Biology and Genetics, Faculty of Agriculture, Iwate University, 020, Morioka, Japan A cDNA library of rice (Oryza sativa L.) has been constructed from anthers at an early stage of pollen development. By differential screening of the library, we have isolated cDNAs of two genes, designated as Osc4 and Osc6, that are abundantly expressed in anthers containing tetrads and uninucleate microspores, but are not expressed in leaves or roots. Expression of Osc4 is absent in mature anthers, while Osc6 is present although the expression decays during pollen maturation. A comparison of the nucleotide and deduced amino acid sequences with those in data banks has not shown significant homology to known molecules. OSC4 Molecular characterization of rice genes specifically expressed in the anther tapetum 1994 Plant Mol Biol Institute for Cell Biology and Genetics, Faculty of Agriculture, Iwate University, 020, Morioka, Japan In situ localization of mRNA was carried out on two cDNAs (Osc4 and Osc6) that had been isolated from rice anthers at the microspore stage. The mRNA corresponding to each cDNA was shown to be localized only in the tapetal cells of the rice immature anthers, but not in the microspores or the mature pollen. The corresponding genomic clone, Osg6B, was isolated, and its 5'-upstream region was found to regulate beta-glucuronidase expression in the tapetum of transgenic tobacco. A set of 5' deletions was also generated and a 1095 bp 5' region was revealed to be necessary for activation of the Osg6B promoter in transgenic tobacco. OSC4 High temperatures cause male sterility in rice plants with transcriptional alterations during pollen development 2009 Plant Cell Physiol Rice Biotechnology Research Team, National Institute of Crop Science, NARO, Tsukuba, Ibaraki 305-8518, Japan. Plant male reproductive development is highly organized and sensitive to various environmental stressors, including high temperature. We have established an experimental procedure to evaluate high temperature injury in japonica rice plants. High temperature treatment (39 degrees C/30 degrees C) starting at the microspore stage repeatedly reduced spikelet fertility in our system. Morphological observations revealed that pollen viability in plants exposed to high temperatures was lower than that in control plants. Most pollen grains in high temperature-treated plants displayed a normal round shape and stained reddish purple with Alexander's reagent; however, the pollen grains were very poorly attached and displayed limited germination on the stigma. To investigate gene regulatory mechanisms in the anther in high temperature environments, DNA microarray analysis was performed by comparing non-treated samples with samples treated with 2-4 d of high heat. Genes responsive to high temperatures were identified from clustering of microarray data. Among these, at least 13 were designated as high temperature-repressed genes in the anther. Expression analyses revealed that these genes were expressed specifically in the immature anther mainly in the tapetum at the microspore stage and down-regulated after 1 d of high temperature. The expression levels of Osc6, OsRAFTIN and TDR, which are tapetum-specific genes, were unaffected by high temperatures. These results suggest that not all tapetal genes are inhibited by increased temperatures and the tapetum itself is not degraded in such an environment. However, high temperatures may disrupt some of the tapetum functions required for pollen adhesion and germination on the stigma. OsC6,TDR The rice tapetum degeneration retardation gene is required for tapetum degradation and anther development 2006 Plant Cell Shanghai Jiao Tong University-Shanghai Institutes for Biological Sciences-Pensylvania State University Joint Center for Life Sciences, Shanghai 200240, China. In flowering plants, tapetum degeneration is proposed to be triggered by a programmed cell death (PCD) process during late stages of pollen development; the PCD is thought to provide cellular contents supporting pollen wall formation and to allow the subsequent pollen release. However, the molecular basis regulating tapetum PCD in plants remains poorly understood. We report the isolation and characterization of a rice (Oryza sativa) male sterile mutant tapetum degeneration retardation (tdr), which exhibits degeneration retardation of the tapetum and middle layer as well as collapse of microspores. The TDR gene is preferentially expressed in the tapetum and encodes a putative basic helix-loop-helix protein, which is likely localized to the nucleus. More importantly, two genes, Os CP1 and Os c6, encoding a Cys protease and a protease inhibitor, respectively, were shown to be the likely direct targets of TDR through chromatin immunoprecipitation analyses and the electrophoretic mobility shift assay. These results indicate that TDR is a key component of the molecular network regulating rice tapetum development and degeneration. OsC6,OsCP1,TDR Expression of a carbonic anhydrase gene is induced by environmental stresses in rice (Oryza sativa L.) 2007 Biotechnol Lett Alkali Soil Natural Environmental Science Center, Stress Molecular Biology Laboratory, Northeast Forestry University, Harbin, PR China. Expression of the gene (OsCA1) coding for carbonic anhydrase (CA) in leaves and roots of rice was induced by environmental stresses from salts (NaCl, NaHCO(3) and Na(2)CO(3)), and osmotic stress (10%, w/v, PEG 6000). CA activity of rice seedlings more than doubled under some of these stresses. Transgenic Arabidopsis over-expressing OsCA1 had a greater salt tolerance at the seedling stage than wild-type plants in 1/2 MS medium with 5 mM NaHCO(3), 50 mM NaCl, on 100 mM NaCl. Thus CA expression responds to environmental stresses and is related to stress tolerance in rice. OsCA1 Light regulation of circadian clock-controlled gene expression in rice 2001 The Plant Journal Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan. Using transgenic rice seedlings expressing a firefly luciferase (luc) gene under the control of a rice CAB (chlorophyll a/b binding protein) promoter, we demonstrated how light affects circadian clocks in the transcription of the CAB gene. Rhythmic luc expression was observed for more than 5 d under constant light and dark (DD) conditions after light/dark entrainment. After a light pulse was applied at different time points in DD various temporal patterns of CAB gene expression were individually observed. We first examined two distinct properties related to the entrainment mechanism of the circadian clock: fluence-rate dependence of free-running periods (FRPs) and phase resetting by a light pulse. Although fluence-rate dependent shortening of FRP was demonstrated, the FRP in DD was almost equal to that in constant light of a middle fluence-rate, indicating that this fluence-rate dependence may not fully describe the entrainment of the circadian clock in rice. Typical phase responses of the circadian clock by a single light pulse were also observed at the transcriptional level in rice seedlings. Thus, the phase resettings upon the light/dark transitions of daily cycles may be sufficient to explain the entrainment mechanisms of rice. We have further demonstrated that, in addition to having a gating effect to acute response, a light pulse can activate the circadian clock-controlled CAB1R gene expression at the first circadian peak in a phase-dependent manner. This suggests that light activates circadian clock activity in the diurnal CAB gene expression under daily light/dark cycles. OsCAB1R Structure and expression analysis of the OsCam1-1 calmodulin gene from Oryza sativa L 2008 BMB Rep Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand. Calmodulin (CaM) proteins, members of the EF-hand family of Ca(2+)- binding proteins, represent important relays in plant calcium signals. Here, OsCam1-1 was isolated by PCR amplification from the rice genome. The gene contains an ORF of 450 base pairs with a single intron at the same position found in other plant Cam genes. A promoter region with a TATA box at position-26 was predicted and fused to a gus reporter gene, and this construct was used to produce transgenic rice by Agrobacterium-mediated transformation. GUS activity was observed in all organs examined and throughout tissues in cross-sections, but activity was strongest in the vascular bundles of leaves and the vascular cylinders of roots. To examine the properties of OsCaM1-1, the encoding cDNA was expressed in Escherichia coli. The electrophoretic mobility shift when incubated with Ca(2+) indicates that recombinant OsCaM1-1 is a functional Ca(2+)-binding protein. In addition, OsCaM1-1 bound the CaMKII target peptide confirming its likely functionality as a calmodulin. OsCam1-1 Exogenous ABA induces salt tolerance in indica rice (Oryza sativa L.): The role of OsP5CS1 and OsP5CR gene expression during salt stress 2013 Environmental and Experimental Botany Environment and Plant Physiology Research Unit, Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Abscisic acid (ABA) applied exogenously at 100 μM prior to and during the salt-stress period induced salt tolerance in both the salt-susceptible (LPT123) and the genetically related salt-resistant (LPT123-TC171) rice lines, enhanced the survival rate by 20%, and triggered proline (Pro) accumulation earlier than that by salt-stress alone, supporting a role for Pro as an osmoprotectant. In both rice lines, salt-stress induced OsP5CS1 gene expression, suggesting that proline accumulation occurs via OsP5CS1 gene expression during salt stress. An increase in the endogenous ABA level was required for the induction of OsP5CS1 gene expression by salt stress. Under salt stress, topical ABA application-induced OsP5CS1 gene expression only in the salt-resistant line but up-regulated OsP5CR gene expression in both rice lines, suggesting that the increased proline accumulation and salt resistance induced by topical ABA application may result from the up-regulation of OsP5CR and not, directly at least, from OsP5CS1. Moreover, exogenous ABA application up-regulates OsCam1-1 (the salt-stress-responsive calmodulin) gene expression, and calmodulin was shown to play a role in the signal transduction cascade in proline accumulation during salt stress. These data suggest the role of the calmodulin signaling cascade and the induction of OsP5CR gene expression in proline accumulation by exogenous ABA application. OsCam1-1,OsP5CR,OsP5CS|OsP5CS1 Genome-wide identification and analyses of the rice calmodulin and related potential calcium sensor proteins 2007 BMC Plant Biol Department of Biochemistry, Faculty of Science, Chulalongkorn University, Payathai Road, Patumwan, Bangkok 10330, Thailand. b.bongkoj@gmail.com BACKGROUND: A wide range of stimuli evoke rapid and transient increases in [Ca2+]cyt in plant cells which are transmitted by protein sensors that contain EF-hand motifs. Here, a group of Oryza sativa L. genes encoding calmodulin (CaM) and CaM-like (CML) proteins that do not possess functional domains other than the Ca2+-binding EF-hand motifs was analyzed. RESULTS: By functional analyses and BLAST searches of the TIGR rice database, a maximum number of 243 proteins that possibly have EF-hand motifs were identified in the rice genome. Using a neighbor-joining tree based on amino acid sequence similarity, five loci were defined as Cam genes and thirty two additional CML genes were identified. Extensive analyses of the gene structures, the chromosome locations, the EF-hand motif organization, expression characteristics including analysis by RT-PCR and a comparative analysis of Cam and CML genes in rice and Arabidopsis are presented. CONCLUSION: Although many proteins have unknown functions, the complexity of this gene family indicates the importance of Ca2+-signals in regulating cellular responses to stimuli and this family of proteins likely plays a critical role as their transducers. OsCam1-1,OsCam1-2|OsCam1,OsCam1-3|OsCaM1-3,OsCam2,OsCam3 Heat shock-induced biphasic Ca(2+) signature and OsCaM1-1 nuclear localization mediate downstream signalling in acquisition of thermotolerance in rice (Oryza sativa L.) 2012 Plant Cell Environ Institute of Plant Biology & Department of Life Science, National Taiwan University, Taipei 10617, Taiwan. We investigated heat shock (HS)-triggered Ca(2+) signalling transduced by a Ca(2+) sensor, calmodulin (CaM), linked to early transcriptome changes of HS-responsive genes in rice. We observed a biphasic [Ca(2+) ](cyt) signature in root cells that was distinct from that in epicotyl and leaf cells, which showed a monophasic response after HS. Treatment with Ca(2+) and A23187 generated an intense and sustained increase in [Ca(2+) ](cyt) in response to HS. Conversely, treatment with Ca(2+) chelator, L-type Ca(2+) channel blocker and CaM antagonist, but not intracellular Ca(2+) release inhibitor, strongly inhibited the increased [Ca(2+) ](cyt) . HS combined with Ca(2+) and A23187 accelerated the expression of OsCaM1-1 and sHSPC/N genes, which suggests that the HS-induced apoplastic Ca(2+) influx is responsible for the [Ca(2+) ](cyt) response and downstream HS signalling. In addition, the biphasic response of OsCaM1-1 in the nucleus followed the Ca(2+) signature, which may provide the information necessary to direct HS-related gene expression. Overexpression of OsCaM1-1 induced the expression of Ca(2+) /HS-related AtCBK3, AtPP7, AtHSF and AtHSP at a non-inducing temperature and enhanced intrinsic thermotolerance in transgenic Arabidopsis. Therefore, HS-triggered rapid increases in [Ca(2+) ](cyt) , together with OsCaM1-1 expression and its nuclear localization, are important in mediating downstream HS-related gene expression for the acquisition of thermotolerance in rice. OsCam1-1,OsCam1-3|OsCaM1-3 Calcium signaling-mediated and differential induction of calmodulin gene expression by stress in Oryza sativa L 2005 J Biochem Mol Biol Department of Biochemistry, Faculty of Science, Chulalongkorn University, Payathai Road, Patumwan, Bangkok 10330, Thailand. Ca(2+)/calmodulin transduction pathways have been implicated in mediating stress response and tolerance in plants. Here, three genes encoding calmodulin (Cam) members of the EF-hand family of Ca(2+)-binding proteins were identified from Oryza sativa L. databases. Complementary DNA for each of the calmodulin genes, OsCam1, OsCam2, and OsCam3 were sequenced. OsCam1 and OsCam2 encode a conventional 148-amino acid calmodulin protein that contains four characteristic Ca(2+)-binding motifs. OsCam3 encode a similar protein with a 38-amino-acid extension containing a putative prenylation site (CVIL) at the carboxyl terminus. RT-PCR showed that each of the genes is expressed in leaves and roots of 2-week old rice seedlings. By RNA gel blot analysis, OsCam1 mRNA levels strongly increased in response to NaCl, mannitol and wounding treatments. In contrast, OsCam2 mRNA levels were relatively unchanged under all conditions investigated. NaCl treatment and wounding also increased the OsCam3 mRNA level, but in a more transient manner. Our results indicate that although the expression of genes encoding different calmodulin isoforms is ubiquitous, they are differentially regulated by various stress signals. In addition, we have demonstrated that the calcium-channel blocker lanthanum chloride inhibited the induction of OsCam1 gene expression by both NaCl and mannitol treatments. These results suggest that osmotic stressinduced expression of OsCam1 gene requires the [Ca(2+)]cyt elevation that is known to occur in response to these stimuli. OsCam1-2|OsCam1,OsCam2,OsCam3 RNAi-directed downregulation of vacuolar H(+) -ATPase subunit a results in enhanced stomatal aperture and density in rice 2013 PLoS One School of Life Science, Chongqing University, Chongqing, China. Stomatal movement plays a key role in plant development and response to drought and salt stress by regulating gas exchange and water loss. A number of genes have been demonstrated to be involved in the regulation of this process. Using inverse genetics approach, we characterized the function of a rice (Oryza sativa L.) vacuolar H(+)-ATPase subunit A (OsVHA-A) gene in stomatal conductance regulation and physiological response to salt and osmotic stress. OsVHA-A was constitutively expressed in different rice tissues, and the fusion protein of GFP-OsVHA-A was exclusively targeted to tonoplast when transiently expressed in the onion epidermal cells. Heterologous expression of OsVHA-A was able to rescue the yeast mutant vma1Delta (lacking subunit A activity) phenotype, suggesting that it partially restores the activity of V-ATPase. Meanwhile, RNAi-directed knockdown of OsVHA-A led to a reduction of vacuolar H(+)-ATPase activity and an enhancement of plasma membrane H(+)-ATPase activity, thereby increasing the concentrations of extracellular H(+) and intracellular K(+) and Na(+) under stress conditions. Knockdown of OsVHA-A also resulted in the upregulation of PAM3 (plasma membrane H(+)-ATPase 3) and downregulation of CAM1 (calmodulin 1), CAM3 (calmodulin 3) and YDA1 (YODA, a MAPKK gene). Altered level of the ion concentration and the gene expression by knockdown of OsVHA-A probably resulted in expanded aperture of stomatal pores and increased stomatal density. In addition, OsVHA-A RNAi plants displayed significant growth inhibition under salt and osmotic stress conditions. Taken together, our results suggest that OsVHA-A takes part in regulating stomatal density and opening via interfering with pH value and ionic equilibrium in guard cells and thereby affects the growth of rice plants. OsCam1-3|OsCaM1-3,YDA1 Biophysical characterization of calmodulin and calmodulin-like proteins from rice, Oryza sativa L 2011 Acta Biochim Biophys Sin (Shanghai) Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. Calmodulin (CaM) transduces the increase in cytosolic Ca(2+) concentrations by binding to and altering the activities of target proteins, thereby affecting the physiological responses to the vast array of stimuli. Here, we examined the purified recombinant proteins encoded by three Cam and eight Cam-like (CML) genes from rice. With the exception of one OsCML, all recombinant proteins could be purified by Ca(2+)-dependent hydrophobic chromatography and exhibited an electrophoretic mobility shift when incubated with Ca(2+). The three CaMs all bound CaM kinase II peptide, but none of the eight CMLs did, suggesting a possible differential target binding between the CaM and CML proteins. In addition, their conformational changes upon Ca(2+)-binding were evaluated by circular dichroism spectroscopy and fluorescence spectroscopy using 8-Anilino-1-naphthalene-sulfonic acid. Taken together, OsCMLs were found exhibiting a spectrum of both structural and functional characteristics that ranged from typical to atypical of CaMs. From structural comparison, the OsCMLs have overall main-chain conformation nearly identical to OsCaMs, but with distinct distribution of some charged and hydrophobic amino acids on their target-binding site. These results suggest that genetic polymorphism has promoted the functional diversity of the OsCML family, whose members possess modes of actions probably different from, though maybe overlapping with, those of OsCaMs. OsCam2,OsCam3 Calmodulin isoform-specific activation of a rice calmodulin-binding kinase conferred by only three amino-acids of OsCaM61 2006 FEBS Lett Key Lab of MOE for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China. The kinase activity of a Ca(2+)/calmodulin (CaM)-binding serine/threonine protein kinase from rice (Oryza sativa) (OsCBK) has been reported to be unaffected by OsCaM1 binding. In this study, we examined whether other rice CaMs can stimulate OsCBK. It was observed that OsCaM61 stimulated OsCBK in a Ca(2+)-dependent manner. In addition, Ala(111), Gly(123) and Ser(127) were identified as critical residues for OsCBK activation. Mutational study and fluorescent spectroscopy analysis indicated that CaM-binding affinity does not correlate with the kinase activity and that these key amino-acids in OsCaM61 play a vital role in suitable changes of OsCBK conformation for kinase activation. OsCaM61,OsCBK Structural analysis of a calmodulin variant from rice: the C-terminal extension of OsCaM61 regulates its calcium binding and enzyme activation properties 2013 J Biol Chem From the Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada and. OsCaM61 is one of five calmodulins known to be present in Oryza sativa that relays the increase of cytosolic [Ca(2+)] to downstream targets. OsCaM61 bears a unique C-terminal extension with a prenylation site. Using nuclear magnetic resonance (NMR) spectroscopy we studied the behavior of the calmodulin (CaM) domain and the C-terminal extension of OsCaM61 in the absence and presence of Ca(2+). NMR dynamics data for OsCaM61 indicate that the two lobes of the CaM domain act together unlike the independent behavior of the lobes seen in mammalian CaM and soybean CaM4. Also, data demonstrate that the positively charged nuclear localization signal region in the tail in apo-OsCaM61 is helical, whereas it becomes flexible in the Ca(2+)-saturated protein. The extra helix in apo-OsCaM61 provides additional interactions in the C-lobe and increases the structural stability of the closed apo conformation. This leads to a decrease in the Ca(2+) binding affinity of EF-hands III and IV in OsCaM61. In Ca(2+)-OsCaM61, the basic nuclear localization signal cluster adopts an extended conformation, exposing the C-terminal extension for prenylation or enabling OsCaM61 to be transferred to the nucleus. Moreover, Ser(172) and Ala(173), residues in the tail, interact with different regions of the protein. These interactions affect the ability of OsCaM61 to activate different target proteins. Altogether, our data show that the tail is not simply a linker between the prenyl group and the protein but that it also provides a new regulatory mechanism that some plants have developed to fine-tune Ca(2+) signaling events. OsCaM61 OsCAND1 is required for crown root emergence in rice 2011 Mol Plant State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, P.R. China. Crown roots are main components of the fibrous root system and important for crops to anchor and absorb water and nutrition. To understand the molecular mechanisms of crown root formation, we isolated a rice mutant defective in crown root emergence designated as Oscand1 (named after the Arabidopsis homologous gene AtCAND1). The defect of visible crown root in the Oscand1 mutant is the result of cessation of the G2/M cell cycle transition in the crown root meristem. Map-based cloning revealed that OsCAND1 is a homolog of Arabidopsis CAND1. During crown root primordium development, the expression of OsCAND1 is confined to the root cap after the establishment of fundamental organization. The transgenic plants harboring DR5::GUS showed that auxin signaling in crown root tip is abnormal in the mutant. Exogenous auxin application can partially rescue the defect of crown root development in Oscand1. Taken together, these data show that OsCAND1 is involved in auxin signaling to maintain the G2/M cell cycle transition in crown root meristem and, consequently, the emergence of crown root. Our findings provide new information about the molecular regulation of the emergence of crown root in rice. OsCAND1 Differential regulation of chlorophyll a oxygenase genes in rice 2005 Plant Mol Biol National Research Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, 790-784, Korea. Chlorophyll b is synthesized from chlorophyll a by chlorophyll a oxygenase. We have identified two genes (OsCAO1 and OsCAO2) from the rice genome that are highly homologous to previously studied chlorophyll a oxygenase (CAO) genes. They are positioned in tandem, probably resulting from recent gene duplications. The proteins they encode contain two conserved functional motifs - the Rieske Fe-sulfur coordinating center and a non-heme mononuclear Fe-binding site. OsCAO1 is induced by light and is preferentially expressed in photosynthetic tissues. Its mRNA level decreases when plants are grown in the dark. In contrast, OsCAO2 mRNA levels are higher under dark conditions, and its expression is down-regulated by exposure to light. To elucidate the physiological function of the CAO genes, we have isolated knockout mutant lines tagged by T-DNA or Tos17. Mutant plants containing a T-DNA insertion in the first intron of the OsCAO1 gene have pale green leaves, indicating chlorophyll b deficiency. We have also isolated a pale green mutant with a Tos17 insertion in that OsCAO1 gene. In contrast, OsCAO2 knockout mutant leaves do not differ significantly from the wild type. These results suggest that OsCAO1 plays a major role in chlorophyll b biosynthesis, and that OsCAO2 may function in the dark. OsCAO1,OsCAO2 Heterologous expression analyses of rice OsCAS in Arabidopsis and in yeast provide evidence for its roles in cyanide detoxification rather than in cysteine synthesis in vivo 2009 J Exp Bot School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, China. While most dicot plants produce little ethylene in their vegetative stage, many monocots such as rice liberate a relatively large amount of ethylene with cyanide as a co-product in their seedling stage when etiolated. One of the known functions of beta-cyanoalanine synthase (CAS) is to detoxify the co-product cyanide during ethylene biosynthesis in higher plants. Based on a tryptic peptide sequence obtained from a partially purified CAS activity protein preparation in etiolated rice seedlings, the full-length putative rice CAS-encoding cDNA sequence (OsCAS), which is homologous to those O-acetylserine sulphydrylase (OASS) genes, was cloned. Unlike most of the CAS genes reported from dicots, the transcription of OsCAS is promoted by auxins but suppressed by ethylene. To address the function and the subcellular localization of this gene product in planta, a binary vector construct consisting of this gene appended with a yellow fluorescent protein-encoding sequence was employed to transform Arabidopsis. Specific activities on CAS and OASS of the purified recombinant protein from transgenic Arabidopsis were 181.04 micromol H(2)S mg(-1) protein min(-1) and 0.92 micromol Cys mg(-1) protein min(-1), respectively, indicating that OsCAS favours CAS activity. The subcellular localization of OsCAS was found mostly in the mitochondria by immunogold electron-microscopy. Chemical cross-linking and in-gel assay on a heterodimer composed of functional and non-functional mutants in a yeast expression system on OsCAS suggested that OsCAS functions as a homodimer, similar to that of OASS. Despite the structural similarity of OsCAS with OASS, it has also been confirmed that OsCAS could not interact with serine-acetyltransferase, indicating that OsCAS mainly functions in cyanide detoxification. OsCAS ABA controls H(2)O(2) accumulation through the induction of OsCATB in rice leaves under water stress 2011 Plant Cell Physiol Department of Biology, Hong Kong Baptist University, Hong Kong, China. The production of both ABA and H(2)O(2) is induced by drought and can act as signals under stress conditions. We investigated the relationships between ABA, H(2)O(2) and catalase (CAT) in rice leaves when rice seedlings were treated with polyethylene glycol as water stress treatment. As a key gene in ABA biosynthesis, OsNCED3 was significantly induced in rice by water stress treatment and such induction preceded the rapid increase in ABA. Water stress inhibited the expression of CATA and CATC but substantially enhanced the expression of CATB. Exogenously applied ABA promoted the expression of CATB also and inhibited the expression of CATC in a concentration-dependent manner. When ABA production was inhibited by using ABA biosynthesis inhibitors nordihydroguaiaretic acid and tungstate, expression of CATB was also subdued while CATC was enhanced under the water stress. Accumulation of H(2)O(2) was also reduced when endogenous ABA production was inhibited and showed a correlation with the total activity of catalases. Our results suggest that water stress-induced ABA prevents the excessive accumulation of H(2)O(2), through the induction of the expression of CATB gene during water stress. OsCATA,OsCATB,OsCATC|noe1,OsNCED3 Nitric oxide and protein S-nitrosylation are integral to hydrogen peroxide-induced leaf cell death in rice 2012 Plant Physiol State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Chinese Academy of Sciences, Beijing 100101, China. Nitric oxide (NO) is a key redox-active, small molecule involved in various aspects of plant growth and development. Here, we report the identification of an NO accumulation mutant, nitric oxide excess1 (noe1), in rice (Oryza sativa), the isolation of the corresponding gene, and the analysis of its role in NO-mediated leaf cell death. Map-based cloning revealed that NOE1 encoded a rice catalase, OsCATC. Furthermore, noe1 resulted in an increase of hydrogen peroxide (H(2)O(2)) in the leaves, which consequently promoted NO production via the activation of nitrate reductase. The removal of excess NO reduced cell death in both leaves and suspension cultures derived from noe1 plants, implicating NO as an important endogenous mediator of H(2)O(2)-induced leaf cell death. Reduction of intracellular S-nitrosothiol (SNO) levels, generated by overexpression of rice S-nitrosoglutathione reductase gene (GSNOR1), which regulates global levels of protein S-nitrosylation, alleviated leaf cell death in noe1 plants. Thus, S-nitrosylation was also involved in light-dependent leaf cell death in noe1. Utilizing the biotin-switch assay, nanoliquid chromatography, and tandem mass spectrometry, S-nitrosylated proteins were identified in both wild-type and noe1 plants. NO targets identified only in noe1 plants included glyceraldehyde 3-phosphate dehydrogenase and thioredoxin, which have been reported to be involved in S-nitrosylation-regulated cell death in animals. Collectively, our data suggest that both NO and SNOs are important mediators in the process of H(2)O(2)-induced leaf cell death in rice. OsCATC|noe1 Expression of the vacuolar Ca2+/H+ exchanger, OsCAX1a, in rice: cell and age specificity of expression, and enhancement by Ca2+ 2006 Plant Cell Physiol Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Japan. Calcium is an essential macronutrient for plants and functions in signal transduction. Regulation of the cytosolic calcium concentration is required for normal cell growth. In calcium homeostasis in plant cells, Ca(2+)/H(+) exchangers are involved in Ca(2+) compartmentalization into intracellular compartments. Here, we examine the intracellular localization of a rice Ca(2+)/H(+) exchanger, OsCAX1a, fused to a green fluorescent protein and transiently expressed in onion epidermis and rice protoplasts. Green fluorescence was observed in the vacuolar membrane. After sucrose gradient centrifugation of the homogenate of rice plants, OsCAX1a was detected in the same fraction as the vacuolar membrane aquaporin gamma-TIP. We then quantified the mRNA and protein of OsCAX1a in plants grown with metal ions. OsCAX1a mRNA was induced in roots by high concentrations of Ca(2+). The protein level in shoots was also increased in the presence of high concentrations of Ca(2+). Furthermore, transgenic rice plants transformed with the OsCAX1a promoter fused to beta-glucuronidase showed reporter expression in vascular bundles, stomata, trichomes, steles, flowers, embryos and aleurone layers. In the case of stomata and trichomes, transcription of OsCAX1a was particularly high in aged organs. These results suggest that OsCAX1a transports Ca(2+) into vacuoles and is involved in Ca(2+) homeostasis in cells that suffer from high concentrations of Ca(2+). OsCAX1a Molecular and biochemical characterization of a calcium/calmodulin-binding protein kinase from rice 2002 Biochem J The Key Lab of MOE for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China. A Ca2+/calmodulin (CaM)-binding protein kinase from rice ( Oryza sativa ), OsCBK, has been characterized that lacks Ca2+-binding EF hands and has Ca2+/CaM-independent autophosphorylation and substrate-phosphorylation activity. OsCBK has all 11 subdomains of a kinase catalytic domain and a putative CaM-binding domain, and shares high identity with Ca2+-dependent-protein-kinase ('CDPK')-related protein kinases in plants. OsCBK bound CaM in a Ca2+-dependent manner as previously reported for Ca2+/calmodulin-dependent protein kinases in animals, but autophosphorylation and phosphorylation of histone IIIs were Ca2+/CaM-independent. Surface plasmon resonance analysis showed that OsCBK specifically bound CaM with high affinity ( K (D)=30 nM). Capillary electrophoresis showed that phosphorylation of OsCBK occurred on serine and threonine residues. These data show that OsCBK is a serine/threonine protein kinase that binds Ca2+/CaM, but whose enzymic activity is independent of Ca2+/CaM. In situ hybridization showed that OsCBK is expressed in reproductive and vegetative tissues of rice and shows temporal and spatial changes during plant growth and development. OsCBK is highly expressed in zones of cell division and it is particularly abundant in sporogenous cells of the anther at meiosis. OsCBK A gibberellin-regulated calcineurin B in rice localizes to the tonoplast and is implicated in vacuole function 2005 Plant Physiol Department of Plant and Microbial Biology, University of California, Berkeley, California 94720-3102, USA. Many developmental and environmental signals are transduced through changes in intracellular calcium concentrations, yet only a few calcium-binding proteins have been identified in plants. Calcineurin B-like (CBL) proteins are calcium-binding proteins that are thought to function as plant signal transduction elements. RNA profiling using a rice (Oryza sativa cv Nipponbare) oligonucleotide microarray was used to monitor gene expression in de-embryonated rice grains. This analysis showed that a putative rice CBL gene responded to gibberellic acid, but not abscisic acid, treatment. The CBL gene family in rice contains at least 10 genes and these have extensive similarity to the CBLs of Arabidopsis (Arabidopsis thaliana). In yeast (Saccharomyces cerevisiae) two-hybrid assays, rice CBLs interact with the kinase partners of Arabidopsis CBLs. Only one rice CBL gene, OsCBL2, is up-regulated by GA in the aleurone layer. A homolog with 91% sequence identity to OsCBL2 was cloned from barley (Hordeum vulgare cv Himalaya), and designated HvCBL2. We examined the localization and function of OsCBL2 and HvCBL2 in rice and barley aleurone because changes in cytosolic calcium have been implicated in the response of the aleurone cell to GA. Green fluorescent protein translational fusions of OsCBL2 and OsCBL3 were localized to the tonoplast of aleurone cell protein storage vacuoles and OsCBL4-green fluorescent protein was localized to the plasma membrane. Data from experiments using antisense expression of OsCBL2 and HvCBL2 are consistent with a role for OsCBL2 in promoting vacuolation of barley aleurone cells following treatment with GA. OsCBL2 Expression analysis of the calcineurin B-like gene family in rice (Oryza sativa L.) under environmental stresses 2008 Gene State key lab of crop genetics and germplasm enhancement, Nanjing Agricultural University, Nanjing, 210095, PR China. Calcium plays a crucial role as a second messenger in mediating various defense responses under environmental stresses. Calcineurin B-like (CBL) proteins have been implicated as important Ca2+ sensors in plant-specific calcium signaling. Based on the similarity of sequence, ten CBL genes were identified by searching the rice japonica genome database, which were randomly distribute on chromosomes 1, 2, 3, 5, 10 and 12. By semi-quantitative RT-PCR approach the expression pattern of each gene was detected in various organs at the adult stage and seedlings treated with NaCl, PEG and cold stresses or exogenous ABA. The results showed that the induction of each rice CBL gene was not only responsive to different stress conditions, but also organ specific. In vivo targeting experiment revealed that OsCBL8 localized to the plasma membrane, which was consistent with OsCBL4 and SOS3 previously reported. To elucidate the putative function of OsCBL8 gene, transgenic rice plants overexpressing OsCBL8 gene were generated by the Agrobacterium-mediated approach. The OsCBL8 transgenic rice seedlings showed more tolerance to salt stress than non-transgenic seedlings. OsCBL8 Preliminary Study on Function of Calcineurin B-Like Protein Gene OsCBL8 in Rice 2010 Rice Science State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China The homozygous T3 transgenic lines with sense OsCBL8 gene and antisense OsCBL8 gene obtained by agro-transformation were used to investigate the function of OsCBL8 in rice. Semi-quantitative RT-PCR showed that the expression of OsCBL8 extremely increased in sense transgenic lines, and decreased to some extents in antisense transgenic lines. Such up- and down-regulation of the OsCBL8 gene in these transgenic lines had little effects on main agronomic traits, but significantly decreased the number of filled grains per panicle and seed setting rate in some of transgenic lines. By evaluation of the tolerance to 150 mmol/L NaCl, 20% PEG6000 and low temperature treatments, and relevant physiological indices, 8F12, a sense transgenic line with high salt tolerance, and 8R14, an antisense transgenic line with high drought tolerance, were obtained, which suggests that the OsCBL8 gene is involved in the response of rice to abiotic stresses. OsCBL8 Overexpression of rice CBS domain containing protein improves salinity, oxidative, and heavy metal tolerance in transgenic tobacco 2012 Mol Biotechnol Plant Molecular Biology, International Center for Genetic Engineering & Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, India. We have recently identified and classified a cystathionine beta-synthase domain containing protein family in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa L.). Based on the microarray and MPSS data, we have suggested their involvement in stress tolerance. In this study, we have characterized a rice protein of unknown function, OsCBSX4. This gene was found to be upregulated under high salinity, heavy metal, and oxidative stresses at seedling stage. Transgenic tobacco plants overexpressing OsCBSX4 exhibited improved tolerance toward salinity, heavy metal, and oxidative stress. This enhanced stress tolerance in transgenic plants could directly be correlated with higher accumulation of OsCBSX4 protein. Transgenic plants could grow and set seeds under continuous presence of 150 mM NaCl. The total seed yield in WT plants was reduced by 80%, while in transgenic plants, it was reduced only by 15-17%. The transgenic plants accumulated less Na+, especially in seeds and maintained higher net photosynthesis rate and Fv/Fm than WT plants under NaCl stress. Transgenic seedlings also accumulated significantly less H2O2 as compared to WT under salinity, heavy metal, and oxidative stress. OsCBSX4 overexpressing transgenic plants exhibit higher abiotic stress tolerance than WT plants suggesting its role in abiotic stress tolerance in plants. OsCBSX4 Isolation of a calmodulin-binding transcription factor from rice (Oryza sativa L.) 2005 J Biol Chem Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660-701, Korea. Calmodulin (CaM) regulates diverse cellular functions by modulating the activities of a variety of enzymes and proteins. However, direct modulation of transcription factors by CaM has been poorly understood. In this study, we isolated a putative transcription factor by screening a rice cDNA expression library by using CaM:horse-radish peroxidase as a probe. This factor, which we have designated OsCBT (Oryza sativa CaM-binding transcription factor), has structural features similar to Arabidopsis AtSRs/AtCAMTAs and encodes a 103-kDa protein because it contains a CG-1 homology DNA-binding domain, three ankyrin repeats, a putative transcriptional activation domain, and five putative CaM-binding motifs. By using a gel overlay assay, gel mobility shift assays, and site-directed mutagenesis, we showed that OsCBT has two different types of functional CaM-binding domains, an IQ motif, and a Ca(2+)-dependent motif. To determine the DNA binding specificity of OsCBT, we employed a random binding site selection method. This analysis showed that OsCBT preferentially binds to the sequence 5'-TWCG(C/T)GTKKKKTKCG-3' (W and K represent A or C and T or G, respectively). OsCBT was able to bind this sequence and activate beta-glucuronidase reporter gene expression driven by a minimal promoter containing tandem repeats of these sequences in Arabidopsis leaf protoplasts. Green fluorescent protein fusions of two putative nuclear localization signals of OsCBT, a bipartite and a SV40 type, were predominantly localized in the nucleus. Most interestingly, the transcriptional activation mediated by OsCBT was inhibited by co-transfection with a CaM gene. Taken together, our results suggest that OsCBT is a transcription activator modulated by CaM. OsCBT The calmodulin-binding transcription factor OsCBT suppresses defense responses to pathogens in rice 2009 Mol Cells Division of Applied Life Science (Brain Korea 21 program), Plant Molecular Biology and Biotechnology Research Center and Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Korea. We previously isolated the OsCBT gene, which encodes a calmodulin (CaM)-binding protein, from a rice expression library constructed from fungal elicitor-treated rice suspension cells. In order to understand the function of OsCBT in rice, we isolated and characterized a T-DNA insertion mutant allele named oscbt-1. The oscbt-1 mutant exhibits reduced levels of OsCBT transcripts and no significant morphological changes compared to wild-type plant although the growth of the mutant is stunted. However, oscbt-1 mutants showed significant resistance to two major rice pathogens. The growth of the rice blast fungus Magnaporthe grisea, as well as the bacterial pathogen Xanthomonas oryzae pv. oryzae was significantly suppressed in oscbt-1 plants. Histochemical analysis indicated that the hypersensitive-response was induced in the oscbt-1 mutant in response to compatible strains of fungal pathogens. OsCBT expression was induced upon challenge with fungal elicitor. We also observed significant increase in the level of pathogenesis-related genes in the oscbt-1 mutant even under pathogen-free condition. Taken together, the results support an idea that OsCBT might act as a negative regulator on plant defense. OsCBT High-level and ubiquitous expression of the rice cytochrome c gene OsCc1 and its promoter activity in transgenic plants provides a useful promoter for transgenesis of monocots 2002 Plant Physiol Department of Biological Science, Myongji University, Yongin 449-728, Korea. Expression patterns of a rice (Oryza sativa) cytochrome c gene OsCc1 and its promoter activity were characterized in transgenic rice plants. OsCc1 transcripts accumulate in most cell types, but to varying levels. Large amounts of OsCc1 transcripts are found in the roots, calli, and suspension cells, but relatively lower in mature leaves, demonstrating its higher levels of expression in non-photosynthetic tissues. Unlike the human cytochrome c gene, which is responsive to cAMP, OsCc1 expression is not enhanced in various rice tissues after dibutyryl cAMP treatments. OsCc1 promoter was linked to the sgfp gene and its activities in different tissues and cell types of transgenic rice plants were analyzed in comparison with the Act1 and RbcS promoters. OsCc1 promoter directs expression in virtually all organs of transgenic plants including roots, leaves, calli, embryos, and suspension cells, showing a particularly high activity in calli and roots. Activity of the OsCc1 promoter was 3-fold higher than Act1 in calli and roots and comparable with RbcS in leaves, representing a useful alternative to the maize (Zea mays) Ubi1 and the rice Act1 promoters for transgene expression in monocots. OsCc1 Cloning and functional characterization of a cation-chloride cotransporter gene OsCCC1 2011 Plant Mol Biol Kay Laborarory of Plant Stress Research, School of Life Science, Shandong Normal University, Jinan, Shandong Province, People's Republic of China. Potassium (K+) and chloride (Cl-) are two essential elements for plant growth and development. While it is known that plants possess specific membrane transporters for transporting K+ and Cl-, it remains unclear if they actively use K+-coupled Cl- cotransporters (KCC), as used in animals, to transport K+ and Cl-. We have cloned an Oryza sativa cDNA encoding for a member of the cation-Cl- cotransporter (CCC) family. Phylogenetic analysis revealed that plant CCC proteins are highly conserved and that they have greater sequence similarity to the sub-family of animal K--Cl- cotransporters than to other cation-Cl- cotransporters. Real-time PCR revealed that the O. sativa cDNA, which was named OsCCC1, can be induced by KCl in the shoot and root and that the expression level was higher in the leaf and root tips than in any other part of the rice plant. The OsCCC1 protein was located not only in onion plasma membrane but also in O. sativa plasma membrane. The OsCCC1 gene-silenced plants grow more slowly than wild-type (WT) plants, especially under the KCl treatment regime. After 1 month of KCl treatment, the leaf tips of the gene-silenced lines were necrosed. In addition, seed germination, root length, and fresh and dry weight were distinctly lower in the gene-silenced lines than in WT plants, especially after KCl treatment. Analysis of Na+, K+, and Cl- contents of the gene-silenced lines and WT plants grown under the NaCl and KCl treatment regimes revealed that the former accumulated relatively less K+ and Cl- than the latter but that they did not differ in terms of Na+ contents, suggesting OsCCC1 may be involved in K+ and Cl- transport. Results from different tests indicated that the OsCCC1 plays a significant role in K+ and Cl- homeostasis and rice plant development. OsCCC1 Overexpression of the rice carotenoid cleavage dioxygenase 1 gene in Golden Rice endosperm suggests apocarotenoids as substrates in planta 2010 Planta Faculty of Biology, Institute of Biology II, Albert-Ludwigs University of Freiburg, Schaenzlestr 1, 79104 Freiburg, Germany. Carotenoids are converted by carotenoid cleavage dioxygenases that catalyze oxidative cleavage reactions leading to apocarotenoids. However, apocarotenoids can also be further truncated by some members of this enzyme family. The plant carotenoid cleavage dioxygenase 1 (CCD1) subfamily is known to degrade both carotenoids and apocarotenoids in vitro, leading to different volatile compounds. In this study, we investigated the impact of the rice CCD1 (OsCCD1) on the pigmentation of Golden Rice 2 (GR2), a genetically modified rice variety accumulating carotenoids in the endosperm. For this purpose, the corresponding cDNA was introduced into the rice genome under the control of an endosperm-specific promoter in sense and anti-sense orientations. Despite high expression levels of OsCCD1 in sense plants, pigment analysis revealed carotenoid levels and patterns comparable to those of GR2, pleading against carotenoids as substrates in rice endosperm. In support, similar carotenoid contents were determined in anti-sense plants. To check whether OsCCD1 overexpressed in GR2 endosperm is active, in vitro assays were performed with apocarotenoid substrates. HPLC analysis confirmed the cleavage activity of introduced OsCCD1. Our data indicate that apocarotenoids rather than carotenoids are the substrates of OsCCD1 in planta. OsCCD1 The interactions among DWARF10, auxin and cytokinin underlie lateral bud outgrowth in rice 2010 J Integr Plant Biol The State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Previous studies have shown that DWARF10 (D10) is a rice ortholog of MAX4/RMS1/DAD1, encoding a carotenoid cleavage dioxygenase and functioning in strigolactones/strigolactone-derivatives (SL) biosynthesis. Here we use D10- RNA interference (RNAi) transgenic plants similar to d10 mutant in phenotypes to investigate the interactions among D10, auxin and cytokinin in regulating rice shoot branching. Auxin levels in node 1 of both decapitated D10-RNAi and wild type plants decreased significantly, showing that decapitation does reduce endogenous auxin concentration, but decapitation has no clear effects on auxin levels in node 2 of the same plants. This implies that node 1 may be the location where a possible interaction between auxin and D10 gene would be detected. D10 expression in node 1 is inhibited by decapitation, and this inhibition can be restored by exogenous auxin application, indicating that D10 may play an important role in auxin regulation of SL. The decreased expression of most OsPINs in shoot nodes of D10-RNAi plants may cause a reduced auxin transport capacity. Furthermore, effects of auxin treatment of decapitated plants on the expression of cytokinin biosynthetic genes suggest that D10 promotes cytokinin biosynthesis by reducing auxin levels. Besides, in D10-RNAi plants, decreased storage cytokinin levels in the shoot node may partly account for the increased active cytokinin contents, resulting in more tillering phenotypes. D10|OsCCD8|OsCCD8b A new lead chemical for strigolactone biosynthesis inhibitors 2010 Plant Cell Physiol Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo, 113-8657 Japan. Several triazole-containing chemicals have previously been shown to act as efficient inhibitors of cytochrome P450 monooxygenases. To discover a strigolactone biosynthesis inhibitor, we screened a chemical library of triazole derivatives to find chemicals that induce tiller bud outgrowth of rice seedlings. We discovered a triazole-type chemical, TIS13 [2,2-dimethyl-7-phenoxy-4-(1H-1,2,4-triazol-1-yl)heptan-3-ol], which induced outgrowth of second tiller buds of wild-type seedlings, as observed for non-treated strigolactone-deficient d10 mutant seedlings. TIS13 treatment reduced strigolactone levels in both roots and root exudates in a concentration-dependent manner. Co-application of GR24, a synthetic strigolactone, with TIS13 canceled the TIS13-induced tiller bud outgrowth. Taken together, these results indicate that TIS13 inhibits strigolactone biosynthesis in rice seedlings. We propose that TIS13 is a new lead compound for the development of specific strigolactone biosynthesis inhibitors. D10|OsCCD8|OsCCD8b Cinnamoyl-CoA reductase, a key enzyme in lignin biosynthesis, is an effector of small GTPase Rac in defense signaling in rice 2006 Proc Natl Acad Sci U S A Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0192, Japan. OsRac1, one of the Rac/Rop family of small GTPases, plays important roles in defense responses, including a role in the production of reactive oxygen species mediated by NADPH oxidase. We have identified an effector of OsRac1, namely rice (Oryza sativa) cinnamoyl-CoA reductase 1 (OsCCR1), an enzyme involved in lignin biosynthesis. Lignin, which is polymerized through peroxidase activity by using H(2)O(2) in the cell wall, is an important factor in plant defense responses, because it presents an undegradable mechanical barrier to most pathogens. Expression of OsCCR1 was induced by a sphingolipid elicitor, suggesting that OsCCR1 participates in defense signaling. In in vitro interaction and two-hybrid experiments, OsRac1 was shown to bind OsCCR1 in a GTP-dependent manner. Moreover, the interaction of OsCCR1 with OsRac1 led to the enzymatic activation of OsCCR1 in vitro. Transgenic cell cultures expressing the constitutively active OsRac1 accumulated lignin through enhanced CCR activity and increased reactive oxygen species production. Thus, it is likely that OsRac1 controls lignin synthesis through regulation of both NADPH oxidase and OsCCR1 activities during defense responses in rice. OsCCR1,OsRac1 Potential role of the rice OsCCS52A gene in endoreduplication 2012 Planta Division of Applied Life Science, BK21 Program, Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660-701, Republic of Korea. In eukaryotes, the cell cycle consists of four distinct phases: G1, S, G2 and M. In certain condition, the cells skip M-phase and undergo endoreduplication. Endoreduplication, occurring during a modified cell cycle, duplicates the entire genome without being followed by M-phase. A cycle of endoreduplication is common in most of the differentiated cells of plant vegetative tissues and it occurs extensively in cereal endosperm cells. Endoreduplication occurs when CDK/Cyclin complex low or inactive caused by ubiquitin-mediated degradation by APC and their activators. In this study, rice cell cycle switch 52 A (OsCCS52A), an APC activator, is functionally characterized using the reverse genetic approach. In rice, OsCCS52A is highly expressed in seedlings, flowers, immature panicles and 15 DAP kernels. Localization studies revealed that OsCCS52A is a nuclear protein. OsCCS52A interacts with OsCdc16 in yeast. In addition, overexpression of OsCCS52A inhibits mitotic cell division and induces endoreduplication and cell elongation in fission yeast. The homozygous mutant exhibits dwarfism and smaller seeds. Further analysis demonstrated that endoreduplication cycles in the endosperm of mutant seeds were disturbed, evidenced by reduced nuclear and cell sizes. Taken together, these results suggest that OsCCS52A is involved in maintaining normal seed size formation by mediating the exit from mitotic cell division to enter the endoreduplication cycles in rice endosperm. OsCCS52A|TAD|TE Functional characterization of a B-type cell cycle switch 52 in rice (OsCCS52B) 2012 Plant Cell, Tissue and Organ Culture (PCTOC) Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, 660-701, Republic of Korea Plant growth and development depend on a precise coordination between cell division and cell expansion. In this study, a rice cell cycle switch 52 B (OsCCS52B) was functionally characterized using two approaches: overexpression of the gene product in fission yeast and characterization of an insertion mutant line 1B-10423. In wild-type plants, OsCCS52B is highly expressed in generative organs such as flowers and kernels. Overexpression of OsCCS52B induces cell elongation and slower cell proliferation in fission yeast. Characterization of the mutant line 1B-10423 revealed that the mutant exhibits semi-dwarf and smaller kernel phenotypes. In addition, microscopic analysis of mutant kernels showed that the reduced kernel size was due to a reduced cell size. However, the nuclear size and ploidy level were unaffected. These results suggest that OsCCS52B may be involved in cell expansion regulation in rice endosperm. OsCCS52B Molecular cloning and characterization of OsCDase, a ceramidase enzyme from rice 2008 Plant J School of Biology and Environmental Science, University College Dublin, Belfield, Dublin 4, Ireland. SUMMARY: Sphingolipids are a structurally diverse group of molecules based on long-chain sphingoid bases that are found in animal, fungal and plant cells. In contrast to the situation in animals and yeast, much less is known about the spectrum of sphingolipid species in plants and the roles they play in mediating cellular processes. Here, we report the cloning and characterization of a plant ceramidase from rice (Oryza sativa spp. Japonica cv. Nipponbare). Sequence analysis suggests that the rice ceramidase (OsCDase) is similar to mammalian neutral ceramidases. We demonstrate that OsCDase is a bona fide ceramidase by heterologous expression in the yeast double knockout mutant Deltaypc1Deltaydc1 that lacks the yeast ceramidases YPC1p and YDC1p. Biochemical characterization of OsCDase showed that it exhibited classical Michaelis-Menten kinetics, with optimum activity between pH 5.7 and 6.0. OsCDase activity was enhanced in the presence of Ca(2+), Mg(2+), Mn(2+) and Zn(2+), but inhibited in the presence of Fe(2+). OsCDase appears to use ceramide instead of phytoceramide as a substrate. Subcellular localization showed that OsCDase is localized to the endoplasmic reticulum and Golgi, suggesting that these organelles are sites of ceramide metabolism in plants. OsCDase OsCDPK13, a calcium-dependent protein kinase gene from rice, is induced in response to cold and gibberellin 2003 Plant Physiology and Biochemistry National Institute of Agrobiological Sciences, Tsukuba, Japan. Calcium-dependent protein kinases (CDPKs) play an important role in rice signal transduction, but the precise role of each individual CDPK is still largely unknown. Recently, a full-length cDNA encoding OsCDPK13 from rice seedling was isolated. To characterize the function of OsCDPK13, its responses to various stresses and hormones were analyzed in this study. OsCDPK13 accumulated in 2-week-old leaf sheath and callus, and became phosphorylated in response to cold and gibberellin (GA). OsCDPK13 gene expression and protein accumulation were up-regulated in response to GA3 treatment, but suppressed in response to abscisic acid and brassinolide. Antisense OsCDPK13 transgenic rice lines were shorter than the vector control lines, and the expression of OsCDPK13 was lower in dwarf mutants of rice than in wild type. Furthermore, OsCDPK13 gene expression and protein accumulation were enhanced in response to cold, but suppressed under salt and drought stresses. Sense OsCDPK13 transgenic rice lines had higher recovery rates after cold stress than vector control rice. The expression of OsCDPK13 was stronger in cold-tolerant rice varieties than in cold-sensitive ones. The results suggest that OsCDPK13 might be an important signaling component in the response of rice to GA and cold stress. OsCDPK13|OsCDPK11|OsCPK11 Molecular cloning of two novel rice cDNA sequences encoding putative calcium-dependent protein kinases 1995 Plant Mol Biol Istituto Biosintesi Vegetali, C.N.R., Milan, Italy. We have isolated, from a cDNA library constructed from rice coleoptiles, two sequences, OSCPK2 and OSCPK11, that encode for putative calcium-dependent protein kinase (CDPK) proteins. OSCPK2 and OSCPK11 cDNAs are related to SPK, another gene encoding a rice CDPK that is specifically expressed in developing seeds [20]. OSCPK2 and OSCPK11-predicted protein sequences are 533 and 542 amino acids (aa) long with a corresponding molecular mass of 59436 and 61079 Da respectively. Within their polypeptide chain, they all contain those conserved features that define a plant CDPK; kinase catalytic sequences are linked to a calmodulin-like regulatory domain through a junction region. The calmodulin-like regulatory domain of the predicted OSCPK2 protein contains 4 EF-hand calcium-binding sites while OSCPK11 has conserved just one canonical EF-hand motif. In addition, OSCPK2- and OSCPK11-predicted proteins contain, at their N-terminal region preceding the catalytic domain, a stretch of 80 or 74 residues highly rich in hydrophilic amino acids. Comparison of the NH2-terminal sequence of all three rice CDPKs so far identified (OSCPK2, OSCPK11 and SPK) indicates the presence of a conserved MGxxC(S/Q)xxT motif that may define a consensus signal for N-myristoylation. OSCPK2 and OSCPK11 proteins are both encoded by a single-copy gene and their polyadenylated transcripts are 2.4 and 3.5 kb long respectively. OSCPK2 and OSCPK11 mRNAs are equally abundant in rice roots and coleoptiles. A 12 h white light treatment of the coleoptiles reduces the amount of OSCPK2 mRNA with only a slight effect on the level of OSCPK11 transcript. With anoxic treatments, OSCPK2 mRNA level declined significantly and promptly while the amount of OSCPK11 transcript remained constant. OsCDPK13|OsCDPK11|OsCPK11,OsCDPK2|OsCPK2 Rice calcium-dependent protein kinase isoforms OsCDPK2 and OsCDPK11 show different responses to light and different expression patterns during seed development 1999 Plant Mol Biol Istituto Biosintesi Vegetali CNR, Via Bassini 15, 20133, Milano, Italy We investigated the spatial and temporal expression patterns of two rice calcium-dependent protein kinases (CDPKs), OsCDPK2 and OSCDPK11, using isoform-specific antisera. Bands of the expected molecular sizes for OsCDPK2 (59 kDa) and OsCDPK11 (61 kDa) were detected on western blots. OsCDPK2 and OsCDPK11 mRNA and protein levels increased in unison during flower development. However, at the onset of seed development, the protein expression profiles diverged significantly. OsCDPK2 protein was expressed at low levels during early seed development, but increased to high levels that were maintained in later stages (20 days after fertilisation, DAF). Conversely, OsCDPK11 protein levels were high at the beginning of seed development, but fell rapidly from 10 DAF onwards. This decrease in the level of OsCDPK11 protein was associated with the abundant synthesis of a truncated mRNA species. OsCDPK2 expression was also closely associated with light perception. OsCDPK2 protein was barely detectable in green leaves exposed to light, but levels increased sharply when plants were shifted to darkness. Initially, this increase reflected a rapid elevation in the levels of OsCDPK2 mRNA, which was normally located in the mesophyll. Conversely, OsCDPK11 mRNA and protein levels were unaffected by light. These data strongly indicate that two rice CDPK isoforms have different functions in seed development and in response to light in leaves. OsCDPK13|OsCDPK11|OsCPK11,OsCDPK2|OsCPK2 OsCDPK13, a calcium-dependent protein kinase gene from rice, is induced by cold and gibberellin in rice leaf sheath 2004 Plant Mol Biol National Institute of Agrobiological Sciences, Tsukuba, Japan. Calcium-dependent protein kinases (CDPKs) play an important role in rice signal transduction, but the precise role of each individual CDPK is still largely unknown. Recently, a full-length cDNA encoding OsCDPK13 from rice seedling was isolated. To characterize the function of OsCDPK13, its responses to various stresses and hormones were analyzed in this study. OsCDPK13 accumulated in 2-week-old leaf sheath and callus, and became phosphorylated in response to cold and gibberellin (GA). OsCDPK13 gene expression and protein accumulation were up-regulated in response to GA3 treatment, but suppressed in response to abscisic acid and brassinolide. Antisense OsCDPK13 transgenic rice lines were shorter than the vector control lines, and the expression of OsCDPK13 was lower in dwarf mutants of rice than in wild type. Furthermore, OsCDPK13 gene expression and protein accumulation were enhanced in response to cold, but suppressed under salt and drought stresses. Sense OsCDPK13 transgenic rice lines had higher recovery rates after cold stress than vector control rice. The expression of OsCDPK13 was stronger in cold-tolerant rice varieties than in cold-sensitive ones. The results suggest that OsCDPK13 might be an important signaling component in the response of rice to GA and cold stress. OsCDPK13|OsCDPK11|OsCPK11 Over-expression of calcium-dependent protein kinase 13 and calreticulin interacting protein 1 confers cold tolerance on rice plants 2007 Mol Genet Genomics National Institute of Crop Science, 2-1-18 Kannondai, Tsukuba 305-8518, Japan. skomatsu@affrc.go.jp Calcium is a ubiquitous signaling molecule and changes in cytosolic calcium concentration are involved in plant responses to various stimuli. The rice calcium-dependent protein kinase 13 (CDPK13) and calreticulin interacting protein 1 (CRTintP1) have previously been reported to be involved in cold stress response in rice. In this study, rice lines transformed with sense CDPK13 or CRTintP1 constructs were produced and used to investigate the function of these proteins. When the plants were incubated at 5 degrees C for 3 days, leaf blades of both the sense transgenic and vector control rice plants became wilted and curled. When the plants were transferred back to non-stress conditions after cold treatment, the leaf blades died, but the sheaths remained green in the sense transgenic rice plants. Expression of CDPK13 or CRTintP1 was further examined in several rice varieties including cold-tolerant rice varieties. Accumulation of these proteins in the cold-tolerant rice variety was higher than that in rice varieties that are intermediate in their cold tolerance. To examine whether over-expression of CDPK13 and CRTintP1 would have any effect on the proteins or not, sense transgenic rice plants were analyzed using proteomics. The 2D-PAGE profiles of proteins from the vector control were compared with those of the sense transgenic rice plants. Two of the proteins that differed between these lines were calreticulins. The results suggest that CDPK13, calreticulin and CRTintP1 might be important signaling components for response to cold stress in rice. OsCDPK13|OsCDPK11|OsCPK11 Cloning and biochemical properties of CDPK gene OsCDPK14 from rice 2005 J Plant Physiol Laboratory of Plant Development Physiology and Molecular Biology, College of Life Sciences, Beijing Normal University, PR China. A rice CDPK gene, OsCDPK14 (AY144497), was cloned from developing caryopses of rice (Oryza sativa cv. Zhonghua 15). Its cDNA sequence (1922 bp) contains an ORF encoding a 514 amino acids protein (56.7kD, pl 5.18). OsCDPK14 shows the typical structural features of the CDPK family, including a conserved catalytic Ser/Thr kinase domain, an autoinhibitory domain and a CaM-like domain with four putative Ca2+-binding EF hands. Subcellular targeting indicated that OsCDPK14 was located in the cytoplasm, probably due to the absence of myristoylation and palmitoylation motifs. OsCDPK14 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using Syntide-2, a synthetic peptide. Kinase activity was shown to be Ca2+-dependent, and this activation was strongly enhanced by Mn2+ and inhibited by W7 in vitro. These results provide significant insights into the regulation and biochemical properties of OsCDPK14, suggesting OsCDPK14 may be a signal factor of cytoplasm in rice plant. OsCPK24|OsCDPK14 Overexpression of the calcium-dependent protein kinase OsCDPK2 in transgenic rice is repressed by light in leaves and disrupts seed development 2000 Transgenic Res Department of Botany, University of Milan, Via Celoria 26, 20133, Milano, Italy Independent transgenic rice lines overexpressing the rice CDPK isoform OsCDPK2 were generated by particle bombardment. High levels of OsCDPK2 were detected in leaves removed from etiolated plants, as well as in stems and flowers. However, there was no overexpression in green leaves that had been exposed to light, confirming that OsCDPK2 protein stability was subject to light regulation. The morphological phenotype of transgenic plants producing high levels of recombinant OsCDPK2 was normal until the onset of seed development. Flowers developed normally, producing well-shaped ovaries and stigmas, and mature anthers filled with pollen grains. However, seed formation in these plants was strongly inhibited, with only 3-7% of the flowers producing seeds. Seed development was arrested at an early stage. We discuss these data with respect to the possible requirement for specific CDPK isoforms during rice seed development. OsCDPK2|OsCPK2 Membrane localization of a rice calcium-dependent protein kinase (CDPK) is mediated by myristoylation and palmitoylation 2000 The Plant Journal Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Centro de Investigaciones Biológicas, Fundación para Investigaciones Biológicas Aplicadas, Mar del Plata, Argentina. Calcium-dependent protein kinases (CDPKs), the most abundant serine/threonine kinases in plants, are found in various subcellular localizations, which suggests that this family of kinases may be involved in multiple signal transduction pathways. A complete analysis to try to understand the molecular basis of the presence of CDPKs in various localizations in the cell has not been accomplished yet. It has been suggested that myristoylation may be responsible for membrane association of CDPKs. In this study, we used a rice CDPK, OSCPK2, which has a consensus sequence for myristoylation at the N-terminus, to address this question. We expressed wild-type OSCPK2 and various mutants in different heterologous systems to investigate the factors that affect its membrane association. The results show that OSCPK2 is myristoylated and palmitoylated and targeted to the membrane fraction. Both modifications are required, myristoylation being essential for membrane localization and palmitoylation for its full association. The fact that palmitoylation is a reversible modification may provide a mechanism for regulation of the subcellular localization. OSCPK2 is the first CDPK shown to be targeted to membranes by an src homology domain 4 (SH4) located at the N-terminus of the molecule. OsCDPK2|OsCPK2 Functional analysis of DNA sequences controlling the expression of the rice OsCDPK2 gene 2006 Planta Istituto Biologia e Biotecnologia Agraria, CNR, Via Bassini 15, 20133 Milano, Italy. Plant calcium-dependent protein kinases (CDPKs) are involved in calcium-mediated signal transduction pathways. Their expression is finely tuned in different tissues and in response to specific signals, but the mechanism of such a regulation is still largely unknown. OsCDPK2 gene expression is modulated in vivo during rice (Oryza sativa L.) flower development and is downregulated by white light in leaves. In order to identify OsCDPK2 regulatory sequences, we amplified and cloned both the 5' and 3'-flanking regions of the gene. Sequence analysis revealed that the leader sequence is interrupted by an intron, whose regulatory role was investigated. Different ss-gucuronidase (GUS) expression vectors, carrying combinations of the putative OsCDPK2 regulatory regions, were generated and GUS expression was analyzed both in transient assays and in transgenic rice plants. The whole 5'-flanking sequence was able to drive GUS expression in rice calli and leaves transiently transformed with the biolistic technique. Analysis of the GUS expression pattern in transgenic plants revealed strong activity in root tips, leaf veins and mesophyll cells, in flower reproductive organs and in mature pollen grains. Expression was also shown to be subject to an intron-mediated enhancement (IME) mechanism, since the deletion of the leader intron sequence from chimeric OsCDPK2::GUS plasmids almost completely abolished GUS activity. Furthermore, in transiently transformed leaves, GUS expression driven by the OsCDPK2 promoter-leader region was constitutively observed regardless of light or dark exposure. Light-regulated expression was restored by inserting the OsCDPK2 3' untranslated region (3'UTR) downstream of the chimeric OsCDPK2::GUS transcription unit, suggesting that light down-regulation is mediated by a mechanism driven by the 3'UTR. OsCDPK2|OsCPK2 Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants 2000 The Plant Journal Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. A rice gene encoding a calcium-dependent protein kinase (CDPK), OsCDPK7, was induced by cold and salt stresses. To elucidate the physiological function of OsCDPK7, we generated transgenic rice plants with altered levels of the protein. The extent of tolerance to cold and salt/drought stresses of these plants correlated well with the level of OsCDPK7 expression. Therefore, OsCDPK7 was shown to be a positive regulator commonly involved in the tolerance to both stresses in rice. Over-expression of OsCDPK7 enhanced induction of some stress-responsive genes in response to salinity/drought, but not to cold. Thus, it was suggested that the downstream pathways leading to the cold and salt/drought tolerance are different from each other. It seems likely that at least two distinct pathways commonly use a single CDPK, maintaining the signalling specificity through unknown post-translational regulation mechanisms. These results demonstrate that simple manipulation of CDPK activity has great potential with regard to plant improvement. OsCDPK7,SALT A Ca2+-Dependent Protein Kinase that Endows Rice Plants with Cold- and Salt-Stress Tolerance Functions in Vascular Bundles 2001 Plant and Cell Physiology Laboratory of Plant Physiology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, 606-8502 Japan A rice Ca2+-dependent protein kinase, OsCDPK7, is a positive regulator commonly involved in the tolerance to cold and salt/drought. We carried out in situ detection of the transcript and immunolocalization of the protein. In the wild-type rice plants under both stress conditions, OsCDPK7 was expressed predominantly in vascular tissues of crowns and roots, vascular bundles and central cylinder, respectively, where water stress occurs most severely. This enzyme was also expressed in the peripheral cylinder of crown vascular bundles and root sclerenchyma. Similar localization patterns with stronger signals were observed in stress-tolerant OsCDPK7 over-expressing transformants with the cauliflower mosaic virus 35S promoter. The transcript of a putative target gene of the OsCDPK7 signaling pathway, rab16A, was also detected essentially in the same tissues upon salt stress, suggesting that the OsCDPK7 pathway operates predominantly in these regions. We propose that the use of the 35S promoter fortuitously strengthened the localized expression of OsCDPK7, resulting in enhancement of the stress signaling in the inherently operating regions leading to improved stress tolerance. OsCDPK7 Heterologous expression and characterization of recombinant OsCDR1, a rice aspartic proteinase involved in disease resistance 2010 Protein Expr Purif Genome Research Centre, Department of Microbiology and Biotechnology Centre, Faculty of Science, The M.S. University of Baroda, Vadodara, Gujarat 390002, India. The Oryza sativa constitutive disease resistance 1 (OsCDR1) gene product is an aspartic proteinase that has been implicated in disease resistance signaling. This apoplastic enzyme is a member of the group of 'atypical' plant aspartic proteinases. Recombinant OsCDR1 expressed in Escherichia coli exhibited protease activity against succinylated-casein substrate. Inactivating the enzyme through modification of an aspartate residue present in the deduced active site completely abolished its proteinase activity. Infiltration of the OsCDR1 fusion protein into leaves of Arabidopsis plants induced PR2 transcripts in both the infiltrated leaf (primary) and in non-treated secondary leaves while the inactive recombinant protein failed to induce either local or systemic PR2. These findings demonstrate that OsCDR1 is capable of inducing systemic defense responses in plants. OsCDR1 Overexpression of rice (Oryza sativa L.) OsCDR1 leads to constitutive activation of defense responses in rice and Arabidopsis 2009 Mol Plant Microbe Interact Genome Research Centre, Department of Microbiology and Biotechnology Centre, Faculty of Science, The M.S. University of Baroda, Vadodara, Gujarat, India. Plant aspartic proteases (AP) play key roles in the regulation of biological processes, such as the recognition of pathogens and pests and the induction of effective defense responses. A large number of AP (>400) have been identified in silico in the rice genome. None have previously been isolated and functionally characterized for their involvement in disease resistance. We describe here the isolation and characterization of a gene (OsCDR1) from rice which encodes a predicted aspartate protease. Expression of OsCDR1 was activated upon treatments with benzothiadiazole and salicylic acid, which are signal molecules in plant disease resistance responses. Ectopic expression of OsCDR1 in Arabidopsis and rice conferred enhanced resistance against bacterial and fungal pathogens. The enhanced disease resistance observed in transgenic plants was correlated with induction of pathogenesis-related gene expression and was shown by mutational analysis to be dependent on AP activity of the transgene-encoded product. OsCDR1 accumulates in intercellular fluids (IF) in transgenic plants. Infiltration of IF from transgenic Arabidopsis plants into leaves of wild-type (WT) Arabidopsis induced the systemic defense response. These results demonstrate the conservation of CDR1 function between rice and Arabidopsis during the disease resistance response. OsCDR1 Novel Cysteine-Rich Peptides from Digitaria ciliaris and Oryza sativa Enhance Tolerance to Cadmium by Limiting its Cellular Accumulation 2008 Plant and Cell Physiology Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan. By means of functional screening using the cadmium (Cd)-sensitive ycf1 yeast mutant, we have isolated a novel cDNA clone, DcCDT1, from Digitaria ciliaris growing in a former mining area in northern Japan, and have shown that it confers Cd tolerance to the yeast cells, which accumulated almost 2-fold lower Cd levels than control cells. The 521 bp DcCDT1 cDNA contains an open reading frame of 168 bp and encodes a deduced peptide, DcCDT1, that is 55 amino acid residues in length, of which 15 (27.3%) are cysteine residues. Five DcCDT1 homologs (here termed OsCDT1-OsCDT5) have been identified in rice, and all of them were up-regulated to varying degrees in the above-ground tissues by CdCl(2) treatment. Localization of green fluorescent protein fusions suggests that DcCDT1 and OsCDT1 are targeted to both cytoplasmic membranes and cell walls of plant cells. Transgenic Arabidopsis thaliana plants overexpressing DcCDT1 or OsCDT1 displayed a Cd-tolerant phenotype and, consistent with our yeast data, accumulated lower amounts of Cd when grown on CdCl(2). Collectively, our data suggest that DcCDT1 and OsCDT1 function to prevent entry of Cd into yeast and plant cells and thereby enhance their Cd tolerance. OsCDT1|OsCDT2 A plasma membrane-localized small peptide is involved in rice aluminum tolerance 2013 Plant J Institute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki, Japan. A transcription factor for Al tolerance, ART1, regulates the expression of at least 30 genes in rice. Here we functionally characterized one of the downstream genes, OsCDT3, which encodes a predicted peptide of only 53 amino acid residues rich in cysteine. Knockdown of this gene resulted in decreased tolerance to Al, but did not affect the tolerance to Cd. The aluminum (Al) content in the root residues including cell wall and the plasma membrane of knockdown lines decreased, but the Al concentration in the root cell sap increased compared with those of the wild-type rice. OsCDT3 was mainly expressed in the roots and its expression was specifically induced by Al exposure, not by low pH and other metals. There was a small genotypic variation in OsCDT3 expression level, but no correlation between Al tolerance and the OsCDT3 variation was found among 17 rice cultivars. Analysis of pOsCDT3::GFP transgenic rice showed that OsCDT3 was expressed at all cells in the root tips. Transient expression of OsCDT3 fused with GFP at both N- and C-termini showed that OsCDT3 was anchored to the plasma membrane. Expression of OsCDT3 in yeast conferred tolerance to Al, but not to Cd. Furthermore, OsCDT3 did not show transport activity for Al in yeast, but was able to directly bind Al in vitro. Taken together, our results indicate that OsCDT3 anchoring to the plasma membrane may play a role in stopping entry of Al into the root cells by binding Al, therefore, contributing to high Al tolerance in rice. OsCDT3 Carbohydrate-binding module of a rice endo-beta-1,4-glycanase, OsCel9A, expressed in auxin-induced lateral root primordia, is post-translationally truncated 2006 Plant Cell Physiol Hydraulic and Bio Engineering Research Section, Technology Center, Taisei Co., 344-1 Nase-cho, Totuka-ku, Yokohama, 245-0051 Japan. kouki.yoshida@sakura.taisei.co.jp We report the cloning of a glycoside hydrolase family (GHF) 9 gene of rice (Oryza sativa L. cv. Sasanishiki), OsCel9A, corresponding to the auxin-induced 51 kDa endo-1,4-beta-glucanase (EGase). This enzyme reveals a broad substrate specificity with respect to sugar backbones (glucose and xylose) in beta-1,4-glycans of type II cell wall. OsCel9A encodes a 640 amino acid polypeptide and is an ortholog of TomCel8, a tomato EGase containing a carbohydrate-binding module (CBM) 2 sequence at its C-terminus. The expression of four rice EGase genes including OsCel9A showed different patterns of organ specificity and responses to auxin. OsCel9A was preferentially expressed during the initiation of lateral roots or subcultured root calli, but was hardly expressed during auxin-induced coleoptile elongation or in seed calli, in contrast to OsCel9D, a KORRIGAN (KOR) homolog. In situ localization of OsCel9A transcripts demonstrated that its expression was specifically up-regulated in lateral root primordia (LRP). Northern blotting analysis showed the presence of a single product of OsCel9A. In contrast, both mass spectrometric analyses of peptide fragments from purified 51 kDa EGase proteins and immunogel blot analysis of EGase proteins in root extracts using two antibodies against internal peptide sequences of OsCel9A revealed that the entire CBM2 region was post-translationally truncated from the 67 kDa nascent protein to generate 51 kDa EGase isoforms. Analyses of auxin concentration and time course dependence of accumulation of two EGase isoforms suggested that the translation and post-translational CBM2 truncation of the OsCel9A gene may participate in lateral root development. OsCel9A|OsGLU5 MULTIPASS, a rice R2R3-type MYB transcription factor, regulates adaptive growth by integrating multiple hormonal pathways 2013 Plant J Institute of Biochemistry and Biology, University of Potsdam, Karl Liebknecht Strasse 24-25, Haus 20, 14476, Potsdam, Germany; Max Planck Institute of Molecular Plant Physiology, Am Muhlenberg 1, 14476, Potsdam, Germany. Growth regulation is an important aspect of plant adaptation during environmental perturbations. Here, the role of MULTIPASS (OsMPS), an R2R3-type MYB transcription factor of rice, was explored. OsMPS is induced by salt stress and expressed in vegetative and reproductive tissues. Over-expression of OsMPS reduces growth under non-stress conditions, while knockdown plants display increased biomass. OsMPS expression is induced by abscisic acid and cytokinin, but is repressed by auxin, gibberellin and brassinolide. Growth retardation caused by OsMPS over-expression is partially restored by auxin application. Expression profiling revealed that OsMPS negatively regulates the expression of EXPANSIN (EXP) and cell-wall biosynthesis as well as phytohormone signaling genes. Furthermore, the expression of OsMPS-dependent genes is regulated by auxin, cytokinin and abscisic acid. Moreover, we show that OsMPS is a direct upstream regulator of OsEXPA4, OsEXPA8, OsEXPB2, OsEXPB3, OsEXPB6 and the endoglucanase genes OsGLU5 and OsGLU14. The multiple responses of OsMPS and its target genes to various hormones suggest an integrative function of OsMPS in the cross-talk between phytohormones and the environment to regulate adaptive growth. OsCel9A|OsGLU5,OsEXPA8,OsMPS A conserved cysteine motif is critical for rice ceramide kinase activity and function 2011 PLoS One State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China. BACKGROUND: Ceramide kinase (CERK) is a key regulator of cell survival in dicotyledonous plants and animals. Much less is known about the roles of CERK and ceramides in mediating cellular processes in monocot plants. Here, we report the characterization of a ceramide kinase, OsCERK, from rice (Oryza sativa spp. Japonica cv. Nipponbare) and investigate the effects of ceramides on rice cell viability. PRINCIPAL FINDINGS: OsCERK can complement the Arabidopsis CERK mutant acd5. Recombinant OsCERK has ceramide kinase activity with Michaelis-Menten kinetics and optimal activity at 7.0 pH and 40 degrees C. Mg2+ activates OsCERK in a concentration-dependent manner. Importantly, a CXXXCXXC motif, conserved in all ceramide kinases and important for the activity of the human enzyme, is critical for OsCERK enzyme activity and in planta function. In a rice protoplast system, inhibition of CERK leads to cell death and the ratio of added ceramide and ceramide-1-phosphate, CERK's substrate and product, respectively, influences cell survival. Ceramide-induced rice cell death has apoptotic features and is an active process that requires both de novo protein synthesis and phosphorylation, respectively. Finally, mitochondria membrane potential loss previously associated with ceramide-induced cell death in Arabidopsis was also found in rice, but it occurred with different timing. CONCLUSIONS: OsCERK is a bona fide ceramide kinase with a functionally and evolutionarily conserved Cys-rich motif that plays an important role in modulating cell fate in plants. The vital function of the conserved motif in both human and rice CERKs suggests that the biochemical mechanism of CERKs is similar in animals and plants. Furthermore, ceramides induce cell death with similar features in monocot and dicot plants. OsCERK The Hop/Sti1-Hsp90 chaperone complex facilitates the maturation and transport of a PAMP receptor in rice innate immunity 2010 Cell Host Microbe Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Takayama, Ikoma, Japan. Recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) represents a critical first step of innate defense in plants and animals. However, maturation and transport of PRRs are not well understood. We find that the rice chitin receptor OsCERK1 interacts with Hsp90 and its cochaperone Hop/Sti1 in the endoplasmic reticulum (ER). Hop/Sti1 and Hsp90 are required for efficient transport of OsCERK1 from the ER to the plasma membrane (PM) via a pathway dependent on Sar1, a small GTPase which regulates ER-to-Golgi trafficking. Further, Hop/Sti1 and Hsp90 are present at the PM in a complex (designated the "defensome") with OsRac1, a plant-specific Rho-type GTPase. Finally, Hop/Sti1 was required for chitin-triggered immunity and resistance to rice blast fungus. Our results suggest that the Hop/Sti1-Hsp90 chaperone complex plays an important and likely conserved role in the maturation and transport of PRRs and may function to link PRRs and Rac/Rop GTPases. OsCERK1,OsRac1,Hsp90|rHsp90 A missense mutation in the transmembrane domain of CESA4 affects protein abundance in the plasma membrane and results in abnormal cell wall biosynthesis in rice 2009 Plant Mol Biol National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 100101 Beijing, China. Cellulose synthase (CESA) is a critical catalytic subunit of the cellulose synthase complex responsible for glucan chain elongation. Our knowledge about how CESA functions is still very limited. Here, we report the functional characterization of a rice mutant, brittle culm11, that shows growth retardation and dramatically reduced plant strength. Map-based cloning revealed that all the mutant phenotypes result from a missense mutation in OsCESA4 (G858R), a highly conserved residue at the end of the fifth transmembrane domain. The aberrant secondary cell wall of the mutant plants is attributed to significantly reduced cellulose content, abnormal secondary wall structure of sclerenchyma cells, and overall altered wall composition, as detected by chemical analyses and immunochemical staining. Importantly, we have found that this point mutation decreases the abundance of OsCESA4 in the plasma membrane, probably due to a defect in the process of CESA complex secretion. The data from our biochemical, genetic, and pharmacological analyses indicate that this residue is critical for maintaining the normal level of CESA proteins in the plasma membrane. OsCesA4|Bc7|bc11 Three Distinct Rice Cellulose Synthase Catalytic Subunit Genes Required for Cellulose Synthesis in the Secondary Wall 2003 Plant Physiol Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305-0854, Japan. Several brittle culm mutations of rice (Oryza sativa) causing fragility of plant tissues have been identified genetically but not characterized at a molecular level. We show here that the genes responsible for three distinct brittle mutations of rice, induced by the insertion of the endogenous retrotransposon Tos17, correspond to CesA (cellulose synthase catalytic subunit) genes, OsCesA4, OsCesA7 and OsCesA9. Three CesA genes were expressed in seedlings, culms, premature panicles, and roots but not in mature leaves, and the expression profiles were almost identical among the three genes. Cellulose contents were dramatically decreased (8.9%-25.5% of the wild-type level) in the culms of null mutants of the three genes, indicating that these genes are not functionally redundant. Consistent with these results, cell walls in the cortical fiber cells were shown to be thinner in all the mutants than in wild-type plants. Based on these observations, the structure of a cellulose-synthesizing complex involved in the synthesis of the secondary cell wall is discussed. OsCesA4|Bc7|bc11,OsCesA7,Bc6|OsCesA9 A missense mutation in the transmembrane domain of CESA9 affects cell wall biosynthesis and plant growth in rice 2012 Plant Sci College of Agronomy and Plant Protection, Qingdao Agricultural University, Qingdao 266109, China. Rice is a model organism in poaceae plants to study cell wall biosynthesis. In this study, a mutant S1-60 isolated from an EMS mutagenized japonica cultivar Nipponbare, is characterized by brittle culms that can be easily broken by bending. The reduction in mechanical strength was due to defect in thickening of the sclerenchyma cell wall. The amount of cellulose in S1-60 culms was reduced to 44.7% of that of wild-type plants. Besides, the mutant also exhibited pleiotropic phenotypes, such as dwarfism and partial sterility. Genetic analysis and map-based cloning showed that all the phenotype of S1-60 mutant was caused by a recessive point mutation in the OsCESA9 gene, which encodes the cellulose synthase A subunit 9. This yet uncharacterized missense mutation changed the highly conserved G905 to D at the beginning of the fifth transmembrane domain. The OsCESA9 gene is predominantly expressed in the culms of mature stage plants, consistent with the brittle phenotype in the culm. These results indicate that OsCESA9 plays an important role in cell wall biosynthesis and plant growth. Bc6|OsCesA9 Identification of an E-box motif responsible for the expression of jasmonic acid-induced chitinase gene OsChia4a in rice 2012 J Plant Physiol Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. The plant hormone jasmonic acid (JA) is known to be involved in multiple defence responses against pathogens, which include the production of pathogenesis-related (PR) proteins. In order to investigate the induction mechanism of the rice defence responses by JA, we performed transcriptome analyses and focused on a chitinase gene, OsChia4a, which was identified to be one of the highest JA-inductive genes. The recombinant protein of His-tagged OsChia4a exhibited an inhibitory effect against the spore germination and hyphal growth of Magnaporthe oryzae. The promoter analysis of OsChia4a revealed that the region from -515 bp to -265 bp upstream of the ATG translation initiation site was required for the responsiveness to JA. A subsequent mutation analysis indicated that an E-box (CANNTG) in this region act as a JA-responsive cis element. These results imply that a basic helix-loop-helix transcription factor is likely to be involved in the regulation of the OsChia4a expression in a JA-dependent manner. OsChia4a Mitochondria provide the main source of cytosolic ATP for activation of outward-rectifying K+ channels in mesophyll protoplast of chlorophyll-deficient mutant rice (OsCHLH) seedlings 2004 J Biol Chem Bionanotechnology Center, Department of Life Science, Pohang University of Science and Technology, Kyungbuk, Korea. gohunse@postech.ac.kr The role of mitochondria in providing intracellular ATP that controls the activity of plasma membrane outward-rectifying K+ channels was evaluated. The OsCHLH rice mutant, which lacks chlorophyll in the thylakoids, was isolated by T-DNA gene trapping (Jung, K.-H., Hur, J., Ryu, C.-H., Choi, Y., Chung, Y.-Y., Miyao, A., Hirochika, H., and An, G. (2003) Plant Cell Physiol. 44, 463-472). The OsCHLH mutant is unable to fix CO2 and exhibits reduced growth. Wild type and mutant plants exhibit similar rates of respiratory O2 uptake in the dark, whereas the rate of photosynthetic O2 evolution by the mutant was negligible during illumination. During dark respiration the wild type and mutant exhibited similar levels of cytoplasmic ATP. In the mutant oligomycin treatment (an inhibitor of mitochondrial F1F0-ATPase) drastically reduced ATP production. The fact that this was reversed by the addition of glucose suggested that the mutant produced ATP exclusively from mitochondria but not from chloroplasts. In whole cell patch clamp experiments, the activity of outward-rectifying K+ channels of rice mesophyll cells showed ATP-dependent currents, which were 1.5-fold greater in wild type than in mutant cells. Channels in both wild type and mutant cells were deactivated by the removal of cytosolic ATP, whereas in the presence of ATP the channels remained active. We conclude that mesophyll cells in the OsCHLH rice mutant derive ATP from mitochondrial respiration, and that this is critical for the normal function of plasma membrane outward-rectifying K+ channels. OsCHLH Characterization of a rice chlorophyll-deficient mutant using the T-DNA gene-trap system 2003 Plant and Cell Physiology National Research Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, 790-784 Republic of Korea. We have previously generated a large pool of T-DNA insertional lines in rice. In this study, we screened those TDNA pools for rice mutants that had defective chlorophylls. Among the 1,995 lines examined in the T2 generation, 189 showed a chlorophyll-deficient phenotype that segregated as a single recessive locus. Among the mutants, 10 lines were P-glucuronidase (GUS)-positive in the leaves. Line 9-07117 has a T-DNA insertion into the gene that is highly homologous to XANTHA-F in barley and CHLH in Arabidopsis. This OsCHLH gene encodes the largest subunit of the rice Mg-chelatase, a key enzyme in the chlorophyll branch of the tetrapyrrole biosynthetic pathway. In the T2 and T3 generations, the chlorina mutant phenotypes are co-segregated with the T-DNA. We have identified two additional chlorina mutants that have a Tos17 insertion in the OsCHLH gene. Those phenotypes were cosegregated with Tos17 in the progeny. GUS assays and RNA blot analysis showed that expression of the OsCHLH gene is light inducible, while TEM analysis revealed that the thylakoid membrane of the mutant chloroplasts is underdeveloped. The chlorophyll content was very low in the OschlH mutants. This is the first report that T-DNA insertional mutagenesis can be used for functional analysis of rice genes. OsCHLH Molecular cloning and characterization of OsCHR4, a rice chromatin-remodeling factor required for early chloroplast development in adaxial mesophyll 2012 Planta State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, People's Republic of China. Mi-2 protein, the central component of the NuRD nucleosome remodeling and histone deacetylase complex, plays a role in transcriptional repression in animals. Mi-2-like genes have been reported in Arabidopsis, though their function in monocots remains largely unknown. In the present study, a rice Mi-2-like gene, OsCHR4 (Oryza sativa Chromatin Remodeling 4, LOC_Os07g03450), was cloned from a rice mutant with adaxial albino leaves. The Oschr4 mutant exhibited defective chloroplasts in adaxial mesophyll, but not in abaxial mesophyll. Ultrastructural observations indicated that proplastid growth and/or thylakoid membrane formation in adaxial mesophyll cells was blocked in the Oschr4 mutant. Subcellular localization revealed that OsCHR4::GFP fusion protein was targeted to the nuclei. OsCHR4 was mainly expressed in the root meristem, flower, vascular bundle, and mesophyll cells by promoter::GUS analysis in transgenic rice. The transcripts of some nuclear- and plastid-encoded genes required for early chloroplast development and photosynthesis were decreased in the adaxial albino mesophyll of the Oschr4 mutant. These observations provide evidence that OsCHR4, the rice Mi-2-like protein, plays an important role in early chloroplast development in adaxial mesophyll cells. The results increase our understanding of the molecular mechanism underlying tissue-specific chloroplast development in plants. OsCHR4 Cell Wall Invertase in Developing Rice Caryopsis: Molecular Cloning of OsCIN1 and Analysis of its Expression in Relation to its Role in Grain Filling 2002 Plant and Cell Physiology Department of Rice Research, National Agricultural Research Center, Joetsu, Niigata, 943-0193 Japan To establish the significance of cell wall invertase in grain filling of rice (Oryza sativa L.), we cloned a cDNA for a cell wall invertase from developing grains of rice. The cDNA, designated OsCIN1, contains an open reading frame of 1731 bp encoding a polypeptide of 577 amino acid residues. The deduced amino acid sequence showed typical features of the cell wall invertases, including a beta-fructosidase motif and a cysteine catalytic site, and shared 78.6 and 73.7% identity with maize cell wall invertases, Incw1 and Incw2, respectively. OsCIN1 is expressed in roots, in sink- and source-leaves, and in panicles. During the course of grain filling in the caryopses, OsCIN1 transcript is detectable only in the very early stage of their development, 1-4 d after flowering, when the cell wall invertase activity is the highest and the increase in caryopsis length is rapid. In situ localization of the mRNA revealed that OsCIN1 is expressed preferentially in the vascular parenchyma of the dorsal vein, integument and its surrounding cells, and is expressed weakly in the nucellar projection and nucellar epidermis. These results suggest that, during the early stage of caryopsis development, OsCIN1 is important for supplying a carbon source to developing filial tissues by cleaving unloaded sucrose in the apoplast. OsCIN1 Regulation of Microbe-Associated Molecular Pattern-Induced Hypersensitive Cell Death, Phytoalexin Production, and Defense Gene Expression by Calcineurin B-Like Protein-Interacting Protein Kinases, OsCIPK14/15, in Rice Cultured Cells 2010 Plant Physiol Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan. Although cytosolic free Ca(2+) mobilization induced by microbe/pathogen-associated molecular patterns is postulated to play a pivotal role in innate immunity in plants, the molecular links between Ca(2+) and downstream defense responses still remain largely unknown. Calcineurin B-like proteins (CBLs) act as Ca(2+) sensors to activate specific protein kinases, CBL-interacting protein kinases (CIPKs). We here identified two CIPKs, OsCIPK14 and OsCIPK15, rapidly induced by microbe-associated molecular patterns, including chitooligosaccharides and xylanase (Trichoderma viride/ethylene-inducing xylanase [TvX/EIX]), in rice (Oryza sativa). Although they are located on different chromosomes, they have over 95% nucleotide sequence identity, including the surrounding genomic region, suggesting that they are duplicated genes. OsCIPK14/15 interacted with several OsCBLs through the FISL/NAF motif in yeast cells and showed the strongest interaction with OsCBL4. The recombinant OsCIPK14/15 proteins showed Mn(2+)-dependent protein kinase activity, which was enhanced both by deletion of their FISL/NAF motifs and by combination with OsCBL4. OsCIPK14/15-RNAi transgenic cell lines showed reduced sensitivity to TvX/EIX for the induction of a wide range of defense responses, including hypersensitive cell death, mitochondrial dysfunction, phytoalexin biosynthesis, and pathogenesis-related gene expression. On the other hand, TvX/EIX-induced cell death was enhanced in OsCIPK15-overexpressing lines. Our results suggest that OsCIPK14/15 play a crucial role in the microbe-associated molecular pattern-induced defense signaling pathway in rice cultured cells. OsCIPK14,OsCIPK15 Coordinated responses to oxygen and sugar deficiency allow rice seedlings to tolerate flooding 2009 Sci Signal Graduate Institute of Life Sciences, National Defense Medical Center, Neihu, Taipei 114, Taiwan, ROC. Flooding is a widespread natural disaster that leads to oxygen (O(2)) and energy deficiency in terrestrial plants, thereby reducing their productivity. Rice is unusually tolerant to flooding, but the underlying mechanism for this tolerance has remained elusive. Here, we show that protein kinase CIPK15 [calcineurin B-like (CBL)-interacting protein kinase] plays a key role in O(2)-deficiency tolerance in rice. CIPK15 regulates the plant global energy and stress sensor SnRK1A (Snf1-related protein kinase 1) and links O(2)-deficiency signals to the SnRK1-dependent sugar-sensing cascade to regulate sugar and energy production and to enable rice growth under floodwater. Our studies contribute to understanding how rice grows under the conditions of O(2) deficiency necessary for growing rice in irrigated lowlands. OsCIPK15,SnRK1A Calcineurin B-like interacting protein kinase OsCIPK23 functions in pollination and drought stress responses in rice (Oryza sativa L.) 2008 Journal of Genetics and Genomics Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, National Centre for Plant Gene Research, Beijing, China. Drought is very harmful to grain yield due to its adverse effect on reproduction, especially on pollination process in rice. However, the molecular basis of such an effect still remains largely unknown. Here, we report the role of a member of CBL (Calcineurin B-Like) Interacting Protein Kinase (CIPK) family, OsCIPK23, in pollination and stress responses in rice. Molecular analyses revealed that it is mainly expressed in pistil and anther but up-regulated by pollination, as well as by treatments of various abiotic stresses and phytohormones. RNA interference-mediated suppression of OsCIPK23 expression significantly reduced seed set and conferred a hypersensitive response to drought stress, indicating its possible roles in pollination and drought stress. In consistent, overexpression of OsCIPK23 induced the expression of several drought tolerance related genes. Taken together, these results indicate that OsCIPK23 is a multistress induced gene and likely mediates a signaling pathway commonly shared by both pollination and drought stress responses in rice. OsCIPK23 Molecular cloning, functional expression and subcellular localization of two putative vacuolar voltage-gated chloride channels in rice (Oryza sativa L.) 2006 Plant Cell Physiol Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan. anakamur@nias.affrc.go.jp We isolated two cDNA clones (OsCLC-1 and OsCLC-2) homologous to tobacco CLC-Nt1, which encodes a voltage-gated chloride channel, from rice (Oryza sativa L. ssp. japonica, cv. Nipponbare). The deduced amino acid sequences were highly conserved (87.9% identity with each other). Southern blot analysis of the rice genomic DNA revealed that OsCLC-1 and OsCLC-2 were single-copy genes on chromosomes 4 and 2, respectively. OsCLC-1 was expressed in most tissues, whereas OsCLC-2 was expressed only in the roots, nodes, internodes and leaf sheaths. The level of expression of OsCLC-1, but not of OsCLC-2, was increased by treatment with NaCl. Both genes could partly substitute for GEF1, which encodes the sole chloride channel in yeast, by restoring growth under ionic stress. These results indicate that both genes are chloride channel genes. The proteins from both genes were immunochemically detected in the tonoplast fraction. Tagged synthetic green fluorescent protein which was fused to OsCLC-1 or OsCLC-2 localized in the vacuolar membranes. These results indicate that the proteins may play a role in the transport of chloride ions across the vacuolar membrane. We isolated loss-of-function mutants of both genes from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17, in the exon region, and found inhibition of growth at all life stages. OsCLC-1,OsCLC-2 Salt-dependent regulation of chloride channel transcripts in rice 2006 Plant Science Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan Rice OsCLC1 homologous to voltage-dependent Cl− channels of the CLC-family was characterized to study the regulation of Cl− homeostasis under salt stress conditions. By transcript analyses, expression of OsCLC1 was found in leaves and roots. Transcriptional regulation during salt stress was compared in the salt-sensitive Cl−-accumulating rice line IR29 and the salt-tolerant Cl−-excluding rice line Pokkali. In response to salt stress OsCLC1 transcript levels were repressed in leaves and roots of IR29 whereas in Pokkali expression was transiently induced. Under same conditions, in IR29 mRNA levels of the Na+/H+ antiporter OsNHX1 and of the vacuolar H+-ATPase subunit OsVHA-B decreased upon salt stress whereas Pokkali showed transient stimulation of OsVHA-B transcripts. Cell-specificity of OsCLC1 transcription was analyzed by in situ PCR. In leaves, signals were detected in mesophyll cells and stomata. In addition, expression occurred in xylem parenchyma cells and in the phloem whereas in salt-treated plants transcript amounts were reduced in mesophyll cells. Our results indicate coordinated regulation of anion and cation homeostasis in salt-treated rice and suggest function of OsCLC1 in osmotic adjustment at high salinity. OsCLC1,OsNHX1 The SNF1-type serine-threonine protein kinase SAPK4 regulates stress-responsive gene expression in rice 2008 BMC Plant Biol Department of Physiology and Biochemistry of Plants, Faculty of Biology, University of Bielefeld, 33615 Bielefeld, Germany. calliste.diedhiou@uni-bielefeld.de BACKGROUND: Plants respond to extracellularly perceived abiotic stresses such as low temperature, drought, and salinity by activation of complex intracellular signaling cascades that regulate acclimatory biochemical and physiological changes. Protein kinases are major signal transduction factors that have a central role in mediating acclimation to environmental changes in eukaryotic organisms. In this study, we characterized the function of the sucrose nonfermenting 1-related protein kinase2 (SnRK2) SAPK4 in the salt stress response of rice. RESULTS: Translational fusion of SAPK4 with the green fluorescent protein (GFP) showed subcellular localization in cytoplasm and nucleus. To examine the role of SAPK4 in salt tolerance we generated transgenic rice plants with over-expression of rice SAPK4 under control of the CaMV-35S promoter. Induced expression of SAPK4 resulted in improved germination, growth and development under salt stress both in seedlings and mature plants. In response to salt stress, the SAPK4-overexpressing rice accumulated less Na+ and Cl- and showed improved photosynthesis. SAPK4-regulated genes with functions in ion homeostasis and oxidative stress response were identified: the vacuolar H+-ATPase, the Na+/H+ antiporter NHX1, the Cl- channel OsCLC1 and a catalase. CONCLUSION: Our results show that SAPK4 regulates ion homeostasis and growth and development under salinity and suggest function of SAPK4 as a regulatory factor in plant salt stress acclimation. Identification of signaling elements involved in stress adaptation in plants presents a powerful approach to identify transcriptional activators of adaptive mechanisms to environmental changes that have the potential to improve tolerance in crop plants. OsCLC1,SAPK4|OSPDK Expression, imprinting, and evolution of rice homologs of the polycomb group genes 2009 Mol Plant CSIRO Plant Industry, GPO BOX 1600, ACT 2601, Australia. ming.luo@csiro.au Polycomb group proteins (PcG) play important roles in epigenetic regulation of gene expression. Some core PcG proteins, such as Enhancer of Zeste (E(z)), Suppressor of Zeste (12) (Su(z)12), and Extra Sex Combs (ESC), are conserved in plants. The rice genome contains two E(z)-like genes, OsiEZ1 and OsCLF, two homologs of Su(z)12, OsEMF2a and OsEMF2b, and two ESC-like genes, OsFIE1 and OsFIE2. OsFIE1 is expressed only in endosperm; the maternal copy is expressed while the paternal copy is not active. Other rice PcG genes are expressed in a wide range of tissues and are not imprinted in the endosperm. The two E(z)-like genes appear to have duplicated before the separation of the dicots and monocots; the two homologs of Su(z)12 possibly duplicated during the evolution of the Gramineae and the two ESC-like genes are likely to have duplicated in the ancestor of the grasses. No homologs of the Arabidopsis seed-expressed PcG genes MEA and FIS2 were identified in the rice genome. We have isolated T-DNA insertion lines in the rice homologs of three PcG genes. There is no autonomous endosperm development in these T-DNA insertion lines. One line with a T-DNA insertion in OsEMF2b displays pleiotropic phenotypes including altered flowering time and abnormal flower organs, suggesting important roles in rice development for this gene. OsCLF,OsEMF2a,OsEMF2b,OsFIE1|Epi-df,OsFIE2,OsiEZ1|OsSET1 Characterization of a newly identified rice chitinase-like protein (OsCLP) homologous to xylanase inhibitor 2013 BMC Biotechnol Division of Applied Life Science (BK21 program), Gyeongsang National University, Jinju, 660-701, South Korea. BACKGROUND: During rice blast fungal attack, plant xylanase inhibitor proteins (XIPs) that inhibit fungal xylanase activity are believed to act as a defensive barrier against fungal pathogens. To understand the role of XIPs in rice, a xylanase inhibitor was cloned from rice. The expression of this gene was examined at the transcriptional/translational levels during compatible and incompatible interactions, and the biochemical activity of this protein was also examined. RESULTS: Sequence alignment revealed that the deduced amino acid sequence of OsCLP shares a high degree of similarity with that of other plant TAXI-type XIPs. However, recombinant OsCLP did not display inhibitory activity against endo-1,4-beta-xylanase enzymes from Aureobasidium pullulans (A. pullulans) or Trichoderma viride (T. viride). Instead, an in-gel activity assay revealed strong chitinase activity. The transcription and translation of OsCLP were highly induced when rice was exposed to pathogens in an incompatible interaction. In addition, exogenous treatment with OsCLP affected the growth of the basidiomycete fungus Rhizoctonia solani through degradation of the hyphal cell wall. These data suggest that OsCLP, which has chitinase activity, may play an important role in plant defenses against pathogens. CONCLUSIONS: Taken together, our results demonstrate that OsCLP may have antifungal activity. This protein may directly inhibit pathogen growth by degrading fungal cell wall components through chitinase activity. OsCLP Molecular characterization of rice hsp101: complementation of yeast hsp104 mutation by disaggregation of protein granules and differential expression in indica and japonica rice types 2003 Plant Mol Biol Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, Dhaula Kuan, New Delhi-110021, India. HSP100 protein is an important component of the heat-shock response in diverse organisms. Using specific primers based on cDNA sequence, rice hsp101 gene was PCR-amplified and sequenced. Southern analysis revealed that there appears to be a single gene per haploid genome coding for HSP101 protein in rice. Northern analysis showed that expression of hsp101 transcript is strictly heat-inducible and induction is transient in nature. In the temperature regime tested, 45 degrees C treatment to intact rice seedlings for 2 h showed maximal levels of hsp101 mRNA. Rice full-length hsp101 cDNA complemented yeast mutant disrupted for its own hsp104 gene by insertional mutagenesis, with efficacy that was comparable with Arabidopsis hsp101 cDNA. Electron micrographic evidence suggested that rice hsp101 cDNA in yeast is active in re-solubilizing the stress-induced protein granules in the post-stress recovery period. Rice hsp101 cDNA expression in hsp104 deficient yeast also caused recovery in tolerance against arsenite. Western analyses showed that this protein is expressed more rapidly during the stress period and retained for longer duration in the post-stress recovery period in japonica rice as compared to indica rice types. This is the first report wherein plant HSP100 protein expression is correlated to disappearance of protein granules in the yeast cells and distinct rice type-dependent protein expression patterns are reported. HSP101|OsClpB-cyt|HSP100 OsHsfA2c and OsHsfB4b are involved in the transcriptional regulation of cytoplasmic OsClpB (Hsp100) gene in rice (Oryza sativa L.) 2012 Cell Stress Chaperones Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi 110021, India. ClpB-cytoplasmic (ClpB-cyt)/Hsp100 is an important chaperone protein in rice. Cellular expression of OsClpB-cyt transcript is governed by heat stress, metal stress, and developmental cues. Transgenic rice plants produced with 2 kb OsClpB-cyt promoter driving Gus reporter gene showed heat- and metal-regulated Gus expression in vegetative tissues and constitutive Gus expression in calli, flowering tissues, and embryonal half of seeds. Rice seedlings regenerated with OsClpB-cyt promoter fragment with deletion of its canonical heat shock element sequence (HSE(-273 to -280)) showed not only heat shock inducibility of Gus transcript/protein but also constitutive expression of Gus in vegetative tissues. It thus emerges that the only classical HSE present in OsClpB-cyt promoter is involved in repressing expression of OsClpB-cyt transcript under unstressed control conditions. Yeast one-hybrid assays suggested that OsHsfA2c specifically interacts with OsClpB-cyt promoter. OsHsfA2c also showed binding with OsClpB-cyt and OsHsfB4b showed binding with OsClpB-cyt; notably, interaction of OsHsfB4b was seen for all three OsClpB/Hsp100 protein isoforms (i.e., ClpB-cytoplasmic, ClpB-mitochondrial, and ClpB-chloroplastic). Furthermore, OsHsfB4b showed interaction with OsHsfA2c. This study suggests that OsHsfA2c may play a role as transcriptional activator and that OsHsfB4b is an important part of this heat shock responsive circuitry. HSP101|OsClpB-cyt|HSP100,OsHSF6|OsHsfA2c,OsHsfB4b Genome-wide analysis of rice ClpB/HSP100, ClpC and ClpD genes 2010 BMC Genomics Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi-110021, India. BACKGROUND: ClpB-cyt/HSP100 protein acts as chaperone, mediating disaggregation of denatured proteins. Previous studies have shown that ClpB-cyt/HSP100 gene belongs to the group class I Clp ATPase proteins and ClpB-cyt/HSP100 transcript is regulated by heat stress and developmental cues. RESULTS: Nine ORFs were noted to constitute rice class I Clp ATPases in the following manner: 3 ClpB proteins (ClpB-cyt, Os05g44340; ClpB-m, Os02g08490; ClpB-c, Os03g31300), 4 ClpC proteins (ClpC1, Os04g32560; ClpC2, Os12g12580; ClpC3, Os11g16590; ClpC4, Os11g16770) and 2 ClpD proteins (ClpD1, Os02g32520; ClpD2, Os04g33210). Using the respective signal sequences cloned upstream to GFP/CFP reporter proteins and transient expression studies with onion epidermal cells, evidence is provided that rice ClpB-m and Clp-c proteins are indeed localized to their respective cell locations mitochondria and chloroplasts, respectively. Associated with their diverse cell locations, domain structures of OsClpB-c, OsClpB-m and OsClpB-cyt proteins are noted to possess a high-level conservation. OsClpB-cyt transcript is shown to be enriched at milk and dough stages of seed development. While expression of OsClpB-m was significantly less as compared to its cytoplasmic and chloroplastic counterparts in different tissues, this transcript showed highest heat-induced expression amongst the 3 ClpB proteins. OsClpC1 and OsClpC2 are predicted to be chloroplast-localized as is the case with all known plant ClpC proteins. However, the fact that OsClpC3 protein appears mitochondrial/chloroplastic with equal probability and OsClpC4 a plasma membrane protein reflects functional diversity of this class. Different class I Clp ATPase transcripts were noted to be cross-induced by a host of different abiotic stress conditions. Complementation assays of Deltahsp104 mutant yeast cells showed that OsClpB-cyt, OsClpB-m, OsClpC1 and OsClpD1 have significantly positive effects. Remarkably, OsClpD1 gene imparted appreciably high level tolerance to the mutant yeast cells. CONCLUSIONS: Rice class I Clp ATPase gene family is constituted of 9 members. Of these 9, only 3 belonging to ClpB group are heat stress regulated. Distribution of ClpB proteins to different cell organelles indicates that their functioning might be critical in different cell locations. From the complementation assays, OsClpD1 appears to be more effective than OsClpB-cyt protein in rescuing the thermosensitive defect of the yeast ScDeltahsp104 mutant cells. HSP101|OsClpB-cyt|HSP100 Complexity of rice Hsp100 gene family: lessons from rice genome sequence data 2007 J Biosci Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi 110 021, India. Elucidation of genome sequence provides an excellent platform to understand detailed complexity of the various gene families. Hsp100 is an important family of chaperones in diverse living systems. There are eight putative gene loci encoding for Hsp100 proteins in Arabidopsis genome. In rice, two full-length Hsp100 cDNAs have been isolated and sequenced so far. Analysis of rice genomic sequence by in silico approach showed that two isolated rice Hsp100 cDNAs correspond to Os05g44340 and Os02g32520 genes in the rice genome database. There appears to be three additional proteins (encoded by Os03g31300, Os04g32560 and Os04g33210 gene loci) that are variably homologous to Os05g44340 and Os02g32520 throughout the entire amino acid sequence. The above five rice Hsp100 genes show significant similarities in the signature sequences known to be conserved among Hsp100 proteins. While Os05g44340 encodes cytoplasmic Hsp100 protein, those encoded by the other four genes are predicted to have chloroplast transit peptides. HSP101|OsClpB-cyt|HSP100 An active DNA transposon nDart causing leaf variegation and mutable dwarfism and its related elements in rice 2006 Plant J National Institute for Basic Biology, Okazaki 444-8585, Japan. While characterized mutable alleles caused by DNA transposons have been abundant in maize since the discovery of Dissociation conferring variegation by Barbara McClintock, only a few mutable alleles have been described in rice even though the rice genome contains various transposons. Here, we show that a spontaneous mutable virescent allele, pyl-v, is caused by the disruption of the nuclear-coded essential chloroplast protease gene, OsClpP5, due to insertion of a 607-bp non-autonomous DNA transposon, non-autonomous DNA-based active rice transposon one (nDart1), belonging to the hAT superfamily. The transposition of nDart1 can be induced by crossing with a line containing an autonomous element, aDart, and stabilized by segregating out of aDart. We also identified a novel mutable dwarf allele thl-m caused by an insertion of nDart1. The japonica cultivar Nipponbare carries no aDart, although it contains epigenetically silenced Dart element(s), which can be activated by 5-azacytidine. Nipponbare bears four subgroups of about 3.6-kb Dart-like sequences, three of which contain potential transposase genes, and around 3.6-kb elements without an apparent transposase gene, as well as three subgroups of about 0.6-kb nDart1-related elements that are all internal deletions of the Dart-like sequences. Both nDart1 and 3.6-kb Dart-like elements were also present in indica varieties 93-11 and Kasalath. nDart1 appears to be the most active mutagen among nDart1-related elements contributing to generating natural variations. A candidate for an autonomous element, aDart, and a possible application of nDart1 for transposon tagging are discussed. OsClpP5 Rice choline monooxygenase (OsCMO) protein functions in enhancing glycine betaine biosynthesis in transgenic tobacco but does not accumulate in rice (Oryza sativa L. ssp. japonica) 2012 Plant Cell Rep Ministry of Education Key Laboratory for Bio-resource and Eco-environment, College of Life Science and State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu, 610064, China. Glycine betaine (GB) is a compatible quaternary amine that enables plants to tolerate abiotic stresses, including salt, drought and cold. In plants, GB is synthesized through two-step of successive oxidations from choline, catalyzed by choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), respectively. Rice is considered as a typical non-GB accumulating species, although the entire genome sequencing revealed rice contains orthologs of both CMO and BADH. Several studies unraveled that rice has a functional BADH gene, but whether rice CMO gene (OsCMO) is functional or a pseudogene remains to be elucidated. In the present study, we report the functional characterization of rice CMO gene. The OsCMO gene was isolated from rice cv. Nipponbare (Oryza sativa L. ssp. japonica) using RT-PCR. Northern blot demonstrated the transcription of OsCMO is enhanced by salt stress. Transgenic tobacco plants overexpressing OsCMO results in increased GB content and elevated tolerance to salt stress. Immunoblotting analysis demonstrates that a functional OsCMO protein with correct size was present in transgenic tobacco but rarely accumulated in wild-type rice plants. Surprisingly, a large amount of truncated proteins derived from OsCMO was induced in the rice seedlings in response to salt stresses. This suggests that it is the lack of a functional OsCMO protein that presumably results in non-GB accumulation in the tested rice plant. KEY MESSAGE: Expression and transgenic studies demonstrate OsCMO is transcriptionally induced in response to salt stress and functions in increasing glycinebetaine accumulation and enhancing tolerance to salt stress. Immunoblotting analysis suggests that no accumulation of glycinebetaine in the Japonica rice plant presumably results from lack of a functional OsCMO protein. OsCMO Heterologous expression of rice calnexin (OsCNX) confers drought tolerance in Nicotiana tabacum 2013 Mol Biol Rep Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India. maryam21_7@yahoo.com Calnexin (CNX) is an integral membrane protein of endoplasmic reticulum (ER) and is a critical component of ER quality control machinery. It acts as a chaperone and ensures proper folding of newly synthesised glycoproteins. CNX shares a considerable homology with its luminal counterpart calreticulin (CRT). Together, they constitute CNX/CRT cycle which is imperative for proper folding of nascent proteins. CNX deficient organisms develop severe complications because of improper folding of proteins and consequently ER stress. CNX maintains calcium homeostasis by binding to the Ca(2+) which is a central node in various signaling pathways. Phosphorylation of cytoplasmic tail of CNX controls the sarco endoplasmic reticulum calcium ATPase and thus the movement of Ca(2+) in and out of its store-house, i.e. ER. Our studies on Oryza sativa CNX (OsCNX) reveal constitutive expression at various developmental stages and various tissues, thereby proving its requirement throughout the plant development. Further, its expression under various stress conditions gives an insight of the crosstalk existing between ER stress and abiotic stress signaling. This was confirmed by heterologous expression of OsCNX (OsCNX-HE) in tobacco and the OsCNX-HE lines were observed to exhibit better germination under mannitol stress and survival under dehydration stress conditions. The dehydration tolerance conferred by OsCNX appears to be ABA-dependent pathway. OsCNX silencing COI1 in rice increases susceptibility to chewing insects and impairs inducible defense 2012 PLoS One State Key Laboratory of Conservation and Utilization of Subtropical Agro-bioresources, Ministry of Agriculture, South China Agricultural University, Wushan, Guangzhou, People's Republic of China. The jasmonic acid (JA) pathway plays a key role in plant defense responses against herbivorous insects. CORONATINE INSENSITIVE1 (COI1) is an F-box protein essential for all jasmonate responses. However, the precise defense function of COI1 in monocotyledonous plants, especially in rice (Oryza sativa L.) is largely unknown. We silenced OsCOI1 in rice plants via RNA interference (RNAi) to determine the role of OsCOI1 in rice defense against rice leaf folder (LF) Cnaphalocrocis medinalis, a chewing insect, and brown planthopper (BPH) Nilaparvata lugens, a phloem-feeding insect. In wild-type rice plants (WT), the transcripts of OsCOI1 were strongly and continuously up-regulated by LF infestation and methyl jasmonate (MeJA) treatment, but not by BPH infestation. The abundance of trypsin protease inhibitor (TrypPI), and the enzymatic activities of polyphenol oxidase (PPO) and peroxidase (POD) were enhanced in response to both LF and BPH infestation, but the activity of lipoxygenase (LOX) was only induced by LF. The RNAi lines with repressed expression of OsCOI1 showed reduced resistance against LF, but no change against BPH. Silencing OsCOI1 did not alter LF-induced LOX activity and JA content, but it led to a reduction in the TrypPI content, POD and PPO activity by 62.3%, 48.5% and 27.2%, respectively. In addition, MeJA-induced TrypPI and POD activity were reduced by 57.2% and 48.2% in OsCOI1 RNAi plants. These results suggest that OsCOI1 is an indispensable signaling component, controlling JA-regulated defense against chewing insect (LF) in rice plants, and COI1 is also required for induction of TrypPI, POD and PPO in rice defense response to LF infestation. OsCOI1|OsCOI1a Involvement of OsJAZ8 in jasmonate-induced resistance to bacterial blight in rice 2012 Plant Cell Physiol Kagawa University, Miki, Kagawa, 761-0795 Japan. The plant hormone jasmonic acid (JA) has a crucial role in both host immunity and development in plants. Here, we report the importance of JA signaling in the defense system of rice. Exogenous application of JA conferred resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) in rice. Expression of OsJAZ8, a rice jasmonate ZIM-domain protein, was highly up-regulated by JA. OsJAZ8 interacted with a putative OsCOI1, which is a component of the SCF(COI1) E3 ubiquitin ligase complex, in a coronatine-dependent manner. OsJAZ8 also formed heterodimers with other OsJAZ proteins but did not form homodimer. JA treatment caused OsJAZ8 degradation and this degradation was dependent on the 26S proteasome pathway. Furthermore, the JA-dependent OsJAZ8 degradation was mediated by the Jas domain. Transgenic rice plants overexpressing OsJAZ8DeltaC, which lacks the Jas domain, exhibited a JA-insensitive phenotype. A large-scale analysis using a rice DNA microarray revealed that overexpression of OsJAZ8DeltaC altered the expression of JA-responsive genes, including defense-related genes, in rice. Furthermore, OsJAZ8DeltaC negatively regulated the JA-induced resistance to Xoo in rice. On the basis of these data, we conclude that JA plays an important role in resistance to Xoo, and OsJAZ8 acts as a repressor of JA signaling in rice. OsCOI1|OsCOI1a,OsJAZ12|OsTIFY11d,OsJAZ7|OsTIFY10b,OsJAZ8|OsTIFY10c,OsJAZ9|OsTIFY11a,OsNINJA1,OsNINJA2 Plant hormone jasmonate prioritizes defense over growth by interfering with gibberellin signaling cascade 2012 Proc Natl Acad Sci U S A National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Plants must effectively defend against biotic and abiotic stresses to survive in nature. However, this defense is costly and is often accompanied by significant growth inhibition. How plants coordinate the fluctuating growth-defense dynamics is not well understood and remains a fundamental question. Jasmonate (JA) and gibberellic acid (GA) are important plant hormones that mediate defense and growth, respectively. Binding of bioactive JA or GA ligands to cognate receptors leads to proteasome-dependent degradation of specific transcriptional repressors (the JAZ or DELLA family of proteins), which, at the resting state, represses cognate transcription factors involved in defense (e.g., MYCs) or growth [e.g. phytochrome interacting factors (PIFs)]. In this study, we found that the coi1 JA receptor mutants of rice (a domesticated monocot crop) and Arabidopsis (a model dicot plant) both exhibit hallmark phenotypes of GA-hypersensitive mutants. JA delays GA-mediated DELLA protein degradation, and the della mutant is less sensitive to JA for growth inhibition. Overexpression of a selected group of JAZ repressors in Arabidopsis plants partially phenocopies GA-associated phenotypes of the coi1 mutant, and JAZ9 inhibits RGA (a DELLA protein) interaction with transcription factor PIF3. Importantly, the pif quadruple (pifq) mutant no longer responds to JA-induced growth inhibition, and overexpression of PIF3 could partially overcome JA-induced growth inhibition. Thus, a molecular cascade involving the COI1-JAZ-DELLA-PIF signaling module, by which angiosperm plants prioritize JA-mediated defense over growth, has been elucidated. OsCOI1|OsCOI1a,OsCOI1b Oryza sativa COI homologues restore jasmonate signal transduction in Arabidopsis coi1-1 mutants 2013 PLoS One Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea. CORONATINE INSENSITIVE 1 (COI1) encodes an E3 ubiquitin ligase complex component that interacts with JAZ proteins and targets them for degradation in response to JA signaling. The Arabidopsis genome has a single copy of COI1, but the Oryza sativa genome has three closely related COI homologs. To examine the functions of the three OsCOIs, we used yeast two-hybrid assays to examine their interactions with JAZ proteins and found that OsCOIs interacted with OsJAZs and with JAZs, in a coronatine dependent manner. We also tested whether OsCOI1a and OsCOI1b could complement Arabidopsis coi1-1 mutants and found that overexpression of either gene in the coi1-1 mutant resulted in restoration of JA signal transduction and production of seeds, indicating successful complementation. Although OsCOI2 interacted with a few OsJAZs, we were not able to successfully complement the coi1-1 mutant with OsCOI2. Molecular modeling revealed that the three OsCOIs adopt 3D structures similar to COI1. Structural differences resulting from amino acid variations, especially among amino acid residues involved in the interaction with coronatine and JAZ proteins, were tested by mutation analysis. When His-391 in OsCOI2 was substituted with Tyr-391, OsCOI2 interacted with a wider range of JAZ proteins, including OsJAZ1, 2, 5 approximately 9 and 11, and complemented coi1-1 mutants at a higher frequency than the other OsCOIs and COI1. These results indicate that the three OsCOIs are orthologues of COI1 and play key roles in JA signaling. OsCOI2 Overexpression of OsCOIN, a putative cold inducible zinc finger protein, increased tolerance to chilling, salt and drought, and enhanced proline level in rice 2007 Planta Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Science, Beijing 100093, China. Rice (Oryza sativa L.) plant is sensitive to chilling, particularly at early stages of seedling development. Here a novel cold-inducible gene, designated OsCOIN (Oryza sativa cold-inducible), was isolated and characterized. Results showed that OsCOIN protein, a RING finger protein, was localized in both nuclear and cytoplasm membrane. OsCOIN is expressed in all rice organs and strongly induced by low temperature, ABA, salt and drought. Over-expression of OsCOIN in transgenic rice lines significantly enhanced their tolerance to cold, salt and drought, accompanied by an up-regulation of OsP5CS expression and an increase of cellular proline level. OsCOIN,OsP5CS|OsP5CS1 The role of OsCOM1 in homologous chromosome synapsis and recombination in rice meiosis 2012 Plant J State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. COM1/SAE2 is a highly conserved gene from yeast to higher eukaryotes. Its orthologs, known to cooperate with the MRX complex (Mre11/Rad50/Xrs2), are required for meiotic DNA double-strand break (DSB) ends resection and specific mitotic DSB repair events. Here, the rice (Oryza sativa, 2n = 2x = 24) COM1/SAE2 homolog was identified through positional cloning, termed OsCOM1. Four independent mutants of OsCOM1 were isolated and characterized. In Oscom1 mutants, synaptonemal complex (SC) formation, homologous pairing and recombination were severely inhibited, whereas aberrant non-homologous chromosome entanglements occurred constantly. Several key meiotic proteins, including ZEP1 and OsMER3, were not loaded normally onto chromosomes in Oscom1 mutants, whereas the localization of OsREC8, PAIR2 and PAIR3 seemed to be normal. Moreover, OsCOM1 was loaded normally onto meiotic chromosomes in Osrec8, zep1 and Osmer3 mutants, but could not be properly loaded in Osam1, pair2 and OsSPO11-1(RNAi) plants. These results provide direct evidence for the functions of OsCOM1 in promoting homologous synapsis and recombination in rice meiosis. OsCOM1,OsRad21-4|OsREC8,OsSPO11-1|OsTOP6A1,PAIR2,PAIR3,MER3|RCK,ZEP1 NARROW LEAF 7 controls leaf shape mediated by auxin in rice 2008 Mol Genet Genomics Agricultural Research Institute, HOKUREN Federation of Agricultural Cooperatives, Higashi-5, Kita-15, Naganuma, Hokkaido 0691317, Japan. fujino-kenji@hokuren.jp Elucidation of the genetic basis of the control of leaf shape could be of use in the manipulation of crop traits, leading to more stable and increased crop production. To improve our understanding of the process controlling leaf shape, we identified a mutant gene in rice that causes a significant decrease in the width of the leaf blade, termed narrow leaf 7 (nal7). This spontaneous mutation of nal7 occurred during the process of developing advanced back-crossed progeny derived from crosses of rice varieties with wild type leaf phenotype. While the mutation resulted in reduced leaf width, no significant morphological changes at the cellular level in leaves were observed, except in bulli-form cells. The NAL7 locus encodes a flavin-containing monooxygenase, which displays sequence homology with YUCCA. Inspection of a structural model of NAL7 suggests that the mutation results in an inactive enzyme. The IAA content in the nal7 mutant was altered compared with that of wild type. The nal7 mutant overexpressing NAL7 cDNA exhibited overgrowth and abnormal morphology of the root, which was likely to be due to auxin overproduction. These results indicate that NAL7 is involved in auxin biosynthesis. OsFMO|OsCOW1 Cloning, characterization and expression of OsFMO(t) in rice encoding a flavin monooxygenase 2013 J Genet Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Province, College of Life Science, South China Agricultural University, Guangzhou 510642, People's Republic of China. jicai@scau.edu.cn. Flavin monooxygenases (FMO) play a key role in tryptophan (Trp)-dependent indole-acetic acid (IAA) biosynthesis in plants and regulate plant growth and development. In this study, the full-length genomic DNA and cDNA of OsFMO(t), a FMO gene that was originally identified from a rolled-leaf mutant in rice, was isolated and cloned from wild type of the rolled-leaf mutant. OsFMO(t) was found to have four exons and three introns, and encode a protein with 422 amino acid residues that contains two basic conserved motifs, with a 'GxGxxG' characteristic structure. OsFMO(t) showed high amino acid sequence identity with FMO proteins from other plants, in particular with YUCCA from Arabidopsis, FLOOZY from Petunia, and OsYUCCA1 from rice. Our phylogenetic analysis showed that OsFMO(t) and the homologous FMO proteins belong to the same clade in the evolutionary tree. Overexpression of OsFMO(t) in transformed rice calli produced IAA-excessive phenotypes that showed browning and lethal effects when exogenous auxins such as naphthylacetic acid (NAA) were added to the medium. These results suggested that the OsFMO(t) protein is involved in IAA biosynthesis in rice and its overexpression could lead to the malformation of calli. Spatio-temporal expression analysis using RT-PCR and histochemical analysis for GUS activity revealed that expression of OsFMO(t) was totally absent in the rolled-leaf mutant. However, in the wild type variety, this gene was expressed at different levels temporally and spatially, with the highest expression observed in tissues with fast growth and cell division such as shoot apexes, tender leaves and root tips. Our results demonstrated that IAA biosynthesis regulated by OsFMO(t) is likely localized and might play an essential role in shaping local IAA concentrations which, in turn, is critical for regulating normal growth and development in rice. OsFMO|OsCOW1,OsYUCCA1 Constitutively wilted 1, a member of the rice YUCCA gene family, is required for maintaining water homeostasis and an appropriate root to shoot ratio 2007 Plant Mol Biol National Research Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Korea. Increasing its root to shoot ratio is a plant strategy for restoring water homeostasis in response to the long-term imposition of mild water stress. In addition to its important role in diverse fundamental processes, indole-3-acetic acid (IAA) is involved in root growth and development. Recent extensive characterizations of the YUCCA gene family in Arabidopsis and rice have elucidated that member's function in a tryptophan-dependent IAA biosynthetic pathway. Through forward- and reverse-genetics screening, we have isolated Tos17 and T-DNA insertional rice mutants in a CONSTITUTIVELY WILTED1 (COW1) gene, which encodes a new member of the YUCCA protein family. Homozygous plants with either a Tos17 or T-DNA-inserted allele of OsCOW1 exhibit phenotypes of rolled leaves, reduced leaf widths, and lower root to shoot ratios. These phenotypes are evident in seedlings as early as 7-10 d after germination, and remain until maturity. When oscow1 seedlings are grown under low-intensity light and high relative humidity, the rolled-leaf phenotype is greatly alleviated. For comparison, in such conditions, the transpiration rate for WT leaves decreases approx. 5- to 10-fold, implying that this mutant trait results from wilting rather than being a morphogenic defect. Furthermore, a lower turgor potential and transpiration rate in their mature leaves indicates that oscow1 plants are water-deficient, due to insufficient water uptake that possibly stems from that diminished root to shoot ratio. Thus, our observations suggest that OsCOW1-mediated IAA biosynthesis plays an important role in maintaining root to shoot ratios and, in turn, affects water homeostasis in rice. OsFMO|OsCOW1 A detrimental mitochondrial-nuclear interaction causes cytoplasmic male sterility in rice 2013 Nat Genet State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou, China. Plant cytoplasmic male sterility (CMS) results from incompatibilities between the organellar and nuclear genomes and prevents self pollination, enabling hybrid crop breeding to increase yields. The Wild Abortive CMS (CMS-WA) has been exploited in the majority of 'three-line' hybrid rice production since the 1970s, but the molecular basis of this trait remains unknown. Here we report that a new mitochondrial gene, WA352, which originated recently in wild rice, confers CMS-WA because the protein it encodes interacts with the nuclear-encoded mitochondrial protein COX11. In CMS-WA lines, WA352 accumulates preferentially in the anther tapetum, thereby inhibiting COX11 function in peroxide metabolism and triggering premature tapetal programmed cell death and consequent pollen abortion. WA352-induced sterility can be suppressed by two restorer-of-fertility (Rf) genes, suggesting the existence of different mechanisms to counteract deleterious cytoplasmic factors. Thus, CMS-related cytoplasmic-nuclear incompatibility is driven by a detrimental interaction between a newly evolved mitochondrial gene and a conserved, essential nuclear gene. COX11 Isolation and characterization of a rice cysteine protease gene, OSCP1, using T-DNA gene-trap system 2004 Plant Mol Biol School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea. The T-DNA gene-trap system has been efficiently used to elucidate gene functions in plants. We report here a functional analysis of a cysteine protease gene, OsCP1, isolated from a pool of T-DNA insertional rice. GUS assay with the T-DNA tagged line indicated that the OsCP1 promoter was highly active in the rice anther. Sequence analysis revealed that the deduced amino acid sequence of OsCP1 was homologous to those of papain family cyteine proteases containing the highly conserved interspersed amino acid motif, ERFNIN. This result suggested that the gene encodes a cysteine protease in rice. We also identified a suppressed mutant from T2 progeny of the T-DNA tagged line. The mutant showed a significant defect in pollen development. Taken together, the results demonstrated that OsCP1 is a cysteine protease gene that might play an important role in pollen development. OsCP1 Protein disulfide isomerase-like protein 1-1 controls endosperm development through regulation of the amount and composition of seed proteins in rice 2012 PLoS One Department of Plant Science, Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Korea. Protein disulfide isomerase (PDI) is a chaperone protein involved in oxidative protein folding by acting as a catalyst and assisting folding in the endoplasmic reticulum (ER). A genome database search showed that rice contains 19 PDI-like genes. However, their functions are not clearly identified. This paper shows possible functions of rice PDI-like protein 1-1 (PDIL1-1) during seed development. Seeds of the T-DNA insertion PDIL1-1 mutant, PDIL1-1Delta, identified by genomic DNA PCR and western blot analysis, display a chalky phenotype and a thick aleurone layer. Protein content per seed was significantly lower and free sugar content higher in PDIL1-1Delta mutant seeds than in the wild type. Proteomic analysis of PDIL1-1Delta mutant seeds showed that PDIL1-1 is post-translationally regulated, and its loss causes accumulation of many types of seed proteins including glucose/starch metabolism- and ROS (reactive oxygen species) scavenging-related proteins. In addition, PDIL1-1 strongly interacts with the cysteine protease OsCP1. Our data indicate that the opaque phenotype of PDIL1-1Delta mutant seeds results from production of irregular starch granules and protein body through loss of regulatory activity for various proteins involved in the synthesis of seed components. OsCP1 The rice RING E3 ligase, OsCTR1, inhibits trafficking to the chloroplasts of OsCP12 and OsRP1, and its overexpression confers drought tolerance in Arabidopsis 2014 Plant Cell Environ Department of Applied Plant Sciences, Kangwon National University, Chuncheon, 200-713, Korea. Plant growth under low water availability adversely affects many key processes with morphological, physiological, biochemical and molecular consequences. Here, we found that a rice gene, OsCTR1, encoding the RING Ub E3 ligase plays an important role in drought tolerance. OsCTR1 was highly expressed in response to dehydration treatment and defense-related phytohormones, and its encoded protein was localized in both the chloroplasts and the cytosol. Intriguingly, the OsCTR1 protein was found predominantly targeted to the cytosol when rice protoplasts transfected with OsCTR1 were treated with abscisic acid (ABA). Several interacting partners were identified, which were mainly targeted to the chloroplasts, and interactions with OsCTR1 were confirmed by using biomolecular fluorescence complementation (BiFC). Interestingly, two chloroplast-localized proteins (OsCP12 and OsRP1) interacted with OsCTR1 in the cytosol, and ubiquitination by OsCTR1 led to protein degradation via the Ub 26S proteasome. Heterogeneous overexpression of OsCTR1 in Arabidopsis exhibited hypersensitive phenotypes with respect to ABA-responsive seed germination, seedling growth and stomatal closure. The ABA-sensitive transgenic plants also showed improvement in their tolerance against severe water deficits. Taken together, our findings lend support to the hypothesis that the molecular functions of OsCTR1 are related to tolerance to water-deficit stress via ABA-dependent regulation and related systems. OsCP12,OsCTR1,OsRP1 qUVR-10, a major quantitative trait locus for ultraviolet-B resistance in rice, encodes cyclobutane pyrimidine dimer photolyase 2005 Genetics National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. uechu@nias.affrc.go.jp Rice qUVR-10, a quantitative trait locus (QTL) for ultraviolet-B (UVB) resistance on chromosome 10, was cloned by map-based strategy. It was detected in backcross inbred lines (BILs) derived from a cross between the japonica variety Nipponbare (UV resistant) and the indica variety Kasalath (UV sensitive). Plants homozygous for the Nipponbare allele at the qUVR-10 locus were more resistant to UVB compared with the Kasalath allele. High-resolution mapping using 1850 F(2) plants enabled us to delimit qUVR-10 to a <27-kb genomic region. We identified a gene encoding the cyclobutane pyrimidine dimer (CPD) photolyase in this region. Activity of CPD photorepair in Nipponbare was higher than that of Kasalath and nearly isogenic with qUVR-10 [NIL(qUVR-10)], suggesting that the CPD photolyase of Kasalath was defective. We introduced a genomic fragment containing the CPD photolyase gene of Nipponbare to NIL(qUVR-10). Transgenic plants showed the same level of resistance as Nipponbare did, indicating that the qUVR-10 encoded the CPD photolyase. Comparison of the qUVR-10 sequence in the Nipponbare and Kasalath alleles revealed one probable candidate for the functional nucleotide polymorphism. It was indicated that single-base substitution in the CPD photolyase gene caused the alteration of activity of CPD photorepair and UVB resistance. Furthermore, we were able to develop a UV-hyperresistant plant by overexpression of the photolyase gene. CPD|OsCPDP|qUVR-10 Spontaneously occurring mutations in the cyclobutane pyrimidine dimer photolyase gene cause different sensitivities to ultraviolet-B in rice 2005 Plant J Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan. j-hidema@ige.tohoku.ac.jp Sensitivity to ultraviolet-B (UVB) radiation (280-320 nm) varies widely among rice cultivars. We previously indicated that UV-resistant rice cultivars are better able to repair cyclobutane pyrimidine dimers (CPDs) through photorepair than are UV-sensitive cultivars. In this paper, we report that UVB sensitivity in rice, in part, is the result of defective CPD photolyase alleles. Surjamkhi (indica) exhibited greater sensitivity to UVB radiation and was more deficient in CPD photorepair ability compared with UV-resistant Sasanishiki (japonica). The deficiency in CPD photorepair in Surjamkhi resulted from changes in two nucleotides at positions 377 and 888 in the photolyase gene, causing alterations of two deduced amino acids at positions 126 and 296 in the photolyase enzyme. A linkage analysis in populations derived from Surjamkhi and Sasanishiki showed that UVB sensitivity is a quantitative inherited trait and that the CPD photolyase locus is tightly linked with a quantitative trait locus that explains a major portion of the genetic variation for this trait. These results suggest that spontaneously occurring mutations in the CPD photolyase gene cause different degrees of sensitivity to UVB in rice, and that the resistance of rice to UVB radiation could be increased by increasing the photolyase function through conventional breeding or bioengineering. CPD|OsCPDP|qUVR-10 Increase in CPD photolyase activity functions effectively to prevent growth inhibition caused by UVB radiation 2007 Plant J Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan. Rice cultivars vary widely in their sensitivity to ultraviolet B (UVB) and this has been correlated with cyclobutane pyrimidine dimer (CPD) photolyase mutations that alter the structure/function of this photorepair enzyme. Here, we tested whether CPD photolyase function determines the UVB sensitivity of rice (Oryza sativa) by generating transgenic rice plants bearing the CPD photolyase gene of the UV-resistant rice cultivar Sasanishiki in the sense orientation (S-B and S-C lines) or the antisense orientation (AS-D line). The S-B and S-C plants had 5.1- and 45.7-fold higher CPD photolyase activities than the wild-type, respectively, were significantly more resistant to UVB-induced growth damage, and maintained significantly lower CPD levels in their leaves during growth under elevated UVB radiation. Conversely, the AS-D plant had little photolyase activity, was severely damaged by elevated UVB radiation, and maintained higher CPD levels in its leaves during growth under UVB radiation. Notably, the S-C plant was not more resistant to UVB-induced growth inhibition than the S-B plant, even though it had much higher CPD photolyase activity. These results strongly indicate that UVB-induced CPDs are one of principal causes of UVB-induced growth inhibition in rice plants grown under supplementary UVB radiation, and that increasing CPD photolyase activity can significantly alleviate UVB-caused growth inhibition in rice. However, further protection from UVB-induced damage may require the genetic enhancement of other systems as well. CPD|OsCPDP|qUVR-10 Cyclobutane pyrimidine dimer (CPD) photolyase repairs ultraviolet-B-induced CPDs in rice chloroplast and mitochondrial DNA 2011 Plant J Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan. Plants use sunlight as energy for photosynthesis; however, plant DNA is exposed to the harmful effects of ultraviolet-B (UV-B) radiation (280-320 nm) in the process. UV-B radiation damages nuclear, chloroplast and mitochondrial DNA by the formation of cyclobutane pyrimidine dimers (CPDs), which are the primary UV-B-induced DNA lesions, and are a principal cause of UV-B-induced growth inhibition in plants. Repair of CPDs is therefore essential for plant survival while exposed to UV-B-containing sunlight. Nuclear repair of the UV-B-induced CPDs involves the photoreversal of CPDs, photoreactivation, which is mediated by CPD photolyase that monomerizes the CPDs in DNA by using the energy of near-UV and visible light (300-500 nm). To date, the CPD repair processes in plant chloroplasts and mitochondria remain poorly understood. Here, we report the photoreactivation of CPDs in chloroplast and mitochondrial DNA in rice. Biochemical and subcellular localization analyses using rice strains with different levels of CPD photolyase activity and transgenic rice strains showed that full-length CPD photolyase is encoded by a single gene, not a splice variant, and is expressed and targeted not only to nuclei but also to chloroplasts and mitochondria. The results indicate that rice may have evolved a CPD photolyase that functions in chloroplasts, mitochondria and nuclei, and that contains DNA to protect cells from the harmful effects of UV-B radiation. CPD|OsCPDP|qUVR-10 UV radiation-sensitive norin 1 rice contains defective cyclobutane pyrimidine dimer photolyase 2000 Plant Cell Institute of Genetic Ecology, Tohoku University, Sendai 980-8577, Japan. Norin 1, a progenitor of many economically important Japanese rice strains, is highly sensitive to the damaging effects of UVB radiation (wavelengths 290 to 320 nm). Norin 1 seedlings are deficient in photorepair of cyclobutane pyrimidine dimers. However, the molecular origin of this deficiency was not known and, because rice photolyase genes have not been cloned and sequenced, could not be determined by examining photolyase structural genes or upstream regulatory elements for mutations. We therefore used a photoflash approach, which showed that the deficiency in photorepair in vivo resulted from a functionally altered photolyase. These results were confirmed by studies with extracts, which showed that the Norin 1 photolyase-dimer complex was highly thermolabile relative to the wild-type Sasanishiki photolyase. This deficiency results from a structure/function alteration of photolyase rather than of nonspecific repair, photolytic, or regulatory elements. Thus, the molecular origin of this plant DNA repair deficiency, resulting from a spontaneously occurring mutation to UV radiation sensitivity, is defective photolyase. CPD|OsCPDP|qUVR-10 Ultraviolet-B sensitivities in Japanese lowland rice cultivars: cyclobutane pyrimidine dimer photolyase activity and gene mutation 2004 Plant Cell Physiol Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, 980-8577 Japan. There is a cultivar difference in the response to ultraviolet-B (UVB: 280-320 nm) in rice (Oryza sativa L.). Among Japanese lowland rice cultivars, Sasanishiki, a leading Japanese rice cultivar, is resistant to the damaging effects of UVB while Norin 1, a close relative, is less resistant. We found previously that Norin 1 was deficient in cyclobutane pyrimidine dimer (CPD) photorepair ability and suggested that the UVB sensitivity in rice depends largely on CPD photorepair ability. In order to verify that suggestion, we examined the correlation between UVB sensitivity and CPD photolyase activity in 17 rice cultivars of progenitors and relatives in breeding of UV-resistant Sasanishiki and UV-sensitive Norin 1. The amino acid at position 126 of the deduced amino acid sequence of CPD photolyase in cultivars including such as Norin 1 was found to be arginine, the CPD photolyase activities of which were lower. The amino acid at that position in cultivars including such as Sasanishiki was glutamine. Furthermore, cultivars more resistant to UVB were found to exhibit higher photolyase activities than less resistant cultivars. These results emphasize that single amino acid alteration from glutamine to arginine leads to a deficit of CPD photolyase activity and that CPD photolyase activity is one of the main factors determining UVB sensitivity in rice. CPD|OsCPDP|qUVR-10 The native cyclobutane pyrimidine dimer photolyase of rice is phosphorylated 2008 Plant Physiol Department of Environmental Life Sciences , Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan. tera@ige.tohoku.ac.jp The cyclobutane pyrimidine dimer (CPD) is a major type of DNA damage induced by ultraviolet B (UVB) radiation. CPD photolyase, which absorbs blue/UVA light as an energy source to monomerize dimers, is a crucial factor for determining the sensitivity of rice (Oryza sativa) to UVB radiation. Here, we purified native class II CPD photolyase from rice leaves. As the final purification step, CPD photolyase was bound to CPD-containing DNA conjugated to magnetic beads and then released by blue-light irradiation. The final purified fraction contained 54- and 56-kD proteins, whereas rice CPD photolyase expressed from Escherichia coli was a single 55-kD protein. Western-blot analysis using anti-rice CPD photolyase antiserum suggested that both the 54- and 56-kD proteins were the CPD photolyase. Treatment with protein phosphatase revealed that the 56-kD native rice CPD photolyase was phosphorylated, whereas the E. coli-expressed rice CPD photolyase was not. The purified native rice CPD photolyase also had significantly higher CPD photorepair activity than the E. coli-expressed CPD photolyase. According to the absorption, emission, and excitation spectra, the purified native rice CPD photolyase possesses both a pterin-like chromophore and an FAD chromophore. The binding activity of the native rice CPD photolyase to thymine dimers was higher than that of the E. coli-expressed CPD photolyase. These results suggest that the structure of the native rice CPD photolyase differs significantly from that of the E. coli-expressed rice CPD photolyase, and the structural modification of the native CPD photolyase leads to higher activity in rice. CPD|OsCPDP|qUVR-10 A gene for a Class II DNA photolyase from Oryza sativa: cloning of the cDNA by dilution-amplification 2003 Mol Genet Genomics Department of Molecular Biosciences, Graduate School of Life Sciences, Tohoku University, 980-8577 Sendai, Japan. Ultraviolet radiation induces the formation of two classes of photoproducts in DNA-the cyclobutane pyrimidine dimer (CPD) and the pyrimidine [6-4] pyrimidone photoproduct (6-4 product). Many organisms produce enzymes, termed photolyases, which specifically bind to these lesions and split them via a UV-A/blue light-dependent mechanism, thereby reversing the damage. These photolyases are specific for either CPDs or 6-4 products. Two classes of photolyases (class I and class II) repair CPDs. A gene that encodes a protein with class II CPD photolyase activity in vitro has been cloned from several plants including Arabidopsis thaliana, Cucumis sativus and Chlamydomonas reinhardtii. We report here the isolation of a homolog of this gene from rice (Oryza sativa), which was cloned on the basis of sequence similarity and PCR-based dilution-amplification. The cDNA comprises a very GC-rich (75%) 5; region, while the 3; portion has a GC content of 50%. This gene encodes a protein with CPD photolyase activity when expressed in E. coli. The CPD photolyase gene encodes at least two types of mRNA, formed by alternative splicing of exon 5. One of the mRNAs encodes an ORF for 506 amino acid residues, while the other is predicted to code for 364 amino acid residues. The two RNAs occur in about equal amounts in O. sativa cells. CPD|OsCPDP|qUVR-10 Overexpression of constitutively active OsCPK10 increases Arabidopsis resistance against Pseudomonas syringae pv. tomato and rice resistance against Magnaporthe grisea 2013 Plant Physiol Biochem State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China. Calcium-dependent protein kinases (CDPKs) are crucial calcium sensors involved in plant responses to pathogen infection. Here, we report isolation and functional characterization of the pathogen-responsive rice OsCPK10 gene. The expression of OsCPK10 was strongly induced following treatment with a Magnaporthe grisea elicitor. Kinase activity assay showed that the functional OsCPK10 protein not only autophosphorylated, but also phosphorylated Casein in a calcium-dependent manner. Overexpression of constitutively active OsCPK10 in Arabidopsis enhanced the resistance to infection with Pseudomonas syringae pv. tomato, associated with elevated expression of both SA- and JA-related defense genes. Similarly, transgenic rice plants containing constitutively active OsCPK10 exhibited enhanced resistance to blast fungus M. grisea. The enhanced resistance in the transgenic lines was associated with activated expression of SA- and JA-related defense genes. Collectively, our results indicate that rice OsCPK10 is a crucial regulator in plant immune responses, and that it may regulate disease resistance by activating both SA- and JA-dependent defense responses. OsCPK10 Expression profile of calcium-dependent protein kinase (CDPKs) genes during the whole lifespan and under phytohormone treatment conditions in rice (Oryza sativa L. ssp. indica) 2009 Plant Mol Biol National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan, People's Republic of China. Calcium-dependent protein kinases (CDPKs) control plant development and response to various stress environments through the important roles in the regulation of Ca(2+) signaling. Thirty-one CDPK genes have been identified in the rice genome by a complete search of the genome based upon HMM profiles. In this study, the expression of this gene family was analyzed using the Affymetrix rice genome array in three rice cultivars: Minghui 63, Zhenshan 97, and their hybrid Shanyou 63 independently. Twenty-seven tissues sampled throughout the entire rice life-span were studied, along with three hormone treatments (GA3, NAA and KT), applied to the seedling at the trefoil stage. All 31 genes were found to be expressed in at least one of the experimental stages studied and revealed diverse expression patterns. We identified differential expression of the OsCPK genes in the stamen (1 day before flowering), the panicle (at the heading stage), the endosperm (days after pollination) and also in callus, in all three cultivars. Eight genes, OsCPK2, OsCPK11, OsCPK14, OsCPK22, OsCPK25, OsCPK26, OsCPK27 and OsCPK29 were found dominantly expressed in the panicle and the stamen, and five genes, OsCPK6, OsCPK7, OsCPK12, OsCPK23 and OsCPK31 were up-regulated in the endosperm stage. The OsCPK genes were also found to be regulated in rice seedlings subjected to different hormone treatment conditions, however their expression were not the same for all varieties. These diverse expression profiles trigger the functional analysis of the CDPK family in rice. OsCPK12 Overexpression of a calcium-dependent protein kinase gene enhances growth of rice under low-nitrogen conditions 2010 Plant Biotechnology Univ Tokyo, Grad Sch Agr & Life Sci, Dept Agr & Environm Biol, Tokyo 1138657, Japan The excessive amounts of nitrogen applied in current farming systems can cause environmental problems. There is therefore a need to improve the ability of crop plants to utilize nitrogenous fertilizers. We screened for nitrogen deficiency-tolerant lines among transgenic rice plants that overexpressed full-length cDNAs (FL-cDNAs) corresponding to low-nitrogen response genes, genes related to nitrogen metabolism, and genes related to carbon metabolism. We found that overexpression of OsCPK12 FL-cDNA, encoding a calcium-dependent protein kinase ( CDPK), conferred tolerance to low-nitrogen stress in rice. After two weeks of low-nitrogen treatment, dry weights of shoots from OsCPK12-overexpressing plants were greater than those from control plants. Furthermore, total nitrogen contents of OsCPK12-overexpressing plants were higher than those of the control plants. Our findings suggest that OsCPK12 is involved in the signal transduction pathway(s) in the low-nitrogen stress response and may be useful in engineering crop plants with improved tolerance to low nitrogen levels. OsCPK12 A rice calcium-dependent protein kinase OsCPK12 oppositely modulates salt-stress tolerance and blast disease resistance 2012 Plant J National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan. tasano@affrc.go.jp Calcium-dependent protein kinases (CDPKs) regulate the downstream components in calcium signaling pathways. We investigated the effects of overexpression and disruption of an Oryza sativa (rice) CDPK (OsCPK12) on the plant's response to abiotic and biotic stresses. OsCPK12-overexpressing (OsCPK12-OX) plants exhibited increased tolerance to salt stress. The accumulation of hydrogen peroxide (H(2) O(2) ) in the leaves was less in OsCPK12-OX plants than in wild-type (WT) plants. Genes encoding reactive oxygen species (ROS) scavenging enzymes (OsAPx2 and OsAPx8) were more highly expressed in OsCPK12-OX plants than in WT plants, whereas the expression of the NADPH oxidase gene, OsrbohI, was decreased in OsCPK12-OX plants compared with WT plants. Conversely, a retrotransposon (Tos17) insertion mutant, oscpk12, and plants transformed with an OsCPK12 RNA interference (RNAi) construct were more sensitive to high salinity than were WT plants. The level of H(2) O(2) accumulation was greater in oscpk12 and OsCPK12 RNAi plants than in the WT. These results suggest that OsCPK12 promotes tolerance to salt stress by reducing the accumulation of ROS. We also observed that OsCPK12-OX seedlings had increased sensitivity to abscisic acid (ABA) and increased susceptibility to blast fungus, probably resulting from the repression of ROS production and/or the involvement of OsCPK12 in the ABA signaling pathway. Collectively, our results suggest that OsCPK12 functions in multiple signaling pathways, positively regulating salt tolerance and negatively modulating blast resistance. OsCPK12,SALT Functional characterisation of OsCPK21, a calcium-dependent protein kinase that confers salt tolerance in rice 2011 Plant Mol Biol Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan. tasano@affrc.go.jp Calcium acts as a messenger in various signal transduction pathways in plants. Calcium-dependent protein kinases (CDPKs) play important roles in regulating downstream components in calcium signaling pathways. In rice, the CDPKs constitute a large multigene family consisting of 29 genes, but the biological functions and functional divergence or redundancy of most of these genes remain unclear. Using a mini-scale full-length cDNA overexpressor (FOX) gene hunting system, we generated 250 independent transgenic rice plants overexpressing individual rice CDPKs (CDPK FOX-rice lines). These CDPK FOX-rice lines were screened for salt stress tolerance. The survival rate of the OsCPK21-FOX plants was higher than that of wild-type (WT) plants grown under high salinity conditions. The inhibition of seedling growth by abscisic acid (ABA) treatment was greater in the OsCPK21-FOX plants than in WT plants. Several ABA- and high salinity-inducible genes were more highly expressed in the OsCPK21-FOX plants than in WT plants. These results suggest that OsCPK21 is involved in the positive regulation of the signaling pathways that are involved in the response to ABA and salt stress. OsCPK21 Characterization and mapping of a shattering mutant in rice that corresponds to a block of domestication genes 2006 Genetics National Institute of Agricultural Biotechnology, Rural Development Administration, Suwon, Korea. Easy shattering reduces yield due to grain loss during harvest in cereals. Shattering is also a hindrance in breeding programs that use wild accessions because the shattering habit is often linked to desirable traits. We characterized a shattering mutant line of rice, Hsh, which was derived from a nonshattering japonica variety, Hwacheong, by N-methyl-N-nitrosourea (MNU) treatment. The breaking tensile strength (BTS) of the grain pedicel was measured using a digital force gauge to evaluate the degree of shattering of rice varieties at 5, 10, 15, 20, 25, 30, 35, and 40 days after heading (DAH). The BTS of Hwacheong did not decrease with increasing DAH, maintaining a level of 180-240 gf, while that of Hsh decreased greatly during 10-20 DAH and finally stabilized at 50 gf. Optical microscopy revealed that Hsh had a well-developed abscission layer similar to the wild rice Oryza nivara (accession IRGC105706), while Hwacheong did not produce an abscission layer, indicating that the shattering of Hsh was caused by differentiation of the abscission layer. On the basis of the BTS value and morphology of the abscission layer of F(1) plants and segregation data in F(2) populations, it was concluded that the easy shattering of Hsh was controlled by the single recessive gene sh-h. The gene sh-h was determined to be located on rice chromosome 7 by bulked segregant analysis. Using 14 SSR markers on rice chromosome 7, the gene sh-h was mapped between the flanking markers RM8262 and RM7161 at distances of 1.6 and 2.0 cM, respectively. An SSR marker Rc17 cosegregated with the gene sh-h. The locus sh-h for shattering was tightly linked to the Rc locus conferring red pericarp, as well as a QTL qSD(s)-7-1 for seed dormancy, implying that this region might represent a domestication block in the evolutionary pathway of rice. OsCPL1|sh-h,Rc|qSD7-1|qPC7 Inactivation of the CTD phosphatase-like gene OsCPL1 enhances the development of the abscission layer and seed shattering in rice 2010 Plant J Department of Agricultural Bio-resources, National Academy of Agricultural Science (NAAS), Suwon 441-707, Korea. Although susceptibility to seed shattering causes severe yield loss during cereal crop harvest, it is an adaptive trait for seed dispersal in wild plants. We previously identified a recessive shattering locus, sh-h, from the rice shattering mutant line Hsh that carries an enhanced abscission layer. Here, we further mapped sh-h to a 34-kb region on chromosome 7 by analyzing 240 F(2) plants and five F(3) lines from the cross between Hsh and Blue&Gundil. Hsh had a point mutation at the 3' splice site of the seventh intron within LOC_Os07g10690, causing a 15-bp deletion of its mRNA as a result of altered splicing. Two transferred DNA (T-DNA) insertion mutants and one point mutant exhibited the enhanced shattering phenotype, confirming that LOC_Os07g10690 is indeed the sh-h gene. RNA interference (RNAi) transgenic lines with suppressed expression of this gene exhibited greater shattering. This gene, which encodes a protein containing a conserved carboxy-terminal domain (CTD) phosphatase domain, was named Oryza sativa CTD phosphatase-like 1 (OsCPL1). Subcellular localization and biochemical analysis revealed that the OsCPL1 protein is a nuclear phosphatase, a common characteristic of metazoan CTD phosphatases involved in cell differentiation. These results demonstrate that OsCPL1 represses differentiation of the abscission layer during panicle development. OsCPL1|sh-h OsCpn60alpha1, encoding the plastid chaperonin 60alpha subunit, is essential for folding of rbcL 2013 Mol Cells Crop Biotech Institute and Department of Genetic Engineering, Kyung Hee University, Yongin, 446-701, Korea. Chaperonins are involved in protein-folding. The rice genome encodes six plastid chaperonin subunits (Cpn60) - three alpha and three beta. Our study showed that they were differentially expressed during normal plant development. Moreover, five were induced by heat stress (42 degrees C) but not by cold (10 degrees C). The oscpn60alpha1 mutant had a pale-green phenotype at the seedling stage and development ceased after the fourth leaf appeared. Transiently expressed OsCpn60alpha1:GFP fusion protein was localized to the chloroplast stroma. Immuno-blot analysis indicated that the level of Rubisco large subunit (rbcL) was severely reduced in the mutant while levels were unchanged for some imported proteins, e.g., stromal heat shock protein 70 (Hsp70) and chlorophyll a/b binding protein 1 (Lhcb1). This demonstrated that OsCpn60alpha1 is required for the folding of rbcL and that failure of that process is seedling-lethal. OsCPn60alpha1|OsCPn60a1 Expression Pattern of the Coparyl Diphosphate Synthase Gene in Developing Rice Anthers 2014 Bioscience, Biotechnology and Biochemistry Department of Applied Biological Sciences, The University of Tokyo Rice anthers contain high concentrations of gibberellins A(4) and A(7). To understand their physiological roles, we examined the site of their biosynthesis by analyzing the expression pattern of a gene (OsCPS) encoding coparyl diphosphate synthase in developing rice flowers. Expression was apparent in the anthers 1-2 days before flowering, and CPS mRNA accumulated in the maturing pollen. OsCPS|OsCPS1 Cloning and Characterization of cDNAs Encodingent-Copalyl Diphosphate Synthases in Wheat: Insight into the Evolution of Rice Phytoalexin Biosynthetic Genes 2014 Bioscience, Biotechnology and Biochemistry Department of Bioresource Engineering, Yamagata University, Tsuruoka, Yamagata, Japan. In vitro assays using recombinant enzymes enabled three cDNAs encoding ent-copalyl diphosphate synthases to be identified in wheat (Triticum aestivum): TaCPS1, TaCPS2, and TaCPS3. The phylogenetic tree and expression analyses suggest that TaCPS3 is responsible for gibberellin biosynthesis, while TaCPS1 and TaCPS2 are possible functional homologs of diterpene cyclase genes OsCPS2 and OsCPS4 involved in phytoalexin biosynthesis in rice. OsCPS|OsCPS1,OsCPS2|OsCyc2,OsCPS4|OsCyc1 gid1, a gibberellin-insensitive dwarf mutant, shows altered regulation of probenazole-inducible protein (PBZ1) in response to cold stress and pathogen attack 2006 Plant, Cell and Environment National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan. A recessive gibberellin (GA)-insensitive dwarf mutant of rice, gibberellin-insensitive dwarf1 (gid1), has been identified, which shows a severe dwarf phenotype and contains high concentrations of endogenous GA. To elucidate the function of gid1, proteins regulated downstream of gid1 were analysed using a proteomic approach. Proteins extracted from suspension-cultured cells of gid1 and its wild type were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Of a total of 962 proteins identified from the suspension-cultured cells, 16 were increased and 14 were decreased in gid1 compared with its wild type. Among the proteins hyper-accumulated in gid1 were osmotin, triosephosphate isomerase, probenazole inducible protein (PBZ1) and pathogenesis-related protein 10. Of these four genes, only the expression of PBZ1 was increased by exogenous GA3 application. Expression of this gene was also enhanced in shoots of the wild type by cold stress or by rice blast fungus infection. Under normal growth conditions, there was more PBZ1 protein in gid1 than in the wild type. In addition, gid1 showed increased tolerance to cold stress and resistance to blast fungus infection. The entcopalyl diphosphate synthase (OsCPS) genes, which encode enzymes at the branch point between GA and phytoalexin biosynthesis, were expressed differentially in gid1 relative to the wild type. Specifically, OsCPS1, which encodes an enzyme in the GA biosynthesis pathway, was down-regulated and OsCPS2 and OsCPS4, which encode enzymes in phytoalexin biosynthesis, were up-regulated in gid1. These results suggest that the expression of PBZ1 is regulated by GA signalling and stress stimuli, and that gid1 is involved in tolerance to cold stress and resistance to blast fungus. OsCPS|OsCPS1,OsCPS2|OsCyc2,OsCPS4|OsCyc1,GID1|OsGID1,OsPR10a|PBZ1 Biological functions of ent- and syn-copalyl diphosphate synthases in rice: key enzymes for the branch point of gibberellin and phytoalexin biosynthesis 2004 Plant J Course of the Science of Bioresources, The United Graduate School of Agricultural Science, Iwate University (Yamagata University), Tsuruoka, Yamagata 997-8555, Japan. Rice (Oryza sativa L.) produces ent-copalyl diphosphate (ent-CDP) and syn-CDP as precursors for several classes of phytoalexins and the phytohormones, gibberellins (GAs). It has recently been shown that a loss-of-function mutation of OsCPS1, a gene encoding a putative ent-CDP synthase, results in a severely GA-deficient dwarf phenotype in rice. To clarify the biological functions of the ent- and syn-CDP synthases involved in the biosynthesis of phytoalexins and/or GAs, we isolated two cDNAs, OsCyc1 and OsCyc2, encoding putative diterpene cyclases from ultraviolet (UV)-irradiated rice leaves (cv. Nipponbare). The production of phytoalexins in rice leaves is known to be highly induced by UV treatment. Using a bacterial expression system, we demonstrated that OsCyc1 encodes syn-CDP synthase and that OsCyc2 and OsCPS1 encode ent-CDP synthase. The level of expression of the OsCyc1 and OsCyc2 transcripts in rice leaves increased drastically in response to UV treatment, whereas expression of the OsCPS1 transcript was not induced by UV light. These results suggest that OsCyc1, OsCyc2 and OsCPS1 are responsible for the biosynthesis of momilactones A and B and oryzalexin S, oryzalexins A-F and phytocassanes A-E, and GAs, respectively. Our results strongly suggest the presence of two ent-CDP synthase isoforms in rice, one that participates in the biosynthesis of GAs and a second that is involved in the biosynthesis of phytoalexins. OsCPS|OsCPS1,OsCPS2|OsCyc2,OsCPS4|OsCyc1 Comparison of the Enzymatic Properties ofent-Copalyl Diphosphate Synthases in the Biosynthesis of Phytoalexins and Gibberellins in Rice 2014 Bioscience, Biotechnology and Biochemistry Biotechnology Research Center, Toyama Prefectural University, Japan. The rice genome contains two ent-copalyl diphosphate synthase genes: OsCPS1 acts in gibberellin (phytohormone) biosynthesis, and OsCPS2/OsCyc2 acts in the synthesis of oryzalexins A-F and phytocassanes A-E (phytoalexins). We characterized the enzymatic properties of recombinant OsCPS2/OsCyc2 fused with a tag-protein at the N-terminus, and compared them to those of OsCPS1. Several enzymatic properties of OsCPS2/OsCyc2, including the optimal pH, optimal temperature, divalent cation requirement, and kinetic values for the geranylgeranyl diphosphate (GGDP) substrate, were almost the same as those of OsCPS1. However, OsCPS2/OsCyc2 activity was not inhibited by 50-60 muM GGDP substrate, by which the OsCPS1 activity was inhibited. Furthermore, the OsCPS1 activity exhibited approximately 70% inhibition by 100 muM Amo-1618 (a gibberellin biosynthetic inhibitor), whereas the OsCPS2/OsCyc2 activity exhibited approximately 10% inhibition. These results indicate that the properties of OsCPS2/OsCyc2 were partially different from those of OsCPS1, although OsCPS2/OsCyc2 catalyzes the same reaction step as OsCPS1. OsCPS2|OsCyc2 Genetic evidence for natural product-mediated plant-plant allelopathy in rice (Oryza sativa) 2012 New Phytol Department of Biochemistry, Biophysics, & Molecular Biology, Iowa State University, Ames, IA 50011, USA. * There is controversy as to whether specific natural products play a role in directly mediating antagonistic plant-plant interactions - that is, allelopathy. If proved to exist, such phenomena would hold considerable promise for agronomic improvement of staple food crops such as rice (Oryza sativa). * However, while substantiated by the presence of phytotoxic compounds at potentially relevant concentrations, demonstrating a direct role for specific natural products in allelopathy has been difficult because of the chemical complexity of root and plant litter exudates. This complexity can be bypassed via selective genetic manipulation to ablate production of putative allelopathic compounds, but such an approach previously has not been applied. * The rice diterpenoid momilactones provide an example of natural products for which correlative biochemical evidence has been obtained for a role in allelopathy. Here, we apply reverse genetics, using knock-outs of the relevant diterpene synthases (copalyl diphosphate synthase 4 (OsCPS4) and kaurene synthase-like 4 (OsKSL4)), to demonstrate that rice momilactones are involved in allelopathy, including suppressing growth of the widespread rice paddy weed, barnyard grass (Echinochloa crus-galli). * Thus, our results not only provide novel genetic evidence for natural product-mediated allelopathy, but also furnish a molecular target for breeding and metabolic engineering of this important crop plant. OsCPS4|OsCyc1,OsDTS2|OsKSL4|OsKS4 Crinkly4 receptor-like kinase is required to maintain the interlocking of the palea and lemma, and fertility in rice, by promoting epidermal cell differentiation 2012 Plant J Institute of Molecular Cell Biology, Hebei Key Laboratory of Molecular and Cellular Biology, Hebei Normal University, Shijiazhuang, Hebei 050016, China. The palea and lemma are unique organs in grass plants that form a protective barrier around the floral organs and developing kernel. The interlocking of the palea and lemma is critical for maintaining fertility and seed yield in rice; however, the molecules that control the interlocking structure remain largely unknown. Here, we showed that when OsCR4 mRNA expression was knocked down in rice by RNA interference, the palea and lemma separated at later spikelet stages and gradually turned brown after heading, resulting in the severe interruption of pistil pollination and damage to the development of embryo and endosperm, with defects in aleurone. The irregular architecture of the palea and lemma was caused by tumour-like cell growth in the outer epidermis and wart-like cell masses in the inner epidermis. These abnormal cells showed discontinuous cuticles and uneven cell walls, leading to organ self-fusion that distorted the interlocking structures. Additionally, the faster leakage of chlorophyll, reduced silica content and elevated accumulation of anthocyanin in the palea and lemma indicated a lesion in the protective barrier, which also impaired seed quality. OsCR4 is an active receptor-like kinase associated with the membrane fraction. An analysis of promoter::GUS reporter plants showed that OsCR4 is specifically expressed in the epidermal cells of paleas and lemmas. Together, these results suggest that OsCR4 plays an essential role in maintaining the interlocking of the palea and lemma by promoting epidermal cell differentiation. OsCR4 Functional and signaling mechanism analysis of rice CRYPTOCHROME 1 2006 Plant J National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai, China. Cryptochromes (CRY) are blue-light photoreceptors that mediate various light responses, such as inhibition of hypocotyl elongation, enhancement of cotyledon expansion, anthocyanin accumulation and stomatal opening in Arabidopsis. The signaling mechanism of Arabidopsis CRY is mediated through direct interaction with COP1, a negative regulator of photomorphogenesis. CRY has now been characterized in tomato, pea, moss and fern, but its function in monocots is largely unknown. Here we report the function and basic signaling mechanism of rice cryptochrome 1 (OsCRY1). Overexpresion of OsCRY1b resulted in a blue light-dependent short hypcotyl phenotype in Arabidopsis, and a short coleoptile, leaf sheath and leaf blade phenotype in rice (Oryza sativa). On fusion with beta-glucuronidase (GUS), the C-terminal domain of either OsCRY1a (OsCCT1a) or OsCRY1b (OsCCT1b) mediated a constitutive photomorphogenic (COP) phenotype in both Arabidopsis and rice, whereas OsCCT1b mutants corresponding to missense mutations in previously described Arabidopsis cry1 alleles failed to confer a COP phenotype. Yeast two-hybrid and subcellular co-localization studies demonstrated that OsCRY1b interacted physically with rice COP1 (OsCOP1). From these results, we conclude that OsCRY1 is implicated in blue-light inhibition of coleoptile and leaf elongation during early seedling development in rice, and that the signaling mechanism of OsCRY1 involves direct interaction with OsCOP1. OsCRY1a,OsCRY1b Involvement of rice cryptochromes in de-etiolation responses and flowering 2006 Plant Cell Physiol Department of Plant Physiology, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8602 Japan. In order to elucidate the function of cryptochromes (cry) in rice, we have characterized all rice CRY genes, including OsCRY1a, OsCRY1b and OsCRY2. Our studies revealed that OsCRY1 genes were mainly expressed in the green plant tissue, while OsCRY2 gene expression was high in the coleoptile, flower and callus. Light treatment affected neither the expression of any of the OsCRY genes nor the stability of their transcripts. However, it was found that Oscry2 protein was negatively regulated by blue light. Moreover, the level of Oscry2 protein also decreased upon red light treatment, and this red light-dependent degradation was shown to be mediated by phytochrome B. Overexpression of OsCRY1 genes resulted in an increased responsiveness to blue light when measuring coleoptile growth inhibition. Moreover, growth of leaf sheaths and blades was also repressed more in OsCRY1 overexpressers than in wild type (WT) under blue light conditions. These results suggest that Oscry1s are responsible for regulating blue light-mediated de-etiolation in rice. In addition, OsCRY2 antisense transgenic rice flowered later than WT under both long-day and short-day conditions, indicating that Oscry2 is involved in the promotion of flowering time in rice. OsCRY2|CRY2 Root hairless 2 (rth2) mutant represents a loss-of-function allele of the cellulose synthase-like gene OsCSLD1 in rice (Oryza sativa L.) 2011 Breeding Science None Root hair is considered to play important roles in water and nutrient uptake and anchoring the plant to the soil. To gain further knowledge of root hair morphogenesis in rice, we isolated root hairless 2 (rth2) mutant from the mutant panel of Nipponbare. Positional cloning and complementation test revealed that the causal gene of rth2 was Cellulose Synthase-Like D1 (OsCSLD1). rth2 has a premature stop codon in exon 1 as a result of two consecutive nucleotide substitutions and is predicted to produce truncated proteins lacking the D, D, D, QxxRW motif and 8 transmembrane domains. In rth2, bulges were normally initiated from asymmetric divisions of root epidermal cells, but bulges did not elongate. Therefore, rth2 shows completely roothairless phenotype. qRT-PCR analysis revealed that OsCSLD1 was expressed not only in root but also in shoot. In situ hybridization showed that OsCSLD1 was expressed not only in root hairs but also in epidermal and cortex cell walls except for stele. Agronomic character evaluation in pot experiments showed that rth2 did not differ significantly from Nipponbare in all characters examined except for root dry weight, which showed a significant increase in rth2. In paddy field experiment, rth2 was significantly inferior compared with Nipponbare in agronomic performance. OsCSLD1|rth-2 OsCSLD1, a cellulose synthase-like D1 gene, is required for root hair morphogenesis in rice 2007 Plant Physiol Division of Applied Life Science, Plant Molecular Biology and Biotechnology Research Center, Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Korea. Root hairs are long tubular outgrowths that form on the surface of specialized epidermal cells. They are required for nutrient and water uptake and interact with the soil microflora. Here we show that the Oryza sativa cellulose synthase-like D1 (OsCSLD1) gene is required for root hair development, as rice (Oryza sativa) mutants that lack OsCSLD1 function develop abnormal root hairs. In these mutants, while hair development is initiated normally, the hairs elongate less than the wild-type hairs and they have kinks and swellings along their length. Because the csld1 mutants develop the same density and number of root hairs along their seminal root as the wild-type plants, we propose that OsCSLD1 function is required for hair elongation but not initiation. Both gene trap expression pattern and in situ hybridization analyses indicate that OsCSLD1 is expressed in only root hair cells. Furthermore, OsCSLD1 is the only member of the four rice CSLD genes that shows root-specific expression. Given that the Arabidopsis (Arabidopsis thaliana) gene KOJAK/AtCSLD3 is required for root hair elongation and is expressed in the root hair, it appears that OsCSLD1 may be the functional ortholog of KOJAK/AtCSLD3 and that these two genes represent the root hair-specific members of this family of proteins. Thus, at least part of the mechanism of root hair morphogenesis in Arabidopsis is conserved in rice. OsCSLD1|rth-2 Rice slender leaf 1 gene encodes cellulose synthase-like D4 and is specifically expressed in M-phase cells to regulate cell proliferation 2013 J Exp Bot Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Cellulose synthase-like (CSL) genes are predicted to catalyse the biosynthesis of non-cellulosic polysaccharides such as the beta-D-glycan backbone of hemicelluloses and are classified into nine subfamilies (CSLA-CSLH and CSLJ). The CSLD subfamily is conserved in all land plants, and among the nine CSL subfamilies, it shows the highest sequence similarity to the cellulose synthase genes, suggesting that it plays fundamental roles in plant development. This study presents a detailed analysis of slender leaf 1 (sle1) mutants of rice that showed rolled and narrow leaf blades and a reduction in plant height. The narrow leaf blade of sle1 was caused by reduced cell proliferation beginning at the P3 primordial stage. In addition to the size reduction of organs, sle1 mutants exhibited serious developmental defects in pollen formation, anther dehiscence, stomata formation, and cell arrangement in various tissues. Map-based cloning revealed that SLE1 encodes the OsCSLD4 protein, which was identified previously from a narrow leaf and dwarf 1 mutant. In situ hybridization experiments showed that OsCSLD4 was expressed in a patchy pattern in developing organs. Double-target in situ hybridization and quantitative RT-PCR analyses revealed that SLE1 was expressed specifically during the M-phase of the cell cycle, and suggested that the cell-cycle regulation was altered in sle1 mutants. These results suggest that the OsCSLD4 protein plays a pivotal role in the M phase to regulate cell proliferation. Further study of OsCSLD4 is expected to yield new insight into the role of hemicelluloses in plant development. OsCD1|OsCSLD4|NRL1|ND1|sle1 Rice cellulose synthase-like D4 is essential for normal cell-wall biosynthesis and plant growth 2009 Plant J National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Cellulose synthase-like (CSL) proteins of glycosyltransferase family 2 (GT2) are believed to be involved in the biosynthesis of cell-wall polymers. The CSL D sub-family (CSLD) is common to all plants, but the functions of CSLDs remain to be elucidated. We report here an in-depth characterization of a narrow leaf and dwarf1 (nd1) rice mutant that shows significant reduction in plant growth due to retarded cell division. Map-based cloning revealed that ND1 encodes OsCSLD4, one of five members of the CSLD sub-family in rice. OsCSLD4 is mainly expressed in tissues undergoing rapid growth. Expression of OsCSLD4 fluorescently tagged at the C- or N-terminus in rice protoplast cells or Nicotiana benthamiana leaves showed that the protein is located in the endoplasmic reticulum or Golgi vesicles. Golgi localization was verified using phenotype-rescued transgenic plants expressing OsCSLD4-GUS under the control of its own promoter. Two phenotype-altered tissues, culms and root tips, were used to investigate the specific wall defects. Immunological studies and monosaccharide compositional and glycosyl linkage analyses explored several wall compositional effects caused by disruption of OsCSLD4, including alterations in the structure of arabinoxylan and the content of cellulose and homogalacturonan, which are distinct in the monocot grass species Oryza sativa (rice). The inconsistent alterations in the two tissues and the observable structural defects in primary walls indicate that OsCSLD4 plays important roles in cell-wall formation and plant growth. OsCD1|OsCSLD4|NRL1|ND1|sle1 OsCD1 encodes a putative member of the cellulose synthase-like D sub-family and is essential for rice plant architecture and growth 2011 Plant Biotechnol J College of Life Science, Tianjin Key Laboratory of Cyto-Genetical and Molecular Regulation, Tianjin Normal University, Tianjin, China. lwjzsq@163.com The cell wall plays important roles in plant architecture and morphogenesis. The cellulose synthase-like super-families were reported to contain glycosyltransferases motif and are required for the biosynthesis of cell wall polysaccharides. Here, we describe a curled leaf and dwarf mutant, cd1, in rice, which exhibits multiple phenotypic traits such as the reduction of plant height and leaf width, curled leaf morphology and a decrease in the number of grains and in the panicle length. Map-based cloning indicates that a member of the cellulose synthase-like D (CSLD) group is a candidate for OsCD1. RNAi transgenic plants with the candidate CSLD gene display a similar phenotype to the cd1 mutant, suggesting that OsCD1 is a member of the CSLD sub-family. Furthermore, sequence analysis indicates that OsCD1 contains the common D,D,D,QXXRW motif, which is a feature of the cellulose synthase-like super-family. Analysis of OsCD1 promoter with GUS fusion expression shows that OsCD1 exhibits higher expression in young meristem tissues such as fresh roots, young panicle and stem apical meristem. Cell wall composition analysis reveals that cellulose content and the level of xylose are significantly reduced in mature culm owing to loss of OsCD1 function. Take together, the work presented here is useful for expanding the understanding of cell wall biosynthesis. OsCD1|OsCSLD4|NRL1|ND1|sle1 Rice plants response to the disruption of OsCSLD4 gene 2010 Plant Signal Behav National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. None OsCD1|OsCSLD4|NRL1|ND1|sle1 Isolation and characterization of a rice mutant with narrow and rolled leaves 2010 Planta State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou, Zhejiang, China. Appropriate leaf shape has proved to be useful in improving photosynthesis and increasing grain yield. To understand the molecular mechanism of leaf morphogenesis, we identified a rice mutant nrl1, which was characterized by a phenotype of narrow and rolled leaves. Microscopic observation showed that the mutation significantly decreased the number of vascular bundles of leaf and stem. Genetic analysis revealed that the mutation was controlled by a single nuclear-encoded recessive gene. To isolate the nrl1 gene, 756 F(2) and F(3) mutant individuals from a cross of the nrl1 mutant with Longtepu were used and a high-resolution physical map of the chromosomal region around the nrl1 gene was made. Finally, the gene was mapped in 16.5 kb region between marker RL21 and marker RL36 within the BAC clone OSJNBa0027H05. Cloning and sequencing of the target region from the mutant showed that there was a 58 bp deletion within the second exon of the cellulose synthase-like D4 gene (TIGR locus Os12g36890). The nrl1 mutation was rescued by transformation with the wild-type cellulose synthase-like D4 gene. Accordingly, the cellulose synthase-like D4 gene was identified as the NRL1 gene. NRL1 was transcribed in various tissues and was mainly expressed in panicles and internodes. NAL7 and SLL1 were found to be upregulated, whereas OsAGO7 were downregulated in the nrl1 mutant. These findings suggested that there might be a functional association between these genes in regulating leaf development. OsCD1|OsCSLD4|NRL1|ND1|sle1,OsAGO7|shl4|SHO2,RL9|SLL1 Identification and characterization of NARROW AND ROLLED LEAF 1, a novel gene regulating leaf morphology and plant architecture in rice 2010 Plant Mol Biol State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, 359 Tiyuchang Road, Hangzhou, China. Leaf morphology is an important agronomic trait in rice breeding. We isolated three allelic mutants of NARROW AND ROLLED LEAF 1 (nrl1) which showed phenotypes of reduced leaf width and semi-rolled leaves and different degrees of dwarfism. Microscopic analysis indicated that the nrl1-1 mutant had fewer longitudinal veins and smaller adaxial bulliform cells compared with the wild-type. The NRL1 gene was mapped to the chromosome 12 and encodes the cellulose synthase-like protein D4 (OsCslD4). Sequence analyses revealed single base substitutions in the three allelic mutants. Genetic complementation and over-expression of the OsCslD4 gene confirmed the identity of NRL1. The gene was expressed in all tested organs of rice at the heading stage and expression level was higher in vigorously growing organs, such as roots, sheaths and panicles than in elsewhere. In the mutant leaves, however, the expression level was lower than that in the wild-type. We conclude that OsCslD4 encoded by NRL1 plays a critical role in leaf morphogenesis and vegetative development in rice. OsCD1|OsCSLD4|NRL1|ND1|sle1 Functional characterization of two cold shock domain proteins from Oryza sativa 2008 Plant Cell Environ West Virginia University, Division of Plant & Soil Sciences, 1090 Agricultural Sciences, Morgantown, WV 26506-6108, USA. Two novel rice cold shock domain (CSD) proteins were cloned and characterized under different stress treatments and during various stages of development. OsCSP1 and OsCSP2 (Oryza sativa CSD protein) encode putative proteins consisting of an N-terminal CSD and glycine-rich regions that are interspersed by 4 and 2 CX(2)CX(4)HX(4)C (CCHC) retroviral-like zinc fingers, respectively. In vivo functional analysis confirmed that OsCSPs can complement a cold-sensitive bacterial strain which lacks four endogenous cold shock proteins. In vitro ssDNA binding assays determined that recombinant OsCSPs are capable of functioning as nucleic acid-binding proteins. Both OsCSP transcripts are transiently up-regulated in response to low-temperature stress and rapidly return to a basal level of gene expression. Protein blot analysis determined that OsCSPs are maintained at a constant level subsequent to a cold treatment lasting over a period of several days. Both the transcript and protein data are in sharp contrast to those previously obtained for winter wheat WCSP1. A time-coursed study through various stages of rice development confirmed that both OsCSP proteins and transcripts are highly accumulated in reproductive tissues and tissues which exhibit meristematic activity. OsCSP1,OsCSP2 Genomic survey, expression profile and co-expression network analysis of OsWD40 family in rice 2012 BMC Genomics National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research Wuhan, Huazhong Agricultural University, Wuhan 430070, China. BACKGROUND: WD40 proteins represent a large family in eukaryotes, which have been involved in a broad spectrum of crucial functions. Systematic characterization and co-expression analysis of OsWD40 genes enable us to understand the networks of the WD40 proteins and their biological processes and gene functions in rice. RESULTS: In this study, we identify and analyze 200 potential OsWD40 genes in rice, describing their gene structures, genome localizations, and evolutionary relationship of each member. Expression profiles covering the whole life cycle in rice has revealed that transcripts of OsWD40 were accumulated differentially during vegetative and reproductive development and preferentially up or down-regulated in different tissues. Under phytohormone treatments, 25 OsWD40 genes were differentially expressed with treatments of one or more of the phytohormone NAA, KT, or GA3 in rice seedlings. We also used a combined analysis of expression correlation and Gene Ontology annotation to infer the biological role of the OsWD40 genes in rice. The results suggested that OsWD40 genes may perform their diverse functions by complex network, thus were predictive for understanding their biological pathways. The analysis also revealed that OsWD40 genes might interact with each other to take part in metabolic pathways, suggesting a more complex feedback network. CONCLUSIONS: All of these analyses suggest that the functions of OsWD40 genes are diversified, which provide useful references for selecting candidate genes for further functional studies. OsCstF50|OsWD40-93,OsKTN80b|OsWD40-111,OsKTN80c|OsWD40-26,RACK1B|OsWD40-122 Survey of rice proteins interacting with OsFCA and OsFY proteins which are homologous to the Arabidopsis flowering time proteins, FCA and FY 2009 Plant Cell Physiol Plant Signaling Network Research Center, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea. The FCA protein is involved in controlling flowering time and plays more general roles in RNA-mediated chromatin silencing in Arabidopsis. It contains two RNA-binding domains and a WW domain. The FCA protein interacts with FY, a polyadenylation factor, via its WW domain. We previously characterized a rice gene, OsFCA, which was homologous to FCA. Here, we found that the OsFCA protein could interact through its WW domain with the following proteins: OsFY, a protein containing a CID domain present in RNA-processing factors such as Pcf11 and Nrd1; a protein similar to splicing factor SF1; a protein similar to FUSE splicing factor; and OsMADS8. The FY protein is associated with the 3' end processing machinery in Arabidopsis. Thus, we examined interactions between OsFY and the rice homologs (OsCstF-50, -64 and -77) of the AtCstF-50, -64 and -77 proteins. We found that OsFY could bind OsCstF50, whereas the OsCstF77 protein could bridge the interaction between OsCstF50 and OsCstF64. Taken together, our data suggest that OsFCA could interact with several proteins other than OsFY through its WW domain and may play several roles in rice. OsCstF50|OsWD40-93,OsCstF64,OsCstF77,FCA|OsFCA,OsFY,OsMADS8|OsMADS24 Characterization of stress and methylglyoxal inducible triose phosphate isomerase (OscTPI) from rice 2012 Plant Signal Behav Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, New Delhi, India. As compared with plant system, triose phosphate isomerase (TPI), a crucial enzyme of glycolysis, has been well studied in animals. In order to characterize TPI in plants, a full-length cDNA encoding OscTPI was cloned from rice and expressed in E. coli. The recombinant OscTPI was purified to homogeneity and it showed Km value of 0.1281 +/- 0.025 microM, and the Vmax value of 138.7 +/- 16 micromol min (-1) mg (-1) which is comparable to the kinetic values studied in other plants. The OscTPI was found to be exclusively present in the cytoplasm when checked with the various methods. Functional assay showed that OscTPI could complement a TPI mutation in yeast. Real time PCR analysis revealed that OscTPI transcript level was regulated in response to various abiotic stresses. Interestingly, it was highly induced under different concentration of methylglyoxal (MG) stress in a concentration dependent manner. There was also a corresponding increase in the protein and the enzyme activity of OscTPI both in shoot and root tissues under MG stress. Our result shows that increases in MG leads to the increase in TPI which results in decrease of DHAP and consequently decrease in the level of toxic MG. OscTPI Rice CONSTITUTIVE TRIPLE-RESPONSE2 is involved in the ethylene-receptor signalling and regulation of various aspects of rice growth and development 2013 J Exp Bot National Key Laboratory of Plant Molecular Genetics and National Center for Plant Gene Research (Shanghai), Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, PR China. In Arabidopsis, the ethylene-receptor signal output occurs at the endoplasmic reticulum and is mediated by the Raf-like protein CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) but is prevented by overexpression of the CTR1 N terminus. A phylogenic analysis suggested that rice OsCTR2 is closely related to CTR1, and ectopic expression of CTR1p:OsCTR2 complemented Arabidopsis ctr1-1. Arabidopsis ethylene receptors ETHYLENE RESPONSE1 and ETHYLENE RESPONSE SENSOR1 physically interacted with OsCTR2 on yeast two-hybrid assay, and green fluorescence protein-tagged OsCTR2 was localized at the endoplasmic reticulum. The osctr2 loss-of-function mutation and expression of the 35S:OsCTR2 (1-513) transgene that encodes the OsCTR2 N terminus (residues 1-513) revealed several and many aspects, respectively, of ethylene-induced growth alteration in rice. Because the osctr2 allele did not produce all aspects of ethylene-induced growth alteration, the ethylene-receptor signal output might be mediated in part by OsCTR2 and by other components in rice. Yield-related agronomic traits, including flowering time and effective tiller number, were altered in osctr2 and 35S:OsCTR2 (1-513) transgenic lines. Applying prolonged ethylene treatment to evaluate ethylene effects on rice without compromising rice growth is technically challenging. Our understanding of roles of ethylene in various aspects of growth and development in japonica rice varieties could be advanced with the use of the osctr2 and 35S:OsCTR2 (1-513) transgenic lines. OsCTR2 OsCYCP1;1, a PHO80 homologous protein, negatively regulates phosphate starvation signaling in the roots of rice (Oryza sativa L.) 2014 Plant Molecular Biology Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, Zhejiang, People's Republic of China Phosphorus is one of the most essential and limiting nutrients in all living organisms, thus the organisms have evolved complicated and precise regulatory mechanisms for phosphorus acquisition, storage and homeostasis. In the budding yeast, Saccharomyces cerevisiae, the modification of PHO4 by the PHO80 and PHO85 complex is a core regulation system. However, the existence and possible functions in phosphate signaling of the homologs of the PHO80 and PHO85 components in plants has yet to be determined. Here we describe the identification of a family of seven PHO80 homologous genes in rice named OsCYCPs. Among these, the OsCYCP1;1 gene was able to partially rescue the pho80 mutant strain of yeast. The OsCYCP1;1 protein was predominantly localized in the nucleus, and was ubiquitously expressed throughout the whole plant and during the entire growth period of rice. Consistent with the negative role of PHO80 in phosphate signaling in yeast, OsCYCP1;1 expression was reduced by phosphate starvation in the roots. This reduction was dependent on PHR2, the central regulator of phosphate signaling in rice. Overexpression and suppression of the expression of OsCYCP1;1 influenced the phosphate starvation signaling response. The inducible expression of phosphate starvation inducible and phosphate transporter genes was suppressed in the OsCYCP1;1 overexpression lines and was relatively enhanced in the OsCYCP1;1 RNAi plants by phosphate starvation. Together, these results demonstrate the role of PHO80 homologs in the phosphate starvation signaling pathway in rice. OsCYCP1;1 Biochemical and structural properties of cyanases from Arabidopsis thaliana and Oryza sativa 2011 PLoS One State Key Laboratory of Protein and Plant Gene Research, Peking-Yale Joint Center for Plant Molecular Genetics and Agrobiotechnology, College of Life Sciences, Peking University, Beijing, People's Republic of China. Cyanate is toxic to all organisms. Cyanase converts cyanate to CO(2) and NH(3) in a bicarbonate-dependent reaction. The biophysical functions and biochemical characteristics of plant cyanases are poorly studied, although it has been investigated in a variety of proteobacteria, cyanobacteria and fungi. In this study, we characterised plant cyanases from Arabidopsis thaliana and Oryza sativa (AtCYN and OsCYN). Prokaryotic-expressed AtCYN and OsCYN both showed cyanase activity in vitro. Temperature had a similar influence on the activity of both cyanases, but pH had a differential impact on AtCYN and OsCYN activity. Homology modelling provided models of monomers of AtCYN and OsCYN, and a coimmunoprecipitation assay and gel filtration indicated that AtCYN and OsCYN formed homodecamers. The analysis of single-residue mutants of AtCYN indicated that the conserved catalytic residues also contributed to the stability of the homodecamer. KCNO treatment inhibited Arabidopsis germination and early seedling growth. Plants containing AtCYN or OsCYN exhibited resistance to KCNO stress, which demonstrated that one role of cyanases in plants is detoxification. Transcription level of AtCYN was higher in the flower than in other organs of Arabidopsis. AtCYN transcription was not significantly affected by KCNO treatment in Arabidopsis, but was induced by salt stress. This research broadens our knowledge on plant detoxification of cyanate via cyanase. OsCYN Classification of rice (Oryza sativa L. Japonica nipponbare) immunophilins (FKBPs, CYPs) and expression patterns under water stress 2010 BMC Plant Biol Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-506, Korea. BACKGROUND: FK506 binding proteins (FKBPs) and cyclophilins (CYPs) are abundant and ubiquitous proteins belonging to the peptidyl-prolyl cis/trans isomerase (PPIase) superfamily, which regulate much of metabolism through a chaperone or an isomerization of proline residues during protein folding. They are collectively referred to as immunophilin (IMM), being present in almost all cellular organs. In particular, a number of IMMs relate to environmental stresses. RESULTS: FKBP and CYP proteins in rice (Oryza sativa cv. Japonica) were identified and classified, and given the appropriate name for each IMM, considering the ortholog-relation with Arabidopsis and Chlamydomonas or molecular weight of the proteins. 29 FKBP and 27 CYP genes can putatively be identified in rice; among them, a number of genes can be putatively classified as orthologs of Arabidopsis IMMs. However, some genes were novel, did not match with those of Arabidopsis and Chlamydomonas, and several genes were paralogs by genetic duplication. Among 56 IMMs in rice, a significant number are regulated by salt and/or desiccation stress. In addition, their expression levels responding to the water-stress have been analyzed in different tissues, and some subcellular IMMs located by means of tagging with GFP protein. CONCLUSION: Like other green photosynthetic organisms such as Arabidopsis (23 FKBPs and 29 CYPs) and Chlamydomonas (23 FKBs and 26 CYNs), rice has the highest number of IMM genes among organisms reported so far, suggesting that the numbers relate closely to photosynthesis. Classification of the putative FKBPs and CYPs in rice provides the information about their evolutional/functional significance when comparisons are drawn with the relatively well studied genera, Arabidopsis and Chlamydomonas. In addition, many of the genes upregulated by water stress offer the possibility of manipulating the stress responses in rice. OsCYP-25|CYP19-3,OsTIG Cyclophilins are encoded by a small gene family in rice 1994 Plant Mol Biol Institute of Developmental and Molecular Biology, Texas A&M University, College Station 77843-3155. cDNA clones were isolated and sequenced that encode two related but distinct rice cyclophilins, Cyp1 and Cyp2. The predicted amino acid sequences of each are 72% identical to human T-cell cyclophilin. Genomic DNA gel blot analysis suggests cyclophilins in rice are encoded by a small, 6-10-member gene family. Both Cyp1 and Cyp2 have seven extra amino acid residues in the N-terminal portion of the proteins that are not found in human or other non-plant cyclophilins, suggesting that this is a characteristic of plant cyclophilins. Cyp2 was expressed as 1000 nt transcripts in leaf and root tissues. Cyp1 was expressed as 800 and 900 nt transcripts. Whereas the 900 nt transcript was present in both root and leaf mRNA, the 800 nt transcript was only detectable in root mRNA. A genomic clone of Cyp2 was isolated, sequenced and shown to lack introns. A single transcriptional start site was identified 27 residues downstream of a putative TATA box. The 5' end of the transcript was shown to contain a region rich in adenyl residues (27 of 35). This region would not be conducive to secondary structure formation, which raises the possibility that Cyp2 might be preferentially translated during stress conditions. OsCYP-25|CYP19-3 Multiple abiotic stress responsive rice cyclophilin: (OsCYP-25) mediates a wide range of cellular responses 2013 Commun Integr Biol Plant Molecular Biology Group; International Centre for Genetic Engineering and Biotechnology; Aruna Asaf Ali Marg, New Delhi, India. Cyclophilins (CYP), a member of immunophillin group of proteins, are more often conserved in all genera including plants. Here, we report on the identification of a new cyclophilin gene OsCYP-25 (LOC_Os09 g39780) from rice which found to be upregulated in response to various abiotic stresses viz., salinity, cold, heat and drought. It has an ORF of 540 bp, encoding a protein of 179 amino acids, consisting of PPIase domain, which is highly conserved. The OsCYP-25 promoter analysis revealed that different cis-regulatory elements (e.g., MYBCORE, MYC, CBFHV, GT1GMSCAM4, DRECRTCOREAT, CCAATBOX1, WRKY71OS and WBOXATNPR1) are involved to mediate OsCYP-25 response under stress. We have also predicted interacting partners by STRING software. In interactome, protein partners includes WD domain containing protein, the 60S ribosome subunit biogenesis protein, the ribosomal protein L10, the DEAD-box helicase, the EIF-2alpha, YT521-B protein, the 60S ribosomal protein and the PPR repeat domain containing protein. The in silico analysis showed that OsCYP-25 interacts with different proteins involved in cell growth, differentiation, ribosome biogenesis, RNA metabolism, RNA editing, gene expression, signal transduction or stress response. These findings suggest that OsCYP-25 might perform an important function in mediating wide range of cellular response under multiple abiotic stresses. OsCYP-25|CYP19-3 LATERAL ROOTLESS2, a cyclophilin protein, regulates lateral root initiation and auxin signaling pathway in rice 2013 Mol Plant State Key Laboratory of Plant Genomics and National Plant Gene Research Center, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. None OsCYP2 OsCYP2, a chaperone involved in degradation of auxin-responsive proteins, plays crucial roles in rice lateral root initiation 2013 Plant J State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou, 310058, China. Auxin plays a pivotal role in many facets of plant development. It acts by inducing the interaction between auxin-responsive [auxin (AUX)/indole-3-acetic acid (IAA)] proteins and the ubiquitin protein ligase SCF(TIR) to promote the degradation of the AUX/IAA proteins. Other cofactors and chaperones that participate in auxin signaling remain to be identified. Here, we characterized rice (Oryza sativa) plants with mutations in a cyclophilin gene (OsCYP2). cyp2 mutants showed defects in auxin responses and exhibited a variety of auxin-related growth defects in the root. In cyp2 mutants, lateral root initiation was blocked after nuclear migration but before the first anticlinal division of the pericycle cell. Yeast two-hybrid and in vitro pull-down results revealed an association between OsCYP2 and the co-chaperone Suppressor of G2 allele of skp1 (OsSGT1). Luciferase complementation imaging assays further supported this interaction. Similar to previous findings in an Arabidopsis thaliana SGT1 mutant (atsgt1b), degradation of AUX/IAA proteins was retarded in cyp2 mutants treated with exogenous 1-naphthylacetic acid. Our results suggest that OsCYP2 participates in auxin signal transduction by interacting with OsSGT1. OsCYP2 Heterologous expression of a salinity and developmentally regulated rice cyclophilin gene (OsCyp2) in E. coli and S. cerevisiae confers tolerance towards multiple abiotic stresses 2009 Mol Biotechnol Jawaharlal Nehru University, New Delhi, India. Cyclophilin 2 (OsCyp2) is a cytosolic member of immunophilin family from rice. We have isolated its full length cDNA (1,056 bp) with an open reading frame of 519 bp encoding a polypeptide of 172 amino acids and an estimated pI of 8.61. Peptidyl prolyl cis-trans isomerase activity of the protein was determined using N-succinyl-ala-ala-pro-phe-p-nitroanilidine as peptide substrate. It has a catalytic efficiency (K (cat)/K (m)) of 4.5 x 10(6)/(mol/l)/s, which is comparable to known cyclophilins from plants. Its activity is specifically inhibited by cyclosporin A, a macrolide drug inhibitor of cyclophilins. Transcript analysis showed it to be a developmentally and differentially regulated gene; showing changes in abundance at seedling, tillering and heading stage under non-stress and salinity stress conditions. Expression of OsCyp2 enhances the ability of Escherichia coli to survive under diverse abiotic stresses viz. salinity, high temperature, osmotic stress (mannitol) and oxidative stress (H(2)O(2)). OsCyp2 was able to complement the yeast mutant lacking native Cyp2 and also improved the growth of wild type yeast under above-mentioned stress conditions. Based on these results, we propose that OsCyp2 may serve as a 'suitable candidate' for raising transgenic plants for enhanced multiple abiotic stress tolerance. OsCYP2 Proteomic identification of OsCYP2, a rice cyclophilin that confers salt tolerance in rice (Oryza sativa L.) seedlings when overexpressed 2011 BMC Plant Biol Plant Molecular Biology & Proteomics Lab, Institute of Biotechnology, Hangzhou Academy of Agricultural Sciences, Hangzhou 310024, PR China Background: High Salinity is a major environmental stress influencing growth and development of rice. Comparative proteomic analysis of hybrid rice shoot proteins from Shanyou 10 seedlings, a salt-tolerant hybrid variety, and Liangyoupeijiu seedlings, a salt-sensitive hybrid variety, was performed to identify new components involved in salt-stress signaling. OsCYP2 The rice thylakoid lumenal cyclophilin OsCYP20-2 confers enhanced environmental stress tolerance in tobacco and Arabidopsis 2012 Plant Cell Rep Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 305-806, Korea. The role that the putative thylakoid lumenal cyclophilin (CYP) CYP20-2 locates in the thylakoid, and whether CYP20-2 is an essential gene, have not yet been elucidated. Here, we show that CYP20-2 is well conserved in several photosynthetic plants and that the transcript level of the rice OsCYP20-2 gene is highly regulated under abiotic stress. We found that ectopic expression of rice OsCYP20-2 in both tobacco and Arabidopsis confers enhanced tolerance to osmotic stress and extremely high light. Based on these results, we suggest that although the exact biochemical function of OsCYP20-2 in the thylakoid lumen (TL) remains unclear, it may be involved in photosynthetic acclimation to help plants cope with environmental stress; the OsCYP20-2 gene may be a candidate for enhancing multiple abiotic stress tolerance. OsCYP20-2 Oscyp71Z2 involves diterpenoid phytoalexin biosynthesis that contributes to bacterial blight resistance in rice 2013 Plant Sci College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China. Bacterial blight is one of the most destructive rice diseases, which caused by Xoo, and results in yield losses, endangering worldwide food security. Diterpenoid phytoalexins, a type of antimicrobials produced in rice, are critical for resistance to fungal and bacterial pathogens. This article reports the characterization of the cytochrome P450 gene Oscyp71Z2, which belongs to the CYP71Z subfamily. Overexpression of Oscyp71Z2 in rice enhanced resistance to Xoo at the booting stage. The accumulation of phytoalexins was rapidly and strongly induced in Oscyp71Z2-overexpressing plants, and the transcript levels of genes related to the phytoalexin biosynthesis pathway were elevated. The H(2)O(2) concentration in Oscyp71Z2-overexpressing plants was reduced in accordance with the increase in ROS-scavenging ability due to the induction of SOD as well as POD and CAT activation. We also showed that suppression of Oscyp71Z2 had no significantly effect on disease resistance to Xoo in rice. These results demonstrated that Oscyp71Z2 plays an important role in bacterial blight resistance by regulating the diterpenoid phytoalexin biosynthesis and H(2)O(2) generation. Oscyp71Z2 A Rice Cytochrome P450OsCYP84AThat May Interact with the UV Tolerance Pathway 2014 Bioscience, Biotechnology and Biochemistry Department of Biological Science and Technology, Tokyo University of Science, Noda, Chiba, Japan. Cytochrome P450s are widespread in the plant kingdom. The functions of plant P450s are dispersed through many aspects of plant metabolisms, which are involved in the biosynthesis of defense compounds and protectants against ultraviolet rays, as well as metabolic pathways for the biosynthesis and/or degradation of fatty acids, hormones, and signaling molecules. We found a gene for rice P450, OsCYP84A, which was classified into CYP84A in the CYP71 clan. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that this gene was ubiquitously expressed without any temporal and spatial specificity under normal growth conditions, but its expression was inducibly and significantly increased by ultraviolet (UV)-B and UV-C irradiation. Rice transformants in which OsCYP84A expression was suppressed by the antisense gene showed apparent growth retardation with obvious symptoms of damage on the plant bodies under UV-B irradiation, although no phenotypic alteration occurred under normal growth conditions. These results suggest the existence of a novel UV-tolerance system involving OsCYP84A. OsCYP84A microRNAs targeting DEAD-box helicases are involved in salinity stress response in rice (Oryza sativa L.) 2012 BMC Plant Biol Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India. BACKGROUND: Rice (Oryza sativa L.), one of the most important food crop in the world, is considered to be a salt-sensitive crop. Excess levels of salt adversely affect all the major metabolic activities, including cell wall damage, cytoplasmic lysis and genomic stability. In order to cope with salt stress, plants have evolved high degrees of developmental plasticity, including adaptation via cascades of molecular networks and changes in gene expression profiles. Posttranscriptional regulation, through the activity of microRNAs, also plays an important role in the plant response to salinity conditions. MicroRNAs are small endogenous RNAs that modulate gene expression and are involved in the most essential physiological processes, including plant development and adaptation to environmental changes. RESULTS: In the present study, we investigated the expression profiles of osa-MIR414, osa-MIR408 and osa-MIR164e along with their targeted genes, under salinity stress conditions in wild type and transgenic rice plants ectopically expressing the PDH45 (Pea DNA Helicase) gene. The present miRNAs were predicted to target the OsABP (ATP-Binding Protein), OsDSHCT (DOB1/SK12/helY-like DEAD-box Helicase) and OsDBH (DEAD-Box Helicase) genes, included in the DEAD-box helicase family. An in silico characterization of the proteins was performed and the miRNAs predicted targets were validated by RLM-5'RACE. The qRT-PCR analysis showed that the OsABP, OsDBH and OsDSHCT genes were up-regulated in response to 100 and 200 mM NaCl treatments. The present study also highlighted an increased accumulation of the gene transcripts in wild type plants, with the exception of the OsABP mRNA which showed the highest level (15.1-fold change compared to control) in the transgenic plants treated with 200 mM NaCl. Salinity treatments also affected the expression of osa-MIR414, osa-MIR164e and osa-MIR408, found to be significantly down-regulated, although the changes in miRNA expression were limited. CONCLUSIONS: Osa-MIR414, osa-MIR164e and osa-MIR408 were experimentally validated for the first time in plants as targeting the OsABP, OsDBH and OsDSHCT genes. Our data showed that that the genes were up-regulated and the miRNAs were down-regulated in relation to salt stress. The negative correlation between the miRNAs and their targets was proven. OsDBH,OsDSHCT,CTD2,DDX47,OsNAM,PRP5,RNAhA Identification of precursor transcripts for 6 novel miRNAs expands the diversity on the genomic organisation and expression of miRNA genes in rice 2008 BMC Plant Biol Laboratoire Genome et Developpement des Plantes, UMR5096, Universite de Perpignan via Domitia - CNRS-IRD, 52, Av, Paul Alduy, 66860 Perpignan Cedex, France. Severine.Lacombe@sainsbury-laboratory.ac.uk BACKGROUND: The plant miRNAs represent an important class of endogenous small RNAs that guide cleavage of an mRNA target or repress its translation to control development and adaptation to stresses. MiRNAs are nuclear-encoded genes transcribed by RNA polymerase II, producing a primary precursor that is subsequently processed by DCL1 an RNase III Dicer-like protein. In rice hundreds of miRNAs have been described or predicted, but little is known on their genes and precursors which are important criteria to distinguish them from siRNAs. Here we develop a combination of experimental approaches to detect novel miRNAs in rice, identify their precursor transcripts and genes and predict or validate their mRNA targets. RESULTS: We produced four cDNA libraries from small RNA fractions extracted from distinct rice tissues. By in silico analysis we selected 6 potential novel miRNAs, and confirmed that their expression requires OsDCL1. We predicted their targets and used 5'RACE to validate cleavage for three of them, targeting a PPR, an SPX domain protein and a GT-like transcription factor respectively. In addition, we identified precursor transcripts for the 6 miRNAs expressed in rice, showing that these precursors can be efficiently processed using a transient expression assay in transfected Nicotiana benthamiana leaves. Most interestingly, we describe two precursors producing tandem miRNAs, but in distinct arrays. We focus on one of them encoding osa-miR159a.2, a novel miRNA produced from the same stem-loop structure encoding the conserved osa-miR159a.1. We show that this dual osa-miR159a.2-osa-miR159a.1 structure is conserved in distant rice species and maize. Finally we show that the predicted mRNA target of osa-miR159a.2 encoding a GT-like transcription factor is cleaved in vivo at the expected site. CONCLUSION: The combination of approaches developed here identified six novel miRNAs expressed in rice which can be clearly distinguished from siRNAs. Importantly, we show that two miRNAs can be produced from a single precursor, either from tandem stem-loops or tandemly arrayed in a single stem-loop. This suggests that processing of these precursors could be an important regulatory step to produce one or more functional miRNAs in plants and perhaps coordinate cleavage of distinct targets in the same plant tissue. OsDCL1,OsDCL2a,OsDCL3a,OsDCL3b Roles of DCL4 and DCL3b in rice phased small RNA biogenesis 2012 The Plant Journal State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China Higher plants have evolved multiple proteins in the RNase III family to produce and regulate different classes of small RNAs with specialized molecular functions. In rice (Oryza sativa), numerous genomic clusters are targeted by one of two microRNAs (miRNAs), miR2118 and miR2275, to produce secondary small interfering RNAs (siRNAs) of either 21 or 24 nucleotides in a phased manner. The biogenesis requirements or the functions of the phased small RNAs are completely unknown. Here we examine the rice Dicer-Like (DCL) family, including OsDCL1, -3a, -3b and -4. By deep sequencing of small RNAs from different tissues of the wild type and osdcl4-1, we revealed that the processing of 21-nucleotide siRNAs, including trans-acting siRNAs (tasiRNA) and over 1000 phased small RNA loci, was largely dependent on OsDCL4. Surprisingly, the processing of 24-nucleotide phased small RNA requires the DCL3 homolog OsDCL3b rather than OsDCL3a, suggesting functional divergence within DCL3 family. RNA ligase-mediated 5′ rapid amplification of cDNA ends and parallel analysis of RNA ends (PARE)/degradome analysis confirmed that most of the 21- and 24-nucleotide phased small RNA clusters were initiated from the target sites of miR2118 and miR2275, respectively. Furthermore, the accumulation of the two triggering miRNAs requires OsDCL1 activity. Finally, we show that phased small RNAs are preferentially produced in the male reproductive organs and are likely to be conserved in monocots. Our results revealed significant roles of OsDCL4, OsDCL3b and OsDCL1 in the 21- and 24-nucleotide phased small RNA biogenesis pathway in rice. OsDCL1,OsDCL3a,OsDCL3b,SHO1|OsDCL4 Loss of function of OsDCL1 affects microRNA accumulation and causes developmental defects in rice 2005 Plant Physiol National Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing. MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are two types of noncoding RNAs involved in developmental regulation, genome maintenance, and defense in eukaryotes. The activity of Dicer or Dicer-like (DCL) proteins is required for the maturation of miRNAs and siRNAs. In this study, we cloned and sequenced 66 candidate rice (Oryza sativa) miRNAs out of 1,650 small RNA sequences (19 to approximately 25 nt), and they could be further grouped into 21 families, 12 of which are newly identified and three of which, OsmiR528, OsmiR529, and OsmiR530, have been confirmed by northern blot. To study the function of rice DCL proteins (OsDCLs) in the biogenesis of miRNAs and siRNAs, we searched genome databases and identified four OsDCLs. An RNA interference approach was applied to knock down two OsDCLs, OsDCL1 and OsDCL4, respectively. Strong loss of function of OsDCL1IR transformants that expressed inverted repeats of OsDCL1 resulted in developmental arrest at the seedling stage, and weak loss of function of OsDCL1IR transformants caused pleiotropic developmental defects. Moreover, all miRNAs tested were greatly reduced in OsDCL1IR but not OsDCL4IR transformants, indicating that OsDCL1 plays a critical role in miRNA processing in rice. In contrast, the production of siRNA from transgenic inverted repeats and endogenous CentO regions were not affected in either OsDCL1IR or OsDCL4IR transformants, suggesting that the production of miRNAs and siRNAs is via distinct OsDCLs. OsDCL1,SHO1|OsDCL4 Knock-down of OsDCL2 in rice negatively affects maintenance of the endogenous dsRNA virus, Oryza sativa endornavirus 2010 Plant Cell Physiol Department of Applied Biological Sciences, Tokyo University of Agriculture and Technology, Tokyo, Japan. An endogenous double-stranded RNA (dsRNA), which has recently been recognized as the dsRNA virus Oryza sativa endornavirus (OsEV), is found in many strains of cultivated rice (Oryza sativa). Small RNAs derived from OsEV dsRNA were detected, indicating that the RNA silencing machinery recognizes OsEV dsRNA. The existence of OsEV in knock-down (KD) lines of five genes of RNA-dependent RNA polymerase (OsRDR1-OsRDR5) or two genes of Dicer-like protein (OsDCL2 or OsDCL3a) was examined to characterize the relationship between the host RNA silencing system and the propagation of this dsRNA virus. OsEV was not detected in OsRDR4-KD or OsDCL2-KD T(1) lines. We attempted to introduce OsEV into these KD lines by crossing them with OsEV-carrying plants because of the efficient transmission of OsEV to F(1) plants via pollen or ova. All OsRDR4-KD but only some OsDCL2-KD F(1) plants contained OsEV. Some OsDCL2-KD F(1) plants consisted of OsEV-carrying and OsEV-free cells. These results suggest that the maintenance of OsEV is unstable in OsDCL2-KD plants. Furthermore, the amount of OsEV-derived small interfering RNA (vsiRNA) in the OsDCL2-KD plants increased relative to the wild type. This increased level of vsiRNA may cause OsEV instability during cell division. OsDCL2a DCL2 is highly expressed in the egg cell in both rice and Arabidopsis 2011 Plant Signal Behav The University of Tokyo, Tokyo, Japan. Small RNAs are riboregulators that play critical roles in eukaryotic cells. They repress gene expression by acting either on DNA to guide sequence elimination and chromatin remodeling, or on RNA to guide cleavage and translation repression. Arabidopsis thaliana and Oryza sativa contain four and six DICER-LIKE (DCL) genes with specialized functions in small RNA biogenesis for RNA interference-related processes. We recently profiled genome-wide gene expression in egg and synergid cells in rice. In this article, we show that OsDCL2, OsDCL4, and OsHEN1 are preferentially expressed in the egg cell. In addition, we revealed that AtDCL2 is also preferentially expressed in the Arabidopsis egg cell. These findings suggest that small RNA pathways are activated in the egg cell in both rice and Arabidopsis. The activation of these pathways in the egg cell might be essential for egg cell maturation, fertilization, or embryogenesis. OsDCL2a,SHO1|OsDCL4,WAF1|OsHEN1 Dicer-like 3 produces transposable element-associated 24-nt siRNAs that control agricultural traits in rice 2014 Proc Natl Acad Sci U S A State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Transposable elements (TEs) and repetitive sequences make up over 35% of the rice (Oryza sativa) genome. The host regulates the activity of different TEs by different epigenetic mechanisms, including DNA methylation, histone H3K9 methylation, and histone H3K4 demethylation. TEs can also affect the expression of host genes. For example, miniature inverted repeat TEs (MITEs), dispersed high copy-number DNA TEs, can influence the expression of nearby genes. In plants, 24-nt small interfering RNAs (siRNAs) are mainly derived from repeats and TEs. However, the extent to which TEs, particularly MITEs associated with 24-nt siRNAs, affect gene expression remains elusive. Here, we show that the rice Dicer-like 3 homolog OsDCL3a is primarily responsible for 24-nt siRNA processing. Impairing OsDCL3a expression by RNA interference caused phenotypes affecting important agricultural traits; these phenotypes include dwarfism, larger flag leaf angle, and fewer secondary branches. We used small RNA deep sequencing to identify 535,054 24-nt siRNA clusters. Of these clusters, approximately 82% were OsDCL3a-dependent and showed significant enrichment of MITEs. Reduction of OsDCL3a function reduced the 24-nt siRNAs predominantly from MITEs and elevated expression of nearby genes. OsDCL3a directly targets genes involved in gibberellin and brassinosteroid homeostasis; OsDCL3a deficiency may affect these genes, thus causing the phenotypes of dwarfism and enlarged flag leaf angle. Our work identifies OsDCL3a-dependent 24-nt siRNAs derived from MITEs as broadly functioning regulators for fine-tuning gene expression, which may reflect a conserved epigenetic mechanism in higher plants with genomes rich in dispersed repeats or TEs. OsDCL3a DDM1 (decrease in DNA methylation) genes in rice (Oryza sativa) 2012 Mol Genet Genomics CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan. Regulation of cytosine methylation in the plant genome is of pivotal in determining the epigenetic states of chromosome regions. Relative tolerance of plant to deficiency in cytosine methylation provides unparalleled opportunities to study the mechanism for regulation of cytosine methylation. The Decrease in DNA Methylation 1 (DDM1) of Arabidopsis thaliana is one of the best characterized plant epigenetic regulators that are necessary for maintenance of cytosine methylation in genomic DNA. Although cytosine methylation could affect various aspects of plant growth and development including those related to agricultural importance, orthologs of DDM1 in plants other than Arabidopsis has not been studied in detail. In this study, we identified two rice genes with similarity to Arabidopsis DDM1 and designated them OsDDM1a and OsDDM1b. Both of the rice DDM1 homologs are transcribed during development and their amino acid sequences are 93 % identical to each other. Transgenic rice lines expressing the OsDDM1a cDNA in the antisense orientation exhibited genomic DNA hypomethylation. In those lines, repeated sequences were more severely affected than a single copy sequence as is the case in Arabidopsis ddm1 mutants. Transcripts derived from endogenous transposon-related loci were up-regulated in the antisense OsDDM1 lines, opening a possibility to identify and utilize potentially active transposons for rice functional genomics. OsDDM1a,OsDDM1b The rice wall-associated receptor-like kinase gene OsDEES1 plays a role in female gametophyte development 2012 Plant Physiol Hebei Key Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hebei Normal University, Shijiazhuang 050016, China. The wall-associated kinase (WAK) gene family is a unique subfamily of receptor-like kinases (RLKs) in plants. WAK-RLKs play roles in cell expansion, pathogen resistance, and metal tolerance in Arabidopsis (Arabidopsis thaliana). Rice (Oryza sativa) has far more WAK-RLK genes than Arabidopsis, but the functions of rice WAK-RLKs are poorly understood. In this study, we found that one rice WAK-RLK gene, DEFECT IN EARLY EMBRYO SAC1 (OsDEES1), is involved in the regulation of early embryo sac development. OsDEES1 silencing by RNA interference caused a high rate of female sterility. Crossing experiments showed that female reproductive organs lacking OsDEES1 carried a functional defect. A detailed investigation of the ovaries from OsDEES1 RNA interference plants indicated that the knockdown of OsDEES1 expression did not affect megasporogenesis but that it disturbed female gametophyte formation, resulting in a degenerated embryo sac and defective seed formation. OsDEES1 exhibited a tissue-specific expression pattern in flowers and seedlings. In the ovary, OsDEES1 was expressed in the megagametophyte region and surrounding nucellus cells in the ovule near the micropylar region. OsDEES1 was found to be a membrane-localized protein with a unique sequence compared with other WAK-RLKs. These data indicate that OsDEES1 plays a role in rice sexual reproduction by regulating female gametophyte development. This study offers new insight into the functions of the WAK-RLK family. OsDEES1 OsDEG10 encoding a small RNA-binding protein is involved in abiotic stress signaling 2009 Biochem Biophys Res Commun Department of Biological Sciences, Pusan National University, San 30, Jangjeon-dong, Geumjeong-gu, Busan 609-735, Republic of Korea. Excessive light can be harmful to photosynthetic apparatus since it causes photoinhibition and photooxidation, and plants often encounter hypoxic or anoxic environments when they become submerged by heavy rain or an ensuing flood. In this study, Oryza sativa Differentially Expressed Genes (OsDEGs) from rice under photooxidation and anoxia conditions were isolated using DD-PCR. Among them, OsDEG10 is predicted to encode a small RNA-binding protein (RBP) and the transcript levels of OsDEG10 strongly increased under most of abiotic stress treatments such as high light, anoxia, NaCl, ABA, MV and cold. However, the transcript levels of two rice OsDEG10 homologs were not changed under those treatments. OsDEG10 RNAi transgenic plants were more sensitive to high light and cold stresses compared to wild-type plants. Our results suggest that OsDEG10 is a small RBP involved in the response to various abiotic stresses. OsDEG10 The ADAXIALIZED LEAF1 gene functions in leaf and embryonic pattern formation in rice 2009 Dev Biol Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. The adaxial-abaxial axis in leaf primordia is thought to be established first and is necessary for the expansion of the leaf lamina along the mediolateral axis. To understand axis information in leaf development, we isolated the adaxialized leaf1 (adl1) mutant in rice, which forms abaxially rolled leaves. adl1 leaves are covered with bulliform-like cells, which are normally distributed only on the adaxial surface. An adl1 double mutant with the adaxially snowy leaf mutant, which has albino cells that specifically appear in the abaxial mesophyll tissue, indicated that adl1 leaves show adaxialization in both epidermal and mesophyll tissues. The expression of HD-ZIPIII genes in adl1 mutant increased in mature leaves, but not in the young primordia or the SAM. This indicated that ADL1 may not be directly involved in determining initial leaf polarity, but rather is associated with the maintenance of axis information. ADL1 encodes a plant-specific calpain-like cysteine proteinase orthologous to maize DEFECTIVE KERNEL1. Furthermore, we identified intermediate and strong alleles of the adl1 mutant that generate shootless embryos and globular-arrested embryos with aleurone layer loss, respectively. We propose that ADL1 plays an important role in pattern formation of the leaf and embryo by promoting proper epidermal development. ADL1|OsDEK1 Regulation of aleurone development in cereal grains 2011 J Exp Bot Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA. becraft@iastate.edu The aleurone layer of cereal grains is important biologically as well as nutritionally and economically. Here, current knowledge on the regulation of aleurone development is reviewed. Recent reports suggest that the control of aleurone development is more complex than earlier models portrayed. Multiple levels of genetic regulation control aleurone cell fate, differentiation, and organization. The hormones auxin and cytokinin can also influence aleurone development. New technical advances promise to facilitate future progress. ADL1|OsDEK1 Introgression of qPE9-1 allele, conferring the panicle erectness, leads to the decrease of grain yield per plant in japonica rice (Oryza sativa L.) 2011 J Genet Genomics Jiangsu Key Laboratory of Crop Genetics and Physiology, Key Laboratory of Plant Functional Genomics, Ministry of Education, Agricultural College of Yangzhou University, 12# East Wenhui Rd., Jiangsu 225009, China. Panicle architecture is closely related to yield formation. The qPE9-1 gene has been proved to be widely used in high-yield rice cultivar developments, conferring erect panicle character in japonica rice. Recently, qPE9-1 has been successfully cloned; however, the genetic effect on grain yield per plant of the erect panicle allele qPE9-1 is controversial yet. In the present study, a drooping panicle parent Nongken 57, carrying qpe9-1 allele, was used as recurrent parent to successively backcross to a typical erect panicle line from the double haploid (DH) population (Wuyunjing 8/Nongken 57), which was previously shown to carry qPE9-1 allele. Thus a pair of near-isogenic lines (NILs) was developed. The comparison of agronomic traits between the NILs showed that, when qpe9-1 was replaced by qPE9-1, the panicle architecture was changed from drooping to erect; moreover, the panicle length, plant height, 1000-grain weight and the tillers were significantly decreased, consequently resulting in the dramatic decrease of grain yield per plant by 30%. Therefore, we concluded that the qPE9-1 was a major factor controlling panicle architecture, and qPE9-1 had pleiotropic nature, with negative effects on grain yield per plant. This result strongly suggests that the erect panicle allele qPE9-1 should be used together with other favorable genes in the high-yield breeding practice. In addition, the effect of qPE9-1 on eating and cooking quality was also discussed in the present study. DEP1|DN1|qPE9-1|OsDEP1 Identification and characterization of a major QTL responsible for erect panicle trait in japonica rice (Oryza sativa L.) 2007 Theor Appl Genet The Key Laboratory of Plant Functional Genomics, Ministry of Education of China, Yangzhou 225009, Jiangsu, People's Republic of China. yichuan@yzu.edu.cn Panicle erectness (PE) is one of the most important traits for high-yielding japonica cultivars. Although several cultivars with PE trait have been developed and released for commercial production in China, there is little information on the inheritance of PE traits in rice. In the present study, 69 widely cultivated japonica cultivars and a double haploid (DH) population derived from a cross between a PE cultivar (Wuyunjing 8) and a drooping panicle cultivar (Nongken 57) were utilized to elucidate the mechanisms of PE formation and to map PE associated genes. Our data suggested that panicle length (PL) and plant height (PH) significantly affected panicle curvature (PC), with shorter PL and PH resulting in smaller PC and consequently more erect. A putative major gene was identified on chromosome 9 by molecular markers and bulk segregant analysis in DH population. In order to finely map the major gene, all simple sequence repeats (SSR) markers on chromosome 9 as well as 100 newly developed sequence-tagged site (STS) markers were used to construct a linkage group for quantitative trait locus (QTL) mapping. A major QTL, qPE9-1, between STS marker H90 and SSR marker RM5652, was detected, and accounted for 41.72% of PC variation with pleiotropic effect on PH and PL. another QTL, qPE9-2, was also found to be adjacent to qPE9-1. In addition, we found that H90, the nearest marker to qPE9-1, used for genotyping 38 cultivars with extremely erect and drooping panicles, segregated in agreement with PC, suggesting the H90 product was possibly part of the qPE9-1 gene or closely related to it. These data demonstrated that H90 could be used for marker-aided selection for the PE trait in breeding and in the cloning of qPE9-1. DEP1|DN1|qPE9-1|OsDEP1 Deletion in a quantitative trait gene qPE9-1 associated with panicle erectness improves plant architecture during rice domestication 2009 Genetics Jiangsu Key Laboratory of Crop Genetics and Physiology/Key Laboratory of the Ministry of Education for Plant Functional Genomics, Yangzhou University, Yangzhou 225009, Jiangsu Province, People's Republic of China. Rice plant architecture is an important agronomic trait and a major determinant in high productivity. Panicle erectness is the preferred plant architecture in japonica rice, but the molecular mechanism underlying domestication of the erect panicle remains elusive. Here we report the map-based cloning of a major quantitative trait locus, qPE9-1, which plays an integral role in regulation of rice plant architecture including panicle erectness. The R6547 qPE9-1 gene encodes a 426-amino-acid protein, homologous to the keratin-associated protein 5-4 family. The gene is composed of three Von Willebrand factor type C domains, one transmembrane domain, and one 4-disulfide-core domain. Phenotypic comparisons of a set of near-isogenic lines and transgenic lines reveal that the functional allele (qPE9-1) results in drooping panicles, and the loss-of-function mutation (qpe9-1) leads to more erect panicles. In addition, the qPE9-1 locus regulates panicle and grain length, grain weight, and consequently grain yield. We propose that the panicle erectness trait resulted from a natural random loss-of-function mutation for the qPE9-1 gene and has subsequently been the target of artificial selection during japonica rice breeding. DEP1|DN1|qPE9-1|OsDEP1 Rice DEP1, encoding a highly cysteine-rich G protein gamma subunit, confers cadmium tolerance on yeast cells and plants 2013 J Exp Bot Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba, Sendai, Miyagi 980-8577, Japan. A rice cDNA, OsDEP1, encoding a highly cysteine (Cys)-rich G protein gamma subunit, was initially identified as it conferred cadmium (Cd) tolerance on yeast cells. Of the 426 aa constituting OsDEP1, 120 are Cys residues (28.2%), of which 88 are clustered in the C-terminal half region (aa 170-426). To evaluate the independent effects of these two regions, two truncated versions of the OsDEP1-expressing plasmids pOsDEP1(1-169) and pOsDEP1(170-426) were used to examine their effects on yeast Cd tolerance. Although OsDEP1(170-426) conferred a similar level of Cd tolerance as the intact OsDEP1, OsDEP1(1-169) provided no such tolerance, indicating that the tolerance effect is localized to the aa 170-426 C-terminal peptide region. The Cd responses of transgenic Arabidopsis plants constitutively expressing OsDEP1, OsDEP1(1-169) or OsDEP1(170-426), were similar to the observations in yeast cells, with OsDEP1 and OsDEP1(170-426) transgenic plants displaying Cd tolerance but OsDEP1(1-169) plants showing no such tolerance. In addition, a positive correlation between the transcript levels of OsDEP1 or OsDEP1(170-426) in the transgenics and the Cd content of these plants upon Cd application was observed. As several Arabidopsis loss-of-function heterotrimeric G protein beta and gamma subunit gene mutants did not show differences in their Cd sensitivity compared with wild-type plants, we propose that the Cys-rich region of OsDEP1 may function directly as a trap for Cd ions. DEP1|DN1|qPE9-1|OsDEP1 Natural variation at the DEP1 locus enhances grain yield in rice 2009 Nat Genet The State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, National Centre for Plant Gene Research, Beijing, China. Grain yield is controlled by quantitative trait loci (QTLs) derived from natural variations in many crop plants. Here we report the molecular characterization of a major rice grain yield QTL that acts through the determination of panicle architecture. The dominant allele at the DEP1 locus is a gain-of-function mutation causing truncation of a phosphatidylethanolamine-binding protein-like domain protein. The effect of this allele is to enhance meristematic activity, resulting in a reduced length of the inflorescence internode, an increased number of grains per panicle and a consequent increase in grain yield. This allele is common to many Chinese high-yielding rice varieties and likely represents a relatively recent introduction into the cultivated rice gene pool. We also show that a functionally equivalent allele is present in the temperate cereals and seems to have arisen before the divergence of the wheat and barley lineages. DEP1|DN1|qPE9-1|OsDEP1 Genome-wide binding analysis of the transcription activator ideal plant architecture1 reveals a complex network regulating rice plant architecture 2013 Plant Cell State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Ideal plant architecture1 (IPA1) is critical in regulating rice (Oryza sativa) plant architecture and substantially enhances grain yield. To elucidate its molecular basis, we first confirmed IPA1 as a functional transcription activator and then identified 1067 and 2185 genes associated with IPA1 binding sites in shoot apices and young panicles, respectively, through chromatin immunoprecipitation sequencing assays. The Squamosa promoter binding protein-box direct binding core motif GTAC was highly enriched in IPA1 binding peaks; interestingly, a previously uncharacterized indirect binding motif TGGGCC/T was found to be significantly enriched through the interaction of IPA1 with proliferating cell nuclear antigen promoter binding factor1 or promoter binding factor2. Genome-wide expression profiling by RNA sequencing revealed IPA1 roles in diverse pathways. Moreover, our results demonstrated that IPA1 could directly bind to the promoter of rice teosinte branched1, a negative regulator of tiller bud outgrowth, to suppress rice tillering, and directly and positively regulate dense and erect panicle1, an important gene regulating panicle architecture, to influence plant height and panicle length. The elucidation of target genes of IPA1 genome-wide will contribute to understanding the molecular mechanisms underlying plant architecture and to facilitating the breeding of elite varieties with ideal plant architecture. DEP1|DN1|qPE9-1|OsDEP1,OsSPL14|IPA1|WFP,OsTB1|FC1 Mutation of OsDET1 increases chlorophyll content in rice 2013 Plant Sci Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, 174 Shazheng Street, Shapingba District, Chongqing 400030, China. As an important agronomic trait, the chlorophyll (Chl) content is closely related to photosynthesis in plants. A rice mutant Gc (Oryza sativa indica) was characterized previously by its enhanced Chl content (Chl b and total Chl) and exaggerated photosynthetic rate. Here, we describe the enhanced Chl content was caused by a mutation in the rice homolog of the DE-ETIOLATED1 (DET1) known to be involved in light transduction and morphogenesis in Arabidopsis and tomato. Sequence analysis revealed that the Gc mutant carried two fragment-insertions and a fragment-deletion upstream of the start codon of OsDET1, which led to enhance mRNA levels of OsDET1. Besides, the Gc mutant harbored a single T-to-C base transversion in the seventh exon of OsDET1, which resulted in leucine(328) to serine(328) localized in the highly conserved region. Genetic complementation demonstrated that OsDET1 mutation conferred the enhanced Chl content in the Gc mutant leaf. OsDET1 was richly expressed in green tissues, and its expression seems to be under circadian control. OsDET1-GFP fusion protein in onion epidermal cells showed that OsDET1 localized to the nucleus. These results indicated that OsDET1 mutation in Gc mutant increases Chl content in rice, which might be fundamental for enhanced photoresponsiveness. OsDET1 The rice OsDG2 encoding a glycine-rich protein is involved in the regulation of chloroplast development during early seedling stage 2014 Plant Cell Rep Development Center of Plant Germplasm Resources, College of Life and Environment Sciences, Shanghai Normal University, Shanghai, 200234, China. KEY MESSAGE: OsDG2 gene encoded a novel chloroplast-targeted GRP in rice. Disruption of the OsDG2 would lead to delayed greening phenotype and affected expression levels of genes associated with chloroplast development at early leaf stage of rice. Glycine-rich proteins (GRPs) participate in various biological processes in plants. However, the evidence of GRPs involved in chloroplast development in plants is quite limited. In this study, we identified a rice GRP gene mutant named osdg2 (O ryza s ativa d elayed g reening 2), which exhibits delayed greening phenotype characterized as bright yellow leaves before the three-leaf stage and thereafter turns to normal green. Further study showed that the mutant phenotype was consistent with changes in chlorophyll content and chloroplast development. The rice OsDG2 gene, encoding a novel GRP protein, was located on chromosome 2 through map-based cloning method and confirmed by molecular complementation tests. Subcellular localization results showed that OsDG2 was targeted in chloroplasts. In addition, the OsDG2 transcripts were highly expressed in leaves and undetectable in other tissues, showing the tissue-specific expression. In osdg2 mutant, the expression levels of most genes associated with chloroplast development were severely decreased in the 3rd leaves, but almost recovered to wild-type level in the 4th leaves. Our findings indicated that the nuclear-encoded OsDG2 plays important roles in chloroplast development at early leaf stage of rice. OsDG2 OsDGL1, a homolog of an oligosaccharyltransferase complex subunit, is involved in N-glycosylation and root development in rice 2013 Plant Cell Physiol The State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, PR China. A leaky rice mutant was isolated from an ethylmethane sulfonate (EMS)-mutagenized rice library based on its short root phenotype. The map-based cloning results showed that the mutant was due to a point mutation in the intron of OsDGL1 (LOC_Os07g10830), which encodes the dolichyl-diphosphooligosaccharide-protein glycosyltransferase 48 kDa subunit precursor. The mutation results in premature termination of protein synthesis. OsDGL1 is an ortholog of Arabidopsis DGL1, human OST48 and yeast WBP1, an essential protein subunit of the oligosaccharyltransferase (OST) complex, which is involved in N-glycosylation in eukaryotes. The leaky rice mutant, Osdgl1, displayed a change of matrix polysaccharides in its root cell wall, shorter root cell length, smaller root meristem and cell death in the root. Consistent with the known function of the OST complex in eukaryotes, the Osdgl1 mutation leads to a defect in N-glycosylation in the root. It was also found that reactive oxygen species (ROS) may be involved in this process. OsDGL1 Drought inducible OsDhn1 promoter is activated by OsDREB1A and OsDREB1D 2013 Journal of Plant Biology Department of Life Science, Sogang University, Seoul, 121-742, Korea In order to understand the stress-responsive mechanism controlling OsDhn1 gene, promoter analysis of OsDhn1 gene was conducted. First, a 1.5 kb-long promoter region of OsDhn1 was isolated and characterized by beta-glucuronidase (GUS) analysis. GUS assay of OsDhn1::gus plants showed that the GUS activity in the leaves was induced by drought stress but not by wound, cold, salt, or ABA treatment. Second, transactivation assay revealed that OsDhn1 promoter is activated by OsDREB1A or OsDREB1D as well as CBF1 but not by OsDREB1B or OsDREB1C. Taken together, these suggest that the drought-inducible OsDhn1 gene is regulated by abiotic stress signaling pathway involving CBF/DREB. OsDhn1,OsDREB1D,OsDREB1A|OsDREBL Characterization of an abiotic stress-inducible dehydrin gene, OsDhn1, in rice (Oryza sativa L.) 2005 Mol Cells Department of Life Science, Sogang University, Seoul 121-742, Korea. A full-length 1.1 kb cDNA, designated Oryza sativa Dehydrin 1 (OsDhn1), was isolated from the seed coat of rice. The deduced protein is hydrophilic and has three K-type and one S-type motifs (SK3-type), indicating that OsDhn1 belongs to the acidic dehydrin family, which includes wheat WCOR410 and Arabidopsis COR47. Expression of OsDhn1 was strongly induced by low temperature as well as by drought. Induction of OsDhn1 by cold stress was clearcut in the roots of seedlings and the epidermis of palea and lemma. OsDhn1 was also up-regulated in UBI::CBF1/DREB1b transgenic plants indicating that it is regulated by the CBF/DREB stress signaling pathway. OsDhn1 The OsDHODH1 gene is involved in salt and drought tolerance in rice 2009 J Integr Plant Biol State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Agriculture, Nanjing Agricultural University, Nanjing 210095, China. In the present paper, we identified and cloned OsDHODH1 encoding a putative cytosolic dihydroorotate dehydrogenase (DHODH) in rice. Expression analysis indicated that OsDHODH1 is upregulated by salt, drought and exogenous abscisic acid (ABA), but not by cold. By prokaryotic expression, we determined the enzymatic activity of OsDHODH1 and found that overproduction of OsDHODH1 significantly improved the tolerance of Escherichia coli cells to salt and osmotic stresses. Overexpression of the OsDHODH1 gene in rice increased the DHODH activity and enhanced plant tolerance to salt and drought stresses as compared with wild type and OsDHODH1-antisense transgenic plants. Our findings reveal, for the first time, that cytosolic dihydroorotate dehydrogenase is involved in plant stress response and that OsDHODH1 could be used in engineering crop plants with enhanced tolerance to salt and drought. OsDHODH1 The SINA E3 ligase OsDIS1 negatively regulates drought response in rice 2011 Plant Physiol Hunan Provincial Key Laboratory of Crop Germplasm Innovation and Utilization, Hunan Agricultural University, Changsha Hunan 410128, China. Ubiquitin-regulated protein degradation is a critical regulatory mechanism that controls a wide range of biological processes in plants. Here, we report that OsDIS1 (for Oryza sativa drought-induced SINA protein 1), a C3HC4 RING finger E3 ligase, is involved in drought-stress signal transduction in rice (O. sativa). The expression of OsDIS1 was up-regulated by drought treatment. In vitro ubiquitination assays showed that OsDIS1 possessed E3 ubiquitin ligase activity and that the conserved region of the RING finger was required for the activity. Transient expression assays in Nicotiana benthamiana leaves and rice protoplasts indicated that OsDIS1 was localized predominantly in the nucleus. Overexpression of OsDIS1 reduced drought tolerance in transgenic rice plants, while RNA interference silencing of OsDIS1 enhanced drought tolerance. Microarray analysis revealed that a large number of drought-responsive genes were induced or suppressed in the OsDIS1 overexpression plants under normal and drought conditions. Yeast two-hybrid screening showed that OsDIS1 interacted with OsNek6 (for O. sativa NIMA-related kinase 6), a tubulin complex-related serine/threonine protein kinase. Coexpression assays in N. benthamiana leaves indicated that OsNek6 was degraded by OsDIS1 via the 26S proteasome-dependent pathway and that this degradation was abolished by the OsDIS1(H71Y) mutation, which is essential for its E3 ligase activity. Together, these results demonstrate that OsDIS1 plays a negative role in drought stress tolerance through transcriptional regulation of diverse stress-related genes and possibly through posttranslational regulation of OsNek6 in rice. OsDIS1 OsDIS1-mediated stress response pathway in rice 2011 Plant Signal Behav State Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China. Ubiquitin-mediated protein degradation has been well demonstrated as a key regulatory mechanism in response to drought stress in Arabidopsis. However, the biological function of most E3 ligase genes in drought response is still unknown in rice. We recently showed that OsDIS1 (Oryza sativa drought-induced SINA protein 1), a SINA type E3 ligase, is involved in the drought-stress signal transduction in rice. OsDIS1 plays a negative role in drought stress tolerance through the transcriptional regulation of diverse stress-related genes and also possibly through the posttranslational regulation of its interacting protein OsNek6 in rice. Here we also show that OsDIS1 interacts with OsSKIPa, a drought and salt stress positive regulator in rice. Based on these results, we propose a working model for the function of OsDIS1 in regulating the stress signaling pathway in rice. OsDIS1,OsSKIPa Isolation and characterization of OsDMC1 , the rice homologue of the yeast DMC1 gene essential for meiosis 2001 Sexual Plant Reproduction Research Center for Plant Molecular and Developmental Biology, Institute of Botany, The Chinese Academy of Sciences, 20 Nanxincum, Xiangshan, Beijing 100093, Yeast DMC1 is a meiosis-specific gene required for homologous chromosome pairing in meiosis. Using degenerate primers designed according to amino acid motifs conserved in yeast Dmc1 and Arabidopsis AtDmc1, we obtained full-length cDNA of a rice homologue of the DMC1 gene (OsDMC1) by RT-PCR and rapid amplification of cDNA ends (RACEs). OsDmc1 exhibited 53% amino acid sequence identity to yeast Dmc1 and 81% to AtDmc1. OsDMC1 was expressed at high-levels in reproductive organs, low-levels in roots, and undetectable levels in leaves and seedlings. Southern blot analyses revealed that OsDMC1 is one of two DMC1 homologues present in rice. OsDMC1 DNA strand exchange activity of rice recombinase OsDmc1 monitored by fluorescence resonance energy transfer and the role of ATP hydrolysis 2006 FEBS J Molecular Biology Division, Bhabha Atomic Research Center, Mumbai, India. Rad51 and disrupted meiotic cDNA1 (Dmc1) are the two eukaryotic DNA recombinases that participate in homology search and strand exchange reactions during homologous recombination mediated DNA repair. Rad51 expresses in both mitotic and meiotic tissues whereas Dmc1 is confined to meiosis. DNA binding and pairing activities of Oryza sativa disrupted meiotic cDNA1 (OsDmc1) from rice have been reported earlier. In the present study, DNA renaturation and strand exchange activities of OsDmc1 have been studied, in real time and without the steps of deproteinization, using fluorescence resonance energy transfer (FRET). The extent as well as the rate of renaturation is the highest in conditions that contain ATP, but significantly less when ATP is replaced by slowly hydrolysable analogues of ATP, namely adenosine 5'-(beta,gamma-imido) triphosphate (AMP-PNP) or adenosine 5'-O-(3-thio triphosphate) (ATP-gamma-S), where the former was substantially poorer than the latter in facilitating the renaturation function. FRET assay results also revealed OsDmc1 protein concentration dependent strand exchange function, where the activity was the fastest in the presence of ATP, whereas in the absence of a nucleotide cofactor it was several fold ( approximately 15-fold) slower. Interestingly, strand exchange, in reactions where ATP was replaced with AMP-PNP or ATP-gamma-S, was somewhat slower than that of even minus nucleotide cofactor control. Notwithstanding the slow rates, the reactions with no nucleotide cofactor or with ATP-analogues did reach the same steady state level as seen in ATP reaction. FRET changes were unaffected by the steps of deproteinization following OsDmc1 reaction, suggesting that the assay results reflected stable events involving exchanges of homologous DNA strands. All these results, put together, suggest that OsDmc1 catalyses homologous renaturation as well as strand exchange events where ATP hydrolysis seems to critically decide the rates of the reaction system. These studies open up new facets of a plant recombinase function in relation to the role of ATP hydrolysis. OsDMC1 DNA binding and pairing activity of OsDmc1, a recombinase from rice 2005 Plant Mol Biol Molecular Biology Division, Bhabha Atomic Research Center, Mumbai, India. A cloned cDNA corresponding to OsDMC1 from rice anther tissue was expressed in Escherichia coli. The OsDmc1 protein was largely present in the inclusion bodies of the cell lysatE., which was solubilized by 8.0 M urea containing buffeR., purified to homogeneity by Ni-CAM agarose column chromatography, followed by renaturation to its native state through stepwise dialysis against reduced concentrations of urea. The purified protein cross-reacted with anti-yeast Dmc1 antibodies. The binding efficiency observed with circular single-stranded DNA (ssDNA) was similar to that with circular double-stranded DNA (dsDNA). The binding to either DNA showed no ATP dependencE., but required 5-10 mM Mg2+ in the presence of ATP. Even though the protein binding to dsDNA was as efficient as it was to ssDNA, the former induced no DNA dependent ATPasE., whereas the binding to ssDNA stimulated a significant level of DNA dependent ATPase activity. OsDmc1-ssDNA complex, with its ATPase proficiency, also mediated renaturation of homologous complementary strands as well as assimilation of single strands into homologous supercoiled duplexes leading to D-loop formation. The D-loop formation was lowered by excess of OsDmc1 protein. This D-loop formation activity was promoted by non-hydrolyzable ATP analog, AMP-PNP and was not observed in absence of ATP or presence of ADP/ATP-gamma-S. These properties reflected the classical hallmarks of a recombinase and represented the first biochemical characterization of a plant Dmc1 protein. OsDMC1 OsDMC1 is required for homologous pairing in Oryza sativa 2007 Plant Mol Biol Research Center of Molecular & Developmental Biology, Key Laboratory of Photosynthesis & Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, 20 Nanxincun, Xiangshan, Haidianqu, Beijing, China. OsDMC1 is the rice homologue of the yeast DMC1 gene. Here, we analyzed the function of OsDMC1 in meiosis using an RNA interference approach. The OsDMC1-RNAi lines grew normally during their vegetative phase but showed spikelet and pollen sterility. The sterility phenotypes were associated with down-regulated OsDMC1 transcript and protein levels mediated by RNAi. Further cytological observations of male meiocytes revealed that knock-down of OsDMC1 led to defects in bivalent formation and subsequent unequal chromosome segregation and irregular spore generation, and induced changes in male meiotic progression. Fluorescent in situ hybridization experiments revealed that the OsDMC1-RNAi lines were defective in homologous pairing. These data indicate that OsDMC1 is essential for rice meiosis and plays an important role in homologous pairing. OsDMC1 A rice gene for microbial symbiosis, Oryza sativa CCaMK, reduces CH4 flux in a paddy field with low nitrogen input 2014 Appl Environ Microbiol Graduate School of Life Sciences, Tohoku University, Katahira, Aoba-ku, Sendai, Miyagi, Japan. Plants have mutualistic symbiotic relationships with rhizobia and fungi by the common symbiosis pathway, of which Ca(2+)/calmodulin-dependent protein kinase (encoded by CCaMK) is a central component. Although Oryza sativa CCaMK (OsCCaMK) is required for fungal accommodation in rice roots, little is known about the role of OsCCaMK in rice symbiosis with bacteria. Here, we report the effect of a Tos17-induced OsCCaMK mutant (NE1115) on CH4 flux in low-nitrogen (LN) and standard-nitrogen (SN) paddy fields compared with wild-type (WT) Nipponbare. The growth of NE1115 was significantly decreased compared with that of the WT, especially in the LN field. The CH4 flux of NE1115 in the LN field was significantly greater (156 to 407% in 2011 and 170 to 816% in 2012) than that of the WT, although no difference was observed in the SN field. The copy number of pmoA (encodes methane monooxygenase in methanotrophs) was significantly higher in the roots and rhizosphere soil of the WT than in those of NE1115. However, the mcrA (encodes methyl coenzyme M reductase in methanogens) copy number did not differ between the WT and NE1115. These results were supported by a (13)C-labeled CH4-feeding experiment. In addition, the natural abundance of (15)N in WT shoots (3.05 per thousand) was significantly lower than in NE1115 shoots (3.45 per thousand), suggesting greater N2 fixation in the WT because of dilution with atmospheric N2 (0.00 per thousand). Thus, CH4 oxidation and N2 fixation were simultaneously activated in the root zone of WT rice in the LN field and both processes are likely controlled by OsCCaMK. OsCCaMK|OsDMI3 OsDMI3 is a novel component of abscisic acid signaling in the induction of antioxidant defense in leaves of rice 2012 Mol Plant College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China. Ca(2+) and calmodulin (CaM) have been shown to play an important role in abscisic acid (ABA)-induced antioxidant defense. However, it is unknown whether Ca(2+)/CaM-dependent protein kinase (CCaMK) is involved in the process. In the present study, the role of rice CCaMK, OsDMI3, in ABA-induced antioxidant defense was investigated in leaves of rice (Oryza sativa) plants. Treatments with ABA, H(2)O(2), and polyethylene glycol (PEG) induced the expression of OsDMI3 and the activity of OsDMI3, and H(2)O(2) is required for the ABA-induced increases in the expression and the activity of OsDMI3 under water stress. Subcellular localization analysis showed that OsDMI3 is located in the nucleus, the cytoplasm, and the plasma membrane. The analysis of the transient expression of OsDMI3 in rice protoplasts and the RNA interference (RNAi) silencing of OsDMI3 in rice protoplasts showed that OsDMI3 is required for ABA-induced increases in the expression and the activities of superoxide dismutase (SOD) and catalase (CAT). Further, the oxidative damage induced by higher concentrations of PEG and H(2)O(2) was aggravated in the mutant of OsDMI3. Moreover, the analysis of the RNAi silencing of OsDMI3 in protoplasts and the mutant of OsDMI3 showed that higher levels of H(2)O(2) accumulation require OsDMI3 activation in ABA signaling, but the initial H(2)O(2) production induced by ABA is not dependent on the activation of OsDMI3 in leaves of rice plants. Our data reveal that OsDMI3 is an important component in ABA-induced antioxidant defense in rice. OsCCaMK|OsDMI3 OsDMI3-mediated activation of OsMPK1 regulates the activities of antioxidant enzymes in abscisic acid signalling in rice 2014 Plant Cell Environ College of Life Sciences, Nanjing Agricultural University, Nanjing, 210095, China; National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, 210095, China. In rice, the Ca(2+) /calmodulin (CaM)-dependent protein kinase (CCaMK) OsDMI3 has been shown to be required for abscisic acid (ABA)-induced antioxidant defence. However, it is not clear how OsDMI3 participates in this process in rice. In this study, the cross-talk between OsDMI3 and the major ABA-activated MAPK OsMPK1 in ABA-induced antioxidant defence was investigated. ABA treatment induced the expression of OsDMI3 and OsMPK1 and the activities of OsDMI3 and OsMPK1 in rice leaves. In the mutant of OsDMI3, the ABA-induced increases in the expression and the activity of OsMPK1 were substantially reduced. But in the mutant of OsMPK1, the ABA-induced increases in the expression and the activity of OsDMI3 were not affected. Pretreatments with MAPKK inhibitors also did not affect the ABA-induced activation of OsDMI3. Further, a transient expression analysis in combination with mutant analysis in rice protoplasts showed that OsMPK1 is required for OsDMI3-induced increases in the activities of antioxidant enzymes and the production of H2 O2 . Our data indicate that there exists a cross-talk between OsDMI3 and OsMPK1 in ABA signalling, in which OsDMI3 functions upstream of OsMPK1 to regulate the activities of antioxidant enzymes and the production of H2 O2 in rice. OsCCaMK|OsDMI3,OsMPK1|OsMAPK6|OsSIPK Fungal symbiosis in rice requires an ortholog of a legume common symbiosis gene encoding a Ca2+/calmodulin-dependent protein kinase 2007 Plant Physiol Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky 40546, USA. In natural ecosystems, many plants are able to establish mutually beneficial symbioses with microorganisms. Of critical importance to sustainable agriculture are the symbioses formed between more than 80% of terrestrial plants and arbuscular mycorrhizal (AM) fungi and between legumes and nitrogen-fixing rhizobial bacteria. Interestingly, the two symbioses share overlapping signaling pathways in legumes, suggesting that the evolutionarily recent root nodule symbiosis may have acquired functions from the ancient AM symbiosis. The Medicago truncatula DMI3 (DOESN'T MAKE INFECTIONS3) gene (MtDMI3) and its orthologs in legumes are required for both bacterial and fungal symbioses. MtDMI3 encodes a Ca(2+)/calmodulin-dependent protein kinase (CCaMK) essential for the transduction of the Ca(2+) signal induced by the perception of Nod factors. Putative orthologs of MtDMI3 are also present in non-legumes, but their function in AM symbiosis has not been demonstrated in any non-legume species. Here, we combine reverse genetic approaches and a cross-species complementation test to characterize the function of the rice (Oryza sativa) ortholog of MtDMI3, namely, OsDMI3, in AM symbiosis. We demonstrate that OsDMI3 is not only required for AM symbiosis in rice but also is able to complement a M. truncatula dmi3 mutant, indicating an equivalent role of MtDMI3 orthologs in non-legumes. OsCCaMK|OsDMI3 CYCLOPS, a mediator of symbiotic intracellular accommodation 2008 Proc Natl Acad Sci U S A Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. The initiation of intracellular infection of legume roots by symbiotic rhizobia bacteria and arbuscular mycorrhiza (AM) fungi is preceded by the induction of calcium signatures in and around the nucleus of root epidermal cells. Although a calcium and calmodulin-dependent kinase (CCaMK) is a key mediator of symbiotic root responses, the decoding of the calcium signal and the molecular events downstream are only poorly understood. Here, we characterize Lotus japonicus cyclops mutants on which microbial infection was severely inhibited. In contrast, nodule organogenesis was initiated in response to rhizobia, but arrested prematurely. This arrest was overcome when a deregulated CCaMK mutant version was introduced into cyclops mutants, conferring the development of full-sized, spontaneous nodules. Because cyclops mutants block symbiotic infection but are competent for nodule development, they reveal a bifurcation of signal transduction downstream of CCaMK. We identified CYCLOPS by positional cloning. CYCLOPS carries a functional nuclear localization signal and a predicted coiled-coil domain. We observed colocalization and physical interaction between CCaMK and CYCLOPS in plant and yeast cell nuclei in the absence of symbiotic stimulation. Importantly, CYCLOPS is a phosphorylation substrate of CCaMK in vitro. Cyclops mutants of rice were impaired in AM, and rice CYCLOPS could restore symbiosis in Lotus cyclops mutants, indicating a functional conservation across angiosperms. Our results suggest that CYCLOPS forms an ancient, preassembled signal transduction complex with CCaMK that is specifically required for infection, whereas organogenesis likely requires additional yet-to-be identified CCaMK interactors or substrates. OsCCaMK|OsDMI3,CYCLOPS|OsIPD3 Sense and antisense OsDof12 transcripts in rice 2008 BMC Mol Biol State Key Laboratory of Plant Genomics & National Plant Gene Research Center (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Chaoyang District, Beijing 100101, PR China. djli@genetics.ac.cn BACKGROUND: Antisense transcription is a widespread phenomenon in plants and mammals. Our previous data on rice gene expression analysis by microarray indicated that the sense and antisense transcripts at the OsDof12 locus were co-expressed in leaves. In current study, we analyzed the expression patterns in detail and looked for the possible mechanism related to their expression patterns. RESULTS: OsDof12, being a single copy gene located on rice chromosome 3, encodes a predicted Dof protein of 440 amino acids with one intron of 945 bp. The antisense transcript, OsDofl2os, overlaps with both the exonic and intronic regions of OsDof12 and encodes a functionally unknown protein of 104 amino acids with no intron. The sense-antisense OsDof12 transcripts were co-expressed within the same tissues, and their expressions were not tissue-specific in general. At different developmental stages in rice, the OsDof12 and OsDof12os transcripts exhibited reciprocal expression patterns. Interestingly, the expression of both genes was significantly induced under drought treatment, and inhibited by dark treatment. In the ProOsDof12-GUS and ProOsDof12os-GUS transgenic rice plants, the expression profiles of GUS were consistent with those of the OsDof12 and OsDof12os transcripts, respectively. In addition, the analysis of cis-regulatory elements indicated that either of the two promoters contained 74 classes of cis-regulatory elements predicted, of which the two promoter regions shared 53 classes. CONCLUSION: Based on the expression profiles of OsDof12 and OsDof12os, the expression patterns of GUS in the ProOsDof12-GUS and ProOsDof12os-GUS transgenic rice plants and the predicted common cis-regulatory elements shared by the two promoters, we suggest that the co-expression patterns of OsDof12 and OsDof12os might be attributed to the basically common nature of the two promoters. OsDof12 A novel nuclear-localized CCCH-type zinc finger protein, OsDOS, is involved in delaying leaf senescence in rice 2006 Plant Physiol Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences and National Centre for Plant Gene Research, Beijing 100080, China. Leaf senescence is a developmentally programmed degeneration process, which is fine tuned by a complex regulatory network for plant fitness. However, molecular regulation of leaf senescence is poorly understood, especially in rice (Oryza sativa), an important staple crop for more than half of the world population. Here, we report a novel nuclear-localized CCCH-type zinc finger protein, Oryza sativa delay of the onset of senescence (OsDOS), involved in delaying leaf senescence in rice. The expression of OsDOS was down-regulated during natural leaf senescence, panicle development, and pollination, although its transcripts were accumulated in various organs. RNAi knockdown of OsDOS caused an accelerated age-dependent leaf senescence, whereas its overexpression produced a marked delay of leaf senescence, suggesting that it acts as a negative regulator for leaf senescence. A genome-wide expression analysis further confirmed its negative regulation for leaf senescence and revealed that, in particular, the jasmonate (JA) pathway was found to be hyperactive in the OsDOS RNAi transgenic lines but impaired in the OsDOS overexpressing transgenic lines, indicating that this pathway is likely involved in the OsDOS-mediated delaying of leaf senescence. Furthermore, methyl JA treatments of both seeds and detached leaves from the RNAi and the overexpressing transgenic lines showed hyper- and hyporesponses, respectively, consistent with the negative regulation of the JA pathway by OsDOS. Together, these results indicate that OsDOS is a novel nuclear protein that delays leaf senescence likely, at least in part, by integrating developmental cues to the JA pathway. OsDOS A rice gene of de novo origin negatively regulates pathogen-induced defense response 2009 PLoS One National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China. How defense genes originated with the evolution of their specific pathogen-responsive traits remains an important problem. It is generally known that a form of duplication can generate new genes, suggesting that a new gene usually evolves from an ancestral gene. However, we show that a new defense gene in plants may evolve by de novo origination, resulting in sophisticated disease-resistant functions in rice. Analyses of gene evolution showed that this new gene, OsDR10, had homologs only in the closest relative, Leersia genus, but not other subfamilies of the grass family; therefore, it is a rice tribe-specific gene that may have originated de novo in the tribe. We further show that this gene may evolve a highly conservative rice-specific function that contributes to the regulation difference between rice and other plant species in response to pathogen infections. Biologic analyses including gene silencing, pathologic analysis, and mutant characterization by transformation showed that the OsDR10-suppressed plants enhanced resistance to a broad spectrum of Xanthomonas oryzae pv. oryzae strains, which cause bacterial blight disease. This enhanced disease resistance was accompanied by increased accumulation of endogenous salicylic acid (SA) and suppressed accumulation of endogenous jasmonic acid (JA) as well as modified expression of a subset of defense-responsive genes functioning both upstream and downstream of SA and JA. These data and analyses provide fresh insights into the new biologic and evolutionary processes of a de novo gene recruited rapidly. OsDR10,CHS,ICS1|OsVDAC1,PAD4|OsPAD4 Dual function of rice OsDR8 gene in disease resistance and thiamine accumulation 2006 Plant Mol Biol National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, 430070, China. The function of OsDR8, a rice disease resistance-responsive gene, was studied. Silencing of OsDR8 using an RNA interference approach resulted in phenotypic alteration of the plants. The transgenic plants with repressed expression of OsDR8 showed reduced resistance or susceptibility to Xanthomonas oryzae pv. oryzae and Magnaporthe grisea causing bacterial blight and blast, which are two of the most devastating diseases in rice worldwide, respectively. The putative product of OsDR8 was highly homologous to an enzyme involved in the biosynthesis of the thiazole precursor of thiamine. Transgenic plants showing repressed expression of OsDR8 and reduced resistance had significantly lower levels of thiamine than the control plants. Exogenous application of thiamine could complement the compromised defense of the OsDR8-silenced plants. The expression level of several defense-responsive genes including the earlier functional genes of defense transduction pathway, OsPOX and OsPAL, and the downstream genes of the pathway, OsPR1a, OsPR1b, OsPR4, OsPR5 and OsPR10, was also decreased in the OsDR8-silenced plants. These results suggest that the impact of OsDR8 on disease resistance in rice may be through the regulation of expression of other defense-responsive genes and the site of OsDR8 function is on the upstream of the signal transduction pathway. In addition, the accumulation of thiamine may be essential for bacterial blight resistance and blast resistance. OsDR8,OsPR5|Pir2|PR-5|PR5-1 OsDREB4 genes in rice encode AP2-containing proteins that bind specifically to the dehydration-responsive element 2005 J Integr Plant Biol Department of Plant Science, College of Biological Sciences, China Agricultural University, Beijing 100094, China Most dehydration-responsive element-binding (DREB) factors interact specifically with the dehydration-responsive element (DRE) and control the expression of many stress-inducible genes in Arabidopsis. In rice (Oryza sativa L. cv. Lansheng), we cloned three DREB homologs: OsDREBI-1, OsDREB4-1, and OsDREB4-2. The deduced amino acid sequences revealed that each protein contained a potential nuclear localization signal, an AP2 DNA-binding domain, and a possible acidic activation domain. The yeast one-hybrid assay indicated that both OsDREB4-1 and OsDREB4-2 proteins specifically bound to DRE and activated expression of the dual reporter genes of histidine (HIS3) and galactosidase (LacZ). In rice seedlings, expression of OsDREB4-1 was induced by dehydration and high salt, whereas OsDREBl-l and OsDREB4-2 were expressed constitutively. Under normal growth conditions, OsDREBI-1 was expressed strongly in the leaf, sheath, and spike, was expressed relatively weak in the stem and only faintly expressed in the roots, whereas expression of transcripts of OsDREB4-1 and OsDREB4-2 was higher in the roots, stem, and spike, lower in the leaf, and undetectable in the sheath. Together, these results imply that expression of the OsDREB genes could be controlled by specific aspects of differentiation or development. Thus, OsDREB4-1 could function as a trans-acting factor in the DRE/DREB regulated stress-responsive pathway. OsDREB1-1|CR350,OsDREB4-1|CR223,OsDREB4-2|CR250 Isolation, optimization, and functional analysis of the cDNA encoding transcription factor OsDREB1B in Oryza Sativa L 2006 Molecular Breeding College of Bioscience and Biotechnology, Yangzhou University, 88 Daxue Road, Yangzhou, 225009, The Peoples’s Republic of China A previous study had indicated that the transcription factors DREB/CBF (DRE-binding protein/C-repeat binding factor) play important roles in the expression of many stress inducible genes under cold-temperature, dehydration and high-salt conditions. In this study, we have isolated a cDNA clone that encoded a DRE-binding protein from rice cDNA library using the yeast one-hybrid system with DRE cis-acting element in the promoter region of rd29A gene as bait. Sequence analysis of the deduced amino acid sequence showed this protein was a putative AP2/EREBP transcription factor with a conserved AP2/EREBP domain and a potential nuclear localization signal (NLS). Expression pattern studies of this DRE-binding protein revealed that this gene was not only strongly induced by cold-temperature as reported by previous study but also induced by high-temperature as well. For the purpose of analyzing this gene conveniently, we attempted to apply the codon optimization method to reconstruct the gene of transcription factor in plants. A new sequence having decreasing GC contents, secondary structures, optimized codons and identical amino acid sequence with native gene was synthesized, which named OsDREB1BI, and then this optimized gene was transformed into Arabidopsis thaliana cv. Columbia by floral dip method. Results indicated that the OsDREB1BI gene was over-expressed in transgenic plants under cold and high-temperature, meanwhile, those transgenic plants also revealed freezing and heat tolerance. These might lay a strong foundation for exploiting the freezing and heat tolerance of rice and other species. OsDREB1B Seven zinc-finger transcription factors are novel regulators of the stress responsive gene OsDREB1B 2012 J Exp Bot Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa and Instituto de Biologia Experimental e Tecnologica, Oeiras, Portugal. Plants have evolved several mechanisms in order to cope with adverse environmental conditions. The transcription factors (TFs) belonging to the DREB1/CBF subfamily have been described as major regulators of the plant responses to different abiotic stresses. This study focused on the rice gene OsDREB1B, initially described as highly and specifically induced by cold. However, here it is shown that OsDREB1B is not only induced by low temperatures, but also by drought and mechanical stress. In order to identify novel TFs that bind to its promoter, a yeast one-hybrid system was used to screen a cold-induced cDNA expression library. Thereby seven novel Zn-finger TFs were identified that bind to the promoter of OsDREB1B. Among them, there were four Zn-finger homeodomain (ZF-HD) and three C(2)H(2)-type Zn-finger TFs. Gene expression studies showed that these TFs are differentially regulated at transcriptional level by different abiotic stress conditions, which is illustrative of the crosstalk between stress signalling pathways. Protein-protein interaction studies revealed the formation of homo- and heterodimers among the ZF-HD TFs identified, but not for the C(2)H(2)-type. Using a transactivation assay in Arabidopsis protoplasts, all the TFs identified repressed the expression of the reporter gene, driven by the promoter of OsDREB1B. This assay also showed that the dimerization observed between the ZF-HD TFs may play a role on their transactivation activity. The results here presented suggest a prominent role of Zn-finger TFs in the regulation of OsDREB1B. OsDREB1B OsDREB genes in rice,Oryza sativaL., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression 2003 The Plant Journal Biological Resources Division, Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan. The transcription factors DREBs/CBFs specifically interact with the dehydration-responsive element/C-repeat (DRE/CRT) cis-acting element (core motif: G/ACCGAC) and control the expression of many stress-inducible genes in Arabidopsis. In rice, we isolated five cDNAs for DREB homologs: OsDREB1A, OsDREB1B, OsDREB1C, OsDREB1D, and OsDREB2A. Expression of OsDREB1A and OsDREB1B was induced by cold, whereas expression of OsDREB2A was induced by dehydration and high-salt stresses. The OsDREB1A and OsDREB2A proteins specifically bound to DRE and activated the transcription of the GUS reporter gene driven by DRE in rice protoplasts. Over-expression of OsDREB1A in transgenic Arabidopsis induced over-expression of target stress-inducible genes of Arabidopsis DREB1A resulting in plants with higher tolerance to drought, high-salt, and freezing stresses. This indicated that OsDREB1A has functional similarity to DREB1A. However, in microarray and RNA blot analyses, some stress-inducible target genes of the DREB1A proteins that have only ACCGAC as DRE were not over-expressed in the OsDREB1A transgenic Arabidopsis. The OsDREB1A protein bound to GCCGAC more preferentially than to ACCGAC whereas the DREB1A proteins bound to both GCCGAC and ACCGAC efficiently. The structures of DREB1-type ERF/AP2 domains in monocots are closely related to each other as compared with that in the dicots. OsDREB1A is potentially useful for producing transgenic monocots that are tolerant to drought, high-salt, and/or cold stresses. OsDREB1B,OsDREB1D,OsDREB2A,OsDREB1A|OsDREBL Colinearity and similar expression pattern of rice DREB1s reveal their functional conservation in the cold-responsive pathway 2012 PLoS One Key Laboratory of Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, China. The clustered genes C-repeat (CRT) binding factor (CBF)1/dehydration-responsive element binding protein (DREB)1B, CBF2/DREB1C, and CBF3/DREB1A play a central role in cold acclimation and facilitate plant resistance to freezing in Arabidopsis thaliana. Rice (Oryza sativa L.) is very sensitive to low temperatures; enhancing the cold stress tolerance of rice is a key challenge to increasing its yield. In this study, we demonstrate chilling acclimation, a phenomenon similar to Arabidopsis cold acclimation, in rice. To determine whether rice CBF/DREB1 genes participate in this cold-responsive pathway, all putative homologs of Arabidopsis DREB1 genes were filtered from the complete rice genome through a BLASTP search, followed by phylogenetic, colinearity and expression analysis. We thereby identified 10 rice genes as putative DREB1 homologs: nine of these were located in rice genomic regions with some colinearity to the Arabidopsis CBF1-CBF4 region. Expression profiling revealed that six of these genes (Os01g73770, Os02g45450, Os04g48350, Os06g03670, Os09g35010, and Os09g35030) were similarly expressed in response to chilling acclimation and cold stress and were co-expressed with genes involved in cold signalling, suggesting that these DREB1 homologs may be involved in the cold response in rice. The results presented here serve as a prelude towards understanding the function of rice homologs of DREB1 genes in cold-sensitive crops. OsDREB1B,OsDREB1D,OsDREB1F|RCBF2,OsDREB1A|OsDREBL Rice DREB1B promoter shows distinct stress-specific responses, and the overexpression of cDNA in tobacco confers improved abiotic and biotic stress tolerance 2008 Plant Mol Biol Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, 500046, India. CBF/DREB (C-repeat binding factor/dehydration responsive element binding factor) family of transcription factors in plants is reported to be associated with regulation of gene expression under stress conditions. Here, we report the functional characterization of a DREB transcription factor, DREB1B gene from rice (Oryza sativa ssp. indica). The OsDREB1B gene was differentially regulated at the transcriptional level by osmotic stress, oxidative stress, salicylic acid, ABA, and cold. A 745 bp promoter region of OsDREB1B cDNA was fused to the beta-glucuronidase (GUS) gene and introduced via Agrobacterium tumifaciens into the genome of Arabidopsis. Histochemical analysis of GUS expression in T(2) transgenic Arabidopsis plants indicated that OsDREB1B shows stress-specific induction pattern in response to a variety of stresses like mannitol, NaCl, PEG, methyl viologen, cold, ABA, and salicylic acid. Leaf-order-dependent induction pattern of the promoter was observed in response to both cold and ABA stresses. Further, OsDREB1B cDNA was introduced into tobacco plants under the control of CaMV35S promoter to investigate the role of DREB1B product in plant stress response. Transgenic tobacco plants have shown improved seed germination, root growth, membrane stability, and 2, 2-diphenyl-1-pycrilhydrazil hydrate (DPPH) free radical scavenging activity under inhibitory concentrations of mannitol. Importantly, transgenic plants accumulated higher fresh weight under long-term osmotic stress, and also have shown retention of more water than the wild type during drought stress. Overexpression of OsDREB1B in tobacco also improved the oxidative and freezing stress tolerance of transgenic plants. In addition, tobacco plants constitutively expressing OsDREB1B have shown decreased sensitivity to tobacco streak virus infection. Constitutive expression of OsDREB1B in tobacco also induced the expression of PR genes in transgenic plants. The data obtained provide strong in vivo evidence that OsDREB1B is involved in both abiotic and biotic stress responses, and confers broad-spectrum stress tolerance to transgenic plants. OsDREB1B Expression of a rice DREB1 gene, OsDREB1D, enhances cold and high-salt tolerance in transgenic Arabidopsis 2009 BMB Rep College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, China. OsDREB1D, a special DREB (dehydration responsive element binding protein) homologous gene, whose transcripts cannot be detected in rice (Oryza sativa L), either with or without stress treatments, was amplified from the rice genome DNA. The yeast one-hybrid assay revealed that OsDREB1D was able to form a complex with the dehydration responsive element/C-repeat motif. It can also bind with a sequence of LTRE (low temperature responsive element). To analyze the function of OsDREB1D, the gene was transformed and over-expressed in Arabidopsis thaliana cv. Columbia. Results indicated that the over-expression of OsDREB1D conferred cold and high-salt tolerance in transgenic plants, and that transgenic plants were also insensitive to ABA (abscisic acid). From these data, we deduced that this OsDREB1D gene functions similarly as other DREB transcription factors. The expression of OsDREB1D in rice may be controlled by a special mechanism for the redundancy of function. OsDREB1D Isolated and characterization of a cDNA encoding ethylene-responsive element binding protein (EREBP)/AP2-type protein, RCBF2, in Oryza sativa L 2007 Biotechnol Lett Department of Horticulture, Nanjing Agricultural University, Weigang, Nanjing, People's Republic of China. A transcription factor RCBF2 which interacts with C-repeat/DRE was isolated from Oryza sativa L. by a yeast one-hybrid method. Analysis of the deduced RCBF2 amino acid sequence revealed that RCBF2 contained a conserved ethylene-responsive element binding protein (EREBP)/AP2 domain of 59 amino acids and a potential nuclear localization sequence. RCBF2 showed a high level of homology with other CBF family members only in AP2 domain. Phylogenetic analysis showed that RCBF2 might be different from other eight DRE-binding proteins on evolutionary relationship. The semi-quantitative RT-PCR (s-Q RT-PCR) analysis indicated the expression of RCBF2 gene was induced by cold, dehydration and high-salinity, but not by abscisic acid, and the transcription of RCBF2 gene accumulated primarily in rice immature seeds, growing point and shoots. OsDREB1F|RCBF2 Overexpression of a rice OsDREB1F gene increases salt, drought, and low temperature tolerance in both Arabidopsis and rice 2008 Plant Mol Biol State Key Laboratory of Plant Genomics and National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China. DREB transcription factors play key roles in plant stress signalling transduction pathway, they can specifically bind to DRE/CRT element (G/ACCGAC) and activate the expression of many stress inducible genes. Here, a novel rice DREB transcription factor, OsDREB1F, was cloned and characterised via subtractive suppression hybridisation (SSH) from upland rice. Expression analysis revealed that OsDREB1F gene was induced by salt, drought, cold stresses, and also ABA application, but not by pathogen, wound, and H2O2. Subcellular localization results indicated that OsDREB1F localizes in nucleus. Yeast activity assay demonstrated that OsDREB1F gene encodes a transcription activator, and can specifically bind to DRE/CRT but not to ABRE element. Transgenic plants harbouring OsDREB1F gene led to enhanced tolerance to salt, drought, and low temperature in both rice and Arabidopsis. The further characterisation of OsDREB1F-overexpressing Arabidopsis showed that, besides activating the expression of COR genes which contain DRE/CRT element in their upstream promoter regions, the expression of rd29B and RAB18 genes were also activated, suggested that OsDREB1F may also participate in ABA-dependent pathway. OsDREB1F|RCBF2 Induced over-expression of the transcription factor OsDREB2A improves drought tolerance in rice 2011 Plant Physiol Biochem Hebei Key Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hebei Normal University, Shijiazhuang, Hebei 050016, China. The DREB and CBF transcription factors play a critical role in plant development and abiotic stress responses and, therefore, represent attractive targets for a molecular plant breeding approach. In this study, the rice OsDREB2A gene was isolated and expressed under the control of a stress-inducible promoter (4ABRC) to improve the abiotic stress tolerance of japonica rice variety TNG67. T(2) and T(3) transgenic lines over-expressing OsDREB2A were found to have improved survival rates under severe drought and salt stress conditions relative to non-transgenic rice plants or rice plants transformed with the empty vector control. OsDREB2A expression was found to be markedly induced by drought and ABA treatment. The results indicate that the induced over-expression of OsDREB2A driven by the 4ABRC promoter in engineered rice plants may protect cells during stress. OsDREB2A Functional analysis of a novel Cys2/His2-type zinc finger protein involved in salt tolerance in rice 2010 J Exp Bot National Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Agriculture, Nanjing Agricultural University, Nanjing, China. The Cys2/His2-type zinc finger proteins have been implicated in different cellular processes involved in plant development and stress responses. Through microarray analysis, a salt-responsive zinc finger protein gene ZFP179 was identified and subsequently cloned from rice seedlings. ZFP179 encodes a 17.95 kDa protein with two C2H2-type zinc finger motifs having transcriptional activation activity. The real-time RT-PCR analysis showed that ZFP179 was highly expressed in immature spikes, and markedly induced in the seedlings by NaCl, PEG 6000, and ABA treatments. Overexpression of ZFP179 in rice increased salt tolerance and the transgenic seedlings showed hypersensitivity to exogenous ABA. The increased levels of free proline and soluble sugars were observed in transgenic plants compared to wild-type plants under salt stress. The ZFP179 transgenic rice exhibited significantly increased tolerance to oxidative stress, the reactive oxygen species (ROS)-scavenging ability, and expression levels of a number of stress-related genes, including OsDREB2A, OsP5CS OsProT, and OsLea3 under salt stress. Our studies suggest that ZFP179 plays a crucial role in the plant response to salt stress, and is useful in developing transgenic crops with enhanced tolerance to salt stress. OsDREB2A,OsLEA3|OsLEA3-1,OsP5CS|OsP5CS1,OsProT|ProT,ZFP179 OsDREB2A, a Rice Transcription Factor, Significantly Affects Salt Tolerance in Transgenic Soybean 2013 PLoS One The Guangdong Subcenter of National Center for Soybean Improvement, State Key Laboratory of Agricultural and Biological Resources Protection and Utilization in Subtropics, College of Agriculture, South China Agricultural University, Guangzhou, China. The dehydration responsive element binding (DREB) transcription factors play an important role in regulating stress-related genes. OsDREB2A, a member of the DREBP subfamily of AP2/ERF transcription factors in rice (Oryza sativa), is involved in the abiotic stress response. OsDREB2A expression is induced by drought, low-temperature and salt stresses. Here, we report the ability of OsDREB2A to regulate high-salt response in transgenic soybean. Overexpressing OsDREB2A in soybeans enhanced salt tolerance by accumulating osmolytes, such as soluble sugars and free proline, and improving the expression levels of some stress-responsive transcription factors and key genes. The phenotypic characterization of transgenic soybean were significantly better than those of wild-type (WT). Electrophoresis mobility shift assay (EMSA) revealed that the OsDREB2A can bind to the DRE core element in vitro. These results indicate that OsDREB2A may participate in abiotic stress by directly binding with DRE element to regulate the expression of downstream genes. Overexpression of OsDREB2A in soybean might be used to improve tolerance to salt stress. OsDREB2A A R2R3-type MYB gene, OsMYB2, is involved in salt, cold, and dehydration tolerance in rice 2012 J Exp Bot State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, PR China. MYB-type transcription factors play a diverse role in plant development and response to abiotic stress. This study isolated a rice R2R3-type MYB gene, OsMYB2, and functionally characterized its role in tolerance to abiotic stress by generating transgenic rice plants with overexpressing and RNA interference OsMYB2. Expression of OsMYB2 was up-regulated by salt, cold, and dehydration stress. OsMYB2 was localized in the nucleus with transactivation activity. No difference in growth and development between the OsMYB2-overexpressing and wild-type plants was observed under normal growth conditions, but the OsMYB2-overexpressing plants were more tolerant to salt, cold, and dehydration stresses and more sensitive to abscisic acid than wild-type plants. The OsMYB2-overexpressing plants accumulated greater amounts of soluble sugars and proline than wild-type plants under salt stress. Overexpression of OsMYB2 enhanced up-regulation of genes encoding proline synthase and transporters. The OsMYB2-overexpressing plants accumulated less amounts of H(2)O(2) and malondialdehyde. The enhanced activities of antioxidant enzymes, including peroxidase, superoxide dismutase, and catalase, may underlie the lower H(2)O(2) contents in OsMYB2-overexpressing plants. There was greater up-regulation of stress-related genes, including OsLEA3, OsRab16A, and OsDREB2A, in the OsMYB2-overexpressing plants. Microarray analysis showed that expression of numerous genes involving diverse functions in stress response was altered in the OsMYB2-overexpressing plants. These findings suggest that OsMYB2 encodes a stress-responsive MYB transcription factor that plays a regulatory role in tolerance of rice to salt, cold, and dehydration stress. OsDREB2A,OsLEA3|OsLEA3-1,Osmyb2 Salt-responsive ERF1 regulates reactive oxygen species-dependent signaling during the initial response to salt stress in rice 2013 Plant Cell Institute of Biochemistry and Biology, University of Potsdam, 14476 Potsdam, Germany. Early detection of salt stress is vital for plant survival and growth. Still, the molecular processes controlling early salt stress perception and signaling are not fully understood. Here, we identified salt-responsive ERF1 (SERF1), a rice (Oryza sativa) transcription factor (TF) gene that shows a root-specific induction upon salt and hydrogen peroxide (H2O2) treatment. Loss of SERF1 impairs the salt-inducible expression of genes encoding members of a mitogen-activated protein kinase (MAPK) cascade and salt tolerance-mediating TFs. Furthermore, we show that SERF1-dependent genes are H2O2 responsive and demonstrate that SERF1 binds to the promoters of MAPK kinase kinase6 (MAP3K6), MAPK5, dehydration-responsive element bindinG2A (DREB2A), and zinc finger protein179 (ZFP179) in vitro and in vivo. SERF1 also directly induces its own gene expression. In addition, SERF1 is a phosphorylation target of MAPK5, resulting in enhanced transcriptional activity of SERF1 toward its direct target genes. In agreement, plants deficient for SERF1 are more sensitive to salt stress compared with the wild type, while constitutive overexpression of SERF1 improves salinity tolerance. We propose that SERF1 amplifies the reactive oxygen species-activated MAPK cascade signal during the initial phase of salt stress and translates the salt-induced signal into an appropriate expressional response resulting in salt tolerance. OsDREB2A,OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1,SERF1,ZFP179 Expression of OsDREB2A transcription factor confers enhanced dehydration and salt stress tolerance in rice (Oryza sativa L.) 2011 Biotechnol Lett Plant Molecular Biology Laboratory, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India. Stress responsive transcriptional regulation is an adaptive strategy of plants that alleviates the adverse effects of environmental stresses. The ectopic overexpression of Dehydration-Responsive Element Binding transcription factors (DREBs) either in homologous or in heterologous plants improved stress tolerance indicating the DRE/DREB regulon is conserved across plants. We developed 30 transgenic T(0) rice plants overexpressing OsDREB2A which were devoid of any growth penalty or phenotypic abnormalities during stressed or non-stressed conditions. Integration of T-DNA in the rice genome and stress inducible overexpression of OsDREB2A had occurred in these transgenic lines. Functional analyses of T(1)-3 and T(1)-10 lines revealed significant tolerance to osmotic, salt and dehydration stresses during simulated stress conditions with enhanced growth performance as compared to wild type. OsDREB2A, thus, confers stress tolerance in homologous rice system that failed in the heterologous Arabidopsis system earlier. OsDREB2A Comprehensive analysis of rice DREB2-type genes that encode transcription factors involved in the expression of abiotic stress-responsive genes 2010 Mol Genet Genomics Laboratory of Plant Molecular Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, 113-8657, Japan. DREB2s (dehydration-responsive element-binding protein 2s) are transcription factors that interact with a cis-acting DRE (dehydration-responsive element)/CRT (C-repeat) sequence and activate the expression of downstream genes involved in water- and heat-shock stress responses and tolerance in Arabidopsis thaliana. In this study, we performed a comprehensive analysis of all five DREB2-type genes in rice (OsDREB2 s: OsDREB2A, OsDREB2B, OsDREB2C, OsDREB2E and OsABI4) to determine which of them contribute to plant stress responses. We analysed the expression patterns of these genes under abiotic stress conditions, and we examined the subcellular localisation and transcriptional activation activity of their translational products in protoplasts. Only OsDREB2A and OsDREB2B showed abiotic stress-inducible gene expression. In addition, OsDREB2B showed nuclear specific localisation and the highest transactivation activity. OsDREB2B has functional and non-functional forms of its transcript similar to its orthologues in the grass family, and the functional form of its transcript was markedly increased during stress conditions. We analysed the splicing mechanism of OsDREB2B with transgenic rice that express the non-functional transcript and we found that the non-functional form is not a precursor of the functional form; thus, stress-inducible alternative splicing of pre-mRNA is an important mechanism for the regulation of OsDREB2B. Transgenic Arabidopsis plants overexpressing OsDREB2B showed enhanced expression of DREB2A target genes and improved drought and heat-shock stress tolerance. These results suggest that OsDREB2B is a key gene that encodes a stress-inducible DREB2-type transcription factor that functions in stress-responsive gene expression in rice. OsDREB2B An AP2/EREBP-type transcription-factor gene from rice is cold-inducible and encodes a nuclear-localized protein 2003 TAG Theoretical and Applied Genetics Plant Biotechnology Laboratory, Institute of Genetics and Development Biology, The Chinese Academy of Sciences, Beijing 100101, China. We cloned an AP2/EREBP gene by dot blotting and named it OsDREBL. Analysis of its deduced amino-acid sequence indicated that this protein had a potential nuclear-localization signal, a possible acidic-activation domain and an AP2 DNA binding domain. Northern analysis showed that the transcripts of OsDREBL accumulated rapidly (within 30 min) in response to low temperature, but not in response to ABA, NaCl and dehydration treatments. Southern analysis indicated the presence of a single-copy of the OsDREBL gene in the Oryza sativa genome. Our research also demonstrated that OsDREBL was localized to the nucleus but did not bind effectively to the C-repeat/dehydration responsive element (CRT/DRE). These results suggested that OsDREBL may function as a transcription factor in the cold-stress response, independent of the DREB signal-transduction pathway. OsDREB1A|OsDREBL Overexpression of a rice defense-related F-box protein gene OsDRF1 in tobacco improves disease resistance through potentiation of defense gene expression 2008 Physiol Plant State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University-Huajiachi Campus, Hangzhou, Zhejiang 310029, China. F-box proteins play important roles in plant growth/development and responses to environmental stimuli through targeting substrates into degradation machinery. A rice defense-related F-box protein gene, OsDRF1, was cloned and identified during a course of study aimed at elucidating the molecular basis of induced immunity in rice. OsDRF1 encodes a protein of 328 amino acids, containing a highly conserved F-box domain. Expression of OsDRF1 was induced upon treatment with benzothiadiazole (BTH), a chemical inducer of defense responses in rice. Moreover, in BTH-treated rice seedlings, expression of OsDRF1 was further induced by infection with Magnaporthe grisea, the rice blast fungus, compared with those in water-treated seedlings. OsDRF1 was also upregulated in rice seedlings after treatment with ABA. Overexpression of OsDRF1 in transgenic tobacco resulted in enhanced disease resistance against tomato mosaic virus (ToMV) and Pseudomonas syringae pv. tabaci and strengthened expression of defense-related genes after salicylic acid treatment or ToMV infection. Root elongation of the OsDRF1-overexpressing transgenic seedlings was significantly inhibited by ABA, indicating that overexpression of OsDRF1 resulted in increased ABA sensitivity. The results suggest that OsDRF1 plays a role in disease resistance via upregulating defense-related gene expression and that OsDRF1 may also be involved in the response to ABA. OsDRF1 Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast 2013 Biochem Biophys Res Commun Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024, PR China. pangjs542@nenu.edu.cn DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5'-CCGG-3' containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92-9.12%, and 2.88-6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA methyltransferase gene with conserved activity in both prokaryotic and eukaryotic non-host species. OsDRM2 De novo methyltransferase, OsDRM2, interacts with the ATP-dependent RNA helicase, OseIF4A, in rice 2013 J Mol Biol University School of Biotechnology, Guru Gobind Singh Indraprastha University, Sector 16C, Dwarka, New Delhi 110078, India. Domains rearranged methyltransferases (DRMs) are the de novo methyltransferases that regulate cytosine methylation in plants in a manner similar to the animal de novo methyltransferases, DNMT3a and DNMT3b. These enzymes catalyze the establishment of new methylation patterns and are guided to target sites by small RNAs through the process of RNA-directed DNA methylation (RdDM). In the current accepted view for RdDM, intricate interactions among transcription factors/chromatin modifying proteins and the large subunits of plant-specific polymerases, Pol IV and Pol V, regulate the 24-nt small interfering RNA guided de novo methylation of cytosines. The RNA-induced silencing complex assembled on Pol-V-transcribed non-coding RNA finally facilitates the recruitment of DRM2 by unknown mechanism/protein interactions to chromatin sites. In an attempt to determine the cellular proteins that specifically interact with DRM2, a yeast two-hybrid screen was performed using young rice panicles. We report that rice DRM2 interacts with the ATP-dependent RNA helicase, eIF4A. Direct interaction between the two proteins is demonstrated in vivo by bimolecular fluorescence complementation method and in vitro by histidine-pull-down assays. Deletion analysis reveals that interaction between OsDRM2 and OseIF4A is specifically mediated through ubiquitin-associated domain of OsDRM2 while, both domains 1 and 2 of OseIF4A are critical for mediating strong interaction with OsDRM2 in vivo. Interaction between Arabidopsis eIF4AI and eIF4AII with OsDRM2 and nuclear localization of these complexes suggests possible conservation of functional interaction between de novo methyltransferases and the translation initiation factor, eIF4A, in RdDM across plant species. OsDRM2,OseIF4A Targeted disruption of an orthologue of DOMAINS REARRANGED METHYLASE 2, OsDRM2, impairs the growth of rice plants by abnormal DNA methylation 2012 Plant J National Institute for Basic Biology, Okazaki 444-8585, Japan. moritoh@nips.ac.jp Recent methylome analyses of the entire Arabidopsis thaliana genome using various mutants have provided detailed information about the DNA methylation pattern and its function. However, information about DNA methylation in other plants is limited, partly because of the lack of mutants. To study DNA methylation in rice (Oryza sativa) we applied homologous recombination-mediated gene targeting to generate targeted disruptants of OsDRM2, a rice orthologue of DOMAINS REARRANGED METHYLASE 1 and 2 (DRM1/2), which encode DNA methyltransferases responsible for de novo and non-CG methylation in Arabidopsis. Whereas Arabidopsis drm1 drm2 double mutants showed no morphological alterations, targeted disruptants of rice OsDRM2 displayed pleiotropic developmental phenotypes in both vegetative and reproductive stages, including growth defects, semi-dwarfed stature, reductions in tiller number, delayed heading or no heading, abnormal panicle and spikelet morphology, and complete sterility. In these osdrm2 disruptants, a 13.9% decrease in 5-methylcytosine was observed by HPLC analysis. The CG and non-CG methylation levels were reduced in RIRE7/CRR1 retrotransposons, and in 5S rDNA repeats. Associated transcriptional activation was detected in RIRE7/CRR1. Furthermore, de novo methylation by an RNA-directed DNA methylation (RdDM) process involving transgene-derived exogenous small interfering RNA (siRNA) was deficient in osdrm2-disrupted cells. Impaired growth and abnormal DNA methylation of osdrm2 disruptants were restored by the complementation of wild-type OsDRM2 cDNA. Our results suggest that OsDRM2 is responsible for de novo, CG and non-CG methylation in rice genomic sequences, and that DNA methylation regulated by OsDRM2 is essential for proper rice development in both vegetative and reproductive stages. OsDRM2 A RING finger E3 ligase gene, Oryza sativa Delayed Seed Germination 1 (OsDSG1), controls seed germination and stress responses in rice 2010 Plant Mol Biol Crop Biotech Institute & Department of Plant Molecular Systems Biotechnology, Kyung Hee University, Yongin, 446-701, Republic of Korea. Seed germination is an important character for plant growth and seed quality. We identified a rice mutant that was delayed in its germination. There, T-DNA was inserted into Oryza sativa Delayed Seed Germination 1 (OsDSG1), causing a recessive null mutation. Overexpression of the gene enhanced seed germination. OsDSG1 is most similar to Arabidopsis AIP2, an E3 ligase targeting ABI3.Yeast two-hybrid experiments showed that our OsDSG1 binds to OsABI3, indicating that OsDSG1 is a rice ortholog of AIP2. Self-ubiquitination assay indicated that bacterially expressed OsDSG1 protein has E3 ubiquitin ligase activity. Real-time PCR analysis revealed that OsDSG1 was expressed in leaves and roots, and strongly in developing seeds. In addition to the delayed-germination phenotype, mutant plants were shorter and had greater tolerance to high-salt and drought stresses. In the osdsg1 mutant, transcript levels of ABA signaling genes and ABA responsive genes were significantly increased. By contrast, expressions of OsGAMYB and its downstream genes that encode hydrolytic enzymes were markedly reduced. These observations support that OsDSG1 is a major regulator of ABA signaling in germinating seeds. Finally, we observed that the germination rates of various rice cultivars depended upon the transcript levels of OsDSG1 and other ABA-signaling genes. OsDSG1,OsMYBGA|OsGAMYB Rice Gene OsDSR-1 Promotes Lateral Root Development in Arabidopsis Under High-Potassium Conditions 2011 Journal of Plant Biology Laboratory for Agro-ecological Process in Subtropical Region, Institute of Subtropical Agriculture, CAS, Changsha, Hunan, 410125, People’s Republic of China Rice gene Oryza sativa Drought Stress Response-1 (OsDSR-1) was one of the genes identified to be responsive to drought stress in the panicle of rice at booting and heading stages by both microarray and quantitative real-time PCR analyses. OsDSR-1 encodes a putative calcium-binding protein, and its overexpression in Arabidopsis rendered transgenic plants to produce much shorter lateral roots (LRs) than wild-type (WT) plants in the medium supplemented with abscisic acid (ABA), suggesting that OsDSR-1 may act as a positive regulator during the process of ABA inhibition of LR development. No significant difference was observed in the total LR length between WT and transgenic plants in the media with the increase of only osmotic stress caused by NaCl, LiCl, and mannitol, while transgenic Arabidopsis seedlings appeared to produce larger root systems with longer total LR lengths under high-potassium conditions than WT seedlings. Further analysis showed that external Ca2+ was required for the production of larger root systems, indicating that the promotion by OsDSR-1 of the LR development of transgenic Arabidopsis seemed to occur in a Ca2+-dependent manner under high-potassium conditions. We propose that OsDSR-1 may function as a calcium sensor of the signal transduction pathway controlling the LR development under high-potassium conditions. OsDSR-1 Molecular cloning and characterization of a cDNA encodingent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin biosynthetic enzyme, from suspension-cultured rice cells treated with a chitin elicitor 2004 The Plant Journal Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. We have isolated and characterized a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspension-cultured rice cells treated with a chitin elicitor. OsDTC1 functions as ent-cassa-12,15-diene synthase, which is considered to play a key role in the biosynthesis of (-)-phytocassanes recently isolated as rice diterpenoid phytoalexins. The expression of OsDTC1 mRNA was also confirmed in ultraviolet (UV)-irradiated rice leaves. In addition, we identified ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (-)-phytocassanes, as an endogenous compound in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves. The OsDTC1 cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of the biosynthesis of (-)-phytocassanes in rice. OsDTC1|OsKS3 Identification of syn-pimara-7,15-diene synthase reveals functional clustering of terpene synthases involved in rice phytoalexin/allelochemical biosynthesis 2004 Plant Physiol Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA. Rice (Oryza sativa) produces momilactone diterpenoids as both phytoalexins and allelochemicals. Accordingly, the committed step in biosynthesis of these natural products is catalyzed by the class I terpene synthase that converts syn-copalyl diphosphate to the corresponding polycyclic hydrocarbon intermediate syn-pimara-7,15-diene. Here, a functional genomics approach was utilized to identify a syn-copalyl diphosphate specific 9beta-pimara-7,15-diene synthase (OsDTS2). To our knowledge, this is the first identified terpene synthase with this particular substrate stereoselectivity and, by comparison with the previously described and closely related ent-copalyl diphosphate specific cassa-12,15-diene synthase (OsDTC1), provides a model system for investigating the enzymatic determinants underlying the observed difference in substrate specificity. Further, OsDTS2 mRNA in leaves is up-regulated by conditions that stimulate phytoalexin biosynthesis but is constitutively expressed in roots, where momilactones are constantly synthesized as allelochemicals. Therefore, transcription of OsDTS2 seems to be an important regulatory point for controlling production of these defensive compounds. Finally, the gene identified here as OsDTS2 has previously been mapped at 14.3 cM on chromosome 4. The class II terpene synthase producing syn-copalyl diphosphate from the universal diterpenoid precursor geranylgeranyl diphosphate was also mapped to this same region. These genes catalyze sequential cyclization steps in momilactone biosynthesis and seem to have been evolutionarily coupled by physical linkage and resulting cosegregation. Further, the observed correlation between physical proximity and common metabolic function indicates that other such class I and class II terpene synthase gene clusters may similarly catalyze consecutive reactions in shared biosynthetic pathways. OsDTC1|OsKS3,OsDTS2|OsKSL4|OsKS4 Rice DUR3 mediates high-affinity urea transport and plays an effective role in improvement of urea acquisition and utilization when expressed in Arabidopsis 2012 New Phytol Department of Plant Nutrition, Key Laboratory of Plant and Soil Interactions of MEoC, College of Resources and Environmental Sciences, China Agricultural University, Beijing, China. * Despite the great agricultural and ecological importance of efficient use of urea-containing nitrogen fertilizers by crops, molecular and physiological identities of urea transport in higher plants have been investigated only in Arabidopsis. * We performed short-time urea-influx assays which have identified a low-affinity and high-affinity (K(m) of 7.55 muM) transport system for urea-uptake by rice roots (Oryza sativa). * A high-affinity urea transporter OsDUR3 from rice was functionally characterized here for the first time among crops. OsDUR3 encodes an integral membrane-protein with 721 amino acid residues and 15 predicted transmembrane domains. Heterologous expression demonstrated that OsDUR3 restored yeast dur3-mutant growth on urea and facilitated urea import with a K(m) of c. 10 muM in Xenopus oocytes. * Quantitative reverse-transcription polymerase chain reaction (qPCR) analysis revealed upregulation of OsDUR3 in rice roots under nitrogen-deficiency and urea-resupply after nitrogen-starvation. Importantly, overexpression of OsDUR3 complemented the Arabidopsis atdur3-1 mutant, improving growth on low urea and increasing root urea-uptake markedly. Together with its plasma membrane localization detected by green fluorescent protein (GFP)-tagging and with findings that disruption of OsDUR3 by T-DNA reduces rice growth on urea and urea uptake, we suggest that OsDUR3 is an active urea transporter that plays a significant role in effective urea acquisition and utilisation in rice. OsDUR3 Divinyl chlorophyll(ide) a can be converted to monovinyl chlorophyll(ide) a by a divinyl reductase in rice 2010 Plant Physiol Rice Research Institute , Sichuan Agricultural University, Chengdu 611130, China. 3,8-Divinyl (proto)chlorophyll(ide) a 8-vinyl reductase (DVR) catalyzes the reduction of 8-vinyl group on the tetrapyrrole to an ethyl group, which is indispensable for monovinyl chlorophyll (Chl) synthesis. So far, three 8-vinyl reductase genes (DVR, bciA, and slr1923) have been characterized from Arabidopsis (Arabidopsis thaliana), Chlorobium tepidum, and Synechocystis sp. PCC6803. However, no 8-vinyl reductase gene has yet been identified in monocotyledonous plants. In this study, we isolated a spontaneous mutant, 824ys, in rice (Oryza sativa). The mutant exhibited a yellow-green leaf phenotype, reduced Chl level, arrested chloroplast development, and retarded growth rate. The phenotype of the 824ys mutant was caused by a recessive mutation in a nuclear gene on the short arm of rice chromosome 3. Map-based cloning of this mutant resulted in the identification of a gene (Os03g22780) showing sequence similarity with the Arabidopsis DVR gene (AT5G18660). In the 824ys mutant, nine nucleotides were deleted at residues 952 to 960 in the open reading frame, resulting in a deletion of three amino acid residues in the encoded product. High-performance liquid chromatography analysis of Chls indicated the mutant accumulates only divinyl Chl a and b. A recombinant protein encoded by Os03g22780 was expressed in Escherichia coli and found to catalyze the conversion of divinyl chlorophyll(ide) a to monovinyl chlorophyll(ide) a. Therefore, it has been confirmed that Os03g22780, renamed as OsDVR, encodes a functional DVR in rice. Based upon these results, we succeeded to identify an 8-vinyl reductase gene in monocotyledonous plants and, more importantly, confirmed the DVR activity to convert divinyl Chl a to monovinyl Chl a. OsDVR One divinyl reductase reduces the 8-vinyl groups in various intermediates of chlorophyll biosynthesis in a given higher plant species, but the isozyme differs between species 2013 Plant Physiol Rice Research Institute, Sichuan Agricultural University, Chengdu 611130, China. Divinyl reductase (DVR) converts 8-vinyl groups on various chlorophyll intermediates to ethyl groups, which is indispensable for chlorophyll biosynthesis. To date, five DVR activities have been detected, but adequate evidence of enzymatic assays using purified or recombinant DVR proteins has not been demonstrated, and it is unclear whether one or multiple enzymes catalyze these activities. In this study, we systematically carried out enzymatic assays using four recombinant DVR proteins and five divinyl substrates and then investigated the in vivo accumulation of various chlorophyll intermediates in rice (Oryza sativa), maize (Zea mays), and cucumber (Cucumis sativus). The results demonstrated that both rice and maize DVR proteins can convert all of the five divinyl substrates to corresponding monovinyl compounds, while both cucumber and Arabidopsis (Arabidopsis thaliana) DVR proteins can convert three of them. Meanwhile, the OsDVR (Os03g22780)-inactivated 824ys mutant of rice exclusively accumulated divinyl chlorophylls in its various organs during different developmental stages. Collectively, we conclude that a single DVR with broad substrate specificity is responsible for reducing the 8-vinyl groups of various chlorophyll intermediates in higher plants, but DVR proteins from different species have diverse and differing substrate preferences, although they are homologous. OsDVR Characterization of plant eukaryotic translation initiation factor 6 (eIF6) genes: The essential role in embryogenesis and their differential expression in Arabidopsis and rice 2010 Biochem Biophys Res Commun Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan. Eukaryotic translation initiation factor 6 (eIF6) is an essential component of ribosome biogenesis. In our present study, we characterize plant eIF6 genes for the first time. Although a single gene encodes eIF6 in yeast and animals, two genes were found to encode proteins homologous to animal and yeast eIF6 in Arabidopsis and rice, denoted At-eIF6;1 and At-eIF6;2, and Os-eIF6;1 and Os-eIF6;2, respectively. Analysis of the yeast eif6 (tif6) mutant suggested that plant eIF6, at least in the case of At-eIF6;1, can complement the essential function of eIF6 in yeast. Evidence for the essential role of eIF6 in plants was also provided by the embryonic-lethal phenotype of the at-eif6;1 mutant. In contrast, At-eIF6;2 appears not to be essential due to its very low expression level and the normal growth phenotype of the eif6;2 mutants. Consistent with the putative role of plant eIF6 in ribosome biogenesis, At-eIF6;1 is predominately expressed in tissues where cell division actively proceeds under the control of intronic cis-regulatory elements. On the other hand, both Os-eIF6;1 and Os-eIF6;2 are probably active genes because they are expressed at significant expression levels. Interestingly, the supply of ammonium nitrate as a plant nutrient was found to induce specifically the expression of Os-eIF6;2. Our present findings indicate that the eIF6 genes have differently evolved in plant and animal kingdoms and also in distinct plant species. Os-eIF6;1,Os-eIF6;2 Cloning of a cDNA encoding an ETR2-like protein (Os-ERL1) from deep water rice (Oryza sativa L.) and increase in its mRNA level by submergence, ethylene, and gibberellin treatments 2004 J Exp Bot Division of Biological Resource Science, Graduate School of Agricultural Science, Tohoku University, Kawatabi, Naruko Miyagi 989-6711, Japan. watanabe@bios.tohoku.ac.jp A cDNA from deep water rice treated with ethylene, encoding an ethylene receptor homologous to Arabidopsis thaliana ETR2 and EIN4, was isolated using differential display and RACE techniques. The cDNA (2880 bp), corresponding to the Os-ERL1 gene (Oryza sativa ETHYLENE RESPONSE 2 like 1; GenBank accession number AB107219), contained an open reading frame of 2289 bp coding for 763 amino acids. The protein Os-ERL1 has 50% and 52% similarity to Arabidopsis ETR2 and EIN4, respectively. The Os-ERL1 gene was up-regulated by flooding, and by treatment with ethylene and gibberellin. These results suggest that deep water rice responds to flooding by increasing the number of its ethylene receptors. ETR2|Os-ERL1 E2F sites that can interact with E2F proteins cloned from rice are required for meristematic tissue-specific expression of rice and tobacco proliferating cell nuclear antigen promoters 2002 The Plant Journal Molecular Genetics Department, National Institute of Agrobiological Sciences, Core Research of Science and Technology (CREST), Japan Science and Technology Corporation, Tsukuba, Ibaraki 305-8602, Japan. Plants have recently been found to have E2F-like and Rb-like proteins, regulators responsible for the G1(G0)-S phase transition of the cell cycle in animals. Here we show that E2F is involved in transcription of plant genes for proliferating cell nuclear antigen (PCNA), which is required for DNA replication. Potential E2F binding sites found in the rice PCNA promoters mediated transcriptional activation in actively dividing cells and tissues of tobacco, but not transcriptional repression in terminally differentiated tissues, as also observed for the PCF binding sites previously found in the rice promoter. Similar results were obtained from analyses for a PCNA promoter isolated from tobacco, which contained two E2F-like sites, each with a different degree of contribution to the promoter activation. These E2F-like sites except for a rice site were indeed bound specifically by recombinant proteins of rice E2F, OsE2F1 and OsE2F2, and complexes of OsE2F1 with Arabidopsis DP proteins. Furthermore, OsE2F1 had the ability to transactivate an E2F-reporter gene containing the tobacco E2F site on co-expression with an Arabidopsis DP, and the transactivation was greatly enhanced by tagging a canonical nuclear localization signal to OsE2F1, suggesting a nuclear import-mediated regulation of the OsE2F1 function. In addition, we found that a large number of replication- and mismatch repair-associated genes in Arabidopsis contain E2F binding sequences conserved in their predicted promoter regions. OsE2F1,OsE2F2 Rice ethylene-response AP2/ERF factor OsEATB restricts internode elongation by down-regulating a gibberellin biosynthetic gene 2011 Plant Physiol State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China. Plant height is a decisive factor in plant architecture. Rice (Oryza sativa) plants have the potential for rapid internodal elongation, which determines plant height. A large body of physiological research has shown that ethylene and gibberellin are involved in this process. The APETALA2 (AP2)/Ethylene-Responsive Element Binding Factor (ERF) family of transcriptional factors is only present in the plant kingdom. This family has various developmental and physiological functions. A rice AP2/ERF gene, OsEATB (for ERF protein associated with tillering and panicle branching) was cloned from indica rice variety 9311. Bioinformatic analysis suggested that this ERF has a potential new function. Ectopic expression of OsEATB showed that the cross talk between ethylene and gibberellin, which is mediated by OsEATB, might underlie differences in rice internode elongation. Analyses of gene expression demonstrated that OsEATB restricts ethylene-induced enhancement of gibberellin responsiveness during the internode elongation process by down-regulating the gibberellin biosynthetic gene, ent-kaurene synthase A. Plant height is negatively correlated with tiller number, and higher yields are typically obtained from dwarf crops. OsEATB reduces rice plant height and panicle length at maturity, promoting the branching potential of both tillers and spikelets. These are useful traits for breeding high-yielding crops. OsEATB The OsEBP-89 gene of rice encodes a putative EREBP transcription factor and is temporally expressed in developing endosperm and intercalary meristem 2002 Plant Mol Biol National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology, Chinese Academy of Sciences, People's Republic of China. The AP2/EREBP transcription factors play important roles in plant development and in the responses of plants to biotic and abiotic stresses. All members of the EREBP subfamily described to date are from dicotyledonous plants. In this paper, we describe the cloning and characterization of a rice gene, OsEBP-89, encoding a protein 326 amino acids long with a typical EREBP domain; this is the first report of an EREBP transcription factor in a monocotyledonous plant. Except for the EREBP domain, the OsEBP-89 protein does not have substantial sequence similarities to other members of the subfamily. The DNA-binding activity of the EREBP domain was confirmed by electrophoretic mobility-shift assays. An activation domain rich in acidic amino acids was identified by using a yeast one-hybrid system. Two putative nuclear-localization signals were also identified. The results of northern blot hybridization experiments showed that the transcript of the OsEBP-89 gene accumulates primarily in immature seeds, roots, and leaves (low levels). More detailed information about the pattern of OsEBP-89 gene expression was obtained by histochemical studies of transgenic rice plants carrying an OsEBP-89 5'/GUS reporter gene. The reporter gene was expressed in the endosperm starting at 7 days after pollination and in the intercalary meristem of plants. Expression of OsEBP-89 was induced in roots of rice seedlings by treatment with ACC, NaCl, or 2,4-D. Two cis-acting elements, an endosperm motif and a primary PERE, are present upstream of the OsEBP-89 coding region and may be involved in regulating its expression. Collectively, these results suggest that the OsEBP-89 gene is a new member of the EREBP subfamily and may be involved in ethylene-dependent seed maturation and shoot development of rice. OsEBP-89 Expressional analysis of an EREBP transcription factor gene OsEBP-89 in rice 2004 Acta Biochim Biophys Sin (Shanghai) The State Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology Ecology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200032, China. OsEBP-89 gene encodes an ethylene responsive element binding protein (EREBP) transcription factor from rice (Oryza sativa). Northern blot analysis revealed that OsEBP-89 was expressed in root, stem, seeds, flowers and leaves of rice. Histochemical assay showed that GUS expressed mainly in phloem of vascular tissues of the root and stem transition region (RST), basal part of sheath roots, stem node and basal part of adventitious roots, also in endosperm of seeds in transgenic rice harboring OsEBP-89/GUS construct (pNSG). A sequence of region from C279 to C97 was found to play an important role for OsEBP-89 genes expression though promoter deletion assay. The possible function of OsEBP-89 gene was discussed. OsEBP-89 Identification and characterization of OsEBS, a gene involved in enhanced plant biomass and spikelet number in rice 2013 Plant Biotechnol J State Key Laboratory of Genetic Engineering, Institute of Genetics, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, China. Common wild rice (Oryza rufipogon Griff.) is an important genetic reservoir for rice improvement. We investigated a quantitative trait locus (QTL), qGP5-1, which is related to plant height, leaf size and panicle architecture, using a set of introgression lines of O. rufipogon in the background of the Indica cultivar Guichao2 (Oryza sativa L.). We cloned and characterized qGP5-1 and confirmed that the newly identified gene OsEBS (enhancing biomass and spikelet number) increased plant height, leaf size and spikelet number per panicle, leading to an increase in total grain yield per plant. Our results showed that the increased size of vegetative organs in OsEBS-expressed plants was enormously caused by increasing cell number. Sequence alignment showed that OsEBS protein contains a region with high similarity to the N-terminal conserved ATPase domain of Hsp70, but it lacks the C-terminal regions of the peptide-binding domain and the C-terminal lid. More results indicated that OsEBS gene did not have typical characteristics of Hsp70 in this study. Furthermore, Arabidopsis (Arabidopsis thaliana) transformed with OsEBS showed a similar phenotype to OsEBS-transgenic rice, indicating a conserved function of OsEBS among plant species. Together, we report the cloning and characterization of OsEBS, a new QTL that controls rice biomass and spikelet number, through map-based cloning, and it may have utility in improving grain yield in rice. OsEBS Homologous expression of gamma-glutamylcysteine synthetase increases grain yield and tolerance of transgenic rice plants to environmental stresses 2013 J Plant Physiol Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, South Korea. cyhmilkyway@hanmail.net Various environmental stresses induce reactive oxygen species (ROS), causing deleterious effects on plant cells. Glutathione (GSH), a critical antioxidant, is used to combat ROS. GSH is produced by gamma-glutamylcysteine synthetase (gamma-ECS) and glutathione synthetase (GS). To evaluate the functional roles of the Oryza sativa L. Japonica cv. Ilmi ECS (OsECS) gene, we generated transgenic rice plants overexpressing OsECS under the control of an inducible promoter (Rab21). When grown under saline conditions (100mM) for 4 weeks, 2-independent transgenic (TGR1 and TGR2) rice plants remained bright green in comparison to control wild-type (WT) rice plants. TGR1 and TGR2 rice plants also showed a higher GSH/GSSG ratio than did WT rice plants in the presence of 100mM NaCl, which led to enhanced redox homeostasis. TGR1 and TGR2 rice plants also showed lower ion leakage and higher chlorophyll-fluorescence when exposed to 10muM methyl viologen (MV). Furthermore, the TGR1 and TGR2 rice seeds had approximately 1.5-fold higher germination rates in the presence of 200mM salt. Under paddy field conditions, OsECS-overexpression in transgenic rice plants increased rice grain yield (TGW) and improved biomass. Overall, our results show that OsECS overexpression in transgenic rice increases tolerance and germination rate in the presence of abiotic stress by improving redox homeostasis via an enhanced GSH pool. Our findings suggest that increases in grain yield by OsECS overexpression could improve crop yields under natural environmental conditions. OsECS Molecular cloning and mRNA expression analysis of a novel rice ( L.) MAPK kinase kinase, , an ortholog of , reveal its role in defense/stress signalling pathways and development 2003 Biochem Biophys Res Commun Department of Molecular Biology, College of Natural Science, Sejong University, Seoul 143-747, Republic of Korea. Mitogen-activated protein kinase (MAPK) cascade(s) is important for plant defense/stress responses. Though MAPKs have been identified and characterized in rice (Oryza sativa L.), a monocot cereal crop research model, the first upstream component of the kinase cascade, namely MAPK kinase kinase (MAPKKK) has not yet been identified. Here we report the cloning of a novel rice gene encoding a MAPKKK, OsEDR1, designated based on its homology with the Arabidopsis MAPKKK, AtEDR1. OsEDR1, a single copy gene in the genome of rice, encodes a predicted protein with molecular mass of 113046.13 and a pI of 9.03. Using our established two-week-old rice seedling in vitro model system, we show that OsEDR1 has a constitutive expression in seedling leaves and is further up-regulated within 15 min upon wounding by cut, treatment with the global signals jasmonic acid (JA), salicylic acid (SA), ethylene (ethephon, ET), abscisic acid, and hydrogen peroxide. In addition, protein phosphatase inhibitors, fungal elicitor chitosan, drought, high salt and sugar, and heavy metals also dramatically induce its expression. Moreover, OsEDR1 expression was altered by co-application of JA, SA, and ET, and required de novo synthesized protein factor(s) in its transient regulation. Furthermore, using an in vivo system we also show that OsEDR1 responds to changes in temperature and environmental pollutants-ozone and sulfur dioxide. Finally, OsEDR1 expression varied significantly in vegetative and reproductive tissues. These results suggest a role for OsEDR1 in defense/stress signalling pathways and development. OsEDR1|OsACDR1 OsEDR1 negatively regulates rice bacterial resistance via activation of ethylene biosynthesis 2011 Plant Cell Environ National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Rice OsEDR1 is a sequence ortholog of Arabidopsis EDR1. However, its molecular function is unknown. We show here that OsEDR1-suppressing/knockout (KO) plants, which developed spontaneous lesions on the leaves, have enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo) causing bacterial blight disease. This resistance was associated with increased accumulation of salicylic acid (SA) and jasmonic acid (JA), induced expression of SA- and JA-related genes and suppressed accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC), the direct precursor of ethylene, and expression of ethylene-related genes. OsEDR1-KO plants also showed suppressed production of ethylene. Knockout of OsEDR1 suppressed the ACC synthase (ACS) gene family, which encodes the rate-limiting enzymes of ethylene biosynthesis by catalysing the formation of ACC. The lesion phenotype and enhanced bacterial resistance of the OsEDR1-KO plants was partly complemented by the treatment with ACC. ACC treatment was associated with decreased SA and JA biosynthesis in OsEDR1-KO plants. In contrast, aminoethoxyvinylglycine, the inhibitor of ethylene biosynthesis, promoted expression of SA and JA synthesis-related genes in OsEDR1-KO plants. These results suggest that ethylene is a negative signalling molecule in rice bacterial resistance. In the rice-Xoo interaction, OsEDR1 transcriptionally promotes the synthesis of ethylene that, in turn, suppresses SA- and JA-associated defence signalling. OsEDR1|OsACDR1 Negative regulation of defense responses in plants by a conserved MAPKK kinase 2001 Proc Natl Acad Sci U S A Department of Biology, Indiana University, Bloomington, IN 47405-3700, USA. The enhanced disease resistance 1 (edr1) mutation of Arabidopsis confers resistance to powdery mildew disease caused by the fungus Erysiphe cichoracearum. Resistance mediated by the edr1 mutation is correlated with induction of several defense responses, including host cell death. Double mutant analysis revealed that all edr1-associated phenotypes are suppressed by mutations that block salicylic acid (SA) perception (nim1) or reduce SA production (pad4 and eds1). The NahG transgene, which lowers endogenous SA levels, also suppressed edr1. In contrast, the ein2 mutation did not suppress edr1-mediated resistance and associated phenotypes, indicating that ethylene and jasmonic acid-induced responses are not required for edr1 resistance. The EDR1 gene was isolated by positional cloning and was found to encode a putative MAP kinase kinase kinase similar to CTR1, a negative regulator of ethylene responses in Arabidopsis. Taken together, these data suggest that EDR1 functions at the top of a MAP kinase cascade that negatively regulates SA-inducible defense responses. Putative orthologs of EDR1 are present in monocots such as rice and barley, indicating that EDR1 may regulate defense responses in a wide range of crop species. OsEDR1|OsACDR1 Rice OsACDR1 (Oryza sativa accelerated cell death and resistance 1) is a potential positive regulator of fungal disease resistance 2009 Mol Cells Department of Molecular Biology, College of Natural Science, Sejong University, Seoul 143-747, Korea. Rice Oryza sativa accelerated cell death and resistance 1 (OsACDR1) encodes a putative Raf-like mitogen-activated protein kinase kinase kinase (MAPKKK). We had previously reported upregulation of the OsACDR1 transcript by a range of environmental stimuli involved in eliciting defense-related pathways. Here we apply biochemical, gain and loss-of-function approaches to characterize OsACDR1 function in rice. The OsACDR1 protein showed autophosphorylation and possessed kinase activity. Rice plants overexpressing OsACDR1 exhibited spontaneous hypersensitive response (HR)-like lesions on leaves, upregulation of defense-related marker genes and accumulation of phenolic compounds and secondary metabolites (phytoalexins). These transgenic plants also acquired enhanced resistance to a fungal pathogen (Magnaporthe grisea) and showed inhibition of appressorial penetration on the leaf surface. In contrast, loss-offunction and RNA silenced OsACDR1 rice mutant plants showed downregulation of defense-related marker genes expressions and susceptibility to M. grisea. Furthermore, transient expression of an OsACDR1:GFP fusion protein in rice protoplast and onion epidermal cells revealed its localization to the nucleus. These results indicate that OsACDR1 plays an important role in the positive regulation of disease resistance in rice. OsEDR1|OsACDR1 Purification and characterization of a novel hypersensitive response-inducing elicitor from Magnaporthe oryzae that triggers defense response in rice 2012 PLoS One Key Laboratory of Integrated Pest Management in Crops, Ministry of Agriculture, Institute of Plant Protection, Chinses Academy of Agricultural Sciences, Beijing, China. BACKGROUND: Magnaporthe oryzae, the rice blast fungus, might secrete certain proteins related to plant-fungal pathogen interactions. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we report the purification, characterization, and gene cloning of a novel hypersensitive response-inducing protein elicitor (MoHrip1) secreted by M. oryzae. The protein fraction was purified and identified by de novo sequencing, and the sequence matched the genomic sequence of a putative protein from M. oryzae strain 70-15 (GenBank accession No. XP_366602.1). The elicitor-encoding gene mohrip1 was isolated; it consisted of a 429 bp cDNA, which encodes a polypeptide of 142 amino acids with a molecular weight of 14.322 kDa and a pI of 4.53. The deduced protein, MoHrip1, was expressed in E. coli. And the expression protein collected from bacterium also forms necrotic lesions in tobacco. MoHrip1 could induce the early events of the defense response, including hydrogen peroxide production, callose deposition, and alkalization of the extracellular medium, in tobacco. Moreover, MoHrip1-treated rice seedlings possessed significantly enhanced systemic resistance to M. oryzae compared to the control seedlings. The real-time PCR results indicated that the expression of some pathogenesis-related genes and genes involved in signal transduction could also be induced by MoHrip1. CONCLUSION/SIGNIFICANCE: The results demonstrate that MoHrip1 triggers defense responses in rice and could be used for controlling rice blast disease. OsEDS1 OsWRKY13 mediates rice disease resistance by regulating defense-related genes in salicylate- and jasmonate-dependent signaling 2007 Mol Plant Microbe Interact National Key Laboratory of Crop Genetic Improvement, National Center for Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China. Although 109 WRKY genes have been identified in the rice genome, the functions of most are unknown. Here, we show that OsWRKY13 plays a pivotal role in rice disease resistance. Overexpression of OsWRKY13 can enhance rice resistance to bacterial blight and fungal blast, two of the most devastating diseases of rice worldwide, at both the seedling and adult stages, and shows no influence on the fertility. This overexpression was accompanied by the activation of salicylic acid (SA) synthesis-related genes and SA-responsive genes and the suppression of jasmonic acid (JA) synthesis-related genes and JA-responsive genes. OsWRKY13 bound to the promoters of its own and at least three other genes in SA- and JA-dependent signaling pathways. Its DNA-binding activity was influenced by pathogen infection. These results suggest that OsWRKY13, as an activator of the SA-dependent pathway and a suppressor of JA-dependent pathways, mediates rice resistance by directly or indirectly regulating the expression of a subset of genes acting both upstream and downstream of SA and JA. Furthermore, OsWRKY13 will provide a transgenic tool for engineering wider-spectrum and whole-growth-stage resistance rice in breeding programs. OsEDS1,OsWRKY13,CHS,ICS1|OsVDAC1,PAD4|OsPAD4 Protein elicitor PemG1 from Magnaporthe grisea induces systemic acquired resistance (SAR) in plants 2011 Mol Plant Microbe Interact State Key Laboratory of Agricultural Microbio,ogy, College of Life Science and Technology, Huazhong Agricultural University. Wuahn 430070, China. Elicitors can stimulate defense responses in plants and have become a popular strategy in plant disease control. Previously, we isolated a novel protein elicitor, PemG1, from Magnaporthe grisea. In the present study, PemG1 protein expressed in and purified from Escherichia coli improved resistance of rice and Arabidopsis to bacterial infection, induced transient expression of pathogenesis-related (PR) genes, and increased accumulation of hydrogen peroxide in rice. The effects of PemG1 on disease resistance and PR gene expression were mobilized systemically throughout the rice plant and persisted for more than 28 days. PemG1-induced accumulation of OsPR-1a in rice was prevented by the calcium channel blockers LaCl(3), BAPTA, EGTA, W7, and TFP. Arabidopsis mutants that are insensitive to jasmonic acid (JA) and ethylene showed increased resistance to bacterial infection after PemG1 treatment but PemG1 did not affect resistance of mutants with an impaired salicylic acid (SA) transduction pathway. In rice, PemG1 induced overexpressions of the SA signal-related genes (OsEDS1, OsPAL1, and OsNH1) but not the JA pathway-related genes (OsLOX2 and OsAOS2). Our findings reveal that PemG1 protein can function as an activator of plant disease resistance, and the PemG1-mediated systemic acquired resistance is modulated by SA- and Ca(2+)-related signaling pathways. OsEDS1 Phytochromes regulate SA and JA signaling pathways in rice and are required for developmentally controlled resistance to Magnaporthe grisea 2011 Mol Plant High-Tech Research Center, Shandong Academy of Agricultural Sciences, Jinan 250100, PR China. xzhxie2010@163.com Old leaves of wild-type rice plants (Oryza sativa L. cv. Nipponbare) are more resistant to blast fungus (Magnaporthe grisea) than new leaves. In contrast, both old and new leaves of the rice phytochrome triple mutant (phyAphyBphyC) are susceptible to blast fungus. We demonstrate that pathogenesis-related class 1 (PR1) proteins are rapidly and strongly induced during M. grisea infection and following exogenous jasmonate (JA) or salicylic acid (SA) exposure in the old leaves, but not in the new leaves of the wild-type. In contrast, the accumulation of PR1 proteins was significantly attenuated in old and new leaves of the phyAphyBphyC mutant. These results suggest that phytochromes are required for the induction of PR1 proteins in rice. Basal transcription levels of PR1a and PR1b were substantially higher in the wild-type as compared to the phyAphyBphyC mutant, suggesting that phytochromes also are required for basal expression of PR1 genes. Moreover, the transcript levels of genes known to function in SA- or JA-dependent defense pathways were regulated by leaf age and functional phytochromes. Taken together, our findings demonstrate that phytochromes are required in rice for age-related resistance to M. grisea and may indirectly increase PR1 gene expression by regulating SA- and JA-dependent defense pathways. OsEDS1,CHS,ICS1|OsVDAC1,PAD4|OsPAD4 Expression of an endo-(1,3;1,4)-beta-glucanase in response to wounding, methyl jasmonate, abscisic acid and ethephon in rice seedlings 2009 J Plant Physiol National Agricultural Research Center for Hokkaido Region, 1 Hitsujigaoka, Toyohira-ku, Sapporo 062-8555, Japan. takiyama@affrc.go.jp We isolated two rice endo-(1,3;1,4)-beta-glucanase genes, denoted OsEGL1 and OsEGL2, which encoded proteins that shared 64% amino acid sequence identity. Both the OsEGL1 and OsEGL2 genes were successfully expressed in Escherichia coli to produce functional proteins. Purified OsEGL1 and OsEGL2 proteins hydrolyzed (1,3;1,4)-beta-glucans, but not (1,3;1,6)-beta-linked or (1,3)-beta-linked glucopolysaccharides nor carboxymethyl cellulose, similar to previously characterized grass endo-(1,3;1,4)-beta-glucanases. RNA blot analysis revealed that the OsEGL1 gene is expressed constitutively not only in young roots of rice seedlings, but also in mature roots of adult rice plants. Little or no expression of the OsEGL2 gene was observed in all tissues or treatments tested, but database and RT-PCR analysis indicated it is expressed in ripening panicle. In rice seedling leaves, OsEGL1 gene expression significantly increased in response to methyl jasmonate, abscisic acid, ethephon and mechanical wounding. Mechanical wounding also increased the leaf elongation rate in rice seedlings by 16% relative to that of control seedlings at day 4 after treatment. The increase in the leaf elongation rate of rice seedlings treated under mechanical wounding was concomitant with an increase in OsEGL1 expression levels in seedling leaves. OsEGL2 Involvement of two rice ETHYLENE INSENSITIVE3-LIKE genes in wound signaling 2009 Mol Genet Genomics National Institute of Crop Science, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, 305-8518, Japan. Ethylene and jasmonic acid (JA) have been proposed as key compounds for wound signaling in plants. In Arabidopsis, ETHYLENE INSENSITIVE3 (EIN3), which is an essential transcription factor for ethylene signaling, is regulated at the post-transcriptional level, while transcriptional regulation of EIN3 or EIN3-LIKE (EIL) genes has not been well documented. The expression of 6 rice EIL genes (OsEIL1-6) was analyzed and only OsEIL1 and 2 were found to be wound-inducible EIL. OsEIL2 was also induced by JA. Electrophoretic mobility shift assays showed that recombinant OsEIL1 and 2 proteins bound to specific DNA sequences that are recognized by a wound-inducible tobacco EIL. Accumulation of OsEIL1 and 2 transcripts reached a maximum at 1 and 0.5 h after wounding, respectively, and the corresponding DNA-binding activity in nuclear extracts of rice leaves was increased at 1 h after wounding. Candidates for OsEIL-target genes were selected by microarray analysis of wounded rice and by promoter sequence analyses of wound-inducible genes identified by microarray analysis. In OsEIL1- and/or 2-suppressed rice plants, the expression of at least four of 18 candidate genes analyzed was down-regulated. These results indicate the importance of inducible OsEILs in wound signaling in rice. OsEIL1|EIN3,OsEIL2 OsEF3, a homologous gene of Arabidopsis ELF3, has pleiotropic effects in rice 2009 Plant Biol (Stuttg) Rice Research Institute, Sichuan Agriculture University, Chengdu, China. Heading date is an important agronomic trait in rice. A rice mutant with a late heading date and no photoperiodic sensitivity in long or short day conditions was obtained from rice T-DNA insertion mutants in Zhonghua11 (ZH11). Through isolation and analysis of the flanking sequence of the T-NDA insertion site, the target sequence of insertion was obtained and found to locate in AP003296, the sequence accession number of rice chromosome 1 of RGP (http://rgp.dna.affrc.go.jp). The putative amino acid sequences of this target gene are homologous to the Arabidopsis protein ELF3 encoded by an early flowering gene. The rice target gene orthologous to Arabidopsis ELF3 is named OsEF3; this encodes a putative nematode responsive protein-like protein. OsEF3 has pleiotropic effects in rice that differ from the effects of Arabidopsis ELF3, which only affects biological rhythms. OsEF3 regulates heading date by influencing the BVG stage and does not affect photoperiodic sensitivity, which suggests that the OsEF3 gene may be involved in an autonomous pathway in rice. OsEF3 may affect root development and kilo-grain weight by delaying cell division or cell elongation. OsEF3|OsELF3-2 Nucleotide sequence of the rice (Oryza sativa) Em protein gene (Emp1) 1992 Plant Mol Biol Department of Genetics, University of California, Davis 95616. None OsEm|EMP1 Gibberellin is not a regulator of miR156 in rice juvenile-adult phase change 2012 Rice (N Y) Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, 113-8657, Japan. antanaka@mail.ecc.u-tokyo.ac.jp. Plant hormone gibberellin (GA) promotes juvenile-adult phase change in higher plants. To confirm the functions of GA in rice, I used dwarf mutant d18-dy. d18-dy is a loss-of-function allele of D18, which encodes GA3ox2. d18-dy mutant exhibited long juvenile phase in morphological traits such as the size of the shoot apical meristem (SAM), shape of leaf blades, presence or absence of midribs and node-internode differentiation in stem. In contrast, expression patterns of juvenile-adult phase change markers miR156 and miR172 were similar between wild type and d18-dy. In addition, d18-dy mutation and GA did not affect expression levels of downstream genes of miR156. GA does not function upstream of miR156 in juvenile-adult phase change. OsEm|EMP1,OSVP1|VP1 Regulation of the Osem gene by abscisic acid and the transcriptional activator VP1: analysis of cis-acting promoter elements required for regulation by abscisic acid and VP1 1995 The Plant Journal Center for Molecular Biology and Genetics, Mie University, Tsu, Japan. Osem, a rice gene homologous to the wheat Em gene, which encodes one of the late-embryogenesis abundant proteins was isolated. The gene was characterized with respect to control of transcription by abscisic acid (ABA) and the transcriptional activator VP1, which is involved in the ABA-regulated gene expression during late embryo-genesis. A fusion gene (Osem-GUS) consisting of the Osem promoter and the bacterial beta-glucuronidase (GUS) gene was constructed and tested in a transient expression system, using protoplasts derived from a suspension-cultured line of rice cells, for activation by ABA and by co-transfection with an expression vector (35S-Osvp1) for the rice VP1 (OSVP1) cDNA. The expression of Osem-GUS was strongly (40- to 150-fold) activated by externally applied ABA and by over-expression of (OS)VP1. The Osem promoter has three ACGTG-containing sequences, motif A, motif B and motif A', which resemble the abscisic acid-responsive element (ABRE) that was previously identified in the wheat Em and the rice Rab16. There is also a CATGCATG sequence, which is known as the Sph box and is shown to be essential for the regulation by VP1 of the maize anthocyanin regulatory gene C1. Focusing on these sequence elements, various mutant derivatives of the Osem promoter in the transient expression system were assayed. The analysis revealed that motif A functions not only as an ABRE but also as a sequence element required for the regulation by (OS)VP1. OsEm|EMP1,OSVP1|VP1 Increased nitrogen-use efficiency in transgenic rice plants over-expressing a nitrogen-responsive early nodulin gene identified from rice expression profiling 2009 Plant Cell Environ Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1. Development of genetic varieties with improved nitrogen-use efficiency (NUE) is essential for sustainable agriculture. In this study, we developed a growth system for rice wherein N was the growth-limiting factor, and identified N-responsive genes by a whole genome transcriptional profiling approach. Some genes were selected to test their functionality in NUE by a transgenic approach. One such example with positive effects on NUE is an early nodulin gene OsENOD93-1. This OsENOD93-1 gene responded significantly to both N induction and N reduction. Transgenic rice plants over-expressing the OsENOD93-1 gene had increased shoot dry biomass and seed yield. This OsENOD93-1 gene was expressed at high levels in roots of wild-type (WT) plants, and its protein product was localized in mitochondria. Transgenic plants accumulated higher concentrations of total amino acids and total N in roots. A higher concentration of amino acids in xylem sap was detected in transgenic plants, especially under N stress. In situ hybridization revealed that OsENOD93-1 is expressed in vascular bundles, as well as in epidermis and endodermis. This work demonstrates that transcriptional profiling, coupled with a transgenic validation approach, is an effective strategy for gene discovery. The knowledge gained from this study could be applied to other important crops. OsENOD93-1 Functional characterization and expression analysis of a gene, OsENT2, encoding an equilibrative nucleoside transporter in rice suggest a function in cytokinin transport 2005 Plant Physiol RIKEN (The Institute of Physical and Chemical Research) Plant Science Center, Tsurumi-ku, Yokohama 230-0045, Japan. We identified four genes for potential equilibrative nucleoside transporters (ENTs) from rice (Oryza sativa; designated OsENT1 through OsENT4). Growth analysis of budding yeast (Saccharomyces cerevisiae) cells expressing OsENTs showed that OsENT2 transported adenosine and uridine with high affinity (adenosine, K(m) = 3.0 microm; uridine, K(m) = 0.7 microm). Purine or pyrimidine nucleosides and 2'-deoxynucleosides strongly inhibited adenosine transport via OsENT2, suggesting that OsENT2 possesses broad substrate specificity. OsENT2-mediated adenosine transport was resistant to the typical inhibitors of mammalian ENTs, nitrobenzylmercaptopurine ribonucleoside, dilazep, and dipyridamole. The transport activity was maximal at pH 5.0 and decreased slightly at lower as well as higher pH. In competition experiments with various cytokinins, adenosine transport by OsENT2 was inhibited by isopentenyladenine riboside (iPR). Direct measurements with radiolabeled cytokinins demonstrated that OsENT2 mediated uptake of iPR (K(m) = 32 microm) and trans-zeatin riboside (K(m) = 660 microm), suggesting that OsENT2 participates in iPR transport in planta. In mature plants, OsENT2 was predominantly expressed in roots. The OsENT2 promoter drove the expression of the beta-glucuronidase reporter gene in the scutellum during germination and in vascular tissues in germinated plants, suggesting a participation of OsENT2 in the retrieval of endosperm-derived nucleosides by the germinating embryo and in the long-distance transport of nucleosides in growing plants, respectively. OsENT2 Differential proteomic analysis of soluble extracellular proteins reveals the cysteine protease and cystatin involved in suspension-cultured cell proliferation in rice 2009 Biochim Biophys Acta Institute of Molecular Cell Biology, Hebei Normal University, Shijiazhuang 050016, PR China. Extracellular matrix proteins play crucial roles in plant development, morphogenesis, cell division, and proliferation. To identify extracellular proteins that regulate cell growth, the soluble proteins of extracellular matrix were extracted from suspension-cultured rice cells for different lengths of time. The extract obtained from 3-d cultures was found to increase cells' fresh weight, while extracts from 6-d and 9-d cultures showed no effect on cells' growth. A comparative proteomic analysis was used to identify soluble extracellular proteins differentially expressed between 3 and 6 days in suspension-cultured cells. Ten unique protein spots were isolated and identified by mass spectrometry. Among them, included a cysteine protease (OsCP) and a putative cysteine protease inhibitor (cystatin, OC-I). OsCP has been down regulated in vivo using RNAi transgenic lines. The fresh and dry weight growth rates of OsCP RNAi cell lines were lower than empty vector control. Recombinant protein of OC-I could inhibit the OsCP protease activity, also it could inhibit the weight increase of suspension-culture cell as well as extracellular protease activity. These results suggest that OsCP and OC-I may be involved in the process of suspension-cultured rice cells proliferation. REP-1|OsEP3A Two cis-acting elements necessary and sufficient for gibberellin-upregulated proteinase expression in rice seeds 2003 The Plant Journal Department of Biological Sciences, Tokyo Metropolitan University, Minami-ohsawa 1-1, Hachioji-shi, Tokyo 192-0397, Japan. In germinating rice seeds, a cysteine proteinase (REP-1), synthesized in aleurone-layer cells, is a key enzyme in the degradation of the major storage protein, glutelin. The expression of the gene for REP-1 (Rep1) is induced by gibberellins (GAs) and repressed by abscisic acid (ABA). To identify GA-responsive elements in the Rep1 promoter, we developed a transient expression system in rice aleurone cells. Deletion and point-mutation analyses indicated that the GA-response complex was composed of TAACAGA, TAACGTA, and two copies of CAACTC. The two former sequences were identical to GAREs conserved in the promoter of genes for alpha-amylase and proteinases in cereals. The latter, termed as CAACTC regulatory elements (CAREs), were novel GAREs. Gain-of-function experiments revealed that two pairs of GARE and CARE were necessary and sufficient to confer GA inducibility. The sequences were also required for effective transactivation by the transcription factor OsGAMyb. Four copies of either GARE or CARE showed transactivation neither by OsGAMyb nor by GA induction. CARE and GARE were also found in the promoters of a rice alpha-amylase gene, RAmy1A, and a barley proteinase gene, EPB1, which are expressed in germinating seeds. Mutations of CARE in their promoters caused a loss of GA inducibility and GAMyb transactivation, suggesting that CARE is the regulatory element for GA-inducible expression of hydrolase genes in the germinating seeds. REP-1|OsEP3A,OsMYBGA|OsGAMYB,AmyI-1|RAmy1A Multiple mode regulation of a cysteine proteinase gene expression in rice 2000 Plant Physiol Department of Biology, National Taiwan Normal University, Taipei 11650, Taiwan, Republic of China. In many plants, cysteine proteinases play essential roles in a variety of developmental and physiological processes. In rice (Oryza sativa), REP-1 is a primary cysteine proteinase responsible for the digestion of seed storage proteins to provide nutrients to support the growth of young seedlings. In the present study, the gene encoding REP-1 was isolated, characterized, and designated as OsEP3A. An OsEP3A-specific DNA probe was used to study the effect of various factors on the expression of OsEP3A in germinating seeds and vegetative tissues of rice. The expression of OsEP3A is hormonally regulated in germinating seeds, spatially and temporally regulated in vegetative tissues, and nitrogen-regulated in suspension-cultured cells. The OsEP3A promoter was linked to the coding sequence of the reporter gene, gusA, which encodes beta-glucuronidase (GUS), and the chimeric gene was introduced into the rice genome. The OsEP3A promoter is sufficient to confer nitrogen regulation of GUS expression in suspension-cultured cells. Histochemical studies also indicate that the OsEP3A promoter is sufficient to confer the hormonal regulation of GUS expression in germinating seeds. These studies demonstrate that in rice the REP-1 protease encoded by OsEP3A may play a role in various physiological responses and processes, and that multiple mechanisms regulate the expression of OsEP3A. REP-1|OsEP3A Analysis of rice ER-resident J-proteins reveals diversity and functional differentiation of the ER-resident Hsp70 system in plants 2013 J Exp Bot Functional Transgenic Crops Research Unit, Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. The heat shock protein 70 (Hsp70) chaperone system participates in protein folding and quality control of unfolded proteins. To examine the roles of co-chaperones in the rice Hsp70 chaperone system in the endoplasmic reticulum (ER), the functions of six ER-resident J-proteins (OsP58A, OsP58B, OsERdj2, OsERdj3A, OsERdj3B, and OsERdj7) in rice were investigated. The expression of OsP58B, OsERdj3A, and OsERdj3B was predominantly up-regulated in roots subjected to ER stress. This response was mediated by signalling through ATF6 orthologues such as OsbZIP39 and OsbZIP60, but not through the IRE1/OsbZIP50 pathway. A co-immunoprecipitation assay demonstrated that OsP58A, OsP58B, and OsERdj3B preferentially interact with the major OsBiP, OsBiP1, while OsERdj3A interacts preferentially with OsBiP5, suggesting that there are different affinities between OsBiPs and J-proteins. In the endosperm tissue, OsP58A, OsP58B, and OsERdj2 were mainly localized in the ER, whereas OsERdj2 was localized around the outer surfaces of ER-derived protein bodies (PB-Is). Furthermore, OsERdj3A was not expressed in wild-type seeds but was up-regulated in transgenic seeds accumulating human interleukin-7 (hIL-7). Since ERdj3A-green fluorescent protein (GFP) was also detected in vacuoles of callus cells under ER stress conditions, OsERdj3A is a bona fide vacuole-localized protein. OsP58A, OsP58B and OsERdj3A were differentially accumulated in transgenic plants expressing various recombinant proteins. These results reveal the functional diversity of the rice ER-resident Hsp70 system. OsERdj3A OsRMC, a negative regulator of salt stress response in rice, is regulated by two AP2/ERF transcription factors 2013 Plant Mol Biol Genomics of Plant Stress Laboratory, Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Oeiras, Portugal. High salinity causes remarkable losses in rice productivity worldwide mainly because it inhibits growth and reduces grain yield. To cope with environmental changes, plants evolved several adaptive mechanisms, which involve the regulation of many stress-responsive genes. Among these, we have chosen OsRMC to study its transcriptional regulation in rice seedlings subjected to high salinity. Its transcription was highly induced by salt treatment and showed a stress-dose-dependent pattern. OsRMC encodes a receptor-like kinase described as a negative regulator of salt stress responses in rice. To investigate how OsRMC is regulated in response to high salinity, a salt-induced rice cDNA expression library was constructed and subsequently screened using the yeast one-hybrid system and the OsRMC promoter as bait. Thereby, two transcription factors (TFs), OsEREBP1 and OsEREBP2, belonging to the AP2/ERF family were identified. Both TFs were shown to bind to the same GCC-like DNA motif in OsRMC promoter and to negatively regulate its gene expression. The identified TFs were characterized regarding their gene expression under different abiotic stress conditions. This study revealed that OsEREBP1 transcript level is not significantly affected by salt, ABA or severe cold (5 degrees C) and is only slightly regulated by drought and moderate cold. On the other hand, the OsEREBP2 transcript level increased after cold, ABA, drought and high salinity treatments, indicating that OsEREBP2 may play a central role mediating the response to different abiotic stresses. Gene expression analysis in rice varieties with contrasting salt tolerance further suggests that OsEREBP2 is involved in salt stress response in rice. OsEREBP1,OsEREBP2|ERF99,OsRMC|OsRLK BWMK1, a Rice Mitogen-Activated Protein Kinase, Locates in the Nucleus and Mediates Pathogenesis-Related Gene Expression by Activation of a Transcription Factor 2003 Plant Physiol Division of Applied Life Science (BK21 Program), Gyeongsang National University, Jinju 660-701, Korea. Mitogen-activated protein kinase (MAPK) cascades are known to transduce plant defense signals, but the downstream components of the MAPK have as yet not been elucidated. Here, we report an MAPK from rice (Oryza sativa), BWMK1, and a transcription factor, OsEREBP1, phosphorylated by the kinase. The MAPK carries a TDY phosphorylation motif instead of the more common TEY motif in its kinase domain and has an unusually extended C-terminal domain that is essential to its kinase activity and translocation to the nucleus. The MAPK phosphorylates OsEREBP1 that binds to the GCC box element (AGCCGCC) of the several basic pathogenesis-related gene promoters, which in turn enhances DNA-binding activity of the factor to the cis element in vitro. Transient co-expression of the BWMK1 and OsEREBP1 in Arabidopsis protoplasts elevates the expression of the beta-glucuronidase reporter gene driven by the GCC box element. Furthermore, transgenic tobacco (Nicotiana tabacum) plants overexpressing BWMK1 expressed many pathogenesis-related genes at higher levels than wild-type plants with an enhanced resistance to pathogens. These findings suggest that MAPKs contribute to plant defense signal transduction by phosphorylating one or more transcription factors. OsEREBP1,OsWJUMK1|OsMPK12|OsBWMK1 AP2/ERF Transcription Factor in Rice: Genome-Wide Canvas and Syntenic Relationships between Monocots and Eudicots 2012 Evol Bioinform Online China-UK HUST-RRes Genetic Engineering and Genomics Joint Laboratory, International Science and Technology Cooperation Base (Genetic Engineering) of Chinese Ministry of Science and Technology, The key laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology (HUST), Luoyu Road 1037, Wuhan 430074, China. The transcription factor family intimately regulates gene expression in response to hormones, biotic and abiotic factors, symbiotic interactions, cell differentiation, and stress signalling pathways in plants. In this study, 170 AP2/ERF family genes are identified by phylogenetic analysis of the rice genome (Oryza sativa l. japonica) and they are divided into a total of 11 groups, including four major groups (AP2, ERF, DREB, and RAV), 10 subgroups, and two soloists. Gene structure analysis revealed that, at position-6, the amino acid threonine (Thr-6) is conserved in the double domain AP2 proteins compared to the amino acid arginine (Arg-6), which is preserved in the single domain of ERF proteins. In addition, the histidine (His) amino acid is found in both domains of the double domain AP2 protein, which is missing in single domain ERF proteins. Motif analysis indicates that most of the conserved motifs, apart from the AP2/ERF domain, are exclusively distributed among the specific clades in the phylogenetic tree and regulate plausible functions. Expression analysis reveals a widespread distribution of the rice AP2/ERF family genes within plant tissues. In the vegetative organs, the transcripts of these genes are found most abundant in the roots followed by the leaf and stem; whereas, in reproductive tissues, the gene expression of this family is observed high in the embryo and lemma. From chromosomal localization, it appears that repetition and tandem-duplication may contribute to the evolution of new genes in the rice genome. In this study, interspecies comparisons between rice and wheat reveal 34 rice loci and unveil the extent of collinearity between the two genomes. It was subsequently ascertained that chromosome-9 has more orthologous loci for CRT/DRE genes whereas chromosome-2 exhibits orthologs for ERF subfamily members. Maximum conserved synteny is found in chromosome-3 for AP2 double domain subfamily genes. Macrosynteny between rice and Arabidopsis, a distant, related genome, uncovered 11 homologs/orthologs loci in both genomes. The distribution of AP2/ERF family gene paralogs in Arabidopsis was most frequent in chromosome-1 followed by chromosome-5. In Arabidopsis, ERF subfamily gene orthologs are found on chromosome-1, chromosome-3, and chromosome-5, whereas DRE subfamily genes are found on chromosome-2 and chromosome-5. Orthologs for RAV and AP2 with double domains in Arabidopsis are located on chromosome-1 and chromosome-3, respectively. In conclusion, the data generated in this survey will be useful for conducting genomic research to determine the precise role of the AP2/ERF gene during stress responses with the ultimate goal of improving crops. OsEREBP2|ERF99 Overexpression of OsERF1, a novel rice ERF gene, up-regulates ethylene-responsive genes expression besides affects growth and development in Arabidopsis 2008 J Plant Physiol Research Center for Molecular & Developmental Biology, Key Laboratory of Photosynthesis & Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China. Ethylene-responsive factors (ERFs), composing the largest group of AP2/EREBP transcription factors, are involved in diverse functions and some of them have been identified in plants. However, even in model plants Arabidopsis and rice, most of the genes in this group are functionally unknown yet. Especially in rice, ERF genes involved in ethylene response have not been reported previously. Here, we report a novel member of ERF group in rice, OsERF1 (Ethylene Response Factor gene in Oryza sativa). OsERF1 expressed consistently in different organs of rice and could be up-regulated by ethylene, a plant hormone associated with stress response. Overexpression of OsERF1 in Arabidopsis up-regulated the expression of two known ethylene-responsive genes, PDF1.2 and b-chitinase, and also significantly affected the growth and development of transgenic Arabidopsis. These results suggest the involvement of OsERF1 in ethylene response. OsERF1 RSOsPR10 expression in response to environmental stresses is regulated antagonistically by jasmonate/ethylene and salicylic acid signaling pathways in rice roots 2011 Plant Cell Physiol Department of Biological Sciences, Tokyo Metropolitan University, Minami-Osawa, Hachioji, Tokyo, 192-0397 Japan. Plant roots play important roles not only in the absorption of water and nutrients, but also in stress tolerance. Previously, we identified RSOsPR10 as a root-specific pathogenesis-related (PR) protein induced by drought and salt treatments in rice. Transcripts and proteins of RSOsPR10 were strongly induced by jasmonate (JA) and the ethylene (ET) precursor 1-aminocyclopropane-1-carboxylic acid (ACC), while salicylic acid (SA) almost completely suppressed these inductions. Immunohistochemical analyses showed that RSOsPR10 strongly accumulated in cortex cells surrounding the vascular system of roots, and this accumulation was also suppressed when SA was applied simultaneously with stress or hormone treatments. In the JA-deficient mutant hebiba, RSOsPR10 expression was up-regulated by NaCl, wounding, drought and exogenous application of JA. This suggested the involvement of a signal transduction pathway that integrates JA and ET signals in plant defense responses. Expression of OsERF1, a transcription factor in the JA/ET pathway, was induced earlier than that of RSOsPR10 after salt, JA and ACC treatments. Simultaneous SA treatment strongly inhibited the induction of RSOsPR10 expression and, to a lesser extent, induction of OsERF1 expression. These results suggest that JA/ET and SA pathways function in the stress-responsive induction of RSOsPR10, and that OsERF1 may be one of the transcriptional factors in the JA/ET pathway. OsERF1,RSOsPR10 An EAR-motif-containing ERF transcription factor affects herbivore-induced signaling, defense and resistance in rice 2011 The Plant Journal National Key Laboratory of Rice Biology, Institute of Insect Science, Zhejiang University, Hangzhou 310029, China. Ethylene responsive factors (ERFs) are a large family of plant-specific transcription factors that are involved in the regulation of plant development and stress responses. However, little to nothing is known about their role in herbivore-induced defense. We discovered a nucleus-localized ERF gene in rice (Oryza sativa), OsERF3, that was rapidly up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis. Antisense and over-expression of OsERF3 revealed that it positively affects transcript levels of two mitogen-activated protein kinases (MAPKs) and two WRKY genes as well as concentrations of jasmonate (JA), salicylate (SA) and the activity of trypsin protease inhibitors (TrypPIs). OsERF3 was also found to mediate the resistance of rice to SSB. On the other hand, OsERF3 was slightly suppressed by the rice brown planthopper (BPH) Nilaparvata lugens (Stål) and increased susceptibility to this piercing sucking insect, possibly by suppressing H(2)O(2) biosynthesis. We propose that OsERF3 affects early components of herbivore-induced defense responses by suppressing MAPK repressors and modulating JA, SA, ethylene and H(2)O(2) pathways as well as plant resistance. Our results also illustrate that OsERF3 acts as a central switch that gears the plant's metabolism towards an appropriate response to chewing or piercing/sucking insects. AP37|OsERF3 Isolation and identification of an AP2/ERF factor that binds an allelic cis-element of rice gene LRK6 2011 Genet Res (Camb) State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai, China. Allelic expression of the rice yield-related gene, leucine-rich receptor-like kinase 6 (LRK6), in the hybrid of 93-11 (Oryza sativa L. subsp. Indica var. 93-11) and Nipponbare (O. sativa L. subsp. Japonica var. Nipponbare) is determined by allelic promoter cis-elements. Using deletion analysis of the LRK6 promoter, we identified two distinct regions that might contribute to LRK6 expression. Sequence alignment revealed differences in these LRK6 promoter regions in 93-11 and Nipponbare. One of the segments, named differential sequence of LRK6 promoter 2 (DSLP2), contains potential transcription factor binding sites. Using a yeast one-hybrid assay, we isolated an ethylene-responsive factor (ERF) protein that binds to DSLP2. Sequence analysis and a GCC-box assay showed that the ERF gene, O. sativa ERF 3 (OsERF3), which belongs to ERF subfamily class II, has a conserved ERF domain and an ERF-associated amphiphilic repression repressor motif. We used an in vivo mutation assay to identify a new motif (5'-TAA(A)GT-3') located in DSLP2, which interacts with OsERF3. These results suggest that OsERF3, an AP2 (APETALA 2 Gene)/ERF transcription factor, binds the LRK6 promoter at this new motif, which might cause differential expression of LRK6 in the 93-11/Nipponbare hybrid. AP37|OsERF3 The rice ERF transcription factor OsERF922 negatively regulates resistance to Magnaporthe oryzae and salt tolerance 2012 J Exp Bot Key Laboratory of Plant Pathology, Department of Plant Pathology, China Agricultural University, Beijing, China. Rice OsERF922, encoding an APETELA2/ethylene response factor (AP2/ERF) type transcription factor, is rapidly and strongly induced by abscisic acid (ABA) and salt treatments, as well as by both virulent and avirulent pathovars of Magnaporthe oryzae, the causal agent of rice blast disease. OsERF922 is localized to the nucleus, binds specifically to the GCC box sequence, and acts as a transcriptional activator in plant cells. Knockdown of OsERF922 by means of RNAi enhanced resistance against M. oryzae. The elevated disease resistance of the RNAi plants was associated with increased expression of PR, PAL, and the other genes encoding phytoalexin biosynthetic enzymes and without M. oryzae infection. In contrast, OsERF922-overexpressing plants showed reduced expression of these defence-related genes and enhanced susceptibility to M. oryzae. In addition, the OsERF922-overexpressing lines exhibited decreased tolerance to salt stress with an increased Na(+)/K(+) ratio in the shoots. The ABA levels were found increased in the overexpressing lines and decreased in the RNAi plants. Expression of the ABA biosynthesis-related genes, 9-cis-epoxycarotenoid dioxygenase (NCED) 3 and 4, was upregulated in the OsERF922-overexpressing plants, and NCED4 was downregulated in the RNAi lines. These results suggest that OsERF922 is integrated into the cross-talk between biotic and abiotic stress-signalling networks perhaps through modulation of the ABA levels. OsERF922,OsNCED3,OsNCED4 ER membrane-localized oxidoreductase Ero1 is required for disulfide bond formation in the rice endosperm 2009 Proc Natl Acad Sci U S A Division of Plant Sciences, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan. The developing endosperm of rice (Oryza sativa, Os) synthesizes a large amount of storage proteins on the rough (r)ER. The major storage proteins, glutelins and prolamins, contain either intra or intermolecular disulfide bonds, and oxidative protein folding is necessary for the sorting of the proteins to the protein bodies. Here, we investigated an electron transfer pathway for the formation of protein disulfide bonds in the rER of the rice endosperm, focusing on the roles of the thiol-disulfide oxidoreductase, OsEro1. Confocal microscopic analysis revealed that N-glycosylated OsEro1 is localized to the rER membrane in the subaleurone cells, and that targeting of OsEro1 to the rER membrane depends on the N-terminal region from Met-1 to Ser-55. The RNAi knockdown of OsERO1 inhibited the formation of native disulfide bonds in the glutelin precursors (proglutelins) and promoted aggregation of the proglutelins through nonnative intermolecular disulfide bonds in the rER. Inhibition of the formation of native disulfide bonds was also observed in the seeds of the esp2 mutant, which lacks protein disulfide isomerase-like (PDIL)1;1, but shows enhanced OsEro1 expression. We detected the generation of H(2)O(2) in the rER of the WT subaleurone cells, whereas the rER-derived H(2)O(2) levels decreased markedly in EM49 homozygous mutant seeds, which have fewer sulfhydryl groups than the WT seeds. Together, we propose that the formation of native disulfide bonds in proglutelins depends on an electron transfer pathway involving OsEro1 and OsPDIL. OsEro1 Genome-wide analysis of the auxin response factors (ARF) gene family in rice (Oryza sativa) 2007 Gene Crop Molecular Breeding Center, Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, P.R. China. Auxin response factors (ARFs) are transcription factors that bind with specificity to TGTCTC-containing auxin response elements (AuxREs) found in promoters of primary/early auxin response genes and mediate responses to the plant hormone auxin. The ARF genes are represented by a large multigene family in plants. A comprehensive genome-wide analysis was carried out in this study to find all ARFs in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa subsp. japonica), 23 and 25 ARF genes, named as AtARFs and OsARFs, were identified, respectively. Chromosomal locations of all OsARFs were presented and it was found that the duplication of OsARFs was associated with only the chromosomal block duplications but not local tandem duplications. A phylogenetic tree was generated from alignments of the full-length protein sequences of 25 OsARFs and 23 AtARFs to examine the phylogenetic relationships of rice and Arabidopsis ARF proteins. All 48 members of ARF gene families fell into three major classes, a total of 13 sister pairs, including 9 OsARF-OsARF, 2 AtARF-AtARF and 2 AtARF-OsARF sister pairs were formed, showing different orthologous relationships between AtARFs and OsARFs. EST analysis and RT-PCR assays demonstrated that 24 of all 25 OsARF genes were active and the transcript abundance of some OsARF genes was affected by auxin treatment or light- and dark-grown conditions. The outcome of the present study provides basic genomic information for the rice ARF gene family and will pave the way for elucidating the precise role of OsARFs in plant growth and development in the future. OsETTIN1|OsARF15 Rice exonuclease-1 homologue, OsEXO1, that interacts with DNA polymerase lambda and RPA subunit proteins, is involved in cell proliferation 2008 Plant Mol Biol Division of Plant Biotechnology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan. Exonuclease 1, a class III member of the RAD2 nuclease family, is a structure-specific nuclease involved in DNA metabolism (replication, repair and recombination). We have identified a homologue to Exonuclease-1 from rice (Oryza sativa L. cv. Nipponbare) and have designated it O. sativa Exonuclease-1 (OsEXO1). The open reading frame of OsEXO1 encodes a predicted product of 836 amino acid residues with a molecular weight of 92 kDa. Two highly conserved nuclease domains (XPG-N and XPG-I) are present in the N-terminal region of the protein. OsEXO1-sGFP fusion protein transiently overexpressed in the onion epidermal cells localized to the nucleus. The transcript of OsEXO1 is highly expressed in meristematic tissues and panicles. Inhibition of cell proliferation by removal of sucrose from the medium or by the addition of cell cycle inhibitors decreased OsEXO1 expression. Functional complementation assays using yeast RAD2 member null mutants demonstrates that OsEXO1 is able to substitute for ScEXO1 and ScRAD27 functions. Yeast two-hybrid analysis shows that OsEXO1 interacted with rice DNA polymerase lambda (OsPol lambda), the 70 kDa subunit b of rice replication protein A (OsRPA70b), and the 32 kDa subunit 1 of rice replication protein A (OsRPA32-1). Irradiation of UV-B induces OsEXO1 expression while hydrogen peroxide treatment represses it. These results suggest that OsEXO1 plays an important role in both cell proliferation and UV-damaged nuclear DNA repair pathway under dark conditions. OsEXO1,OsRPA2|OsRPA32-1 Overexpression of OsRecQl4 and/or OsExo1 enhances DSB-induced homologous recombination in rice 2012 Plant Cell Physiol Plant Genome Engineering Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan. During homologous recombination (HR)-mediated DNA double-strand break (DSB) repair in eukaryotes, an initial step is the creation of a 3'-single-stranded DNA (ssDNA) overhang via resection of a 5' end. Rad51 polymerizes on this ssDNA to search for a homologous sequence, and the gapped sequence is then repaired using an undamaged homologous DNA strand as template. Recent studies in eukaryotes indicate that resection of the DSB site is promoted by the cooperative action of RecQ helicase family proteins: Bloom helicase (BLM) in mammals or Sgs1 in yeast, and exonuclease 1 (Exo1). However, the role of RecQ helicase and exonuclease during the 5'-resection process of HR in plant cells has not yet been defined. Here, we demonstrate that overexpression of rice proteins OsRecQl4 (BLM counterpart) and/or OsExo1 (Exo1 homolog) can enhance DSB processing, as evaluated by recombination substrate reporter lines in rice. These results could be applied to construct an efficient gene targeting system in rice. OsEXO1,OsRecQl4 Identification of specific fragments of HpaG Xooc, a harpin from Xanthomonas oryzae pv. oryzicola, that induce disease resistance and enhance growth in plants 2008 Phytopathology Plant Growth and Defense Signaling Laboratory, Group of Key Laboratory of Monitoring and Management of Plant Pathogens and Insect Pests, Ministry of Agriculture of P.R. China, Nanjing Agricultural University, Nanjing, China. Harpin proteins from gram-negative plant-pathogenic bacteria can stimulate hypersensitive cell death (HCD) and pathogen defense as well as enhance growth in plants. Two of these diverse activities clearly are beneficial and may depend on particular functional regions of the proteins. Identification of beneficial and deleterious regions might facilitate the beneficial use of harpin-related proteins on crops without causing negative effects like cell death. Here, we report the identification and testing of nine functional fragments of HpaG(Xooc), a 137-amino-acid harpin protein from Xanthomonas oryzae pv. oryzicola, the pathogen that causes bacterial leaf streak of rice. Polymerase chain reaction-based mutagenesis generated nine proteinaceous fragments of HpaG(Xooc); these caused different responses following their application to Nicotiana tabacum (tobacco) and Oryza sativa (rice). Fragment HpaG62-137, which spans the indicated amino acid residues of the HpaG, induced more intense HCD; in contrast, HpaG10-42 did not cause evident cell death in tobacco. However, both fragments stimulated stronger defense responses and enhanced more growth in rice than the full-length parent protein, HpaG(Xooc). Of the nine fragments, the parent protein and one deletion mutant of HpaG(Xooc) tested, HpaG10-42, stimulated higher levels of rice growth and resulted in greater levels of resistance to X. oryzae pv. oryzae and Magnaporthe grisea. These pathogens cause bacterial leaf blight and rice blast, respectively, the two most important diseases of rice world-wide. HpaG10-42 was more active than HpaG(Xooc) in inducing expression of several genes that regulate rice defense and growth processes and activating certain signaling pathways, which may explain the greater beneficial effects observed from treatment with that fragment. Overall, our results suggest that HpaG10-42 holds promise for practical agricultural use to induce disease resistance and enhance growth of rice. OsEXP1 Expression of alpha-expansin and expansin-like genes in deepwater rice 2002 Plant Physiol Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824-1312, USA. Previously, we have studied the expression and regulation of four alpha- and 14 beta-expansin genes in deepwater rice (Oryza sativa). We now report on the structure, expression, and regulation of 22 additional alpha-expansin (Os-EXP) genes, four expansin-like (Os-EXPL) genes, and one expansin-related (Os-EXPR) gene, which have recently been identified in the expressed sequence tag and genomic databases of rice. Alpha-expansins are characterized by a series of conserved Cys residues in the N-terminal half of the protein, a histidine-phenylalanine-aspartate (HFD) motif in the central region, and a series of tryptophan residues near the carboxyl terminus. Of the 22 additional alpha-expansin genes, five are expressed in internodes and leaves, three in coleoptiles, and nine in roots, with high transcript levels in the growing regions of these organs. Transcripts of five alpha-expansin genes were found in roots only. Expression of five alpha-expansin genes was induced in the internode by treatment with gibberellin (GA) and by wounding. The wound response resulted from excising stem sections or from piercing pinholes into the stem of intact plants. EXPL proteins lack the HFD motif and have two additional Cys residues in their C- and N-terminal regions. The positions of conserved tryptophan residues at the C-terminal region are different from those of alpha- and beta-expansins. Expression of the Os-EXPL3 gene is correlated with elongation and slightly induced by applied GA. However, the expression of the Os-EXPL1 and Os-EXPL2 genes showed limited correlation with cell elongation and was not induced by GA. We found no expression of the Os-EXPR1 gene in the organs examined. OsEXP1 Root hair-specific expansins modulate root hair elongation in rice 2011 Plant J State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, China. Root hair growth requires intensive cell-wall modification. This study demonstrates that root hair-specific expansin As, a sub-clade of the cell wall-loosening expansin proteins, are required for root hair elongation in rice (Oryza sativa L.). We identified a gene encoding EXPA17 (OsEXPA17) from a rice mutant with short root hairs. Promoter::reporter transgenic lines exhibited exclusive OsEXPA17 expression in root hair cells. The OsEXPA17 mutant protein (OsexpA17) contained a point mutation, causing a change in the amino acid sequence (Gly104-->Arg). This amino acid alteration is predicted to disrupt a highly conserved disulfide bond in the mutant. Suppression of OsEXPA17 by RNA interference further confirmed requirement for the gene in root hair elongation. Complementation of the OsEXPA17 mutant with other root hair EXPAs (OsEXPA30 and Arabidopsis EXPA7) can restore root hair elongation, indicating functional conservation of these root hair EXPAs in monocots and dicots. These results demonstrate that members of the root hair EXPA sub-clade play a crucial role in root hair cell elongation in Graminaceae. OsEXPA17,OsEXPA30 Overexpression of OsEXPA8, a root-specific gene, improves rice growth and root system architecture by facilitating cell extension 2013 PLoS One Bioengineering College, Chongqing University, Chongqing, China. Expansins are unique plant cell wall proteins that are involved in cell wall modifications underlying many plant developmental processes. In this work, we investigated the possible biological role of the root-specific alpha-expansin gene OsEXPA8 in rice growth and development by generating transgenic plants. Overexpression of OsEXPA8 in rice plants yielded pleiotropic phenotypes of improved root system architecture (longer primary roots, more lateral roots and root hairs), increased plant height, enhanced leaf number and enlarged leaf size. Further study indicated that the average cell length in both leaf and root vascular bundles was enhanced, and the cell growth in suspension cultures was increased, which revealed the cellular basis for OsEXPA8-mediated rice plant growth acceleration. Expansins are thought to be a key factor required for cell enlargement and wall loosening. Atomic force microscopy (AFM) technology revealed that average wall stiffness values for 35S::OsEXPA8 transgenic suspension-cultured cells decreased over six-fold compared to wild-type counterparts during different growth phases. Moreover, a prominent change in the wall polymer composition of suspension cells was observed, and Fourier-transform infrared (FTIR) spectra revealed a relative increase in the ratios of the polysaccharide/lignin content in cell wall compositions of OsEXPA8 overexpressors. These results support a role for expansins in cell expansion and plant growth. OsEXPA8 Biochemical properties and localization of the beta-expansin OsEXPB3 in rice (Oryza sativa L.) 2005 Mol Cells Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824-1312, USA. Alpha-expansins are bound to the cell wall of plants and can be solubilized with an extraction buffer containing 1 M NaCl. Localization of alpha-expansins in the cell wall was confirmed by immunogold labeling and electron microscopy. The subcellular localization of vegetative beta-expansins has not yet been studied. Using antibodies specific for OsEXPB3, a vegetative beta-expansin of rice (Oryza sativa L.), we found that OsEXPB3 is tightly bound to the cell wall and, unlike alpha-expansins, cannot be solubilized with extraction buffer containing 1 M NaCl. OsEXPB3 protein could only be extracted with buffer containing SDS. The subcellular localization of the OsEXPB3 protein was confirmed by immunogold labeling and electron microscopy. Gold particles were mainly distributed over the primary cell walls. Immunohistochemistry showed that OsEXPB3 is present in all regions of the coleoptile and root tissues tested. OsEXPB3 Root hair-specific EXPANSIN B genes have been selected for Graminaceae root hairs 2010 Mol Cells Department of Biological Sciences and Plant Genomics and Breeding Institute, Seoul National University, Seoul, 151-742, Korea. Cell differentiation ultimately relies on the regulation of cell type-specific genes. For a root hair cell to undergo morphogenesis, diverse cellular processes including cell-wall loosening must occur in a root hair cell-specific manner. Previously, we identified and characterized root hairspecific cis-elements (RHE) from the genes encoding the cell wall-loosening protein EXPANSIN A (EXPA) which functions preferentially on dicot cell walls. This study reports two root hair-specific grass EXPB genes that contain RHEs. These genes are thought to encode proteins that function more efficiently on grass cell walls. The proximal promoter regions of two orthologous EXPB genes from rice (Oryza sativa; OsEXPB5) and barley (Hordeum vulgare; HvEXPB1) included RHE motifs. These promoters could direct root hair-specific expression of green fluorescent protein (GFP) in the roots of rice and Arabidopsis (Arabidopsis thaliana). Promoter deletion analyses demonstrated that the RHE motifs are necessary for root hairspecific expression of these EXPB promoters. Phylogenetic analysis of EXP protein sequences indicated that grass EXPBs are the only orthologs to these root hair-specific EXPBs, separating dicot EXPBs to distal branches of the tree. These results suggest that RHE-containing root hair-specific EXPB genes have evolved for grass-specific cell wall modification during root hair morphogenesis. OsEXPB5 A rice microsomal delta-12 fatty acid desaturase can enhance resistance to cold stress in yeast and Oryza sativa 2011 Molecular Breeding State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, 220 Handan Road, Shanghai, 200433, People’s Republic of China A full-length cDNA clone of OsFAD2, which encodes a Δ-12 fatty acid desaturase, the key enzyme for the conversion of oleic acid (18:1) into linoleic acid (18:2), was isolated from rice (Oryza sativa ssp. japonica) leaves. The deduced amino acid sequence of OsFAD2 displayed three histidine boxes characteristic of all membrane-bound desaturases, and possessed a C-terminal signal for endoplasmic reticulum retention. Phylogenetic analysis showed that OsFAD2 is grouped within plant housekeeping FAD2 sequences. Expression analysis by real-time PCR showed that the gene is expressed in all tissues of rice tested, including root, seed, stem, and leaf. In situ hybridization showed that OsFAD2 mRNA accumulated in leaf mesophyll cells and in root epidermis cells when exposed to 15°C for 4 days in dark conditions. When OsFAD2 was expressed in Saccharomyces cerevisiae, the cells could convert oleic acid to linoleic acid, which wild-type yeast cells cannot do, suggesting that the isolated gene encoded a functional FAD2 enzyme. Heterologous expression of OsFAD2 enhanced the yeast cells’ cold tolerance capacity compared to wild-type yeast. OsFAD2 was also shown to be a highly active desaturase when expressed in Xenopus oocytes. In addition, when the OsFAD2 gene was transferred into an Arabidopsis thaliana fad2-1 mutant, it effectively restored wild-type fatty acid composition and growth characteristics. Stress tolerance and light regulatory elements were identified in the predicted promoter of the OsFAD2 gene. Exogenously supplied hormone affected the level of FAD2 mRNA accumulation, accompanied by a change of content of di-unsaturated fatty acid species in rice leaves. Furthermore, OsFAD2 enhanced tolerance to low temperature when overexpressed in rice at the vegetative stage. More importantly, the 35S::OsFAD2 plants showed significantly enhanced cold tolerance at the reproductive stage, increasing grain yield by 46% over controls in the greenhouse under cold conditions. These results indicated that OsFAD2 is involved in fatty acid desaturation and maintenance of the membrane lipids balance in cells, and could improve the tolerance of yeast and rice to low temperature stress. OsFAD2 Structure, chromosomal location and expression of a rice gene encoding the microsome omega-3 fatty acid desaturase 1997 Plant Mol Biol Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan. The omega-3 fatty acid desaturases are membrane-bound enzymes catalyzing the conversion of linoleic acid to linolenic acid in lipids, and are located both in the microsome and plastid envelopes as two different isoforms. A cDNA encoding the microsome omega-3 fatty acid desaturase (OsFAD3) and the corresponding genomic clone were isolated from rice (Oryza sativa L.). The OsFAD3 gene was composed of 8 exons and 7 introns. A microsatellite was present in the second exon of the OsFAD3 gene, showing polymorphism between Indica and Japonica rice varieties. The mapping of this microsatellite showed that the OsFAD3 gene was located on chromosome 11. Expression of the OsFAD3 cDNA in tobacco hairy root tissues and subsequent analysis of fatty acid compositions demonstrated the activity of the microsome omega-3 fatty acid desaturase. The OsFAD3 mRNA was abundant in root tissues, but was hardly detectable in leaves. In root tissues, a high level of the OsFAD3 mRNA was observed at 15 degrees C and 20 degrees C, with its level decreasing markedly at temperatures below 10 degrees C. The accumulation of the OsFAD3 mRNA in leaf tissues remained at quite low levels, both at normal growth temperatures and at chilling temperatures. Similar temperature responses of the OsFAD3 gene were observed both in chilling- tolerant and in chilling-intolerant rice cultivars. OsFAD3 Identification and evaluation of omega-3 fatty acid desaturase genes for hyperfortifying alpha-linolenic acid in transgenic rice seed 2012 J Exp Bot Key Laboratory of Plant Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China. alpha-Linolenic acid (ALA) deficiency and a skewed of omega6:omega3 fatty acid ratio in the diet are a major explanation for the prevalence of cardiovascular diseases and inflammatory/autoimmune diseases. There is a need to enhance the ALA content and to reduce the ratio of linoleic acid (LA) to ALA. Six omega-3 (Delta-15) fatty acid desaturase (FAD) genes were cloned from rice and soybean. The subcellular localizations of the proteins were identified. The FAD genes were introduced into rice under the control of an endosperm-specific promoter, GluC, or a Ubi-1 promoter to evaluate their potential in increasing the ALA content in seeds. The ALA contents in the seeds of endoplasmic reticulum (ER)-localized GmFAD3-1 and OsFAD3 overexpression lines increased from 0.36 mg g(-)(1) to 8.57 mg g(-)(1) and 10.06 mg g(-)(1), respectively, which was 23.8- and 27.9-fold higher than that of non-transformants. The trait of high ALA content was stably inheritable over three generations. Homologous OsFAD3 is more active than GmFAD3-1 in catalysing LA conversion to ALA in rice seeds. Overexpression of ER-localized GmFAD3-2/3 and chloroplast-localized OsFAD7/8 had less effect on increasing the ALA content in rice seeds. The GluC promoter is advantageous compared with Ubi-1 in this experimental system. The enhanced ALA was preferentially located at the sn-2 position in triacylglycerols. A meal-size portion of high ALA rice would meet >80% of the daily adult ALA requirement. The ALA-rich rice could be expected to ameliorate much of the global dietary ALA deficiency. OsFAD3,OsFAD7,OsFAD8 Characterization of a rice (Oryza sativa L.) gene encoding a temperature-dependent chloroplast omega-3 fatty acid desaturase 2006 Biochem Biophys Res Commun Institute of Genetics, Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, School of Life Science, Fudan University, Shanghai, China. A cDNA, designated Osfad8, encoding a chloroplast omega-3 fatty acid desaturase responsible for trienoic fatty acid formation, was isolated from the leaves of Oryza sativa L. by RT-PCR. Southern blot hybridization indicated that a small gene family composed of two copies or closely linked genes exists. RNA in situ hybridization showed that the accumulation of Osfad8 mRNA was abundant in leaves but hardly detectable in roots. The Osfad8 transcript level in leaves was much higher at 15 degrees C than at normal temperature (25 degrees C). In situ hybridization also showed particularly prominent expression of Osfad8 in the palisade layer and spongy parenchyma cells of leaves when exposed to 15 degrees C conditions for 5 days and 10 days. Two transgenic lines (8S-52 and 8S-101) harboring the Osfad8 ORF in sense orientation under the control of the CaMV 35S promoter contained increased levels of hexadecatrienoic (16:3) and linolenic (18:3) fatty acids. When exposed to 2 degrees C for 7 days, the damage observed to the control plants was significantly alleviated in the 8S-52 and 8S-101 lines. The amounts of trienoic fatty acids in an Osfad8 antisense line (8A-35) declined 40.2% compared to the control plants. The 8A-35 plants survived after growth at 44 degrees C for 3 days while the control plants died. These data suggest that Osfad8 encodes a temperature-dependent chloroplast omega-3 fatty acid desaturase. OsFAD8 Effects of low temperature stress on rice (Oryza sativa L.) plastid ω-3 desaturase gene, OsFAD8 and its functional analysis using T-DNA mutants 2009 Plant Cell, Tissue and Organ Culture (PCTOC) Department of Biological Sciences, Pusan National University, Pusan, Republic of Korea Expression of two rice plastidial omega-3 desaturase genes, OsFAD7 and OsFAD8 under different temperatures has been investigated. Transcript levels of OsFAD7 increased at high temperature; while those of OsFAD8 increased at low temperatures. Fatty acid analysis of homozygous OsFAD8 T-DNA knockout mutant and wild type plants have exhibited changes in fatty acid composition after cold stress, thus confirming that OsFAD8 gene codes for omega-3 fatty acid desaturase activity at low temperature. Photosynthetic efficiency and recovery of OsFAD8 knockout mutants are significantly reduced after cold stress as compared to those of wild type plants. Characterization of OsFAD8 suggests that it has a functional role in maintaining levels of trienoic fatty acids and stress tolerance at low temperatures. OsFAD8 The F-box protein OsFBK12 targets OsSAMS1 for degradation and affects pleiotropic phenotypes, including leaf senescence, in rice 2013 Plant Physiol Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. Leaf senescence is related to the grain-filling rate and grain weight in cereals. Many components involved in senescence regulation at either the genetic or physiological level are known. However, less is known about molecular regulation mechanisms. Here, we report that OsFBK12 (an F-box protein containing a Kelch repeat motif) interacts with S-ADENOSYL-l-METHIONINE SYNTHETASE1 (SAMS1) to regulate leaf senescence and seed size as well as grain number in rice (Oryza sativa). Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays indicate that OsFBK12 interacts with Oryza sativa S-PHASE KINASE-ASSOCIATED PROTEIN1-LIKE PROTEIN and with OsSAMS1. Biochemical and physiological data showed that OsFBK12 targets OsSAMS1 for degradation. OsFBK12-RNA interference lines and OsSAMS1 overexpression lines showed increased ethylene levels, while OsFBK12-OX lines and OsSAMS1-RNA interference plants exhibited decreased ethylene. Phenotypically, overexpression of OsFBK12 led to a delay in leaf senescence and germination and increased seed size, whereas knockdown lines of either OsFBK12 or OsSAMS1 promoted the senescence program. Our results suggest that OsFBK12 is involved in the 26S proteasome pathway by interacting with Oryza sativa S-PHASE KINASE-ASSOCIATED PROTEIN1-LIKE PROTEIN and that it targets the substrate OsSAMS1 for degradation, triggering changes in ethylene levels for the regulation of leaf senescence and grain size. These data have potential applications in the molecular breeding of rice. OsFBK12,OsSAMS1 A rice F-box gene, OsFbx352, is involved in glucose-delayed seed germination in rice 2012 J Exp Bot State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, the Chinese Academy of Sciences,Beijing 100093 PR China. F-box proteins play diverse roles in regulating numerous physiological processes in plants. This study isolated a gene (OsFbx352) from rice encoding an F-box domain protein and characterized its role in seed germination. Expression of OsFbx352 was upregulated by abscisic acid (ABA). The transcripts of OsFbx352 were increased upon imbibition of rice seeds and the increase was markedly suppressed by glucose. Germination of seeds with overexpression of OsFbx352 was less suppressed by glucose than that of wild-type seeds, while glucose had greater inhibition for germination of seeds with knockdown of OsFbx352 by RNA interference (RNAi) than that of wild-type seeds. The differential response of germination of the transgenic and wild-type seeds to glucose may be accounted for by differences in ABA content among overexpressing, RNAi, and wild-type seeds such that overexpression of OsFbx352 and knockdown of OsFbx352 led to lower and higher ABA contents, respectively, than that of wild-type seeds in the presence of glucose. Overexpression of OsFbx352 led to a reduction in expression of genes responsible for ABA synthesis (OsNced2, OsNced3) and an increase in expression of genes encoding ABA catabolism (OsAba-ox2, OsAba-ox3) in the presence of glucose. These findings indicate that OsFbx352 plays a regulatory role in the regulation of glucose-induced suppression of seed germination by targeting ABA metabolism. OsFbx352,OsNCED2,OsNCED3 Overexpression of the rFCA RNA recognition motif affects morphologies modifications in rice (Oryza sativa L.) 2007 Biosci Rep Institute of Genetics, State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, PR China. RNA recognition motifs as important regulators of gene expression are highly conserved in animals and plants. The FCA floral promotion gene in Arabidopsis encodes a protein, containing two RNA recognition motifs (RRM) and a WW protein interaction domain. Here we isolated FCA cDNA from rice. FCA in rice (rFCA) was homologous to FCA-gamma of Arabidopsis and contained conserved domains. To investigate the function of RRM domain, fragment RRM1 and RRM2 of rFCA were introduced into rice subspecies Oryza sativa L. subsp. Indica var. 9311 and another rice subspecies Oryza sativa L. subsp. Japonica var. zhonghua11 transformation. Two transgenic lines exhibited similar phenotypes, flowering time delay, seed size and cell volume of transgenic plants was increased. These results showed that constitutive expression of RRMs could regulate cellular size. The patterns of overexpression of two RRM domains and their similar morphologies indicate they may play a same role. FCA|OsFCA Alternative splicing and expression analysis of OsFCA (FCA in Oryza sativa L.), a gene homologous to FCA in Arabidopsis 2006 DNA Seq Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China. A subtracted cDNAs library was constructed using rice (Oryza sativa L.) calli cDNA as driver and differentiating calli cDNA as tester. A novel gene homologous with FCA in Arabidopsis was cloned from rice by screening the SSH (suppression subtractive hybridization) library followed by RACE. Four alternative transcripts of OsFCA were cloned from the leaves of rice, and designated as OsFCA-1, OsFCA-2, OsFCA-3 and OsFCA-4 respectively. OsFCA-1 was homologous to FCA-gamma of Arabidopsis and contained several conserved domains (two RNA Recognition Motifs and one WW-domain). OsFCA-2 was 102 bp shorter than OsFCA-1 which caused the WW-domain deletion. The proteins encoded by OsFCA-3 and OsFCA-4 were 101 amino acids shorter than OsFCA-1 at the N-terminal which is a glycine-rich region. The fluorescence quantitative PCR analysis showed that the mRNA of OsFCA-1 is the most abundant in the four splicing variants of rice FCA, and its expression level is much higher in differentiating calli than in calli. The expression of OsFCA-1 is steady in the leaves of three different stage, but up-regulated in young spikelet of primary branch-differentiating stage and down-regulated in young spikelet of pistil and stamen-differentiating stage. FCA|OsFCA OsFY, a Homolog of AtFY, Encodes a Protein that Can Interact with OsFCA-γ in Rice (Oryza sativa L.) 2006 Acta Biochimica et Biophysica Sinica Shanghai Key Laboratory of Bio-energy Crop, School of Life Sciences, Shanghai University, Shanghai 200444, China. FCA and FY are flowering time related genes involved in the autonomous flowering pathway in Arabidopsis. FCA interacts with FY to regulate the alternative processing of FCA pre-mRNA. The FCA/FY interaction is also required for the regulation of FLC expression, a major floral repressor in Arabidopsis. However, it is not clear if the regulation of this autonomous flowering pathway is also present in monocot plants, such as rice. Recently, alternative RNA processing of OsFCA was observed in rice, which strongly suggested the existence of an autonomous flowering pathway in rice. In this work, we cloned the cDNA of the autonomous flowering pathway gene OsFY from rice. The predicted OsFY protein contained a conserved 7 WD-repeat region and at least two Pro-Pro-Leu-Pro motifs compared to Arabidopsis FY. The protein-protein interaction between OsFY and OsFCA-gamma, the key feature of their gene function, was also demonstrated using the yeast two-hybrid system. The GenBank database search provided evidence of expression for other autonomous pathway gene homologs in rice. These results indicate that the autonomous flowering pathway is present in monocots, and the regulation through FY and FCA interaction is conserved between monocots and dicots. FCA|OsFCA,OsFY Conservation and divergence of FCA function between Arabidopsis and rice 2005 Plant Mol Biol School of Life sciences and Biotechnology, Korea University, 136-701 Seoul, Korea. Although several genes have been identified in rice which are functionally equivalent to the flowering time genes in Arabidopsis, primarily genes involved in the photoperiod pathway, little data is available regarding the genes that function in the autonomous pathway in rice. In order to acquire further insight into the control of heading dates in rice, we isolated and conducted an expression analysis on OsFCA, which exhibited 38% sequence homology with Arabidopsis FCA. The N-terminal region of the OsFCA protein appears to be unusually rich in glycine-residues, unlike the N-terminal region found in FCA. However, the genetic structure of OsFCA is, in general, similar to that of FCA. RT-PCR and in silico analyses also showed that alternative splicing and polyadenylation at intron3 were conserved in the genetic expression of OsFCA. We were able to detect alpha, beta, and gamma transcripts, but not the delta transcript, of the OsFCA gene. The beta and gamma transcripts of the OsFCA gene were detected via Northern analysis in the leaves, roots, and flowers of the plant. Flowers in younger stages exhibited higher transcript levels. These data suggest that intron3 may constitute a primary control point in the OsFCA pre-mRNA processing of rice. The overexpression of OsFCA cDNA, driven by the 35S promoter, was shown to partially rescue the late flowering phenotype of the fca mutant, suggesting that the functions of the OsFCA and the FCA are partially overlapped, despite the lack of an apparent FLC homologue in the rice genome. The constitutive expression of OsFCA resulted in no downregulation of FLC, but did result in the weak upregulation of SOC1 in the transgenic Arabidopsis. OsFCA overexpression did not result in a reduction of the gamma transcript levels of FCA in the transgenic Arabidopsis either, thereby suggesting that OsFCA had no effects on the autoregulation of Arabidopsis FCA. All of these results imply conservation and divergence in the functions of FCA between rice and Arabidopsis. FCA|OsFCA Plant homologue of flap endonuclease-1: molecular cloning, characterization, and evidence of expression in meristematic tissues 2000 Plant Mol Biol Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, Chiba-ken, Japan. Flap endonuclease-1 (FEN-1) is an important enzyme involved in DNA replication and repair. We isolated a 1.4 kb cDNA from rice (Oryza sativa), termed OsFEN-1, encoding a protein which shows homology with the eukaryotic FEN-1 proteins. OsFEN-1 protein was overexpressed in Escherichia coli and purified to near homogeneity. DNA cleavage analysis using different branched DNA structures indicated that OsFEN-1 protein possesses both 5'-flap endonuclease and 5' to 3' double-stranded DNA exonuclease activities. OsFEN-1 protein incises a 5'-flap and 5'-pseudo Y structure one base 3' of the branched point in the duplex region. The enzymatic properties indicated that we succeeded in obtaining the gene and the protein of a plant counterpart of FEN-1. OsFEN-1 transcripts were expressed strongly in proliferating tissues such as root tips and young leaves that contain root apical meristem and marginal meristem, respectively. No expression was detected in mature leaves although the leaves were exposed to UV. We analyzed the spatial distribution pattern of OsFEN-1 transcripts by in situ hybridization. In the shoot apex, OsFEN-1 mRNA was abundant in the shoot apical meristem, tiller bud, leaf primordia, ligule primordia and marginal meristem of young leaves. In the roots, the transcript accumulated to high levels in the root apical meristem. Our results indicate that OsFEN-1 is expressed in tissues rich in proliferating cells, and its expression may be required for cell growth and organ formation. OsFEN-1 Differential regulation of the two rice ferritin genes (OsFER1 and OsFER2) 2009 Plant Science Universidade Federal do Rio Grande do Sul, Centro de Biotecnologia, Porto Alegre, RS, Brazil Iron is essential to plants. However, when free and in excess, iron can catalyze the formation of oxygen free radicals. Ferritin, a protein capable of storing up to 4500 atoms of iron, can act as an iron buffer inside plant cells. Using a strategy based in amplicon size difference, we were able to analyze the expression profile of the two rice ferritin genes (OsFER1 and OsFER2). Both genes are expressed, although with different regulation and organ distribution. Exposure to copper, Paraquat, SNP and excess iron led to accumulation of ferritin mRNA, remarkably of OsFER2. The iron-induced expression was abolished by treatment with GSH, indicating that the induction observed is dependent of an oxidative step. OsFER2 mRNA levels in rice flag leaves and panicles at different reproductive stages were higher than OsFER1 mRNA levels. No ferritin mRNA was detected in rice seeds. However, imbibition under light led to ferritin expression, which was abolished when seeds were kept in the dark, suggesting a light-regulated induction. Ferritin mRNA accumulation was seen in the dark only when seeds were germinated in the presence of externally supplied iron. We suggest that the primary role of rice ferritins is related to defense against iron-mediated oxidative stress. OsFER1,OsFER2 Formin homology 1 (OsFH1) regulates root-hair elongation in rice (Oryza sativa) 2013 Planta Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center (PMBBRC), Gyeongsang National University, Jinju, 660-701, Korea. The outgrowth of root hairs from the epidermal cell layer is regulated by a strict genetic regulatory system and external growth conditions. Rice plants cultivated in water-logged paddy land are exposed to a soil ecology that differs from the environment surrounding upland plants, such as Arabidopsis and maize. To identify genes that play important roles in root-hair growth, a forward genetics approach was used to screen for short-root-hair mutants. A short-root-hair mutant was identified, and the gene was isolated using map-based cloning and sequencing. The mutant harbored a point mutation at a splicing acceptor site, which led to truncation of OsFH1 (rice formin homology 1). Subsequent analysis of two additional T-DNA mutants verified that OsFH1 is important for root-hair elongation. Further studies revealed that the action of OsFH1 on root-hair growth is dependent on growth conditions. The mutant Osfh1 exhibited root-hair defects when roots were grown submerged in solution, and mutant roots produced normal root hairs in the air. However, root-hair phenotypes of mutants were not influenced by the external supply of hormones or carbohydrates, a deficiency of nutrients, such as Fe or P i , or aeration. This study shows that OsFH1 plays a significant role in root-hair elongation in a growth condition-dependent manner. OsFH1 Identification and characterization of an epi-allele of FIE1 reveals a regulatory linkage between two epigenetic marks in rice 2012 Plant Cell State Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, China. DNA methylation and histone H3 Lys 9 dimethylation (H3K9me2) are important epigenetic repression marks for silencing transposons in heterochromatin and for regulating gene expression. However, the mechanistic relationship to other repressive marks, such as histone H3 Lys 27 trimethylation (H3K27me3) is unclear. FERTILIZATION-INDEPENDENT ENDOSPERM1 (FIE1) encodes an Esc-like core component of the Polycomb repressive complex 2, which is involved in H3K27me3-mediated gene repression. Here, we identify a gain-of-function epi-allele (Epi-df) of rice (Oryza sativa) FIE1; this allele causes a dwarf stature and various floral defects that are inherited in a dominant fashion. We found that Epi-df has no changes in nucleotide sequence but is hypomethylated in the 5' region of FIE1 and has reduced H3K9me2 and increased H3K4me3. In Epi-df, FIE1 was ectopically expressed and its imprinting was disrupted. FIE1 interacted with rice Enhancer of Zeste homologs, consistent with its role in H3K27me3 repression. Ectopic expression of FIE1 in Epi-df resulted in alteration of H3K27me3 levels in hundreds of genes. In summary, this work identifies an epi-allele involved in H3K27me3-mediated gene repression that itself is highly regulated by DNA methylation and histone H3K9me2, thereby shedding light on the link between DNA methylation and histone methylation, the two important epigenetic marks regulating rice development. OsFIE1|Epi-df Involvement of rice Polycomb protein OsFIE2 in plant growth and seed size 2012 Plant Biotechnology Reports Bio-crop Development Division, Department of Agricultural Bio-resources, National Academy of Agricultural Science, RDA, Suwon, 441-701, Republic of Korea Seed development is a complex but orchestrated process that requires the fine-tuning of parentally governed gene expression, which is regulated by Polycomb proteins. Over the last decade, various Polycomb proteins have been identified and functionally characterized in plants, and it has been found that they form the PRC2 suppressor complex, which is involved in various developmental programs, including seed development. In this study, the function of the rice fertilization-independent endosperm gene OsFIE2, which expresses a protein homologous to the Arabidopsis Polycomb protein FIE, was characterized. We also characterized OsEZ1/OsiEZ1, another key component of the PRC2 complex. Both the OsFIE2 and OsEZ11 genes are strongly expressed in leaf and stem compared to other tissues, including root, anther, ovary, and ovule. We further examined whether OsFIE2 interacted with OsEZ1 using a yeast two-hybrid system. Interaction analysis showed that OsFIE2 interacted with OsEZ1 but not with Arabidopsis MEA protein. To examine the physiological roles of OsFIE2, 35S:OsFIE2 Arabidopsis lines were generated. Transgenic plants with 35S:OsFIE2 grew faster than wild-type plants during early development. Importantly, they produced bigger seed than the wild type, indicating that OsFIE2 may play an important role in seed size. In addition, we generated pOsFIE2:GUS plants to examine the spatial expression pattern of OsFIE2. GUS expression was detected in cotyledon but not in any other tissues, suggesting that OsFIE2 expression may be required to suppress homeotic genes in cotyledon. OsFIE2,OsiEZ1|OsSET1 Polycomb group gene OsFIE2 regulates rice (Oryza sativa) seed development and grain filling via a mechanism distinct from Arabidopsis 2013 PLoS Genet Department of Biochemistry and Molecular Biology, Mississippi State University, Mississippi State, Mississippi, United States of America. Cereal endosperm represents 60% of the calories consumed by human beings worldwide. In addition, cereals also serve as the primary feedstock for livestock. However, the regulatory mechanism of cereal endosperm and seed development is largely unknown. Polycomb complex has been shown to play a key role in the regulation of endosperm development in Arabidopsis, but its role in cereal endosperm development remains obscure. Additionally, the enzyme activities of the polycomb complexes have not been demonstrated in plants. Here we purified the rice OsFIE2-polycomb complex using tandem affinity purification and demonstrated its specific H3 methyltransferase activity. We found that the OsFIE2 gene product was responsible for H3K27me3 production specifically in vivo. Genetic studies showed that a reduction of OsFIE2 expression led to smaller seeds, partially filled seeds, and partial loss of seed dormancy. Gene expression and proteomics analyses found that the starch synthesis rate limiting step enzyme and multiple storage proteins are down-regulated in OsFIE2 reduction lines. Genome wide ChIP-Seq data analysis shows that H3K27me3 is associated with many genes in the young seeds. The H3K27me3 modification and gene expression in a key helix-loop-helix transcription factor is shown to be regulated by OsFIE2. Our results suggest that OsFIE2-polycomb complex positively regulates rice endosperm development and grain filling via a mechanism highly different from that in Arabidopsis. OsFIE2 A Rice Immunophilin Gene, OsFKBP16-3, Confers Tolerance to Environmental Stress in Arabidopsis and Rice 2013 Int J Mol Sci Green Bio Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-506, Korea. hscho@kribb.re.kr. The putative thylakoid lumen immunophilin, FKBP16-3, has not yet been characterized, although this protein is known to be regulated by thioredoxin and possesses a well-conserved CxxxC motif in photosynthetic organisms. Here, we characterized rice OsFKBP16-3 and examined the role of this gene in the regulation of abiotic stress in plants. FKBP16-3s are well conserved in eukaryotic photosynthetic organisms, including the presence of a unique disulfide-forming CxxxC motif in their N-terminal regions. OsFKBP16-3 was mainly expressed in rice leaf tissues and was upregulated by various abiotic stresses, including salt, drought, high light, hydrogen peroxide, heat and methyl viologen. The chloroplast localization of OsFKBP16-3-GFP was confirmed through the transient expression of OsFKBP16-3 in Nicotiana benthamiana leaves. Transgenic Arabidopsis and transgenic rice plants that constitutively expressed OsFKBP16-3 exhibited increased tolerance to salinity, drought and oxidative stresses, but showed no change in growth or phenotype, compared with vector control plants, when grown under non-stressed conditions. This is the first report to demonstrate the potential role of FKBP16-3 in the environmental stress response, which may be regulated by a redox relay process in the thylakoid lumen, suggesting that artificial regulation of FKBP16-3 expression is a candidate for stress-tolerant crop breeding. OsFKBP16-3 Isolation and characterization of two fructokinase cDNA clones from rice 2003 Phytochemistry State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, 310029, PR China. Two cDNA clones, OsFKI and OsFKII, encoding fructokinase (EC 2.7.1.4) were isolated from immature seeds of rice (Oryza sativa L.) by PCR. OsFKI cDNA encoded a deduced protein of 323 amino acids that was 59-71% identical to previously characterized plant fructokinases. In contrast, OsFKII cDNA encoded a deduced protein of 336 amino acids that shared only 64% amino acid identity with OsFKI. The deduced proteins both possessed an ATP-binding motif and putative substrate recognition site sequences that were previously identified in bacterial fructokinases. Genomic DNA blot analysis also revealed that each fructokinase gene exists as a single copy in the rice genome. The identity of OsFKI and OsFKII as fructokinases was confirmed by the expression of enzyme activity in E. coli. Although both OsFKI and OsFKII utilized fructose as substrate, only OsFKII activity was strongly inhibited at a high fructose concentration. The mRNA corresponding to OsFKII accumulated at high levels in developing rice grains, whereas there were only low levels of OsFKI transcripts in immature seeds. These results indicate that fructokinase in rice endosperm is encoded by two divergent genes, which play different roles in rice grains for starch storage based on their sensitivity to substrate inhibition and level of transcripts in endosperm. OsFKI,OsFKII An XA21-associated kinase (OsSERK2) regulates immunity mediated by the XA21 and XA3 immune receptors 2014 Mol Plant Department of Plant Pathology and the Genome Center, University of California, Davis, CA 95616, USA. The rice XA21 immune receptor kinase and the structurally related XA3 receptor confer immunity to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight. Here we report the isolation of OsSERK2 (rice somatic embryogenesis receptor kinase 2) and demonstrate that OsSERK2 positively regulates immunity mediated by XA21 and XA3 as well as the rice immune receptor FLS2 (OsFLS2). Rice plants silenced for OsSerk2 display altered morphology and reduced sensitivity to the hormone brassinolide. OsSERK2 interacts with the intracellular domains of each immune receptor in the yeast two-hybrid system in a kinase activity-dependent manner. OsSERK2 undergoes bidirectional transphosphorylation with XA21 in vitro and forms a constitutive complex with XA21 in vivo. These results demonstrate an essential role for OsSERK2 in the function of three rice immune receptors and suggest that direct interaction with the rice immune receptors is critical for their function. Taken together, our findings suggest that the mechanism of OsSERK2-meditated regulation of rice XA21, XA3, and FLS2 differs from that of AtSERK3/BAK1-mediated regulation of Arabidopsis FLS2 and EFR. OsFLS2,OsSERK2,xa21,Xa26|Xa3 Analysis of flagellin perception mediated by flg22 receptor OsFLS2 in rice 2008 Mol Plant Microbe Interact Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5, Takayama Ikoma, Nara 630-0191, Japan. Plants have sensitive perception systems that recognize various pathogen-derived molecules. We previously reported that rice detects flagellin from a rice-incompatible strain of gram-negative phytopathogenic bacterium, Acidovorax avenae, which induces subsequent immune responses involving cell death. The mechanism of flagellin perception in rice, however, has remained obscure. In this study, we found that flg22, a peptide derived from the flagellin N-terminus, induced weak immune responses without cell death in cultured rice cells. To elucidate the mechanism by which flg22 induced signaling in rice, we characterized OsFLS2, the rice ortholog of AtFLS2, which mediates flg22 perception. Heterologous expression of OsFLS2 functions in Arabidopsis, showing the conservation of the flg22 signaling pathway across divergent plant taxa. OsFLS2-overexpressing rice cultured cells generated stronger immune responses with the induction of cell death following stimulation with flg22 and flagellin. However, examination of the growth rate of the compatible strain in inoculated OsFLS2-overexpressing rice could not confirm bacterial growth suppression compared with wild-type rice. These results suggest that rice possesses a conserved flagellin perception system utilizing the FLS2 receptor which, when upregulated, hardly affects resistance against compatible A. avenae. OsFLS2 The OsFOR1 gene encodes a polygalacturonase-inhibiting protein (PGIP) that regulates floral organ number in rice 2003 Plant Mol Biol National Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Korea We have isolated a cDNA clone, OsFOR1, from the immature panicles of rice. The OsFOR1 (Oryza sativa floral organ regulator 1) gene encodes a protein that contains a leucine-rich repeat (LRR) domain. This domain comprises 10 tandem repeats of a canonical 24-amino acid LRR sequence. The structure and the number of LRRs for OsFOR1 are similar to those of polygalacturonase-inhibiting proteins (PGIPs) from various other plant species. Moreover, the OsFOR1 recombinant protein, when fused to maltose-binding protein (MBP), shows PGIP activity against the Aspergillus niger polygalacturonase. OsFOR1 is highly expressed in the calli and immature and mature panicles, while detectable at only low levels in seedling roots and mature stems. In situ hybridization experiments showed the transcripts of OsFOR1 are present in young spikelet primordia and in almost all of the young floral organs. Transgenic approaches were used to study in vivo functioning. Antisense expression of OsFOR1 resulted in an increase in the numbers of floral organs, including the stamen, carpel, palea/lemma, stigma, and lodicule. OsFOR1 transcript was not detected in the frizzy panicle mutant, which is defective in its spikelet formation but normal in inflorescence-meristem initiation and maintenance. Therefore, we suggest that OsFOR1 plays a role in the formation and/or maintenance of floral organ primordia. OsFOR1|PGIP Two solanesyl diphosphate synthases with different subcellular localizations and their respective physiological roles in Oryza sativa 2010 J Exp Bot Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji, Kyoto, Japan. Long chain prenyl diphosphates are crucial biosynthetic precursors of ubiquinone (UQ) in many organisms, ranging from bacteria to humans, as well as precursors of plastoquinone in photosynthetic organisms. The cloning and characterization of two solanesyl diphosphate synthase genes, OsSPS1 and OsSPS2, in Oryza sativa is reported here. OsSPS1 was highly expressed in root tissue whereas OsSPS2 was found to be high in both leaves and roots. Enzymatic characterization using recombinant proteins showed that both OsSPS1 and OsSPS2 could produce solanesyl diphosphates as their final product, while OsSPS1 showed stronger activity than OsSPS2. However, an important biological difference was observed between the two genes: OsSPS1 complemented the yeast coq1 disruptant, which does not form UQ, whereas OsSPS2 only very weakly complemented the growth defect of the coq1 mutant. HPLC analyses showed that both OsSPS1 and OsSPS2 yeast transformants produced UQ9 instead of UQ6, which is the native yeast UQ. According to the complementation study, the UQ9 levels in OsSPS2 transformants were much lower than that of OsSPS1. Green fluorescent protein fusion analyses showed that OsSPS1 localized to mitochondria, while OsSPS2 localized to plastids. This suggests that OsSPS1 is involved in the supply of solanesyl diphosphate for ubiquinone-9 biosynthesis in mitochondria, whereas OsSPS2 is involved in providing solanesyl diphosphate for plastoquinone-9 formation. These findings indicate that O. sativa has a different mechanism for the supply of isoprenoid precursors in UQ biosynthesis from Arabidopsis thaliana, in which SPS1 provides a prenyl moiety for UQ9 at the endoplasmic reticulum. OsFPPS1|FPPS1|FPPS,OsSPS2,OsFPPS2|FPPS2,OsFPPS3,OsGGPPS1,OsGGPPS2 Localization of farnesyl diphosphate synthase in chloroplasts 1999 Plant Cell Physiol BioScience Center, Nagoya University, Japan. The subcellular localization of plant farnesyl diphosphate synthase (FPPS) was examined. Immunocytochemical staining using anti-FPPS1 antibody followed by electron microscopy showed that FPPS1 was localized to chloroplasts of rice mesophyll cells. Subcellular fractions from wheat leaves were examined by immunoblot analysis. FPPS was detected in the chloroplast fraction in wheat, and was protected from proteolysis following trypsin treatment of chloroplasts. FPPS was also detected in the chloroplast fraction of a dicot plant, tobacco. OsFPPS1|FPPS1|FPPS,OsFPPS2|FPPS2 OsFRDL1 is a citrate transporter required for efficient translocation of iron in rice 2009 Plant Physiol Research Institute for Bioresources, Okayama University, Kurashiki 710-0046, Japan. Multidrug and toxic compound extrusion (MATE) transporters represent a large family in plants, but their functions are poorly understood. Here, we report the function of a rice (Oryza sativa) MATE gene (Os03g0216700, OsFRDL1), the closest homolog of barley (Hordeum vulgare) HvAACT1 (aluminum [Al]-activated citrate transporter 1), in terms of metal stress (iron [Fe] deficiency and Al toxicity). This gene was mainly expressed in the roots and the expression level was not affected by either Fe deficiency or Al toxicity. Knockout of this gene resulted in leaf chlorosis, lower leaf Fe concentration, higher accumulation of zinc and manganese concentration in the leaves, and precipitation of Fe in the root's stele. The concentration of citrate and ferric iron in the xylem sap was lower in the knockout line compared to the wild-type rice. Heterologous expression of OsFRDL1 in Xenopus oocytes showed transport activity for citrate. Immunostaining showed that OsFRDL1 was localized at the pericycle cells of the roots. On the other hand, there was no difference in the Al-induced secretion of citrate from the roots between the knockout line and the wild-type rice. Taken together, our results indicate that OsFRDL1 is a citrate transporter localized at the pericycle cells, which is necessary for efficient translocation of Fe to the shoot as a Fe-citrate complex. OsFRDL1 A rice FRD3-like (OsFRDL1) gene is expressed in the cells involved in long-distance transport 2004 Soil Science and Plant Nutrition Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences , The University of Tokyo , Tokyo, 113-8657, Japan We identified four putative AtFRD3-like genes (OsFRDL) in the rice genome that exhibited 39.1 to 56.7% amino acid sequence similarities to Arabidopsis FRD3. Of these, we cloned three OsFRDL genes from a cDNA library prepared from iron-deficient rice roots: OsFRDL1, OsFRDL2, and OsFRDL3. OsFRDL1 was expressed weakly in Fe-sufficient roots, and slight expression was induced in the roots of Fe-deficient plants. OsFRDL2 was expressed constitutively in both roots and leaves, and Fe deficiency reduced its expression in leaves. OsFRDL3 was expressed in leaves, but not in roots; Fe deficiency induced slight expression in leaves. An OsFRDL1-sGFP fusion protein was localized in the plasma membrane in onion epidermal cells. The promoter GUS analysis showed that OsFRDL1 was localized in the cells involved in long-distance transport, in both Fe-sufficient and Fe-deficient plants. Furthermore, OsFRDL1 expression was observed during the reproductive stage. These results suggest that OsFRDL1 is a transporter that resides in the plasma membrane of cells involved in long-distant transport. OsFRDL1 A rice beta-1,3-glucanase gene Osg1 is required for callose degradation in pollen development 2011 Planta Key Laboratory of Ministry of Education for Plant Development Biology, College of Life Sciences, Wuhan University, Wuhan 430072, People's Republic of China. Plant beta-1,3-glucanases are involved in plant defense and development. In rice (Oryza sativa), 14 genes encoding putative beta-1,3-glucanases have been isolated and sequenced. However, only limited information is available on the function of these beta-1,3-glucanase genes. In this study, we report a detailed functional characterization of one of these genes, Osg1. Osg1 encodes a glucanase carrying no C-terminal extension. Osg1 was found to be expressed throughout the plant and highly expressed in florets, leaf sheaths, and leaf blades. Investigations using real-time PCR, immunocytochemical analysis, and a GUS-reporter gene driven by the Osg1 promoter indicated that Osg1 was mainly expressed at the late meiosis, early microspore, and middle microspore stages in the florets. To elucidate the role of Osg1, we suppressed expression of the Osg1 gene by RNA interference in transgenic rice. The silencing of Osg1 resulted in male sterility. The pollen mother cells appeared to be normal in Osg1-RI plants, but callose degradation was disrupted around the microspores in the anther locules of the Osg1-RI plants at the early microspore stage. Consequently, the release of the young microspores into the anther locules was delayed, and the microspores began to degenerate later. These results provide evidence that Osg1 is essential for timely callose degradation in the process of tetrad dissolution. Osg1 Molecular Cloning and Characterization of a Novel beta-1,3-Glucanase Gene from Rice 2014 Bioscience, Biotechnology and Biochemistry Plant Physiology Laboratory, National Agricultural Research Center for Tohoku Region Beta-1,3-glucanases are referred to as pathogenesis-related proteins and they are also involved in several developmental processes. We isolated a cDNA for beta-1,3-glucanase from rice anther and named it Oryza sativa glucanase 1 (Osg1). Phylogenetic analysis showed that Osg1 belonged to monocotyledonous endo-beta-1,3-glucanase subgroup A. RT-PCR analysis revealed that Osg1 transcripts were present in leaves, roots, and anthers. Osg1 The rare sugar D-allose acts as a triggering molecule of rice defence via ROS generation 2013 J Exp Bot Faculty of Agriculture, Rare Sugar Research Center, and Gene Research Center, Kagawa University, Miki, Kagawa 761-0795, Japan. Only D-allose, among various rare monosaccharides tested, induced resistance to Xanthomonas oryzae pv. oryzae in susceptible rice leaves with defence responses: reactive oxygen species, lesion mimic formation, and PR-protein gene expression. These responses were suppressed by ascorbic acid or diphenylene iodonium. Transgenic rice plants overexpressing OsrbohC, encoding NADPH oxidase, were enhanced in sensitivity to D-allose. D-Allose-mediated defence responses were suppressed by the presence of a hexokinase inhibitor. 6-Deoxy-D-allose, a structural derivative of D-allose unable to be phosphorylated, did not confer resistance. Transgenic rice plants expressing Escherichia coli AlsK encoding D-allose kinase to increase D-allose 6-phosphate synthesis were more sensitive to D-allose, but E. coli AlsI encoding D-allose 6-phosphate isomerase expression to decrease D-allose 6-phosphate reduced sensitivity. A D-glucose 6-phosphate dehydrogenase-defective mutant was also less sensitive, and OsG6PDH1 complementation restored full sensitivity. These results reveal that a monosaccharide, D-allose, induces rice resistance to X. oryzae pv. oryzae by activating NADPH oxidase through the activity of D-glucose 6-phosphate dehydrogenase, initiated by hexokinase-mediated conversion of D-allose to D-allose 6-phosphate, and treatment with D-allose might prove to be useful for reducing disease development in rice. OsG6PDH1,OsG6PDH2,OsG6PDH3,OsG6PDH5 OsGA20ox1, a candidate gene for a major QTL controlling seedling vigor in rice 2012 Theor Appl Genet Iwate Agricultural Research Center, 20-1, Narita, Kitakami, Iwate 024-0003, Japan. a-abe@pref.iwate.jp Seedling vigor is among the major determinants of stable stand establishment in direct-seeded rice (Oryza sativa L.) in temperate regions. Quantitative trait loci (QTL) for seedling vigor were identified using 250 recombinant inbred lines (RILs) derived from a cross between two japonica rice cultivars Kakehashi and Dunghan Shali. Seedling heights measured at 14 days after sowing were 20.3 and 29.4 cm for Kakehashi and Dunghan Shali, respectively. For the RILs, the height ranged from 14.1 to 31.7 cm. Four putative QTLs associated with seedling height were detected. qPHS3-2, the major QTL that was located on the long arm of chromosome 3, accounted for 26.2 % of the phenotypic variance. Using progeny of the near isogenic lines (NILs) produced by the backcross introduction of a chromosome segment carrying this major QTL into an elite cultivar Iwatekko, we fine-mapped qPHS3-2 to a 81-kb interval between two markers, ID_CAPS_01 and RM16227. Within this mapped region, we identified the gene OsGA20ox1, which is related to gibberellin (GA) biosynthesis. The relative expression levels of GA20ox1 in seedlings of Dunghan Shali and NILs were higher than that of Iwatekko. Concomitantly, the amount of endogenous active GA was higher in Dunghan Shali and the NILs compared to the level detected in Iwatekko. These results indicate that OsGA20ox1 is a strong candidate gene for major QTL controlling seedling vigor in rice. OsGA20ox1 A role of OsGA20ox1 , encoding an isoform of gibberellin 20-oxidase, for regulation of plant stature in rice 2004 Plant Mol Biol Laboratory of Bio-control, Graduate School of Natural Science and Technology, Niigata University, 2-8050 Ikarashi, Niigata 950-2181, Japan. Gibberellin (GA) 20-oxidase (GA20ox) is a key enzyme that normally catalyzes the penultimate steps in GA biosynthesis. One of the GA20ox genes in rice (Oryza sativa L.), OsGA20ox2 ( SD1 ), is well known as the "Green Revolution gene", and loss-of function mutation in this locus causes semi-dwarfism. Another GA20ox gene, OsGA20ox1, has also been identified, but its contribution to plant stature has remained unclear because no suitable mutants have been available. We isolated a mutant, B142, tagged with a T-DNA containing three CaMV 35S promoters, which showed a tall, GA-overproduction phenotype. The final stature of the B142 mutant reflects internode overgrowth and is approximately twice that of its wild-type parent. This mutant responds to application of both GA3 and a GA biosynthesis inhibitor, indicating that it is a novel tall mutant of rice distinct from GA signaling mutants such as slr1 . The integrated T-DNAs, which contain three CaMV 35S promoters, are located upstream of the OsGA20ox1 open reading frame (ORF) in the B142 mutant genome. Analysis of mRNA and the endogenous GAs reveal that biologically active GA level is increased by up-regulation of the OsGA20ox1 gene in B142. Introduction of OsGA20ox1 cDNA driven by 35S promoter into the wild type phenocopies the morphological characteristics of B142. These results indicate that the elongated phenotype of the B142 mutant is caused by up-regulation of the OsGA20ox1 gene. Moreover, the final stature of rice was reduced by specific suppression of the OsGA20ox1 gene expression. This result indicates that not only OsGA20ox2 but also OsGA20ox1 affects plant stature. OsGA20ox1,sd1|GA20ox2 Gibberellin 20-oxidase gene OsGA20ox3 regulates plant stature and disease development in rice 2013 Mol Plant Microbe Interact State Key Laboratory of Agrobiotechnology, China Agricultural University, Yuanmingyan West Road 2, Beijing 100193, China. Gibberellin (GA) 20-oxidase (GA20ox) catalyses consecutive steps of oxidation in the late part of the GA biosynthetic pathway. A T-DNA insertion mutant (17S-14) in rice, with an elongated phenotype, was isolated. Analysis of the flanking sequences of the T-DNA insertion site revealed that an incomplete T-DNA integration resulted in enhanced constitutively expression of downstream OsGA20ox3 in the mutant. The accumulation of bioactive GA(1) and GA(4) were increased in the mutant in comparison with the wild-type plant. Transgenic plants overexpressing OsGA20ox3 showed phenotypes similar to those of the 17S-14 mutant, and the RNA interference (RNAi) lines that had decreased OsGA20ox3 expression exhibited a semidwarf phenotype. Expression of OsGA20ox3 was detected in the leaves and roots of young seedlings, immature panicles, anthers, and pollens, based on beta-glucuronidase (GUS) activity staining in transgenic plants expressing the OsGA20ox3 promoter fused to the GUS gene. The OsGA20ox3 RNAi lines showed enhanced resistance against rice pathogens Magnaporthe oryzae (causing rice blast) and Xanthomonas oryzae pv. oryzae (causing bacterial blight) and increased expression of defense-related genes. Conversely, OsGA20ox3-overexpressing plants were more susceptible to these pathogens comparing with the wild-type plants. The susceptibility of wild-type plants to X. oryzae pv. oryzae was increased by exogenous application of GA(3) and decreased by S-3307 treatment. Together, the results provide direct evidence for a critical role of OsGA20ox3 in regulating not only plant stature but also disease resistance in rice. OsGA20ox3|GA20ox3 Reduction of gibberellin by low temperature disrupts pollen development in rice 2014 Plant Physiol Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan. Microsporogenesis in rice (Oryza sativa) plants is susceptible to moderate low temperature (LT; approximately 19°C) that disrupts pollen development and causes severe reductions in grain yields. Although considerable research has been invested in the study of cool-temperature injury, a full understanding of the molecular mechanism has not been achieved. Here, we show that endogenous levels of the bioactive gibberellins (GAs) GA4 and GA7, and expression levels of the GA biosynthesis genes GA20ox3 and GA3ox1, decrease in the developing anthers by exposure to LT. By contrast, the levels of precursor GA12 were higher in response to LT. In addition, the expression of the dehydration-responsive element-binding protein DREB2B and SLENDER RICE1 (SLR1)/DELLA was up-regulated in response to LT. Mutants involved in GA biosynthetic and response pathways were hypersensitive to LT stress, including the semidwarf mutants sd1 and d35, the gain-of-function mutant slr1-d, and gibberellin insensitive dwarf1. The reduction in the number of sporogenous cells and the abnormal enlargement of tapetal cells occurred most severely in the GA-insensitive mutant. Application of exogenous GA significantly reversed the male sterility caused by LT, and simultaneous application of exogenous GA with sucrose substantially improved the extent of normal pollen development. Modern rice varieties carrying the sd1 mutation are widely cultivated, and the sd1 mutation is considered one of the greatest achievements of the Green Revolution. The protective strategy achieved by our work may help sustain steady yields of rice under global climate change. OsGA20ox3|GA20ox3 Activation of gibberellin 2-oxidase 6 decreases active gibberellin levels and creates a dominant semi-dwarf phenotype in rice (Oryza sativa L.) 2010 Journal of Genetics and Genomics State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Gibberellin (GA) 2-oxidase plays a key role in the GA catabolic pathway through 2beta-hydroxylation. In the present study, we isolated a CaMV 35S-enhancer activation tagged mutant, H032. This mutant exhibited a dominant dwarf and GA-deficient phenotype, with a final stature that was less than half of its wild-type counterpart. The endogenous bioactive GAs are markedly decreased in the H032 mutant, and application of bioactive GAs (GA(3) or GA(4)) can reverse the dwarf phenotype. The integrated T-DNA was detected 12.8 kb upstream of the OsGA2ox6 in the H032 genome by TAIL-PCR. An increased level of OsGA2ox6 mRNA was detected at a high level in the H032 mutant, which might be due to the enhancer role of the CaMV 35S promoter. RNAi and ectopic expression analysis of OsGA2ox6 indicated that the dwarf trait and the decreased levels of bioactive GAs in the H032 mutant were a result of the up-regulation of the OsGA2ox6 gene. BLASTP analysis revealed that OsGA2ox6 belongs to the class III of GA 2-oxidases, which is a novel type of GA2ox that uses C20-GAs (GA(12) and/or GA(53)) as the substrates. Interestingly, we found that a GA biosynthesis inhibitor, paclobutrazol, positively regulated the OsGA2ox6 gene. Unlike the over-expression of OsGA2ox1, which led to a high rate of seed abortion, the H032 mutant retained normal flowering and seed production. These results indicate that OsGA2ox6 mainly affects plant stature, and the dominant dwarf trait of the H032 mutant can be used as an efficient dwarf resource in rice breeding. OsGA2ox1,OsGA2ox6 Expression of a Gibberellin 2-Oxidase Gene around the Shoot Apex Is Related to Phase Transition in Rice 2001 Plant Physiol Institute of Agriculture and Forestry, University of Tsukuba, Tsukuba 305-8572, Japan A major catabolic pathway for gibberellin (GA) is initiated by 2beta-hydroxylation, a reaction catalyzed by GA 2-oxidase. We have isolated and characterized a cDNA, designated Oryza sativa GA 2-oxidase 1 (OsGA2ox1) from rice (Oryza sativa L. cv Nipponbare) that encodes a GA 2-oxidase. The encoded protein, produced by heterologous expression inEscherichia coli, converted GA1, GA4, GA9, GA20, and GA44 to the corresponding 2beta-hydroxylated products GA8, GA34, GA51, GA29, and GA98, respectively. Ectopic expression of theOsGA2ox1 cDNA in transgenic rice inhibited stem elongation and the development of reproductive organs. These transgenic plants were deficient in endogenous GA1. These results indicate that OsGA2ox1 encodes a GA 2-oxidase, which is functional not only in vitro but also in vivo. OsGA2ox1was expressed in shoot apex and roots but not in leaves and stems. In situ hybridization analysis revealed that OsGA2ox1 mRNA was localized in a ring at the basal region of leaf primordia and young leaves. This ring-shaped expression around the shoot apex was drastically decreased after the phase transition from vegetative to reproductive growth. It was absent in the floral meristem, but it was still present in the lateral meristem that remained in the vegetative phase. These observations suggest that OsGA2ox1 controls the level of bioactive GAs in the shoot apical meristem; therefore, reduction in its expression may contribute to the early development of the inflorescence meristem. OsGA2ox1 Evolutionary analysis of three gibberellin oxidase genes in rice, Arabidopsis, and soybean 2011 Gene Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. GAs are plant hormones that play fundamental roles in plant growth and development. GA2ox, GA3ox, and GA20ox are three key enzymes in GA biosynthesis. These enzymes belong to the 2OG-Fe (II) oxygenase superfamily and are independently encoded by different gene families. To date, genome-wide comparative analyses of GA oxidases in plant species have not been thoroughly carried out. In the present work, 61 GA oxidase family genes from rice (Oryza sativa), Arabidopsis, and soybean (Glycine max) were identified and a full study of these genes including phylogenetic tree construction, gene structure, gene family expansion and analysis of functional motifs was performed. Based on phylogeny, most of the GA oxidases were divided into four subgroups that reflected functional classifications. Intron/intron average length of GA oxidase genes in rice analysis revealed that GA oxidase genes in rice experienced substantial evolutionary divergence. Segmental duplication events were mainly found in soybean genome. However, in rice and Arabidopsis, no single expansion pattern exhibited dominance, indicating that GA oxidase genes from these species might have been subjected to a more complex evolutionary mechanism. In addition, special functional motifs were discovered in GA20ox, GA3ox, and GA2ox, which suggested that different functional motifs are associated with differences in protein function. Taken together our results suggest that GA oxidase family genes have undergone divergent evolutionary routes, especially at the monocot-dicot split, with dynamic evolution occurring in Arabidopsis thaliana and soybean. OsGA2ox5 OsGA2ox5, a gibberellin metabolism enzyme, is involved in plant growth, the root gravity response and salt stress 2014 PLoS One Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. Gibberellin (GA) 2-oxidases play an important role in the GA catabolic pathway through 2beta-hydroxylation. There are two classes of GA2oxs, i.e., a larger class of C(1)(9)-GA2oxs and a smaller class of C(2)(0)-GA2oxs. In this study, the gene encoding a GA 2-oxidase of rice, Oryza sativa GA 2-oxidase 5 (OsGA2ox5), was cloned and characterized. BLASTP analysis showed that OsGA2ox5 belongs to the C(2)(0)-GA2oxs subfamily, a subfamily of GA2oxs acting on C(2)(0)-GAs (GA(1)(2), GA(5)(3)). Subcellular localization of OsGA2ox5-YFP in transiently transformed onion epidermal cells revealed the presence of this protein in both of the nucleus and cytoplasm. Real-time PCR analysis, along with GUS staining, revealed that OsGA2ox5 is expressed in the roots, culms, leaves, sheaths and panicles of rice. Rice plants overexpressing OsGA2ox5 exhibited dominant dwarf and GA-deficient phenotypes, with shorter stems and later development of reproductive organs than the wild type. The dwarfism phenotype was partially rescued by the application of exogenous GA3 at a concentration of 10 microM. Ectopic expression of OsGA2ox5 cDNA in Arabidopsis resulted in a similar phenotype. Real-time PCR assays revealed that both GA synthesis-related genes and GA signaling genes were expressed at higher levels in transgenic rice plants than in wild-type rice; OsGA3ox1, which encodes a key enzyme in the last step of the bioactive GAs synthesis pathway, was highly expressed in transgenic rice. The roots of OsGA2ox5-ox plants exhibited increased starch granule accumulation and gravity responses, revealing a role for GA in root starch granule development and gravity responses. Furthermore, rice and Arabidopsis plants overexpressing OsGA2ox5 were more resistant to high-salinity stress than wild-type plants. These results suggest that OsGA2ox5 plays important roles in GAs homeostasis, development, gravity responses and stress tolerance in rice. OsGA2ox5 A novel class of gibberellin 2-oxidases control semidwarfism, tillering, and root development in rice 2008 Plant Cell Institute of Molecular Biology, National Chung-Hsing University, Taichung 402, Taiwan, Republic of China. Gibberellin 2-oxidases (GA2oxs) regulate plant growth by inactivating endogenous bioactive gibberellins (GAs). Two classes of GA2oxs inactivate GAs through 2beta-hydroxylation: a larger class of C(19) GA2oxs and a smaller class of C(20) GA2oxs. In this study, we show that members of the rice (Oryza sativa) GA2ox family are differentially regulated and act in concert or individually to control GA levels during flowering, tillering, and seed germination. Using mutant and transgenic analysis, C(20) GA2oxs were shown to play pleiotropic roles regulating rice growth and architecture. In particular, rice overexpressing these GA2oxs exhibited early and increased tillering and adventitious root growth. GA negatively regulated expression of two transcription factors, O. sativa homeobox 1 and TEOSINTE BRANCHED1, which control meristem initiation and axillary bud outgrowth, respectively, and that in turn inhibited tillering. One of three conserved motifs unique to the C(20) GA2oxs (motif III) was found to be important for activity of these GA2oxs. Moreover, C(20) GA2oxs were found to cause less severe GA-defective phenotypes than C(19) GA2oxs. Our studies demonstrate that improvements in plant architecture, such as semidwarfism, increased root systems and higher tiller numbers, could be induced by overexpression of wild-type or modified C(20) GA2oxs. OsGA2ox6 Molecular cloning and differential expression of an gamma-aminobutyrate transaminase gene, OsGABA-T, in rice (Oryza sativa) leaves infected with blast fungus 2006 J Plant Res Department of Plant Pathology, College of Agriculture and Biotechnology, China Agricultural University, Beijing, People's Republic of China. gamma-Aminobutyrate transaminase (GABA-T) catalyzes the conversion of GABA to succinic semialdehyde. Using differential display PCR and cDNA library screening, a full-length GABA-T cDNA (OsGABA-T) was isolated from rice (Oryza sativa) leaves infected with an incompatible race of Magnaporthe grisea. The deduced amino acid sequence comprises 483 amino acid residues and shares 85-69% identity with GABA-T sequences from other plants. OsGABA-T expression is induced by blast fungus infection, mechanical wounding and ultraviolet radiation in rice leaves and is not detected in normal rice organs. This gene is also induced by defense signal molecules such as salicylic acid and abscisic acid, but not by jasmonic acid. Our data suggest that OsGABA-T (GABA shunt) may play a role in restricting the levels of cell death during the host-pathogen interaction. OsGABA-T Rice (Oryza sativa) contains a novel isoform of glutamate decarboxylase that lacks an authentic calmodulin-binding domain at the C-terminus 2001 Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression Department of Biological Sciences, Shimane University, Matsue, Japan. We have isolated full-length cDNAs for two distinct isoforms of glutamate decarboxylase (GAD), designated OsGAD1 and OsGAD2 from a rice shoot cDNA library. Open reading frames found in OsGAD1 and OsGAD2 cDNAs encode putative proteins of 501 (56.7 kDa) and 500 amino acids (55.6 kDa), respectively. They show 69% identity to each other and 67-78% identity to dicotyledonous counterpart sequences determined so far. Comparative analysis of relevant genomic clones obtained from the rice genomic library with these cDNAs as probes demonstrated that the number and sizes of introns deduced for these two genes differ considerably. Interestingly, in the regions in the putative gene products corresponding to the C-terminal 30-amino-acid peptide known as the calmodulin-binding domain of plant GADs, OsGAD1 possesses a typical motif, while OsGAD2 contains several substitutions of amino acids that contribute strongly to the binding of calmodulin (CaM). An in vitro CaM-binding assay of these proteins over-expressed in Escherichia coli revealed that OsGAD1 can in fact bind specifically to bovine CaM but OsGAD2 cannot. RNA analysis showed that transcripts of OsGAD1 and OsGAD2 were present in all tissues examined, but their expression was differentially regulated, at least in roots and maturing seeds. OsGAD1,OsGAD2 Seed-specific expression of truncated OsGAD2 produces GABA-enriched rice grains that influence a decrease in blood pressure in spontaneously hypertensive rats 2009 Transgenic Res Department of Biological Science, Shimane University, Nishikawatsu 1060, Matsue, Shimane 690-8504, Japan. akama@life.shimane-u.ac.jp Gamma-aminobutyric acid (GABA) is a four-carbon amino acid that is commonly present in living organisms and functions as a major inhibitory neurotransmitter in mammals. It is understood to have a potentially anti-hypertensive effect in mammals. GABA is synthesized from glutamate by glutamate decarboxylase (GAD). In plants, GAD is regulated via its calmodulin-binding domain (CaMBD) by Ca(2+)/CaM. We have previously reported that a C-terminal truncated version of one of the five rice GAD isoforms, GAD2DeltaC, revealed higher enzymatic activity in vitro and that its over-expression resulted in exceptionally high GABA accumulation (Akama and Takaiwa, J Exp Bot 58:2699-2607, 2007). In this study, GAD2DeltaC, under the control of the rice glutelin promoter (GluB-1), was introduced into rice cells via Agrobacterium-mediated transformation to produce transgenic rice lines. Analysis of the free amino acid content of rice grains revealed up to about a 30-fold higher level of GABA than in non-transformed rice grains. There were also very high levels of various free protein amino acids in the seeds. GABA-enriched rice grains were milled to a fine powder for oral administration to spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto rats (WKYs). Six weeks of administration showed that transgenic rice brings about a 20 mmHg decrease in blood pressure in two different kinds of SHRs, while there was no significant hypotensive effect in WKYs. These results suggest an alternative way to control and/or cure hypertension in humans with GABA-enriched rice as part of a common daily diet. OsGAD2 C-terminal extension of rice glutamate decarboxylase (OsGAD2) functions as an autoinhibitory domain and overexpression of a truncated mutant results in the accumulation of extremely high levels of GABA in plant cells 2007 J Exp Bot Department of Biological Science, Shimane University, Nishikawatsu 1060, Matsue, Shimane, Japan. akama@life.shimane-u.ac.jp Glutamate decarboxylase (GAD) converts L-glutamate to gamma-aminobutyric acid (GABA), which is a non-protein amino acid present in all organisms. Plant GADs carry a C-terminal extension that binds to Ca(2+)/calmodulin (CaM) to modulate enzyme activity. However, rice possesses two distinct types of GAD, OsGAD1 and OsGAD2. Although they both have a C-terminal extension, the former peptide contains an authentic CaM-binding domain (CaMBD), which is common to dicotyledonous plants, while the latter does not. Therefore, the role of the C-terminal extension in functional expression of OsGAD2 was investigated. An in vitro enzyme assay using recombinant OsGAD2 proteins revealed low activity in the presence or absence of Ca(2+)/CaM. However, a truncated version of GAD2 (OsGAD2DeltaC) had over 40-fold higher activity than wild-type GAD at physiological pH. These two DNA constructs were introduced simultaneously into rice calli via Agrobacterium to establish transgenic cell lines. Free amino acids were isolated from several lines for each construct to determine GABA content. Calli overexpressing OsGAD2 and OsGAD2DeltaC had about 6-fold and 100-fold the GABA content of wild-type calli, respectively. Regenerated OsGAD2DeltaC rice plants had aberrant phenotypes such as dwarfism, etiolated leaves, and sterility. These data suggest that the C-terminal extension of OsGAD2 plays a role as a strong autoinhibitory domain, and that truncation of this domain causes the enzyme to act constitutively, with higher activity both in vitro and in vivo. OsGAD2 The rice OsGAE1 is a novel gibberellin-regulated gene and involved in rice growth 2006 Plant Mol Biol Department of Molecular Biology, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba 305-8602, Japan. Gibberellins (GAs) are a class of phytohormones that regulate many aspects of plant growth and development processes including stem elongation, flowering, and seed germination. A novel GA-enhanced gene, designated as OsGAE1, was identified using microarray analysis of GA-regulated genes. OsGAE1 expressed in a dose- and time course-dependent manner with minimum expression at 1 microM GA(3) and maximum expression at 50 microM GA(3) starting from 30 min and peaked at 24 h after GA(3) treatment. OsGAE1 expression was up-regulated by GA(3) at transcript level while no significant effect was observed for other hormones. OsGAE1 was expressed in Escherichia coli with N-terminal His(6) tag and the recombinant protein migrated at 38 kDa, slightly larger than the predicted 29 kDa, during SDS-PAGE. Anti-OsGAE1 antibodies immunoreacted with a protein of 40 kDa in rice leaf sheath. OsGAE1 expressed mainly in growing leaf sheath and callus compared to leaf and root. In situ hybridization and OsGAE1 promoter analysis revealed that OsGAE1 expressed in shoot apex meristem and young primary leaves. Northern blot, Western blot, and GUS activities revealed that OsGAE1 is up-regulated by GA(3). Transgenic rice expressing OsGAE1 in antisense orientation exhibited severely affected vegetative and reproductive growth. The transgenic plants were 55-70% short compared to control. These results suggest that OsGAE1 is differentially expressed in rice leaf sheath in relation to GA(3) and it encodes a functional protein which is involved in GA-regulated growth and development of rice. OsGAE1 Functional dissections between GAMYB and Dof transcription factors suggest a role for protein-protein associations in the gibberellin-mediated expression of the RAmy1A gene in the rice aleurone 2003 Plant Physiol Laboratory of Environmental Molecular Biology, Division of Bioscience, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan. washi@ees.hokudai.ac.jp In the germinated cereal aleurone layer, gibberellic acids (GA) induce expression of a number of genes encoding hydrolytic enzymes that participate in the mobilization of stored molecules. Previous analyses suggest that the key events controlling the GA-regulated gene expression in the aleurone are formation of active transcription machinery referred to as the GA responsive complex, followed by recruiting GAMYB. In general, bipartite promoter contexts composed of the GA-responsive element and the pyrimidine box are observed within the regulatory regions of cereal GA-responsive genes. Protein factors that recognize each promoter sequence were identified and distinct effects on the GA-mediated activation of gene expression have been also investigated; however, the connection and intercalation between two promoter motifs remain obscure. In this study, I have evaluated cooperative function of GAMYB and a pyrimidine box-binding protein OsDOF3 that influenced the promoter activity of the most predominant GA-responsive gene (RAmy1A) of rice (Oryza sativa). Transient expression of OsDOF3 in the germinated aleurone prolonged GAMYB function on the reporter expression in the absence of GA. The synergistic effect required a set of DNA bindings of two proteins on the RAmy1A promoter region. The yeast two-hybrid assay showed the physical interaction of GAMYB and OsDOF3 in yeast cells, indicating that the association of GAMYB and OsDOF3 may be a functional unit in transcription regulation. The results showed the accessory function of OsDOF3 responsible for a dosage-dependent mediation of GA signaling that leads to high-level expression of physiological target genes. OsMYBGA|OsGAMYB,AmyI-1|RAmy1A,RPBF|OsDof3 Convergent starvation signals and hormone crosstalk in regulating nutrient mobilization upon germination in cereals 2012 Plant Cell Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 115, Taiwan, Republic of China. Germination is a unique developmental transition from metabolically quiescent seed to actively growing seedling that requires an ensemble of hydrolases for coordinated nutrient mobilization to support heterotrophic growth until autotrophic photosynthesis is established. This study reveals two crucial transcription factors, MYBS1 and MYBGA, present in rice (Oryza sativa) and barley (Hordeum vulgare), that function to integrate diverse nutrient starvation and gibberellin (GA) signaling pathways during germination of cereal grains. Sugar represses but sugar starvation induces MYBS1 synthesis and its nuclear translocation. GA antagonizes sugar repression by enhancing conuclear transport of the GA-inducible MYBGA with MYBS1 and the formation of a stable bipartite MYB-DNA complex to activate the alpha-amylase gene. We further discovered that not only sugar but also nitrogen and phosphate starvation signals converge and interconnect with GA to promote the conuclear import of MYBS1 and MYBGA, resulting in the expression of a large set of GA-inducible but functionally distinct hydrolases, transporters, and regulators associated with mobilization of the full complement of nutrients to support active seedling growth in cereals. OsMYBGA|OsGAMYB,OsMYBS1 GAMYB controls different sets of genes and is differentially regulated by microRNA in aleurone cells and anthers 2006 Plant J Bioscience and Biotechnology Center, Nagoya University, Furocho, Chikusa, Nagoya 464-8601, Japan. GAMYB is a component of gibberellin (GA) signaling in cereal aleurone cells, and has an important role in flower development. However, it is unclear how GAMYB function is regulated. We examined the involvement of a microRNA, miR159, in the regulation of GAMYB expression in cereal aleurone cells and flower development. In aleurone cells, no miR159 expression was observed with or without GA treatment, suggesting that miR159 is not involved in the regulation of GAMYB and GAMYB-like genes in this tissue. miR159 was expressed in tissues other than aleurone, and miR159 over-expressors showed similar but more severe phenotypes than the gamyb mutant. GAMYB and GAMYB-like genes are co-expressed with miR159 in anthers, and the mRNA levels for GAMYB and GAMYB-like genes are negatively correlated with miR159 levels during anther development. Thus, OsGAMYB and OsGAMYB-like genes are regulated by miR159 in flowers. A microarray analysis revealed that OsGAMYB and its upstream regulator SLR1 are involved in the regulation of almost all GA-mediated gene expression in rice aleurone cells. Moreover, different sets of genes are regulated by GAMYB in aleurone cells and anthers. GAMYB binds directly to promoter regions of its target genes in anthers as well as aleurone cells. Based on these observations, we suggest that the regulation of GAMYB expression and GAMYB function are different in aleurone cells and flowers in rice. OsMYBGA|OsGAMYB,SLR1|OsGAI Identification of gamyb-4 and analysis of the regulatory role of GAMYB in rice anther development 2010 J Integr Plant Biol School of Life Sciences, Shanghai University, Shanghai 200240, China. In higher plants, male reproductive development is a complex biological process that includes cell division and differentiation, cell to cell communication etc., while the mechanism underlying plant male reproductive development remains less understood. GAMYB encodes a gibberellins acid (GA) inducible transcription factor that is required for the early anther development in rice (Oryza sativa L.). Here, we report the isolation and characterization of a new allele gamyb-4 with a C base deletion in the second exon (+2308), causing a frame shift and premature translational termination. Histological analysis showed that gamyb-4 developed abnormal enlarged tapetum and could not undergo normal meiosis. To understand the regulatory role of GAMYB, we carried out quantitative reverse transcription-polymerase chain reaction analysis and comparison of microarray data. These results revealed that the expression of TDR (TAPETUM DEGENERATION RETARDATION), a tapetal cell death regulator, was downregulated in gamyb-4 and udt1 (undeveloped tapetum1). While the GAMYB expression was not obviously changed in tdr and udt1-1, and no apparent expression fold change of UDT1 in tdr and gamyb-4, suggesting that TDR may act downstream of GAMYB and UDT1, and GAMYB and UDT1 work in parallel to regulate rice early anther development. This work is helpful in understanding the regulatory network in rice anther development. OsMYBGA|OsGAMYB,TDR,Udt1 Loss-of-function mutations of the rice GAMYB gene impair alpha-amylase expression in aleurone and flower development 2004 Plant Cell BioScience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan. GAMYB was first isolated as a positive transcriptional regulator of gibberellin (GA)-dependent alpha-amylase expression in barley aleurone cells, and its molecular and biochemical properties have been well characterized. However, the role of GAMYB elsewhere in the plant is not well understood. To investigate the molecular function of GAMYB outside of the aleurone cells, we isolated loss-of-function mutants from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17. Through PCR screening using primers for rice GAMYB (OsGAMYB) and Tos17, we isolated three independent mutant alleles that contained Tos17 inserted in the exon region. No alpha-amylase expression in the endosperm was induced in these mutants in response to GA treatment, indicating that the Tos17 insertion had knocked out OsGAMYB function. We found no significant defects in the growth and development of the mutants at the vegetative stage. After the phase transition to the reproductive stage, however, shortened internodes and defects in floral organ development, especially a defect in pollen development, were observed. On the other hand, no difference was detected in flowering time. High-level OsGAMYB expression was detected in the aleurone cells, inflorescence shoot apical region, stamen primordia, and tapetum cells of the anther, but only low-level expression occurred in organs at the vegetative stage or in the elongating stem. These results demonstrate that, in addition to its role in the induction of alpha-amylase in aleurone, OsGAMYB also is important for floral organ development and essential for pollen development. OsMYBGA|OsGAMYB A rice WRKY gene encodes a transcriptional repressor of the gibberellin signaling pathway in aleurone cells 2004 Plant Physiol Department of Biological Sciences, University of Nevada, 4505 Maryland Parkway, Las Vegas, Nevada 89154, USA. The molecular mechanism by which GA regulates plant growth and development has been a subject of active research. Analyses of the rice (Oryza sativa) genomic sequences identified 77 WRKY genes, among which OsWRKY71 is highly expressed in aleurone cells. Transient expression of OsWRKY71 by particle bombardment specifically represses GA-induced Amy32b alpha-amylase promoter but not abscisic acid-induced HVA22 or HVA1 promoter activity in aleurone cells. Moreover, OsWRKY71 blocks the activation of the Amy32b promoter by the GA-inducible transcriptional activator OsGAMYB. Consistent with its role as a transcriptional repressor, OsWRKY71 is localized to nuclei of aleurone cells and binds specifically to functionally defined TGAC-containing W boxes of the Amy32b promoter in vitro. Mutation of the two W boxes prevents the binding of OsWRKY71 to the mutated promoter, and releases the suppression of the OsGAMYB-activated Amy32b expression by OsWRKY71, suggesting that OsWRKY71 blocks GA signaling by functionally interfering with OsGAMYB. Exogenous GA treatment decreases the steady-state mRNA level of OsWRKY71 and destabilizes the GFP:OsWRKY71 fusion protein. These findings suggest that OsWRKY71 encodes a transcriptional repressor of GA signaling in aleurone cells. OsMYBGA|OsGAMYB,OsWRKY71 Identification of Fe-excess-induced genes in rice shoots reveals a WRKY transcription factor responsive to Fe, drought and senescence 2010 Mol Biol Rep Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, Porto Alegre, RS, 91501-970, Brazil. Fe participates in several important reactions in plant metabolism. However, Fe homeostasis in plants is not completely understood, and molecular studies on Fe-excess stress are scarce. Rice (Oryza sativa L. ssp. indica) is largely cultivated in submerged conditions, where the extremely reductive environment can lead to severe Fe overload. In this work, we used representational difference analysis (RDA) to isolate sequences up-regulated in rice shoots after exposure to Fe-excess. We isolated 24 sequences which have putative functions in distinct cellular processes, such as transcription regulation (OsWRKY80), stress response (OsGAP1, DEAD-BOX RNA helicase), proteolysis (oryzain-alpha, rhomboid protein), photosynthesis (chlorophyll a/b binding protein), sugar metabolism (beta glucosidase) and electron transport (NADH ubiquinone oxireductase). We show that the putative WRKY transcription factor OsWRKY80 is up-regulated in rice leaves, stems and roots after Fe-excess treatment. This up-regulation is also observed after dark-induced senescence and drought stress, indicating that OsWRKY80 could be a general stress-responsive gene. To our knowledge, this is the first report of an Fe-excess-induced transcription factor in plants. OsGAP1,OsWRKY80 Antagonistic, overlapping and distinct responses to biotic stress in rice (Oryza sativa) and interactions with abiotic stress 2013 BMC Genomics Centre for Computational Systems Biology, Bayliss Building M316 University of Western Australia, 35 Stirling Highway, Crawley 6009, Western Australia, Australia. reena.narsai@uwa.edu.au BACKGROUND: Every year, substantial crop loss occurs globally, as a result of bacterial, fungal, parasite and viral infections in rice. Here, we present an in-depth investigation of the transcriptomic response to infection with the destructive bacterial pathogen Xanthomonas oryzae pv. oryzae(Xoo) in both resistant and susceptible varieties of Oryza sativa. A comparative analysis to fungal, parasite and viral infection in rice is also presented. RESULTS: Within 24 h of Xoo inoculation, significant reduction of cell wall components and induction of several signalling components, membrane bound receptor kinases and specific WRKY and NAC transcription factors was prominent, providing a framework for how the presence of this pathogen was signalled and response mounted. Extensive comparative analyses of various other pathogen responses, including in response to infection with another bacterium (Xoc), resistant and susceptible parasite infection, fungal, and viral infections, led to a proposed model for the rice biotic stress response. In this way, a conserved induction of calcium signalling functions, and specific WRKY and NAC transcription factors, was identified in response to all biotic stresses. Comparison of these responses to abiotic stress (cold, drought, salt, heat), enabled the identification of unique genes responsive only to bacterial infection, 240 genes responsive to both abiotic and biotic stress, and 135 genes responsive to biotic, but not abiotic stresses. Functional significance of a number of these genes, using genetic inactivation or over-expression, has revealed significant stress-associated phenotypes. While only a few antagonistic responses were observed between biotic and abiotic stresses, e.g. for a number of endochitinases and kinase encoding genes, some of these may be crucial in explaining greater pathogen infection and damage under abiotic stresses. CONCLUSIONS: The analyses presented here provides a global view of the responses to multiple stresses, further validates known resistance-associated genes, and highlights new potential target genes, some lineage specific to rice, that play important roles in response to stress, providing a roadmap to develop varieties of rice that are more resistant to multiple biotic and abiotic stresses, as encountered in nature. OsGAP1 Constitutive expression of a rice GTPase-activating protein induces defense responses 2008 New Phytologist Department of Biology, The Chinese University of Hong Kong, Shatin, Hong Kong Special Administrative Region. G-proteins (guanine nucleotide-binding proteins that usually exhibit GTPase activities) and related signal transduction processes play important roles in mediating plant defense responses; here, a rice (Oryza sativa) cDNA clone, OsGAP1, encoding a GTPase-activating protein (GAP) that also contains a protein kinase C conserved region 2 (C2) domain is reported. An interacting G-protein partner for the OsGAP1 protein was identified by yeast two-hybrid library screening and confirmed by co-immunoprecipitation; the GTPase-activation activity of OsGAP1 on this interacting G-protein was demonstrated using in vitro assays. OsGAP1 was induced by wounding in rice and the presence of the R locus Xa14 enhances such induction. Gain-of-function tests in transgenic rice and Arabidopsis thaliana showed that constitutive expression of OsGAP1 led to increased resistance to bacterial pathogens in both monocots and dicots. OsGAP1 An ancient P-loop GTPase in rice is regulated by a higher plant-specific regulatory protein 2010 J Biol Chem State Key Laboratory of Agrobiotechnology and School of Life Sciences, Chinese University of Hong Kong, Shatin, Hong Kong. YchF is a subfamily of the Obg family in the TRAFAC class of P-loop GTPases. The wide distribution of YchF homologues in both eukarya and bacteria suggests that they are descendents of an ancient protein, yet their physiological roles remain unclear. Using the OsYchF1-OsGAP1 pair from rice as the prototype, we provide evidence for the regulation of GTPase/ATPase activities and RNA binding capacity of a plant YchF (OsYchF1) by its regulatory protein (OsGAP1). The effects of OsGAP1 on the subcellular localization/cycling and physiological functions of OsYchF1 are also discussed. The finding that OsYchF1 and OsGAP1 are involved in plant defense response might shed light on the functional roles of YchF homologues in plants. This work suggests that during evolution, an ancestral P-loop GTPase/ATPase may acquire new regulation and function(s) by the evolution of a lineage-specific regulatory protein. OsGAP1,OsYchF1 OsGAP1 functions as a positive regulator of OsRab11-mediated TGN to PM or vacuole trafficking 2005 Plant Cell Physiol Division of Applied Life Sciences, Graduate School of Gyeongsang National University, Jinju 660-701, Korea. The Ypt/Rab family of small G-proteins is important in regulating vesicular transport. Rabs hydrolyze GTP very slowly on their own and require GTPase-activating proteins (GAPs). Here we report the identification and characterization of OsGAP1, a Rab-specific rice GAP. OsGAP1 strongly stimulated OsRab8a and OsRab11, which are homologs of the mammalian Rab8 and Rab11 proteins that are essential for Golgi to plasma membrane (PM) and trans-Golgi network (TGN) to PM trafficking, respectively. Substitution of two invariant arginines within the catalytic domain of Oryza sativa GTPase-activating protein 1 (OsGAP1) with alanines significantly inhibited its GAP activity. In vivo targeting experiments revealed that OsGAP1 localizes to the TGN or pre-vacuolar compartment (PVC). A yeast expression system demonstrated that wild-type OsGAP1 facilitates O. sativa dissociation inhibitor 3 (OsGDI3)-catalyzed OsRab11 recycling at an early stage, but the OsGAP1(R385A) and (R450A) mutants do not. Thus, GTP hydrolysis is essential for Rab recycling. Moreover, expression of the OsGAP1 mutants in Arabidopsis protoplasts inhibited the trafficking of some cargo proteins, including the PM-localizing H+-ATPase-green fluorescent protein (GFP) and Ca2+-ATPase8-GFP and the central vacuole-localizing Arabidopsis aleurain-like protein (AALP)-GFP. The OsGAP1 mutants caused these proteins to accumulate at the Golgi apparatus. Surprisingly, OsRab11 overproduction relieved the inhibitory effect of the OsGAP1 mutants on vesicular trafficking. OsRab8a had no such effect. Thus, the OsGAP1 mutants may inhibit TGN to PM or central vacuole trafficking because they induce the sequestration of endogenous Rab11. We propose that OsGAP1 facilitates vesicular trafficking from the TGN to the PM or central vacuole by both stimulating the GTPase activity of OsRab11 and increasing the recycling of inactive OsRab11. OsGAP1,OsRab11 Crystal structures of rice (Oryza sativa) glyceraldehyde-3-phosphate dehydrogenase complexes with NAD and sulfate suggest involvement of Phe37 in NAD binding for catalysis 2012 Plant Mol Biol Life Science Group, Scientific Research Division, National Synchrotron Radiation Research Center, Hsinchu, Taiwan. Cytosolic Oryza sativa glyceraldehyde-3-phosphate dehydrogenase (OsGAPDH), the enzyme involved in the ubiquitous glycolysis, catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-biphosphoglycerate (BPG) using nicotinamide adenine dinucleotide (NAD) as an electron acceptor. We report crystal structures of OsGAPDH in three conditions of NAD-free, NAD-bound and sulfate-soaked forms to discuss the molecular determinants for coenzyme specificity. The structure of OsGAPDH showed a homotetramer form with each monomer comprising three domains-NAD-binding, catalytic and S-loop domains. NAD binds to each OsGAPDH subunits with some residues forming positively charged grooves that attract sulfate anions, as a simulation of phosphate groups in the product BPG. Phe37 not only forms a bottleneck to improve NAD-binding but also combines with Pro193 and Asp35 as key conserved residues for NAD-specificity in OsGAPDH. The binding of NAD alters the side-chain conformation of Phe37 with a 90 degrees rotation related to the adenine moiety of NAD, concomitant with clamping the active site about 0.6 A from the "open" to "closed" form, producing an increased affinity specific for NAD. Phe37 exists only in higher organisms, whereas it is replaced by other residues (Thr or Leu) with smaller side chains in lower organisms, which makes a greater distance between Leu34 and NAD of E. coli GAPDH than that between Phe37 and NAD of OsGAPDH. We demonstrated that Phe37 plays a crucial role in stabilizing NAD binding or intermediating of apo-holo transition, resulting in a greater NAD-dependent catalytic efficiency using site-directed mutagenesis. Phe37 might be introduced by evolution generating a catalytic advantage in cytosolic GAPDH. OsGAPDH Molecular cloning, characterization, expression and chromosomal location of OsGAPDH, a submergence responsive gene in rice ( Oryza sativa L.) 2002 Theor Appl Genet Department of Low Temperature Science, Hokkaido National Agricultural Experiment Station, 1 Hitsujigaoka, Toyohira, Sapporo, Hokkaido 062-8555, Japan, mapillai1@hotmail.com Differential clones from submergence stress and control treatment from rice seedlings were isolated by the differential screening method. One of the clones, OsGAPDH, represented a gene that was expressed at high level during 12-h submergence. A homology search of GenBank databases showed that OsGAPDH had significant sequence homology with maize non-reversible glyceraldehyde-3-Phosphate dehydrogenase. The OsGAPDH sequence consists of 1,772 bp with the longest open reading frame encoding 499 amino acids with a calculated relative mass of 54.2 kDa. Genomic Southern analysis indicated that one or two copies of the OsGAPDH gene occur in the Yukihikari genome. The chromosomal location of the OsGAPDH gene was identified by RFLP analysis indicating that OsGAPDH was located on chromosome 8. Tissue-specific expression of OsGAPDH indicated that the high level of mRNA was detected in the panicle. Plants exposed to drought, submergence and ABA treatment showed an increased accumulation of OsGAPDH transcripts. The induction of Escherichia coli cells containing the pGST-OsGAPDH plasmid resulted in the accumulation of a large amount of the 83.2-kDa recombinant protein. The purified GAPDH enzyme showed an optimum activity at pH 8.5 and 50 degrees C, and was strongly inhibited by ATP and ADP. OsGAPDH Two OsGASR genes, rice GAST homologue genes that are abundant in proliferating tissues, show different expression patterns in developing panicles 2006 Genes Genet Syst Division of Plant Biotechnology, Tissue Engineering Research Center, Tokyo University of Science Two different types of genes for rice GA-stimulated transcript (GAST) homologue genes, Oryza sativa GA-stimulated transcript-related gene 1 (OsGASR1) and gene 2 (OsGASR2), were found. Both OsGASR proteins contain a cysteine-rich domain highly conserved among GAST family proteins in their C-terminal regions. Gibberellin A3 (GA3) stimulated expression of both OsGASRs in the wild-type Nipponbare and GA3 synthesis-deficient mutant. Expression of both OsGASRs apparently increased when cell proliferation entered the logarithmic phase, and rapidly reduced when cell proliferation was temporarily halted. RT-PCR analysis indicated different expression patterns of these genes in developing panicles. OsGASR1 was limitedly but strongly expressed in florets while OsGASR2 was expressed in both florets and branches. In situ hybridization showed that they were strongly expressed in the root apical meristem (RAM) and shoot apical meristem (SAM), but little signals were detected in mature leaves. Transient expression of OsGASR-GFP fusion proteins in onion epidermal cells revealed that both OsGASR proteins localized to the apoplasm or cell wall. These results suggest that OsGASR1 and OsGASR2 were involved in cell division and might play diverse roles in differentation of panicles. OsGASR1,OsGASR2 Cloning and characterization of the granule-bound starch synthase II gene in rice: gene expression is regulated by the nitrogen level, sugar and circadian rhythm 2003 Planta State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, 310029, Hangzhou, China. A full-length coding domain sequence of a gene analogous to granule-bound starch synthase (GBSS; ADP-glucose-starch glucosyltransferase, EC 2.4.1.21) was cloned and defined as OsGBSSII based on a Nitrogen (N)-starvation-induced cDNA library constructed using the rapid subtraction hybridization method. The deduced amino acid sequence of OsGBSSII was 62-85% identical to those of GBSS proteins from other plant species. The exon/intron organization of OsGBSSII was similar to that of OsGBSSI. OsGBSSII was mainly expressed in leaves and its protein was exclusively bound to starch granules in rice leaves, which suggests that the amylose in rice leaves is synthesized by OsGBSSII. N-starvation-induced expression of OsGBSSII could be repressed by supplying nitrate, ammonia or amino acid (glutamic acid or glutamine), glucosamine (an inhibitor of hexokinase) or dark conditions. These results indicate that N-starvation induction was dependent on the photosynthetic product and hexokinase in rice leaves. Sugars induced the accumulation of OsGBSSII transcripts in excised leaves through glycolysis-dependent pathways. OsGBSSII gene expression is regulated by the circadian rhythm in rice leaves. OsGBSSII Knockdown of GDCH gene reveals reactive oxygen species-induced leaf senescence in rice 2013 Plant Cell Environ State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China. Glycine decarboxylase complex (GDC) is a multi-protein complex, comprising P-, H-, T- and L-protein subunits, which plays a major role in photorespiration in plants. While structural analysis has demonstrated that the H subunit of GDC (GDCH) plays a pivotal role in GDC, research on the role of GDCH in biological processes in plants is seldom reported. Here, the function of GDCH, stresses resulting from GDCH-knockdown and the interactions of these stresses with other cellular processes were studied in rice plants. Under high CO(2), the OsGDCH RNA interference (OsGDCH-RNAi) plants grew normally, but under ambient CO(2), severely suppressed OsGDCH-RNAi plants (SSPs) were non-viable, which displayed a photorespiration-deficient phenotype. Under ambient CO(2), chlorophyll loss, protein degradation, lipid peroxidation and photosynthesis decline occurred in SSPs. Electron microscopy studies showed that chloroplast breakdown and autophagy took place in these plants. Reactive oxygen species (ROS), including O2(-) and H(2)O(2), accumulated and the antioxidant enzyme activities decreased in the leaves of SSPs under ambient CO(2). The expression of transcription factors and senescence-associated genes (SAGs), which was up-regulated in SSPs after transfer to ambient CO(2), was enhanced in wild-type plants treated with H(2)O(2). Evidences demonstrate ROS induce senescence in SSPs, and transcription factors OsWRKY72 may mediate the ROS-induced senescence. OsGDCH,OsWRKY72 Effects of salt stress on ion balance and nitrogen metabolism of old and young leaves in rice (Oryza sativa L.) 2012 BMC Plant Biol Key laboratory of Molecular Epigenetics of MOE, Northeast Normal University, Changchun 130024 Jilin Province, China. BACKGROUND: It is well known that salt stress has different effects on old and young tissues. However, it remains largely unexplored whether old and young tissues have different regulatory mechanism during adaptation of plants to salt stress. The aim of this study was to investigate whether salt stress has different effects on the ion balance and nitrogen metabolism in the old and young leaves of rice, and to compare functions of both organs in rice salt tolerance. RESULTS: Rice protected young leaves from ion harm via the large accumulation of Na+ and Cl- in old leaves. The up-regulation of OsHKT1;1, OsHAK10 and OsHAK16 might contribute to accumulation of Na+ in old leaves under salt stress. In addition, lower expression of OsHKT1;5 and OsSOS1 in old leaves may decrease frequency of retrieving Na+ from old leaf cells. Under salt stress, old leaves showed higher concentration of NO3- content than young leaves. Up-regulation of OsNRT1;2, a gene coding nitrate transporter, might contribute to the accumulation of NO3- in the old leaves of salt stressed-rice. Salt stress clearly up-regulated the expression of OsGDH2 and OsGDH3 in old leaves, while strongly down-regulated expression of OsGS2 and OsFd-GOGAT in old leaves. CONCLUSIONS: The down-regulation of OsGS2 and OsFd-GOGAT in old leaves might be a harmful response to excesses of Na+ and Cl-. Under salt stress, rice might accumulate Na+ and Cl- to toxic levels in old leaves. This might influence photorespiration process, reduce NH4+ production from photorespiration, and immediately down-regulate the expression of OsGS2 and OsFd-GOGAT in old leaves of salt stressed rice. Excesses of Na+ and Cl- also might change the pathway of NH4+ assimilation in old leaves of salt stressed rice plants, weaken GOGAT/GS pathway and elevate GDH pathway. OsGDH2,OsGS2|lambdaGS31,OsHAK10,OsHAK16,OsHKT1;1|OsHKT4,OsHKT1;5|SKC1|OsHKT8,OsNHA1|OsSOS1 Localization of NAD-isocitrate dehydrogenase and glutamate dehydrogenase in rice roots: candidates for providing carbon skeletons to NADH-glutamate synthase 2005 Plant Cell Physiol Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai, Japan. In rice roots, transient and cell-type-specific accumulation of both mRNA and protein for NADH-dependent glutamate synthase (NADH-GOGAT) occurs after the supply of NH(4) (+) ions. In order to better understand the origin of 2-oxoglutarate for this reaction, we focused on mitochondrial NAD-dependent isocitrate dehydrogenase (IDH) and glutamate dehydrogenase (GDH) in rice roots. Six rice cDNAs encoding a single catalytic (OsIDHa) and two regulatory (OsIDHc;1, OsIDHc;2) IDH subunits and three GDH proteins (OsGDH1-3) were isolated. These genes, except OsGDH3, were expressed in the roots. Real-time PCR analysis showed that OsIDHa and OsIDHc;1 transcripts, but not OsGDH1 and OsGDH2 transcripts, accumulated in a similar manner to NADH-GOGAT mRNA along the crown roots after the supply of different forms of inorganic nitrogen. Furthermore, immunolocalization studies revealed the NH(4) (+) induction of IDHa protein in two cell layers of the root surface, i.e. epidermis and exodermis, where NADH-GOGAT also accumulated. The possible relationship between NADH-GOGAT, IDH and GDH is discussed. OsGDH2,OsGDH3 Characterization of two fungal-elicitor-induced rice cDNAs encoding functional homologues of the rab-specific GDP-dissociation inhibitor 1999 Planta Department of Biochemistry, Gyeongsang National University, Chinju, Korea. By using the mRNA differential display approach to isolate defense signaling genes active at the early stage of fungal infection two cDNA fragments with high sequence homology to rab-specific GDP-dissociation inhibitors (GDIs) were identified in rice (Oryza sativa L.) suspension cells. Using polymerase-chain-reaction products as probes, two full-length cDNA clones were isolated from a cDNA library of fungal-elicitor-treated rice, and designated as OsGDI1 and OsGDI2. The deduced amino acid sequences of the isolated cDNAs exhibited substantial homology to Arabidopsis rab-GDIs. Northern analysis revealed that transcripts detected with the 3'-gene-specific DNA probes accumulated to high levels within 30 min after treatment with a fungal elicitor derived from Magnaporthe grisea. The functionality of the OsGDIs was demonstrated by their ability to rescue the Sec19 mutant of Saccharomyces cerevisiae which is defective in vesicle transport. The proteins, expressed in Escherchia coli, cross-reacted with a polyclonal antibody prepared against bovine rab-GDI. Like bovine rab-GDI, the OsGDI proteins efficiently dissociated rab3A from bovine synaptic membranes. Using the two-hybrid system, it was shown that the OsGDIs specifically interact with the small GTP-binding proteins belonging to the rab subfamily. The specific interaction was also demonstrated in vitro by glutathione S-transferase resin pull-down assay. OsGDI1,OsGDI2 Rice GDP dissociation inhibitor 3 inhibits OsMAPK2 activity through physical interaction 2011 Biochem Biophys Res Commun Department of Molecular Biotechnology, Dong-A University, Busan 604-714, Republic of Korea. GDP dissociation inhibitor (GDI) plays an essential role in regulating the state of bound nucleotides and subcellular localizations of Rab proteins. In our previous study, we showed that OsGDI3 facilitates the recycling of OsRab11 with a help of OsGAP1. In this study, we show that OsGDI3 complement the yeast sec19-1 mutant, a temperature-sensitive allele of the yeast GDI gene, suggesting that OsGDI3 is a functional ortholog of yeast GDI. To obtain further knowledge on the function of OsGDI3, candidate OsGDI3-interacting proteins were identified by yeast two-hybrid screens. OsMAPK2 is one of OsGDI3 interacting proteins from yeast two-hybrid screens and subject to further analysis. A kinase assay showed that the autophosphorylation activity of OsMAPK2 is inhibited by OsGDI3 in vitro. In addition, ectopic expressions of OsGDI3-in Arabidopsis cause reductions at the level of phosphorylated AtMPK in phosphorylation activity. Taken together, OsGDI3 functions as a negative regulator of OsMAPK2 through modulating its kinase activity. OsGDI3,OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1 The OsGEN-L protein from Oryza sativa possesses Holliday junction resolvase activity as well as 5 '-flap endonuclease activity 2012 J Biochem Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, Fukuoka 812-8581, Japan. OsGEN-L has a 5'-flap endonuclease activity and plays an essential role in rice microspore development. The Class 4 RAD2/XPG family nucleases, including OsGEN-L, were recently found to have resolving activity for the Holliday junction (HJ), the intermediate of DNA strand recombination. In this study, we performed a detailed characterization of OsGEN-L, as a structure-specific endonuclease. Highly purified OsGEN-L was prepared as the full-length protein for in vitro endonuclease assays using various structured DNAs, and the 5'-flap endonuclease activity, which is stimulated in a PCNA-dependent manner, was demonstrated. In addition, the in vitro HJ resolving activity of OsGEN-L represents the first such activity originating from plant cells. OsGEN-L cleaved HJ at symmetrically related sites of the branch point. However, the two branched strands seemed to be cleaved individually, and not cooperatively, by each OsGEN-L monomer protein. The substrate specificity suggests that OsGEN-L functions in multiple processes of DNA metabolism in rice cells. OsGEN-L RNAi-mediated silencing of OsGEN-L (OsGEN-like), a new member of the RAD2/XPG nuclease family, causes male sterility by defect of microspore development in rice 2005 Plant Cell Physiol Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Takayama 8916-5, Ikoma, Nara, 630-0192 Japan. We have cloned a new member of the RAD2/XPG nuclease family, OsGEN-L (OsGEN-like), from rice (Oryza sativa L.). OsGEN-L possesses two domains, the N- and I-regions, that are conserved in the RAD2/XPG nuclease family. Database searches and phylogenetic analyses revealed that OsGEN-L belongs to class 4 of the RAD2/XPG nuclease family, and OsGEN-L homologs were found in animals and higher plants. To elucidate the function of OsGEN-L, we generated rice OsGEN-L-RNAi transgenic plants in which OsGEN-L expression was silenced. Most of the OsGEN-L-RNAi plants displayed low fertility, and some of them were male-sterile. OsGEN-L-RNAi plants lacked mature pollen, resulting from a defect in early microspore development. A OsGEN-L-green fluorescent protein (GFP) fusion protein was localized in the nucleus, and the OsGEN-L promoter was specifically active in the anthers. Furthermore, a recombinant OsGEN-L protein possessed flap endonuclease activity and both single-stranded and double-stranded DNA-binding activities. Our results suggest that OsGEN-L plays an essential role in DNA metabolism required for early microspore development in rice. OsGEN-L A germin-like protein gene family functions as a complex quantitative trait locus conferring broad-spectrum disease resistance in rice 2009 Plant Physiol Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, Colorado 80523-1177, USA. Plant disease resistance governed by quantitative trait loci (QTL) is predicted to be effective against a broad spectrum of pathogens and long lasting. Use of these QTL to improve crop species, however, is hindered because the genes contributing to the trait are not known. Five disease resistance QTL that colocalized with defense response genes were accumulated by marker-aided selection to develop blast-resistant varieties. One advanced backcross line carrying the major-effect QTL on chromosome (chr) 8, which included a cluster of 12 germin-like protein (OsGLP) gene members, exhibited resistance to rice (Oryza sativa) blast disease over 14 cropping seasons. To determine if OsGLP members contribute to resistance and if the resistance was broad spectrum, a highly conserved portion of the OsGLP coding region was used as an RNA interference trigger to silence a few to all expressed chr 8 OsGLP family members. Challenge with two different fungal pathogens (causal agents of rice blast and sheath blight diseases) revealed that as more chr 8 OsGLP genes were suppressed, disease susceptibility of the plants increased. Of the 12 chr 8 OsGLPs, one clustered subfamily (OsGER4) contributed most to resistance. The similarities of sequence, gene organization, and roles in disease resistance of GLP family members in rice and other cereals, including barley (Hordeum vulgare) and wheat (Triticum aestivum), suggest that resistance contributed by the chr 8 OsGLP is a broad-spectrum, basal mechanism conserved among the Gramineae. Natural selection may have preserved a whole gene family to provide a stepwise, flexible defense response to pathogen invasion. OsGER1 GER1, a GDSL motif-encoding gene from rice is a novel early light- and jasmonate-induced gene 2007 Plant Biol (Stuttg) Botanisches Institut 1, Technische Universitat Karlsruhe, Germany. michael.riemann@bio.uni-karlsruhe.de The reaction of the rice mutant HEBIBA differs from that of wild-type rice in that the mutant responds inversely to red light and is defective in the light-triggered biosynthesis of jasmonic acid (JA). Using the wild type and the HEBIBA mutant of rice in a differential display screen, we attempted to identify genes that act in or near the convergence point of light and JA signalling. We isolated specifically regulated DNA fragments from approximately 10 000 displayed bands, and identified a new early light- and JA-induced gene. This gene encodes an enzyme containing a GDSL motif, showing 38 % identity at the amino acid level to lipase Arab-1 in Arabidopsis thaliana. The GDSL CONTAINING ENZYME RICE 1 gene (GER1) is rapidly induced by both red (R) and far-red (FR) light and by JA. The results are discussed with respect to a possible role for GER1 as a negative regulator of coleoptile elongation in the context of recent findings on the impact of JA on light signalling. OsGER1 Identification and cloning of a submergence-induced gene OsGGT (glycogenin glucosyltransferase) from rice (Oryza sativa L.) by suppression subtractive hybridization 2005 Planta Laboratory of Plant Biotechnology, Faculty of Agriculture, Tottori University, Koyama, Tottori, 680-8553, Japan. A submergence-induced gene, OsGGT, was cloned from 7-day submerged rice (Oryza sativa L. plants, FR13A (a submergence-tolerant cultivar, Indica), using suppression subtractive hybridization and both 5'- and 3'-rapid amplification of cDNA ends (RACE). The full-length OsGGT cDNA contains 1,273 bp with an open reading frame of 1,140 bp (17-1,156) that encodes 379 amino acids. Its deduced amino acid sequence is homologous with glycogenin glucosyltransferase. We found that the OsGGT gene is located in the 17,970-20,077 bp region of genome fragment AAAA01002475.1 of the Indica cultivar and in the 53,293-51,186 bp region of genome fragment AC037426.12 of chromosome 10 of the Japanica cultivar. A time-course study showed that OsGGT-gene expression increased in FR13A during submergence but decreased in IR42 (submergence-intolerant cultivar, Indica). The expression of the OsGGT gene in FR13A was induced by salicylic acid and benzyladenine. The accumulation of OsGGT mRNA in FR13A also increased in response to ethylene, gibberellin, abscisic acid, drought and salt treatment, but methyl jasmonate treatment and cold stress had no effect on expression. These results suggest that the OsGGT gene could be related to submergence stress and associated with a general defensive response to various environmental stresses. OsGGT The auxin-responsive GH3 gene family in rice (Oryza sativa) 2006 Funct Integr Genomics Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India. Auxin regulates plant growth and development by altering the expression of diverse genes. Among these, the genes of Aux/IAA, SAUR, and GH3 classes have been extensively studied in dicots, but little information is available on monocots. We have identified 12 members of GH3 gene family in rice using sequences of full-length cDNA clones available from KOME and analysis of the whole genome sequence of rice. The genomic organization as well as chromosomal location of all the OsGH3 genes is reported. The rice GH3 proteins can be classified in two groups (groups I and II) on the basis of their phylogenetic relationship with Arabidopsis GH3 proteins. Based upon the sequences available in the database, not a single group III GH3 protein could be identified in rice. An extensive survey of EST sequences of other monocots led to the conclusion that although GH3 gene family is highly conserved in both dicots and monocots but the group III is conspicuous by its absence in monocots. The in silico analysis has been complemented with experimental data to quantify transcript levels of all GH3 gene family members. Using real-time polymerase chain reaction, the organ-specific expression of individual OsGH3 genes in light- and dark-grown seedlings/plants has been examined. The transcript abundance of nearly all OsGH3 genes is enhanced on auxin treatment, with the effect more pronounced on OsGH3-1, -2, and -4. The functional validation of these genes in transgenics or analysis of gene-specific insertional mutants will help in elucidating their precise role in auxin signal transduction. OsGH3-2,OsGH3.1|OsGH3-1|LC1,OsGH3.13|TLD1,OsGH3.8|OsGH3-8|OsMGH3,OsJar1|OsGH3.5|OsGH3-5 A GH3 family member, OsGH3-2, modulates auxin and abscisic acid levels and differentially affects drought and cold tolerance in rice 2012 J Exp Bot National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, PR China. Plant responses to abiotic stresses are coordinated by arrays of growth and developmental processes. Indole-3-acetic acid (IAA) and abscisic acid (ABA) play critical roles in developmental programmes and environmental responses, respectively, through complex signalling and metabolism networks. However, crosstalk between the two phytohormones in the stress responses remains largely unknown. Here, it is reported that a GH3 family gene, OsGH3-2, encoding an enzyme catalysing IAA conjugation to amino acids, is involved in the modulation of ABA level and stress tolerance. Expression of OsGH3-2 was induced by drought but was suppressed by cold. Overexpression of OsGH3-2 in rice caused significant morphological aberrations related to IAA deficiency, such as dwarfism, smaller leaves, and fewer crown roots and root hairs. The overexpressing line showed significantly reduced carotene, ABA, and free IAA levels, greater stomata aperture, and faster water loss, and was hypersensitive to drought stress. However, the overexpressing line showed increased cold tolerance, which was due to the combined effects of reduced free IAA content, alleviated oxidative damage, and decreased membrane penetrability. Furthermore, expression levels of some ABA synthesis- and stress-related genes were significantly changed in the overexpression line. It was conclude that OsGH3-2 modulates both endogenous free IAA and ABA homeostasis and differentially affects drought and cold tolerance in rice. OsGH3-2,OsPSY|OsPSY3 Manipulating broad-spectrum disease resistance by suppressing pathogen-induced auxin accumulation in rice 2011 Plant Physiol National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research , Huazhong Agricultural University, Wuhan 430070, China. Breeding crops with the quality of broad-spectrum disease resistance using genetic resources is one of the principal goals of crop improvement. However, the molecular mechanism of broad-spectrum resistance remains largely unknown. Here, we show that GH3-2, encoding an indole-3-acetic acid (IAA)-amido synthetase, mediates a broad-spectrum resistance to bacterial Xanthomonas oryzae pv oryzae and Xanthomonas oryzae pv oryzicola and fungal Magnaporthe grisea in rice (Oryza sativa). IAA, the major form of auxin in rice, results in rice more vulnerable to the invasion of different types of pathogens, which is at least partly due to IAA-induced loosening of the cell wall, the natural protective barrier of plant cells to invaders. X. oryzae pv oryzae, X. oryzae pv oryzicola, and M. grisea secrete IAA, which, in turn, may induce rice to synthesize its own IAA at the infection site. IAA induces the production of expansins, the cell wall-loosening proteins, and makes rice vulnerable to pathogens. GH3-2 is likely contributing to a minor quantitative trait locus for broad-spectrum resistance. Activation of GH3-2 inactivates IAA by catalyzing the formation of an IAA-amino acid conjugate, which results in the suppression of expansin genes. Thus, GH3-2 mediates basal resistance by suppressing pathogen-induced IAA accumulation. It is expected that, regulated by a pathogen-induced strong promoter, GH3-2 alone may be used for breeding rice with a broad-spectrum disease resistance. OsGH3-2 Rice GH3 gene family: regulators of growth and development 2011 Plant Signal Behav Huazhong Agricultural University, Wuhan, China. Auxin is an indispensable hormone throughout the lifetime of nearly all plant species. Several aspects of plant growth and development are rigidly governed by auxin, from micro to macro hierarchies; auxin also has a close relationship with plant-pathogen interactions. Undoubtedly, precise auxin levels are vitally important to plants, which have many effective mechanisms to maintain auxin homeostasis. One mechanism is conjugating amino acid to excessive indole-3-acetic acid (IAA; main form of auxin) through some GH3 family proteins to inactivate it. Our previous study demonstrated that GH3-2 mediated broad-spectrum resistance in rice (Oryza sativa L.) by suppressing pathogen-induced IAA accumulation and downregulating auxin signaling. Here, we further investigated the expression pattern of GH3-2 and other GH3 family paralogues in the life cycle of rice and presented the possible function of GH3-2 on rice root development by histochemical analysis of GH3-2 promoter:GUS reporter transgenic plants. OsGH3-2 Studies on the rice LEAF INCLINATION1 (LC1), an IAA-amido synthetase, reveal the effects of auxin in leaf inclination control 2013 Mol Plant National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. The angle of rice leaf inclination is an important agronomic trait and closely related to the yields and architecture of crops. Although few mutants with altered leaf angles have been reported, the molecular mechanism remains to be elucidated, especially whether hormones are involved in this process. Through genetic screening, a rice gain-of-function mutant leaf inclination1, lc1-D, was identified from the Shanghai T-DNA Insertion Population (SHIP). Phenotypic analysis confirmed the exaggerated leaf angles of lc1-D due to the stimulated cell elongation at the lamina joint. LC1 is transcribed in various tissues and encodes OsGH3-1, an indole-3-acetic acid (IAA) amido synthetase, whose homolog of Arabidopsis functions in maintaining the auxin homeostasis by conjugating excess IAA to various amino acids. Indeed, recombinant LC1 can catalyze the conjugation of IAA to Ala, Asp, and Asn in vitro, which is consistent with the decreased free IAA amount in lc1-D mutant. lc1-D is insensitive to IAA and hypersensitive to exogenous BR, in agreement with the microarray analysis that reveals the altered transcriptions of genes involved in auxin signaling and BR biosynthesis. These results indicate the crucial roles of auxin homeostasis in the leaf inclination control. OsGH3.1|OsGH3-1|LC1 Constitutive expression of OsGH3.1 reduces auxin content and enhances defense response and resistance to a fungal pathogen in rice 2009 Mol Plant Microbe Interact Instituto Valenciano de Investigaciones Agrarias. Carretera Moncada-Naquera, Moncada, Spain. Domingo_concar@gva.es GH3 genes are main components of the hormonal mechanism regulating growth and development and, hence, are deeply involved in a broad range of physiological processes. They are implicated in hormonal homeostasis through the conjugation to amino acids of the free form of essential plant growth regulators such as indoleacetic and jasmonic acids. In this work, we showed that OsGH3.1 overexpression in rice caused dwarfism and significantly reduced both free auxin content and cell elongation. Functional classification of the transcriptomic profiling revealed that most genes involved in auxin biosynthesis and auxin signaling inhibition were induced and repressed, respectively. Many genes related to cell organization and biogenesis were also significantly downregulated. The survey also showed that, although the response to abiotic stresses was not clearly stimulated, OsGH3.1 overexpression did activate a significant number of defense-related genes. In successive bioassays, it was demonstrated that the resistance of rice plants to pathogen infection, evaluated with two different Magnaporthe grisea strains, was higher in the transformants overexpressing OsGH3.1. Taken together, these results indicate that OsGH3.1 overexpression reduces auxin content, inhibits cell growth and cell wall loosening, and enhances resistance to a fungal pathogen. Our results provide evidence that auxin homeostasis can regulate the activation of the defense response in rice. OsGH3.1|OsGH3-1|LC1 Altered architecture and enhanced drought tolerance in rice via the down-regulation of indole-3-acetic acid by TLD1/OsGH3.13 activation 2009 Plant Physiol Institute of Molecular Cell Biology, Hebei Normal University, Shijiazhuang, Hebei, China 050016. Plant architecture is determined by genetic and developmental programs as well as by environmental factors. Sessile plants have evolved a subtle adaptive mechanism that allows them to alter their growth and development during periods of stress. Phytohormones play a central role in this process; however, the molecules responsible for integrating growth- and stress-related signals are unknown. Here, we report a gain-of-function rice (Oryza sativa) mutant, tld1-D, characterized by (and named for) an increased number of tillers, enlarged leaf angles, and dwarfism. TLD1 is a rice GH3.13 gene that encodes indole-3-acetic acid (IAA)-amido synthetase, which is suppressed in aboveground tissues under normal conditions but which is dramatically induced by drought stress. The activation of TLD1 reduced the IAA maxima at the lamina joint, shoot base, and nodes, resulting in subsequent alterations in plant architecture and tissue patterning but enhancing drought tolerance. Accordingly, the decreased level of free IAA in tld1-D due to the conjugation of IAA with amino acids greatly facilitated the accumulation of late-embryogenesis abundant mRNA compared with the wild type. The direct regulation of such drought-inducible genes by changes in the concentration of IAA provides a model for changes in plant architecture via the process of drought adaptation, which occurs frequently in nature. OsGH3.13|TLD1 Kinetic basis for the conjugation of auxin by a GH3 family indole-acetic acid-amido synthetase 2010 J Biol Chem National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. The GH3 family of acyl-acid-amido synthetases catalyze the ATP-dependent formation of amino acid conjugates to modulate levels of active plant hormones, including auxins and jasmonates. Initial biochemical studies of various GH3s show that these enzymes group into three families based on sequence relationships and acyl-acid substrate preference (I, jasmonate-conjugating; II, auxin- and salicylic acid-conjugating; III, benzoate-conjugating); however, little is known about the kinetic and chemical mechanisms of these enzymes. Here we use GH3-8 from Oryza sativa (rice; OsGH3-8), which functions as an indole-acetic acid (IAA)-amido synthetase, for detailed mechanistic studies. Steady-state kinetic analysis shows that the OsGH3-8 requires either Mg(2+) or Mn(2+) for maximal activity and is specific for aspartate but accepts asparagine as a substrate with a 45-fold decrease in catalytic efficiency and accepts other auxin analogs, including phenyl-acetic acid, indole butyric acid, and naphthalene-acetic acid, as acyl-acid substrates with 1.4-9-fold reductions in k(cat)/K(m) relative to IAA. Initial velocity and product inhibition studies indicate that the enzyme uses a Bi Uni Uni Bi Ping Pong reaction sequence. In the first half-reaction, ATP binds first followed by IAA. Next, formation of an adenylated IAA intermediate results in release of pyrophosphate. The second half-reaction begins with binding of aspartate, which reacts with the adenylated intermediate to release IAA-Asp and AMP. Formation of a catalytically competent adenylated-IAA reaction intermediate was confirmed by mass spectrometry. These mechanistic studies provide insight on the reaction catalyzed by the GH3 family of enzymes to modulate plant hormone action. OsGH3.8|OsGH3-8|OsMGH3 OsMADS1, a rice MADS-box factor, controls differentiation of specific cell types in the lemma and palea and is an early-acting regulator of inner floral organs 2005 Plant J Department of MCB, Indian Institute of Science, Bangalore 560012, India. Grass flowers are highly derived compared to their eudicot counterparts. To delineate OsMADS1 functions in rice floret organ development we have examined its evolution and the consequences of its knockdown or overexpression. Molecular phylogeny suggests the co-evolution of OsMADS1 with grass family diversification. OsMADS1 knockdown perturbs the differentiation of specific cell types in the lemma and palea, creating glume-like features, with severe derangements in lemma differentiation. Conversely, ectopic OsMADS1 expression suffices to direct lemma-like differentiation in the glume. Strikingly, in many OsMADS1 knockdown florets glume-like organs occupy all the inner whorls. Such effects in the second and third whorl are unexplained, as wild-type florets do not express OsMADS1 in these primordia and because transcripts for rice B and C organ-identity genes are unaffected by OsMADS1 knockdown. Through a screen for OsMADS1 targets we identify a flower-specific Nt-gh3 type gene, OsMGH3, as a downstream gene. The delayed transcription activation of OsMGH3 by dexamethasone-inducible OsMADS1 suggests indirect activation. The OsMGH3 floret expression profile suggests a novel role for OsMADS1 as an early-acting regulator of second and third whorl organ fate. We thus demonstrate the differential contribution of OsMADS1 for lemma versus palea development and provide evidence for its regulatory function in patterning inner whorl organs. OsGH3.8|OsGH3-8|OsMGH3,OsMADS1|LHS1|AFO Activation of the indole-3-acetic acid-amido synthetase GH3-8 suppresses expansin expression and promotes salicylate- and jasmonate-independent basal immunity in rice 2008 Plant Cell National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. New evidence suggests a role for the plant growth hormone auxin in pathogenesis and disease resistance. Bacterial infection induces the accumulation of indole-3-acetic acid (IAA), the major type of auxin, in rice (Oryza sativa). IAA induces the expression of expansins, proteins that loosen the cell wall. Loosening the cell wall is key for plant growth but may also make the plant vulnerable to biotic intruders. Here, we report that rice GH3-8, an auxin-responsive gene functioning in auxin-dependent development, activates disease resistance in a salicylic acid signaling- and jasmonic acid signaling-independent pathway. GH3-8 encodes an IAA-amino synthetase that prevents free IAA accumulation. Overexpression of GH3-8 results in enhanced disease resistance to the rice pathogen Xanthomonas oryzae pv oryzae. This resistance is independent of jasmonic acid and salicylic acid signaling. Overexpression of GH3-8 also causes abnormal plant morphology and retarded growth and development. Both enhanced resistance and abnormal development may be caused by inhibition of the expression of expansins via suppressed auxin signaling. OsGH3.8|OsGH3-8|OsMGH3 Auxin-responsive OsMGH3, a common downstream target of OsMADS1 and OsMADS6, controls rice floret fertility 2011 Plant Cell Physiol Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore-560012, India. GH3 proteins control auxin homeostasis by inactivating excess auxin as conjugates of amino acids and sugars and thereby controlling cellular bioactive auxin. Since auxin regulates many aspects of plant growth and development, regulated expression of these genes offers a mechanism to control various developmental processes. OsMGH3/OsGH3-8 is expressed abundantly in rice florets and is regulated by two related and redundant transcription factors, OsMADS1 and OsMADS6, but its contribution to flower development is not known. We functionally characterize OsMGH3 by overexpression and knock-down analysis and show a partial overlap in these phenotypes with that of mutants in OsMADS1 and OsMADS6. The overexpression of OsMGH3 during the vegetative phase affects the overall plant architecture, whereas its inflorescence-specific overexpression creates short panicles with reduced branching, resembling in part the effects of OsMADS1 overexpression. In contrast, the down-regulation of endogenous OsMGH3 caused phenotypes consistent with auxin overproduction or activated signaling, such as ectopic rooting from aerial nodes. Florets in OsMGH3 knock-down plants were affected in carpel development and pollen viability, both of which reduced fertility. Some of these floret phenotypes are similar to osmads6 mutants. Taken together, we provide evidence for the functional significance of auxin homeostasis and its transcriptional regulation during rice panicle branching and floret organ development. OsGH3.8|OsGH3-8|OsMGH3,OsMADS1|LHS1|AFO,OsMADS6|MFO1 Global identification of multiple OsGH9 family members and their involvement in cellulose crystallinity modification in rice 2013 PLoS One National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research (Wuhan), Biomass and Bioenergy Research Centre, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, People's Republic of China. Plant glycoside hydrolase family 9 (GH9) comprises typical endo-beta-1,4-glucanase (EGases, EC3.2.1.4). Although GH9A (KORRIGAN) family genes have been reported to be involved in cellulose biosynthesis in plants, much remains unknown about other GH9 subclasses. In this study, we observed a global gene co-expression profiling and conducted a correlation analysis between OsGH9 and OsCESA among 66 tissues covering most periods of life cycles in 2 rice varieties. Our results showed that OsGH9A3 and B5 possessed an extremely high co-expression with OsCESA1, 3, and 8 typical for cellulose biosynthesis in rice. Using two distinct rice non-GH9 mutants and wild type, we performed integrative analysis of gene expression level by qRT-PCR, cellulase activities in situ and in vitro, and lignocellulose crystallinity index (CrI) in four internodes of stem tissues. For the first time, OsGH9B1, 3, and 16 were characterized with the potential role in lignocellulose crystallinity alteration in rice, whereas OsGH9A3 and B5 were suggested for cellulose biosynthesis. In addition, phylogenetic analysis and gene co-expression comparison revealed GH9 function similarity in Arabidopsis and rice. Hence, the data can provide insights into GH9 function in plants and offer the potential strategy for genetic manipulation of plant cell wall using the five aforementioned novel OsGH9 genes. OsGH9A3,OsGH9B1,OsGLU1|OsGH9A1 Os-GIGANTEA confers robust diurnal rhythms on the global transcriptome of rice in the field 2011 Plant Cell Photosynthesis and Photobiology Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Japan. tizawa@nias.affrc.go.jp The circadian clock controls physiological traits such as flowering time, photosynthesis, and growth in plants under laboratory conditions. Under natural field conditions, however, little is known about the significance of the circadian clock in plants. By time-course transcriptome analyses of rice (Oryza sativa) leaves, using a newly isolated rice circadian clock-related mutant carrying a null mutation in Os-GIGANTEA (Os-GI), we show here that Os-GI controlled 75% (false discovery rate = 0.05) of genes among 27,201 genes tested and was required for strong amplitudes and fine-tuning of the diurnal rhythm phases of global gene expression in the field. However, transcripts involved in primary metabolism were not greatly affected by osgi. Time-course metabolome analyses of leaves revealed no trends of change in primary metabolites in osgi plants, and net photosynthetic rates and grain yields were not affected. By contrast, some transcripts and metabolites in the phenylpropanoid metabolite pathway were consistently affected. Thus, net primary assimilation of rice was still robust in the face of such osgi mutation-related circadian clock defects in the field, unlike the case with defects caused by Arabidopsis thaliana toc1 and ztl mutations in the laboratory. OsGI Circadian regulation of rice (Oryza sativa L.) CONSTANS-like gene transcripts 2004 Mol Cells School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea. We identified three rice cDNA clones showing amino acid similarity to the Arabidopsis CONSTANS-like proteins from a database search (S12569, S3574, and C60910), to examine if their transcript abundances were under circadian control. Unlike the other two proteins, the protein encoded by the S12569 cDNA contains only one CONSTANS-like zinc finger B box, and a CCT region. We found that the transcript levels of these rice CONSTANS-like (COL) genes were under circadian control. The oscillation phase of the S12569 gene transcript was more or less opposite to those of OsGI (rice GIGANTEA homolog) and Hd1 (rice COSTANS homolog), whereas the phases of the other two gene transcripts were similar to that of the Hd1 transcript. S12569 mRNA started to increase about 3 h after the onset of the dark period, with a peak about 3 h after its end. The S3574 and C60910 genes were expressed to similar extents during the vegetative and reproductive phases, like OsGI. Higher levels of S12569 transcripts, however, like those of Hd1, were detected in the earlier stages of panicle development. Unlike Hd1 transcripts, S12569, S3574, and C60910 transcripts were present at similar levels in the aerial parts of plants and in their roots during the vegetative phase. In conclusion, the rice COL genes showed distinctive expression patterns from the CO and COL genes, as well as Hd1, a rice CO homolog. OsGI Adaptation of photoperiodic control pathways produces short-day flowering in rice 2003 Nature Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan. The photoperiodic control of flowering is one of the important developmental processes of plants because it is directly related to successful reproduction. Although the molecular genetic analysis of Arabidopsis thaliana, a long-day (LD) plant, has provided models to explain the control of flowering time in this species, very little is known about its molecular mechanisms for short-day (SD) plants. Here we show how the photoperiodic control of flowering is regulated in rice, a SD plant. Overexpression of OsGI, an orthologue of the Arabidopsis GIGANTEA (GI) gene in transgenic rice, caused late flowering under both SD and LD conditions. Expression of the rice orthologue of the Arabidopsis CONSTANS (CO) gene was increased in the transgenic rice, whereas expression of the rice orthologue of FLOWERING LOCUS T (FT) was suppressed. Our results indicate that three key regulatory genes for the photoperiodic control of flowering are conserved between Arabidopsis, a LD plant, and rice, a SD plant, but regulation of the FT gene by CO was reversed, resulting in the suppression of flowering in rice under LD conditions. OsGI Physiological significance of the plant circadian clock in natural field conditions 2012 Plant Cell Environ Functional Plant Research Unit, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Japan. tizawa@nias.affrc.go.jp For many decades, researchers have focused on the self-sustainable oscillations of plant circadian clocks, which can only be observed under artificial constant environmental conditions. However, plants have evolved under natural diurnal conditions where several major environmental cues such as light, temperature and humidity are dramatically changing and interacting with each other. Therefore, little is known about the roles of the plant circadian clock in natural field conditions. Molecular genetic analyses in Arabidopsis thaliana have revealed that some core circadian clock genes are required for the establishment of robust circadian rhythms under artificial diurnal conditions, and that others function only as self-oscillators. However, it is largely unknown yet how those robust rhythms can be obtained under natural diurnal conditions. Recently, an extensive time-course transcriptome analysis of rice (Oryza sativa) leaves in natural field conditions revealed that OsGIGANTEA, the sole rice ortholog of the Arabidopsis GIGANTEA gene, governs the robust diurnal rhythm of rice leaf transcriptomes even under natural diurnal conditions; rice Osgi mutants exhibited severely defective transcriptome rhythms under strong diurnal changes in environmental cues. This review focuses on the physiological significance of the plant circadian clock in natural field conditions. OsGI Isolation of Rice Genes Possibly Involved in the Photoperiodic Control of Flowering by a Fluorescent Differential Display Method 2002 Plant and Cell Physiology Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, 630-0101 Japan To better understand the molecular mechanisms of the photoperiodic regulation of rice, a short-day plant, we isolated 27 cDNAs that were differentially expressed in the photoperiod-insensitive se5 mutant from approximately 8,400 independent mRNA species by the use of a fluorescent differential display (FDD). For this screening, we isolated mRNAs at five different time points during the night and compared their expression patterns between se5 and the wild type. Of 27 cDNAs isolated, 12 showed diurnal expression patterns often associated with genes involved in the determination of the flowering time. In se5, expression of nine cDNAs was increased. Five of these cDNAs were up-regulated under SD, suggesting that they may promote flowering under SD. They included genes encoding a cDNA containing a putative NAC domain, the fructose-bisphosphate aldolase, and a protease inhibitor. Expression of three cDNAs was decreased in se5 but not photoperiodically regulated. These cDNAs included a rice homolog of Arabidopsis GIGANTEA (GI), lir1, and a gene for myo-inositol 1-phosphate synthase, all of which were previously shown to be under the control of circadian clocks. The expression patterns of the rice homolog of GI, OsGI, were similar to those of the Arabidopsis GI, suggesting the conservation of some mechanisms for the photoperiodic regulation of flowering between these two species. OsGI A study of phytohormone biosynthetic gene expression using a circadian clock-related mutant in rice 2011 Plant Signal Behav Functional Plant Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan. We have recently isolated a rice circadian clock-related mutant carrying a null mutation in Os-GIGANTEA(GI) gene, the solo ortholog of Arabidopsis GI. Time-course global transcriptome analyses of leaves from wild-type and osgi mutant grown in the field have revealed that Os-GI affects gene expression of more than half of genes on rice 44k microarray. To better understand the biological significance of circadian clock function in growth and development of rice, we here investigated the gene expression involved in phytohormone biosynthesis. Here we found that mRNA levels of a few major genes encoding GA2-oxidase which can inactivate bioactive gibberellins (GAs) were remarkably increased in osgi-1 plants. This suggests that Os-GI functions to maintain bioactive GA level through the regulation of the GA-deactivating enzyme genes in rice. Consistently, osgi-1 plants showed semi-dwarf phenotype with reduced internode and leaf sheath elongation. OsGI Thermodynamic characterization of OsGID1-gibberellin binding using calorimetry and docking simulations 2011 J Mol Recognit Division of Plant Research, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. Gibberellins (GAs) are phytohormones regulating various developmental processes in plants. In rice, the initial GA-signaling events involve the binding of a GA to the soluble GA receptor protein, GID1. Although X-ray structures for certain GID1/GA complexes have recently been determined, an examination of the complexes does not fully clarify how GID1s discriminate among different GAs. Herein, we present a study aimed at defining the types of forces important to binding via a combination of isothermal titration calorimetry (ITC) and computational docking studies that employed rice GID1 (OsGID1), OsGID1 mutants, which were designed to have a decreased possible number of hydrogen bonds with bound GA, and GA variants. We find that, in general, GA binding is enthalpically driven and that a hydrogen bond between the phenolic hydroxyl of OsGID1 Tyr134 and the C-3 hydroxyl of a GA is a defining structural element. A hydrogen-bond network that involves the C-6 carboxyl of a GA that directly hydrogen bonds the hydroxyl of Ser198 and indirectly, via a two-water-molecule network, the phenolic hydroxyl of Tyr329 and the NH of the amide side-chain of Asn255 is also important for GA binding. The binding of OsGID1 by GA(1) is the most enthalpically driven association found for the biologically active GAs evaluated in this study. This observation might be a consequence of a hydrogen bond formed between the hydroxyl at the C-13 position of GA(1) and the main chain carbonyl of OsGID1 Phe245. Our results demonstrate that by combining ITC experiments and computational methods much can be learned about the thermodynamics of ligand/protein binding. GID1|OsGID1 The suppressive function of the rice DELLA protein SLR1 is dependent on its transcriptional activation activity 2012 Plant J Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan. When the gibberellin (GA) receptor GIBBERELLIN INSENSITIVE DWARF 1 (GID1) binds to GA, GID1 interacts with DELLA proteins, repressors of GA signaling. This interaction inhibits the suppressive function of DELLA protein and thereby activates the GA response. However, how DELLA proteins exert their suppressive function and how GID1s inhibit suppressive function of DELLA proteins is unclear. By yeast one-hybrid experiments and transient expression of the N-terminal region of rice DELLA protein (SLR1) in rice callus, we established that the N-terminal DELLA/TVHYNP motif of SLR1 possesses transactivation activity. When SLR1 proteins with various deletions were over-expressed in rice, the severity of dwarfism correlated with the transactivation activity observed in yeast, indicating that SLR1 suppresses plant growth through transactivation activity. This activity was suppressed by the GA-dependent GID1-SLR1 interaction, which may explain why GA responses are induced in the presence of GA. The C-terminal GRAS domain of SLR1 also exhibits a suppressive function on plant growth, possibly by directly or indirectly interacting with the promoter region of target genes. Our results indicate that the N-terminal region of SLR1 has two roles in GA signaling: interaction with GID1 and transactivation activity. GID1|OsGID1,SLR1|OsGAI GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin 2005 Nature Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan. Gibberellins (GAs) are phytohormones that are essential for many developmental processes in plants. It has been postulated that plants have both membrane-bound and soluble GA receptors; however, no GA receptors have yet been identified. Here we report the isolation and characterization of a new GA-insensitive dwarf mutant of rice, gid1. The GID1 gene encodes an unknown protein with similarity to the hormone-sensitive lipases, and we observed preferential localization of a GID1-green fluorescent protein (GFP) signal in nuclei. Recombinant glutathione S-transferase (GST)-GID1 had a high affinity only for biologically active GAs, whereas mutated GST-GID1 corresponding to three gid1 alleles had no GA-binding affinity. The dissociation constant for GA4 was estimated to be around 10(-7) M, enough to account for the GA dependency of shoot elongation. Moreover, GID1 bound to SLR1, a rice DELLA protein, in a GA-dependent manner in yeast cells. GID1 overexpression resulted in a GA-hypersensitive phenotype. Together, our results indicate that GID1 is a soluble receptor mediating GA signalling in rice. GID1|OsGID1,SLR1|OsGAI Molecular interactions of a soluble gibberellin receptor, GID1, with a rice DELLA protein, SLR1, and gibberellin 2007 Plant Cell Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan. mueguchi@agr.nagoya-u.ac.jp GIBBERELLIN INSENSITIVE DWARF1 (GID1) encodes a soluble gibberellin (GA) receptor that shares sequence similarity with a hormone-sensitive lipase (HSL). Previously, a yeast two-hybrid (Y2H) assay revealed that the GID1-GA complex directly interacts with SLENDER RICE1 (SLR1), a DELLA repressor protein in GA signaling. Here, we demonstrated, by pull-down and bimolecular fluorescence complementation (BiFC) experiments, that the GA-dependent GID1-SLR1 interaction also occurs in planta. GA(4) was found to have the highest affinity to GID1 in Y2H assays and is the most effective form of GA in planta. Domain analyses of SLR1 using Y2H, gel filtration, and BiFC methods revealed that the DELLA and TVHYNP domains of SLR1 are required for the GID1-SLR1 interaction. To identify the important regions of GID1 for GA and SLR1 interactions, we used many different mutant versions of GID1, such as the spontaneous mutant GID1s, N- and C-terminal truncated GID1s, and mutagenized GID1 proteins with conserved amino acids replaced with Ala. The amino acid residues important for SLR1 interaction completely overlapped the residues required for GA binding that were scattered throughout the GID1 molecule. When we plotted these residues on the GID1 structure predicted by analogy with HSL tertiary structure, many residues were located at regions corresponding to the substrate binding pocket and lid. Furthermore, the GA-GID1 interaction was stabilized by SLR1. Based on these observations, we proposed a molecular model for interaction between GA, GID1, and SLR1. GID1|OsGID1 A rice gid1 suppressor mutant reveals that gibberellin is not always required for interaction between its receptor, GID1, and DELLA proteins 2010 Plant Cell Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan. To investigate gibberellin (GA) signaling using the rice (Oryza sativa) GA receptor GIBBERELLIN-INSENSITIVE DWARF1 (GID1) mutant gid1-8, we isolated a suppressor mutant, Suppressor of gid1-1 (Sgd-1). Sgd-1 is an intragenic mutant containing the original gid1-8 mutation (L45F) and an additional amino acid substitution (P99S) in the loop region. GID1(P99S) interacts with the rice DELLA protein SLENDER RICE1 (SLR1), even in the absence of GA. Substitution of the 99th Pro with other amino acids revealed that substitution with Ala (P99A) caused the highest level of GA-independent interaction. Physicochemical analysis using surface plasmon resonance revealed that GID1(P99A) has smaller K(a) (association) and K(d) (dissociation) values for GA(4) than does wild-type GID1. This suggests that the GID1(P99A) lid is at least partially closed, resulting in both GA-independent and GA-hypersensitive interactions with SLR1. One of the three Arabidopsis thaliana GID1s, At GID1b, can also interact with DELLA proteins in the absence of GA, so we investigated whether GA-independent interaction of At GID1b depends on a mechanism similar to that of rice GID1(P99A). Substitution of the loop region or a few amino acids of At GID1b with those of At GID1a diminished its GA-independent interaction with GAI while maintaining the GA-dependent interaction. Soybean (Glycine max) and Brassica napus also have GID1s similar to At GID1b, indicating that these unique GID1s occur in various dicots and may have important functions in these plants. GID1|OsGID1,SLR1|OsGAI Rice OsGL1-6 Is Involved in Leaf Cuticular Wax Accumulation and Drought Resistance 2013 PLoS One Laboratory Center of Basic Biology and Biotechnology, Education Department of Guangdong Province, Cuticular wax is a class of organic compounds that comprises the outermost layer of plant surfaces. Plant cuticular wax, the last barrier of self-defense, plays an important role in plant growth and development. The OsGL1-6 gene, a member of the fatty aldehyde decarbonylase gene family, is highly homologous to Arabidopsis CER1, which is involved in cuticular wax biosynthesis. However, whether OsGL1-6 participates in cuticular wax biosynthesis remains unknown. In this study, an OsGL1-6 antisense-RNA vector driven by its own promoter was constructed and introduced into the rice variety Zhonghua11 by Agrobacterium-mediated transformation to obtain several independent transgenic plants with decreased OsGL1-6 expression. These OsGL1-6 antisense-RNA transgenic plants showed droopy leaves at the booting stage, significantly decreased leaf cuticular wax deposition, thinner cuticle membrane, increased chlorophyll leaching and water loss rates, and enhanced drought sensitivity. The OsGL1-6 gene was constitutively expressed in all examined organs and was very highly expressed in leaf epidermal cells and vascular bundles. The transient expression of OsGL1-6-GFP fusion indicated that OsGL1-6 is localized in the endoplasmic reticulum. Qualitative and quantitative analysis of the wax composition using gas chromatography-mass spectrometry revealed a significantly reduced total cuticular wax load on the leaf blades of the OsGL1-6 antisense-RNA transgenic plants as well as markedly decreased alkane and aldehyde contents. Their primary alcohol contents increased significantly compared with those in the wild type plants, suggesting that OsGL1-6 is associated with the decarbonylation pathways in wax biosynthesis. We propose that OsGL1-6 is involved in the accumulation of leaf cuticular wax and directly impacts drought resistance in rice. OsGL1-6 Characterization of Glossy1-homologous genes in rice involved in leaf wax accumulation and drought resistance 2009 Plant Mol Biol National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China. The outermost surfaces of plants are covered with an epicuticular wax layer that provides a primary waterproof barrier and protection against different environmental stresses. Glossy 1 (GL1) is one of the reported genes controlling wax synthesis. This study analyzed GL1-homologous genes in Oryza sativa and characterized the key members of this family involved in wax synthesis and stress resistance. Sequence analysis revealed 11 homologous genes of GL1 in rice, designated OsGL1-1 to OsGL1-11. OsGL1-1, -2 and -3 are closely related to GL1. OsGL1-4, -5, -6, and -7 are closely related to Arabidopsis CER1 that is involved in cuticular wax biosynthesis. OsGL1-8, -9, -10 and -11 are closely related to SUR2 encoding a putative sterol desaturase also involved in epicuticular wax biosynthesis. These genes showed variable expression levels in different tissues and organs of rice, and most of them were induced by abiotic stresses. Compared to the wild type, the OsGL1-2-over-expression rice exhibited more wax crystallization and a thicker epicuticular layer; while the mutant of this gene showed less wax crystallization and a thinner cuticular layer. Chlorophyll leaching experiment suggested that the cuticular permeability was decreased and increased in the over-expression lines and the mutant, respectively. Quantification analysis of wax composition by GC-MS revealed a significant reduction of total cuticular wax in the mutant and increase of total cuticular wax in the over-expression plants. Compared to the over-expression and wild type plants, the osgl1-2 mutant was more sensitive to drought stress at reproductive stage, suggesting an important role of this gene in drought resistance. OsGL1-6,OsGL1-1|WSL2 Ectopic overexpression of the transcription factor OsGLK1 induces chloroplast development in non-green rice cells 2009 Plant Cell Physiol Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan. hnakamura@pgr1.ch.a.u-tokyo.ac.jp For systematic and genome-wide analyses of rice gene functions, we took advantage of the full-length cDNA overexpresser (FOX) gene-hunting system and generated >12 000 independent FOX-rice lines from >25 000 rice calli treated with the rice-FOX Agrobacterium library. We found two FOX-rice lines generating green calli on a callus-inducing medium containing 2,4-D, on which wild-type rice calli became ivory yellow. In both lines, OsGLK1 cDNA encoding a GARP transcription factor was ectopically overexpressed. Using rice expression-microarray and northern blot analyses, we found that a large number of nucleus-encoded genes involved in chloroplast functions were highly expressed and transcripts of plastid-encoded genes, psaA, psbA and rbcL, increased in the OsGLK1-FOX calli. Transmission electron microscopy showed the existence of differentiated chloroplasts with grana stacks in OsGLK1-FOX calli cells. However, in darkness, OsGLK1-FOX calli did not show a green color or develop grana stacks. Furthermore, we found developed chloroplasts in vascular bundle and bundle sheath cells of coleoptiles and leaves from OsGLK1-FOX seedlings. The OsGLK1-FOX calli exhibited high photosynthetic activity and were able to grow on sucrose-depleted media, indicating that developed chloroplasts in OsGLK1-FOX rice calli are functional and active. We also observed that the endogenous OsGLK1 mRNA level increased synchronously with the greening of wild-type calli after transfer to plantlet regeneration medium. These results strongly suggest that OsGLK1 regulates chloroplast development under the control of light and phytohormones, and that it is a key regulator of chloroplast development. OsGLK1 Molecular cloning, characterization and in vitro expression of a novel endo-1,3-beta-glucanase up-regulated by ABA and drought stress in rice (Oryza sativa L.) 2001 Plant Science Department of Low Temperature Science, National Agricultural Research Center for Hokkaido Region, 1 Hitsujigaoka, Toyohira, Sapporo, Hokkaido 062-8555, Japan We report the identification of a full length cDNA encoding endo-1,3-beta-glucanase (OsGLN1) from a rice cDNA library by using barley endo-1,3-beta-glucanase isoenzyme GII gene as probe. The OsGLN1 has an open reading frame of 954 bp that encodes a polypeptide of 318 amino acid residues with the calculated Mr of 34,723 and the predicted pI of 8.38. The deduced amino acid sequence of OsGLN1 exhibits 71% highest positional identity with the barley endo-1,3-beta-glucanase isoenzyme GV, which does not have the N-terminal signal peptide. Northern blot analysis revealed that the expression of OsGLN1 is up-regulated by drought stress and abscisic acid (ABA) treatment and the accumulation of OsGLN1 transcript is more in the roots of rice seedlings. Immunoblot analysis with antibody raised against GST-OsGLN1 recombinant protein demonstrated that there is a proportional increase between the 34-kDa OsGLN1 protein and OsGLN1 transcript. The GST-OsGLN1 recombinant protein rapidly hydrolyzed the cell wall beta-glucans of rice fungal pathogen, Pyricularia oryzae, other than typical substrates for endo-1,3-beta-glucanase. These results clearly indicated that the endo-1,3-beta-glucanase encoded by OsGLN1 plays an important role associated with plant defence against abiotic and biotic stresses particularly for drought and fungal pathogen in rice seedlings. OsGLN1 Cloning, characterization and expression of OsGLN2, a rice endo-1,3-beta-glucanase gene regulated developmentally in flowers and hormonally in germinating seeds 2004 Planta Department of Low-Temperature Science, National Agricultural Research Center for Hokkaido Region, 1 Hitsujigaoka, Toyohira-ku, Sapporo, 062-8555 Hokkaido, Japan. takiyama@affrc.go.jp We report here the isolation and characterization of a new endo-1,3-beta-glucanase (1,3-beta-GLU) cDNA, OsGLN2, that is expressed both in flowers and in germinating seeds of rice (Oryza sativa L.). The isolated OsGLN2 gene encoded a protein which displayed 72%, 93% and 92% identity at the amino acid level with those encoded by barley GII, rice Gns4 and glu1 1,3-beta-GLU genes, respectively. A GST-OsGLN2 recombinant protein expressed in Escherichia coli preferentially hydrolyzed Laminaria digitata 1,3;1,6-beta-glucan and liberated only oligosaccharides, suggesting that the enzyme can be classified as a 1,3-beta-GLU. Northern analysis with a 3'-UTR gene-specific probe revealed that OsGLN2 is expressed exclusively in the paleae and lemmas during flowering, and no expression of OsGLN2 was detected in other tissues such as leaf blades, leaf sheaths, stems, nodes and roots in mature rice plants. The OsGLN2 gene is also expressed in germinating seeds, where its expression is predominant in endosperms rather than embryos. In de-embryonated rice half-seeds, addition of gibberellin A3 (GA) greatly enhanced expression of the OsGLN2 gene, while the GA-induced gene expression was suppressed strongly by abscisic acid (ABA). This is the first report, to our knowledge, that OsGLN2 encodes a 1,3-beta-GLU and is expressed specifically in paleae and lemmas during flowering and in germinating seeds, where its expression is enhanced by GA and suppressed by ABA. OsGLN2 Interaction of N-acetylglutamate kinase with a PII-like protein in rice 2004 Plant Cell Physiol Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555 Japan. PII protein in bacteria is a sensor for 2-oxoglutarate and a transmitter for glutamine signaling. We identified an OsGlnB gene that encoded a bacterial PII-like protein in rice. Yeast two-hybrid analysis showed that an OsGlnB gene product interacted with N-acetylglutamate kinase 1 (OsNAGK1) and PII-like protein (OsGlnB) itself in rice. In cyanobacteria, NAGK is a key enzyme in arginine biosynthesis. Transient expression of OsGlnB cDNA or OsNAGK1 cDNA fused with sGFP in rice leaf blades strongly suggested that the PII-like protein as well as OsNAGK1 protein is located in chloroplasts. Both OsGlnB and OsNAGK1 genes were expressed in roots, leaf blades, leaf sheaths and spikelets of rice, and these two genes were coordinately expressed in leaf blades during the life span. Thus, PII-like protein in rice plants is potentially able to interact with OsNAGK1 protein in vivo. This finding will provide a clue to the precise physiological function of PII-like protein in rice. OsGlnB,OsNAGK1|NAGK1,OsNAGK2|NAGK2 Functional role of rice germin-like protein1 in regulation of plant height and disease resistance 2010 Biochem Biophys Res Commun Department of Biotechnology and Adv Lab for Plant Genetic Engineering, Advanced Technology Development Center, Indian Institute of Technology, Kharagpur 721302, India. The functional role of rice (Oryza sativa) germin-like protein1 (OsGLP1) was elucidated through development of transgenic plants involving endogenous gene silencing in rice and heterologous gene expression in tobacco. Usually, the single copy OsGLP1 gene in rice plant was found to be expressed predominantly in green vegetative tissues. The transgenic rice lines showed significant reduction in endogenous OsGLP1 expression due to 26nt siRNA-mediated gene silencing, displayed semi-dwarfism and were affected seriously by fungal diseases, compared to the untransformed plant. Structural homology modeling predicted a superoxide dismutase (SOD) domain in OsGLP1 protein which upon over-expression in transgenic tobacco plant clearly documented SOD activity. Our observations on the maintenance of cell dimension, cell wall-associated localization particularly in the sub-epidermal tissues and the SOD activity of OsGLP1 could explain its functional role in regulation of plant height and disease resistance in rice plant. OsGLP1 Transgenically expressed rice germin-like protein1 in tobacco causes hyper-accumulation of H2O2 and reinforcement of the cell wall components 2010 Biochem Biophys Res Commun Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, India. Our recent report documented that the rice germin-like protein1 (OsGLP1), being a cell wall-associated protein involves in disease resistance in rice and possesses superoxide dismutase (SOD) activity as recognized by heterologous expression in tobacco. In the present study, the transgenic tobacco plants were analyzed further to decipher the detailed physiological and biochemical functions of the OsGLP1 and its associated SOD activity. The transgenic tobacco lines expressing SOD-active OsGLP1 showed tolerance against biotic and abiotic stresses mitigated by hyper-accumulating H(2)O(2) upon infection by fungal pathogen (Fusarium solani) and treatment to chemical oxidizing agent (ammonium persulfate), respectively. Histological staining revealed enhanced cross-linking of the cell wall components in the stem tissues of the transgenic plants. Fourier transform infrared spectroscopy (FTIR) analysis of the biopolymer from the stem tissues of the transgenic and untransformed plants revealed differential banding pattern of the spectra corresponding to various functional groups. Our findings demonstrate that the OsGLP1 with its inherent SOD activity is responsible for hyper-accumulation of H(2)O(2) and reinforcement of the cell wall components. OsGLP1 OsGLU1, a putative membrane-bound endo-1,4-beta-D-glucanase from rice, affects plant internode elongation 2006 Plant Mol Biol National Key Lab of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 100101, Beijing, People's Republic of China. A dwarf mutant glu was identified from screening of T-DNA tagged rice population. Genetic analysis of the T1 generation of glu revealed that a segregation ratio of wild-type:dwarf phenotype was 3:1, suggesting that the mutated phenotype was controlled by a single recessive nuclear locus. The mutated gene OsGLU1, identified by Tail-PCR, encodes a putative membrane-bound endo-1,4-beta-D-glucanase, which is highly conserved between mono- and dicotyledonous plants. Mutation of OsGLU1 resulted in a reduction in cell elongation, and a decrease in cellulose content but an increase in pectin content, suggesting that OsGLU1 affects the internode elongation and cell wall components of rice plants. Transgenic glu mutants harboring the OsGLU1 gene complemented the mutation and displayed the wild-type phenotype. In addition, OsGLU1 RNAi plants showed similar phenotype as the glu mutant has. These results indicate that OsGLU1 plays important roles in plant cell growth. Gibberellins and brassinosteroids induced OsGLU1 expression. In rice genome, endo-1,4-beta-D-glucanases form a multiple gene family with 15 members, and each may have a distinct expression pattern in different organs. These results indicate that endo-1,4-beta-D-glucanases may play diverse roles in growth and developmental process of rice plants. OsGLU1|OsGH9A1 OsGLU3, a putative membrane-bound endo-1,4-beta-glucanase, is required for root cell elongation and division in rice (Oryza sativa L.) 2012 Mol Plant The State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, China. Plant roots move through the soil by elongation. This is vital to their ability to anchor the plant and acquire water and minerals from the soil. In order to identify new genes involved in root elongation in rice, we screened an ethyl methane sulfonate (EMS)-mutagenized rice library, and isolated a short root mutant, Osglu3-1. The map-based cloning results showed that the mutant was due to a point mutation in OsGLU3, which encodes a putative membrane-bound endo-1,4-beta-glucanase. Osglu3-1 displayed less crystalline cellulose content in its root cell wall, shorter root cell length, and a slightly smaller root meristem as visualized by restricted expression of OsCYCB1,1:GUS. Exogenous application of glucose can suppress both the lower root cell wall cellulose content and short root phenotypes of Osglu3-1. Consistently, OsGLU3 is ubiquitously expressed in various tissues with strong expression in root tip, lateral root, and crown root primodia. The fully functional OsGLU3-GFP was detected in plasma membrane, and FM4-64-labeled compartments in the root meristem and elongation zones. We also found that phosphate starvation, an environmental stress, altered cell wall cellulose content to modulate root elongation in a OsGLU3-dependant way. OsGLU3|RT Modulating the root elongation by phosphate/nitrogen starvation in an OsGLU3 dependant way in rice 2012 Plant Signal Behav State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou, China. Previously, we showed that OsGLU3, a beta-1,4-endoglucanase, can affect the cellulose synthesis for root elongation in rice. And the phosphate starvation induced root elongation in rice depends on the function of OsGLU3. Here, we further showed that OsGLU3 is also dispensable for nitrogen starvation induced root elongation in rice. OsGLU3|RT ROOT GROWTH INHIBITING, a Rice Endo-1,4-beta-d-Glucanase, Regulates Cell Wall Loosening and is Essential for Root Elongation 2012 Journal of Plant Growth Regulation Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi, 464-8601, Japan The molecular mechanism involved in cell wall dynamics has not been well clarified, although it is quite important for organ growth. We characterized a rice mutant, root growth inhibiting (rt), which is defective in root elongation. The rt mutant showed a severe defect in cell elongation at the root-elongating zone with additional collapse of epidermal and cortex cells at the root tip caused by the defect in the smooth exfoliation of root cap cells. Consistent with these phenotypes, expression of the RT gene, which encodes a member of the membrane-anchored endo-1,4-beta-d-glucanase, was specifically localized in the root-elongating zone and at the junction between epidermal and root cap cells. The enzymatic analysis of root extracts from the wild-type and rt mutant indicated that RT hydrolyzes noncrystalline amorphous cellulose. The cellulose content was slightly increased but the crystallinity of cellulose was decreased in the rt root. In addition, the hemicellulose composition was different between wild-type and rt roots. The total extensibility was significantly lower in the rt root explants. Based on these results, we concluded that RT is involved in the disassembly of the cell wall for cell elongation in roots as well as for root cap exfoliation from the epidermal cell layer by hydrolyzing the noncrystalline amorphous cellulose fibers of cellulose microfibrils resulting in loosening of the hemicellulose and cellulose interaction. OsGLU3|RT Identification and fine mapping of a thermo-sensitive chlorophyll deficient mutant in rice (Oryza sativa L.) 2007 Planta College of Life Sciences, Zhejiang University, 310029 Hangzhou, ZJ, China. A thermo-sensitive chlorophyll deficient mutant was isolated from more than 15,000 transgenic rice lines. The mutant displayed normal phenotype at 23 degrees C or lower temperature (permissive temperature). However, when grown at 26 degrees C or higher (nonpermissive temperature) the plant exhibited an abnormal phenotype characterized by yellow green leaves. Genetic analysis revealed that a single nuclear-encoded recessive gene is responsible for the mutation, which is tentatively designed as cde1(t) (chlorophyll deficient 1, temporally). PCR analysis and hygromycin resistance assay indicated the mutation was not caused by T-DNA insertion. To isolate the cde1(t) gene, a map-based cloning strategy was employed and 15 new markers (five SSR and ten InDels markers) were developed. A high-resolution physical map of the chromosomal region around the cde1(t) gene was made using F(2) and F(3) population consisting of 1,858 mutant individuals. Finally, the cde1(t) gene was mapped in 7.5 kb region between marker ID10 and marker ID11 on chromosome 2. Sequence analysis revealed only one candidate gene, OsGluRS, in the 7.5 kb region. Cloning and sequencing of the target region from the cde1(t) mutant showed that a missense mutation occurred in the mutant. So the OsGluRS gene (TIGR locus Os02 g02860) which encode glutamyl-tRNA synthetase was identified as the Cde1(t) gene. OsGluRS|Cde1(t) Reduced expression of a gene encoding a Golgi localized monosaccharide transporter (OsGMST1) confers hypersensitivity to salt in rice (Oryza sativa) 2011 J Exp Bot Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. Sugar transport is critical for normal plant development and stress responses. However, functional evidence for the roles of monosaccharide transporters in rice (Oryza sativa) has not previously been presented. In this study, reversed genetics was used to identify OsGMST1 as a member of the monosaccharide transporter family in rice. The predicted 481 amino acid protein has the typical features of a sugar transporter in the plastid glucose transporter subfamily consistent with reduced monosaccharide accumulation in plants with reduced OsGMST1 expression. OsGMST1-green fluorescent protein is localized to the Golgi apparatus. OsGMST1 expression is induced by salt treatment and reduced expression confers hypersensitivity to salt stress in rice. OsGMST1 may play a direct or an indirect role in tolerance to salt stress in rice. OsGMST1 A novel short-root gene encodes a glucosamine-6-phosphate acetyltransferase required for maintaining normal root cell shape in rice 2005 Plant Physiol State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310029, People's Republic of China. Glycosylation is a posttranslational modification occurring in many secreted and membrane-associated proteins in eukaryotes. It plays important roles in both physiological and pathological processes. Most of these protein modifications depend on UDP-N-acetylglucosamine. In this study, a T-DNA insertional rice (Oryza sativa) mutant exhibiting a temperature-sensitive defect in root elongation was isolated. Genetic and molecular analysis indicated that the mutated phenotype was caused by loss of function of a gene encoding a glucosamine-6-P acetyltransferase (designated OsGNA1), which is involved in de novo UDP-N-acetylglucosamine biosynthesis. The aberrant root morphology of the gna1 mutant includes shortening of roots, disruption of microtubules, and shrinkage of cells in the root elongation zone. Our observations support the idea that protein glycosylation plays a key role in cell metabolism, microtubule stabilization, and cell shape in rice roots. OsGNA1 CRL4 regulates crown root formation through auxin transport in rice 2008 Plant Root None Adventitious (crown) roots account for the majority of the root system of monocots. It is reported that auxin plays an important role in the formation of crown roots, but the underlying molecular mechanisms are still unknown. We characterized a rice (Oryza sativa L.) mutant crown rootless4 (crl4) that was found to have defective crown root formation. Besides reduced crown root number, the crl4 mutant showed auxin-related abnormal phenotypical character-istics such as reduced lateral root number and impaired root gravitropism. CRL4 encodes a protein highly homologous with Arabidopsis GNOM, which mediates auxin-dependent plant growth by coordinating the polar localization of auxin efflux carrier PIN1. In the crl4 mutant, auxin transport was impaired in shoots and roots. Besides, the GUS staining controlled by DR5 promoter in the node of crl4 mutant was fainter and spread wider than that of wild-type. These results indicate that maintaining an appropriate auxin accumulation and gradient through CRL4 in the basal portion of shoots is essential for crown root formation in rice. CRL4|OsGNOM1 Adventitious root formation in rice requires OsGNOM1 and is mediated by the OsPINs family 2009 Cell Res State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, China. The fibrous root system in cereals comprises primarily adventitious roots (ARs), which play important roles in nutrient and water uptake. Current knowledge regarding the molecular mechanism underlying AR development is still limited. We report here the isolation of four rice (Oryza sativa L.) mutants, from different genetic backgrounds, all of which were defective in AR formation. These mutants exhibited reduced numbers of lateral roots (LRs) and partial loss of gravitropism. The mutants also displayed enhanced sensitivity to N-1-naphthylphthalamic acid, an inhibitor of polar auxin transport (PAT), indicating that the mutations affected auxin transport. Positional cloning using one of the four mutants revealed that it was caused by loss-of-function of a guanine nucleotide exchange factor for ADP-ribosylation factor (OsGNOM1). RT-PCR and analysis of promoter::GUS transgenic plants showed that OsGNOM1 is expressed in AR primordia, vascular tissues, LRs, root tips, leaves, anthers and lemma veins, with a distribution pattern similar to that of auxin. In addition, the expressions of OsPIN2, OsPIN5b and OsPIN9 were altered in the mutants. Taken together, these findings indicate that OsGNOM1 affects the formation of ARs through regulating PAT. CRL4|OsGNOM1,OsPIN2 Cloning and characterization of a glucose 6-phosphate/phosphate translocator fromOryza sativa 2003 Journal of Zhejiang University SCIENCE A The State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310029, China. Plastids of nongreen tissues import carbon as a source of biosynthetic pathways and energy, and glucose 6-phosphate is the preferred hexose phosphate taken up by nongreen plastids. A cDNA clone encoding glucose 6-phosphate/phosphate translocator (GPT) was isolated from a cDNA library of immature seeds of rice and named as OsGPT. The cDNA has one uninterrupted open reading frame encoding a 42 kDa polypeptide possessing transit peptide consisting of 70 amino acid residues. The OsGPT gene maps on chromosome 8 of rice and is linked to the quantitative trait locus for 1000-grain weight. The expression of OsCPT is mainly restricted to heterotrophic tissues. These results suggest that glucose 6-phosphate imported via GPT can be used for starch biosynthesis in rice nongreen plastids. OsGPT Molecular characterization of a carbon transporter in plastids from heterotrophic tissues: the glucose 6-phosphate/phosphate antiporter 1998 Plant Cell Botanisches Institut der Universitat zu Koln, Lehrstuhl II, Cologne, Germany. Plastids of nongreen tissues import carbon as a source of biosynthetic pathways and energy. Within plastids, carbon can be used in the biosynthesis of starch or as a substrate for the oxidative pentose phosphate pathway, for example. We have used maize endosperm to purify a plastidic glucose 6-phosphate/phosphate translocator (GPT). The corresponding cDNA was isolated from maize endosperm as well as from tissues of pea roots and potato tubers. Analysis of the primary sequences of the cDNAs revealed that the GPT proteins have a high degree of identity with each other but share only approximately 38% identical amino acids with members of both the triose phosphate/phosphate translocator (TPT) and the phosphoenolpyruvate/phosphate translocator (PPT) families. Thus, the GPTs represent a third group of plastidic phosphate antiporters. All three classes of phosphate translocator genes show differential patterns of expression. Whereas the TPT gene is predominantly present in tissues that perform photosynthetic carbon metabolism and the PPT gene appears to be ubiquitously expressed, the expression of the GPT gene is mainly restricted to heterotrophic tissues. Expression of the coding region of the GPT in transformed yeast cells and subsequent transport experiments with the purified protein demonstrated that the GPT protein mediates a 1:1 exchange of glucose 6-phosphate mainly with inorganic phosphate and triose phosphates. Glucose 6-phosphate imported via the GPT can thus be used either for starch biosynthesis, during which process inorganic phosphate is released, or as a substrate for the oxidative pentose phosphate pathway, yielding triose phosphates. OsGPT Role of peroxidases in the compensation of cytosolic ascorbate peroxidase knockdown in rice plants under abiotic stress 2011 Plant Cell Environ Departamento de Bioquimica e Biologia Molecular, Universidade Federal do Ceara, Ceara, Brasil. Current studies, particularly in Arabidopsis, have demonstrated that mutants deficient in cytosolic ascorbate peroxidases (APXs) are susceptible to the oxidative damage induced by abiotic stress. In contrast, we demonstrate here that rice mutants double silenced for cytosolic APXs (APx1/2s) up-regulated other peroxidases, making the mutants able to cope with abiotic stress, such as salt, heat, high light and methyl viologen, similar to non-transformed (NT) plants. The APx1/2s mutants exhibited an altered redox homeostasis, as indicated by increased levels of H(2)O(2) and ascorbate and glutathione redox states. Both mutant and NT plants exhibited similar photosynthesis (CO(2)) assimilation and photochemical efficiency) under both normal and stress conditions. Overall, the antioxidative compensatory mechanism displayed by the mutants was associated with increased expression of OsGpx genes, which resulted in higher glutathione peroxidase (GPX) activity in the cytosolic and chloroplastic fractions. The transcript levels of OsCatA and OsCatB and the activities of catalase (CAT) and guaiacol peroxidase (GPOD; type III peroxidases) were also up-regulated. None of the six studied isoforms of OsApx were up-regulated under normal growth conditions. Therefore, the deficiency in cytosolic APXs was effectively compensated for by up-regulation of other peroxidases. We propose that signalling mechanisms triggered in rice mutants could be distinct from those proposed for Arabidopsis. OsGpx3|GPX3|OsGPx03 Glutathione peroxidase family - an evolutionary overview 2008 FEBS J Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil. Glutathione peroxidases (EC 1.11.1.9 and EC 1.11.1.12) catalyze the reduction of H(2)O(2) or organic hydroperoxides to water or corresponding alcohols using reduced glutathione. Some glutathione peroxidase isozymes have a selenium-dependent glutathione peroxidase activity and present a selenocysteine encoded by the opal TGA codon. In the present study, insights into the evolution of the whole glutathione peroxidase gene family were obtained after a comprehensive phylogenetic analysis using the improved number of glutathione peroxidase sequences recorded in the PeroxiBase database (http://peroxidase.isb-sib.ch/index.php). The identification of a common ancestral origin for the diverse glutathione peroxidase clusters was not possible. The complex relationships and evolutionary rates of this gene family suggest that basal glutathione peroxidase classes, present in all kingdoms, have originated from independent evolutionary events such as gene duplication, gene losses, lateral gene transfer among invertebrates and vertebrates or plants. In addition, the present study also emphasizes the possibility of some members being submitted to strong selective forces that probably dictated functional convergences of taxonomically distant groups. OsGpx3|GPX3|OsGPx03 The mitochondrial glutathione peroxidase GPX3 is essential for H2O2 homeostasis and root and shoot development in rice 2013 Plant Sci Department of Genetics, Federal University of Rio Grande do Sul, RS, Brazil. Glutathione (GSH) peroxidases (GPXs: EC 1.11.1.9 and EC1.11.1.12) are non-heme thiol peroxidases that catalyze the reduction of H2O2 or organic hydroperoxides to water, and they have been identified in almost all kingdoms of life. The rice glutathione peroxidase (OsGPX) gene family is comprised of 5 members spread throughout a range of sub cellular compartments. The OsGPX gene family is induced in response to exogenous H2O2 and cold stress. In contrast, they are down regulated in response to drought and UV-B light treatments. Transgenic rice plants have been generated that lack mitochondrial OsGPX3. These GPX3s plants showed shorter roots and shoots compared to non-transformed (NT) plants, and higher amounts of H2O2 mitochondrial release were observed in the roots of these plants cultivated under normal conditions. This accumulation of H2O2 is positively associated with shorter root length in GPX3s plants compared to NT ones. Moreover, GPX3 promoter analysis indicated that it is mainly expressed in root tissue. These results suggest that silencing the mitochondrial OsGPX3 gene impairs normal plant development and leads to a stress-induced morphogenic response via H2O2 accumulation. OsGpx3|GPX3|OsGPx03 Na(+) but not Cl(-) or osmotic stress is involved in NaCl-induced expression of Glutathione reductase in roots of rice seedlings 2009 J Plant Physiol Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan, Republic of China. Glutathione reductase (GR, EC 1,6.4.2) is an important reactive oxygen species-scavenging enzyme. The present study examined the relative importance of Na(+), Cl(-), and the osmotic component in NaCl-induced expression of Oryza sativa glutathione reductase (OsGR) genes in rice roots. Semi-quantitative RT-PCR was used to quantify the mRNA levels for one cytosolic (OsGR2) and two chloroplastic (OsGR1 and OsGR3) isoforms of GR identified in the rice genome. The expression of OsGR2 and OsGR3 but not OsGR1 was increased in rice roots treated with NaCl. Treatment with 150 mM NaCl and 150 mM NaNO(3) affected OsGR2 and OsGR3 induction similarly, which suggests that Na(+) but not Cl(-) is required for the NaCl-induced expression of OsGR2 and OsGR3. We also show that Na(+) but not Cl(-) is required for NaCl-enhanced GR activity and hydrogen peroxide (H(2)O(2)) production in rice roots. In addition to its component of ion toxicity, salt concentration in soil results in an osmotic effect. Here, we show that OsGR2 and OsGR3 expression, GR activity, and H(2)O(2) content were not affected at a concentration of mannitol iso-osmotic with 150 mM NaCl. NaCl-induced OsGR2 and OsGR3 in rice roots could be associated with Na(+) but not an osmotic component. OsGR1,OsGR2|RGRC2,OsGR3 NaCl-induced expression of glutathione reductase in roots of rice (Oryza sativa L.) seedlings is mediated through hydrogen peroxide but not abscisic acid 2009 Plant and Soil Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan, Republic of China Reactive oxygen species (ROS) play an important role in NaCl stress. Plants tolerant to NaCl stress may evolve certain strategies to remove these ROS, thus reducing their toxic effects. Therefore, the expression patterns of the gene family encoding glutathione reductase (GR, EC 1.6.4.2) were analyzed in roots of etiolated rice (Oryza sativa L.) seedlings in response to NaCl stress. Semi-quantitative RT-PCR was applied to quantify the mRNA levels for one cytosolic (OsGR2) and two chloroplastic (OsGR1 and OsGR3) isoforms of glutathione reductase identified in the rice genome. The expression of OsGR2 and OsGR3 but not OsGR1 was increased in rice roots treated with 150 mM NaCl. The Rab16A is an abscisic acid (ABA)-responsive rice gene. Increasing concentrations of ABA, from 1 to 12 μM, progressively increased the expression of OsRab16A in rice roots. In the present study, the ABA level was judged by the expression of OsRab16A in rice roots. Treatment with 150 mM NaCl induced the expression of OsRab16A, and the expression increased with increasing concentrations of ABA, which suggests that ABA may be involved in this response in rice roots. In fact, exogenous application of ABA enhanced the expression of OsGR2 and OsGR3 in rice roots. On inhibiting ABA accumulation with sodium tungstate (Tu), an inhibitor of ABA biosynthesis, the expression of OsGR2 and OsGR3 was still induced by NaCl; therefore, NaCl-triggered expression of OsGR2 and OsGR3 in rice roots is not mediated by accumulation of ABA. However, NaCl treatment could induce H2O2 production in rice roots, and H2O2 treatment resulted in enhanced OsGR2 and OsGR3 induction. On inhibiting the NaCl-induced accumulation of H2O2 with diphenylene iodonium, the expression of OsGR2 and OsGR3 was also suppressed. Moreover, the increase in H2O2 level was prior to the induction of OsGR2 and OsGR3 in NaCl-treated rice roots. Thus, H2O2, but not ABA, is involved in regulation of OsGR2 and OsGR3 expression in NaCl-treated rice roots. OsGR1,OsGR2|RGRC2,OsGR3 Gene cloning and expression of cytosolic glutathione reductase in rice (Oryza sativa L.) 1998 Plant Cell Physiol Laboratory of Genetic Engineering, Faculty of Agriculture, Kyoto Prefectural University, Japan. We have isolated a cDNA (RGRC2) encoding glutathione reductase (GR) from rice (Oryza sativa L.). The comparison of deduced amino acid sequences from RGRC2 and other plant GR cDNAs indicated that RGRC2 encodes a putative cytosolic isoform. The recombinant RGRC2 protein had enzymatic properties comparable to those of GR from rice embryo. Subcellular fractionation showed that the RGRC2 protein is localized primarily in cytosol. mRNA and protein of RGRC2 were observed mainly in roots and calli but little in leaf tissues. Southern blot analysis showed that the RGRC2 gene exists as a single copy gene. Here, we have also isolated a genomic clone completely corresponding to RGRC2. The RGRC2 gene is split into 16 exons spread about 7.4 kb of chromosomal DNA, with coding sequence beginning in the 2nd exon and ending in the 16th exon. From the presence of two ABA-responsive elements in the 5'-flanking region of RGRC2, we examined the expression in rice seedlings treated with ABA and the ABA-related environmental stresses, chilling, drought and salinity. The expression of RGRC2 was strongly induced by all these treatments. We suggest that the expression of the rice cytosolic GR gene is regulated via ABA-mediated signal transduction pathway under environmental stresses. OsGR2|RGRC2 Identification and characterization of a novel chloroplast/mitochondria co-localized glutathione reductase 3 involved in salt stress response in rice 2013 Plant Mol Biol Department of Agricultural Chemistry, National Taiwan University, Taipei, 10617, Taiwan. Glutathione reductases (GRs) are important components of the antioxidant machinery that plants use to respond against abiotic stresses. In rice, one cytosolic and two chloroplastic GR isoforms have been identified. In this work, we describe the cloning and characterization of the full-length cDNA encoding OsGR3, a chloroplast-localized GR that up to now was considered as a non-functional enzyme because of assumed lack of N-terminal conserved domains. The expression of OsGR3 in E. coli validated that it can be translated as a protein with GR activity. OsGR3 shows 76 and 53 % identity with OsGR1 (chloroplastic) and OsGR2 (cytosolic), respectively. Phylogenetic analysis revealed 2 chloroplastic GRs in Poaceae species, including rice, sorghum and brachypodium, but only one chloroplastic GR in dicots. A plastid transit peptide is located at the N terminus of OsGR3, and genetic transformation of rice with a GR3-GFP fusion construct further confirmed its localization in chloroplasts. Furthermore, OsGR1 and OsGR3 are also targeted to mitochondria, which suggest a combined antioxidant mechanism in both chloroplasts and mitochondria. However, both isoforms showed a distinct response to salinity: the expression of OsGR3 but not OsGR1 was induced by salt stress. In addition, the transcript level of OsGR3 was greatly increased with salicylic acid treatment but was not significantly affected by methyl jasmonate, dehydration or heat shock stress. Our results provide new clues about the possible roles of functional OsGR3 in salt stress and biotic stress tolerance. OsGR3 OsGRAS19 may be a novel component involved in the brassinosteroid signaling pathway in rice 2013 Mol Plant State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China None OsGRAS19 Down-regulation of OsGRF1 gene in rice rhd1 mutant results in reduced heading date 2005 J Integr Plant Biol Institute of Genetics and Developmental Biology, the Chinese Academy of Sciences, Beijing 100101, China; A rice mutant with reduced heading date (designated rhd1) found in a transgenic line of cultivar Teqing 2 (Oryza sativa L. ssp. indica) was used to identify the genes related to rice heading and thereby to study its molecular mechanism. Genetic analysis showed that rhd1 was a dominant mutation and did not result from T-DNA insertion. By using the differential display polymerase chain reaction (DD-PCR) technique, differential gene expression between rhd1 and Teqing 2 was compared and a rhd1-down-regulated cDNA fragment was identified. Sequence analysis showed that this fragment shared 99% similarity to the OsGRF1 (O. sativa growth-regulating factor 1) gene. The OsGRF1 gene encodes a putative transcription factor, which contains two conserved regions: the QLQ (Gln, Leu, Gln) and WRC (Trp, Arg, Cys) domains. Southern analysis indicates that OsGRF1 may be encoded by single copy gene in the rice genome. RNA interference results revealed that transgenic lines with reduced OsGRF1 transcript displayed delayed growth and development, developed small leaves, and had delayed heading. The extent of the phenotypes developed was well-correlated with the OsGRF1 gene transcript. Our results clearly demonstrate that the OsGRF1 gene is not only involved in regulating growth at the juvenile stage, but that it may also be involved in the regulation of heading in rice. OsGRF1|rhd1 A novel gibberellin-induced gene from rice and its potential regulatory role in stem growth 2000 Plant Physiol Michigan State University-Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824-1312, USA. Os-GRF1 (Oryza sativa-GROWTH-REGULATING FACTOR1) was identified in a search for genes that are differentially expressed in the intercalary meristem of deepwater rice (Oryza sativa L.) internodes in response to gibberellin (GA). Os-GRF1 displays general features of transcription factors, contains a functional nuclear localization signal, and has three regions with similarities to sequences in the database. One of these regions is similar to a protein interaction domain of SWI2/SNF2, which is a subunit of a chromatin-remodeling complex in yeast. The two other domains are novel and found only in plant proteins of unknown function. To study its role in plant growth, Os-GRF1 was expressed in Arabidopsis. Stem elongation of transformed plants was severely inhibited, and normal growth could not be recovered by the application of GA. Our results indicate that Os-GRF1 belongs to a novel class of plant proteins and may play a regulatory role in GA-induced stem elongation. OsGRF1|rhd1 Structure, expression pattern and chromosomal localization of the rice Osgrp-2 gene 2003 Sci China C Life Sci Institute of Microbiology, Chinese Academy of Sciences, 100080, Beijing, China. Glycine-rich proteins (GRPs) belong to a kind of important structural proteins of plant cell walls. The expression of GRP genes is regulated spatially and developmentally as well as by various environmental stresses, thus providing a good model for the study of plant gene expression. We obtained the genomic sequence of a new GRP gene (Osgrp- 2) from a rice genomic library. The transcription start site of Osgrp- 2 was determined by 5'-rapid amplification of cDNA ends (RACE) and a 2.4-kb promoter sequence was thus delimited. The spatial and developmental expression pattern as well as the wound-inducible character of Osgrp- 2 in rice plants was analyzed in detail. Furthermore, the gene was mapped onto rice chromosome 10 by analysis of restriction fragment length polymorphism (RFLP). Osgrp-2 The promoter of a rice glycine-rich protein gene, Osgrp-2, confers vascular-specific expression in transgenic plants 2003 Planta Laboratory of Plant Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China. The genomic sequence of a rice (Oryza sativa L.) glycine-rich protein (GRP) gene, designated Osgrp-2, has been previously determined (GenBank U40708). Primer extension analysis indicated that transcription starts 47 bp upstream of the translation start codon. To gain an insight into the transcriptional regulation of this gene, the 2,401-bp promoter sequence and a series of its 5' deletions were transcriptionally fused to the beta-glucuronidase (GUS) gene. GUS activity was subsequently assayed in a transient expression system of tobacco ( Nicotiana tabacum L.) protoplasts, which revealed the presence of a positive regulatory region (-2290 to -1406) and two negative regulatory regions (-2401 to -2291 and -1405 to -1022) in the Osgrp-2 promoter for the promoter activity. The positive regulatory region displayed an enhancer-like activity when fused to the cauliflower mosaic virus (CaMV) 35S minimal promoter (-89 to +6) to drive GUS expression and assayed on tobacco leaves by the Agrobacterium-mediated transient expression technique (agroinfiltration). Histochemical staining for GUS activity on transgenic tobacco plants has further indicated a preferential expression in vascular tissues of stems and leaves conferred by the positive regulatory region. A 1,023-bp fragment of the Osgrp-2 promoter (-1021 to +2) fused with GUS was transformed into tobacco and proved to be capable of conferring vascular-specific expression. Further 5' and 3' deletion analysis of the 1,023-bp promoter revealed that a 99-bp fragment located from -497 to -399 contained cis-elements responsible for vascular-specific expression. Osgrp-2 Molecular characterization of a novel isoform of rice (Oryza sativa L.) glycine rich-RNA binding protein and evidence for its involvement in high temperature stress response 2007 Plant Science Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, Dhaula Kuan, New Delhi 110021, India A novel full-length cDNA encoding for glycine rich (GR)-RNA binding protein (RBP) (Osgr-rbp4) is isolated from rice heat shock cDNA library. Amino acid sequence of the deduced protein reveals existence of RNA recognition motif (RRM) comprising of highly conserved RNA binding RNPI and RNPII domains at the N-terminus. C-terminus of this protein is rich in arginine and glycine residues. Blast search analysis on rice genome sequence database shows that GR-RBP protein family is constituted of multiple members with high level of amino acid conservation in RNA recognition motif and glycine domain regions. Similar analysis across wider biological systems from NCBI database indicated that rice GR-RBP4 has homologs in different living genera. Osgr-rbp4 transcript in rice seedlings is constitutively expressed as well as regulated by different abiotic stresses including high temperature stress. Ectopic over-expression of Osgr-rbp4 cDNA imparts high temperature stress tolerance to wild type yeast cells. It is shown that OsGR-RBP4 in rice leaf cells and its immunologically homologous protein in tobacco BY2 protoplasts are nuclear proteins. Upon heat shock, bulk of these proteins appears to be localized in the cytoplasm. We suggest that OsGR-RBP4 probably bind and stabilize the stress-inducible transcripts under HS conditions. Osgr-rbp4|Osgrp1 Expression of the rice Osgrp1 promoter-Gus reporter gene is specifically associated with cell elongation/expansion and differentiation 1995 Plant Mol Biol Field of Plant Biology, Cornell University, Ithaca, NY 14853, USA. To study the expression and regulation of a rice glycine-rich cell wall protein gene, Osgrp1, transgenic rice plants were regenerated that contain the Osgrp1 promoter or its 5' deletions fused with the bacterial beta-glucuronidase (GUS) reporter gene. We report here a detailed histochemical analysis of the Osgrp1-Gus expression patterns in transgenic rice plants. In roots of transgenic rice plants, GUS expression was specifically located in cell elongation and differentiation regions, and no GUS expression was detectable in the apical meristem and the mature region. In shoots, GUS activity was expressed only in young leaves or in the growing basal parts of developing leaves, and little GUS activity was expressed in mature leaves or mature parts of developing leaves. In shoot apices, GUS activity was detected only in those leaf cells which were starting to expand and differentiate, and GUS expression was not detected in the apical meristem and the young meristematic leaf primordia. GUS activity was highly expressed in the young stem tissue, particularly in the developing vascular bundles and epidermis. Thus, the expression of the Osgrp1 gene is closely associated with cell elongation/expansion during the post-mitotic cell differentiation process. The Osgrp1-Gus gene was also expressed in response to wounding and down-regulated by water-stress conditions in the elongation region of roots. Promoter deletion analysis indicates that both positive and negative mechanisms are involved in regulating the specific expression patterns. We propose a simple model for the developmental regulation of the Osgrp1 gene expression. Osgr-rbp4|Osgrp1 GSH-dependent peroxidase activity of the rice (Oryza sativa) glutaredoxin, a thioltransferase 2002 Biochem Biophys Res Commun Division of Applied Life Sciences (BK21 program), Department of Biochemistry, PMBBRC, Gyeongsang National University, 660-701, Chinju, Republic of Korea. Glutaredoxin (Grx) is a 12-kDa thioltransferase that reduces disulfide bonds of other proteins and maintains the redox potential of cells. In addition to its oxidoreductase activity, we report here that a rice Grx (OsGrx) can also function as a GSH-dependent peroxidase. Because of this antioxidant activity, OsGrx protects glutamine synthetase from oxidative damage. Individually replacing the conserved Cys residues in OsGrx with Ser shows that Cys(23), but not Cys(26), is essential for the thioltransferase and GSH-dependent peroxidase activities. Kinetic characterization of OsGrx reveals that the maximal catalytic efficiency (V(max)/K(m)) is obtained with cumene hydroperoxide rather than H(2)O(2) or t-butyl hydroperoxide. OsGrx Genome-wide survey and expression analysis suggest diverse roles of glutaredoxin gene family members during development and response to various stimuli in rice 2010 DNA Res National Institute of Plant Genome Research (NIPGR), Aruna Asaf Ali Marg, New Delhi 110 067, India. Glutaredoxins (GRXs) are glutathione-dependent oxidoreductase enzymes involved in a variety of cellular processes. In this study, our analysis revealed the presence of 48 genes encoding GRX proteins in the rice genome. GRX proteins could be classified into four classes, namely CC-, CGFS-, CPYC- and GRL-type, based on phylogenetic analysis. The classification was supported with organization of predicted conserved putative motifs in GRX proteins. We found that expansion of this gene family has occurred largely via whole genome duplication events in a species-specific manner. We explored rice oligonucleotide array data to gain insights into the function of GRX gene family members during various stages of development and in response to environmental stimuli. The comprehensive expression analysis suggested diverse roles of GRX genes during growth and development in rice. Some of the GRX genes were expressed in specific organs/developmental stages only. The expression of many of rice GRX genes was influenced by various phytohormones, abiotic and biotic stress conditions, suggesting an important role of GRX proteins in response to these stimuli. The identification of GRX genes showing differential expression in specific tissues or in response to environmental stimuli provide a new avenue for in-depth characterization of selected genes of importance. OsGRX19|MIL1,OsGRX8 Somatic and reproductive cell development in rice anther is regulated by a putative glutaredoxin 2012 Plant Cell State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. The switch from mitosis to meiosis is one of the most pivotal events in eukaryotes undergoing sexual reproduction. However, the mechanisms orchestrating meiosis initiation remain elusive, particularly in plants. Flowering plants are heterosporous, with male and female spore genesis adopting different developmental courses. We show here that plant pollen mother cells contain a specific meiosis initiation machinery through characterization of a rice (Oryza sativa) gene, MICROSPORELESS1 (MIL1). The mil1 mutant does not produce microspores in anthers but has the normal female fertility. Detailed molecular and cytological investigations demonstrate that mil1 anthers are defective in the meiotic entry of sporogenous cell progenies and in the differentiation of surrounding somatic cell layers, resulting in locules filled with somatic cells instead of microspores. Furthermore, analysis of mil1 msp1 double mutants reveals that due to the absence of MIL1, the cells in their anther locule center do not activate meiotic cell cycle either, generating a similar anther phenotype to mil1. MIL1 encodes a plant-specific CC-type glutaredoxin, which could interact with TGA transcription factors. These results suggest meiotic entry in microsporocytes is directed by an anther-specific mechanism, which requires MIL1 activity, and redox regulation might play important roles in this process. OsGRX19|MIL1,MEL2 Modified expression of an auxin-responsive rice CC-type glutaredoxin gene affects multiple abiotic stress responses 2013 Planta Functional and Applied Genomics Laboratory, National Institute of Plant Genome Research (NIPGR), Aruna Asaf Ali Marg, New Delhi, 110067, India. Glutaredoxins (GRXs) are the ubiquitous oxidoreductase enzymes, which play an important role in defense against various stresses. Here, we report the role of a CC-type GRX gene from rice, OsGRX8, in abiotic stress tolerance. OsGRX8 protein was found to be localized in nucleus and cytosol and its gene expression is induced by various stress conditions and plant hormone auxin. The over-expression of OsGRX8 in Arabidopsis plants conferred reduced sensitivity to auxin and stress hormone, abscisic acid. In addition, the transgenic Arabidopsis plants exhibited enhanced tolerance to various abiotic stresses, including salinity, osmotic and oxidative stress. Further, the transgenic RNAi rice plants exhibited increased susceptibility to various abiotic stresses, which further confirmed the role of OsGRX8 in abiotic stress responses. The microarray data analysis revealed that expression of a large number of auxin-responsive, known stress-associated and transcription factor encoding genes was altered in GRX transgenic Arabidopsis plants in response to exogenous auxin and stress conditions as compared to wild-type plants. Altogether, these findings suggest the role of OsGRX8 in regulating abiotic stress response and may be used to engineer stress tolerance in crop plants. OsGRX8 Regulation of glutamine synthetase isoforms in two differentially drought-tolerant rice (Oryza sativa L.) cultivars under water deficit conditions 2013 Plant Cell Rep Department of Biotechnology, University of North Bengal, Raja Rammohunpur, Siliguri, West Bengal, 734013, India. KEY MESSAGE : The regulation of GS isoforms by WD was organ specific. Two GS isoforms i.e. OsGS1;1 and OsGS2 were differentially regulated in IR-64 (drought-sensitive) and Khitish (drought-tolerant) cultivars of rice. Water deficit (WD) has adverse effect on rice (Oryza sativa L.) and acclimation requires essential reactions of primary metabolism to continue. Rice plants utilize ammonium as major nitrogen source, which is assimilated into glutamine by the reaction of Glutamine synthetase (GS, EC 6.3.1.2). Rice plants possess one gene (OsGS2) for chloroplastic GS2 and three genes (OsGS1;1, OsGS1;2 and OsGS1;3) for cytosolic GS1. Here, we report the effect of WD on regulation of GS isoforms in drought-sensitive (cv. IR-64) and drought-tolerant (cv. Khitish) rice cultivars. Under WD, total GS activity in root and leaf decreased significantly in IR-64 seedlings in comparison to Khitish seedlings. The reduced GS activity in IR-64 leaf was mainly due to decrease in GS2 activity, which correlated with decrease in corresponding transcript and polypeptide contents. GS1 transcript and polypeptide accumulated in leaf during WD, however, GS1 activity was maintained at a constant level. Total GS activity in stem of both the varieties was insensitive to WD. Among GS1 genes, OsGS1;1 expression was differently regulated by WD in the two rice varieties. Its transcript accumulated more abundantly in IR-64 leaf than in Khitish leaf. Following WD, OsGS1;1 mRNA level in stem and root tissues declined in IR-64 and enhanced in Khitish. A steady OsGS1;2 expression patterns were noted in leaf, stem and root of both the cultivars. Results suggest that OsGS2 and OsGS1;1 expression may contribute to drought tolerance of Khitish cultivar under WD conditions. OsGS|OsGS1|GS1|OsGS1;1|OsGLN1;1,OsGS2|lambdaGS31 Mapping of QTLs associated with cytosolic glutamine synthetase and NADH-glutamate synthase in rice (Oryza sativa L.) 2001 J Exp Bot Department of Applied Plant Science, Graduate School of Agricultural Sciences, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. Ninety‐eight backcross inbred lines (BC1F6) developed between Nipponbare, a japonica rice, and Kasalath, an indica rice were employed to detect putative quantitative trait loci (QTLs) associated with the contents of cytosolic glutamine synthetase (GS1; EC 6.3.1.2) and NADH‐glutamate synthase (NADH‐GOGAT; EC 1.4.1.14) in leaves. Immunoblotting analyses showed transgressive segregations toward lower or greater contents of these enzyme proteins in these backcross inbred lines. Seven chromosomal QTL regions for GS1 protein content and six for NADH‐GOGAT protein content were detected. Some of these QTLs were located in QTL regions for various biochemical and physiological traits affected by nitrogen recycling. These findings suggested that the variation in GS1 and NADH‐GOGAT protein contents in this population is related to the changes in the rate of nitrogen recycling from senescing organs to developing organs, leading to changes in these physiological traits. Furthermore, a structural gene for GS1 was mapped between two RFLP markers, C560 and C1408, on chromosome 2 and co‐located in the QTL region for one‐spikelet weight. A QTL region for NADH‐GOGAT protein content was detected at the position mapped for the NADH‐GOGAT structural gene on chromosome 1. A QTL region for soluble protein content in developing leaves was also detected in this region. Although fine mapping is required to identify individual genes in the future, QTL analysis could be a useful post‐genomic tool to study the gene functions for regulation of nitrogen recycling in rice. OsGS|OsGS1|GS1|OsGS1;1|OsGLN1;1 Three cDNA sequences coding for glutamine synthetase polypeptides in Oryza sativa L 1989 Plant Mol Biol Laboratory of Biochemistry, Faculty of Agriculture, Kyoto Prefectural University, Japan. None OsGS|OsGS1|GS1|OsGS1;1|OsGLN1;1,OsGS2|lambdaGS31 Identification and characterization of a QTL on chromosome 2 for cytosolic glutamine synthetase content and panicle number in rice 2004 Theor Appl Genet Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidoori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. A quantitative trait locus (QTL) associated with the protein content of cytosolic glutamine synthetase (GS1; EC 6.3.1.2) in senescing leaves, panicle number, and panicle weight was characterized in rice (Oryza sativa L.). A near-isogenic line (NIL), C-22, developed by marker-assisted selection was grown under different nitrogen levels in the greenhouse and in a paddy field. Chromosome 2 of C-22 had an approximately 50-cM segment substituted from the Kasalath (indica) chromosome in a Koshihikari (japonica) genetic background. C-22 showed a 12-37% lower content of GS1 protein in leaf blades than Koshihikari, which was in good agreement with a QTL region positively affected by the japonica chromosome. At an early vegetative stage, C-22 had more active tillers than Koshihikari in the greenhouse. At the reproductive stage, both panicle number and total panicle weight of C-22 were significantly higher than those of Koshihikari, particularly when the plants were grown under a low-nitrogen condition. These traits of C-22 were further confirmed in a paddy field. Thus, tiller development was positively affected by the Kasalath chromosome at an early vegetative stage, which resulted in an increased panicle number and panicle weight at the mature stage in C-22. These data indicate that the target QTL (Pnn1; panicle number 1) is important in the development of tillers and panicles in rice. Linkage analyses for panicle number and ratio of developing tiller formation in the second axil (RDT) revealed that Pnn1 was delimited at the 6.7-cM region. OsGS|OsGS1|GS1|OsGS1;1|OsGLN1;1 Overexpression of the glutamine synthetase gene modulates oxidative stress response in rice after exposure to cadmium stress 2013 Plant Cell Rep Department of Crop Science, Chungbuk National University, Cheongju, 361-763, Korea. KEY MESSAGE: Overexpression of OsGS gene modulates oxidative stress response in rice after exposure to cadmium stress. Our results describe the features of transformants with enhanced tolerance to Cd and abiotic stresses. Glutamine synthetase (GS) (EC 6.3.1.2) is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine. Exposure of plants to cadmium (Cd) has been reported to decrease GS activity in maize, pea, bean, and rice. To better understand the function of the GS gene under Cd stress in rice, we constructed a recombinant pART vector carrying the GS gene under the control of the CaMV 35S promoter and OCS terminator and transformed using Agrobacterium tumefaciens. We then investigated GS overexpressing rice lines at the physiological and molecular levels under Cd toxicity and abiotic stress conditions. We observed a decrease in GS enzyme activity and mRNA expression among transgenic and wild-type plants subjected to Cd stress. The decrease, however, was significantly lower in the wild type than in the transgenic plants. This was further validated by the high GS mRNA expression and enzyme activity in most of the transgenic lines. Moreover, after 10 days of exposure to Cd stress, increase in the glutamine reductase activity and low or no malondialdehyde contents were observed. These results showed that overexpression of the GS gene in rice modulated the expression of enzymes responsible for membrane peroxidation that may result in plant death. OsGS|OsGS1|GS1|OsGS1;1|OsGLN1;1 Co-suppressed glutamine synthetase2 gene modifies nitrogen metabolism and plant growth in rice 2010 Chinese Science Bulletin National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, 430070, China A full-length cDNA that encodes the rice chloroplastic glutamine synthetase 2 gene was isolated from a Minghui 63-normalized cDNA library; and GS2 rice transformants were obtained by an Agrobacterium tumefaciens-mediated transformation method. Transcripts of the GS2 gene were shown to accumulate at higher levels in the primary transgenic plants in the T0 generation; whereas plants in the T1 generation exhibited a co-suppressed chlorosis phenotype (yellow leaves) accompanied by decreased plant height, few tillers and decreased dry weight. The plants with yellow leaves also displayed a significant decline in GS2 messenger RNA (mRNA) transcriptional level and chlorophyll content; a decrease in total GS activities of ˜50% was also found. Although there was no decrease in the concentration of total free amino acids, a change in the concentration of individual amino acids was observed. Our result also indicates a decreased metabolic level (soluble protein content and ammonium concentration) in GS2 co-suppressed plants. A correlation between chlorophyll content and GS2 mRNA expression level was also observed. The GS2 co-suppressed plants showed better performance when complemented with exogenous glutamine, indicating that the lack of an organic nitrogen pool inside the cell is the possible reason for the chlorosis phenotype of the transformants. OsGS2|lambdaGS31 T-DNA tagged knockout mutation of rice OsGSK1, an orthologue of Arabidopsis BIN2, with enhanced tolerance to various abiotic stresses 2007 Plant Mol Biol Department of Life Science, Sogang University, Seoul 121-742, Korea. T-DNA-tagged rice plants were screened under cold- or salt-stress conditions to determine the genes involved in the molecular mechanism for their abiotic-stress response. Line 0-165-65 was identified as a salt-responsive line. The gene responsible for this GUS-positive phenotype was revealed by inverse PCR as OsGSK1 (Oryza sativa glycogen synthase kinase3-like gene 1), a member of the plant GSK3/SHAGGY-like protein kinase genes and an orthologue of the Arabidopsis brassinosteroid insensitive 2 (BIN2), AtSK21. Northern blot analysis showed that OsGSK1 was most highly detected in the developing panicles, suggesting that its expression is developmental stage specific. Knockout (KO) mutants of OsGSK1 showed enhanced tolerance to cold, heat, salt, and drought stresses when compared with non-transgenic segregants (NT). Overexpression of the full-length OsGSK1 led to a stunted growth phenotype similar to the one observed with the gain-of-function BIN/AtSK21 mutant. This suggests that OsGSK1 might be a functional rice orthologue that serves as a negative regulator of brassinosteroid (BR)-signaling. Therefore, we propose that stress-responsive OsGSK1 may have physiological roles in stress signal-transduction pathways and floral developmental processes. OsGSK1 Cloning, molecular characterization and heterologous expression of a glutathione S-transferase gene in rice 2011 Russian Journal of Bioorganic Chemistry Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, China. OsGSTL2 is one of three tandem-arranged glutathione S-transferase, lambda class genes in chromosome 3 of rice (Oryza sativa L.). It includes 9 introns and 10 exons, and encodes a protein of 244 amino acid residues with a calculated molecular mass of 27.37 kDa. The predicted three-dimensional structure of OsGSTL2 showed a typical glutathione S-transferase fold. Using semi-quantitative RT-PCR analysis, OsGSTL2 transcript was detected in the roots and leaves of seedling stage and tillering stage, and the roots, leaves and panicles of heading stage from rice plants, and the expression level of OsGSTL2 mRNA in rice roots show significant change under chlorsulfuron stress. The OsGSTL2 gene was cloned into pYTV vector and was transformed into yeast strain PEP4. Western blot analysis showed the exogenous OsGSTL2 was expressed in transformed yeast. GST activity of crude extracts of yeast showed the OsGSTL2 transgenic yeast had higher levels of GST activities than control yeasts. These findings suggested that the OsGSTL2 is a glutathione S-transferase and has potential use in detoxification. OsGSTL2 Biochemical and physiological characterization of a tau class glutathione transferase from rice (Oryza sativa) 2009 Plant Physiol Biochem State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. The classical phase II detoxification glutathione transferases (GSTs) are key metabolic enzymes that catalyze the conjugation of glutathione to various electrophilic compounds. A tau class GST gene (OsGSTU17) was cloned from rice, which encodes a protein of 223 amino acid residues with a calculated molecular mass of 25.18 kDa. The recombinant OsGSTU17 formed a homodimer protein and showed GSH-conjugating activity with various xenobiotics. Kinetic analysis with respect to NBD-Cl as substrate revealed a K(m) of 0.324 mM and V(max) of 0.219 micromol/min per mg of protein. The enzyme had a maximum activity at pH 7.5, and a high thermal stability with 81% of its initial activity at 55 degrees C for 15 min. Site-directed mutagenesis revealed that Ser15 in the N-terminal domain is a critical catalytic residue, responsible for stabilisation of the thiolate anion of enzyme-bound glutathione. OsGSTU17 mRNA was expressed in different tissues of rice, both above and below ground. The relative transcript levels of OsGSTU17 mRNA varied significantly among the tissues in response to CDNB, hydrogen peroxide and atrazine treatments, indicating the gene has diverse regulation mechanisms in response to abiotic stresses. OsGSTU17 Osgstu3 and osgtu4, encoding tau class glutathione S-transferases, are heavy metal- and hypoxic stress-induced and differentially salt stress-responsive in rice roots 2003 FEBS Letters Department of Biology, Faculty of Science, University of Ottawa, 30 Marie Curie, K1N 6N5 ON, Ottawa, Canada. Glutathione S-transferases (GSTs) have poorly understood roles in plant responses to environmental stresses. A polyethylene glycol (PEG)-induced tau class GST was identified in rice roots by protein microsequencing. PEG and the heavy metals Cd (20 microM), Zn (30 microM), Co and Ni rapidly and markedly induced osgstu4 and osgstu3 in rice seedling roots. Osgstu4 and osgstu3 were also induced in roots by hypoxic stress but not by cold nor heat shock. Salt stress and abscisic acid (ABA) rapidly induced osgstu3 in rice roots, whereas osgstu4 exhibited a late salt stress and no ABA response. Salicylic acid, jasmonic acid and the auxin alpha-naphthalene acetic acid triggered osgstu4 and osgstu3 expression. Osgstu4 and osgstu3 were rapidly and markedly induced by the antioxidant dithiothreitol and the strong oxidant hydrogen peroxide, which suggested that redox perturbations and reactive oxygen species are involved in their stress response regulations. Osgstu3,Osgstu4 Cold sensitivity in rice (Oryza sativa L.) is strongly correlated with a naturally occurring I99V mutation in the multifunctional glutathione transferase isoenzyme GSTZ2 2011 Biochem J USDA-ARS Crops Pathology and Genetics Research Unit, Department of Plant Sciences, Mail Stop 1, University of California at Davis, Davis, CA 95616, USA. GSTZs [Zeta class GSTs (glutathione transferases)] are multifunctional enzymes that belong to a highly conserved subfamily of soluble GSTs found in species ranging from fungi and plants to animals. GSTZs are known to function as MAAIs [MAA (maleylacetoacetate) isomerases], which play a role in tyrosine catabolism by catalysing the isomerization of MAA to FAA (fumarylacetoacetate). As tyrosine metabolism in plants differs from animals, the significance of GSTZ/MAAI is unclear. In rice (Oryza sativa L.), a major QTL (quantitative trait locus) for seedling cold tolerance has been fine mapped to a region containing the genes OsGSTZ1 and OsGSTZ2. Sequencing of tolerant (ssp. japonica cv. M-202) and sensitive (ssp. indica cv. IR50) cultivars revealed two SNPs (single nucleotide polymorphisms) in OsGSTZ2 that result in amino acid differences (I99V and N184I). Recombinant OsGSTZ2 containing the Val99 residue found in IR50 had significantly reduced activity on MAA and DCA (dichloroacetic acid), but the Ile184 residue had no effect. The distribution of the SNP (c.295A>G) among various rice accessions indicates a significant association with chilling sensitivity in rice seedlings. The results of the present study show that naturally occurring OsGSTZ2 isoforms differ in their enzymatic properties, which may contribute to the differential response to chilling stress generally exhibited by the two major rice subspecies. OsGSTZ2 Rice glycosyltransferase1 encodes a glycosyltransferase essential for pollen wall formation 2013 Plant Physiol Crop Biotech Institute and Department of Plant Molecular Systems Biotechnology, Kyung Hee University, Yongin 446-701, Korea. The pollen wall consists of an exine and an intine. The mechanism underlying its formation is not well understood. Glycosyltransferases catalyze the modification of biological molecules by attaching a single or multiple sugars and play key roles in a wide range of biological processes. We examined the role of GLYCOSYLTRANSFERASE1 (OsGT1) in pollen wall development in rice (Oryza sativa). This gene is highly expressed in mature pollen, and plants containing alleles caused by transfer DNA insertion do not produce homozygous progeny. Reciprocal crosses between OsGT1/osgt1 and the wild type indicated that the mutation leads to a male gametophyte defect. Microscopic analyses revealed that osgt1 pollen developed normally to the pollen mitosis stage but failed to produce mature grains. In osgt1 pollen, intine structure was disrupted. In addition, starch and protein levels were much lower in the mutant grains. Recombinant OsGT1 transferred glucose from UDP-glucose to the third and seventh positions of quercetin, a universal substrate of glycosyltransferases. Consistent with the role of OsGT1, an OsGT1-green fluorescent protein fusion protein was localized to the Golgi apparatus. Taken together, our results suggest that OsGT1 is a Golgi-localized glycosyltransferase essential for intine construction and pollen maturation, providing new insight into male reproductive development. OsGT1 Transcript expression and regulatory characteristics of a rice glycosyltransferase OsGT61-1 gene 2010 Plant Science Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, Dhaula Kuan, New Delhi 110021, Indias Glycosyltransferases (GTs) catalyze the reaction of glycosylation of various cellular macromolecules. Rice genome contains 609 potential GT genes which have been grouped into 40 gene families according to CAZy (Carbohydrate Active Enzyme) database. Phylogenetic tree showed that the members of GT61 family are related across genera. Close relationship among various rice GT61 proteins was also reflected from MEME analysis. Full-length cDNA corresponding to MSU rice locus Os02g22380 and encoding for xylosyltransferase was obtained (referred to as OsGT61-1). OsGT61-1 transcript was noted to be ABA- and NaCl-responsive and its expression was more prominent in root than in shoot tissues. Experiments involving fusion of OsGT61-1 promoter (1410 bp upstream to ATG) with GUS reporter gene showed that GUS expression was in consonance with the transcript expression. 1410 bp OsGT61-1 promoter contains two miniature inverted repeat transposable element sequences namely Gaijin (144 bp) and Wanderer (211 bp). Using yeast as trans-host, we noted that these sequences negatively influence the downstream transcript expression. OsGT61-1|XAX1 XAX1 from glycosyltransferase family 61 mediates xylosyltransfer to rice xylan 2012 Proc Natl Acad Sci U S A Department of Plant Pathology and the Genome Center, University of California, Davis, CA 95616, USA. Xylan is the second most abundant polysaccharide on Earth and represents an immense quantity of stored energy for biofuel production. Despite its importance, most of the enzymes that synthesize xylan have yet to be identified. Xylans have a backbone of beta-1,4-linked xylose residues with substitutions that include alpha-(1-->2)-linked glucuronosyl, 4-O-methyl glucuronosyl, and alpha-1,2- and alpha-1,3-arabinofuranosyl residues. The substitutions are structurally diverse and vary by taxonomy, with grass xylan representing a unique composition distinct from dicots and other monocots. To date, no enzyme has yet been identified that is specific to grass xylan synthesis. We identified a xylose-deficient loss-of-function rice mutant in Os02g22380, a putative glycosyltransferase in a grass-specific subfamily of family GT61. We designate the mutant xax1 for xylosyl arabinosyl substitution of xylan 1. Enzymatic fingerprinting of xylan showed the specific absence in the mutant of a peak, which was isolated and determined by (1)H-NMR to be (beta-1,4-Xyl)(4) with a beta-Xylp-(1-->2)-alpha-Araf-(1-->3). Rice xax1 mutant plants are deficient in ferulic and coumaric acid, aromatic compounds known to be attached to arabinosyl residues in xylan substituted with xylosyl residues. The xax1 mutant plants exhibit an increased extractability of xylan and increased saccharification, probably reflecting a lower degree of diferulic cross-links. Activity assays with microsomes isolated from tobacco plants transiently expressing XAX1 demonstrated xylosyltransferase activity onto endogenous acceptors. Our results provide insight into grass xylan synthesis and how substitutions may be modified for increased saccharification for biofuel generation. OsGT61-1|XAX1 C-terminal residues of oryza sativa GUN4 are required for the activation of the ChlH subunit of magnesium chelatase in chlorophyll synthesis 2012 FEBS Lett National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China. Oryza sativa GUN4 together with the magnesium chelatase subunits ChlI, ChlD, and ChlH have been heterologously expressed and purified to reconstitute magnesium chelatase activity in vitro. Maximum magnesium chelatase activity requires pre-activation of OsChlH with OsGUN4, Mg(2+) and protoporphyrin-IX. OsGUN4 and OsChlH preincubated without protoporphyrin-IX yields magnesium chelatase activity similar to assays without OsGUN4, suggesting formation of a dead-end complex. Either 9 or 10 C-terminal amino acids of OsGUN4 are slowly hydrolyzed to yield a truncated OsGUN4. These truncated OsGUN4 still bind protoporphyrin-IX and Mg-protoporphyrin-IX but are unable to activate OsChlH. This suggests the mechanism of GUN4 activation of magnesium chelatase is different in eukaryotes compared to cyanobacteria as the orthologous cyanobacterial GUN4 proteins lack this C-terminal extension. OsGUN4 Disruption of a rice gene for alpha-glucan water dikinase, OsGWD1, leads to hyperaccumulation of starch in leaves but exhibits limited effects on growth 2013 Front Plant Sci NARO Agricultural Research Center Niigata, Japan ; Graduate School of Agricultural and Life Sciences, The University of Tokyo Tokyo, Japan. To identify potential regulators of photoassimilate partitioning, we screened for rice mutant plants that accumulate high levels of starch in the leaf blades, and a mutant line leaf starch excess 1 (LSE1) was obtained and characterized. The starch content in the leaf blades of LSE1 was more than 10-fold higher than that in wild-type plants throughout the day, while the sucrose content was unaffected. The gene responsible for the LSE1 phenotype was identified by gene mapping to be a gene encoding alpha-glucan water dikinase, OsGWD1 (Os06g0498400), and a 3.4-kb deletion of the gene was found in the mutant plant. Despite the hyperaccumulation of starch in their leaf blades, LSE1 plants exhibited no significant change in vegetative growth, presenting a clear contrast to the reported mutants of Arabidopsis thaliana and Lotus japonicus in which disruption of the genes for alpha-glucan water dikinase leads to marked inhibition of vegetative growth. In reproductive growth, however, LSE1 exhibited fewer panicles per plant, lower percentage of ripened grains and smaller grains; consequently, the grain yield was lower in LSE1 plants than in wild-type plants by 20~40%. Collectively, although alpha-glucan water dikinase was suggested to have universal importance in leaf starch degradation in higher plants, the physiological priority of leaf starch in photoassimilate allocation may vary among plant species. OsGWD1 Two transcripts with different sizes derived from a rice homeobox gene, OSH1 1996 Biochem Biophys Res Commun BioScience Center, Nagoya University, Japan. A rice homeobox gene, OSH1, contains two functionally independent promoters which generate a larger transcript and a smaller transcript. In Arabidopsis, each promoter can drive the expression of a reporter gene in a different manner, indicating that the expression of different sized transcripts is independently regulated by each promoter. Over-expression of the larger transcript in transformed plants caused altered morphologies (Matsuoka et al., Plant Cell, 1993, 5, 1039-1048); in contrast, over-expression of the smaller transcript did not cause any morphological changes. The results suggest that the product of the smaller transcript fails to alter the expression of its target gene(s) in the transformants, while that of the larger transcript is capable of altering the expression of its target gene(s). OSH1|Oskn1 Decreased GA1 Content Caused by the Overexpression of OSH1 Is Accompanied by Suppression of GA 20-Oxidase Gene Expression 1998 Plant Physiol Division of Pomology, National Institute of Fruit Tree Science, Tsukuba, Ibaraki 305-8605, Japan. We previously reported that overexpression of the rice homeobox gene OSH1 led to altered morphology and hormone levels in transgenic tobacco (Nicotiana tabacum L.) plants. Among the hormones whose levels were changed, GA1 was dramatically reduced. Here we report the results of our analysis on the regulatory mechanism(s) ofOSH1 on GA metabolism. GA53 and GA20, precursors of GA1, were applied separately to transgenic tobacco plants exhibiting severely changed morphology due to overexpression of OSH1. Only treatment with the end product of GA 20-oxidase, GA20, resulted in a striking promotion of stem elongation in transgenic tobacco plants. The internal GA1 and GA20 contents inOSH1-transformed tobacco were dramatically reduced compared with those of wild-type plants, whereas the level of GA19, a mid-product of GA 20-oxidase, was 25% of the wild-type level. We have isolated a cDNA encoding a putative tobacco GA 20-oxidase, which is mainly expressed in vegetative stem tissue. RNA-blot analysis revealed that GA 20-oxidase gene expression was suppressed in stem tissue of OSH1-transformed tobacco plants. Based on these results, we conclude that overexpression ofOSH1 causes a reduction of the level of GA1by suppressing GA 20-oxidase expression. OSH1|Oskn1 A rice tryptophan deficient dwarf mutant, tdd1, contains a reduced level of indole acetic acid and develops abnormal flowers and organless embryos 2009 Plant J Bioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi, Japan. sazuka@agr.nagoya-u.ac.jp Indole-3-acetic acid (IAA) plays a critical role in many aspects of plant growth and development; however, complete pathways of biosynthesis, localization and many aspects of functions of IAA in rice remain unclear. Here, we report the analysis of a rice tryptophan- (Trp-) and IAA-deficient mutant, tryptophan deficient dwarf1 (tdd1), which is embryonic lethal because of a failure to develop most organs during embryogenesis. Regenerated tdd1 plants showed pleiotropic phenotypes: dwarfing, narrow leaves, short roots and abnormal flowers. TDD1 encodes a protein homologous to anthranilate synthase beta-subunit, which catalyses the first step of the Trp biosynthesis pathway and functions upstream of Trp-dependent IAA biosynthesis. TDD1-uidA and DR5-uidA expression overlapped at many sites in WT plants but was lacking in tdd1, indicating that TDD1 is involved in auxin biosynthesis. Both Trp and IAA levels in flowers and embryos were much lower in tdd1 than in wild type (WT). Trp feeding completely rescued the mutant phenotypes and moderate expression of OsYUCCA1, which encodes a key enzyme in Trp-dependent IAA biosynthesis, also rescued plant height and root length, indicating that the abnormal phenotypes of tdd1 are caused predominantly by Trp and IAA deficiency. In tdd1 embryos, the expression patterns of OSH1 and OsSCR, which mark the presumptive apical region and the L2 layer, respectively, are identical to those in WT, suggesting a possibility either that different IAA levels are required for basic pattern formation than for organ formation or that an orthologous gene compensates for TDD1 deficiency during pattern formation. OSH1|Oskn1,OsSCR,OsYUCCA1,TDD1|OASB1 Shoot organization genes regulate shoot apical meristem organization and the pattern of leaf primordium initiation in rice 2000 Plant Cell Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. The mechanism regulating the pattern of leaf initiation was analyzed by using shoot organization (sho) mutants derived from three loci (SHO1, SHO2, and SHO3). In the early vegetative phase, sho mutants show an increased rate of leaf production with random phyllotaxy. The resulting leaves are malformed, threadlike, or short and narrow. Their shoot apical meristems are relatively low and wide, that is, flat shaped, although their shape and size are highly variable among plants of the same genotype. Statistical analysis reveals that the shape of the shoot meristem rather than its size is closely correlated with the variations of plastochron and phyllotaxy. Rapid and random leaf production in sho mutants is correlated with the frequent and disorganized cell divisions in the shoot meristem and with a reduction of expression domain of a rice homeobox gene, OSH1. These changes in the organization and behavior of the shoot apical meristems suggest that sho mutants have fewer indeterminate cells and more determinate cells than wild type, with many cells acting as leaf founder cells. Thus, the SHO genes have an important role in maintaining the proper organization of the shoot apical meristem, which is essential for the normal initiation pattern of leaf primordia. OSH1|Oskn1,OsAGO7|shl4|SHO2,SHO1|OsDCL4 Expression of a rice homeobox gene causes altered morphology of transgenic plants 1993 Plant Cell National Institute of Agrobiological Resources, Tsukuba, Ibaraki, Japan. We have isolated a cDNA clone encoding a homeobox sequence from rice. DNA sequence analysis of this clone, which was designated as Oryza sativa homeobox 1 (OSH1), and a genomic clone encoding the OSH1 sequence have shown that the OSH1 gene consists of five exons and encodes a polypeptide of 361 amino acid residues. Restriction fragment length polymorphism analysis has shown that OSH1 is a single-copy gene located near the phytochrome gene on chromosome 3. Introduction of the cloned OSH1 gene into rice resulted in altered leaf morphology, which was similar to that of the maize morphological mutant Knotted-1 (Kn1), indicating that OSH1 is a rice gene homologous to the maize Kn1 gene. RNA gel blot analysis has shown that the gene is primarily expressed in the shoot apices of young rice seedlings. This finding is supported by results of transformation experiments in which the 5' flanking region of the gene directed expression of a reporter gene in the shoot apex, particularly in stipules, of transgenic Arabidopsis. To elucidate the biological function of the OSH1 gene product, the coding region was introduced into Arabidopsis under the control of the cauliflower mosaic virus 35S promoter. Almost all transformants showed abnormal morphology. The typical phenotype was the formation of clumps of abundant vegetative and reproductive shoot apices containing meristems and leaf primordia, which did not form elongated shoots. Some transformants with a less severe phenotype formed elongated shoots but had abnormally shaped leaves and flowers with stunted sepals, petals, and stamens. The abnormal phenotypes were inherited, and the level of expression of the introduced OSH1 correlates with the severity of the phenotype. These findings indicate that the abnormal morphologies of the transgenic plants are caused by the expression of the OSH1 gene product and, therefore, that OSH1 is related to the plant development process. OSH1|Oskn1 A rice homeobox gene, OSH1, is expressed before organ differentiation in a specific region during early embryogenesis 1996 Proc Natl Acad Sci U S A Nagoya Universiity, Bioscience Center, Chikusa, Japan. Homeobox genes encode a large family of homeodomain proteins that play a key role in the pattern formation of animal embryos. By analogy, homeobox genes in plants are thought to mediate important processes in their embryogenesis, but there is very little evidence to support this notion. Here we described the temporal and spatial expression patterns of a rice homeobox gene, OSH1, during rice embryogenesis. In situ hybridization analysis revealed that in the wild-type embryo, OSH1 was first expressed at the globular stage, much earlier than organogenesis started, in a ventral region where shoot apical meristem and epiblast would later develop. This localized expression of OSH1 indicates that the cellular differentiation has already occurred at this stage. At later stages after organogenesis had initiated, OSH1 expression was observed in shoot apical meristem [except in the L1 (tunica) layer], epiblast, radicle, and their intervening tissues in descending strength of expression level with embryonic maturation. We also performed in situ hybridization analysis with a rice organless embryo mutant, orl1, that develops no embryonic organs. In the orl1 embryo, the expression pattern of OSH1 was the same as that in the wild-type embryo in spite of the lack of embryonic organs. This shows that OSH1 is not directly associated with organ differentiation, but may be related to a regulatory process before or independent of the organ determination. The results described here strongly suggest that, like animal homeobox genes, OSH1 plays an important role in regionalization of cell identity during early embryogenesis. OSH1|Oskn1 Genome-Wide Study of KNOX Regulatory Network Reveals Brassinosteroid Catabolic Genes Important for Shoot Meristem Function in Rice 2014 The Plant Cell Plant Gene Expression Center, U.S. Department of Agriculture-Agricultural Research Service, Plant and Microbial Biology Department, University of California at Berkeley, Berkeley, California 94720 In flowering plants, knotted1-like homeobox (KNOX) transcription factors play crucial roles in establishment and maintenance of the shoot apical meristem (SAM), from which aerial organs such as leaves, stems, and flowers initiate. We report that a rice (Oryza sativa) KNOX gene Oryza sativa homeobox1 (OSH1) represses the brassinosteroid (BR) phytohormone pathway through activation of BR catabolism genes. Inducible overexpression of OSH1 caused BR insensitivity, whereas loss of function showed a BR-overproduction phenotype. Genome-wide identification of loci bound and regulated by OSH1 revealed hormonal and transcriptional regulation as the major function of OSH1. Among these targets, BR catabolism genes CYP734A2, CYP734A4, and CYP734A6 were rapidly upregulated by OSH1 induction. Furthermore, RNA interference knockdown plants of CYP734A genes arrested growth of the SAM and mimicked some osh1 phenotypes. Thus, we suggest that local control of BR levels by KNOX genes is a key regulatory step in SAM function. OSH1|Oskn1 Mutations that cause amino acid substitutions at the invariant positions in homeodomain of OSH3 KNOX protein suggest artificial selection during rice domestication 2001 Genes Genet Syst University of California, Berkeley, USDA/ARS-Plant Gene Expression Center KNOX homeodomain (HD) proteins encoded by KNOTTED1-like homeobox genes (KNOX genes) are considered to work as important regulators for plant developmental and morphogenetic events. We found that OSH3, one of the KNOX genes isolated from a cultivar of Oryza sativa (Nipponbare), encodes a novel HD, which has two amino acid substitutions at invariant positions. Sequence analysis of OSH3 from various domesticated and wild species of rice has revealed that these substitutions are distributed only in Japonica and Javanica type of O. sativa, two groups of domesticated rice in Asia. Surprisingly, nucleotide sequences in the first intron are almost conserved in the rice strains that have the substitutions at the invariant amino acids. Overexpression studies revealed that these invariant amino acids are critical for the function of OSH3 in vivo. The facts that these substitutions occurred specifically at the functionally important amino acids and the sequences are conserved in intron where neutral mutations accumulate suggest the substitutions at the invariant positions of OSH3 have been fixed by artificial selections during domestication. Based on these observations, we hypothesize that OSH3 is responsible for one of the traits that are selectively introduced during the domestication of most of Japonica and a part of Javanica type of rice. OSH3 A Loss-of-Function Mutation in the Rice KNOX Type Homeobox Gene, OSH3 2002 Plant and Cell Physiology BioScience Center, Nagoya University, Chikusa, Nagoya, 464-8601 Japan KNOX homeodomain (HD) proteins encoded by KNOTTED1-like homeobox genes (KNOX genes) are thought to work as switches for cells to change from an indeterminate to a determinate state, although their direct functions are not clear. In the process of isolating KNOX genes from rice, we found that one gene, named OSH3, has two amino acid substitutions in three of the invariant amino acid residues in the HD of KNOX proteins. These amino acid substitutions are not universal in rice: two of the cultivars from the Indica variety of rice do not carry those substitutions but two of the cultivars from Japonica variety do. We tested the effect of these amino acid substitutions on their ability to form dimers and to induce abnormal morophologies when overexpressed in transgenic plants. We found that OSH3 without those substitutions can form dimers and can induce an abnormal phenotype in overexpression studies, and that OSH3 with those amino acid substitutions is defective in both. Based on these observations, we concluded that OSH3 from two of the cultivars from the Japonica variety could have lost its original function, or could have acquired a novel function by modifying the action of HD, or both. OSH3 Alternative RNA products from a rice homeobox gene 1995 Plant J BioScience Center, Nagoya University, Aichi, Japan. Three cDNA clones were isolated from rice, OSH42, OSH44 and OSH45, which encode homeodomain sequences in the C-terminal region. The sequences of these cDNAs differ in the N- and C-termini, but they share an identical homeodomain and an acidic amino acid-rich region. The transcripts corresponding to these cDNAs are encoded by a single gene on rice chromosome 8. Differential transcription initiation results in a large transcript comprised of exons 1 and 3-7 and a smaller transcript comprised of exons 2-7. The larger transcript is constitutively expressed in all tissues tested, while the smaller transcript is expressed in leaves, stems and rachis but not in roots, flowers, or suspension callus cells. Alternative splicing also occurs at three different acceptor sites in intron 6 in all tissues tested. The GAL4 DNA-binding domain of yeast was used to study the function of various protein domains. The acidic amino acid-rich region activates the expression of a reporter gene controlled by the GAL4 target sequence, indicating that it functions as a transactivation domain. The larger transcript encodes a unique alanine and glycine-rich region on the N-terminal side of the acidic region, which is not encoded by the smaller transcript. This region completely suppresses the transactivation activity of the acidic region. This suggests that the product of the larger transcript fails to activate the expression of the target gene(s) while the product of the smaller transcript activates the expression of its target gene(s). OSH45 A Ds-insertion mutant of OSH6 (Oryza sativa Homeobox 6) exhibits outgrowth of vestigial leaf-like structures, bracts, in rice 2007 Planta Division of Applied Life Science, Plant Molecular Biology and Biotechnology Research Center, Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju, South Korea. OSH6 (Oryza sativa Homeobox6) is an ortholog of lg3 (Liguleless3) in maize. We generated a novel allele, termed OSH6-Ds, by inserting a defective Ds element into the third exon of OSH6, which resulted in a truncated OSH6 mRNA. The truncated mRNA was expressed ectopically in leaf tissues and encoded the N-terminal region of OSH6, which includes the KNOX1 and partial KNOX2 subdomains. This recessive mutant showed outgrowth of bracts or produced leaves at the basal node of the panicle. These phenotypes distinguished it from the OSH6 transgene whose ectopic expression led to a "blade to sheath transformation" phenotype at the midrib region of leaves, similar to that seen in dominant Lg3 mutants. Expression of a similar truncated OSH6 cDNA from the 35S promoter (35S::DeltaOSH6) confirmed that the ectopic expression of this product was responsible for the aberrant bract development. These data suggest that OSH6-Ds interferes with a developmental mechanism involved in bract differentiation, especially at the basal nodes of panicles. OSH6 Inventory and functional characterization of the HAK potassium transporters of rice 2002 Plant Physiol Departamento de Biotecnologia, Universidad Politecnica de Madrid, 28040 Madrid, Spain. Plants take up large amounts of K(+) from the soil solution and distribute it to the cells of all organs, where it fulfills important physiological functions. Transport of K(+) from the soil solution to its final destination is mediated by channels and transporters. To better understand K(+) movements in plants, we intended to characterize the function of the large KT-HAK-KUP family of transporters in rice (Oryza sativa cv Nipponbare). By searching in databases and cDNA cloning, we have identified 17 genes (OsHAK1-17) encoding transporters of this family and obtained evidence of the existence of other two genes. Phylogenetic analysis of the encoded transporters reveals a great diversity among them, and three distant transporters, OsHAK1, OsHAK7, and OsHAK10, were expressed in yeast (Saccharomyces cerevisiae) and bacterial mutants to determine their functions. The three transporters mediate K(+) influxes or effluxes, depending on the conditions of the experiment. A comparative kinetic analysis of HAK-mediated K(+) influx in yeast and in roots of K(+)-starved rice seedlings demonstrated the involvement of HAK transporters in root K(+) uptake. We discuss that all HAK transporters may mediate K(+) transport, but probably not only in the plasma membrane. Transient expression of the OsHAK10-green fluorescent protein fusion protein in living onion epidermal cells targeted this protein to the tonoplast. OsHAK10,OsHAK16,OsHAK2,OsHAK5 Rice sodium-insensitive potassium transporter, OsHAK5, confers increased salt tolerance in tobacco BY2 cells 2011 J Biosci Bioeng Group of Molecular and Functional Plant Biology, Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan. Potassium ion (K(+)) plays vital roles in many aspects of cellular homeostasis including competing with sodium ion (Na(+)) during potassium starvation and salt stress. Therefore, one way to engineer plant cells with improved salt tolerance is to enhance K(+) uptake activity of the cells, while keeping Na(+) out during salt stress. Here, in search for Na(+)-insensitive K(+) transporter for this purpose, bacterial expression system was used to characterize two K(+) transporters, OsHAK2 and OsHAK5, isolated from rice (Oryza sativa cv. Nipponbare). The two OsHAK transporters are members of a KT/HAK/KUP transporter family, which is one of the major K(+) transporter families in bacteria, fungi and plants. When expressed in an Escherichia coli K(+) transport mutant strain LB2003, both OsHAK transporters rescued the growth defect in K(+)-limiting conditions by significantly increasing the K(+) content of the cells. Under the condition with a large amount of extracellular Na(+), we found that OsHAK5 functions as a Na(+)-insensitive K(+) transporter, while OsHAK2 is sensitive to extracellular Na(+) and exhibits higher Na(+) over K(+) transport activities. Moreover, constitutive expression of OsHAK5 in cultured-tobacco BY2 (Nicotiana tabacum cv. Bright Yellow 2) cells enhanced the accumulation of K(+) but not Na(+) in the cells during salt stress and conferred increased salt tolerance to the cells. Transient expression experiment indicated that OsHAK5 is localized to the plant plasma membrane. These results suggest that the plasma-membrane localized Na(+) insensitive K(+) transporters, similar to OsHAK5 identified here, could be used as a tool to enhance salt tolerance in plant cells. OsHAK2,OsHAK5 OsHAL3 mediates a new pathway in the light-regulated growth of rice 2009 Nat Cell Biol Shanghai Institute of Plant Physiology and Ecology, China. Plants show distinct morphologies in different light conditions through a process called photomorphogenesis. A predominant feature of photomorphogenesis is the reduced growth of seedlings under light conditions compared with darkness. For this adaptive event, the most well-known molecular mechanism involves photoreceptor-mediated inhibition of cell elongation. However, it is not known whether additional pathways exist. Here, we describe a newly discovered pathway of light-modulated plant growth mediated by the halotolerance protein HAL3, a flavin mononucleotide (FMN)-binding protein involved in cell division. We found that light, especially blue light, suppresses growth of rice seedlings by reducing the activity of Oryza sativa (Os) HAL3. Both in vitro and in vivo studies showed that OsHAL3 is structurally inactivated by light through photo-oxidation and by direct interaction with photons. In addition, the transcriptional expression of OsHAL3 is synergistically regulated by different light conditions. Further investigation suggested that OsHAL3 promotes cell division by recruiting a ubiquitin system, rather than by its 4'-phosphopantothenoylcysteine (PPC) decarboxylase activity. Our results uncover a new mechanism for light-regulated plant growth, namely, light not only inhibits cell elongation but also suppresses cell division through HAL3 and E3 ubiquitin ligase. This study thus brings new insights into our understanding of plant photomorphogenesis. OsHAL3,OsHIP1 Role of OsHAL3 protein, a putative 4'-phosphopantothenoylcysteine decarboxylase in rice 2009 Biochemistry (Moscow) State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, 100094, China. In this study, we cloned the OsHAL3 gene from rice Oryza sativa. Alignment analysis revealed that OsHAL3 has a high sequence identity to Dfp protein in Escherichia coli and AtHAL3a protein in Arabidopsis thaliana, which have 4'-phosphopantothenoylcysteine decarboxylase (PPC-DC) activity. OsHAL3 can complement mutation in the E. coli dfp gene encoding PPC-DC, so that the mutant strains with OsHAL3 can grow on rich media at 42 degrees C and on VB minimal media at 30 degrees C. Complementation tests with point mutations of OsHAL3 suggested that the conserved Cys176 residue of OsHAL3 is a key active-site residue. The mutant OsHAL3 G180A has a partly reduced activity. Related mRNA-level analysis showed that the OsHAL3 gene is induced by calcium pantothenate in rice. OsHAL3 OsHAP3 genes regulate chloroplast biogenesis in rice 2003 The Plant Journal Plant Genetics Laboratory, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka-ken 411-8540, Japan. We have isolated three genes that potentially encode a HAP3/nuclear factor-YB (NF-YB)/CCAAT binding factor-A (CBF-A) subunit of a CCAAT-box binding complex in rice (Oryza sativa), and named them OsHAP3A, OsHAP3B and OsHAP3C. These genes were expressed in various organs including leaves. In the transgenic rice plants with antisense or RNAi construct of OsHAP3A, reduced expression of not only OsHAP3A but also OsHAP3B and OsHAP3C was observed. These plants had pale green leaves, in which the amount of chlorophyll was reduced and chloroplasts were degenerated. Lamella was not well developed and accumulation of starch was not detected. The degenerated chloroplast formation was accompanied by reduced expression of nuclear-encoded photosynthesis genes such as RBCS and CAB, while expression of chloroplast-encoded genes was not affected or rather increased. These results suggest that one or more OsHAP3 genes regulate the expression of nuclear-encoded chloroplast-targeted genes and normal development of chloroplasts. OsHAP3A,OsHAP3B,OsHAP3C Aberrant vegetative and reproductive development by overexpression and lethality by silencing of OsHAP3E in rice 2011 Plant Sci Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. yukito@bios.tohoku.ac.jp We generated transgenic rice plants overexpressing OsHAP3E which encodes a subunit of a CCAAT-motif binding HAP complex. The OsHAP3E-overexpressing plants showed various abnormal morphologies both in their vegetative and reproductive phases. The OsHAP3E-overexpressing plants were dwarf with erected leaves and similar to brassinosteroid mutants in the vegetative phase. In the reproductive phase, dense panicle was developed, and occasionally successive generation of lateral rachises and formation of double flowers were observed. These phenotypes indicate association of OsHAP3E with determination of floral meristem identity. On the other hand, repression of OsHAP3E by RNAi or by overexpressing chimeric repressor fusion constructs brought about lethality to transformed cells, and almost no transformant was obtained. This suggests that the OsHAP3E function is essential for rice cells. Altogether, our loss-of-function and gain-of-function analyses suggest that OsHAP3E plays important pleiotropic roles in vegetative and reproductive development or basic cellular processes in rice. OsHAP3E OsLEC1/OsHAP3E participates in the determination of meristem identity in both vegetative and reproductive developments of rice 2013 J Integr Plant Biol National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. In the vegetative phase of plant development, the shoot apical meristem (SAM) produces leaf primordia in regular phyllotaxy, and transforms to the inflorescence meristem when the plant enters reproductive growth, which will undergo a series of identity differentiations and will finally form a complete and fertile panicle. Our previous studies indicated a tissue-specific expression pattern of the OsLEC1 (leafy cotyledon) gene, which is homologous to the Arabidopsis AtLEC1 gene and belongs to the CCAAT-binding protein HAP3 subfamily, during embryo development. Expression of additional OsLEC1 genomic sequences resulted in abnormalities in the development of leaves, panicles and spikelets. The spikelets in particular presented abnormities, including panicle and spikelet-like structures that occurred reiteratively inside prior spikelets, and the occasional spikelet structures that completely transformed into plantlets (a reproductive habit alteration from sexual to asexual called "pseudovivipary"). Analysis showed that OsLEC1 interacts with several SEPALLATA-like MADS transcription factors, suggesting that increased levels of the OsLEC1 protein might interfere with the normal interaction network of these MADS proteins and lead to defective spikelet development. The expression of OsMADS1 was dramatically reduced, and the DNA methylation level of cytosine in certain regions of the OsMADS1 promoter was increased under OsLEC1 overexpression. These results indicate that OsLEC1 affects the development of leaves, panicles and spikelets, and is a key regulator of meristem identity determination in both rice (Oryza sativa) vegetative and reproductive development. OsHAP3E,OsMADS1|LHS1|AFO The small interfering RNA production pathway is required for shoot meristem initiation in rice 2007 Proc Natl Acad Sci U S A Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan. The shoot apical meristem (SAM) is a group of stem cells that are responsible for plant development. Mutations in rice SHOOTLESS2 (SHL2), SHL4/SHOOT ORGANIZATION2 (SHO2), and SHO1 cause complete deletion or abnormal formation of the SAM. In this study we showed that defects in SAM formation in shl mutants are associated with the loss of expression of the homeodomain-leucine zipper (HD-ZIPIII) family genes. Rice SHL2, SHL4/SHO2, and SHO1 encoded orthologues of Arabidopsis RNA-dependent RNA polymerase 6, ARGONAUTE (AGO) 7, and DICER-like 4, respectively, whose mutations affect leaf development through the trans-acting siRNA (ta-siRNA) pathway. This suggested that the ta-siRNA pathway regulates the critical step of SAM formation during rice embryogenesis. The gain-of-function experiment by the ectopic expression of SHL4 resulted in reduced accumulation of an microRNA, miR166, and partial adaxialization of leaves, supporting a role for the ta-siRNA pathway in the maintenance of leaf polarity as previously reported in maize. Analysis of the spatiotemporal expression patterns of HD-ZIPIII and miR166 in wild-type and shl mutant embryos suggested that the loss of HD-ZIPIII expression in the SAM region of the developing embryo is the result of ectopic expression of miR166. Our analysis of shl mutants demonstrated that HD-ZIPIII expression regulated by miR166 is sensitive to the ta-siRNA pathway during SAM formation in rice embryogenesis. OSHB1,OsRDR6|shl-2,OsAGO7|shl4|SHO2,SHO1|OsDCL4 Developmental role and auxin responsiveness of Class III homeodomain leucine zipper gene family members in rice 2008 Plant Physiol Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. Members of the Class III homeodomain leucine zipper (Class III HD-Zip) gene family are central regulators of crucial aspects of plant development. To better understand the roles of five Class III HD-Zip genes in rice (Oryza sativa) development, we investigated their expression patterns, ectopic expression phenotypes, and auxin responsiveness. Four genes, OSHB1 to OSHB4, were expressed in a localized domain of the shoot apical meristem (SAM), the adaxial cells of leaf primordia, the leaf margins, and the xylem tissue of vascular bundles. In contrast, expression of OSHB5 was observed only in phloem tissue. Plants ectopically expressing microRNA166-resistant versions of the OSHB3 gene exhibited severe defects, including the ectopic production of leaf margins, shoots, and radialized leaves. The treatment of seedlings with auxin quickly induced ectopic OSHB3 expression in the entire region of the SAM, but not in other tissues. Furthermore, this ectopic expression of OSHB3 was correlated with leaf initiation defects. Our findings suggest that rice Class III HD-Zip genes have conserved functions with their homologs in Arabidopsis (Arabidopsis thaliana), but have also acquired specific developmental roles in grasses or monocots. In addition, some Class III HD-Zip genes may regulate the leaf initiation process in the SAM in an auxin-dependent manner. OSHB1,OSHB2,OSHB4,OSHB5,OSHB3|OsHox33 The rice RING finger E3 ligase, OsHCI1, drives nuclear export of multiple substrate proteins and its heterogeneous overexpression enhances acquired thermotolerance 2013 J Exp Bot Department of Applied Plant Sciences, Kangwon National University, Chuncheon 200-713, Korea. Thermotolerance is very important for plant survival when plants are subjected to lethally high temperature. However, thus far little is known about the functions of RING E3 ligase in response to heat shock in plants. This study found that one rice gene encoding the RING finger protein was specifically induced by heat and cold stress treatments but not by salinity or dehydration and named it OsHCI1 (Oryza sativa heat and cold induced 1). Subcellular localization results showed that OsHCI1 was mainly associated with the Golgi apparatus and moved rapidly and extensively along the cytoskeleton. In contrast, OsHCI1 may have accumulated in the nucleus under high temperatures. OsHCI1 physically interacted with nuclear substrate proteins including a basic helix-loop-helix transcription factor. Transient co-overexpression of OsHCI1 and each of three nuclear proteins showed that their fluorescent signals moved into the cytoplasm as punctuate formations. Heterogeneous overexpression of OsHCI1 in Arabidopsis highly increased survival rate through acquired thermotolerance. It is proposed that OsHCI1 mediates nuclear-cytoplasmic trafficking of nuclear substrate proteins via monoubiquitination and drives an inactivation device for the nuclear proteins under heat shock. OsHCI1 Structure and expression of the rice class-I type histone deacetylase genesOsHDAC1-3:OsHDAC1overexpression in transgenic plants leads to increased growth rate and altered architecture 2003 The Plant Journal Department of Biological Science, Myongji University, Yongin 449-728, Korea. Histone deacetylases (HDACs) modulate chromatin structure and transcription. HDACs have been studied as negative regulators in eukaryotic transcription. We isolated the rice OsHDAC1-3 genes for class I-type histone deacetylases, which are related to the RPD3 family. The OsHDAC1 gene encoded a protein of approximately 57 kDa that shared 73.5, 72.7, 79.9, and 57.1% amino acid sequence identity with the OsHDAC2, OsHDAC3, maize RPD3, and human HDAC1 proteins, respectively. Genomic structures and Southern blot analyses revealed that OsHDAC1-3 contained seven, six, and seven exons, respectively, and constituted a class I-type family in the rice genome. OsHDAC1 was expressed at similar levels in the leaves, roots, and callus cells, whereas OsHDAC2 and 3 were expressed in the roots and callus cells, but not in the leaves, exhibiting distinct tissue specificity. To explore the role of histone deacetylases in transgenic plants, we inserted the OsHDAC1 cDNA fragment into the expression vector Ai::OsHDAC1 under the control of the ABA-inducible promoter Ai, and transformed the construct into rice. Levels of mRNA, protein, and HDAC activity were significantly increased in Ai::OsHDAC1 callus cells. The amount of tetra-acetylated H4 in the transgenic cells was greatly reduced, and the reduction was abolished upon treatment with trichostatin A. These results demonstrate that OsHDAC1 overexpression in transgenic cells both yields enzymatically active HDAC complexes and induces changes in histone acetylation in vivo. The overexpression leads to a range of novel phenotypes, involving increased growth rate and altered plant architecture, suggesting that OsHDAC1 functions in the genome-wide programming of gene expression. OsHDAC1 The histone deacetylase OsHDAC1 epigenetically regulates theOsNAC6gene that controls seedling root growth in rice 2009 The Plant Journal School of Biotechnology and Environmental Engineering, Myongji University, Yongin, Korea. We have previously isolated a rice gene encoding a histone deacetylase, OsHDAC1, and observed that its transgenic overexpression increases seedling root growth. To identify the transcriptional repression events that occur as a result of OsHDAC1 overexpression (OsHDAC1(OE)), a global profiling of root-expressed genes was performed on OsHDAC1(OE) or HDAC inhibitor-treated non-transgenic (NT) roots, in comparison with untreated NT roots. We selected 39 genes that are induced and repressed in HDAC inhibitor-treated NT and OsHDAC1(OE) roots, compared with NT roots, respectively. Interestingly, OsNAC6, a member of the NAM-ATAF-CUC (NAC) family, was identified as a key component of the OsHDAC1 regulon, and was found to be epigenetically repressed by OsHDAC1 overexpression. The root phenotype of OsNAC6 knock-out seedlings was observed to be similar to that of the OsHDAC1(OE) seedlings. Conversely, the root phenotype of the OsNAC6 overexpressors was similar to that of the OsHDAC1 knock-out seedlings. These observations indicate that OsHDAC1 negatively regulates the OsNAC6 gene that primarily mediates the alteration in the root growth of the OsHDAC1(OE) seedlings. Chromatin immunoprecipitation assays of the OsNAC6 promoter region using antibodies specific to acetylated histones H3 and H4 revealed that OsHDAC1 epigenetically represses the expression of OsNAC6 by deacetylating K9, K14 and K18 on H3 and K5, K12 and K16 on H4. OsHDAC1,OsNAC6|SNAC2 Altered levels of histone deacetylase OsHDT1 affect differential gene expression patterns in hybrid rice 2011 PLoS One National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China. Hybrids between different inbred varieties display novel patterns of gene expression resulted from parental variation in allelic nucleotide sequences. To study the function of chromatin regulators in hybrid gene expression, the histone deacetylase gene OsHDT1 whose expression displayed a circadian rhythm was over-expressed or inactivated by RNAi in an elite rice parent. Increased OsHDT1 expression did not affect plant growth in the parent but led to early flowering in the hybrid. Nonadditive up-regulation of key flowering time genes was found to be related to flowering time of the hybrid. Over-expression of OsHDT1 repressed the nonadditive expression of the key flowering repressors in the hybrid (i.e. OsGI and Hd1) inducing early flowering. Analysis of histone acetylation suggested that OsHDT1 over-expression might promote deacetylation on OsGI and Hd1 chromatin during the peak expression phase. High throughput differential gene expression analysis revealed that altered OsHDT1 levels affected nonadditive expression of many genes in the hybrid. These data demonstrate that nonadditive gene expression was involved in flowering time control in the hybrid rice and that OsHDT1 level was important for nonadditive or differential expression of many genes including the flowering time genes, suggesting that OsHDT1 may be involved in epigenetic control of parental genome interaction for differential gene expression. OsHDT1|HDT701 HDT701, a histone H4 deacetylase, negatively regulates plant innate immunity by modulating histone H4 acetylation of defense-related genes in rice 2012 Plant Cell Department of Plant Pathology, Ohio State University, Columbus, OH 43210, USA. Histone acetylation and deacetylation play an important role in the modification of chromatin structure and regulation of gene expression in eukaryotes. Chromatin acetylation status is modulated antagonistically by histone acetyltransferases and histone deacetylases (HDACs). In this study, we characterized the function of histone deacetylase701 (HDT701), a member of the plant-specific HD2 subfamily of HDACs, in rice (Oryza sativa) innate immunity. Transcription of HDT701 is increased in the compatible reaction and decreased in the incompatible reaction after infection by the fungal pathogen Magnaporthe oryzae. Overexpression of HDT701 in transgenic rice leads to decreased levels of histone H4 acetylation and enhanced susceptibility to the rice pathogens M. oryzae and Xanthomonas oryzae pv oryzae (Xoo). By contrast, silencing of HDT701 in transgenic rice causes elevated levels of histone H4 acetylation and elevated transcription of pattern recognition receptor (PRR) and defense-related genes, increased generation of reactive oxygen species after pathogen-associated molecular pattern elicitor treatment, as well as enhanced resistance to both M. oryzae and Xoo. We also found that HDT701 can bind to defense-related genes to regulate their expression. Taken together, these results demonstrate that HDT701 negatively regulates innate immunity by modulating the levels of histone H4 acetylation of PRR and defense-related genes in rice. OsHDT1|HDT701 A structurally novel hemopexin fold protein of rice plays role in chlorophyll degradation 2012 Biochem Biophys Res Commun Adv. Lab. for Plant Genetic Engineering, Advanced Technology Development Center, Indian Institute of Technology Kharagpur, Kharagpur 721302, India. Proteins containing hemopexin fold domain are suggested to have diverse functions in various living organisms. In order to investigate the structure and function of this type of protein in rice plant (Oryza sativa), the gene encoding a hemopexin fold protein (OsHFP) was cloned, analyzed in silico and characterized. Molecular modeling revealed that the OsHFP is closely related to other hemopexin fold proteins, but is unique with a cylindrical central tunnel as well as extended N- and C-terminal domains. The recombinant OsHFP was found to bind hemin, the oxidized form of heme in vitro. The expression of the single copy OsHFP gene was detected in rice flower buds. Heterologous expression of OsHFP in green leaf tissues resulted in chlorophyll degradation; however, stable expression of OsHFP was observed in transgenic hairy roots, a non-green tissue. The possible role of OsHFP in regulating programmed cell death in anther green tissues of rice is proposed. OsHFP Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content 2003 Nat Biotechnol Crop Genetics Research and Development, Pioneer Hi-Bred, A DuPont Company, Experimental Station, Wilmington, Delaware 19880, USA. ecahoon@danforthcenter.org Tocotrienols are the primary form of vitamin E in seeds of most monocot plants, including cereals such as rice and wheat. As potent antioxidants, tocotrienols contribute to the nutritive value of cereal grains in human and livestock diets. cDNAs encoding homogentisic acid geranylgeranyl transferase (HGGT), which catalyzes the committed step of tocotrienol biosynthesis, were isolated from barley, wheat and rice seeds. Transgenic expression of the barley HGGT in Arabidopsis thaliana leaves resulted in accumulation of tocotrienols, which were absent from leaves of nontransformed plants, and a 10- to 15-fold increase in total vitamin E antioxidants (tocotrienols plus tocopherols). Overexpression of the barley HGGT in corn seeds resulted in an increase in tocotrienol and tocopherol content of as much as six-fold. These results provide insight into the genetic basis for tocotrienol biosynthesis in plants and demonstrate the ability to enhance the antioxidant content of crops by introduction of an enzyme that redirects metabolic flux. OsHGGT|HGGT Silencing OsHI-LOX makes rice more susceptible to chewing herbivores, but enhances resistance to a phloem feeder 2009 Plant J Institute of Insect Sciences, Zhejiang University, Hangzhou 310029, China. The jasmonic acid (JA) pathway plays a central role in plant defense responses against insects. Some phloem-feeding insects also induce the salicylic acid (SA) pathway, thereby suppressing the plant's JA response. These phenomena have been well studied in dicotyledonous plants, but little is known about them in monocotyledons. We cloned a chloroplast-localized type 2 13-lipoxygenase gene of rice, OsHI-LOX, whose transcripts were up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis and the rice brown planthopper (BPH) Niaparvata lugens, as well as by mechanical wounding and treatment with JA. Antisense expression of OsHI-LOX (as-lox) reduced SSB- or BPH-induced JA and trypsin protease inhibitor (TrypPI) levels, improved the larval performance of SBB as well as that of the rice leaf folder (LF) Cnaphalocrocis medinalis, and increased the damage caused by SSB and LF larvae. In contrast, BPH, a phloem-feeding herbivore, showed a preference for settling and ovipositing on WT plants, on which they consumed more and survived better than on as-lox plants. The enhanced resistance of as-lox plants to BPH infestation correlated with higher levels of BPH-induced H(2)O(2) and SA, as well as with increased hypersensitive response-like cell death. These results imply that OsHI-LOX is involved in herbivore-induced JA biosynthesis, and plays contrasting roles in controlling rice resistance to chewing and phloem-feeding herbivores. The observation that suppression of JA activity results in increased resistance to an insect indicates that revision of the generalized plant defense models in monocotyledons is required, and may help develop novel strategies to protect rice against insect pests. OsHI-LOX Rice hypersensitive induced reaction protein 1 (OsHIR1) associates with plasma membrane and triggers hypersensitive cell death 2010 BMC Plant Biol State Key Laboratory of Agrobiotechnology and School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, PR China. BACKGROUND: In plants, HIR (Hypersensitive Induced Reaction) proteins, members of the PID (Proliferation, Ion and Death) superfamily, have been shown to play a part in the development of spontaneous hypersensitive response lesions in leaves, in reaction to pathogen attacks. The levels of HIR proteins were shown to correlate with localized host cell deaths and defense responses in maize and barley. However, not much was known about the HIR proteins in rice. Since rice is an important cereal crop consumed by more than 50% of the populations in Asia and Africa, it is crucial to understand the mechanisms of disease responses in this plant. We previously identified the rice HIR1 (OsHIR1) as an interacting partner of the OsLRR1 (rice Leucine-Rich Repeat protein 1). Here we show that OsHIR1 triggers hypersensitive cell death and its localization to the plasma membrane is enhanced by OsLRR1. RESULT: Through electron microscopy studies using wild type rice plants, OsHIR1 was found to mainly localize to the plasma membrane, with a minor portion localized to the tonoplast. Moreover, the plasma membrane localization of OsHIR1 was enhanced in transgenic rice plants overexpressing its interacting protein partner, OsLRR1. Co-localization of OsHIR1 and OsLRR1 to the plasma membrane was confirmed by double-labeling electron microscopy. Pathogen inoculation studies using transgenic Arabidopsis thaliana expressing either OsHIR1 or OsLRR1 showed that both transgenic lines exhibited increased resistance toward the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. However, OsHIR1 transgenic plants produced more extensive spontaneous hypersensitive response lesions and contained lower titers of the invading pathogen, when compared to OsLRR1 transgenic plants. CONCLUSION: The OsHIR1 protein is mainly localized to the plasma membrane, and its subcellular localization in that compartment is enhanced by OsLRR1. The expression of OsHIR1 may sensitize the plant so that it is more prone to HR and hence can react more promptly to limit the invading pathogens' spread from the infection sites. OsHIR1 A novel simple extracellular leucine-rich repeat (eLRR) domain protein from rice (OsLRR1) enters the endosomal pathway and interacts with the hypersensitive-induced reaction protein 1 (OsHIR1) 2009 Plant Cell Environ Department of Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, Hong Kong. Receptor-like protein kinases (RLKs) containing an extracellular leucine-rich repeat (eLRR) domain, a transmembrane domain and a cytoplasmic kinase domain play important roles in plant disease resistance. Simple eLRR domain proteins structurally resembling the extracellular portion of the RLKs may also participate in signalling transduction and plant defence response. Yet the molecular mechanisms and subcellular localization in regulating plant disease resistance of these simple eLRR domain proteins are still largely unclear. We provided the first experimental evidence to demonstrate the subcellular localization and trafficking of a novel simple eLRR domain protein (OsLRR1) in the endosomal pathway, using both confocal and electron microscopy. Yeast two-hybrid and in vitro pull-down assays show that OsLRR1 interacts with the rice hypersensitive-induced response protein 1 (OsHIR1) which is localized on plasma membrane. The interaction between LRR1 and HIR1 homologs was shown to be highly conserved among different plant species, suggesting a close functional relationship between the two proteins. The function of OsLRR1 in plant defence response was examined by gain-of-function tests using transgenic Arabidopsis thaliana. The protective effects of OsLRR1 against bacterial pathogen infection were shown by the alleviating of disease symptoms, lowering of pathogen titres and higher expression of defence marker genes. OsHIR1,OsLRR1 Expressions of OsHKT1, OsHKT2, and OsVHA are differentially regulated under NaCl stress in salt-sensitive and salt-tolerant rice (Oryza sativa L.) cultivars 2006 J Exp Bot Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Box 7080, SE 75007 Uppsala, Sweden. abdul.kader@vbsg.slu.se Under NaCl-dominated salt stress, the key to plant survival is maintaining a low cytosolic Na(+) level or Na(+)/K(+) ratio. The OsHKT1, OsHKT2, and OsVHA transporter genes might play important roles in maintaining cytosolic Na(+) homeostasis in rice (Oryza sativa L. indica cvs Pokkali and BRRI Dhan29). Upon NaCl stress, the OsHKT1 transcript was significantly down-regulated in salt-tolerant cv. Pokkali, but not in salt-sensitive cv. BRRI Dhan29. NaCl stress induced the expression of OsHKT2 and OsVHA in both Pokkali and BRRI Dhan29. In cv. Pokkali, OsHKT2 and OsVHA transcripts were induced immediately after NaCl stress. However, in cv. BRRI Dhan29, the induction of OsHKT2 was quite low and of OsVHA was low and delayed, compared with that in cv. Pokkali. OsHKT2 and OsVHA induction mostly occurred in the phloem, in the transition from phloem to mesophyll cells, and in the mesophyll cells of the leaves. The vacuolar area in cv. Pokkali did not change under either short- (5-10 min) or long-term (24 h) salt stress, although it significantly increased 24 h after the stress in cv. BRRI Dhan29. When expressional constructs of VHA-c and VHA-a with YFP and CFP were introduced into isolated protoplasts of cvs Pokkali and BRRI Dhan29, the fluorescence resonance energy transfer (FRET) efficiency between VHA-c and VHA-a upon salt stress decreased slightly in cv. Pokkali, but increased significantly in cv. BRRI Dhan29. The results suggest that the salt-tolerant cv. Pokkali regulates the expression of OsHKT1, OsHKT2, and OsVHA differently from how the salt-sensitive cv. BRRI Dhan29 does. Together, these proteins might confer salt tolerance in Pokkali by maintaining a low cytosolic Na(+) level and a correct ratio of cytosolic Na(+)/K(+). OsHKT1;1|OsHKT4,OsHKT2;1|OsHKT1,OsHKT2;2,OsVHA A conserved primary salt tolerance mechanism mediated by HKT transporters: a mechanism for sodium exclusion and maintenance of high K(+)/Na(+) ratio in leaves during salinity stress 2010 Plant Cell Environ Center for Molecular Genetics, University of California, San Diego, La Jolla, 92093-0116, USA. Increasing soil salinity is a serious threat to agricultural productions worldwide in the 21st century. Several essential Na(+) transporters such as AtNHX1 and AtSOS1 function in Na(+) tolerance under salinity stress in plants. Recently, evidence for a new primary salt tolerance mechanism has been reported, which is mediated by a class of HKT transporters both in dicots such as Arabidopsis and monocot crops such as rice and wheat. Here we present a review on vital physiological functions of HKT transporters including AtHKT1;1 and OsHKT1;5 in preventing shoot Na(+) over-accumulation by mediating Na(+) exclusion from xylem vessels in the presence of a large amount of Na(+) thereby protecting leaves from salinity stress. Findings of the HKT2 transporter sub-family are also updated in this review. Subjects regarding function and regulation of HKT transporters, which need to be elucidated in future research, are discussed. OsHKT1;1|OsHKT4,OsHKT1;5|SKC1|OsHKT8 Sodium transport and HKT transporters: the rice model 2003 The Plant Journal Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, 28040 Madrid, Spain. Na+ uptake in the roots of K+-starved seedlings of barley, rice, and wheat was found to exhibit fast rate, low Km, and high sensitivity to K+. Sunflower plants responded in a similar manner but the uptake was not K+ sensitive. Ba2+ inhibited Na+ uptake, but not K+ uptake in rice roots. This demonstrated that Na+ and K+ uptake are mediated by different transporters, and that K+ blocked but was not transported by the Na+ transporter. The genome of rice cv. Nipponbare contains seven HKT genes, which may encode Na+ transporters, plus two HKT pseudogenes. Yeast expressions of OsHKT1 and OsHKT4 proved that they are Na+ transporters of high and low affinity, respectively, which are sensitive to K+ and Ba2+. Parallel experiments of K+ and Na+ uptake in yeast expressing the wheat or rice HKT1 transporters proved that they were very different; TaHKT1 transported K+ and Na+, and OsHKT1 only Na+. Transcript expressions in shoots of the OsHKT genes were fairly constant and insensitive to changes in the K+ and Na+ concentrations of the nutrient solution. In roots, the expressions were much lower than in shoots, except for OsHKT4 and OsHKT1 in K+-starved plants. We propose that OsHKT transporters are involved in Na+ movements in rice, and that OsHKT1 specifically mediates Na+ uptake in rice roots when the plants are K+ deficient. The incidence of HKT ESTs in several plant species suggests that the rice model with many HKT genes applies to other plants. OsHKT1;1|OsHKT4,OsHKT1;3,OsHKT1;4,OsHKT2;1|OsHKT1 A two-staged model of Na+ exclusion in rice explained by 3D modeling of HKT transporters and alternative splicing 2012 PLoS One Australian Centre for Plant Functional Genomics, University of Adelaide, Adelaide, South Australia, Australia. oliv.cotsaftis@gmail.com The HKT family of Na(+) and Na(+)/K(+) transporters is implicated in plant salinity tolerance. Amongst these transporters, the cereal HKT1;4 and HKT1;5 are responsible for Na(+) exclusion from photosynthetic tissues, a key mechanism for plant salinity tolerance. It has been suggested that Na(+) is retrieved from the xylem transpiration stream either in the root or the leaf sheath, protecting the leaf blades from excessive Na(+) accumulation. However, direct evidence for this scenario is scarce. Comparative modeling and evaluation of rice (Oryza sativa) HKT-transporters based on the recent crystal structure of the bacterial TrkH K(+) transporter allowed to reconcile transcriptomic and physiological data. For OsHKT1;5, both transcript abundance and protein structural features within the selectivity filter could control shoot Na(+) accumulation in a range of rice varieties. For OsHKT1;4, alternative splicing of transcript and the anatomical complexity of the sheath needed to be taken into account. Thus, Na(+) accumulation in a specific leaf blade seems to be regulated by abundance of a correctly spliced OsHKT1;4 transcript in a corresponding sheath. Overall, allelic variation of leaf blade Na(+) accumulation can be explained by a complex interplay of gene transcription, alternative splicing and protein structure. OsHKT1;4,OsHKT1;5|SKC1|OsHKT8 QTLs for Na+ and K+ uptake of the shoots and roots controlling rice salt tolerance 2004 Theor Appl Genet SHARF Laboratory, Shanghai Institute of Plant Physiology and Ecology, The Chinese Academy of Sciences, 300 Fenglin Road, 200032, Shanghai, P.R. China. hxlin@iris.sipp.ac.cn An F2 and an equivalent F3 population derived from a cross between a high salt-tolerance indica variety, Nona Bokra, and a susceptible elite japonica variety, Koshihikari, were produced. We performed QTL mapping for physiological traits related to rice salt-tolerance. Three QTLs for survival days of seedlings (SDSs) under salt stress were detected on chromosomes 1, 6 and 7, respectively, and explained 13.9% to 18.0% of the total phenotypic variance. Based on the correlations between SDSs and other physiological traits, it was considered that damage of leaves was attributed to accumulation of Na+ in the shoot by transport of Na+ from the root to the shoot in external high concentration. We found eight QTLs including three for three traits of the shoots, and five for four traits of the roots at five chromosomal regions, controlled complex physiological traits related to rice salt-tolerance under salt stress. Of these QTLs, the two major QTLs with the very large effect, qSNC-7 for shoot Na+ concentration and qSKC-1 for shoot K+ concentration, explained 48.5% and 40.1% of the total phenotypic variance, respectively. The QTLs detected between the shoots and the roots almost did not share the same map locations, suggesting that the genes controlling the transport of Na+ and K+ between the shoots and the roots may be different. OsHKT1;5|SKC1|OsHKT8 Salinity tolerance, Na+ exclusion and allele mining of HKT1;5 in Oryza sativa and O. glaberrima: many sources, many genes, one mechanism? 2013 BMC Plant Biol International Rice Research Institute, Los Banos, Philippines. BACKGROUND: Cultivated rice species (Oryza sativa L. and O. glaberrima Steud.) are generally considered among the crop species most sensitive to salt stress. A handful of lines are known to be tolerant, and a small number of these have been used extensively as donors in breeding programs. However, these donors use many of the same genes and physiological mechanisms to confer tolerance. Little information is available on the diversity of mechanisms used by these species to cope with salt stress, and there is a strong need to identify varieties displaying additional physiological and/or genetic mechanisms to confer higher tolerance. RESULTS: Here we present data on 103 accessions from O. sativa and 12 accessions from O. glaberrima, many of which are identified as salt tolerant for the first time, showing moderate to high tolerance of high salinity. The correlation of salinity-induced senescence (as judged by the Standard Evaluation System for Rice, or SES, score) with whole-plant and leaf blade Na+ concentrations was high across nearly all accessions, and was almost identical in both O. sativa and O. glaberrima. The association of leaf Na+ concentrations with cultivar-groups was very weak, but association with the OsHKT1;5 allele was generally strong. Seven major and three minor alleles of OsHKT1;5 were identified, and their comparisons with the leaf Na+ concentration showed that the Aromatic allele conferred the highest exclusion and the Japonica allele the least. A number of exceptions to this association with the Oryza HKT1;5 allele were identified; these probably indicate the existence of additional highly effective exclusion mechanisms. In addition, two landraces were identified, one from Thailand and the other from Senegal, that show high tissue tolerance. CONCLUSIONS: Significant variation in salinity tolerance exists within both cultivated Oryza species, and this is the first report of significant tolerance in O. glaberrima. The majority of accessions display a strong quantitative relationship between tolerance and leaf blade Na+ concentration, and thus the major tolerance mechanisms found in these species are those contributing to limiting sodium uptake and accumulation in active leaves. However, there appears to be genetic variation for several mechanisms that affect leaf Na+ concentration, and rare cases of accessions displaying different mechanisms also occur. These mechanisms show great promise for improving salt tolerance in rice over that available from current donors. OsHKT1;5|SKC1|OsHKT8 A rice quantitative trait locus for salt tolerance encodes a sodium transporter 2005 Nat Genet National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Sciences, The Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China. Many important agronomic traits in crop plants, including stress tolerance, are complex traits controlled by quantitative trait loci (QTLs). Isolation of these QTLs holds great promise to improve world agriculture but is a challenging task. We previously mapped a rice QTL, SKC1, that maintained K(+) homeostasis in the salt-tolerant variety under salt stress, consistent with the earlier finding that K(+) homeostasis is important in salt tolerance. To understand the molecular basis of this QTL, we isolated the SKC1 gene by map-based cloning and found that it encoded a member of HKT-type transporters. SKC1 is preferentially expressed in the parenchyma cells surrounding the xylem vessels. Voltage-clamp analysis showed that SKC1 protein functions as a Na(+)-selective transporter. Physiological analysis suggested that SKC1 is involved in regulating K(+)/Na(+) homeostasis under salt stress, providing a potential tool for improving salt tolerance in crops. OsHKT1;5|SKC1|OsHKT8 Rice OsHKT2;1 transporter mediates large Na+ influx component into K+ -starved roots for growth 2007 EMBO J Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California-San Diego, 9500 Gilman Street, La Jolla, CA 92093, USA. Excessive accumulation of sodium in plants causes toxicity. No mutation that greatly diminishes sodium (Na+) influx into plant roots has been isolated. The OsHKT2;1 (previously named OsHKT1) transporter from rice functions as a relatively Na+-selective transporter in heterologous expression systems, but the in vivo function of OsHKT2;1 remains unknown. Here, we analyzed transposon-insertion rice lines disrupted in OsHKT2;1. Interestingly, three independent oshkt2;1-null alleles exhibited significantly reduced growth compared with wild-type plants under low Na+ and K+ starvation conditions. The mutant alleles accumulated less Na+, but not less K+, in roots and shoots. OsHKT2;1 was mainly expressed in the cortex and endodermis of roots. (22)Na+ tracer influx experiments revealed that Na+ influx into oshkt2;1-null roots was dramatically reduced compared with wild-type plants. A rapid repression of OsHKT2;1-mediated Na+ influx and mRNA reduction were found when wild-type plants were exposed to 30 mM NaCl. These analyses demonstrate that Na+ can enhance growth of rice under K+ starvation conditions, and that OsHKT2;1 is the central transporter for nutritional Na+ uptake into K+-starved rice roots. OsHKT2;1|OsHKT1 HKT2;2/1, a K(+)-permeable transporter identified in a salt-tolerant rice cultivar through surveys of natural genetic polymorphism 2012 Plant J Biochimie et Physiologie Moleculaire des Plantes, Institut de Biologie Integrative des Plantes, UMR 5004 CNRS/UMR 0386 INRA/Montpellier SupAgro/Universite Montpellier 2, 34060 Montpellier Cedex 2, France. We have investigated OsHKT2;1 natural variation in a collection of 49 cultivars with different levels of salt tolerance and geographical origins. The effect of identified polymorphism on OsHKT2;1 activity was analysed through heterologous expression of variants in Xenopus oocytes. OsHKT2;1 appeared to be a highly conserved protein with only five possible amino acid substitutions that have no substantial effect on functional properties. Our study, however, also identified a new HKT isoform, No-OsHKT2;2/1 in Nona Bokra, a highly salt-tolerant cultivar. No-OsHKT2;2/1 probably originated from a deletion in chromosome 6, producing a chimeric gene. Its 5' region corresponds to that of OsHKT2;2, whose full-length sequence is not present in Nipponbare but has been identified in Pokkali, a salt-tolerant rice cultivar. Its 3' region corresponds to that of OsHKT2;1. No-OsHKT2;2/1 is essentially expressed in roots and displays a significant level of expression at high Na(+) concentrations, in contrast to OsHKT2;1. Expressed in Xenopus oocytes or in Saccharomyces cerevisiae, No-OsHKT2;2/1 exhibited a strong permeability to Na(+) and K(+), even at high external Na(+) concentrations, like OsHKT2;2, and in contrast to OsHKT2;1. Our results suggest that No-OsHKT2;2/1 can contribute to Nona Bokra salt tolerance by enabling root K(+) uptake under saline conditions. OsHKT2;1|OsHKT1,OsHKT2;2 Differential sodium and potassium transport selectivities of the rice OsHKT2;1 and OsHKT2;2 transporters in plant cells 2010 Plant Physiol Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0116, USA. Na(+) and K(+) homeostasis are crucial for plant growth and development. Two HKT transporter/channel classes have been characterized that mediate either Na(+) transport or Na(+) and K(+) transport when expressed in Xenopus laevis oocytes and yeast. However, the Na(+)/K(+) selectivities of the K(+)-permeable HKT transporters have not yet been studied in plant cells. One study expressing 5' untranslated region-modified HKT constructs in yeast has questioned the relevance of cation selectivities found in heterologous systems for selectivity predictions in plant cells. Therefore, here we analyze two highly homologous rice (Oryza sativa) HKT transporters in plant cells, OsHKT2;1 and OsHKT2;2, that show differential K(+) permeabilities in heterologous systems. Upon stable expression in cultured tobacco (Nicotiana tabacum) Bright-Yellow 2 cells, OsHKT2;1 mediated Na(+) uptake, but little Rb(+) uptake, consistent with earlier studies and new findings presented here in oocytes. In contrast, OsHKT2;2 mediated Na(+)-K(+) cotransport in plant cells such that extracellular K(+) stimulated OsHKT2;2-mediated Na(+) influx and vice versa. Furthermore, at millimolar Na(+) concentrations, OsHKT2;2 mediated Na(+) influx into plant cells without adding extracellular K(+). This study shows that the Na(+)/K(+) selectivities of these HKT transporters in plant cells coincide closely with the selectivities in oocytes and yeast. In addition, the presence of external K(+) and Ca(2+) down-regulated OsHKT2;1-mediated Na(+) influx in two plant systems, Bright-Yellow 2 cells and intact rice roots, and also in Xenopus oocytes. Moreover, OsHKT transporter selectivities in plant cells are shown to depend on the imposed cationic conditions, supporting the model that HKT transporters are multi-ion pores. OsHKT2;1|OsHKT1,OsHKT2;2 K+ transport by the OsHKT2;4 transporter from rice with atypical Na+ transport properties and competition in permeation of K+ over Mg2+ and Ca2+ ions 2011 Plant Physiol Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano 386-8567, Japan. Members of class II of the HKT transporters, which have thus far only been isolated from grasses, were found to mediate Na(+)-K(+) cotransport and at high Na(+) concentrations preferred Na(+)-selective transport, depending on the ionic conditions. But the physiological functions of this K(+)-transporting class II of HKT transporters remain unknown in plants, with the exception of the unique class II Na(+) transporter OsHKT2;1. The genetically tractable rice (Oryza sativa; background Nipponbare) possesses two predicted K(+)-transporting class II HKT transporter genes, OsHKT2;3 and OsHKT2;4. In this study, we have characterized the ion selectivity of the class II rice HKT transporter OsHKT2;4 in yeast and Xenopus laevis oocytes. OsHKT2;4 rescued the growth defect of a K(+) uptake-deficient yeast mutant. Green fluorescent protein-OsHKT2;4 is targeted to the plasma membrane in transgenic plant cells. OsHKT2;4-expressing oocytes exhibited strong K(+) permeability. Interestingly, however, K(+) influx in OsHKT2;4-expressing oocytes did not require stimulation by extracellular Na(+), in contrast to other class II HKT transporters. Furthermore, OsHKT2;4-mediated currents exhibited permeabilities to both Mg(2+) and Ca(2+) in the absence of competing K(+) ions. Comparative analyses of Ca(2+) and Mg(2+) permeabilities in several HKT transporters, including Arabidopsis thaliana HKT1;1 (AtHKT1;1), Triticum aestivum HKT2;1 (TaHKT2;1), OsHKT2;1, OsHKT2;2, and OsHKT2;4, revealed that only OsHKT2;4 and to a lesser degree TaHKT2;1 mediate Mg(2+) transport. Interestingly, cation competition analyses demonstrate that the selectivity of both of these class II HKT transporters for K(+) is dominant over divalent cations, suggesting that Mg(2+) and Ca(2+) transport via OsHKT2;4 may be small and would depend on competing K(+) concentrations in plants. OsHKT2;4,HKT3|OsHKT2;3 The rice monovalent cation transporter OsHKT2;4: revisited ionic selectivity 2012 Plant Physiol Biochimie et Physiologie Moleculaire des Plantes, Unite Mixte de Recherche, 5004 Centre National de la Recherche Scientifique/386 Institut National de la Recherche Agronomique/SupAgro-M/Universite Montpellier 2, Campus SupAgro-M, Montpellier cedex 2, France. The family of plant membrane transporters named HKT (for high-affinity K(+) transporters) can be subdivided into subfamilies 1 and 2, which, respectively, comprise Na(+)-selective transporters and transporters able to function as Na(+)-K(+) symporters, at least when expressed in yeast (Saccharomyces cerevisiae) or Xenopus oocytes. Surprisingly, a subfamily 2 member from rice (Oryza sativa), OsHKT2;4, has been proposed to form cation/K(+) channels or transporters permeable to Ca(2+) when expressed in Xenopus oocytes. Here, OsHKT2;4 functional properties were reassessed in Xenopus oocytes. A Ca(2+) permeability through OsHKT2;4 was not detected, even at very low external K(+) concentration, as shown by highly negative OsHKT2;4 zero-current potential in high Ca(2+) conditions and lack of sensitivity of OsHKT2;4 zero-current potential and conductance to external Ca(2+). The Ca(2+) permeability previously attributed to OsHKT2;4 probably resulted from activation of an endogenous oocyte conductance. OsHKT2;4 displayed a high permeability to K(+) compared with that to Na(+) (permeability sequence: K(+) > Rb(+) approximately Cs(+) > Na(+) approximately Li(+) approximately NH(4)(+)). Examination of OsHKT2;4 current sensitivity to external pH suggested that H(+) is not significantly permeant through OsHKT2;4 in most physiological ionic conditions. Further analyses in media containing both Na(+) and K(+) indicated that OsHKT2;4 functions as K(+)-selective transporter at low external Na(+), but transports also Na(+) at high (>10 mm) Na(+) concentrations. These data identify OsHKT2;4 as a new functional type in the K(+) and Na(+)-permeable HKT transporter subfamily. Furthermore, the high permeability to K(+) in OsHKT2;4 supports the hypothesis that this system is dedicated to K(+) transport in the plant. OsHKT2;4 A rice high-affinity potassium transporter (HKT) conceals a calcium-permeable cation channel 2010 Proc Natl Acad Sci U S A Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA. Plant high-affinity K(+) transport (HKT) proteins are so named because of their relation to bacterial and fungal transporters that mediate high-affinity K(+) uptake. The view that HKT family members are sodium-selective uniporters or sodium-potassium symporters is widely held. We have found that one of the rice HKT proteins also functions as a Ca(2+)-permeable cation channel that conducts current carried by a wide range of monovalent and divalent cations. The HKT rice gene, named OsHKT2;4, is expressed in several cell types, including root hairs and vascular parenchyma cells. The protein is localized to the plasma membrane, thereby providing a mechanism for cation uptake and extrusion. This finding goes against firmly entrenched dogma in showing that HKT proteins can function as both ion carriers and channels. The study further extends the function of HKT proteins to Ca(2+)-linked processes and, in so doing, defines a previously undescribed type of Ca(2+)-permeable cation channels in plants. The work also raises questions about the evolutionary changes in this protein family following the divergence of monocots and dicots. OsHKT2;4 Cadmium retention in rice roots is influenced by cadmium availability, chelation and translocation 2011 Plant Cell Environ Dipartimento di Produzione Vegetale, Universita degli Studi di Milano, 20133 Milan, Italy. fabio.nocito@unimi.it Analysis of rice plants exposed to a broad range of relatively low and environmentally realistic Cd concentrations showed that the root capacity to retain Cd ions rose from 49 to 79%, corresponding to increases in the external Cd(2)+ concentration in the 0.01-1 microM range. Fractioning of Cd ions retained by roots revealed that different events along the metal sequestration pathway (i.e. chelation by thiols, vacuolar compartmentalization, adsorption) contributed to Cd immobilization in the roots. However, large amounts of Cd ions (around 24% of the total amount) predictable as potentially mobile were still found in all conditions, while the amount of Cd ions loaded in the xylem seemed to have already reached saturation at 0.1 microM Cd(2)+, suggesting that Cd translocation may also play an indirect role in determining Cd root retention, especially at the highest external concentrations. In silico search and preliminary analyses in yeast suggest OsHMA2 as a good candidate for the control of Cd xylem loading in rice. Taken as a whole, data indicate Cd chelation, compartmentalization, adsorption and translocation processes as components of a complex 'firewall system' which acts in limiting Cd translocation from the root to the shoot and which reaches different equilibrium positions depending on Cd external concentration. OsHMA2|OsHMA2v The OsHMA2 transporter is involved in root-to-shoot translocation of Zn and Cd in rice 2012 Plant Cell Environ Graduate School of Agricultural & Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan. Zinc (Zn) is an essential micronutrient for plants and humans. Cadmium (Cd) is a Zn analog and one of the most toxic heavy metals to humans. Here we investigated the role of the Zn/Cd transporter OsHMA2. OsHMA2:GFP fusion protein localized to the plasma membrane in onion epidermal cells. The yeast expressing OsHMA2 was able to reverse the growth defect in the presence of excess Zn. The expression of OsHMA2 in rice was observed mainly in the roots where OsHMA2 transcripts were abundant in vascular bundles. Furthermore, Zn and Cd concentrations of OsHMA2-suppressed rice decreased in the leaves, while the Zn concentration increased in the roots compared with the wild type (WT). These results suggest that OsHMA2 plays a role in Zn and Cd loading to the xylem and participates in root-to-shoot translocation of these metals in rice. Furthermore, the Cd concentration in the grains of OsHMA2-overexpressing rice as well as in OsSUT1-promoter OsHMA2 rice decreased to about half that of the WT, although the other metal concentrations were the same as in the WT. A phenotype that reduces only the Cd concentration in rice grains will be very useful for transgenic approaches to food safety. OsHMA2|OsHMA2v Mutations in rice (Oryza sativa) heavy metal ATPase 2 (OsHMA2) restrict the translocation of zinc and cadmium 2012 Plant Cell Physiol Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, Shimoshinjyo-Nakano, Akita, Japan. satohnagasawa@akita-pu.ac.jp Widespread soil contamination with heavy metals has fostered the need for plant breeders to develop new crops that do not accumulate heavy metals. Metal-transporting transmembrane proteins that transport heavy metals across the plant plasma membrane are key targets for developing these new crops. Oryza sativa heavy metal ATPase 3 (OsHMA3) is known to be a useful gene for limiting cadmium (Cd) accumulation in rice. OsHMA2 is a close homolog of OsHMA3, but the function of OsHMA2 is unknown. To gain insight into the function of OsHMA2, we analyzed three Tos17 insertion mutants. The translocation ratios of zinc (Zn) and Cd were clearly lower in all mutants than in the wild type, suggesting that OsHMA2 is a major transporter of Zn and Cd from roots to shoots. By comparing each allele in the OsHMA2 protein structure and measuring the Cd translocation ratio, we identified the C-terminal region as essential for Cd translocation into shoots. Two alleles were identified as good material for breeding rice that does not contain Cd in the grain but does contain some Zn, and that grows normally. OsHMA2|OsHMA2v Preferential delivery of zinc to developing tissues in rice is mediated by P-type heavy metal ATPase OsHMA2 2013 Plant Physiol Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan. Developing tissues such as meristems and reproductive organs require high zinc, but the molecular mechanisms of how zinc taken up by the roots is preferentially delivered to these tissues with low transpiration are unknown. Here, we report that rice (Oryza sativa) heavy metal ATPase2 (OsHMA2), a member of P-type ATPases, is involved in preferential delivery of zinc to the developing tissues in rice. OsHMA2 was mainly expressed in the mature zone of the roots at the vegetative stage, but higher expression was also found in the nodes at the reproductive stage. The expression was unaffected by either zinc deficiency or zinc excess. OsHMA2 was localized at the pericycle of the roots and at the phloem of enlarged and diffuse vascular bundles in the nodes. Heterologous expression of OsHMA2 in yeast (Saccharomyces cerevisiae) showed influx transport activity for zinc as well as cadmium. Two independent Tos17 insertion lines showed decreased zinc concentration in the crown root tips, decreased concentration of zinc and cadmium in the upper nodes and reproductive organs compared with wild-type rice. Furthermore, a short-term labeling experiment with (67)Zn showed that the distribution of zinc to the panicle and uppermost node I was decreased, but that, to the lower nodes, was increased in the two mutants. Taken together, OsHMA2 in the nodes plays an important role in preferential distribution of zinc as well as cadmium through the phloem to the developing tissues. OsHMA2|OsHMA2v A member of the heavy metal P-type ATPase OsHMA5 is involved in xylem loading of copper in rice 2013 Plant Physiol Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan. Heavy metal-transporting P-type ATPase (HMA) has been implicated in the transport of heavy metals in plants. Here, we report the function and role of an uncharacterized member of HMA, OsHMA5 in rice (Oryza sativa). Knockout of OsHMA5 resulted in a decreased copper (Cu) concentration in the shoots but an increased Cu concentration in the roots at the vegetative stage. At the reproductive stage, the concentration of Cu in the brown rice was significantly lower in the mutants than in the wild-type rice; however, there was no difference in the concentrations of iron, manganese, and zinc between two independent mutants and the wild type. The Cu concentration of xylem sap was lower in the mutants than in the wild-type rice. OsHMA5 was mainly expressed in the roots at the vegetative stage but also in nodes, peduncle, rachis, and husk at the reproductive stage. The expression was up-regulated by excess Cu but not by the deficiency of Cu and other metals, including zinc, iron, and manganese, at the vegetative stage. Analysis of the transgenic rice carrying the OsHMA5 promoter fused with green fluorescent protein revealed that it was localized at the root pericycle cells and xylem region of diffuse vascular bundles in node I, vascular tissues of peduncle, rachis, and husk. Furthermore, immunostaining with an antibody against OsHMA5 revealed that it was localized to the plasma membrane. Expression of OsHMA5 in a Cu transport-defective mutant yeast (Saccharomyces cerevisiae) strain restored the growth. Taken together, OsHMA5 is involved in loading Cu to the xylem of the roots and other organs. OsHMA5 P(1B)-ATPases--an ancient family of transition metal pumps with diverse functions in plants 2005 Trends Plant Sci School of Biological Sciences, University of Southampton, Southampton, UK SO16 7PX. lew@soton.ac.uk P(1B)-ATPases form a distinct evolutionary sub-family of P-type ATPases, transporting transition metals such as Cu, Zn, Cd, Pb and Co across membranes in a wide range of organisms, including plants. Structurally they are distinct from other P-types, possessing eight transmembrane helices, a CPx/SPC motif in transmembrane domain six, and putative transition metal-binding domains at the N- and/or C-termini. Arabidopsis has eight P(1B)-ATPases (AtHMA1-AtHMA8), which differ in their structure, function and regulation. They perform a variety of important physiological tasks relating to transition metal transport and homeostasis. The crucial roles of plant P(1B)-ATPases in micronutrient nutrition, delivery of essential metals to target proteins, and toxic metal detoxification are discussed. OsHMA9,qCdT7|OsHMA3 Rice P1B-type heavy-metal ATPase, OsHMA9, is a metal efflux protein 2007 Plant Physiol Division of Molecular and Life Science, Biotechnology Research Center, Pohang University of Science and Technology, Pohang 790-784, Korea. P(1B)-type heavy-metal ATPases (HMAs) are transmembrane metal-transporting proteins that play a key role in metal homeostasis. Despite their importance, very little is known about their functions in monocot species. We report the characterization of rice (Oryza sativa) OsHMA9, a member of the P(1B)-type ATPase family. Semiquantitative reverse transcription-polymerase chain reaction analyses of seedlings showed that OsHMA9 expression was induced by a high concentration of copper (Cu), zinc (Zn), and cadmium. We also determined, through promoterbeta-glucuronidase analysis, that the main expression was in the vascular bundles and anthers. The OsHMA9:green fluorescence protein fusion was localized to the plasma membrane. Heterologous expression of OsHMA9 partially rescued the Cu sensitivity of the Escherichia coli copA mutant, which is defective in Cu-transporting ATPases. It did not rescue the Zn sensitivity of the zntA mutant, which is defective in Zn-transporting ATPase. To further elucidate the functional roles of OsHMA9, we isolated two independent null alleles, oshma9-1 and oshma9-2, from the T-DNA insertion population. Mutant plants exhibited the phenotype of increased sensitivity to elevated levels of Cu, Zn, and lead. These results support a role for OsHMA9 in Cu, Zn, and lead efflux from the cells. This article is the first report on the functional characterization of a P(1B)-type metal efflux transporter in monocots. OsHMA9 Cobalt chloride-induced lateral root formation in rice: the role of heme oxygenase 2013 J Plant Physiol Department of Agronomy, National Taiwan University, Taipei, Taiwan, ROC. Lateral roots (LRs) perform the essential tasks of providing water, nutrients, and physical support to plants. Therefore, understanding the regulation of LR development is of agronomic importance. Recent findings suggest that heme oxygenase (HO) plays an important role in LR development. In this study, we examined the effect of cobalt chloride (CoCl2) on LR formation and HO expression in rice. Treatment with CoCl2 induced LR formation and HO activity. We further observed that CoCl2 could induce the expression of OsHO1 but not OsHO2. CoCl2-increased HO activity occurred before LR formation. Zinc protoporphyrin IX (ZnPPIX, the specific inhibitor of HO) and hemoglobin (the carbon monoxide/nitric oxide scavenger) reduced LR formation, HO activity, and OsHO1 expression. Application of biliverdin, a product of HO-catalyzed reaction, to CoCl2-treated rice seedlings reversed the ZnPPIX-inhibited LR formation and ZnPPIX-decreased HO activity. CoCl2 had no effect on H2O2 content and nitric oxide production. Moreover, application of ascorbate, a H2O2 scavenger, failed to affect CoCl2-promoted LR formation and HO activity. It is concluded that HO is required for CoCl2-promoted LR formation in rice. OsHO2|HO2|OsYLC2 Methyl jasmonate-induced lateral root formation in rice: the role of heme oxygenase and calcium 2013 J Plant Physiol Department of Agronomy, National Taiwan University, Taipei, Taiwan, ROC. Lateral roots (LRs) play important roles in increasing the absorptive capacity of roots as well as to anchor the plant in the soil. Therefore, understanding the regulation of LR development is of agronomic importance. In this study, we examined the effect of methyl jasmonate (MJ) on LR formation in rice. Treatment with MJ induced LR formation and heme oxygenase (HO) activity. As well, MJ could induce OsHO1 mRNA expression. Zinc protoporphyrin IX (the specific inhibitor of HO) and hemoglobin [the carbon monoxide/nitric oxide (NO) scavenger] reduced LR formation, HO activity and OsHO1 expression. LR formation and HO activity induced by MJ was reduced by the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-oxide. The effects of Ca(2+) chelators, Ca(2+)-channel inhibitors, and calmodulin (CaM) antagonists on LR formation induced by MJ were also examined. All these inhibitors were effective in reducing the action of MJ. However, Ca(2+) chelators and Ca(2+) channel inhibitors induced HO activity when combining with MJ further. It is concluded that Ca(2+) may regulate MJ action mainly through CaM-dependent mechanism. OsHO2|HO2|OsYLC2 Young Leaf Chlorosis 2 encodes the stroma-localized heme oxygenase 2 which is required for normal tetrapyrrole biosynthesis in rice 2014 Planta State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Crops and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China. Rice heme oxygenase 2 (OsHO2) mutants are chlorophyll deficient with distinct tetrapyrrole metabolite and transcript profiles, suggesting a potential regulatory role of the stromal-localized OsHO2 in tetrapyrrole biosynthesis. In plants, heme oxygenases (HOs) are classified into the subfamilies HO1 and HO2. HO1 are highly conserved plastid enzymes required for synthesizing the chromophore in phytochromes which mediate a number of light-regulated responses. However, the physiological and biochemical functions of HO2, which are distantly related to HO1, are not well understood, especially in crop plants. From a population of (60)Co-irradiated rice mutants, we identified the ylc2 (young leaf chlorosis 2) mutant which displays a chlorosis phenotype in seedlings with substantially reduced chlorophyll content. Normal leaf pigmentation is gradually restored in older plants while newly emerged leaves remain yellow. Transmission electron microscopy further revealed defective chloroplast structures in the ylc2 seedlings. Map-based cloning located the OsYLC2 gene on chromosome 3 and it encodes the OsHO2 protein. The gene identification was confirmed by complementation and T-DNA mutant analyses. Subcellular localization and chloroplast fractionation experiments indicated that OsHO2 resides in the stroma. However, recombinant enzyme assay demonstrated that OsHO2 is not a functional HO enzyme. Analysis of tetrapyrrole metabolites revealed the reduced levels of most chlorophyll and phytochromobilin precursors in the ylc2 mutant. On the other hand, elevated accumulation of 5-aminolevulinic acid and Mg-protoporphyrin IX was observed. These unique metabolite changes are accompanied by consistent changes in the expression levels of the corresponding tetrapyrrole biosynthesis genes. Taken together, our work suggests that OsHO2 has a potential regulatory role for tetrapyrrole biosynthesis in rice. OsHO2|HO2|OsYLC2 Isolation and characterization of rice (Oryza sativa L.) E3-ubiquitin ligase OsHOS1 gene in the modulation of cold stress response 2013 Plant Mol Biol Genomics of Plant Stress laboratory, Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Av. da Republica, 2780-157, Oeiras, Portugal. Plants can cope with adverse environmental conditions through the activation of stress response signalling pathways, in which the proteasome seems to play an important role. However, the mechanisms underlying the proteasome-mediated stress response in rice are still not fully understood. To address this issue, we have identified a rice E3-ubiquitin ligase, OsHOS1, and characterized its role in the modulation of the cold stress response. Using a RNA interference (RNAi) transgenic approach we found that, under cold conditions, the RNAi::OsHOS1 plants showed a higher expression level of OsDREB1A. This was correlated with an increased amount of OsICE1, a master transcription factor of the cold stress signalling. However, the up-regulation of OsDREB1A was transient and the transgenic plants did not show increased cold tolerance. Nevertheless, we could confirm the interaction of OsHOS1 with OsICE1 by Yeast-Two hybrid and bi-molecular fluorescence complementation in Arabidopsis protoplasts. Moreover, we could also determine through an in vitro degradation assay that the higher amount of OsICE1 in the transgenic plants was correlated with a lower amount of OsHOS1. Hence, we could confirm the involvement of the proteasome in this response mechanism. Taken together our results confirm the importance of OsHOS1, and thus of the proteasome, in the modulation of the cold stress signalling in rice. OsHOS1 A role for the rice homeobox gene Oshox1 in provascular cell fate commitment 2000 Development Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, PO Box 9505, The Netherlands. The vascular tissues of plants form a network of interconnected cell files throughout the plant body. The transition from a genetically totipotent meristematic precursor to different stages of a committed procambial cell, and its subsequent differentiation into a mature vascular element, involves developmental events whose molecular nature is still mostly unknown. The rice protein Oshox1 is a member of the homeodomain leucine zipper family of transcription factors. Here we show that the strikingly precise onset of Oshox1 gene expression marks critical, early stages of provascular ontogenesis in which the developmental fate of procambial cells is specified but not yet stably determined. This suggests that the Oshox1 gene may be involved in the establishment of the conditions required to restrict the developmental potential of procambial cells. In support of this hypothesis, ectopic expression of Oshox1 in provascular cells that normally do not yet express this gene results in anticipation of procambial cell fate commitment, eventually culminating in premature vascular differentiation. Oshox1 represents the first example of a transcription factor whose function can be linked to specification events mediating provascular cell fate commitment. OsHox1 Transcriptional repression by Oshox1, a novel homeodomain leucine zipper protein from rice 1997 The Plant Journal Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, The Netherlands. This paper describes the characterization of Oshox1, a cDNA clone from rice encoding a member of the homeodomain-leucine zipper (HD-Zip) class of putative transcription factors. Oshox1 maps to chromosome 10 and belongs to a family of related rice genes. Two-hybrid assays showed that Oshox1 protein can homodimerize, but can also form heterodimers with an Arabidopsis HD-Zip protein. This suggests that protein-protein interactions may also occur between different HD-Zip proteins in rice, which would provide enormous versatility for generating specific gene-control mechanisms. Oshox1 mRNA could be detected in various rice tissues at different developmental stages, with highest levels in embryos, shoots of seedlings, and leaves of mature plants. Transgenic expression of Oshox1 in Arabidopsis retarded growth and affected leaf size and shape, indicative of a role as developmental regulator. In vitro and in vivo DNA-binding studies revealed that Oshox1 interacts with the pseudopalindromic sequence CAAT(C/G)ATTG, confirming that the protein represents a transcription factor. Oshox1 was found to repress reporter gene activity in rice suspension cells, most likely by a mechanism of active transcriptional repression. Repression was strictly dependent on the presence of upstream Oshox1 binding sites in the reporter gene constructs and a function of the N-terminal region of Oshox1, preceding the homeodomain. OsHox1 The procambium specification gene Oshox1 promotes polar auxin transport capacity and reduces its sensitivity toward inhibition 2002 Plant Physiol Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, P.O. Box 9505, 2300 RA Leiden, The Netherlands. The auxin-inducible homeobox gene Oshox1 of rice (Oryza sativa) is a positive regulator of procambial cell fate commitment, and its overexpression reduces the sensitivity of polar auxin transport (PAT) to the PAT inhibitor 1-N-naphthylphthalamic acid (NPA). Here, we show that wild-type rice leaves formed under conditions of PAT inhibition display vein hypertrophy, reduced distance between longitudinal veins, and increased distance between transverse veins, providing experimental evidence for a role of PAT in vascular patterning in a monocot species. Furthermore, we show that Oshox1 overexpression confers insensitivity to these PAT inhibitor-induced vascular-patterning defects. Finally, we show that in the absence of any overt phenotypical change, Oshox1 overexpression specifically reduces the affinity of the NPA-binding protein toward NPA and enhances PAT and its sensitivity toward auxin. These results are consistent with the hypothesis that Oshox1 promotes fate commitment of procambial cells by increasing their auxin conductivity properties and stabilizing this state against modulations of PAT by an endogenous NPA-like molecule. OsHox1 Function of the HD-Zip I gene Oshox22 in ABA-mediated drought and salt tolerances in rice 2012 Plant Mol Biol Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. Oshox22 belongs to the homeodomain-leucine zipper (HD-Zip) family I of transcription factors, most of which have unknown functions. Here we show that the expression of Oshox22 is strongly induced by salt stress, abscisic acid (ABA), and polyethylene glycol treatment (PEG), and weakly by cold stress. Trans-activation assays in yeast and transient expression analyses in rice protoplasts demonstrated that Oshox22 is able to bind the CAAT(G/C)ATTG element and acts as a transcriptional activator that requires both the HD and Zip domains. Rice plants homozygous for a T-DNA insertion in the promoter region of Oshox22 showed reduced Oshox22 expression and ABA content, decreased sensitivity to ABA, and enhanced tolerance to drought and salt stresses at the seedling stage. In contrast, transgenic rice over-expressing Oshox22 showed increased sensitivity to ABA, increased ABA content, and decreased drought and salt tolerances. Based on these results, we conclude that Oshox22 affects ABA biosynthesis and regulates drought and salt responses through ABA-mediated signal transduction pathways. Oshox22 Knockdown of OsHox33, a member of the class III homeodomain-leucine zipper gene family, accelerates leaf senescence in rice 2013 Sci China Life Sci College of Life Sciences, Tianjin Key Laboratory of Animal and Plant Resistance, Tianjin Normal University, Tianjin, 300387, China, lwjzsq@163.com. The class III homeodomain-leucine zipper (HD-Zip III) gene family plays important roles in plant growth and development, including regulation of apical embryo patterning, embryonic shoot meristem formation, leaf polarity, vascular development, and meristem function, with a particularly crucial function in leaf development. Although HD-Zip III members are highly conserved in land plants, previous studies, such as genetic analyses based on multiple mutants in Arabidopsis and other plants, suggest that various HD-Zip III family genes have evolved with distinct functions and pleiotropic effects on plant growth and development. In this study, we analyzed a HD-Zip III member, OsHox33, and demonstrated that it plays an important role in age-dependent leaf senescence in rice. We constructed two specific RNAi vectors derived from the 5'-end region and 3'-UTR of OsHox33 to knockdown its expression. Transgenic plants harboring either RNAi construct displayed similar phenotypes of precocious leaf senescence symptoms, suggesting that knockdown of OsHox33 accelerates leaf senescence in rice. pOsHox33::GUS fusion expression and RT-PCR revealed that OsHox33 is highly expressed in young organs, especially in young meristems such as shoot apical meristems, intercalary meristems, and young callus. In addition, real-time PCR indicated that OsHox33 was more highly expressed in young leaves than in old leaves. To further investigate OsHox33 function, we analyzed chloroplast ultrastructure in different-aged leaves of RNAi plants, and found that OsHox33 knockdown accelerated chloroplast degradation, which is consistent with RNAi phenotypes. Finally, real-time PCR studies showed that OsHox33 can regulate the expression of GS1 and GS2, two senescence-associated genes. Taken together, the work presented here provides new insights into the function of HD-Zip III members in plants. OSHB3|OsHox33 Functional analysis of rice HOMEOBOX4 (Oshox4) gene reveals a negative function in gibberellin responses 2008 Plant Mol Biol National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China. The homeodomain-leucine zipper (HD-Zip) putative transcription factor genes are divided into 4 families. In this work, we studied the function of a rice HD-Zip I gene, H OME O BO X4 (Oshox4). Oshox4 transcripts were detected in leaf and floral organ primordia but excluded from the shoot apical meristem and the protein was nuclear localized. Over-expression of Oshox4 in rice induced a semi-dwarf phenotype that could not be complemented by applied GA3. The over-expression plants accumulated elevated levels of bioactive GA, while the GA catabolic gene GA2ox3 was upregulated in the transgenic plants. In addition, over-expression of Oshox4 blocked GA-dependent alpha-amylase production. However, down-regulation of Oshox4 in RNAi transgenic plants induced no phenotypic alteration. Interestingly, the expression of YAB1 that is involved in the negative feedback regulation of the GA biosynthesis was upregulated in the Oshox4 over-expressing plants. One-hybrid assays showed that Oshox4 could interact with YAB1 promoter in yeast. In addition, Oshox4 expression was upregulated by GA. These data together suggest that Oshox4 may be involved in the negative regulation of GA signalling and may play a role to fine tune GA responses in rice. Oshox4,OsYABBY1|OsYAB1 Role of hydroperoxide lyase in white-backed planthopper (Sogatella furcifera Horvath)-induced resistance to bacterial blight in rice, Oryza sativa L 2010 Plant J National Institute of Agrobiological Sciences, NIAS, Tsukuba, Ibaraki 305-8602, Japan. A pre-infestation of the white-backed planthopper (WBPH), Sogatella furcifera Horvath, conferred resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) in rice (Oryza sativa L.) under both laboratory and field conditions. The infestation of another planthopper species, the brown planthopper (BPH) Nilaparvata lugens Stal, did not significantly reduce the incidence of bacterial blight symptoms. A large-scale screening using a rice DNA microarray and quantitative RT-PCR revealed that WBPH infestation caused the upregulation of more defence-related genes than did BPH infestation. Hydroperoxide lyase 2 (OsHPL2), an enzyme for producing C(6) volatiles, was upregulated by WBPH infestation, but not by BPH infestation. One C(6) volatile, (E)-2-hexenal, accumulated in rice after WBPH infestation, but not after BPH infestation. A direct application of (E)-2-hexenal to a liquid culture of Xoo inhibited the growth of the bacterium. Furthermore, a vapour treatment of rice plants with (E)-2-hexenal induced resistance to bacterial blight. OsHPL2-overexpressing transgenic rice plants exhibited increased resistance to bacterial blight. Based on these data, we conclude that OsHPL2 and its derived (E)-2-hexenal play some role in WBPH-induced resistance in rice. OsAOS3|OsHPL2 The rice hydroperoxide lyase OsHPL3 functions in defense responses by modulating the oxylipin pathway 2012 Plant J College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China. As important signal molecules, jasmonates (JAs) and green leaf volatiles (GLVs) play diverse roles in plant defense responses against insect pests and pathogens. However, how plants employ their specific defense responses by modulating the levels of JA and GLVs remains unclear. Here, we describe identification of a role for the rice HPL3 gene, which encodes a hydroperoxide lyase (HPL), OsHPL3/CYP74B2, in mediating plant-specific defense responses. The loss-of-function mutant hpl3-1 produced disease-resembling lesions spreading through the whole leaves. A biochemical assay revealed that OsHPL3 possesses intrinsic HPL activity, hydrolyzing hydroperoxylinolenic acid to produce GLVs. The hpl3-1 plants exhibited enhanced induction of JA, trypsin proteinase inhibitors and other volatiles, but decreased levels of GLVs including (Z)-3-hexen-1-ol. OsHPL3 positively modulates resistance to the rice brown planthopper [BPH, Nilaparvata lugens (Stal)] but negatively modulates resistance to the rice striped stem borer [SSB, Chilo suppressalis (Walker)]. Moreover, hpl3-1 plants were more attractive to a BPH egg parasitoid, Anagrus nilaparvatae, than the wild-type, most likely as a result of increased release of BPH-induced volatiles. Interestingly, hpl3-1 plants also showed increased resistance to bacterial blight (Xanthomonas oryzae pv. oryzae). Collectively, these results indicate that OsHPL3, by affecting the levels of JA, GLVs and other volatiles, modulates rice-specific defense responses against different invaders. OsHPL3 Two hydroxypyruvate reductases encoded by OsHPR1 and OsHPR2 are involved in photorespiratory metabolism in rice 2014 J Integr Plant Biol State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agricultural University, Guangzhou, China; Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China. Mutations in the photorespiration pathway display a lethal phenotype in atmospheric air, which can be fully recovered by elevated CO2 . An exception is that mutants of peroxisomal hydroxypyruvate reductase (HPR1) do not have this phenotype, indicating the presence of cytosolic bypass in the photorespiration pathway. In this study, we constructed overexpression of the OsHPR1 gene and RNA interference plants of OsHPR1 and OsHPR2 genes in rice (Oryza sativa L. cv. Zhonghua 11). Results from reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and enzyme assays showed that HPR1 activity changed significantly in corresponding transgenic lines without any effect on HPR2 activity, which is the same for HPR2. However, metabolite analysis and the serine glyoxylate aminotransferase (SGAT) activity assay showed that the metabolite flux of photorespiration was disturbed in RNAi lines of both HPR genes. Furthermore, HPR1 and HPR2 proteins were located to the peroxisome and cytosol, respectively, by transient expression experiment. Double mutant hpr1 x hpr2 was generated by crossing individual mutant of hpr1 and hpr2. The phenotypes of all transgenic lines were determined in ambient air and CO2 -elevated air. The phenotype typical of photorespiration mutants was observed only where activity of both HPR1 and HPR2 were downregulated in the same line. These findings demonstrate that two hydroxypyruvate reductases encoded by OsHPR1 and OsHPR2 are involved in photorespiratory metabolism in rice. OsHPR1,OsHPR2 Iron-binding Haemerythrin RING ubiquitin ligases regulate plant iron responses and accumulation 2013 Nat Commun Japan Science and Technology Agency, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan Iron is essential for most living organisms. Plants transcriptionally induce genes involved in iron acquisition under conditions of low iron availability, but the nature of the deficiency signal and its sensors are unknown. Here we report the identification of new iron regulators in rice, designated Oryza sativa Haemerythrin motif-containing Really Interesting New Gene (RING)- and Zinc-finger protein 1 (OsHRZ1) and OsHRZ2. OsHRZ1, OsHRZ2 and their Arabidopsis homologue BRUTUS bind iron and zinc, and possess ubiquitination activity. OsHRZ1 and OsHRZ2 are susceptible to degradation in roots irrespective of iron conditions. OsHRZ-knockdown plants exhibit substantial tolerance to iron deficiency, and accumulate more iron in their shoots and grains irrespective of soil iron conditions. The expression of iron deficiency-inducible genes involved in iron utilization is enhanced in OsHRZ-knockdown plants, mostly under iron-sufficient conditions. These results suggest that OsHRZ1 and OsHRZ2 are iron-binding sensors that negatively regulate iron acquisition under conditions of iron sufficiency. OsHRZ1,OsHRZ2,OsHORZ1 Functional analysis of OsHSBP1 and OsHSBP2 revealed their involvement in the heat shock response in rice (Oryza sativa L.) 2012 J Exp Bot State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China. The heat shock response (HSR) induces the production of heat shock proteins (HSPs) through the activation of heat shock factors (HSF). HSF binding protein (HSBP) is reported to modulate the function of HSF by binding to their trimer and hence to regulate HSR. This report describes the role of OsHSBP1 and OsHSBP2 in the regulation of the HSR and seed development of rice. Both genes expressed ubiquitously in all tissues under normal growth conditions while their expression levels were significantly increased during recovery after heat shock treatment. Subcellular localization revealed the cytosol-nuclear localization of both OsHSBP1 and OsHSBP2 in onion epidermal cells. The yeast two-hybrid assay depicted the self-binding ability of both genes. Both genes were also important for seed development, as their knock-down lines were associated with significant seed abortion. The thermotolerance assay revealed that OsHSBP1 and OsHSBP2 are negative regulators of HSR and involved in acquired thermotolerance but not in basal thermotolerance since their over-expression transgenic lines pre-heated at sublethal temperature, showed significantly decreased seedling survival after heat shock treatment. Furthermore, antioxidant activity and gene expression of catalase and peroxidase was significantly increased in knock-down transgenic seedlings of OsHSBP1 and OsHSBP2 after heat stress compared with the wild type. The expression of heat specific HSPs was also increased significantly in knockdown line of both genes but in a specific manner, suggesting the involvement of HSBP genes in different pathways. Overall, the present study reveals the role of OsHSBP1 and OsHSBP2 in the regulation of the HSR and seed development of rice. OsHSBP1,OsHSBP2,OsPOD|prx11 OsHSF7 gene in rice, Oryza sativa L., encodes a transcription factor that functions as a high temperature receptive and responsive factor 2009 BMB reports Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences, 2901 Beidi Rd, Shanghai 201106, The People's Republic of China. Three novel Class A genes that encode heat shock transcription factor (HSF) were cloned from Oryza Sativa L using a yeast hybrid method. The OsHSF7 gene was found to be rapidly expressed in high levels in response to temperature, which indicates that it may be involved in heat stress reception and response. Over-expression of OsHSF7 in transgenic Arabidopsis could not induced over the expression of most target heat stress-inducible genes of HSFs; however, the transcription of some HSF target genes was more abundant in transgenic plants following two hours of heat stress treatment. In addition, those transgenic plants also had a higher basal thermotolerance, but not acquired thermotolerance. Collectively, the results of this study indicate that OsHSF7 might play an important role in the response to high temperature. Specifically, these findings indicate that OsHSF7 may be useful in the production of transgenic monocots that can over-express protective genes such as HSPs in response to heat stress, which will enable such plants to tolerate high temperatures. [BMB reports 2009; 42(1): 16-21] OsHSF7 Isolation and characterization of a cDNA encoding two novel heat-shock factor OsHSF6 and OsHSF12 in Oryza sativa L 2005 J Biochem Mol Biol Agro-Biotechnology Research Center of Shanghai Academy of Agricultural Sciences, The People's Republic of China. As a crucial transcription factor family, heat-shock factors were mainly analyzed and characterized in tomato and Arabidopsis. In this study, we isolated two putative heatshock factors OsHSF6 and OsHSF12 that interact specifically with heat-shock element (HSE) from Oryza sativa L by yeast one-hybrid method. The full-length cDNA of OsHSF6 and OsHSF12 have 1074bp and 920bp open reading frame (ORF), respectively. Analysis of the deduced amino acid sequences revealed that OsHSF6 was a class A heat shock factor (HSF) with all the conserved sequence elements characteristic of heat stress transcription factor, while OsHSF12 was a class B HSF with C-terminal domain (CTD) lacking of AHA motif. Bioinformatic analysis showed that the sequences and structures of two HSFs' DNA binding domain (DBD) had a high similarity with LpHSF24. The results of RT-PCR indicated OsHSF6 gene was expressed immediately after rice plants exposure to heat stress, and the transcription of OsHSF6 gene accumulated primarily in immature seeds, roots and leaves. However, we did not find the transcription of OsHSF12 gene in different organs and growth periods. Our results implied that OsHSF6 might be function as a HSF regulating early expression of stress genes in response to heat shock, and OsHSF12 might be act as a synergistic factor to regulate the expression of down-stream genes. OsHSF6|OsHsfA2c Identification and expression analysis of OsHsfs in rice 2009 J Zhejiang Univ Sci B State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, China. Heat stress transcription factors (Hsfs) are the central regulators of defense response to heat stress. We identified a total of 25 rice Hsf genes by genome-wide analysis of rice (Oryza sativa L.) genome, including the subspecies of O. japonica and O. indica. Proteins encoded by OsHsfs were divided into three classes according to their structures. Digital Northern analysis showed that OsHsfs were expressed constitutively. The expressions of these OsHsfs in response to heat stress and oxidative stress differed among the members of the gene family. Promoter analysis identified a number of stress-related cis-elements in the promoter regions of these OsHsfs. No significant correlation, however, was found between the heat-shock responses of genes and their cis-elements. Overall, our results provide a foundation for future research of OsHsfs function. OsHSF6|OsHsfA2c,OsHsfA2e,OsHsfA4a|OsHsfA4b,OsHsfB2b,OsHsfB4b,OsHsfC1b Expression of rice heat stress transcription factor OsHsfA2e enhances tolerance to environmental stresses in transgenic Arabidopsis 2008 Planta Research Institute for Biological Sciences (RIBS), Okayama, Kaga-Gun, Okayama, Japan. Plant growth and crop yields are limited by high-temperature stresses. In this study, we attempted to isolate the rice genes responsible for high-temperature stress tolerance using a transformed Arabidopsis population expressing a full-length cDNA library of rice. From approximately 20,000 lines of transgenic Arabidopsis, we isolated a thermotolerant line, R04333, that could survive transient heat stress at the cotyledon stage. The rice cDNA inserted in R04333 encodes OsHsfA2e, a member of the heat stress transcription factors. The thermotolerant phenotype was observed in newly constructed transgenic Arabidopsis plants expressing OsHsfA2e. Among 5 A2-type HSF genes encoded in the rice genome, four genes, including OsHsfA2e, are induced by high temperatures in rice seedlings. The OsHsfA2e protein was localized to the nuclear region and exhibited transcription activation activity in the C-terminal region. Microarray analysis demonstrated that under unstressed conditions transgenic Arabidopsis overexpressing OsHsfA2e highly expressed certain stress-associated genes, including several classes of heat-shock proteins. The thermotolerant phenotype was observed not only in the cotyledons but also in rosette leaves, inflorescence stems and seeds. In addition, transgenic Arabidopsis exhibited tolerance to high-salinity stress. These observations suggest that the OsHsfA2e may be useful in molecular breeding designed to improve the environmental stress tolerance of crops. OsHsfA2e A 9 bp cis-element in the promoters of class I small heat shock protein genes on chromosome 3 in rice mediates L-azetidine-2-carboxylic acid and heat shock responses 2010 J Exp Bot Institute of Plant Biology, National Taiwan University, Taipei, Taiwan. In rice, the class I small heat shock protein (sHSP-CI) genes were found to be selectively induced by L-azetidine-2-carboxylic acid (AZC) on chromosome 3 but not chromosome 1. Here it is shown that a novel cis-responsive element contributed to the differential regulation. By serial deletion and computational analysis, a 9 bp putative AZC-responsive element (AZRE), GTCCTGGAC, located between nucleotides -186 and -178 relative to the transcription initiation site of Oshsp17.3 was revealed. Deletion of this putative AZRE from the promoter abolished its ability to be induced by AZC. Moreover, electrophoretic mobility shift assay (EMSA) revealed that the AZRE interacted specifically with nuclear proteins from AZC-treated rice seedlings. Two AZRE-protein complexes were detected by EMSA, one of which could be competed out by a canonical heat shock element (HSE). Deletion of the AZRE also affected the HS response. Furthermore, transient co-expression of the heat shock factor OsHsfA4b with the AZRE in the promoter of Oshsp17.3 was effective. The requirement for the putative AZRE for AZC and HS responses in transgenic Arabidopsis was also shown. Thus, AZRE represents an alternative form of heat HSE, and its interaction with canonical HSEs through heat shock factors may be required to respond to HS and AZC. OsHsfA4a|OsHsfA4b Orthologs of the class A4 heat shock transcription factor HsfA4a confer cadmium tolerance in wheat and rice 2009 Plant Cell POSTECH-UZH Global Research Laboratory, Division of Integrative Biology and Biotechnology, Pohang University of Science and Technology, Pohang, 790-784, Korea. Cadmium (Cd) is a widespread soil pollutant; thus, the underlying molecular controls of plant Cd tolerance are of substantial interest. A screen for wheat (Triticum aestivum) genes that confer Cd tolerance to a Cd hypersensitive yeast strain identified Heat shock transcription factor A4a (HsfA4a). Ta HsfA4a is most similar to the class A4 Hsfs from monocots. The most closely related rice (Oryza sativa) homolog, Os HsfA4a, conferred Cd tolerance in yeast, as did Ta HsfA4a, but the second most closely related rice homolog, Os HsfA4d, did not. Cd tolerance was enhanced in rice plants expressing Ta HsfA4a and decreased in rice plants with knocked-down expression of Os HsfA4a. An analysis of the functional domain using chimeric proteins constructed from Ta HsfA4a and Os HsfA4d revealed that the DNA binding domain (DBD) of HsfA4a is critical for Cd tolerance, and within the DBD, Ala-31 and Leu-42 are important for Cd tolerance. Moreover, Ta HsfA4a-mediated Cd resistance in yeast requires metallothionein (MT). In the roots of wheat and rice, Cd stress caused increases in HsfA4a expression, together the MT genes. Our findings thus suggest that HsfA4a of wheat and rice confers Cd tolerance by upregulating MT gene expression in planta. OsHsfA4a|OsHsfA4b Over-expression of OsHsfA7 enhanced salt and drought tolerance in transgenic rice 2013 BMB reports Key Laboratory for Crop Germplasm Innovation and Utilization of Hunan Province, Hunan Agricultural University, Changsha, China. Heat shock proteins play an important role in plant stress tolerance and are mainly regulated by heat shock transcription factors (Hsfs). In this study, we generated transgenic rice over-expressing OsHsfA7 and carried out morphological observation and stress tolerance assays. Transgenic plants exhibited less, shorter lateral roots and root hair. Under salt treatment, over-expressing OsHsfA7 rice showed alleviative appearance of damage symptoms and higher survival rate, leaf electrical conductivity and malondialdehyde content of transgenic plants were lower than those of wild type plants. Meanwhile, transgenic rice seedlings restored normal growth but wild type plants could not be rescued after drought and re-watering treatment. These findings indicate that over-expression of OsHsfA7 gene can increase tolerance to salt and drought stresses in rice seedlings. OsHsfA7 Heat shock factor OsHsfB2b negatively regulates drought and salt tolerance in rice 2013 Plant Cell Rep Hunan Provincial Key Laboratory for Germplasm Innovation and Utilization of Crop, Hunan Agricultural University, Changsha, 410128, Hunan, China. KEY MESSAGE: Expression of OsHsfB2b was strongly induced by heat, salt, ABA and PEG treatments. Drought and salt tolerances were significantly decreased by OsHsfB2b overexpression, but were enhanced by RNA interference. ABSTRACT: Plants have more than 20 heat shock factors (Hsfs) that were designated class A, B, and C. Many members of Class A Hsfs were characterized as activators of transcription, but the functional roles of class B and C Hsfs have not been fully recognized. OsHsfB2b is a member of class B Hsfs in rice (Oryza sativa). Expression of OsHsfB2b was strongly induced by heat, salt, abscisic acid (ABA) and polyethylene glycol (PEG) treatments but was almost not affected by cold stress. Drought and salt tolerances were significantly decreased in OsHsfB2b-overexpressing transgenic rice, but were enhanced in the OsHsfB2b-RNAi transgenic rice. Under drought stress, the OsHsfB2b-overexpressing transgenic rice exhibited increased relative electrical conductivity (REC) and content of malondialdehyde (MDA) and decreased proline content compared with the wild type, while the lower REC and MDA content and increased proline content were found in the OsHsfB2b-RNAi transgenic rice. These results suggest that OsHsfB2b functions as a negative regulator in response to drought and salt stresses in rice, with its existing B3 repression domain (BRD) that might be necessary for the repressive activity. The present study revealed the potential value of OsHsfB2b in genetic improvement of rice. OsHsfB2b Transcription factor OsHsfC1b regulates salt tolerance and development in Oryza sativa ssp. japonica 2012 AoB Plants Institute of Biochemistry and Biology , University of Potsdam , Karl-Liebknecht-Str. 24-25, 14476 Potsdam , Germany. BACKGROUND AND AIMS: Salt stress leads to attenuated growth and productivity in rice. Transcription factors like heat shock factors (HSFs) represent central regulators of stress adaptation. Heat shock factors of the classes A and B are well established as regulators of thermal and non-thermal stress responses in plants; however, the role of class C HSFs is unknown. Here we characterized the function of the OsHsfC1b (Os01g53220) transcription factor from rice. METHODOLOGY: We analysed the expression of OsHsfC1b in the rice japonica cultivars Dongjin and Nipponbare exposed to salt stress as well as after mannitol, abscisic acid (ABA) and H(2)O(2) treatment. For functional characterization of OsHsfC1b, we analysed the physiological response of a T-DNA insertion line (hsfc1b) and two artificial micro-RNA (amiRNA) knock-down lines to salt, mannitol and ABA treatment. In addition, we quantified the expression of small Heat Shock Protein (sHSP) genes and those related to signalling and ion homeostasis by quantitative real-time polymerase chain reaction in roots exposed to salt. The subcellular localization of OsHsfC1b protein fused to green fluorescent protein (GFP) was determined in Arabidopsis mesophyll cell protoplasts. PRINCIPAL RESULTS: Expression of OsHsfC1b was induced by salt, mannitol and ABA, but not by H(2)O(2). Impaired function of OsHsfC1b in the hsfc1b mutant and the amiRNA lines led to decreased salt and osmotic stress tolerance, increased sensitivity to ABA, and temporal misregulation of salt-responsive genes involved in signalling and ion homeostasis. Furthermore, sHSP genes showed enhanced expression in knock-down plants under salt stress. We observed retarded growth of hsfc1b and knock-down lines in comparison with control plants under non-stress conditions. Transient expression of OsHsfC1b fused to GFP in protoplasts revealed nuclear localization of the transcription factor. CONCLUSIONS: OsHsfC1b plays a role in ABA-mediated salt stress tolerance in rice. Furthermore, OsHsfC1b is involved in the response to osmotic stress and is required for plant growth under non-stress conditions. OsHsfC1b Molecular characterization of Oryza sativa 16.9 kDa heat shock protein 1999 Biochem J Department of Botany, National Taiwan University, Taipei, 106, Taiwan, R.O.C. A rice class I low-molecular-mass heat shock protein (LMM HSP) Oshsp 16.9 was overexpressed in Escherichia coli. Oligomerized complexes of Oshsp16.9 were harvested and electron microscopic observations of purified complexes revealed globular structures of 10-20 nm in diameter (with majority of 15-18 nm) and calculated to comprise approx. 12 monomers per complex. In comparison, complexes from native rice class I LMM HSPs were observed as larger ellipsoid- or globular-like random aggregated hetero-oligomers. To characterize the biochemical functions of the hydrophobic N-terminal region of Oshsp16.9, a truncation in the N-terminal region was constructed and introduced into E. coli. Results showed that the N-terminal truncated Oshsp16.9 mutant was capable of forming complexes similar to the full-length Oshsp16.9; however, the deletion protein failed to confer in vitro protein thermostability under elevated temperatures. Protein assays from in vivo treatments at higher temperatures exhibited that non-specific interactions of E. coli cellular proteins only occurred with full-length Oshsp16.9 complexes but not with the mutant complex. In vitro immunoprecipitation of cellular proteins from E. coli overexpressing full-length Oshsp16.9 showed that interactions between plant LMM HSP and E. coli cellular proteins are temperature-dependent. Taken together, the hydrophobic N-terminal region of rice class I LMM HSP is critical in the ability of the protein to interact/bind with its potential substrates. Oshsp16.9 Functional regions of rice heat shock protein, Oshsp16.9, required for conferring thermotolerance in Escherichia coli 2002 Plant Physiol Department of Botany, National Taiwan University, Taipei, Taiwan, Republic of China. Rice (Oryza sativa) class I low-molecular mass (LMM) heat shock protein (HSP), Oshsp16.9, has been shown to be able to confer thermotolerance in Escherichia coli. To define the regions for this intriguing property, deletion mutants of this hsp have been constructed and overexpressed in E. coli XL1-blue cells after isopropyl beta-D-thioglactopyranoside induction. The deletion of amino acid residues 30 through 36 (PATSDND) in the N-terminal domain or 73 through 78 (EEGNVL) in the consensus II domain of Oshsp16.9 led to the loss of chaperone activities and also rendered the E. coli incapable of surviving at 47.5 degrees C. To further investigate the function of these two domains, we determined the light scattering changes of Oshsp16.9 mutant proteins at 320 nm under heat treatment either by themselves or in the presence of a thermosensitive enzyme, citrate synthase. It was observed that regions of amino acid residues 30 through 36 and 73 through 78 were responsible for stability of Oshsp16.9 and its interactions with other unfolded protein substrates, such as citrate synthase. Studies of two-point mutants of Oshsp16.9, GST-N74E73K and GST-N74E74K, indicate that amino acid residues 73 and 74 are an important part of the substrate-binding site of Oshsp16.9. Non-denaturing gel analysis of purified Oshsp16.9 revealed that oligomerization of Oshsp16.9 was necessary but not sufficient for its chaperone activity. Oshsp16.9 Expression of a gene encoding a 16.9-kDa heat-shock protein, Oshsp16.9, in Escherichia coli enhances thermotolerance 1997 Proc Natl Acad Sci U S A Department of Botany, National Taiwan University, Taipei, Taiwan, Republic of China. A gene encoding the rice 16.9-kDa class I low-molecular-mass (LMM) heat-shock protein (HSP), Oshsp16.9, was introduced into Escherichia coli using the pGEX-2T expression vector to analyze the possible function of this LMM HSP under heat stress. It is known that E. coli does not normally produce class I LMM HSPs. We compared the survivability of E. coli XL1-Blue cells transformed with a recombinant plasmid containing a glutathione S-transferase (GST)-Oshsp16.9 fusion protein (pGST-FL cells) with the control E. coli cells transformed with the pGEX-2T vector (pGST cells) under heat-shock (HS) after isopropyl beta-D-thiogalactopyranoside induction. The pGST-FL cells demonstrated thermotolerance at 47.5 degrees C, a treatment that was lethal to the pGST cells. When the cell lysates from these two E. coli transformants were heated at 55 degrees C, the amount of protein denatured in the pGST-FL cells was 50% less than that of the pGST cells. Similar results as pGST-FL cells were obtained in pGST-N78 cells (cells produced a fusion protein with only the N-terminal 78 aa in the Oshsp16.9 portion) but not in pGST-C108 cells (cells produced a fusion protein with C-terminal 108 aa in the Oshsp16.9 portion). The acquired thermotolerant pGST-FL cells synthesized three types of HSPs, including the 76-, 73-, and 64-kDa proteins according to their abundance at a lethal temperature of 47.5 degrees C. This finding indicates that a plant class I LMM HSP, when effectively expressed in transformed prokaryotic cells that do not normally synthesize this class of LMM HSPs, may directly or indirectly increase thermotolerance. Oshsp16.9 Expression analysis of nine rice heat shock protein genes under abiotic stresses and ABA treatment 2009 J Plant Physiol Crop Gene Engineering Key Laboratory of Hunan Province, Hunan Agricultural University, Changsha 410128, China; College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China. Expression profiles of nine rice heat shock protein genes (OsHSPs) were analyzed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The nine genes exhibited distinctive expression in different organs. Expression of nine OsHSP genes was affected differentially by abiotic stresses and abscisic acid (ABA). All nine OsHSP genes were induced strongly by heat shock treatment, whereas none of them were induced by cold. The transcripts of OsHSP80.2, OsHSP71.1 and OsHSP23.7 were increased during salt tress treatment. Expression of OsHSP80.2 and OsHSP24.1 genes were enhanced while treated with 10% PEG. Only OsHSP71.1 was induced by ABA while OsHSP24.1 was suppressed by ABA. These observations imply that the nine OsHSP genes may play different roles in plant development and abiotic stress responses. OsHSP17.0,OsHSP23.7 Overexpression of OsHsp17.0 and OsHsp23.7 enhances drought and salt tolerance in rice 2012 J Plant Physiol Hunan Provincial Key Laboratory for Germplasm Innovation and Utilization of Crop, Hunan Agricultural University, Changsha 410128, China. Heat shock proteins (Hsps) play an important role in plant stress tolerance. We previously reported that expression of OsHsp17.0 and OsHsp23.7 could be enhanced by heat shock treatment and/or other abiotic stresses. In this paper, stress tolerance assays of transgenic rice plants overexpressing OsHsp17.0 and OsHsp23.7 have been carried out. Both OsHsp17.0-OE and OsHsp23.7-OE transgenic lines demonstrated higher germination ability compared to wild-type (WT) plants when subjected to mannitol and NaCl. Phenotypic analysis showed that transgenic rice lines displayed a higher tolerance to drought and salt stress compared to WT plants. In addition, transgenic rice lines showed significantly lower REC, lower MDA content and higher free proline content than WT under drought and salt stresses. These results suggest that OsHsp17.0 and OsHsp23.7 play an important role in rice acclimation to salt and drought stresses and are useful for engineering drought and salt tolerance rice. OsHSP17.0,OsHSP23.7 Enhanced tolerance to drought stress in transgenic rice plants overexpressing a small heat-shock protein, sHSP17.7 2008 Plant Cell Rep National Agricultural Research Center for Hokkaido Region, Hitsujigaoka 1, Toyohira-ku, Sapporo 062-8555, Japan. yutaka@affrc.go.jp Exposure of rice (Oryza sativa L.) seedlings to a high temperature (42 degrees C) for 24 h resulted in a significant increase in tolerance to drought stress. To try to determine the mechanisms of acquisition of tolerance to drought stress by heat shock, the rice small heat-shock protein gene, sHSP17.7, the product of which was shown to act as molecular chaperones in vitro and in vivo in our previous study, was overexpressed in the rice cultivar "Hoshinoyume". Western and Northern blot analyses showed higher expression levels of sHSP17.7 protein in three transgenic lines than in one transgenic line. Drought tolerance was assessed in these transgenic lines and wild-type plants by withholding water for 6 days for evaluation of the ability of plants to continue growth after water-stress treatments. Although no significant difference was found in water potential of seedlings between transgenic lines and wild-type plants at the end of drought treatments, only transgenic seedlings with higher expression levels of sHSP17.7 protein could regrow after rewatering. Similar results were observed in survival rates after treatments with 30% polyethylene glycol (PEG) 3640 for 3 days. These results suggest that overproduction of sHSP17.7 could increase drought tolerance in transgenic rice seedlings. OsHSP17.7 Over-expression of a small heat shock protein, sHSP17.7, confers both heat tolerance and UV-B resistance to rice plants 2004 Molecular Breeding National Agricultural Research Center for Hokkaido Region, Hitsujigaoka 1, Toyohira-ku, Sapporo, 062-8555, Japan Exposure of rice (Oryza sativa L.) seedlings to a high temperature (42degreesC) for 24 h resulted in a significant increase in resistance to UV-B damage. UV-B resistance was enhanced in parallel with the period of heat treatment. sHSP17.7 was isolated from heated rice seedlings, and the influence of rice sHSP17.7 expression on the viability of E. coli under heat-shock conditions was assessed. After heating, the survival rate of sHSP17.7 cells was 2-fold higher than that of the control cells. The molecular chaperone activity of sHSP17.7 was investigated using catalase as a substrate. Recombinant sHSP17.7 had heat-stable chaperone properties that were capable of protecting stressed catalase from precipitation. sHSP17.7 was overexpressed in the rice cultivar 'Hoshinoyume', by Agrobacterium-mediated transformation, under the control of a CaMV 35S promoter. Transgenic rice plants with increased levels of sHSP17.7 protein exhibited significantly increased thermotolerance compared to untransformed control plants. The level of increased thermotolerance was correlated with the level of increased sHSP17.7 protein in the transgenic plants. The transgenic rice plant with the highest constitutive expression of sHSP17.7 had significantly greater resistance to UV-B stress than untransformed control plants. Increase in the degree of resistance of transgenic plants to UV-B was accompanied by an increase in production of sHSP17.7 protein. OsHSP17.7 Induction of a cDNA clone from rice encoding a class II small heat shock protein by heat stress, mechanical injury, and salicylic acid 2007 Plant Science Department of Plant Pathology, National Chung Hsing University, Taichung 402, Taiwan This is the first report of a full-length cDNA clone for a class II small heat shock protein (sHSP) isolated from rice (Oryza sativa L., cv. Tainong No. 67) etiolated seedlings heat shocked at 41 °C for 2 h. The coding sequence consists of 501 bp, and the clone encodes a protein of 18.0 kDa with a predicted pI value of 5.61. The obtained full-length cDNA clone, designated Oshsp18.0-CII, is almost identical to a putative class II sHSP gene located on rice (cv. Nipponbare) chromosome one and another putative class II sHSP rice gene. Oshsp18.0-CII was induced by mechanical injury and salicylic acid treatment, which is not common in this class of sHSP genes. Only one copy of class II sHSP genes is present in the rice genome, and western blot analysis with anti-PsHSP17.7 (a class II pea sHSP) also showed only one protein of ~18 kDa in the 2D gel of heat-shocked rice proteins. Oshsp18.0-CII is GC-rich and contains a secondary structure in its RNA sequence. Oshsp18.0-CII Cloning and characterization of a cDNA encoding an 18.0-kDa class-I low-molecular-weight heat-shock protein from rice 1995 Gene Department of Botany, National Taiwan University, Taipei, Taiwan. A novel cDNA clone, Oshp18.0 cDNA, encoding a rice (Oryza sativa L. cv. Tainong 67) 18.0-kDa heat-shock protein (HSP), was isolated from a cDNA library of heat-shocked rice seedlings by use of the rice HSP cDNA, Oshsp17.3 cDNA, as a probe. The sequence showed that Oshsp18.0 cDNA contains a 749-bp insert encoding an ORF of 160 amino acids, with a predicted molecular mass of 18.0 kDa and a pI of 7.3. Sequence comparison reveals that Oshsp18.0 cDNA is highly homologous to other low-molecular-weight (LMW) HSP cDNAs. Also, the results of hybrid-selected in vitro translation clearly establish that Oshsp18.0 cDNA is the rice 18.0-kDa LMW HSP-encoding cDNA clone. The recombinant Oshsp18.0 fusion protein produced in Escherichia coli was of the size predicted, and was recognized by the class-I rice 16.9-kDa HSP antiserum. The results suggest that Oshsp18.0 cDNA is an 18.0-kDa class-I LMW HSP- encoding cDNA clone from rice. Oshsp18.0 Expression of the chloroplast-localized small heat shock protein by oxidative stress in rice 2000 Gene Department of Animal Science, College of Agriculture, Kyungpook National University, Sankyukdong, Pukku, Taegu, South Korea. A rice (Oryza sativa L. cv. Nakdong) cDNA clone, Oshsp26, encoding the chloroplast-localized small heat shock protein (smHSP) was isolated. Southern blot analysis of genomic DNA and the result of screening of a cDNA library indicated that the Oshsp26 gene is encoded by a single gene in the rice genome. The Oshsp26 gene was expressed following heat stress: the transcript level was highest when rice leaves were treated at high temperatures for 2h at 42 degrees C, and the transcripts became detectable after 20min and reached a maximum level after 2h. It was also found that the Oshsp26 gene was expressed following oxidative stress even in the absence of heat stress. Treatment of rice plants with methyl viologen (MV) in the light and treatment with hydrogen peroxide (H(2)O(2)), either in the light or in the dark, both caused a significant accumulation of the transcripts and the protein. Since MV treatment in the light leads to the generation of H(2)O(2) inside the chloroplast, it is likely that H(2)O(2) by itself acts to induce the expression of the Oshsp26 gene. These results suggest that the chloroplast smHSP plays an important role in protecting the chloroplast against damage caused by oxidative stress as well as by heat stress. Oshsp26 Overexpression of a chloroplast-localized small heat shock protein OsHSP26 confers enhanced tolerance against oxidative and heat stresses in tall fescue 2012 Biotechnol Lett Division of Applied Life Sciences (BK21), IALS, PMBBRC, Gyeongsang National University, Jinju, 660-701, Korea. coquf300@hotmail.com Small heat shock proteins are involved in stress tolerance. We previously isolated and characterized a rice cDNA clone, Oshsp26, encoding a chloroplast-localized small heat shock protein that is expressed following oxidative or heat stress. In this study, we transferred this gene to tall fescue plants by an Agrobacterium-mediated transformation system. The integration and expression of the transgene was confirmed by PCR, Southern, northern, and immunoblot analyzes. Compared to the control plants, the transgenic plants had significantly lower electrolyte leakage and accumulation of thiobarbituric acid-reactive substances when exposed to heat or methyl viologen. The photochemical efficiency of photosystem II (PSII) (Fv/Fm) in the transgenic tall fescue plants was higher than that in the control plants during heat stress (42 degrees C). These results suggest that the OsHSP26 protein plays an important role in the protection of PSII during heat and oxidative stress in vivo. Oshsp26 Rice chloroplast-localized heat shock protein 70, OsHsp70CP1, is essential for chloroplast development under high-temperature conditions 2013 J Plant Physiol Crop Biotech Institute & Department of Genetic Engineering, Kyung Hee University, Yongin 446-701, Republic of Korea. Heat is a primary abiotic stress that reduces crop yields. At the seedling stage, we identified heat-sensitive mutants that carried T-DNA inserted into a heat shock protein 70 gene, OsHsp70CP1. When grown under a constant high temperature (40 degrees C), the seedling leaves developed severe chlorosis whereas plants grown at a constant 27 degrees C showed a normal phenotype. This indicated that OsHsp70CP1 is essential for chloroplast differentiation from the proplastids under high temperatures. Transient expression analyses revealed that OsHsp70CP1 was localized to the stroma. OsHsp70CP1 was dominantly expressed in photosynthetic tissues; transcripts were greatly increased by heat stress. Some transcripts for plastid RNA metabolism were impaired in the mutant while others were not, demonstrating that a subset of nuclear-encoded proteins are substrates of OsHsp70CP1. OsHsp70CP1 OsHT, a rice gene encoding for a plasma-membrane localized histidine transporter 2005 J Integr Plant Biol State Key Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing 100871, China Using a degenerative probe designed according to the most conservative region of a known Lys- and His-specific amino acid transporter (LHT1) from Arabidopsis, we isolated a full-length cDNA named OsHT (histidine transporter of Oryza sativa L.) by screening the rice cDNA library. The cDNA is 1.3 kb in length and the open reading frame encodes for a 441 amino acid protein with a calculated molecular mass of 49 kDa. Multiple sequence alignments showed that OsHT shares a high degree of sequence conservation at the deduced amino acid level with the Arabidopsis LHT1 and six putative lysine and histidine transporters. Computational analysis indicated that OsHT is an integral membrane protein with 11 putative transmembrane helices. This was confirmed by the transient expression assay because the OsHT-GFP fusion protein was, indeed, localized mainly in the plasma membrane of onion epidermal cells. Functional complementation experiments demonstrated that OsHT was able to work as a histidine transporter in Saccharomyces cerevisiae, suggesting that OsHT is a gene that encodes for a histidine transporter from rice. This is the first time that an LHT-type amino acid transporter gene has been cloned from higher plants other than Arabidopsis. OsHT A dominant major locus in chromosome 9 of rice (Oryza sativa L.) confers tolerance to 48 degrees C high temperature at seedling stage 2013 J Hered Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, Yuhangtang Road 388, Hangzhou 310058, China. In an earlier greenhouse screening, we identified a local indica cultivar HT54 tolerant to high temperature at both seedling and grain-filling stages. In this study, we develop an optimized procedure for fine assessment of this heat tolerance. The results indicated that HT54 seedlings could tolerate high temperature up to 48 degrees C for 79h. The genetic analysis of F(1) and F(2) offspring derived from the cross between HT54 and HT13, a heat-sensitive breeding line, reveals that the heat tolerance of HT54 was controlled by a dominant major locus, which has been designated as OsHTAS (Oryza sativa heat tolerance at seedling stage). This locus was mapped on rice chromosome 9 within an interval of 420kb between markers of InDel5 and RM7364. The determined candidate ZFP gene has been confirmed to be cosegregated with a single nucleotide polymorphism (SNP) developed PCR-restriction fragment length polymorphism (RFLP) marker RBsp1407 in its promoter region. Another heat tolerance-associated SNP was identified in the first intron of its 5'-untranslated region. The existence of these SNPs thereby indicated that the OsHTAS locus contains at least two alleles. We named the one from HT54 as OsHTAS ( a ) and the one from HT13 as OsHTAS ( b ). Further dynamic expression analysis demonstrated that OsHTAS ( a ) was actively responsive to 45 degrees C high temperature stress compared with the OsHTAS ( b ) allele. OsHTAS OsHUS1 facilitates accurate meiotic recombination in rice 2014 PLoS Genet State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. Meiotic recombination normally takes place between allelic sequences on homologs. This process can also occur between non-allelic homologous sequences. Such ectopic interaction events can lead to chromosome rearrangements and are normally avoided. However, much remains unknown about how these ectopic interaction events are sensed and eliminated. In this study, using a screen in rice, we characterized a homolog of HUS1 and explored its function in meiotic recombination. In Oshus1 mutants, in conjunction with nearly normal homologous pairing and synapsis, vigorous, aberrant ectopic interactions occurred between nonhomologous chromosomes, leading to multivalent formation and subsequent chromosome fragmentation. These ectopic interactions relied on programmed meiotic double strand breaks and were formed in a manner independent of the OsMER3-mediated interference-sensitive crossover pathway. Although early homologous recombination events occurred normally, the number of interference-sensitive crossovers was reduced in the absence of OsHUS1. Together, our results indicate that OsHUS1 might be involved in regulating ectopic interactions during meiosis, probably by forming the canonical RAD9-RAD1-HUS1 (9-1-1) complex. OsHUS1 RNAi-mediated suppression of hexokinase gene OsHXK10 in rice leads to non-dehiscent anther and reduction of pollen germination 2008 Plant Science Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China Hexokinase (HXK) has been implicated to be a key player in sugar signaling and metabolism. However, little is known how HXKs regulate growth and development in planta. In rice, there are 10 members of the HXK gene family. One of them, OsHXK10, is preferentially expressed in stamen. This study aims to determine the in vivo role of OsHXK10. The promoter of OsHXK10 was found to drive the expression of beta-glucuronidase (GUS) specifically in the anther wall and mature and germinating pollens as well as in palea of rice flower. Interestingly, similar promoter expression pattern was also found in Arabidopsis. Analyses of plants transformed with OsHXK10–green fluorescent protein (GFP) fusion construct suggest that OsHXK10 is localized in the cytoplasm. Suppression of OsHXK10 expression in rice by using RNAi approach showed that the anthers of some flowers in the RNAi lines were unable to dehisce, probably due to inhibition in cell wall thickening of the anthers. The non-dehiscent anther flower (ndaF) failed to shed pollens. Real-time RT-PCR and in situ hybridization analysis revealed that OsHXK10 mRNA level was significantly reduced in the ndaF, particularly in their anther walls, as compared to that of the normal flower. Pollens collected from the RNAi lines appeared to be morphologically normal with similar level of cell wall polysaccharides and starch deposition, as compared with that of the wild-type. However, their germination capacity was markedly decreased. By seed maturity, the proportion of empty seed was significantly increased in the OsHXK10 RNAi lines than that of the wild-type. The results demonstrate that OsHXK10 plays an essential role in anther dehiscence, pollen germination and hence grain filling in rice. OsHXK10 Structure, expression, and functional analysis of the hexokinase gene family in rice (Oryza sativa L.) 2006 Planta Plant Metabolism Research Center & Graduate School of Biotechnology, Kyung Hee University, 449-701 Yongin, Republic of South Korea. Hexokinase (HXK) is a dual-function enzyme that both phosphorylates hexose to form hexose 6-phosphate and plays an important role in sugar sensing and signaling. To investigate the roles of hexokinases in rice growth and development, we analyzed rice sequence databases and isolated ten rice hexokinase cDNAs, OsHXK1 (Oryza sativa Hexokinase 1) through OsHXK10. With the exception of the single-exon gene OsHXK1, the OsHXKs all have a highly conserved genomic structure consisting of nine exons and eight introns. Gene expression profiling revealed that OsHXK2 through OsHXK9 are expressed ubiquitously in various organs, whereas OsHXK10 expression is pollen-specific. Sugars induced the expression of three OsHXKs, OsHXK2, OsHXK5, and OsHXK6, in excised leaves, while suppressing OsHXK7 expression in excised leaves and immature seeds. The hexokinase activity of the OsHXKs was confirmed by functional complementation of the hexokinase-deficient yeast strain YSH7.4-3C (hxk1, hxk2, glk1). OsHXK4 was able to complement this mutant only after the chloroplast-transit peptide was removed. The subcellular localization of OsHXK4 and OsHXK7, observed with green fluorescent protein (GFP) fusion constructs, indicated that OsHXK4 is a plastid-stroma-targeted hexokinase while OsHXK7 localizes to the cytosol. OsHXK10,OsHXK5,OsHXK6 Role of the rice hexokinases OsHXK5 and OsHXK6 as glucose sensors 2009 Plant Physiol Plant Metabolism Research Center and Graduate School of Biotechnology, Department of Horticultural Biotechnology, Kyung Hee University, Yongin 446-701, Korea. The Arabidopsis (Arabidopsis thaliana) hexokinase 1 (AtHXK1) is recognized as an important glucose (Glc) sensor. However, the function of hexokinases as Glc sensors has not been clearly demonstrated in other plant species, including rice (Oryza sativa). To investigate the functions of rice hexokinase isoforms, we characterized OsHXK5 and OsHXK6, which are evolutionarily related to AtHXK1. Transient expression analyses using GFP fusion constructs revealed that OsHXK5 and OsHXK6 are associated with mitochondria. Interestingly, the OsHXK5DeltamTP-GFP and OsHXK6DeltamTP-GFP fusion proteins, which lack N-terminal mitochondrial targeting peptides, were present mainly in the nucleus with a small amount of the proteins seen in the cytosol. In addition, the OsHXK5NLS-GFP and OsHXK6NLS-GFP fusion proteins harboring nuclear localization signals were targeted predominantly in the nucleus, suggesting that these OsHXKs retain a dual-targeting ability to mitochondria and nuclei. In transient expression assays using promoterluciferase fusion constructs, these two OsHXKs and their catalytically inactive alleles dramatically enhanced the Glc-dependent repression of the maize (Zea mays) Rubisco small subunit (RbcS) and rice alpha-amylase genes in mesophyll protoplasts of maize and rice. Notably, the expression of OsHXK5, OsHXK6, or their mutant alleles complemented the Arabidopsis glucose insensitive2-1 mutant, thereby resulting in wild-type characteristics in seedling development, Glc-dependent gene expression, and plant growth. Furthermore, transgenic rice plants overexpressing OsHXK5 or OsHXK6 exhibited hypersensitive plant growth retardation and enhanced repression of the photosynthetic gene RbcS in response to Glc treatment. These results provide evidence that rice OsHXK5 and OsHXK6 can function as Glc sensors. OsHXK5,OsHXK6 Characterization of OsIAA1 gene, a member of rice Aux/IAA family involved in auxin and brassinosteroid hormone responses and plant morphogenesis 2009 Plant Mol Biol National Center of Plant Gene Research (Wuhan), National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China. Aux/IAA and auxin response factor (ARF) are two important families that have been well recognized for their roles in auxin-mediated responses. Aux/IAA proteins are short-lived transcriptional regulators that mediate the auxin responses through interaction with ARF transcription factors. Although quite a few members of the Aux/IAA family have been functionally characterized in dicotyledonous plants such as Arabidopsis, but relatively limited information is available in important crops such as rice. This work focused on isolation and characterization of a member of Aux/IAA family in rice named OsIAA1. The results indicated that OsIAA1 was constitutively expressed in all the tissues and organs investigated. The expression of this gene was induced by various phytohormones including IAA, 2,4-D, kinetin, 24-epibrassinolide, and jasmonic acid. Over-expression of OsIAA1 in rice resulted in reduced inhibition of root elongation to auxin treatment, but increased sensitivity to 24-epiBL treatment. In addition, the OsIAA1-overexpression transgenic plants showed distinctive morphological changes such as decreased plant height and loose plant architecture. Protein interaction analysis suggested that OsIAA1 may act through interaction with OsARF1. T-DNA insertion mutant of OsARF1 showed reduced sensitivity to BR treatment, resembling the phenotype of OsIAA1-overexpression plants. In addition, expression patterns of some genes responsive to brassinosteroid and auxin were changed in the OsIAA1-overexpression plants. These data suggested that OsIAA1 may play important roles in the cross-talk of auxin and brassinosteroid signaling pathways and plant morphogenesis. OsIAA1,OsIAA4 OsIAA1, an Aux/IAA cDNA from Rice, and Changes in Its Expression as Influenced by Auxin and Light 2001 DNA Research Centre for Plant Molecular Biology and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, India. The Aux/IAA class of genes are rapidly induced by exogenous auxins and have been characterized extensively from many dicot species like Arabidopsis, Glycine max and Pisum sativum. We report here the isolation and characterization of rice (Oryza sativa L. subsp. Indica) OsIAA1 cDNA as a monocot member of the Aux/IAA gene family. The predicted amino acid sequence of OsIAA1 corresponds to a protein of ca. 26 kDa, which harbors all four characteristic domains known to be conserved in Aux/IAA proteins. The conservation of these Aux/IAA genes indicates that auxins have essentially a similar mode of action in monocots and dicots. Northern blot analysis revealed that the OsIAA1 transcript levels decrease in the excised coleoptile segments on auxin starvation, and the level is restored when auxin is supplemented; the increase in OsIAA1 transcript level was apparent within 15 to 30 min of auxin application. Auxin-induced OsIAA1 expression appears to be correlated with the elongation of excised coleoptile segments. In light-grown rice seedlings, OsIAA1 is preferentially expressed in roots and basal segment of the seedling, whereas in the etiolated rice seedlings, the OsIAA1 transcripts are most abundant in the coleoptile. A comparative analysis in light- and dark-grown seedling tissues indicates that the OsIAA1 transcript levels decrease on illumination. OsIAA1 Ectopic Overexpression of an AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) Gene OsIAA4 in Rice Induces Morphological Changes and Reduces Responsiveness to Auxin 2013 Int J Mol Sci Key Laboratory of Tropical Plant Resource and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla 666303, Yunnan, China. songyaling@gmail.com. Auxin has pleiotropic effects on plant growth and development. AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins are short-lived transcriptional regulators that mediate auxin responses through interaction with an auxin receptor, the F-box protein transport inhibitor response 1 (TIR1). Most functions of Aux/IAA proteins have been identified in Arabidopsis by studying the gain-of-function mutants in domain II. In this study, we isolated and identified an Aux/IAA protein gene from rice, OsIAA4, whose protein contains a dominant mutation-type domain II. OsIAA4 has very low expression in the entire life cycle of rice. OsIAA4-overexpressing rice plants show dwarfism, increased tiller angles, reduced gravity response, and are less sensitive to synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D). OsIAA1,OsIAA4 A gain-of-function mutation in OsIAA11 affects lateral root development in rice 2012 Mol Plant State Key Laboratory of Plant Physiology and Biochemistry, Zhejiang University, Hangzhou 310058, People's Republic of China. Lateral roots are important to plants for the uptake of nutrients and water. Several members of the Aux/IAA family have been shown to play crucial roles in lateral root development. Here, a member of the rice Aux/IAA family genes, OsIAA11 (LOC_Os03g43400), was isolated from a rice mutant defective in lateral root development. The gain-of-function mutation in OsIAA11 strictly blocks the initiation of lateral root primordia, but it does not affect crown root development. The expression of OsIAA11 is defined in root tips, lateral root caps, steles, and lateral root primordia. The auxin reporter DR5-GUS (beta-glucuronidase) was expressed at lower levels in the mutant than in wild-type, indicating that OsIAA11 is involved in auxin signaling in root caps. The transcript abundance of both OsPIN1b and OsPIN10a was diminished in root tips of the Osiaa11 mutant. Taken together, the results indicate that the gain-of-function mutation in OsIAA11 caused the inhibition of lateral root development in rice. OsIAA11 OsIAA23-mediated auxin signaling defines postembryonic maintenance of QC in rice 2011 Plant J State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, China. Although the quiescent center (QC) is crucial to root development, the molecular mechanisms that regulate its postembryonic maintenance remain obscure. In this study, a semi-dominant mutant that exhibits pleiotropic defects in root tissues, which includes the root cap, lateral and crown roots, was isolated. The mutant is characterized by a loss of QC identity during postembryonic development, and the displayed defects result from a stabilizing mutation in domain II of OsIAA23 (Os06g39590). Expression of OsIAA23 is specific to the QC of the root tip during the development of primary, lateral and crown roots. Consistent with OsIAA23 expression in the QC, the auxin signaling marked by DR5p::GUS (ss-glucuronidase) was absent in the QC region of Osiaa23. Transgenic rice plants harboring Osiaa23 under the control of the QHB promoter mimic partially the defects of Osiaa23. These results indicate that the maintenance of the QC is dependent on OsIAA23-mediated auxin signaling in the QC. These findings provide insight into Aux/IAA-based auxin signaling during postembryonic maintenance of the QC in plants. OsIAA23 Structure and expression analysis of early auxin-responsive Aux/IAA gene family in rice (Oryza sativa) 2006 Funct Integr Genomics Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India. Auxin exerts pleiotropic effects on plant growth and development by regulating the expression of early auxin-responsive genes of auxin/indoleacetic acid (Aux/IAA), small auxin-up RNA, and GH3 classes. These genes have been studied extensively in dicots like soybean and Arabidopsis. We had earlier characterized a cDNA of the first monocot member of Aux/IAA family from rice. The achievement of the large scale rice genome sequencing combined with the availability of full-length cDNA sequences from Knowledge-based Oryza Molecular Biological Encyclopedia provided us the opportunity to draw up the first comprehensive list of Aux/IAA genes in a monocot. By screening the available databases, we have identified 31 Aux/IAA genes having high sequence identity within the conserved domains I, II, III, and IV. The genomic organization as well as chromosomal location of all the Oryza sativa indoleacetic acid (OsIAA) genes is reported. The rice Aux/IAA proteins can be classified in two groups (A and B) on the basis of their phylogenetic relationship with Arabidopsis Aux/IAA proteins. An evolutionary pattern of the rice Aux/IAA genes has been discussed by analyzing their structure (exon/intron organization) and duplications. Interestingly, the duplication of rice Aux/IAA genes was found to be associated with chromosomal block duplication events in rice. The in-silico analysis has been complemented with real-time polymerase chain reaction analysis to quantify transcript levels of all Aux/IAA family members. OsIAA genes showed differential and overlapping organ-specific expression patterns in light- and dark-grown seedlings/plants. Although auxin enhanced the transcript abundance of most of the OsIAA genes, the effect was more pronounced on OsIAA9, 14, 19, 20, 24, and 31. These results provide a foundation for future studies on elucidating the precise role of rice Aux/IAA genes in early steps of auxin signal transduction. OsIAA3 Production and characterization of auxin-insensitive rice by overexpression of a mutagenized rice IAA protein 2006 Plant J Bioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya, Aichi 464-8601, Japan. Since auxin was first isolated and characterized as a plant hormone, the underlying molecular mechanism of auxin signaling has been elucidated primarily in dicot plants represented by Arabidopsis. In monocot plants, the molecular mechanism of auxin signaling has remained unclear, despite various physiological experiments. To understand the function and mechanism of auxin signaling in rice (Oryza sativa), we focused on the IAA gene, a well-studied gene in Arabidopsis that serves as a negative regulator of auxin signaling. We found 24 IAA gene family members in the rice genome. OsIAA3 is one of these family members whose expression is rapidly increased in response to auxin. We produced transgenic rice harboring mOsIAA3-GR, which can overproduce mutant OsIAA3 protein containing an amino acid change in domain II to cause a gain-of-function phenotype, by treatment with dexamethasone. The transgenic rice was insensitive to auxin and gravitropic stimuli, and exhibited short leaf blades, reduced crown root formation, and abnormal leaf formation. These results suggest that, in rice, auxin is important for development and its signaling is mediated by IAA genes. OsIAA3 Tiller formation in rice is altered by overexpression of OsIAGLU gene encoding an IAA-conjugating enzyme or exogenous treatment of free IAA 2013 Journal of Plant Biology Department of Plant Science, Research Institute for Agriculture and Life Sciences, and Plant Genomics and Breeding Institute, Seoul National University, Seoul, 151-921, Korea Optimization of plant architecture is important for cultivation and yield of cereal crops in the field. Tillering is an essential factor used to determine the overall architecture of cereal crops. It has long been recognized that the development of branching patterns is controlled by the level and distribution of auxin within a plant. To better understand the relationship between auxin levels and tillering in rice, we examined rice plants with increased or decreased levels of free IAA. To decrease IAA levels, we selected the rice IAA-glucose synthase gene (OsIAGLU) from the rice genome database based on high sequence homology with IAA-glucose synthase from maize (ZmIAGLU), which is known to generate IAAglucose conjugate from free IAA. The OsIAGLU gene driven by the Cauliflower Mosaic Virus 35S promoter was transformed into a rice cultivar to generate transgenic rice plants constitutively over-expressing this gene. The number of tillers and panicles significantly increased in the transgenic lines compared to the wild-type plants, while plant height and panicle length decreased. These results indicate that decreased levels of free IAA likely enhance tiller formation in rice. To increase levels of free IAA, we treated rice plants with three different concentrations of exogenous IAA (1 μM, 10 μM and 100 μM) twice a week by spraying. Exogenous IAA treatment at concentrations of 10 μM and 100 μM significantly reduced tiller number in three different rice cultivars. These results indicate that exogenously applied IAA inhibits shoot branching in rice. Overall, auxin tightly controls tiller formation in rice in a negative way. OsIAGLU Characterization of a pollen-preferential gene OSIAGP from rice (Oryza sativa L. subspecies indica) coding for an arabinogalactan protein homologue, and analysis of its promoter activity during pollen development and pollen tube growth 2010 Transgenic Res Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, 110021, India. During differential screening of inflorescence-specific cDNA libraries from Oryza sativa indica, an arabinogalactan protein (OSIAGP) cDNA (586 bp) expressing preferentially in the inflorescence has been isolated. It encodes an arabinogalactan protein of 59 amino acids (6.4 kDa) with a transmembrane domain and a secretory domain at the N terminus. The protein shows homology with AGP23 from Arabidopsis, and its homologue in japonica rice is located on chromosome 6. OSIAGP transcripts also accumulate in shoots and roots of rice seedling grown in the dark, but light represses expression of the gene. Analysis of a genomic clone of OSIAGP revealed that its promoter contains several pollen-specificity and light-regulatory elements. The promoter confers pollen-preferential activity on gus, starting from the release of microspores to anther dehiscence in transgenic tobacco, and is also active during pollen tube growth. Analysis of pollen preferential activity of the promoter in the transgenic rice system revealed that even the approximately 300 bp fragment has activity in pollen and the anther wall and further deletion down to approximately 100 bp completely abolishes this activity, which is consistent with in-silico analysis of the promoter. Arabinogalactan proteins have been shown to be involved in the cell elongation process. The homology of OSIAGP with AGP23 and the fact that seedling growth in the dark and pollen tube growth are events based on cell elongation strengthen the possibility of OSIAGP performing a similar function. OSIAGP Molecular characterization of a light-responsive gene, breast basic conserved protein 1 (OsiBBC1), encoding nuclear-localized protein homologous to ribosomal protein L13 from Oryza sativa indica 2004 Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression Centre for Plant Molecular Biology and Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India. Rice (Oryza sativa L. subsp. indica) cDNA for the gene OsiBBC1, encoding homologue of the breast basic conserved protein 1 (BBC1), similar to ribosomal protein L13, has been identified and characterized. OsiBBC1 codes for a 24 kDa highly basic protein with two potential bipartite nuclear localization signals (NLS) and a transcriptional activation domain (TAD). The structural part of the gene is interrupted by four introns. The OsiBBC1 gene is represented by two copies in the rice genome and both of them are expressed. Northern analysis showed that OsiBBC1 is expressed more in the young root, post-fertilized influorescence, leaf base and callus tissue, which are comprised of actively dividing cells, indicating its role in cell division. The OsiBBC1 transcript accumulated more in the root of light-grown seedlings as compared to the shoot while its levels were higher in the shoot as compared to root of the etiolated seedlings, indicating its down-regulation by light. The western analysis, carried out using antibodies raised against a recombinant fusion protein, 6xHis-OsiBBC1, corroborated its tissue-specific expression profile observed by northern analysis. In addition, OsiBBC1/RPL13 protein could be targetted to the nucleus by particle bombardment of OsiBBC1::GUS fusion construct in the onion epidermal cells. OsiBBC1 Rice MAPK phosphatase IBR5 negatively regulates drought stress tolerance in transgenic Nicotiana tabacum 2012 Plant Sci Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, 510275, Guangzhou, PR China. liyugezi@yahoo.com.cn The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) are important negative regulators in the MAPK signaling pathways, which play crucial roles in plant growth, regulation of development and response to environment stresses. Several MAPKs have been reported to be involved in the drought stress response, however, there is no evidence for the specific function of MKPs in drought stress. Here, a putative MKP in rice (Oryza sativa), OsIBR5, was characterized. Expression of OsIBR5 was induced by PEG6000, abscisic acid (ABA) and hydrogen peroxide (H(2)O(2)). Overexpression of OsIBR5 in tobacco plants resulted in hypersensitivity to drought and H(2)O(2) treatments. Drought and ABA-induced stomatal closure was significantly reduced in OsIBR5-overexpressing tobacco plants compared with controls. Moreover, OsIBR5 was found to interact with tobacco MAPKs SIPK and WIPK, and drought-induced WIPK activity was impaired in OsIBR5-overexpressing tobacco plants. These results indicated that OsIBR5 is a MKP which was induced by abiotic stresses and decreased tolerance to drought stress in transgenic tobacco plants. OsIBR5 Rice homologs of inducer of CBF expression (OsICE) are involved in cold acclimation 2011 Plant Biotechnology None Cold stress on crops results in severe yield losses through growth retardation and irreversible damage. Recently, Inducer of CBF Expression 1 (ICE1) was identified as the master regulator inducing dehydration responsive element binding protein/C-repeat binding factor (DREB/CBF)-type transcriptional factors involved in the signaling of cold and osmotic stress in Arabidopsis. To examine whether rice ICE homologs function in cold acclimation via regulation of rice DREB homologs in response to cold stress, we assessed a polypeptide epitope containing an ICE-specific motif. Chilling stress on rice seedlings induced two ICE-related proteins with molecular masses of approximately 55 and 40 kDa. These sizes are consistent with those predicted for OsICE1 and OsICE2, respectively. In contrast to the proteins, cold stress had little or no effect on the expression of OsICE1 and OsICE2. Semi-quantitative RT-PCR indicated that both were constantly expressed, but that cold stress sequentially upregulated OsDREB1B, rice heat shock factor A3 (OsHsfA3), and trehalose-6-phosphate phosphatase (OsTPP1). Trehalose treatment enhanced the cold tolerance of seedlings. These results suggest that OsICE homologs function in transcriptional regulation at upstream of a cold-stress-induced transcription factor cascade involving OsDREB1B and OsHsfA3, leading to cold acclimation, possibly involving trehalose synthesis. OsICE1,OsbHLH001|OsICE2 Overexpression of OrbHLH001, a putative helix-loop-helix transcription factor, causes increased expression of AKT1 and maintains ionic balance under salt stress in rice 2013 J Plant Physiol Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. The basic helix-loop-helix family of proteins, which function as transcription factors, have been intensively studied in plants and animals. However, the molecular mechanism of these factors contributing to stress tolerance is unknown. Here, we report on the overexpression of OrbHLH001 from Dongxiang wild rice (Oryza rufipogon) conferring salt tolerance in transgenic rice plants. The expression of OrbHLH001 was tissue specific, mainly in phloem tissues throughout the plant. Ion assay with the scanning ion-selective electrode technique showed that NaCl stress has a greater influence on Na(+) efflux and K(+) influx in OrbHLH001-overexpressed plants than the wild type. OrbHLH001 protein can induce the expression of OsAKT1 to regulate the Na(+)/K(+) ratio in OrbHLH001-overexpressed plants by specifically binding to an E-box motif in the promoter region of OsAKT1. The mechanism may have potential use in rice molecular breeding. OsbHLH001|OsICE2 Overexpression of a homopeptide repeat-containing bHLH protein gene (OrbHLH001) from Dongxiang Wild Rice confers freezing and salt tolerance in transgenic Arabidopsis 2010 Plant Cell Rep Graduate University of the Chinese Academy of Sciences, 100093, Beijing, P. R. China. Dongxiang Wild Rice (Oryza rufipogon) is the northernmost wild rice in the world known to date and has extremely high cold tolerance and many other adversity-resistant properties. To identify the genes responsible for the high stress tolerance, we isolated and characterized a basic helix-loop-helix (bHLH) protein gene OrbHLH001 from Dongxiang Wild Rice. The gene encodes an ICE1-like protein containing multiple homopeptide repeats. Expression of OrbHLH001 is induced by salt stress and is predominant in the shoots of wild rice seedlings. Overexpression of OrbHLH001 enhanced the tolerance to freezing and salt stresses in transgenic Arabidopsis. Examination of the expression of cold-responsive genes in transgenic Arabidopsis showed that the function of OrbHLH001 differs from that of ICE1 and is independent of a CBF/DREB1 cold-response pathway. OsbHLH001|OsICE2 A POLYCOMB group gene of rice (Oryza sativa L. subspecies indica), OsiEZ1, codes for a nuclear-localized protein expressed preferentially in young seedlings and during reproductive development 2003 Gene Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, 110021, New Delhi, India. The SET domains are conserved amino acid sequences present in chromosomal proteins that contribute to the epigenetic control of gene expression by altering regional organization of the chromatin structure. The SET domain proteins are divided into four subgroups as categorized by their Drosophila members; enhancer of zeste (E(Z)), trithorax (TRX), absent small or homeotic 1 (ASH1) and supressor of variegation (SU(VAR)3-9). Homologs of all four classes have been characterized in yeast, mammals and plants. We report here the isolation and characterization of rice (Oryza sativa L. subspecies indica) cDNA, OsiEZ1, as a monocot member of this family. The OsiEZ1 cDNA is 3133 bp long with an ORF of 2799 bp, and the predicted amino acid sequence (895 residues) corresponds to a protein of ca. 98 kDa. All the characteristic domains known to be conserved in E(Z) homologs (subgroup I) of SET domain containing proteins are present in OsiEZ1. In the rice genome, a 7499 bp long OsiEZ1 sequence is split into 17 exons interrupted by 16 introns. Southern analysis indicates that OsiEZ1 is represented as single copy in the rice genome. Expression studies revealed that the OsiEZ1 transcript level was highest in rice flowers, almost undetectable in developing seeds of 1-2 days post-fertilization but increased significantly in young seeds of 3-5 days post-fertilization. The OsiEZ1 transcript was barely detectable in mature zygotic embryos, but its levels were significantly higher in callus derived from rice scutellum, somatic embryos and young seedlings. The OsiEZ1/GUS recombinant protein was confined to the nucleus in living cells of particle-bombarded onion peels. The expression of OsiEZ1 complemented a set1Delta Saccharomyces cerevisiae mutant that is impaired in telomeric silencing. We suggest that the nuclear-localized OsiEZ1 has a role in regulating various aspects of plant development, and this control is most likely brought about by repressing the activity of downstream regulatory genes. OsiEZ1|OsSET1 OsSET1, a novel SET-domain-containing gene from rice 2003 J Exp Bot PKU-Yale Joint Research Center of Agricultural and Plant Molecular Biology, National Key Laboratory of Protein Engineering and Plant Gene Engineering, College of Life Sciences, Peking University, 5 Yiheyuan Road, Beijing 100871, PR China. A novel SET-domain-containing gene OsSET1 was isolated from rice (Oryza sativa L.). Its deduced protein consists of 895 amino acids. OsSET1 has a high degree of structure similarity to other SET-domain-containing genes such as CLF in higher plants and E(z) in animals. RT-PCR showed that the gene expresses throughout the entire plant. A transient expression assay in onion epidermis revealed that the OsSET1 protein is localized in nuclei. Over-expression of the SET domain of OsSET1 in Arabidopsis resulted in altered shoot development at seedling stages. OsiEZ1|OsSET1 Down-regulation of a LBD-like gene, OsIG1, leads to occurrence of unusual double ovules and developmental abnormalities of various floral organs and megagametophyte in rice 2014 J Exp Bot Ministry of Education Key Laboratory for Bio-resource and Eco-environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064, China. The indeterminate gametophyte1 (ig1) mutation was first characterized to modulate female gametophyte development in maize (Zea mays). However, the function of its rice orthologue, OsIG1, remains unknown. For this, we first analysed OsIG1 localization from differential tissues in rice. Real-time quantitative PCR (qRT-PCR) and histochemical staining results demonstrated that the expression signal of OsIG1 was strongly detected in young inflorescence, moderately in mature flower and weakly in leaf. Furthermore, RNA in situ hybridization analyses exhibited that OsIG1 was strongly expressed in inflorescence meristems, floral meristems, empty-glume- and floret- primordia, especially in the primordia of stamens and immature ovules, and the micropylar side of the mature ovary. In OsIG1-RNAi lines, wrinkled blade formation was accompanied by increased leaf inclination angle. Cross-section further showed that the number of bulliform cells located between the vasculatures was significantly increased, indicating that OsIG1 is involved in division and differentiation of bulliform cell and lateral growth during leaf development. OsIG1-RNAi suppression lines showed pleiotropic phenotypes, including degenerated palea, glume-like features and open hull. In addition, a single OsIG1-RNAi floret is characterized by frequently developing double ovules with abnormal embryo sac development. Additionally, down-regulation of OsIG1 differentially affected the expression of genes associated with the floral organ development including EG1, OsMADS6 and OsMADS1. Taken together, these results demonstrate that OsIG1 plays an essential role in the regulation of empty-glume identity, floral organ number control and female gametophyte development in rice. OsIG1 Biosynthesis and emission of insect herbivory-induced volatile indole in rice 2012 Phytochemistry Department of Plant Sciences, University of Tennessee, Knoxville, TN 37996, USA. Insect-damaged rice plants emit a complex mixture of volatiles that are highly attractive to parasitic wasps. Indole is one constituent of insect-induced rice volatiles, and is produced in plants by the enzyme indole-3-glycerol phosphate lyase (IGL). The alpha-subunit of tryptophan synthase (TSA) is the IGL that catalyses the conversion of indole-3-glycerol phosphate to indole in the alpha-reaction of tryptophan synthesis; however, TSA is only active in the complex with the beta-subunit of tryptophan synthase and is not capable of producing free indole. In maize a TSA homolog, ZmIgl, is the structural gene responsible for volatile indole biosynthesis. Bioinformatic analysis based on the ZmIgl-sequence indicated that the rice genome contains five homologous genes. Three homologs Os03g58260, Os03g58300 and Os07g08430, have detectable transcript levels in seedling tissue and were expressed in both insect-damaged and control rice plants. Only Os03g58300, however, was up-regulated by insect feeding. Recombinant proteins of the three rice genes were tested for IGL activity. Os03g58300 had a low K(m) for indole-3-glycerol phosphate and a high k(cat), and hence can efficiently produce indole. Os07g08430 exhibited biochemical properties resembling characterized TSAs. In contrast, Os03g58260 was inactive as a monomer. Analysis of Os03g58300 expression and indole emission provides further support that Os03g58300 is the bona fide rice IGL for biosynthesis of indole, in analogy to maize, this gene is termed OsIgl. Phylogenetic analysis showed that the rice genes are localized in two distinct clades together with the maize genes ZmIgl and ZmBx1 (Os03g58300) and ZmTSA (Os03g58260 and Os07g08430). The genes in the two clades have distinct enzyme activities and gene structures in terms of intron/exon organization. These results suggest that OsIgl evolved after the split of monocot and dicot lineages and before the diversification of the Poaceae. OsIGL|OsIgl,OsTSA|pOsTSA Exogenous auxin enhances the degradation of a light down-regulated and nuclear-localized OsiIAA1, an Aux/IAA protein from rice, via proteasome 2005 Biochim Biophys Acta Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India. Auxin regulates many aspects of plant growth and development by altering the expression of diverse genes. Among these, the early auxin-responsive genes of Aux/IAA class have been extensively studied in dicots but little information is available on monocots. Earlier, we reported the isolation of OsiIAA1 cDNA, first monocot member of Aux/IAA gene family from rice. Extending this work further, we have isolated the OsiIAA1 gene from rice localized on chromosome 3. The transcriptional start site was mapped to 158 bp upstream to the translational start site. The increased accumulation of OsiIAA1 transcript in auxin-treated rice coleoptiles even in the presence of a protein synthesis inhibitor, cycloheximide, suggested that OsiIAA1 is a primary auxin response gene; the expression of OsiIAA1 gene was also upregulated in the presence of cycloheximide alone. The OsiIAA1 transcript levels were down-regulated in etiolated rice coleoptiles irradiated with far-red, red and blue light, suggesting the existence of a cross-talk between auxin and light signaling. The antibodies raised against His6-OsiIAA1 recombinant protein could detect the OsiIAA1 protein in the plant extract only in the presence of a proteasome inhibitor, MG132, indicating that OsiIAA1 is rapidly degraded by proteasome complex. The degradation of the protein was enhanced by the application of exogenous auxin. Also, the proteasome inhibitor MG132 stabilized the purified His6-OsiIAA1 protein to some extent in the cell-free extracts of rice coleoptiles. The OsiIAA1 protein harbors two nuclear localization signals (NLSs), one bipartite and the other resembling SV40 type NLS. Although both the NLSs were able to target the protein to the nucleus, the bipartite NLS was more effective. These studies indicate that nuclear localization of OsiIAA1 could be a prerequisite for its role in auxin signal transduction. OsIAA13|OsiIAA1 OsIAA13-mediated auxin signaling is involved in lateral root initiation in rice 2012 Plant Sci Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi 464-8601, Japan. kitomi.yuka@d.mbox.nagoya-u.ac.jp The plant hormone auxin is essential for root formation. After auxin perception, transmission of the auxin signal progresses through the degradation of Aux/IAA proteins. In this study, we newly isolated and characterized a rice gain-of-function mutant, Osiaa13, containing a single amino acid substitution in the core sequence required for the degradation of the OsIAA13 protein. The Osiaa13 mutant displayed typical auxin-related phenotypes: the number of lateral roots was significantly reduced and the root gravitropic response was defective. Osiaa13 mutants also exhibited altered GUS staining controlled by the DR5 promoter in lateral root initiation sites. Furthermore, expression levels of several genes that might be associated with lateral root initiation were altered in Osiaa13. Taken together, our results indicate that OsIAA13 is involved in auxin signaling and controls the expression of genes that are required for lateral root initiation in rice. OsIAA13|OsiIAA1 Analyses of two rice (Oryza sativa) cyclin-dependent kinase inhibitors and effects of transgenic expression of OsiICK6 on plant growth and development 2011 Ann Bot Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, Kaixuan Road 268, Hangzhou, China. BACKGROUND AND AIMS: Plants have a family of proteins referred to as ICKs (inhibitors of cyclin-dependent kinase, CDK) or KRPs (Kip-related proteins) that function to regulate the activities of CDK. Knowledge of these plant CDK inhibitors has been gained mostly from studies of selected members in dicotyledonous plants, particularly Arabidopsis. Much remains to be learned regarding the differences among various members of the ICK/KRP family, and regarding the function and regulation of these proteins in monocotyledonous plants. METHODS: We analysed ICK-related sequences in the rice (Orysa sativa L. subsp. indica) genome and determined that there are six members with the conserved C-terminal signature region for ICK/KRP proteins. They are referred to as OsiICKs and further analyses were performed. The interactions with CDKs and cyclins were determined by a yeast two-hybrid assay, and cellular localization by fusion with the enhanced green fluorescence protein (EGFP). The expression of OsiICK6 in different tissues and in response to several treatments was analysed by reverse transcriptase-mediated polymerase chain reaction (RT-PCR) and real-time PCR. Furthermore, OsiICK6 was over-expressed in transgenic rice plants and significant phenotypes were observed. KEY RESULTS AND CONCLUSIONS: Based on putative protein sequences, the six OsiICKs are grouped into two classes, with OsiICK1 and OsiICK6 in each of the two classes, respectively. Results showed that OsiICK1 and OsiICK6 interacted with OsCYCD, but differed in their interactions with CDKA. Both EGFP:OsiICK1 and EGFP:OsiICK6 were localized in the nucleus. Whereas EGFP:OsiICK6 showed a punctuate subnuclear distribution, OsiICK1 had a homogeneous pattern. Over-expression of OsiICK6 resulted in multiple phenotypic effects on plant growth, morphology, pollen viability and seed setting. In OsiICK6-over-expressing plants, leaves rolled toward the abaxial side, suggesting that cell proliferation is critical in maintaining an even growth along the dorsal-ventral plane of leaf blades. OsiICK1,OsiICK6 A new AOX homologous gene OsIM1 from rice (Oryza sativa L.) with an alternative splicing mechanism under salt stress 2003 Theor Appl Genet Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. A differentially expressed OsIM1 gene was isolated from rice salt-tolerant mutant M-20 by differential display. Sequence analysis revealed that the amino-acid sequence of OsIM1 showed 66% and 62% identity with PTOX from tomato ( Capsicum annuum) and AtIM from Arabidopsis, both of which encoded chloroplast-orientated terminal oxidase. Comparison of the nucleotide sequence of the OsIM1 cDNA with its genomic sequence revealed that OsIM1 genomic DNA contained nine exons and eight introns. A pseudo-transcript ( OsIM2), which probably resulted from the abnormal splicing of the OsIM1 pre-mRNA, was also identified. Southern-blot analysis showed that there existed only one copy of the OsIM1 gene in the rice genome. RFLP analysis located it on rice chromosome 3. The Northern blot revealed that OsIM1 was up-regulated by NaCl and ABA treatment. RT-PCR analysis indicated that OsIM1 and OsIM2 co-existed in the OsIM transcript pool, and the ratio of OsIM1/ OsIM2 was differentially regulated by salt stress in the salt-sensitive variety and the salt-tolerant varieties. OsIM1 Rice Importin beta1 gene affects pollen tube elongation 2011 Mol Cells Crop Biotech Institute and Department of Plant Molecular Systems Biotechnology, Kyung Hee University, Yongin, 446-701, Korea. Importin beta1 interacts with nuclear transport factors and mediates the import of nuclear proteins. We isolated a pollen-expressed gene, rice Importin beta1 (OsImpbeta1), from a T-DNA insertional population that was trapped by a promoterless beta-glucuronidase (GUS) gene. The GUS reporter was expressed in the anthers and ovaries from early through mature developmental stages. Its expression was also observed in all floral organs. However, these patterns changed as the spikelet developed. T-DNA was inserted into the OsImpbeta1 gene at 339 bp downstream from the translation initiation site. We obtained another T-DNA insertional allele by searching the flanking sequence tag database. In both lines, the wild-type and T-DNA-carrying progeny segregated at a ratio close to 1:1. The latter genotype was heterozygous (OsImpbeta1/osimpbeta1). Reciprocal crosses between WT and heterozygous plants demonstrated that the mutant alleles could not be transmitted through the male gametophyte. Close examination of the heterozygous anthers revealed that the mutant pollen matured normally. However, in vitro assays showed that tube elongation was hampered in the mutant grains. These results indicate that OsImpbeta1 is specifically required for pollen tube elongation. OsImpbeta1 Identification and organization of chloroplastic and cytosolic L-myo-inositol 1-phosphate synthase coding gene(s) in Oryza sativa: comparison with the wild halophytic rice, Porteresia coarctata 2010 Planta Plant Molecular and Cellular Genetics, Bose Institute (Centenary Campus), Kolkata, India. The gene coding for rice chloroplastic L-myo-inositol-1-phosphate synthase (MIPS; EC 5.5.1.4) has been identified by matrix-assisted laser desorption time-of-flight mass spectrometry analysis of the purified and immunologically cross-reactive approximately 60 kDa chloroplastic protein following two-dimensional polyacrylamide gel electrophoresis, which exhibited sequence identity with the cytosolic MIPS coded by OsINO1-1 gene. A possible chloroplastic transit peptide sequence was identified upstream of the OsINO1-1 gene upon analysis of rice genome. RT-PCR and confocal microscope studies confirmed transcription, effective translation and its functioning as a chloroplast transit peptide. Bioinformatic analysis mapped the chloroplastic MIPS (OsINO1-1) gene on chromosome 3, and a second MIPS gene (OsINO1-2) on chromosome 10 which lacks conventional chloroplast transit peptide sequence as in OsINO1-1. Two new PcINO1 genes, with characteristic promoter activity and upstream cis-elements were identified and cloned, but whether these proteins can be translocated to the chloroplast or not is yet to be ascertained. Electrophoretic mobility shift assay carried out with nuclear extract of Porteresia coarctata leaves grown under both control and stressed condition shows binding of nuclear proteins with the upstream elements. Nucleotide divergence among the different Oryza and Porteresia INO1 genes were calculated and compared. OsINO1-2,OsINO1-1|OsINO1 An insight into the molecular basis of salt tolerance of L-myo-inositol 1-P synthase (PcINO1) from Porteresia coarctata (Roxb.) Tateoka, a halophytic wild rice 2006 Plant Physiol Plant Molecular and Cellular Genetics, Bose Institute, Calcutta Improvement Trust Scheme-VIIM, Calcutta 700 054, India. The molecular basis of salt tolerance of L-myo-inositol 1-P synthase (MIPS; EC 5.5.1.4) from Porteresia coarctata (Roxb.) Tateoka (PcINO1, AF412340) earlier reported from this laboratory, has been analyzed by in vitro mutant and hybrid generation and subsequent biochemical and biophysical studies of the recombinant proteins. A 37-amino acid stretch between Trp-174 and Ser-210 has been confirmed as the salt-tolerance determinant domain in PcINO1 both by loss or gain of salt tolerance by either deletion or by addition to salt-sensitive MIPS(s) of Oryza (OsINO1) and Brassica juncea (BjINO1). This was further verified by growth analysis under salt environment of Schizosaccharomyces pombe transformed with the various gene constructs and studies on the differential behavior of mutant and wild proteins by Trp fluorescence, aggregation, and circular dichroism spectra in the presence of salt. 4,4'-Dianilino-1,1'-binaphthyl-5,5-disulfonic acid binding experiments revealed a lower hydrophobic surface on PcINO1 than OsINO1, contributed by this 37-amino acid stretch explaining the differential behavior of OsINO1 and PcINO1 both with respect to their enzymatic functions and thermodynamic stability in high salt environment. Detailed amino acid sequence comparison and modeling studies revealed the interposition of polar and charged residues and a well-connected hydrogen-bonding network formed by Ser and Thr in this stretch of PcINO1. On the contrary, hydrophobic residues clustered in two continuous stretches in the corresponding region of OsINO1 form a strong hydrophobic patch on the surface. It is conceivable that salt-tolerant MIPS proteins may be designed out of the salt-sensitive plant MIPS proteins by replacement of the corresponding amino acid stretch by the designated 37-amino acid stretch of PcINO1. OsINO1-1|OsINO1 Cold-induced repression of the rice anther-specific cell wall invertase gene OSINV4 is correlated with sucrose accumulation and pollen sterility 2005 Plant Cell and Environment CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia. Low temperatures during rice (Oryza sativa L.) pollen development cause pollen sterility and decreased grain yield. We show that the time of highest sensitivity to cold coincides with the time of peak tapetal activity: the transition of the tetrad to early uni-nucleate stage (young microspore, YM stage). Low temperatures at this stage of pollen development result in an accumulation of sucrose in the anthers, accompanied by decreased activity of cell wall bound acid invertase and depletion of starch in mature pollen grains. Expression analysis of two cell wall (OSINV1, 4) and one vacuolar (OSINV2) acid invertase genes showed that OSINV4 is anther-specific and downregulated by cold treatment. OSINV4 is transiently expressed in the tapetum cell layer at the YM stage, and later from the early binucleate stage in the maturing microspores. The down-regulation of OSINV4 expression in the tapetum at YM may cause a disruption in hexose production and starch formation in the pollen grains. In a cold-tolerant cultivar, OSINV4 expression was not reduced by cold; sucrose did not accumulate in the anthers and starch formation in the pollen grains was not affected. OSINV1,OSINV3,OSINV4 Effects of chilling on male gametophyte development in rice 2006 Cell Biol Int Cooperative Research Centre for Sustainable Rice Production, The University of Sydney, NSW 2006, Australia. emamun@bio.mq.edu.au Chilling during male gametophyte development in rice inhibits development of microspores, causing male sterility. Changes in cellular ultrastructure that have been exposed to mild chilling include microspores with poor pollen wall formation, abnormal vacuolation and hypertrophy of the tapetum and unusual starch accumulation in the plastids of the endothecium in post-meiotic anthers. Anthers observed during tetrad release also have callose (1,3-beta-glucan) wall abnormalities as shown by immunocytochemical labelling. Expression of rice anther specific monosaccharide transporter (OsMST8) is greatly affected by chilling treatment. Perturbed carbohydrate metabolism, which is particularly triggered by repressed genes OsINV4 and OsMST8 during chilling, causes unusual starch storage in the endothecium and this also contributes to other symptoms such as vacuolation and poor microspore wall formation. Premature callose breakdown apparently restricts the basic framework of the future pollen wall. Vacuolation and hypertrophy are also symptoms of osmotic imbalance triggered by the reabsorption of callose breakdown products due to absence of OsMST8 activity. OSINV4,OsMST8 Pollen-specific expression of Oryza sativa indica pollen allergen gene (OSIPA) promoter in rice and Arabidopsis transgenic systems 2011 Mol Biotechnol Centre for Plant Molecular Biology, Osmania University, Hyderabad 500007, Andhra Pradesh, India. Earlier, a pollen-specific Oryza sativa indica pollen allergen gene (OSIPA), coding for expansins/pollen allergens, was isolated from rice, and its promoter--upon expression in tobacco and Arabidopsis--was found active during the late stages of pollen development. In this investigation, to analyze the effects of different putative regulatory motifs of OSIPA promoter, a series of 5' deletions were fused to beta-glucuronidase gene (GUS) which were stably introduced into rice and Arabidopsis. Histochemical GUS analysis of the transgenic plants revealed that a 1631 bp promoter fragment mediates maximum GUS expression at different stages of anther/pollen development. Promoter deletions to -1272, -966, -617, and -199 bp did not change the expression profile of the pollen specificity. However, the activity of promoter was reduced as the length of promoter decreased. The region between -1567 and -199 bp was found adequate to confer pollen-specific expression in both rice and Arabidopsis systems. An approximate 4-fold increase in the GUS activity was observed in the pollen of rice when compared to that of Arabidopsis. As such, the OSIPA promoter seems promising for generation of stable male-sterile lines required for the production of hybrids in rice and other crop plants. OSIPA Promoters of two anther-specific genes confer organ-specific gene expression in a stage-specific manner in transgenic systems 2007 Plant Cell Rep Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi 110021, India. Differential screening of a stage-specific cDNA library of Indica rice has been used to identify two genes expressed in pre-pollination stage panicles, namely OSIPA and OSIPK coding for proteins similar to expansins/pollen allergens and calcium-dependent protein kinases (CDPK), respectively. Northern analysis and in situ hybridizations indicate that OSIPA expresses exclusively in pollen while OSIPK expresses in pollen as well as anther wall. Promoters of these two anther-specific genes show the presence of various cis-acting elements (GTGA and AGAAA) known to confer anther/pollen-specific gene expression. Organ/tissue-specific activity and strength of their regulatory regions have been determined in transgenic systems, i.e., tobacco and Arabidopsis. A unique temporal activity of these two promoters was observed during various developmental stages of anther/pollen. Promoter of OSIPA is active during the late stages of pollen development and remains active till the anthesis, whereas, OSIPK promoter is active to a low level in developing anther till the pollen matures. OSIPK promoter activity diminishes before anthesis. Both promoters show a potential to target expression of the gene of interest in developmental stage-specific manner and can help engineer pollen-specific traits like male-sterility in plants. OSIPA,OSIPK OsIPD3, an ortholog of the Medicago truncatula DMI3 interacting protein IPD3, is required for mycorrhizal symbiosis in rice 2008 New Phytol Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY 40546, USA. Medicago truncatula IPD3 (MtIPD3) is an interacting protein of DMI3 (does not make infections 3), a Ca(2+)/calmodulin-dependent protein kinase (CCaMK) essential for both arbuscular mycorrhizal (AM) and rhizobial symbioses. However, the function of MtIPD3 in root symbioses has not been demonstrated in M. truncatula, because of a lack of knockout mutants for functional analysis. In this study, the availability of IPD3 knockout mutants in rice (Oryza sativa) was exploited to test the function of OsIPD3 in AM symbiosis. Three independent retrotransposon Tos17 insertion lines of OsIPD3 were selected and the phenotypes characterized upon inoculation with the AM fungus Glomus intraradices. Phenotypic and genetic analyses revealed that the Osipd3 mutants were unable to establish a symbiotic association with G. intraradices. In conclusion, IPD3 represents a novel gene required for root symbiosis with AM fungi in plants. CYCLOPS|OsIPD3 IP6K gene identification in plant genomes by tag searching 2011 BMC Proc DEIS, Universita della Calabria, Via Pietro Bucci 41C Rende (CS) Italy. simona.rombo@deis.unical.it. BACKGROUND: Plants have played a special role in inositol polyphosphate (IP) research since in plant seeds was discovered the first IP, the fully phosphorylated inositol ring of phytic acid (IP6). It is now known that phytic acid is further metabolized by the IP6 Kinases (IP6Ks) to generate IP containing pyro-phosphate moiety. The IP6K are evolutionary conserved enzymes identified in several mammalian, fungi and amoebae species. Although IP6K has not yet been identified in plant chromosomes, there are many clues suggesting its presences in vegetal cells. RESULTS: In this paper we propose a new approach to search for the plant IP6K gene, that lead to the identification in plant genome of a nucleotide sequence corresponding to a specific tag of the IP6K family. Such a tag has been found in all IP6K genes identified up to now, as well as in all genes belonging to the Inositol Polyphosphate Kinases superfamily (IPK). The tag sequence corresponds to the inositol-binding site of the enzyme, and it can be considered as characterizing all IPK genes. To this aim we applied a technique based on motif discovery. We exploited DLSME, a software recently proposed, which allows for the motif structure to be only partially specified by the user. First we applied the new method on mitochondrial DNA (mtDNA) of plants, where such a gene could have been nested, possibly encrypted and hidden by virtue of the editing and/or trans-splicing processes. Then we looked for the gene in nuclear genome of two model plants, Arabidopsis thaliana and Oryza sativa. CONCLUSIONS: The analysis we conducted in plant mitochondria provided the negative, though we argue relevant, result that IP6K does not actually occur in vegetable mtDNA. Very interestingly, the tag search in nuclear genomes lead us to identify a promising sequence in chromosome 5 of Oryza sativa. Further analyses are in course to confirm that this sequence actually corresponds to IP6K mammalian gene. OsIPK2|IPK2|IP6K Expression pattern of inositol phosphate-related enzymes in rice (Oryza sativa L.): Implications for the phytic acid biosynthetic pathway 2007 Gene Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan. Phytic acid, myo-inositol-hexakisphosphate (InsP(6)), is a storage form of phosphorus in plants. Despite many physiological investigations of phytic acid accumulation and storage, little is known at the molecular level about its biosynthetic pathway in plants. Recent work has suggested two pathways. One is an inositol lipid-independent pathway that occurs through the sequential phosphorylation of 1D-myo-inositol 3-phosphate (Ins(3)P). The second is a phospholipase C (PLC)-mediated pathway, in which inositol 1,4,5-tris-phosphate (Ins(1,4,5)P(3)) is sequentially phosphorylated to InsP(6). We identified 12 genes from rice (Oryza sativa L.) that code for the enzymes that may be involved in the metabolism of inositol phosphates. These enzymes include 1D-myo-inositol 3-phosphate synthase (MIPS), inositol monophosphatase (IMP), inositol 1,4,5-tris-phosphate kinase/inositol polyphosphate kinase (IPK2), inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1), and inositol 1,3,4-triskisphosphate 5/6-kinase (ITP5/6K). The quantification of absolute amounts of mRNA by real-time RT-PCR revealed the unique expression patterns of these genes. Outstanding up-regulation of the four genes, a MIPS, an IPK1, and two ITP5/6Ks in embryos, suggested that they play a significant role in phytic acid biosynthesis and that the lipid-independent pathway was mainly active in developing seeds. On the other hand, the up-regulation of a MIPS, an IMP, an IPK2, and an ITP5/6K in anthers suggested that a PLC-mediated pathway was active in addition to a lipid-independent pathway in the anthers. OsIPK2|IPK2|IP6K A 286 bp upstream regulatory region of a rice anther-specific gene, OSIPP3, confers pollen-specific expression in Arabidopsis 2013 Biotechnol Lett Department of Plant Molecular Biology, Interdisciplinary Centre for Plant Genomics, University of Delhi South Campus, New Delhi, 110021, India. OSIPP3 gene (coding for pectin methylesterase inhibitor protein) was isolated from a pre-pollinated inflorescence-specific cDNA library by differential screening of stage-specific libraries from Oryza sativa. OSIPP3 is present in the genome of rice as a single copy gene. OSIPP3 gene was expressed exclusively in the pre-pollinated spikelets of rice. Upstream regulatory region (URR) of OSIPP3 was isolated and a series of 5'-deletions were cloned upstream of GUS reporter gene and were used to transform Arabidopsis. OSIPP3_del1 and del2 transgenic plants showed GUS expression in root, anther and silique, while OSIPP3_del3 showed GUS activity only in anthers and siliques. Pollen-specific expression was observed in case of plants harboring OSIPP3_del4 construct. It can, therefore, be concluded that the OSIPP3 URR between -178 and +108 bp is necessary for conferring pollen-specific expression in Arabidopsis. OSIPP3 OsMYB2P-1, an R2R3 MYB transcription factor, is involved in the regulation of phosphate-starvation responses and root architecture in rice 2012 Plant Physiol State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, People's Republic of China. An R2R3 MYB transcription factor, OsMYB2P-1, was identified from microarray data by monitoring the expression profile of rice (Oryza sativa ssp. japonica) seedlings exposed to phosphate (Pi)-deficient medium. Expression of OsMYB2P-1 was induced by Pi starvation. OsMYB2P-1 was localized in the nuclei and exhibited transcriptional activation activity. Overexpression of OsMYB2P-1 in Arabidopsis (Arabidopsis thaliana) and rice enhanced tolerance to Pi starvation, while suppression of OsMYB2P-1 by RNA interference in rice rendered the transgenic rice more sensitive to Pi deficiency. Furthermore, primary roots of OsMYB2P-1-overexpressing plants were shorter than those in wild-type plants under Pi-sufficient conditions, while primary roots and adventitious roots of OsMYB2P-1-overexpressing plants were longer than those of wild-type plants under Pi-deficient conditions. These results suggest that OsMYB2P-1 may also be associated with the regulation of root system architecture. Overexpression of OsMYB2P-1 led to greater expression of Pi-responsive genes such as Oryza sativa UDP-sulfoquinovose synthase, OsIPS1, OsPAP10, OsmiR399a, and OsmiR399j. In contrast, overexpression of OsMYB2P-1 suppressed the expression of OsPHO2 under both Pi-sufficient and Pi-deficient conditions. Moreover, expression of OsPT2, which encodes a low-affinity Pi transporter, was up-regulated in OsMYB2P-1-overexpressing plants under Pi-sufficient conditions, whereas expression of the high-affinity Pi transporters OsPT6, OsPT8, and OsPT10 was up-regulated by overexpression of OsMYB2P-1 under Pi-deficient conditions, suggesting that OsMYB2P-1 may act as a Pi-dependent regulator in controlling the expression of Pi transporters. These findings demonstrate that OsMYB2P-1 is a novel R2R3 MYB transcriptional factor associated with Pi starvation signaling in rice. OsIPS1,OsMYB2P-1,OsPht1;10|OsPT10,OsPht1;2|OsPT2,OsLPT1|OsPht1;6|OsPT6,OsPht1;8|OsPT8 Regulation of the expression of OsIPS1 and OsIPS2 in rice via systemic and local Pi signalling and hormones 2005 Plant Cell and Environment State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hua Jiachi Campus, Hangzhou, 310029, China Two genes with a common region that is characteristic of the TPSI1/Mt4 family were cloned from a Pi-starvation-induced cDNA library of rice roots using suppression subtracted hybridization (SSH). Based on the consensus sequence of these two genes, members of the TPSI1/Mt4 family were found in maize, wheat and barley. BLAST and a cluster analysis in the eight members of the TPSI1/Mt4 family showed two classes of four genes each among monocots. The first gene from rice was designated OsIPS1 based on a comparison of the consensus sequence with AtIPS1, and consequently the second gene, which has been previously reported as OsPI1, was designated OsIPS2. Accumulation of the mRNA of OsIPS1/2 was examined by northern blotting and quantitative reverse transcriptase-polymerase chain reaction in whole-root and split-root experiments under treatment with phosphate (Pi) and the Pi analogue phosphite (Phi). OsIPS1 showed much higher mRNA accumulation in roots than OsIPS2, and an opposite trend was seen in shoots. OsIPS1/2 showed both systemic and local responses to Pi starvation, and less than 10% of the overall induced mRNA level was due to the local Pi concentration in roots. The results indicate that Phi may interfere with earlier events in roots that are associated with a local Pi signalling pathway. An analysis of transgenic plants showed that OsIPS1/2 are independently responsive to Pi signalling and are mainly expressed in lateral roots and in the vascular cylinder in the primary root. Exogenous cytokinin (6-BA) almost completely suppressed systemic Pi starvation signalling and partially suppressed local Pi signalling. Exogenous abscisic acid remarkably reduced Pi starvation signalling. In contrast, exogenous auxin enhanced Pi signalling, especially local Pi signalling in roots. Exogenous ethylene (ethyphon) and the ratio of auxin to cytokinins did not appear to affect the expression of these two genes. OsIPS1 Isolation and Characterization of a Putative Transducer of Endoplasmic Reticulum Stress in Oryza sativa 2002 Plant and Cell Physiology Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Ikoma, 630-0101 Japan Following endoplasmic reticulum (ER) stress that prevents correct folding or assembly of ER proteins, at least three responses occur to maintain cell homeostasis: induction of chaperones, attenuation of protein synthesis, and enhancement of lipid synthesis. Transducers that transmit ER stress to the nucleus have already been identified in yeast and mammals. We report here isolation of a cDNA, OsIre1, from rice encoding a putative homolog of Ire1p, a yeast transducer of ER stress. OsIre1 encodes a polypeptide consisting of 893 amino acids, in which two hydrophobic stretches are present in the amino-terminal (N-terminal) and middle regions, possibly serving as a signal peptide and a transmembrane domain, respectively. The carboxyl-terminal (C-terminal) domain was found to possess serine/threonine protein kinase and ribonuclease-like domains showing high similarities with regions in Ire1 homologs from other organisms. A fusion protein of OsIre1 and green fluorescent protein (GFP) expressed in tobacco BY2 cells could be demonstrated to localize to the ER and the N-terminal domain of OsIre1 could substitute for yeast Ire1p in yeast cells. When produced in bacteria as a fusion protein, the C-terminal region of OsIre1 showed autophosphorylation activity. These results thus indicate that OsIre1 encodes a putative plant transducer of ER stress. IRE1|OsIRE1 A rice isoflavone reductase-like gene, OsIRL, is induced by rice blast fungal elicitor 2003 Mol Cells Division of Applied Life Science (Brain Korea 21 Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660-701, Korea. We have isolated and characterized a rice isoflavone reductase-like gene, OsIRL, whose expression is induced by a fungal elicitor. The OsIRL cDNA contains 1203 bp with an open reading frame of 942 nucleotides encoding 314 amino acids. The deduced amino acid sequence of OsIRL has a putative pyridine nucleotide binding domain and is 68% homologous with the maize isoflavone reductase-like gene. Southern blot analysis revealed that OsIRL belongs to a small multigene family. Expression of OsIRL was induced by treatment with a fungal elicitor and jasmonic acid as well as by inoculation with rice blast fungus. Cycloheximide (1 muM), strongly inhibited the induction of OsIRL by the fungal elicitor, indicating that new protein synthesis is required. The protein kinase inhibitor, staurosporine (1 muM), had little effect, but the phosphatase inhibitor, calyculin A (1 muM), strongly inhibited induction. Treatment with salicylic acid (SA, 5 mM) strongly inhibited expression of OsIRL in response to fungal elicitor and JA, while abscisic acid (ABA, 200 muM) also strongly antagonized OsIRL induction by JA, but had only a weak effect on induction by the fungal elicitor. These results suggest that the expression of OsIRL is positively regulated by phytohormones such as JA, and negatively by phytohormones such as SA, ABA. OsIRL Overexpression of rice isoflavone reductase-like gene (OsIRL) confers tolerance to reactive oxygen species 2010 Physiol Plant Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju, Korea. Isoflavone reductase is an enzyme involved in isoflavonoid biosynthesis in plants. However, rice isoflavone reductase-like gene (OsIRL, accession no. AY071920) has not been unraveled so far. Here, we have characterized its behavior in response to oxidizing agents. Using Northern and Western blot analyses, the OsIRL gene and protein were shown to be down-regulated in young seedling roots treated with reduced glutathione (GSH) and diphenyleneiodonium (DPI), known quenchers of reactive oxygen species (ROS). The OsIRL transcript level in rice suspension-cultured cells was also found to be induced by oxidants such as hydrogen peroxide (H(2)O(2)), ferric chloride (FeCl(3)), methyl viologen (MV) and glucose/glucose oxidase (G/GO), but down-regulated when co-treated with GSH. Furthermore, to investigate whether overexpression of OsIRL in transgenic rice plants promotes resistance to ROS, we generated transgenic rice lines overexpressing the OsIRL gene under an abscisic acid (ABA) inducible promoter. Results showed that the OsIRL transgenic rice line activated by ABA treatment was tolerant against MV and G/GO-induced stress in rice leave and suspension-cultured cells. Our results strongly suggest the involvement of OsIRL in homeostasis of ROS. OsIRL Ethylene is involved in the regulation of iron homeostasis by regulating the expression of iron-acquisition-related genes in Oryza sativa 2011 J Exp Bot State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, 310058, PR China. Plants employ two distinct strategies to obtain iron (Fe) from the soil. In Strategy I but not Strategy II plants, Fe limitation invokes ethylene production which regulates Fe deficiency responses. Oryza sativa (rice) is the only graminaceous plant described that possesses a Strategy I-like system for iron uptake as well as the classic Strategy II system. Ethylene production of rice roots was significantly increased when grown under Fe-depleted conditions. Moreover, 1-aminocyclopropane-1-carboxylic acid (ACC) treatment, a precursor of ethylene, conferred tolerance to Fe deficiency in rice by increasing internal Fe availability. Gene expression analysis of rice iron-regulated bHLH transcription factor OsIRO2, nicotianamine synthases 1 and 2 (NAS1 and NAS2), yellow-stripe like transporter 15 (YSL15) and iron-regulated transporter (IRT1) indicated that ethylene caused an increase in transcript abundance of both Fe (II) and Fe (III)-phytosiderophore uptake systems. RNA interference of OsIRO2 in transgenic rice showed that ethylene acted via this transcription factor to induce the expression of OsNAS1, OsNAS2, OsYSL15, and OsIRT1. By contrast, in Hordeum vulgare L. (barley), no ethylene production or ethylene-mediated effects of Fe response could be detected. In conclusion, Fe-limiting conditions increased ethylene production and signalling in rice, which is novel in Strategy II plant species. OsIRO2,OsIRT1,OsNAS1,OsNAS2,OsYSL15 The rice bHLH protein OsIRO2 is an essential regulator of the genes involved in Fe uptake under Fe-deficient conditions 2007 Plant J Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Iron (Fe) deficiency is a major abiotic stress in crop production. Although responses to Fe deficiency in graminaceous plants, such as increased production and secretion of mugineic acid family phytosiderophores (MAs), have been described, the gene regulation mechanisms related to these responses are largely unknown. To elucidate the regulation mechanisms of the genes related to Fe acquisition in graminaceous plants, we characterized the Fe-deficiency-inducible basic helix-loop-helix transcription factor OsIRO2 in rice. In yeast cells, OsIRO2 functioned as a transcriptional activator. In rice, overexpression of OsIRO2 resulted in increased MAs secretion, whereas repression of OsIRO2 resulted in lower MAs secretion and hypersensitivity to Fe deficiency. Northern blots revealed that the expression of the genes involved in the Fe(III)-MAs transport system was dependent on OsIRO2. The expression of the genes for nicotianamine synthase, a key enzyme in MAs synthesis, was notably affected by the level of OsIRO2 expression. Microarray analysis demonstrated that OsIRO2 regulates 59 Fe-deficiency-induced genes in roots. Some of these genes, including two transcription factors upregulated by Fe deficiency, possessed the OsIRO2 binding sequence in their upstream regions. OsIRO2 possesses a homologous sequence of the Fe-deficiency-responsive cis-acting elements (IDEs) in its upstream region. We propose a novel gene regulation network for Fe-deficiency responses, including OsIRO2, IDEs and the two transcription factors. OsIRO2 OsIRO2 is responsible for iron utilization in rice and improves growth and yield in calcareous soil 2011 Plant Mol Biol Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan. Iron (Fe) deficiency, a worldwide agricultural problem on calcareous soil with low Fe availability, is also a major human nutritional deficit. Plants induce Fe acquisition systems under conditions of low Fe availability. Previously, we reported that an Fe-deficiency-inducible basic helix-loop-helix (bHLH) transcription factor, OsIRO2, is responsible for regulation of the genes involved in Fe homeostasis in rice. Using promoter-GUS transformants, we showed that OsIRO2 is expressed throughout a plant's lifetime in a spatially and temporally similar manner to the genes OsNAS1, OsNAS2 and TOM1, which is involved in Fe absorption and translocation. During germination, OsIRO2 expression was detected in embryos. OsIRO2 expression in vegetative tissues was restricted almost exclusively to vascular bundles of roots and leaves, and to the root exodermis under Fe-sufficient conditions, and expanded to all tissues of roots and leaves in response to Fe deficiency. OsIRO2 expression was also detected in flowers and developing seeds. Plants overexpressing OsIRO2 grew better, and OsIRO2-repressed plants showed poor growth compared to non-transformant rice after germination. OsIRO2 overexpression also resulted in improved tolerance to low Fe availability in calcareous soil. In addition to increased Fe content in shoots, the overexpression plants accumulated higher amounts of Fe in seeds than non-transformants when grown on calcareous soil. These results suggest that OsIRO2 is synchronously expressed with genes involved in Fe homeostasis, and performs a crucial function in regulation not only of Fe uptake from soil but also Fe transport during germination and Fe translocation to grain during seed maturation. OsIRO2,OsNAS1,OsNAS2 Identification of a novel iron regulated basic helix-loop-helix protein involved in Fe homeostasis in Oryza sativa 2010 BMC Plant Biol Joint Research Laboratory in Genomics and Nutriomics, College of Life Sciences, Zhejiang University, Hangzhou 310058, PR China. BACKGROUND: Iron (Fe) is the most limiting micronutrient element for crop production in alkaline soils. A number of transcription factors involved in regulating Fe uptake from soil and transport in plants have been identified. Analysis of transcriptome data from Oryza sativa grown under limiting Fe conditions reveals that transcript abundances of several genes encoding transcription factors are altered by Fe availability. These transcription factors are putative regulators of Fe deficiency responses. RESULTS: Transcript abundance of one nuclear located basic helix-loop-helix family transcription factor, OsIRO3, is up-regulated from 25- to 90-fold under Fe deficiency in both root and shoot respectively. The expression of OsIRO3 is specifically induced by Fe deficiency, and not by other micronutrient deficiencies. Transgenic rice plants over-expressing OsIRO3 were hypersensitive to Fe deficiency, indicating that the Fe deficiency response was compromised. Furthermore, the Fe concentration in shoots of transgenic rice plants over-expressing OsIRO3 was less than that in wild-type plants. Analysis of the transcript abundances of genes normally induced by Fe deficiency in OsIRO3 over-expressing plants indicated their induction was markedly suppressed. CONCLUSION: A novel Fe regulated bHLH transcription factor (OsIRO3) that plays an important role for Fe homeostasis in rice was identified. The inhibitory effect of OsIRO3 over-expression on Fe deficiency response gene expression combined with hypersensitivity of OsIRO3 over-expression lines to low Fe suggest that OsIRO3 is a negative regulator of the Fe deficiency response in rice. OsIRO3 Rice plants take up iron as an Fe3+-phytosiderophore and as Fe2+ 2006 Plant J Department of Global Agricultural Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Only graminaceous monocots possess the Strategy II iron (Fe)-uptake system in which Fe is absorbed by roots as an Fe3+-phytosiderophore. In spite of being a Strategy II plant, however, rice (Oryza sativa) contains the previously identified Fe2+ transporter OsIRT1. In this study, we isolated the OsIRT2 gene from rice, which is highly homologous to OsIRT1. Real-time PCR analysis revealed that OsIRT1 and OsIRT2 are expressed predominantly in roots, and these transporters are induced by low-Fe conditions. When expressed in yeast (Saccharomyces cerevisiae) cells, OsIRT2 cDNA reversed the growth defects of a yeast Fe-uptake mutant. This was similar to the effect of OsIRT1 cDNA. OsIRT1- and OsIRT2-green fluorescent protein fusion proteins localized to the plasma membrane when transiently expressed in onion (Allium cepa L.) epidermal cells. OsIRT1 promoter-GUS analysis revealed that OsIRT1 is expressed in the epidermis and exodermis of the elongating zone and in the inner layer of the cortex of the mature zone of Fe-deficient roots. OsIRT1 expression was also detected in the ccompanion cells. Analysis using the positron-emitting tracer imaging system showed that rice plants are able to take up both an Fe3+-phytosiderophore and Fe2+. This result indicates that, in addition to absorbing an Fe3+-phytosiderophore, rice possesses a novel Fe-uptake system that directly absorbs the Fe2+, a strategy that is advantageous for growth in submerged conditions. OsIRT1,OsIRT2,OsZIP1,OsZIP3,OsZIP6 OsZIP4, a novel zinc-regulated zinc transporter in rice 2005 J Exp Bot Department of Global Agricultural Sciences, Graduate School of Agricultural and Life Science, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Zinc (Zn) is an essential element for the normal growth of plants but information is scarce on the mechanisms whereby Zn is transported in rice (Oryza sativa L.) plants. Four distinct genes, OsZIP4, OsZIP5, OsZIP6, and OsZIP7 that exhibit sequence similarity to the rice ferrous ion transporter, OsIRT1, were isolated. Microarray and northern blot analysis revealed that OsZIP4 was highly expressed under conditions of Zn deficiency in roots and shoots. Real-time-PCR revealed that the OsZIP4 transcripts were more abundant than those of OsZIP1 or OsZIP3 in Zn-deficient roots and shoots. OsZIP4 complemented a Zn-uptake-deficient yeast (Saccharomyces cerevisiae) mutant, Deltazrt1,Deltazrt2, indicating that OsZIP4 is a functional transporter of Zn. OsZIP4-synthetic green fluorescent protein (sGFP) fusion protein was transiently expressed in onion epidermal cells localized to the plasma membrane. In situ hybridization analysis revealed that OsZIP4 in Zn-deficient rice was expressed in shoots and roots, especially in phloem cells. Furthermore, OsZIP4 transcripts were detected in the meristem of Zn-deficient roots and shoots. These results suggested that OsZIP4 is a Zn transporter that may be responsible for the translocation of Zn within rice plants. OsIRT1,OsZIP4,OsZIP5,OsZIP1,OsZIP3,OsZIP6 Cloning an iron-regulated metal transporter from rice 2002 J Exp Bot Laboratory of Plant Molecular Physiology, Department of Applied Biological Chemistry, The University of Tokyo, 1‐1 Yayoi, Bunkyo‐ku, 113‐8657 Tokyo, Japan Rice cDNA and genomic libraries were screened in order to clone an Fe(II) transporter gene. A cDNA clone highly homologous to the Arabidopsis Fe(II) transporter gene IRT1 was isolated from Fe‐deficient rice roots. The cDNA clone was named OsIRT1. A genomic clone corresponding to the cDNA was also obtained, sequenced and analysed. When expressed in yeast cells, OsIRT1 cDNA reversed the growth defects of the yeast iron‐uptake mutant. Northern blot analysis revealed that OsIRT1 mRNA was predominantly expressed in roots and was induced by Fe‐ and Cu‐deficiency. This suggests that OsIRT1 is a functional metal transporter for iron, and is actively engaged in Fe uptake from soils, especially under limiting conditions. OsIRT1 Over-expression of OsIRT1 leads to increased iron and zinc accumulations in rice 2009 Plant Cell Environ Department of Life Science and Functional Genomic Center, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Korea. Uptake and translocation of micronutrients are essential for plant growth. These micronutrients are also important food components. We generated transgenic rice plants over-expressing OsIRT1 to evaluate its functional roles in metal homeostasis. Those plants showed enhanced tolerance to iron deficiency at the seedling stage. In paddy fields, this over-expression caused plant architecture to be altered. In addition, those plants were sensitive to excess Zn and Cd, indicating that OsIRT1 also transports those metals. As expected, iron and zinc contents were elevated in the shoots, roots and mature seeds of over-expressing plants. This demonstrates that OsIRT1 can be used for enhancing micronutrient levels in rice grains. OsIRT1 Iron deficiency enhances cadmium uptake and translocation mediated by the Fe2+transporters OsIRT1 and OsIRT2 in rice 2006 Soil Science and Plant Nutrition Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657 Cadmium (Cd) accumulation in rice grains is enhanced if ponded water is released from paddy fields during the reproductive stage (intermittent irrigation). The release of ponded water creates aerobic soil conditions under which Cd becomes soluble and iron (Fe) solubility decreases. We hypothesized that Fe shortage in rice induces Fe uptake and translocation and that Cd is also taken up and translocated throughout this process. Hydroponically cultured Fe-deficient rice absorbed more Cd than did Fe-sufficient rice, and the presence of Fe enhanced the translocation of Cd to the shoots. Yeast mutants expressing OsIRT1 and OsIRT2, which encode the rice Fe2+ transporter, became more sensitive to Cd, suggesting that Cd was absorbed by OsIRT1 and OsIRT2. We discuss the possibility that Cd accumulation in rice grains during the reproductive stage is mediated by the Fe transport system. OsIRT1,OsIRT2 Mutational reconstructed ferric chelate reductase confers enhanced tolerance in rice to iron deficiency in calcareous soil 2007 Proc Natl Acad Sci U S A Department of Global Agricultural Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Tokyo 113-8657, Japan. Iron (Fe) deficiency is a worldwide agricultural problem on calcareous soils with low-Fe availability due to high soil pH. Rice plants use a well documented phytosiderophore-based system (Strategy II) to take up Fe from the soil and also possess a direct Fe2+ transport system. Rice plants are extremely susceptible to low-Fe supply, however, because of low phytosiderophore secretion and low Fe3+ reduction activity. A yeast Fe3+ chelate-reductase gene refre1/372, selected for better performance at high pH, was fused to the promoter of the Fe-regulated transporter, OsIRT1, and introduced into rice plants. The transgene was expressed in response to a low-Fe nutritional status in roots of transformants. Transgenic rice plants expressing the refre1/372 gene showed higher Fe3+ chelate-reductase activity and a higher Fe-uptake rate than vector controls under Fe-deficient conditions. Consequently, transgenic rice plants exhibited an enhanced tolerance to low-Fe availability and 7.9x the grain yield of nontransformed plants in calcareous soils. This report shows that enhancing the Fe3+ chelate-reductase activity of rice plants that normally have low endogenous levels confers resistance to Fe deficiency. OsIRT1 Inactivation of OsIRX10 leads to decreased xylan content in rice culm cell walls and improved biomass saccharification 2013 Mol Plant Department of Plant Pathology and The Genome Center, University of California, Davis, CA 95616, USA None OsIRX10 Three Novel Rice Genes Closely Related to the Arabidopsis IRX9, IRX9L, and IRX14 Genes and Their Roles in Xylan Biosynthesis 2013 Front Plant Sci Department of Plant Pathology, The Genome Center, University of California Davis, CA, USA ; Joint BioEnergy Institute Emeryville, CA, USA. Xylan is the second most abundant polysaccharide on Earth, and represents a major component of both dicot wood and the cell walls of grasses. Much knowledge has been gained from studies of xylan biosynthesis in the model plant, Arabidopsis. In particular, the irregular xylem (irx) mutants, named for their collapsed xylem cells, have been essential in gaining a greater understanding of the genes involved in xylan biosynthesis. In contrast, xylan biosynthesis in grass cell walls is poorly understood. We identified three rice genes Os07g49370 (OsIRX9), Os01g48440 (OsIRX9L), and Os06g47340 (OsIRX14), from glycosyltransferase family 43 as putative orthologs to the putative beta-1,4-xylan backbone elongating Arabidopsis IRX9, IRX9L, and IRX14 genes, respectively. We demonstrate that the over-expression of the closely related rice genes, in full or partly complement the two well-characterized Arabidopsis irregular xylem (irx) mutants: irx9 and irx14. Complementation was assessed by measuring dwarfed phenotypes, irregular xylem cells in stem cross sections, xylose content of stems, xylosyltransferase (XylT) activity of stems, and stem strength. The expression of OsIRX9 in the irx9 mutant resulted in XylT activity of stems that was over double that of wild type plants, and the stem strength of this line increased to 124% above that of wild type. Taken together, our results suggest that OsIRX9/OsIRX9L, and OsIRX14, have similar functions to the Arabidopsis IRX9 and IRX14 genes, respectively. Furthermore, our expression data indicate that OsIRX9 and OsIRX9L may function in building the xylan backbone in the secondary and primary cell walls, respectively. Our results provide insight into xylan biosynthesis in rice and how expression of a xylan synthesis gene may be modified to increase stem strength. OsIRX14,OsIRX9,OsIRX9L Characterization of expression of the OsPUL gene encoding a pullulanase-type debranching enzyme during seed development and germination in rice 2009 Plant Physiol Biochem Yangzhou University, Jiangsu, China. Starch-debranching enzymes (DBEs) are key enzymes involved in starch metabolism in cereals, having a dual function, in both starch synthesis and degradation. However, their precise roles in this pathway, particularly their expression profiles, remain unclear. In the present study, we performed a quantitative real-time PCR (Q-PCR) analysis of the expression pattern of the OsPUL gene encoding a pullulanase-type DBE in different tissues as well as in seeds at different developmental stages. The results showed that this gene was expressed only in seeds. In addition, the 1177-bp OsPUL promoter sequence was cloned, and some endosperm-specific motifs such as the GCN4 and AACA motifs were observed to exist in this region. The promoter was then fused with the GUS reporter gene and its expression was carefully investigated in transgenic rice. The data from both histochemical and fluorometric analyses showed that the OsPUL promoter was capable of driving the target gene to have a high level of endosperm-specific expression. The OsPUL gene maintained a relatively high expression level during the entire period of seed development, and peaked in the middle and late stages. This observation was very consistent with that of the endogenous transcription analysis by Q-PCR. Furthermore, the seed germination experiment showed that the OsPUL promoter actively functions in the late stage of seed germination. The expression of the OsPUL gene was maintained at a significant level during the entire grain filling period and in the late stage of seed germination, which coincided with its involvement in starch anabolism and catabolism. OsISA1,OsPUL Structural and enzymatic characterization of the isoamylase1 homo-oligomer and the isoamylase1-isoamylase2 hetero-oligomer from rice endosperm 2006 Planta Department of Biological Production, Akita Prefectural University, 241-7 Kaidobata-Nishi, Shimoshinjyo-Nakano, Akita-city, 010-0195, Japan. The present study established that there are two distinct polymeric forms of isoamylase1 (ISA1) in rice endosperm: presumably a homo-pentamer of ISA1 and a hetero-hexamer composed of five ISA1 and one ISA2. The molecular sizes of the homo- and hetero-oligomers, which could be fractionated by hydrophobic chromatography, were approximately 420-480 and 510-550 kDa, respectively. The hetero-oligomer exhibited higher affinities for various branched polyglucans, especially for phytoglycogen, which had a K(m) value that was approximately 12 times lower relative to that with the homo-oligomer, although no marked differences were found in chain preferences for debranching of amylopectin and phytoglycogen between these forms. The hetero-oligomer was active even when incubated at 50 degrees C for 10 min, while the homo-multimer was completely inactivated at 40 degrees C in 10 min. When the ISA1 homo-oligomer was incubated with the ISA2 protein expressed in Escherichia coli and applied onto a nondenature polyacrylamide gel, additional debranching activity bands which were specific for the purified ISA1-ISA2 preparation were also detected, indicating that ISA1 and ISA2 combine to form a hetero-oligomer. These results suggest that the hetero-oligomer plays a predominant role in the amylopectin biosynthesis in rice endosperm although the homo-oligomer can complement the function of the hetero-oligomer at least to some extent. OsISA1,OsISA2,OsPUL Overexpression of a zinc-finger protein gene from rice confers tolerance to cold, dehydration, and salt stress in transgenic tobacco 2004 Proc Natl Acad Sci U S A Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110 021, India. Stress perception and signal transduction leading to tolerance involve a complex interplay of different gene products. We describe here the isolation and characterization of an intronless gene (OSISAP1) from rice encoding a zinc-finger protein that is induced after different types of stresses, namely cold, desiccation, salt, submergence, and heavy metals as well as injury. The gene is also induced by stress hormone abscisic acid. Overexpression of the gene in transgenic tobacco conferred tolerance to cold, dehydration, and salt stress at the seed-germination/seedling stage as reflected by the percentage of germination/green seedlings, the fresh weight of seedlings, and their developmental pattern. Thus, OSISAP1 seems to be an important determinant of stress response in plants. OSISAP1 Overexpression of OsiSAP8, a member of stress associated protein (SAP) gene family of rice confers tolerance to salt, drought and cold stress in transgenic tobacco and rice 2008 Plant Mol Biol Department of Plant Biotechnology, Madurai Kamaraj University, Madurai, 625021, TamilNadu, India. We describe here the isolation and characterization of OsiSAP8, a member of stress Associated protein (SAP) gene family from rice characterized by the presence of A20 and AN1 type Zinc finger domains. OsiSAP8 is a multiple stress inducible gene, induced by various stresses, namely heat, cold, salt, desiccation, submergence, wounding, heavy metals as well as stress hormone Abscisic acid. OsiSAP8 protein fused to GFP was localized towards the periphery of the cells in the epidermal cells of infiltrated Nicotiana benthamiana leaves. Yeast two hybrid analysis revealed that A20 and AN1 type zinc-finger domains of OsiSAP8 interact with each other. Overexpression of the gene in both transgenic tobacco and rice conferred tolerance to salt, drought and cold stress at seed germination/seedling stage as reflected by percentage of germination and gain in fresh weight after stress recovery. Transgenic rice plants were tolerant to salt and drought during anthesis stage without any yield penalty as compared to unstressed transgenic plants. OsiSAP8 Stage- and tissue-specific expression of rice OsIsu1 gene encoding a scaffold protein for mitochondrial iron-sulfur-cluster biogenesis 2009 Biotechnol Lett Asian Natural Environmental Science Center, The University of Tokyo, 1-1-1 Midori-cho, Nishitokyo-shi, Tokyo, 188-0002, Japan. Isu is a scaffold protein involved in mitochondrial iron-sulfur-cluster biogenesis, which affects redox and iron homeostasis in human and yeast cells. A BLASTP search identified two putative Isu genes in rice, and we designated one of them as OsIsu1. When expressed in onion epidermal cells, OsIsu1::GFP was localized to the mitochondria. Northern analysis showed that OsIsu1 was down-regulated in iron-deficient rice root. OsIsu1 promoter-GUS was introduced into Arabidopsis thaliana and histochemical GUS-staining showed that OsIsu1 expression was regulated in a stage- and tissue-specific manner. OsIsu1 was expressed ectopically in Arabidopsis under the control of the CaMV35S promoter, which increased weight of plants. OsIsu1 characterization of an inositol 1,3,4-trisphosphate 5/6-kinase gene that is essential for drought and salt stress responses in rice 2011 Plant Mol Biol National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, 430070 Wuhan, China. Drought and salt stresses are major limiting factors for crop production. To identify critical genes for stress resistance in rice (Oryza sativa L.), we screened T-DNA mutants and identified a drought- and salt-hypersensitive mutant dsm3. The mutant phenotype was caused by a T-DNA insertion in a gene encoding a putative inositol 1,3,4-trisphosphate 5/6-kinase previously named OsITPK2 with unknown function. Under drought stress conditions, the mutant had significantly less accumulation of osmolytes such as proline and soluble sugar and showed significantly reduced root volume, spikelet fertility, biomass, and grain yield; however, malondialdehyde level was increased in the mutant. Interestingly, overexpression of DSM3 (OsITPK2) in rice resulted in drought- and salt-hypersensitive phenotypes and physiological changes similar to those in the mutant. Inositol trisphosphate (IP3) level was decreased in the overexpressors under normal condition and drought stress. A few genes related to osmotic adjustment and reactive oxygen species scavenging were down-regulated in the mutant and overexpression lines. The expression level of DSM3 promoter-driven beta-glucuronidase (GUS) reporter gene in rice was induced by drought, salt and abscisic acid. Protoplast transient expression assay indicated that DSM3 is an endoplasmic reticulum protein. Sequence analysis revealed six putative ITPKs in rice. Transcript level analysis of OsITPK genes revealed that they had different tempo-spatial expression patterns, and the responses of DSM3 to abiotic stresses, including drought, salinity, cold, and high temperature, were distinct from the other five members in rice. These results together suggest that DSM3/OsITPK2 is an important member of the OsITPK family for stress responses, and an optimal expression level is essential for drought and salt tolerance in rice. OsITPK1 RNAi mediated silencing of a wall associated kinase, OsiWAK1 in Oryza sativa results in impaired root development and sterility due to anther indehiscence: Wall Associated Kinases from Oryza sativa 2011 Physiol Mol Biol Plants Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, 625021 TamilNadu India ; Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel. The Wall-Associated Kinase, one of the receptor-like kinase (RLK) gene families in plant, plays important roles in cell expansion, pathogen resistance and heavy metal stress tolerance in Arabidopsis thaliana. Here, we isolated a cDNA encoding a novel WAK from indica rice and designated as OsiWAK1 (Oryza sativa indica WAK-1). In this study, the RNAi construct with OsiWAK1 gene cloned in sense and antisense orientation separated by a functional intron under constitutive promoter, was introduced through biolistic gene gun method into the rice cultivar "IR-50" to determine the effect of OsiWAK1 transcript silencing on rice plant development. Examination of the transgenic plants reveals that OsiWAK1 transcript silencing in rice results in dwarf plants because of the reduction in the size of leaves, flag-leaves, internodes and panicle. The development of root primordia during germination, root hairs and lateral rooting was also effected. Microscopic analysis revealed that the decrease in size is due to reduction in the cell size but not the number of cells. In addition, the transgenic plants also exhibited sterile phenotype due to anther indehiscence and 40 % reduction in pollen viability. These data suggest that OsiWAK1 may play an important role in rice plant growth and development. OsiWAK1 Molecular cloning and functional characterization of OsJAG gene based on a complete-deletion mutant in rice (Oryza sativa L.) 2010 Plant Mol Biol Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fujian Agriculture and Forestry University, 350002, Fuzhou, Fujian, People's Republic of China. In this article, we report an independent work of positional cloning and functional characterization of OsJAG gene in rice. The merit of our work is that we used a genuine null mutant, in which the wild-type allele was completely deleted. This allowed us to identify the mutant phenotypes accurately without the interference of residual function of the target gene. OsJAG is an important gene with pleiotropy, expressing almost throughout the plant and acting in both vegetative phase and reproductive phase. But its main and crucial roles are in regulating the development of all floral organs, especially in specifying the identity of stamens. Interestingly, OsJAG does not affect the number of floral organ primordial and so of floral organs in each whorl, suggesting that OsJAG does not influence the initiation of floral organ primordia, but affect the developmental fate of all floral organs after their primordia have initiated. Loss of OsJAG function results in maldevelopment of all floral organs, such as degenerated lemma and palea, elongated lodicules and deformed and sterile pistil. The stamen appears to be more sensitive to the mutation. All the six stamens in a mutant floret were thoroughly transformed into six pistil-like organs developed at the presumptive positions of the stamens in whorl 3. SL1|OsJAG Rice open beak is a negative regulator of class 1 knox genes and a positive regulator of class B floral homeotic gene 2009 Plant J Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan. Numerous genes are involved in the regulation of plant development, including those that regulate floral homeotic genes, We identified two recessive allelic rice mutants, open beak-1 (opb-1) and opb-2, which exhibited pleiotropic defects in leaf morphogenesis, inflorescence architecture, and floral organ identity. Abnormal cell proliferation was observed in the leaves and spikelets, and ectopic or overexpression of several class 1 knox genes was detected; thus, the abnormal cell proliferation in opb mutants is probably caused by ectopic class 1 knox gene expression. The opb mutants also had defects in floral organ identity, resulting in the development of mosaic organs, including gluminous lodicules, staminoid lodicules, and pistiloid stamens. These results, together with the reduced expression of a class B gene, indicate that OPB positively regulates the expression of class B genes. Map-based cloning revealed that OPB encodes a transcription factor that is orthologous to the Arabidopsis JAGGED gene and is expressed in leaf primordia, inflorescence meristem, rachis branch meristems, floral meristem, and floral organ primordia. Taken together, our data suggest that the OPB gene affects cellular proliferation and floral organ identity through the regulation of class 1 knox genes and floral homeotic genes. SL1|OsJAG STAMENLESS 1, encoding a single C2H2 zinc finger protein, regulates floral organ identity in rice 2009 Plant J State Key Laboratory of Plant Genomics & National Plant Gene Research Centre (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing 100101, China. Floral organ identity is defined by organ homoetic genes whose coordinated expression is crucial with respect to the time and place of floral organ formation. Here, we report molecular cloning and characterization of the rice STAMENLESS 1 (SL1) gene that is involved in floral development. The sl1 mutant largely resembles the rice B-class gene mutant spw1; both exhibit homeotic conversions of lodicules and stamens to palea/lemma-like organs and carpels. Additionally, sl1 produces flowers with varied numbers of inner floral organs, and amorphous tissues without floral organ identity were frequently formed in whorls 3 and 4. We also show that SL1 specifies lodicule and stamen identities through positive transcriptional regulation of SPW1/OsMADS16 expression. SL1 encodes a member of the C2H2 family of zinc finger proteins, closely related to JAG of Arabidopsis. The functional divergence between SL1 and JAG implies that SL1 was co-opted for its distinctive roles in specification of floral organ identity in rice after the lineage split from Arabidopsis. SL1|OsJAG,OsMADS16|SPW1 OsJAR1 and OsJAR2 are jasmonyl-L-isoleucine synthases involved in wound- and pathogen-induced jasmonic acid signalling 2011 Biochem Biophys Res Commun Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. The synthesis of JA-Ile was catalysed by JA-Ile synthase, which is a member of the group I GH3 family of proteins. Here, we showed evidence that OsGH3.5 (OsJAR1) and OsGH3.3 (OsJAR2) are the functional JA-Ile synthases in rice, using recombinant proteins. The expression levels of OsJAR1 and OsJAR2 were induced in response to wounding with the concomitant accumulation of JA-Ile. In contrast, only the expression of OsJAR1 was associated with the accumulation of JA-Ile after blast infection. Our data suggest that these two JA-Ile synthases are differentially involved in the activation of JA signalling in response to wounding and pathogen challenge in rice. OsJar1|OsGH3.5|OsGH3-5,OsJAR2|OsGH3.3|OsGH3-3 OsJAR1 Contributes Mainly to Biosynthesis of the Stress-Induced Jasmonoyl-Isoleucine Involved in Defense Responses in Rice 2014 Bioscience, Biotechnology and Biochemistry Biotechnology Research Center, The University of Tokyo, Tokyo 113-8657, Japan. Jasmonate plays key roles in plant growth and stress responses, as in defense against pathogen attack. Jasmonoyl-isoleucine (JA-Ile), a major active form of jasmonates, is thought to play a pivotal role in plant defense responses, but the involvement of JA-Ile in rice defense responses, including phytoalexin production, remains largely unknown. Here we found that OsJAR1 contributes mainly to stress-induced JA-Ile production by the use of an osjar1 Tos17 mutant. The osjar1 mutant was impaired in JA-induced expression of JA-responsive genes and phytoalexin production, and these defects were restored genetically. Endogenous JA-Ile was indispensable to the production of a flavonoid phytoalexin, sakuranetin, but not to that of diterpenoid phytoalexins in response to heavy metal stress and the rice blast fungus. The osjar1 mutant was also found to be more susceptible to the blast fungus than the parental wild type. These results suggest that JA-Ile production makes a contribution to rice defense responses with a great impact on stress-induced sakuranetin production. OsJar1|OsGH3.5|OsGH3-5 Rice JASMONATE RESISTANT 1 is involved in phytochrome and jasmonate signalling 2008 Plant Cell Environ National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. michael.riemann@bio.uka.de Jasmonic acid (JA) is an important negative regulator of light-regulated coleoptile elongation in rice. We isolated rice JASMONATE RESISTANT 1 (osjar1) mutants from the Tos17 mutant panel by BLAST search. In far-red and blue lights, osjar1 coleoptiles were longer if compared with the wild type (WT), indicating that OsJar1 participates in the suppression of coleoptile elongation in these light conditions, while the mutant did not show a clear phenotype in red light. The analysis of OsJar1 expression in phytochrome (phy) mutants revealed that phytochrome A (phyA) and phytochrome B (phyB) act redundantly to induce this gene by red light, presumably. Unexpectedly, blue light-induced expression of OsJar1 gene was impaired in phyA-deficient mutants, indicating the involvement of phyA in the blue light signalling. In WT seedlings, OsJar1 transcripts were up-regulated transiently in response to treatment with exogenous methyl-jasmonic acid (MeJA). The dose-response curve of the MeJA treatment showed a characteristic pattern: concentrations as low as 4.5 nM could induce OsJar1 transcription, while the gene was superinduced at a concentration of 450 microM MeJA. In summary, this paper demonstrated that OsJar1 modulates light and JA signalling in the photomorphogenesis of rice. OsJar1|OsGH3.5|OsGH3-5,PHYA,PHYB|OsphyB Response of rice to insect elicitors and the role of OsJAR1 in wound and herbivory-induced JA-Ile accumulation 2013 J Integr Plant Biol Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan. Plants produce jasmonic acid (JA) and its amino acid conjugate, jasmonoyl-L-isoleucine (JA-Ile) as major defense signals in response to wounding and herbivory. In rice (Oryza sativa), JA and JA-Ile rapidly increased after mechanical damage, and this increase was further amplified when the wounds were treated with oral secretions from generalist herbivore larvae, lawn armyworms (Spodoptera mauritia), revealing for the first time active perception mechanisms of herbivore-associated elicitor(s) in rice. In the rice genome, two OsJAR genes can conjugate JA and Ile and form JA-Ile in vitro; however, their function in herbivory-induced accumulation of JA-Ile has not been investigated. By functional characterization of TOS17 retrotransposon-tagged Osjar1 plants and their response to simulated herbivory, we show that OsJAR1 is essential for JA-Ile production in herbivore-attacked, field-grown plants. In addition, OsJAR1 was required for normal seed development in rice under field conditions. Our results suggest that OsJAR1 possesses at least two major functions in rice defense and development that cannot be complemented by the additional OsJAR2 gene function, although this gene previously showed overlapping enzyme activity in vitro. OsJar1|OsGH3.5|OsGH3-5 Identification and expression profiling analysis of TIFY family genes involved in stress and phytohormone responses in rice 2009 Plant Mol Biol National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, 430070 Wuhan, China. The TIFY family is a novel plant-specific gene family involved in the regulation of diverse plant-specific biologic processes, such as development and responses to phytohormones, in Arabidopsis. However, there is limited information about this family in monocot species. This report identifies 20 TIFY genes in rice, the model monocot species. Sequence analysis indicated that rice TIFY proteins have conserved motifs beyond the TIFY domain as was previously shown in Arabidopsis. On the basis of their protein structures, members of the TIFY family can be divided into two groups. Transcript level analysis of OsTIFY genes in tissues and organs revealed different tempo-spatial expression patterns, suggesting that expression and function vary by stage of plant growth and development. Most of the OsTIFY genes were predominantly expressed in leaf. Nine OsTIFY genes were responsive to jasmonic acid and wounding treatments. Interestingly, almost all the OsTIFY genes were responsive to one or more abiotic stresses including drought, salinity, and low temperature. Over-expression of OsTIFY11a, one of the stress-inducible genes, resulted in significantly increased tolerance to salt and dehydration stresses. These results suggest that the OsTIFY family may have important roles in response to abiotic stresses. The data presented in this report provide important clues for further elucidating the functions of the genes in the OsTIFY family. OsJAZ1|OsTIFY3|EG2,OsJAZ12|OsTIFY11d,OsJAZ7|OsTIFY10b,OsJAZ8|OsTIFY10c,OsJAZ9|OsTIFY11a Methyl jasmonate reduces grain yield by mediating stress signals to alter spikelet development in rice 2009 Plant Physiol School of Biotechnology and Environmental Engineering, Myongji University, Yongin 449-728, Korea. Jasmonic acid (JA) is involved in plant development and the defense response. Transgenic overexpression of the Arabidopsis (Arabidopsis thaliana) jasmonic acid carboxyl methyltransferase gene (AtJMT) linked to the Ubi1 promoter increased levels of methyl jasmonate (MeJA) by 6-fold in young panicles. Grain yield was greatly reduced in Ubi1:AtJMT plants due to a lower numbers of spikelets and lower filling rates than were observed for nontransgenic (NT) controls. Ubi1:AtJMT plants had altered numbers of spikelet organs, including the lemma/palea, lodicule, anther, and pistil. The loss of grain yield and alteration in spikelet organ numbers were reproduced by treating NT plants with exogenous MeJA, indicating that increased levels of MeJA in Ubi1:AtJMT panicles inhibited spikelet development. Interestingly, MeJA levels were increased by 19-fold in young NT panicles upon exposure to drought conditions, resulting in a loss of grain yield that was similar to that observed in Ubi1:AtJMT plants. Levels of abscisic acid (ABA) were increased by 1.9- and 1.4-fold in Ubi1:AtJMT and drought-treated NT panicles, respectively. The ABA increase in Ubi1:AtJMT panicles grown in nondrought conditions suggests that MeJA, rather than drought stress, induces ABA biosynthesis under drought conditions. Using microarray and quantitative polymerase chain reaction analyses, we identified seven genes that were regulated in both Ubi1:AtJMT and drought-treated NT panicles. Two genes, OsJMT1 and OsSDR (for short-chain alcohol dehydrogenase), are involved in MeJA and ABA biosynthesis, respectively, in rice (Oryza sativa). Overall, our results suggest that plants produce MeJA during drought stress, which in turn stimulates the production of ABA, together leading to a loss of grain yield. OsJMT1,OsSDR Structure and function of florigen and the receptor complex 2013 Trends Plant Sci Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara 630-0192, Japan. In the 1930s, the flowering hormone, florigen, was proposed to be synthesized in leaves under inductive day length and transported to the shoot apex, where it induces flowering. More recently, generated genetic and biochemical data suggest that florigen is a protein encoded by the gene, FLOWERING LOCUS T (FT). A rice (Oryza sativa) FT homolog, Hd3a, interacts with the rice FD homolog, OsFD1, via a 14-3-3 protein. Formation of this tri-protein complex is essential for flowering promotion by Hd3a in rice. In addition, the multifunctionality of FT homologs, other than for flowering promotion, is an emerging concept. Here we review the structural and biochemical features of the florigen protein complex and discuss the molecular basis for the multifunctionality of FT proteins. OsKANADI1|OsKANADI2,OsBIP116b Expression of a gibberellin-induced leucine-rich repeat receptor-like protein kinase in deepwater rice and its interaction with kinase-associated protein phosphatase 1999 Plant Physiol Michigan State University-Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824-1312, USA. We identified in deepwater rice (Oryza sativa L.) a gene encoding a leucine-rich repeat receptor-like transmembrane protein kinase, OsTMK (O. sativa transmembrane kinase). The transcript levels of OsTMK increased in the rice internode in response to gibberellin. Expression of OsTMK was especially high in regions undergoing cell division and elongation. The kinase domain of OsTMK was enzymatically active, autophosphorylating on serine and threonine residues. A cDNA encoding a rice ortholog of a kinase-associated type 2C protein phosphatase (OsKAPP) was cloned. KAPPs are putative downstream components in kinase-mediated signal transduction pathways. The kinase interaction domain of OsKAPP was phosphorylated in vitro by the kinase domain of OsTMK. RNA gel-blot analysis indicated that the expression of OsTMK and OsKAPP was similar in different tissues of the rice plant. In protein-binding assays, OsKAPP interacted with a receptor-like protein kinase, RLK5 of Arabidopsis, but not with the protein kinase domains of the rice and maize receptor-like protein kinases Xa21 and ZmPK1, respectively. OsKAPP,OsTMK Rice shaker potassium channel OsKAT1 confers tolerance to salinity stress on yeast and rice cells 2007 Plant Physiol Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan. We screened a rice (Oryza sativa L. 'Nipponbare') full-length cDNA expression library through functional complementation in yeast (Saccharomyces cerevisiae) to find novel cation transporters involved in salt tolerance. We found that expression of a cDNA clone, encoding the rice homolog of Shaker family K(+) channel KAT1 (OsKAT1), suppressed the salt-sensitive phenotype of yeast strain G19 (Deltaena1-4), which lacks a major component of Na(+) efflux. It also suppressed a K(+)-transport-defective phenotype of yeast strain CY162 (Deltatrk1Deltatrk2), suggesting the enhancement of K(+) uptake by OsKAT1. By the expression of OsKAT1, the K(+) contents of salt-stressed G19 cells increased during the exponential growth phase. At the linear phase, however, OsKAT1-expressing G19 cells accumulated less Na(+) than nonexpressing cells, but almost the same K(+). The cellular Na(+) to K(+) ratio of OsKAT1-expressing G19 cells remained lower than nonexpressing cells under saline conditions. Rice cells overexpressing OsKAT1 also showed enhanced salt tolerance and increased cellular K(+) content. These functions of OsKAT1 are likely to be common among Shaker K(+) channels because OsAKT1 and Arabidopsis (Arabidopsis thaliana) KAT1 were able to complement the salt-sensitive phenotype of G19 as well as OsKAT1. The expression of OsKAT1 was restricted to internodes and rachides of wild-type rice, whereas other Shaker family genes were expressed in various organs. These results suggest that OsKAT1 is involved in salt tolerance of rice in cooperation with other K(+) channels by participating in maintenance of cytosolic cation homeostasis during salt stress and thus protects cells from Na(+). OsKAT1,OsAKT2 Unique features of two potassium channels, OsKAT2 and OsKAT3, expressed in rice guard cells 2013 PLoS One Department of Molecular Breeding, National Academy of Agricultural Science, Rural Development Administration, Suwon, Korea. Potassium is the most abundant cation and a myriad of transporters regulate K(+) homeostasis in plant. Potassium plays a role as a major osmolyte to regulate stomatal movements that control water utility of land plants. Here we report the characterization of two inward rectifying shaker-like potassium channels, OsKAT2 and OsKAT3, expressed in guard cell of rice plants. While OsKAT2 showed typical potassium channel activity, like that of Arabidopsis KAT1, OsKAT3 did not despite high sequence similarity between the two channel proteins. Interestingly, the two potassium channels physically interacted with each other and such interaction negatively regulated the OsKAT2 channel activity in CHO cell system. Furthermore, deletion of the C-terminal domain recovered the channel activity of OsKAT3, suggesting that the C-terminal region was regulatory domain that inhibited channel activity. Two homologous channels with antagonistic interaction has not been previously reported and presents new information for potassium channel regulation in plants, especially in stomatal regulation. OsKAT2,OsKAT3 Dynamic bridges--a calponin-domain kinesin from rice links actin filaments and microtubules in both cycling and non-cycling cells 2009 Plant Cell Physiol Institute of Botany 1 and Center for Functional Nanostructures (CFN), University of Karlsruhe, Kaiserstrasse 2, D-76131 Karlsruhe, Germany. Interaction and cross-talk between microtubules and actin microfilaments are important for the cell axis and polarity during plant cell growth and development, but little is known about the molecular components of this interaction. Plant kinesins with a calponin-homology domain (KCHs) were recently identified and associated with a putative role in microtubule-microfilament cross-linking. KCHs belong to a distinct branch of the minus end-directed kinesin subfamily and so far have only been identified in land plants including the mosses. Here we report the identification of a new KCH from rice (Oryza sativa), OsKCH1, and show that OsKCH1 is associated with cortical microtubules and actin microfilaments in vivo. Furthermore, OsKCH1 is shown to bind to micro-tubules and actin microfilaments in vitro in a domain-dependent way. Additionally, this unique type of kinesin is shown to oligomerize both in vivo and in vitro. These findings are discussed with respect to a general role for KCHs as linkers between actin filaments and microtubules in both cell elongation and division. OsKCH1 Heterologous expression of a rice syntaxin-related protein KNOLLE gene (OsKNOLLE) in yeast and its functional analysis in the role of abiotic stress 2011 Hereditas (Beijing) College of Life Sciences, China Jiliang University, Hangzhou, China. Syntaxin-related protein KNOLLE, a multifunctional protein family belonging to the SNARE superfamily, plays an important role in many physiological processes in plants. In order to understand the function of the syntaxin-related protein KNOLLE (OsKNOLLE) in rice (Oryza sativa), the CDS sequence of OsKNOLLE gene isolated from a japonica rice cultivar "Zhonghua 11" was fused into an expression vector pYX212 and transformed into the S. cerevisiae strain BY4741 by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. The transformants with OsKNOLLE showed better survival abilities than the transformants with the empty vectors based on their phenotypes in responses to different abiotic stresses such as salt, Cu2+, H2O2, Cd2+, and Hg2+. These data suggests that OsKNOLLE plays a crucial role in re-sponses to abiotic stresses. This experimental system sets up a method for studying functions of the OsKNOLLE gene in the future and clarifies the relationship between OsKNOLLE and abiotic stresses. OsKNOLLE A rice semi-dwarf gene, Tan-Ginbozu (D35), encodes the gibberellin biosynthesis enzyme, ent-kaurene oxidase 2004 Plant Mol Biol Bioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi 464-8601, Japan. A rice (Oryza sativa L.) semi-dwarf cultivar, Tan-Ginbozu (d35Tan-Ginbozu), contributed to the increase in crop productivity in Japan in the 1950s. Previous studies suggested that the semi-dwarf stature of d35Tan-Ginbozu is caused by a defective early step of gibberellin biosynthesis, which is catalyzed by ent-kaurene oxidase (KO). To study the molecular characteristics of d35Tan-Ginbozu, we isolated 5 KO-like (KOL) genes from the rice genome, which encoded proteins highly homologous to Arabidopsis and pumpkin KOs. The genes (OsKOL1 to 5) were arranged as tandem repeats in the same direction within a 120 kb sequence. Expression analysis revealed that OsKOL2 and OsKOL4 were actively transcribed in various organs, while OsKOL1 and OsKOL5 were expressed only at low levels; OsKOL3 may be a pseudogene. Sequence analysis and complementation experiments demonstrated that OsKOL2 corresponds to D35. Homozygote with null alleles of D35 showed a severe dwarf phenotype; therefore, d35Tan-Ginbozu is a weak allele of D35. Introduction of OsKOL4 into d35Tan-Ginbozu did not rescue its dwarf phenotype, indicating that OsKOL4 is not involved in GA biosynthesis. OsKOL4 and OsKOL5 are likely to take part in phytoalexin biosynthesis, because their expression was promoted by UV irradiation and/or elicitor treatment. Comparing d35Tan-Ginbozu with other high yielding cultivars, we discuss strategies to produce culm architectures suitable for high crop yield by decreasing GA levels. OsKOS1|OsKOL4|CYP701A8,OsKOS2|OsKOL5,D35|OsKOS3|OsKO2 The rice dwarf virus P2 protein interacts with ent-kaurene oxidases in vivo, leading to reduced biosynthesis of gibberellins and rice dwarf symptoms 2005 Plant Physiol Peking-Yale Joint Center for Plant Molecular Genetics and Agrobiotechnology, National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Science, Peking University, Beijing 100871, China. The mechanisms of viral diseases are a major focus of biology. Despite intensive investigations, how a plant virus interacts with host factors to cause diseases remains poorly understood. The Rice dwarf virus (RDV), a member of the genus Phytoreovirus, causes dwarfed growth phenotypes in infected rice (Oryza sativa) plants. The outer capsid protein P2 is essential during RDV infection of insects and thus influences transmission of RDV by the insect vector. However, its role during RDV infection within the rice host is unknown. By yeast two-hybrid and coimmunoprecipitation assays, we report that P2 of RDV interacts with ent-kaurene oxidases, which play a key role in the biosynthesis of plant growth hormones gibberellins, in infected plants. Furthermore, the expression of ent-kaurene oxidases was reduced in the infected plants. The level of endogenous GA1 (a major active gibberellin in rice vegetative tissues) in the RDV-infected plants was lower than that in healthy plants. Exogenous application of GA3 to RDV-infected rice plants restored the normal growth phenotypes. These results provide evidence that the P2 protein of RDV interferes with the function of a cellular factor, through direct physical interactions, that is important for the biosynthesis of a growth hormone leading to symptom expression. In addition, the interaction between P2 and rice ent-kaurene oxidase-like proteins may decrease phytoalexin biosynthesis and make plants more competent for virus replication. Moreover, P2 may provide a novel tool to investigate the regulation of GA metabolism for plant growth and development. OsKOS1|OsKOL4|CYP701A8,OsKOS2|OsKOL5,D35|OsKOS3|OsKO2,OsKOS4|OsKO1 CYP701A8: a rice ent-kaurene oxidase paralog diverted to more specialized diterpenoid metabolism 2012 Plant Physiol Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA. All higher plants contain an ent-kaurene oxidase (KO), as such a cytochrome P450 (CYP) 701 family member is required for gibberellin (GA) phytohormone biosynthesis. While gene expansion and functional diversification of GA-biosynthesis-derived diterpene synthases into more specialized metabolism has been demonstrated, no functionally divergent KO/CYP701 homologs have been previously identified. Rice (Oryza sativa) contains five CYP701A subfamily members in its genome, despite the fact that only one (OsKO2/CYP701A6) is required for GA biosynthesis. Here we demonstrate that one of the other rice CYP701A subfamily members, OsKOL4/CYP701A8, does not catalyze the prototypical conversion of the ent-kaurene C4alpha-methyl to a carboxylic acid, but instead carries out hydroxylation at the nearby C3alpha position in a number of related diterpenes. In particular, under conditions where OsKO2 catalyzes the expected conversion of ent-kaurene to ent-kaurenoic acid required for GA biosynthesis, OsKOL4 instead efficiently reacts with ent-sandaracopimaradiene and ent-cassadiene to produce the corresponding C3alpha-hydroxylated diterpenoids. These compounds are expected intermediates in biosynthesis of the oryzalexin and phytocassane families of rice antifungal phytoalexins, respectively, and can be detected in rice plants under the appropriate conditions. Thus, it appears that OsKOL4 plays a role in the more specialized diterpenoid metabolism of rice, and our results provide evidence for divergence of a KO/CYP701 family member from GA biosynthesis. This further expands the range of enzymes recruited from the ancestral GA primary pathway to the more complex and specialized labdane-related diterpenoid metabolic network found in rice. OsKOS1|OsKOL4|CYP701A8,D35|OsKOS3|OsKO2 Functional Identification of a Riceent-Kaurene Oxidase, OsKO2, Using thePichia pastorisExpression System 2014 Bioscience, Biotechnology and Biochemistry Biotechnology Research Center, The University of Tokyo, Tokyo, Japan. Rice ent-kaurene oxidase 2 (OsKO2) perhaps functions in the early steps of gibberellin biosynthesis. We found that microsomes from the methylotropic yeast Pichia pastoris expressing both OsKO2 and a fungal cytochrome P450 monooxygenase (P450) reductase converted ent-kaurene to ent-kaurenoic acid. This is direct evidence that OsKO2 is involved in the sequential oxidation of ent-kaurene to ent-kaurenoic acid in gibberellin biosynthesis in rice. D35|OsKOS3|OsKO2 Isolation and characterization of a Ds-tagged rice (Oryza sativa L.) GA-responsive dwarf mutant defective in an early step of the gibberellin biosynthesis pathway 2005 Plant Cell Rep Plant Industry, Commonwealth Scientific and Industrial Research Organization (CSIRO), GPO Box 1600, Canberra, ACT 2601, Australia. We have isolated a severe dwarf transposon (Ds) insertion mutant in rice (Oryza sativa L.), which could be differentiated early in the seedling stage by reduced shoot growth and dark green leaves, and later by severe dwarfism and failure to initiate flowering. These mutants, however, showed normal seed germination and root growth. One of the sequences flanking Ds, rescued from the mutant, was of a chromosome 4-located putative ent-kaurene synthase (KS) gene, encoding the enzyme catalyzing the second step of the gibberellin (GA) biosynthesis pathway. Dwarf mutants were always homozygous for this Ds insertion and no normal plants homozygous for this mutation were recovered in the segregating progeny, indicating that the Ds insertion mutation is recessive. As mutations in three recently reported rice GA-responsive dwarf mutant alleles and the dwarf mutation identified in this study mapped to the same locus, we designate the corresponding gene OsKS1. The osks1 mutant seedlings were responsive to exogenous gibberellin (GA3). OsKS1 transcripts of about 2.3 kb were detected in leaves and stem of wild-type plants, but not in germinating seeds or roots, suggesting that OsKS1 is not involved in germination or root growth. There are at least five OsKS1-like genes in the rice genome, four of which are also represented in rice expressed sequence tag (EST) databases. All OsKS1-like genes are transcribed with different expression patterns. ESTs corresponding to all six OsKS genes are represented in other cereal databases including barley, wheat and maize, suggesting that they are biologically active. OsKS1 The rice OsLOL2 gene encodes a zinc finger protein involved in rice growth and disease resistance 2007 Mol Genet Genomics State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100080, People's Republic of China. Arabidopsis LSD1-related proteins that contain LSD1-like zinc finger domains have been identified to be involved in disease resistance and programmed cell death. To investigate the potential role of LSD1-related gene in rice (Oryza sativa L.), we cloned an LSD1 ortholog, OsLOL2, from the rice cDNA plasmid library. The OsLOL2 gene is predicted to encode a polypeptide of 163 amino acids with two LSD1-like zinc finger domains with 74.5% identity to those of LSD1. Southern blot analysis indicated that OsLOL2 was a single-copy gene in the rice genome. Transgenic rice lines carrying the antisense strand of OsLOL2 with decreased expression of OsLOL2 had dwarf phenotypes, and the dwarfism could be restored by exogenous GA(3) treatment, suggesting that the dwarfism was the result of a deficiency in bioactive gibberellin (GA). In agreement with this possibility, the content of endogenous bioactive GA(1) decreased in the antisense transgenic lines. Expression of OsKS1, one of the genes encoding for GA biosynthetic enzymes, was suppressed in the antisense transgenic lines. Sense transgenic lines with increased expression of OsLOL2 were more resistant to rice bacterial blight, while antisense transgenic lines were less resistant to rice bacterial blight. The OsLOL2-GFP (green fluorescence protein) fusion protein was localized in the nucleus of cells of transgenic BY2 tobacco (Nicotiana tabacum L.). These data suggest that OsLOL2 is involved in rice growth and disease resistance. OsKS1,OsLOL2 Diterpene Cyclases Responsible for the Biosynthesis of Phytoalexins, Momilactones A, B, and Oryzalexins A–F in Rice 2014 Bioscience, Biotechnology and Biochemistry Department of Bioresource Engineering, Yamagata University Rice (Oryza sativa L.) produces diterpene phytoalexins, such as momilactones, oryzalexins, and phytocassanes. Using rice genome information and in vitro assay with recombinant enzymes, we identified genes (OsKS4 and OsKS10) encoding the type-A diterpene cyclases 9beta-pimara-7,15-diene synthase and ent-sandaracopimaradiene synthase which are involved in the biosynthesis of momilactones A, B and oryzalexins A–F respectively. Transcript levels of these two genes increased remarkably after ultraviolet (UV) treatment, which is consistent with elevated production of phytoalexins by UV. These two genes might prove powerful tools for understanding plant defense mechanisms in rice. OsKS10,OsDTS2|OsKSL4|OsKS4 An unexpected diterpene cyclase from rice: functional identification of a stemodene synthase 2006 Arch Biochem Biophys Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA. We have cloned a novel diterpene synthase (OsKSL11) from rice that produces stemod-13(17)-ene from syn-copalyl diphosphate. Notably, this gene sequence was not predicted from the extensive sequence information available for rice, nor, despite extensive phytochemical investigations, has this diterpene or any derived natural product previously been reported in rice plants. OsKSL11 represents the first identified stemodene synthase, which catalyzes the committed step in biosynthesis of the stemodane family of diterpenoid natural products, some of which possess antiviral activity. In addition, OsKSL11 is highly homologous to the mechanistically similar stemarene synthase recently identified from rice, making this pair of diterpene cyclases an excellent model system for investigating the enzymatic determinants for differential product outcome. The unexpected nature of this cyclase and its product parallels recent observations of previously unrecognized natural products metabolism in Arabidopsis thaliana, suggesting that many, if not all, plant species will prove to have extensive biosynthetic capacity. OsDTC2|OsKSL11 Stemar-13-ene synthase, a diterpene cyclase involved in the biosynthesis of the phytoalexin oryzalexin S in rice 2004 FEBS Lett Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. In suspension-cultured rice cells, diterpenoid phytoalexins are produced in response to exogenously applied elicitors. We isolated a cDNA encoding a diterpene cyclase, OsDTC2, from suspension-cultured rice cells treated with a chitin elicitor. The OsDTC2 cDNA was overexpressed in Escherichia coli as a fusion protein with glutathione S-transferase, and the recombinant OsDTC2 was indicated to function as stemar-13-ene synthase that converted syn-copalyl diphosphate to stemar-13-ene, a putative diterpene hydrocarbon precursor of the phytoalexin oryzalexin S. The level of OsDTC2 mRNA in suspension-cultured rice cells began to increase 3 h after addition of the elicitor and reached the maximum after 8 h. The expression of OsDTC2 was also induced in UV-irradiated rice leaves. In addition, we indicated that stemar-13-ene accumulated in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves. OsDTC2|OsKSL11 Promoter analysis of the rice stemar-13-ene synthase gene OsDTC2, which is involved in the biosynthesis of the phytoalexin oryzalexin S 2007 Biochim Biophys Acta Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. A rice diterpene cyclase, OsDTC2, functions as a stemar-13-ene synthase that converts syn-copalyl diphosphate into stemar-13-ene, a putative diterpene hydrocarbon precursor of the phytoalexin oryzalexin S. The transcriptional expression of OsDTC2 is induced by treatment of suspension-cultured rice cells with a chitin oligosaccharide elicitor. To investigate the molecular mechanisms of the elicitor signaling pathway that leads to OsDTC2 expression, we carried out deletion and mutation analysis of the region -1939 bp upstream of the transcription start site of OsDTC2 in rice cells using dual luciferase assays. The region between -1709 and -1450 bp was found to contain six W-box motifs, which are putative recognition sites for WRKY transcription factors, as cis elements involved in elicitor-responsiveness and/or basic promoter activity of OsDTC2. OsDTC2|OsKSL11 OsKu70 is associated with developmental growth and genome stability in rice 2010 Plant Physiol Department of Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea. The cellular functions of Ku70 in repair of DNA double-stranded breaks and telomere regulation have been described in a wide range of organisms. In this study, we identified the rice (Oryza sativa) Ku70 homolog (OsKu70) from the rice genome database. OsKu70 transcript was detected constitutively in every tissue and developmental stage examined and also in undifferentiated callus cells in rice. Yeast two-hybrid and in vitro pull-down experiments revealed that OsKu70 physically interacts with OsKu80. We obtained loss-of-function osku70 T-DNA knockout mutant lines and constructed transgenic rice plants that overexpress the OsKu70 gene in the sense (35S:OsKu70) or antisense (35S:anti-OsKu70) orientation. The homozygous G2 osku70 mutant lines were more sensitive than wild-type plants to a DNA-damaging agent (0.01%-0.05% methyl-methane sulfonate), consistent with the notion that OsKu70 participates in the DNA repair mechanism. Terminal restriction fragment analysis revealed that telomeres in homozygous G2 osku70 mutants were markedly longer (10-20 kb) than those in wild-type plants (5-10 kb), whereas telomere length in heterozygous G2 osku70 mutant and T2 OsKu70-overexpressing transgenic (35S:OsKu70) rice resembled that of the wild-type plant. In contrast to what was observed in Arabidopsis (Arabidopsis thaliana) atku70 mutants, homozygous G2 osku70 rice plants displayed severe developmental defects in both vegetative and reproductive organs under normal growth conditions, resulting in sterile flowers. Analysis of meiotic progression in pollen mother cells demonstrated that up to 11.1% (seven of 63) of G2 mutant anaphase cells displayed one or more chromosomal fusions. These results suggest that OsKu70 is required for the maintenance of chromosome stability and normal developmental growth in rice plants. OsKu70 Suppression of Ku70/80 or Lig4 leads to decreased stable transformation and enhanced homologous recombination in rice 2012 New Phytol Plant Genome Engineering Research Unit, National Institute of Agrobiological Sciences, Ibaraki, Japan. Evidence for the involvement of the nonhomologous end joining (NHEJ) pathway in Agrobacterium-mediated transferred DNA (T-DNA) integration into the genome of the model plant Arabidopsis remains inconclusive. Having established a rapid and highly efficient Agrobacterium-mediated transformation system in rice (Oryza sativa) using scutellum-derived calli, we examined here the involvement of the NHEJ pathway in Agrobacterium-mediated stable transformation in rice. Rice calli from OsKu70, OsKu80 and OsLig4 knockdown (KD) plants were infected with Agrobacterium harboring a sensitive emerald luciferase (LUC) reporter construct to evaluate stable expression and a green fluorescent protein (GFP) construct to monitor transient expression of T-DNA. Transient expression was not suppressed, but stable expression was reduced significantly, in KD plants. Furthermore, KD-Ku70 and KD-Lig4 calli exhibited an increase in the frequency of homologous recombination (HR) compared with control calli. In addition, suppression of OsKu70, OsKu80 and OsLig4 induced the expression of HR-related genes on treatment with DNA-damaging agents. Our findings suggest strongly that NHEJ is involved in Agrobacterium-mediated stable transformation in rice, and that there is a competitive and complementary relationship between the NHEJ and HR pathways for DNA double-strand break repair in rice. OsKu80|Ku80,OsLig4|Lig4 Metabolomic screening applied to rice FOX Arabidopsis lines leads to the identification of a gene-changing nitrogen metabolism 2010 Mol Plant RIKEN Plant Science Center, Tsurumi-ku, Yokohama, Japan. Plant metabolomics developed as a powerful tool to examine gene functions and to gain deeper insight into the physiology of the plant cell. In this study, we screened Arabidopsis lines overexpressing rice full-length (FL) cDNAs (rice FOX Arabidopsis lines) using a gas chromatography-time-of-flight mass spectrometry (GC-TOF/MS)-based technique to identify rice genes that caused metabolic changes. This screening system allows fast and reliable identification of candidate lines showing altered metabolite profiles. We performed metabolomic and transcriptomic analysis of a rice FOX Arabidopsis line that harbored the FL cDNA of the rice ortholog of the Lateral Organ Boundaries (LOB) Domain (LBD)/Asymmetric Leaves2-like (ASL) gene of Arabidopsis, At-LBD37/ASL39. The investigated rice FOX Arabidopsis line showed prominent changes in the levels of metabolites related to nitrogen metabolism. The transcriptomic data as well as the results from the metabolite analysis of the Arabidopsis At-LBD37/ASL39-overexpressor plants were consistent with these findings. Furthermore, the metabolomic and transcriptomic analysis of the Os-LBD37/ASL39-overexpressing rice plants indicated that Os-LBD37/ASL39 is associated with processes related to nitrogen metabolism in rice. Thus, the combination of a metabolomics-based screening method and a gain-of-function approach is useful for rapid characterization of novel genes in both Arabidopsis and rice. Os-LBD37 Overexpression of microRNA OsmiR397 improves rice yield by increasing grain size and promoting panicle branching 2013 Nat Biotechnol Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory for Biocontrol, School of Life Science, Sun Yat-Sen University, Guangzhou, PR China. Increasing grain yields is a major focus of crop breeders around the world. Here we report that overexpression of the rice microRNA (miRNA) OsmiR397, which is naturally highly expressed in young panicles and grains, enlarges grain size and promotes panicle branching, leading to an increase in overall grain yield of up to 25% in a field trial. To our knowledge, no previous report has shown a positive regulatory role of miRNA in the control of plant seed size and grain yield. We determined that OsmiR397 increases grain yield by downregulating its target, OsLAC, whose product is a laccase-like protein that we found to be involved in the sensitivity of plants to brassinosteroids. As miR397 is highly conserved across different species, our results suggest that manipulating miR397 may be useful for increasing grain yield not only in rice but also in other cereal crops. OsLAC Cloning and characterization of two rice long-chain base kinase genes and their function in disease resistance and cell death 2013 Mol Biol Rep State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, Zhejiang, People's Republic of China. Sphingolipid metabolites such as long-chain base 1-phosphates (LCBPs) have been shown to play an important role in plants; however, little is known about their function in plant disease resistance and programmed cell death (PCD). In the present study, we cloned and identified two rice long-chain base kinase (LCBK) genes (OsLCBK1 and OsLCBK2), which are involved in biosynthesis of LCBPs, and performed functional analysis in transgenic tobacco. Expression of OsLCBK1 and OsLCBK2 was induced in rice seedlings after treatments with defense signaling molecules and after infection by Magnaporthe grisea, the causal agent of blast disease. Transgenic tobacco plants overexpressing OsLCBK1 were generated and disease resistance assays indicate that the OsLCBK1-overexpressing plants showed enhanced disease resistance against Pseudmonas syringae pv. tabacci, the causal agent of wildfire disease, and tobacco mosaic virus. Expression levels of some defense-related genes were constitutively up-regulated and further induced after pathogen infection in the OsLCBK1-overexpressing plants. Treatment with fungal toxin fumonisin B1, an effective inducer of PCD in plants, resulted in reduced level of cell death in the OsLCBK1-overexpressing plants, as indicated by cell death staining, leakage of electrolyte and expression of hypersensitive response indicator genes. These data suggest that rice LCBKs, probably through regulation of endogenous LCBP level, play important roles in disease resistance response and PCD in plants. OsLCBK1,OsLCBK2 Low cadmium (LCD), a novel gene related to cadmium tolerance and accumulation in rice 2011 J Exp Bot Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan. The contamination of food crops by cadmium (Cd) is a major concern in food production because it can reduce crop yields and threaten human health. In this study, knockout rice plants (Oryza sativa) tagged with the gene trap vector pGA2707 were screened for Cd tolerance, and the tolerant line lcd was obtained. The lcd mutant showed tolerance to Cd on agar plates and in hydroponic culture during early plant development. Metal concentration measurements in hydroponically grown plants revealed significantly less Cd in the shoots of lcd plants compared with wild-type (WT) shoots. When cultured in the field in soil artificially contaminated with low levels of Cd, lcd showed no significant difference in the Cd content of its leaf blades; however, the Cd concentration in the grains was 55% lower in 2009 and 43% lower in 2010. There were no significant differences in plant dry weight or seed yield between lcd and wild-type plants. LCD, a novel gene, is not homologous to any other known gene. LCD localized to the cytoplasm and nucleus, and was expressed mainly in the vascular tissues in the roots and phloem companion cells in the leaves. These data indicate that lcd may be useful for understanding Cd transport mechanisms and is a promising candidate rice line for use in combating the threat of Cd to human health. OsLCD Low-affinity cation transporter (OsLCT1) regulates cadmium transport into rice grains 2011 Proc Natl Acad Sci U S A Biotechnology Research Center and Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. Accumulation of cadmium (Cd) in rice (Oryza sativa L.) grains poses a potential health problem, especially in Asia. Most Cd in rice grains accumulates through phloem transport, but the molecular mechanism of this transport has not been revealed. In this study, we identified a rice Cd transporter, OsLCT1, involved in Cd transport to the grains. OsLCT1-GFP was localized at the plasma membrane in plant cells, and OsLCT1 showed Cd efflux activity in yeast. In rice plants, strong OsLCT1 expression was observed in leaf blades and nodes during the reproductive stage. In the uppermost node, OsLCT1 transcripts were detected around large vascular bundles and in diffuse vascular bundles. RNAi-mediated knockdown of OsLCT1 did not affect xylem-mediated Cd transport but reduced phloem-mediated Cd transport. The knockdown plants of OsLCT1 accumulated approximately half as much Cd in the grains as did the control plants. The content of other metals in rice grains and plant growth were not negatively affected by OsLCT1 suppression. These results suggest that OsLCT1 functions at the nodes in Cd transport into grains and that in a standard japonica cultivar, the regulation of OsLCT1 enables the generation of "low-Cd rice" without negative effects on agronomical traits. These findings identify a transporter gene for phloem Cd transport in plants. OsLCT1 Reaction specificities of the epsilon-ionone-forming lycopene cyclase from rice (Oryza sativa) elucidated in vitro 2012 FEBS Lett Faculty of Biology, Centre for Biological Signaling Studies (BIOSS), University of Freiburg, D-79104 Freiburg, Germany. Lycopene cyclases responsible for the formation of epsilon-ionone rings (LCYe) mark a plant-specific bifurcation of carotenogenesis. We investigated purified rice LCYe (OsLCYe) in a liposome-based biphasic assay system. OsLCYe depends on reduced flavin cofactors stabilizing a transient state formed during the non-redox cyclization reaction. In contrast to OsLCYb, OsLCYe produces predominantly monocyclic products and monocyclic carotene intermediates are not suitable substrates. Determination of the OsLCYe reaction specificities and the combined use of OsLCYb allow the characterization of the reaction sequence leading to heterocyclic carotenoids. It was also found that 5-cis-lycopene, which was thought to be decisive for epsilon-cyclization, was not involved in the reaction, with OsLCYe acting as an exclusion filter for this naturally occurring isomer. OsLCYe|OsLCYepsilon Increased polyamine biosynthesis enhances stress tolerance by preventing the accumulation of reactive oxygen species: T-DNA mutational analysis of Oryza sativa lysine decarboxylase-like protein 1 2012 Mol Cells Department of Biology, Sunchon National University, Sunchon, 540-742, Korea. A highly oxidative stress-tolerant japonica rice line was isolated by T-DNA insertion mutation followed by screening in the presence of 50 mM H(2)O(2). The T-DNA insertion was mapped to locus Os09g0547500, the gene product of which was annotated as lysine decarboxylase-like protein (GenBank accession No. AK062595). We termed this gene OsLDC-like 1, for Oryza sativa lysine decarboxylase-like 1. The insertion site was in the second exon and resulted in a 27 amino acid N-terminal deletion. Despite this defect in OsLDC-like 1, the mutant line exhibited enhanced accumulation of the polyamines (PAs) putrescine, spermidine, and spermine under conditions of oxidative stress. The generation of reactive oxygen species (ROS) in the mutant line was assessed by qRT-PCR analysis of NADPH oxidase (RbohD and RbohF), and by DCFH-DA staining. Cellular levels of ROS in osldc-like 1 leaves were significantly lower than those in the wild-type (WT) rice after exposure to oxidative, high salt and acid stresses. Exogenously-applied PAs such as spermidine and spermine significantly inhibited the stress-induced accumulation of ROS and cell damage in WT leaves. Additionally, the activities of ROS-detoxifying enzymes were increased in the homozygous mutant line in the presence or absence of H(2)O(2). Thus, mutation of OsLDC-like 1 conferred an oxidative stress-tolerant phenotype. These results suggest that increased cellular PA levels have a physiological role in preventing stress-induced ROS and ethylene accumulation and the resultant cell damage. OsLDC-like1 OsLEA1a, a new Em-like protein of cereal plants 2010 Plant Cell Physiol Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan, ROC. Proteins abundant in seeds during the late stages of development, late embryogenesis abundant (LEA) proteins, are associated with desiccation tolerance. More than 100 of the group I LEA genes, also termed Em genes, have been identified from plants, bacteria and animals. The wide distribution indicates the functional importance of these genes. In the present study, we characterized a novel Em-like gene, OsLEA1a of rice (Oryza sativa). The encoded OsLEA1a protein has an N-terminal sequence similar to that of other plant Em proteins but lacks a 20-mer motif that is the most significant feature of typical Em proteins. The location of the sole intron indicates that the second exon of OsLEA1a is the mutated product of a typical Em gene. Transcriptome analysis revealed OsLEA1a mainly expressed in embryos, with no or only a few transcripts in osmotic stress-treated vegetative tissues. Structural analysis revealed that the OsLEA1a protein adopts high amounts of disordered conformations in solution and undergoes desiccation-induced conformational changes. Macromolecular interaction studies revealed that OsLEA1a protein interacts with non-reducing sugars and phospholipids but not poly-l-lysine. Thus, although the OsLEA1a protein lost its 20-mer motif, it is still involved in the formation of bioglasses with non-reducing sugars or plasma membrane. However, the protein does not function as a chaperone as do other groups of hydrophilic LEA proteins. The orthologs of the OsLEA1a gene had been identified from various grasses but not in dicot plants. Genetic analysis indicated that rice OsLEA1a locates at a 193 kb segment in chromosome 1 and is conserved in several published cereal genomes. Thus, the ancestor of Em-like genes might have evolved after the divergence of monocot plants. OsLEA1a OsLEA3-2, an abiotic stress induced gene of rice plays a key role in salt and drought tolerance 2012 PLoS One Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People's Republic of China. Late embryogenesis abundant (LEA) proteins are involved in tolerance to drought, cold and high salinity in many different organisms. In this report, a LEA protein producing full-length gene OsLEA3-2 was identified in rice (Oryza sativa) using the Rapid Amplification of cDNA Ends (RACE) method. OsLEA3-2 was found to be only expressed in the embryo and can be induced by abiotic stresses. The coding protein localizes to the nucleus and overexpression of OsLEA3-2 in yeast improved growth performance compared with control under salt- and osmotic-stress conditions. OsLEA3-2 was also inserted into pHB vector and overexpressed in Arabidopsis and rice. The transgenic Arabidopsis seedlings showed better growth on MS media supplemented with 150 mM mannitol or 100 mM NaCl as compared with wild type plants. The transgenic rice also showed significantly stronger growth performance than control under salinity or osmotic stress conditions and were able to recover after 20 days of drought stress. In vitro analysis showed that OsLEA3-2 was able to protect LDH from aggregation on freezing and inactivation on desiccation. These results indicated that OsLEA3-2 plays an important role in tolerance to abiotic stresses. OsLEA3-2 A group 3 LEA cDNA of rice, responsive to abscisic acid, but not to jasmonic acid, shows variety-specific differences insaltstressresponse 1997 Gene Laboratorium voor Genetica, Department of Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), Universiteit Gent, Belgium. A cDNA clone oslea3, encoding a group 3 late-embryogenesis abundant (LEA) protein was isolated from roots of rice seedlings (Oryza sativa L.). The encoded OSLEA3 protein has previously been found to accumulate to higher levels in roots of two salt-tolerant compared to a salt-sensitive rice variety in response to abscisic acid (ABA) [Moons et al., 1995. Plant Physiol. 107, 177-186]. The OSLEA3 protein (Mr 20.5, pI 6.5) characteristically contains ten imperfect 11-mer amino acid repeats. Exogenous application of ABA and exposure to salt shock (150 mM NaCl) rapidly induces a de novo, abundant oslea3 transcript accumulation in seedling roots, whereas application of jasmonic acid (9 microM) does not induce oslea3 expression. The stress-induced oslea3 transcript gradually declined upon prolonged salt shock, as wilting-induced damage became irreversible. oslea3 expression was compared for the salt-tolerant variety Pokkali and the salt-sensitive cultivar Taichung N1. Higher maximal mRNA levels were found in roots of the tolerant variety, also declining less rapidly upon sustained salt shock, concomitant with a delayed drop in shoot water content. DNA blot analysis indicated the existence of a small oslea3 gene family in rice with an equal gene number in both ecotypes. The results suggest that a differential regulation of oslea3 expression is an aspect of the varietal differences in salt stress tolerance. OsLEA3|OsLEA3-1 The abiotic stress-responsive NAC-type transcription factor OsNAC5 regulates stress-inducible genes and stress tolerance in rice 2010 Mol Genet Genomics Laboratory of Plant Molecular Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan. The transcription factor OsNAC5 in rice is a member of the plant-specific NAC family that regulates stress responses. Expression of OsNAC5 is induced by abiotic stresses such as drought, cold, high salinity, abscisic acid and methyl jasmonic acid. Transactivation assays using rice protoplasts demonstrated that OsNAC5 is a transcriptional activator, and subcellular localization studies using OsNAC5-GFP fusion proteins showed that it is localized to the nucleus. Pull-down assays revealed that OsNAC5 interacts with OsNAC5, OsNAC6 and SNAC1. To analyze the function of OsNAC5 in rice plants, we generated transgenic plants that overexpressed OsNAC5. The growth of these plants was similar to that of control plants, whereas the growth of OsNAC6-overexpressing transgenic plants was retarded. OsNAC5-overexpressing transgenic plants also had improved tolerance to high salinity compared to control plants. By microarray analysis, many stress-inducible genes, including the "late embryogenesis abundant" gene OsLEA3, were upregulated in rice plants that overexpressed OsNAC5. By gel mobility shift assay, OsNAC5 and OsNAC6 were shown to bind to the OsLEA3 promoter. Collectively, our results indicate that the stress-responsive proteins OsNAC5 and OsNAC6 are transcriptional activators that enhance stress tolerance by upregulating the expression of stress-inducible rice genes such as OsLEA3, although the effects of these proteins on growth are different. Furthermore, because OsNAC5 overexpression did not retard growth, OsNAC5 may be a useful gene that can improve the stress tolerance of rice without affecting its growth. OsLEA3|OsLEA3-1,OsNAC5,OsNAC19|SNAC1|OsNAC9,OsNAC6|SNAC2 Overexpression of OsMYB48-1, a novel MYB-related transcription factor, enhances drought and salinity tolerance in rice 2014 PLoS One Key Lab of Crop Heterosis and Utilization of Ministry of Education and Beijing Key Lab of Crop Genetic Improvement, China Agricultural University, Beijing, People's Republic of China. MYB-type transcription factors (TFs) play essential roles in plant growth, development and respond to environmental stresses. Role of MYB-related TFs of rice in drought stress tolerance is not well documented. Here, we report the isolation and characterization of a novel MYB-related TF, OsMYB48-1, of rice. Expression of OsMYB48-1 was strongly induced by polyethylene glycol (PEG), abscisic acid (ABA), H2O2, and dehydration, while being slightly induced by high salinity and cold treatment. The OsMYB48-1 protein was localized in the nucleus with transactivation activity at the C terminus. Overexpression of OsMYB48-1 in rice significantly improved tolerance to simulated drought and salinity stresses caused by mannitol, PEG, and NaCl, respectively, and drought stress was caused by drying the soil. In contrast to wild type plants, the overexpression lines exhibited reduced rate of water loss, lower malondialdehyde (MDA) content and higher proline content under stress conditions. Moreover, overexpression plants were hypersensitive to ABA at both germination and post-germination stages and accumulated more endogenous ABA under drought stress conditions. Further studies demonstrated that overexpression of OsMYB48-1 could regulate the expression of some ABA biosynthesis genes (OsNCED4, OsNCED5), early signaling genes (OsPP2C68, OSRK1) and late responsive genes (RAB21, OsLEA3, RAB16C and RAB16D) under drought stress conditions. Collectively, these results suggested that OsMYB48-1 functions as a novel MYB-related TF which plays a positive role in drought and salinity tolerance by regulating stress-induced ABA synthesis. OsLEA3|OsLEA3-1,OsMYB48-1,OsNCED4,OsNCED5,RAB21|Rab16A,SAPK6|OSRK1 Over-expression of a LEA gene in rice improves drought resistance under the field conditions 2007 Theor Appl Genet National Center of Plant Gene Research (Wuhan), National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China. Late embryogenesis abundant (LEA) proteins have been implicated in many stress responses of plants. In this report, a LEA protein gene OsLEA3-1 was identified and over-expressed in rice to test the drought resistance of transgenic lines under the field conditions. OsLEA3-1 is induced by drought, salt and abscisic acid (ABA), but not by cold stress. The promoter of OsLEA3-1 isolated from the upland rice IRAT109 exhibits strong activity under drought- and salt-stress conditions. Three expression constructs consisting of the full-length cDNA driven by the drought-inducible promoter of OsLEA3-1 (OsLEA3-H), the CaMV 35S promoter (OsLEA3-S), and the rice Actin1 promoter (OsLEA3-A) were transformed into the drought-sensitive japonica rice Zhonghua 11. Drought resistance pre-screening of T(1) families at anthesis stage revealed that the over-expressing families with OsLEA3-S and OsLEA3-H constructs had significantly higher relative yield (yield under drought stress treatment/yield under normal growth conditions) than the wild type under drought stress conditions, although a yield penalty existed in T(1) families under normal growth conditions. Nine homozygous families, exhibiting over-expression of a single-copy of the transgene and relatively low yield penalty in the T(1) generation, were tested in the field for drought resistance in the T(2) and T(3) generations and in the PVC pipes for drought tolerance in the T(2) generation. Except for two families (transformed with OsLEA3-A), all the other families (transformed with OsLEA3-S and OsLEA3-H constructs) had higher grain yield than the wild type under drought stress in both the field and the PVC pipes conditions. No significant yield penalty was detected for these T(2 )and T(3) families. These results indicate that transgenic rice with significantly enhanced drought resistance and without yield penalty can be generated by over-expressing OsLEA3-1 gene with appropriate promoters and following a bipartite (stress and non-stress) in-field screening protocol. OsLEA3|OsLEA3-1 Molecular analysis of OsLEA4 and its contributions to improve E. coli viability 2012 Appl Biochem Biotechnol Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, People's Republic of China. tzhu2002@yahoo.com.cn OsLEA4, a late embryogenesis abundant (LEA) protein gene from rice (Oryza sativa L.), contains a 312-bp open reading frame encoding a putative polypeptide of 103 amino acids with a calculated molecular mass of 11.19 kDa and a theoretical pI of 10.04. OsLEA4 polypeptide is rich in Ala (22%), Lys (15%), Glu (9%), His (8%), Thr (8%), and Arg (7%) and lacking in Trp, Cys, Asn, and Phe residues. OsLEA4 protein contains a Pfam:LEA_1 domain architecture at positions 1-73 with three alpha-helical domains and without beta-sheet domain. In silico predictions showed that OsLEA4 protein was strongly hydrophilic with the grand average of hydropathy value of -0.816 and instability index of 27.31. The hydrophilic regions were found in the conserved motif of OsLEA4. OsLEA4 gene was introduced into Escherichia coli, and a fusion protein ( approximately 29.4 kDa) was expressed after isopropylthio-beta-D: -galactoside inducting by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. OsLEA4 protein enhanced the tolerance of E. coli recombinant to high salinity, heat, freezing, and UV radiation, which suggested that OsLEA4 protein may play a protective role under stressed conditions. This is the first successful use of E. coli as a prokaryotic system for LEA production from rice. OsLEA4 Molecular characterization and functional analysis by heterologous expression in E. coli under diverse abiotic stresses for OsLEA5, the atypical hydrophobic LEA protein from Oryza sativa L 2012 Mol Genet Genomics Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, China. In this study, we report the molecular characterization and functional analysis of OsLEA5 gene, which belongs to the atypical late embryogenesis abundant (LEA) group 5C from Oryza sativa L. The cDNA of OsLEA5 contains a 456 bp ORF encoding a polypeptide of 151 amino acids with a calculated molecular mass of 16.5 kDa and a theoretical pI of 5.07. The OsLEA5 polypeptide is rich in Leu (10%), Ser (8.6%), and Asp (8.6%), while Cys, Trp, and Gln residue contents are very low, which are 2, 1.3, and 1.3%, respectively. Bioinformatic analysis revealed that group 5C LEA protein subfamily contains a Pfam:LEA_2 domain architecture and is highly hydrophobic, intrinsically ordered with largely beta-sheet and specific amino acid composition and distribution. Real-time PCR analysis showed that OsLEA5 was expressed in different tissue organs during different development stages of rice. The expression levels of OsLEA5 in the roots and panicles of full ripe stage were dramatically increased. The results of stress tolerance and cell viability assay demonstrated that recombinant E. coli cells producing OsLEA5 fusion protein exhibited improved resistance against diverse abiotic stresses: high salinity, osmotic, freezing, heat, and UV radiation. The OsLEA5 protein confers stabilization of the LDH under different abiotic stresses, such as heating, freeze-thawing, and drying in vitro. The combined results indicated that OsLEA5 protein was a hydrophobic atypical LEA and closely associated with resistance to multiple abiotic stresses. This research offered the valuable information for the development of crops with enhanced resistance to diverse stresses. OsLEA5 A rice lectin receptor-like kinase that is involved in innate immune responses also contributes to seed germination 2013 The Plant Journal State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China Seed germination and innate immunity both have significant effects on plant life spans because they control the plant's entry into the ecosystem and provide defenses against various external stresses, respectively. Much ecological evidence has shown that seeds with high vigor are generally more tolerant of various environmental stimuli in the field than those with low vigor. However, there is little genetic evidence linking germination and immunity in plants. Here, we show that the rice lectin receptor-like kinase OslecRK contributes to both seed germination and plant innate immunity. We demonstrate that knocking down the OslecRK gene depresses the expression of α–amylase genes, reducing seed viability and thereby decreasing the rate of seed germination. Moreover, it also inhibits the expression of defense genes, and so reduces the resistance of rice plants to fungal and bacterial pathogens as well as herbivorous insects. Yeast two-hybrid and co-immunoprecipitation experiments revealed that OslecRK interacts with an actin-depolymerizing factor (ADF) in vivo via its kinase domain. Moreover, the rice adf mutant exhibited a reduced seed germination rate due to the suppression of α–amylase gene expression. This mutant also exhibited depressed immune responses and reduced resistance to biotic stresses. Our results thus provide direct genetic evidence for a common physiological pathway connecting germination and immunity in plants. They also partially explain the common observation that high-vigor seeds often perform well in the field. The dual effects of OslecRK may be indicative of progressive adaptive evolution in rice. OslecRK Overexpression of transcription factor OsLFL1 delays flowering time in Oryza sativa 2008 J Plant Physiol National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China. Flowering time is regulated by genetic programs and environment signals in plants. Genetic analysis of flowering time mutants is instrumental in dissecting the regulatory pathways of flower induction. Genotype W378 is a rice (Oryza sativa) late-flowering mutant selected from our collections of T-DNA insertion line. The T-DNA flanking gene in mutant W378 codes OsLFL1 (O. sativa LEC2 and FUSCA3 Like 1), a putative B3 DNA-binding domain-containing transcription factor. In wild-type rice OsLFL1 is expressed exclusively in spikes and young embryos, while in mutant W378 it is ectopically expressed. Introduction of OsLFL1-RNAi into mutant W378 successfully down-regulated OsLFL1 expression and restored flowering to almost normal time, indicating that overexpression of OsLFL1 confers late flowering for mutant W378. The flowering-promoting gene Ehd1 and its downstream genes are all down-regulated in W378. Thus, overexpression of OsLFL1 might delay the flowering of W378 by repressing the expression of Ehd1. OsLFL1 Functional analysis of two isoforms of leaf-type ferredoxin-NADP(+)-oxidoreductase in rice using the heterologous expression system of Arabidopsis 2011 Plant Physiol Plant Functional Genomics Research Group, RIKEN, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. Ferredoxin-NADP(+)-oxidoreductase (FNR) mediates electron transfer between ferredoxin (Fd) and NADP(+); therefore, it is a key enzyme that provides the reducing power used in the Calvin cycle. Other than FNR, nitrite reductase, sulfite reductase, glutamate synthase, and Fd-thioredoxin reductase also accept electrons from Fd, an electron carrier protein in the stroma. Therefore, the regulation of electron partitioning in the chloroplast is important for photosynthesis and other metabolic pathways. The regulatory mechanism of electron partitioning, however, remains to be elucidated. We found, by taking advantage of a gain-of-function approach, that expression of two rice (Oryza sativa) full-length cDNAs of leaf-type FNRs (OsLFNR1 and OsLFNR2) led to altered chlorophyll fluorescence and growth in Arabidopsis (Arabidopsis thaliana) and rice. We revealed that overexpression of the OsLFNR1 and OsLFNR2 full-length cDNAs resulted in distinct phenotypes despite the high sequence similarity between them. Expression of OsLFNR1 affected the nitrogen assimilation pathway without inhibition of photosynthesis under normal conditions. On the other hand, OsLFNR2 expression led to the impairment of photosynthetic linear electron transport as well as Fd-dependent cyclic electron flow around photosystem I. The endogenous protein level of OsLFNR was found to be suppressed in both OsLFNR1- and OsLFNR2-overexpressing rice plants, leading to changes in the stoichiometry of the two LFNR isoforms within the thylakoid and soluble fractions. Thus, we propose that the stoichiometry of two LFNR isoforms plays an important role in electron partitioning between carbon fixation and nitrogen assimilation. OsLFNR1|LFNR1,OsLFNR2|LFNR2,ASCAB9-A|CP26|Lhcb5,CAB2R,chlH,CP24,CP29,Fd1 Mutations in the rice liguleless gene result in a complete loss of the auricle, ligule, and laminar joint 2007 Plant Mol Biol National Research Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Hyo-ja dong, Pohang, Kyungbuk 790-784, Korea. The area between the upper part of the leaf sheath and the basal portion of the leaf blade contains several specialized organs, such as the laminar joint, auricle and ligule. Here we report the identification of T-DNA insertional mutant lines that lack all of these organs. The gene knocked out in the mutant lines encodes a protein that contains a SBP (SQUAMOSA promoter Binding Protein)-domain and is highly homologous to the maize LIGULELESS1 (LG1) gene. At the amino acid sequence level, the OsLG1 protein is 69% identical to maize LG1 and 78% identical to barley LG1. We named the rice gene OsLIGULELESS1 (OsLG1). Transient expression of an OsLG1:RFP (Red Fluorescent Protein) fusion protein indicated that the protein is localized to the nucleus. Transgenic plants harboring the OsLG1 promoter:GUS (beta-glucuronidase) reporter gene construct display preferential expression in developing laminar joint regions and meristemic regions. The gene is also weakly expressed in the ligule, auricles, and leaf sheaths at the basal region. These results indicate that OsLG1 is a transcriptional factor that plays an important role in building the laminar joint between leaf blade and leaf sheath boundary, thereby controlling ligule and auricle development. OsLG1 Genetic control of inflorescence architecture during rice domestication 2013 Nat Commun State Key Laboratory of Plant Physiology and Biochemistry, National Center for Evaluation of Agricultural Wild Plants (Rice), China Agricultural University, Beijing 100193, China. Inflorescence architecture is a key agronomical factor determining grain yield, and thus has been a major target of cereal crop domestication. Transition from a spread panicle typical of ancestral wild rice (Oryza rufipogon Griff.) to the compact panicle of present cultivars (O. sativa L.) was a crucial event in rice domestication. Here we show that the spread panicle architecture of wild rice is controlled by a dominant gene, OsLG1, a previously reported SBP-domain transcription factor that controls rice ligule development. Association analysis indicates that a single-nucleotide polymorphism-6 in the OsLG1 regulatory region led to a compact panicle architecture in cultivars during rice domestication. We speculate that the cis-regulatory mutation can fine-tune the spatial expression of the target gene, and that selection of cis-regulatory mutations might be an efficient strategy for crop domestication. OsLG1 OsLG1 regulates a closed panicle trait in domesticated rice 2013 Nat Genet Graduate School of Agricultural Science, Kobe University, Kobe, Japan. tishii@kobe-u.ac.jp Reduction in seed shattering was an important phenotypic change during cereal domestication. Here we show that a simple morphological change in rice panicle shape, controlled by the SPR3 locus, has a large impact on seed-shedding and pollinating behaviors. In the wild genetic background of rice, we found that plants with a cultivated-like type of closed panicle had significantly reduced seed shedding through seed retention. In addition, the long awns in closed panicles disturbed the free exposure of anthers and stigmas on the flowering spikelets, resulting in a significant reduction of the outcrossing rate. We localized the SPR3 locus to a 9.3-kb genomic region, and our complementation tests suggest that this region regulates the liguleless gene (OsLG1). Sequencing analysis identified reduced nucleotide diversity and a selective sweep at the SPR3 locus in cultivated rice. Our results suggest that a closed panicle was a selected trait during rice domestication. OsLG1 The evolutionarily conserved OsPRR quintet: rice pseudo-response regulators implicated in circadian rhythm 2003 Plant Cell Physiol Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya, 464-8601 Japan. In Arabidopsis thaliana, a number of circadian-associated factors have been identified, including TOC1 (TIMING OF CAB EXPRESSION 1) that is believed to be a component of the central oscillator. TOC1 is a member of a small family of proteins, designated as ARABIDOPSIS PSEUDO-RESPONSE REGULATORS (APRR1/TOC1, APRR3, APRR5, APRR7, and APRR9). As demonstrated previously, these APRR1/TOC1 quintet members are crucial for a better understanding of the molecular links between circadian rhythms, control of flowering time through photoperiodic pathways, and also photosensory signal transduction in this dicotyledonous plant. In this respect, both the dicotyledonous (e.g. A. thaliana) and monocotyledonous (e.g. Oryza sativa) plants might share the evolutionarily conserved molecular mechanism underlying the circadian rhythm. Based on such an assumption, and as the main objective of this study, we asked the question of whether rice also has a set of pseudo-response regulators, and if so, whether or not they are associated with the circadian rhythm. Here we showed that rice has five members of the OsPRR family (Oryza sativa Pseudo-Response Regulator), and also that the expressions of these OsPRR genes are under the control of circadian rhythm. They are expressed in a diurnal and sequential manner in the order of OsPRR73 (OsPRR37)-->OsPRR95 (OsPRR59)-->OsPRR1, which is reminiscent of the circadian waves of the APRR1/TOC1 quintet in A. thaliana. These and other results of this study suggested that the OsPRR quintet, including the ortholog of APRR1/TOC1, might play important roles within, or close to, the circadian clock of rice. OsLHY OsLIC, a Novel CCCH-Type Zinc Finger Protein with Transcription Activation, Mediates Rice Architecture via Brassinosteroids Signaling 2008 PLoS One Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, the Chinese Academy of Sciences, Beijing, China. Rice architecture is an important agronomic trait and a major limiting factor for its high productivity. Here we describe a novel CCCH-type zinc finger gene, OsLIC (Oraza sativaleaf and tiller angle increased controller), which is involved in the regulation of rice plant architecture. OsLIC encoded an ancestral and unique CCCH type zinc finge protein. It has many orthologous in other organisms, ranging from yeast to humane. Suppression of endogenous OsLIC expression resulted in drastically increased leaf and tiller angles, shortened shoot height, and consequently reduced grain production in rice. OsLIC is predominantly expressed in rice collar and tiller bud. Genetic analysis suggested that OsLIC is epistatic to d2-1, whereas d61-1 is epistatic to OsLIC. Interestingly, sterols were significantly higher in level in transgenic shoots than in the wild type. Genome-wide expression analysis indicated that brassinosteroids (BRs) signal transduction was activated in transgenic lines. Moreover, transcription of OsLIC was induced by 24-epibrassinolide. OsLIC, with a single CCCH motif, displayed binding activity to double-stranded DNA and single-stranded polyrA, polyrU and polyrG but not polyrC. It contains a novel conserved EELR domain among eukaryotes and displays transcriptional activation activity in yeast. OsLIC may be a transcription activator to control rice plant architecture. LIC|OsLIC1 OsLIS-L1 encoding a lissencephaly type-1-like protein with WD40 repeats is required for plant height and male gametophyte formation in rice 2012 Planta National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research-Wuhan, Huazhong Agricultural University, Wuhan 430070, China. Although a large number of genes encoding the WD40 motif have been identified as being involved in various developmental processes in Arabidopsis, little is known about the function of these genes in rice (Oryza sativa). Here, we report the cloning and functional characterization of a novel rice gene OsLIS-L1 (Lissencephaly type-1-like 1), which is required for normal fertility and the first internode elongation. OsLIS-L1 encodes a lissencephaly type-1-like protein containing the WD40 motif that is required for brain development in human. SMART algorithm analysis indicated that OsLIS-L1 contains a LIS1 homology (LisH) domain, a C terminus to LisH (CTLH) domain, a five WD40-repeat domain in the middle, and a domain with four WD40 repeats which is homologous to the beta subunit of trimeric G-proteins (G(beta)). OsLIS-L1 transcript is relatively highly abundant in stem and panicle and has a dynamic expression pattern at different panicle developmental stages. Two independent alleles, designated oslis-l1-1 and oslis-l1-2, exhibited similar abnormal developmental phenotypes, including semi-dwarf, shorter panicle length, and reduced male fertility. Cytological examination confirmed that OsLIS-L1 does not affect the meiosis in pollen mother cells. Compared with wild type, the oslis-l1 mutant had abnormal male gametophyte formation, but anther cell wall and pollen wall development were not affected. Histological analysis revealed that OsLIS-L1 regulates the cell proliferation in the first internode under the panicle. Our results indicate that OsLIS-L1 plays an important role in male gametophyte formation and the first internode elongation in rice. ASP1|OsLIS-L1 Aberrant spikelet and panicle1, encoding a TOPLESS-related transcriptional co-repressor, is involved in the regulation of meristem fate in rice 2012 Plant J Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8657, Japan. Post-embryonic development depends on the activity of meristems in plants, and thus control of cell fate in the meristem is crucial to plant development and its architecture. In grasses such as rice and maize, the fate of reproductive meristems changes from indeterminate meristems, such as inflorescence and branch meristems, to determinate meristems, such as the spikelet meristem. Here we analyzed a recessive mutant of rice, aberrant spikelet and panicle1 (asp1), that showed pleiotropic phenotypes such as a disorganized branching pattern, aberrant spikelet morphology, and disarrangement of phyllotaxy. Close examination revealed that regulation of meristem fate was compromised in asp1: degeneration of the inflorescence meristem was delayed, transition from the branch meristem to the spikelet meristem was accelerated, and stem cell maintenance in both the branch meristem and the spikelet meristem was compromised. The genetic program was also disturbed in terms of spikelet development. Gene isolation revealed that ASP1 encodes a transcriptional co-repressor that is related to TOPLESS (TPL) in Arabidopsis and RAMOSA ENHANCER LOCUS2 (REL2) in maize. It is likely that the pleiotropic defects are associated with de-repression of multiple genes related to meristem function in the asp1 mutant. The asp1 mutant also showed de-repression of axillary bud growth and disturbed phyllotaxy in the vegetative phase, suggesting that the function of this gene is closely associated with auxin action. Consistent with these observations and the molecular function of Arabidopsis TPL, auxin signaling was also compromised in the rice asp1 mutant. Taken together, these results indicate that ASP1 regulates various aspects of developmental processes and physiological responses as a transcriptional co-repressor in rice. ASP1|OsLIS-L1 Differences in transcriptional regulatory mechanisms functioning for free lysine content and seed storage protein accumulation in rice grain 2010 Plant Cell Physiol National Institute of Agrobiological Sciences, Transgenic Crop Research & Development Center, Tsukuba, Ibaraki, Japan. Lysine is the most deficient essential amino acid in cereal grains. A bifunctional lysine-degrading enzyme, lysine ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH), is one of the key regulators determining free lysine content in plants. In rice (Oryza sativa. L), a bifunctional OsLKR/SDH is predominantly present in seeds. Here, we show that OsLKR/SDH is directly regulated by major transcriptional regulators of seed storage protein (SSP) genes: the basic leucine zipper (bZIP) transcription factor (TF), RISBZ1, and the DNA-binding with one finger (DOF) transcription factor, RPBF. OsLKR/SDH was highly expressed in the aleurone and subaleurone layers of the endosperm. Mutation analyses in planta, trans-activation reporter assays in vivo and electrophorestic mobility shift assays in vitro showed that the RPBF-recognizing prolamin box (AAAG) and the RISBZ1-recognizing GCN4 motif (TGAG/CTCA) act as important cis-elements for proper expression of OsLKR/SDH like SSP genes. However, mutation of the GCN4 motif within ProOsLKR/SDH did not alter the spatial expression pattern, whereas mutation of the GCN4 motif within ProGluB-1 did alter spatial expression. Reducing either RISBZ1 or RPBF decreased OsLKR/SDH levels, resulting in an increase in free lysine content in rice grain. This result was in contrast to the fact that a significant reduction of SSP was observed only when these transcription factors were simultaneously reduced, suggesting that RISBZ1 and RPBF regulate SSP genes and OsLKR/SDH with high and limited redundancy, respectively. The same combinations of TF and cis-elements are involved in the regulation of OsLKR/SDH and SSP genes, but there is a distinct difference in their regulation mechanisms. OsLKR|SDH,RISBZ1|OsbZIP58,RPBF|OsDof3 Mutation in OsLMS, a gene encoding a protein with two double-stranded RNA binding motifs, causes lesion mimic phenotype and early senescence in rice (Oryza sativa L.) 2012 Genes Genet Syst Iwate Biotechnology Research Center, Kitakami, Narita 22-174-4, Iwate 024-0003, Japan. The rice (Oryza sativa L.) lesion mimic and senescence (lms) EMS-mutant, identified in a japonica cultivar Hitomebore, is characterized by a spontaneous lesion mimic phenotype during its vegetative growth, an accelerated senescence after flowering, and enhanced resistance to rice blast (Magnaporthe oryzae). To isolate the OsLMS gene, we crossed the lms mutant to Kasalath (indica), and used mutant F(2) plants to initially map the candidate region to about 322-kb on the long arm of chromosome 2. Illumina whole-genome re-sequencing of the mutant and aligning the reads to Hitomebore reference sequence within the candidate region delineated by linkage analysis identified a G to A nucleotide substitution. The mutation corresponded to the exon-intron splicing junction of a novel gene that encodes a carboxyl-terminal domain (CTD) phosphatase domain and two double stranded RNA binding motifs (dsRBM) containing protein. By PCR amplification, we confirmed that the mutation causes splicing error that is predicted to introduce a premature stop codon. RNA interference (RNAi) transgenic lines with suppressed expression of LMS gene exhibited the lesion mimic phenotype, confirming that the mutation identified in LMS is responsible for the mutant phenotype. OsLMS shares a moderate amino-acid similarity to the Arabidopsis FIERY2/CPL1 gene, which is known to control many plant processes such as stress response and development. Consistence with this similarity, the lms mutant shows sensitivity to cold stress at the early growth stage, suggesting that LMS is a negative regulator of stress response in rice. OsLMS Molecular cloning and expression of a cDNA encoding Lon protease from rice (Oryza sativa) 2006 Biotechnol Lett Institute of Genetics, State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China. The ATP-dependent Lon protease is a highly conserved enzyme that is present in archeae, eubacteria, and eukaryotes, and plays an important role in intracellular protein degradation. We have isolated a Lon protease gene, OsLon1, from Oryza sativa. The cDNA contained a 2,655 bp ORF. Comparative analysis showed that OsLon1 shared significant similarity with the previously reported Lon proteases from maize, Arabidopsis, human, and bacteria. Tissue expression pattern analysis revealed that OsLon1 was highly expressed in young leaves, mature leaves, and leaf sheaths but only weakly in young roots, mature roots, and young panicles. The OsLon1 gene was successfully expressed in E. coli and the detected protein size, about 120 kDa, matched the expected molecular mass of the His-tagged OsLon1 protein. OsLon1 A novel lipoxygenase gene from developing rice seeds confers dual position specificity and responds to wounding and insect attack 2008 Plant Mol Biol State Key Laboratory for Crop Genetics & Germplasm Enhancement, Nanjing Agricultural University; Research Center of Plant Gene Engineering, Nanjing, Jiangsu Province 210095, PR China. OsLOX1 is a novel full-length cDNA isolated from developing rice seeds. We have examined its biochemical properties and expression patterns. The protein has dual positional specificity, as it releases both C-9 and C-13 oxidized products in a 4:3 ratio. OsLOX1 transcripts were detected at low abundance in immature seeds and newly germinated seedlings, but accumulate rapidly and transiently in response to wounding or brown planthopper (BPH) attack, reaching a peak 3 h after wounding and 6 h after insect feeding. We produced transgenic rice lines carrying either sense or antisense constructs under the control of a cauliflower mosaic virus 35S promoter, and these rice lines showed altered OsLOX1 activity. In all of the antisense lines and more than half of the sense lines the expression levels of OsLOX1, the levels of enzyme activity, and the levels of the endogenous OsLOX1 products (jasmonic acid, (Z)-3-hexenal and colneleic acid) at 6, 48, and 48 h after BPH feeding respectively, were below the levels found in non-transgenic control plants; yet, the levels in the remaining sense transformants were enhanced relative to controls. Transformants with a lower level of OsLOX1 expression were less able to tolerate BPH attack, while those with enhanced OsLOX1 expression were more resistant. Our data suggest that the OsLOX1 product is involved in tolerance of the rice plant to wounding and BPH attack. OsLOX1 The rice OsLpa1 gene encodes a novel protein involved in phytic acid metabolism 2008 Theor Appl Genet USDA-ARS Crops Pathology and Genetics Research Unit, Department of Plant Sciences, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA. The rice low phytic acid 1 (lpa1) mutant was originally identified using a forward genetics approach. This mutant exhibits a 45% reduction in rice seed phytic acid with a molar-equivalent increase in inorganic phosphorus; however, it does not appear to differ significantly in productivity from its wild-type progenitor. A second lpa1 mutant was identified from additional screening for high seed inorganic phosphorus phenotypes. Using a positional cloning strategy, we identified a single candidate gene at the rice Lpa1 locus. Sequence analysis of the candidate gene from the lpa1 mutants revealed two independent mutations (a single base pair substitution and a single base pair deletion) that confirmed the identification of this candidate as the rice low phytic acid 1 gene, OsLpa1. The OsLpa1 gene has three splice variants. The location and nature of the two mutations suggests that these lesions only affect the translation of the predicted protein derived from the longest transcript. The proteins encoded by OsLpa1 do not have homology to any of the inositol phosphate metabolism genes recently characterized in plants, although there is homology to 2-phosphoglycerate kinase, an enzyme found in hyperthermophilic methanogens that catalyzes the formation of 2,3-bisphosphoglycerate from 2-phosphoglycerate. OsLpa1 represents a novel gene involved in phytic acid metabolism. OsLpa1 Characterization of a lipoate-protein ligase A gene of rice (Oryza sativa L.) 2007 Gene School of Biotechnology, Institute of Biotechnology, Yeungnam University, Gyeongsan 712-749, Republic of Korea. kangsg@ynu.ac.kr Lipoic acid is an essential disulfide cofactor required for the lipoate-dependent enzymes including pyruvate dehydrogenase (PDH), alpha-ketoglutarate dehydrogenase (KGDH), and glycine cleavage enzymes that function in key metabolic pathways in most prokaryotes and eukaryotes. Lipoic acid is covalently bound to lipoate-dependent enzymes by lipoate-protein ligase or lipoate transferase. Here, we characterized a lipoyl-protein ligase A (OsLPLA) gene of rice. The OsLPLA gene, which encoded 270 amino acids, was located on an approximately 21 Mb of chromosome 8 on the physical map of Oryza sativa Japonica type. OsLPLA transcripts were abundantly expressed in leaves and developing seeds. The OsLPLA gene functionally complemented an Escherichia coli lplA null mutant. Furthermore, the protein expressed from the OsLPLA gene in an E. coli lplA mutant successfully transferred exogenous lipoate to lipoate-dependent enzymes, including the E2 subunits of the PDH, the E2 subunit of KGDH and the H-protein of glycine decarboxylase, confirming that rice OsLPLA successfully catalyzed covalent attachment of lipoate onto lipoate-dependent enzymes. OsLPLA Ectopic expression of the rice lumazine synthase gene contributes to defense responses in transgenic tobacco 2010 Phytopathology Growth and Defense Signaling Laboratory, Ministry of Agriculture, P.R. China. Lumazine synthase (LS) catalyzes the penultimate reaction in the multistep riboflavin biosynthesis pathway, which is involved in plant defenses. Plant defenses are often subject to synergistic effects of jasmonic acid and ethylene whereas LS is a regulator of jasmonic acid signal transduction. However, little is known about whether the enzyme contributes to defense responses. To study the role of LS in plant pathogen defenses, we generated transgenic tobacco expressing the rice (Oryza sativa) LS gene, OsLS. OsLS was cloned and found to have strong identity with its homologues in higher plants and less homology to microbial orthologues. The OsLS protein localized to chloroplasts in three OsLS-expressing transgenic tobacco (LSETT) lines characterized as enhanced in growth and defense. Compared with control plants, LSETT had higher content of both riboflavin and the cofactors flavin mononucleotide and flavin adenine dinucleotide. In LSETT, jasmonic acid and ethylene were elevated, the expression of defense-related genes was induced, levels of resistance to pathogens were enhanced, and resistance was effective to viral, bacterial, and oomycete pathogens. Extents of OsLS expression correlated with increases in flavin, jasmonic acid, and ethylene content, and correlated with increases in resistance levels, suggesting a role for OsLS in defense responses. OsLS OsLSD1, a rice zinc finger protein, regulates programmed cell death and callus differentiation 2005 Mol Plant Microbe Interact National Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, P R. China. The Arabidopsis LSD1 and LOL1 proteins both contain three conserved zinc finger domains and have antagonistic effects on plant programmed cell death (PCD). In this study, a rice (Oryza sativa) functional homolog of LSD1, designated OsLSD1, was identified. The expression of OsLSD1 was light-induced or dark-suppressed. Overexpression of OsLSD1 driven by the cauliflower mosaic virus 35S promoter accelerated callus differentiation in transformed rice tissues and increased chlorophyll b content in transgenic rice plants. Antisense transgenic rice plants exhibited lesion mimic phenotype, increased expression of PR-1 mRNA, and an accelerated hypersensitive response when inoculated with avirulent isolates of blast fungus. Both sense and antisense transgenic rice plants conferred significantly enhanced resistance against a virulent isolate of blast fungus. Moreover, ectopic overexpression of OsLSD1 in transgenic tobacco (Nicotiana tabacum) enhanced the tolerance to fumonisins B1 (FB1), a PCD-eliciting toxin. OsLSD1 green fluorescent protein fusion protein was located in the nucleus of tobacco cells. Our results suggest that OsLSD1 plays a negative role in regulating plant PCD, whereas it plays a positive role in callus differentiation. OsLSD1 Characterization of two plasma membrane protein 3 genes (PutPMP3) from the alkali grass, Puccinellia tenuiflora, and functional comparison of the rice homologues, OsLti6a/b from rice 2008 BMB Rep College of Agronomy and Biotechnology, China Agricultural University, China. Two full-length cDNAs, PutPMP3-1 and PutPMP3-2, encoding PMP3 family proteins were isolated from Puccinellia tenuiflora, a monocotyledonous halophyte. Expression of both genes was induced by low temperature, salt stress, dehydration, ABA, and NaHCO(3). Transcripts of PutPMP3-2 were more strongly induced by these stresses relative to those of PutPMP3-1, particularly under low temperature and dehydration conditions. Expression of PutPMP3-1 and PutPMP3-2 in yeast mutants lacking the PMP3 gene can functionally complement the membrane hyperpolarization and salt sensitivity phenotypes resulting from PMP3 deletion. To compare the functions of PutPMP3-1 and PutPMP3-2, the orthologous genes in rice (OsLti6a and OsLti6b) were isolated. Both OsLti6a and OsLti6b could functionally complement the loss of PMP3 in yeast. PutPMP3-2 and OsLti6a were more effective in reversing membrane hyperpolarization than PutPMP3-1 and OsLti6b. However, the four yeast transformants each showed similar levels of salt tolerance. These results imply that these PMP3 family members don't function identically under different stress tolerance conditions. OsLti6a,OsLti6b|ddOs32 The OsLti6 genes encoding low-molecular-weight membrane proteins are differentially expressed in rice cultivars with contrasting sensitivity to low temperature 2005 Gene Department of Crop, Soil and Environmental Sciences, University of Arkansas, Fayetteville, AR 72701, USA. Rice (Oryza sativa L.) is sensitive to chilling particularly at early stages of seedling establishment. Two closely related genes (OsLti6a, OsLti6b), which are induced by low temperature during seedling emergence were isolated from a cold tolerant temperate japonica rice cultivar. These genes are closely related to the Arabidopsis rare cold-inducible (RCI2) and barley low-temperature-inducible (BLT101) genes. Based on direct biochemical and indirect physiological evidence and similarity with a conserved protein domain in the Cluster of Orthologous Groups (COG) database (e.g., yeast PMP3), the rice genes belong to a class of low-molecular-weight hydrophobic proteins involved in maintaining the integrity of the plasma membrane during cold, dehydration and salt stress conditions. Both genes exhibit a genotype-specific expression signature characterized by early and late stress-inducible expression in tolerant and intolerant genotypes, respectively. The differences in temporal expression profiles are consistent with cultivar differences in cold-induced membrane leakiness and seedling vigor. The presence of CRT/DRE promoter cis-elements is consistent with the synchronized expression of OsLti6 genes with the C-repeat binding factor/drought responsive element-binding protein (CBF/DREB) transcriptional activator. The present results indicate that the Oslti6 genes are part of a battery of cold stress defense-related genes regulated by a common switch. OsLti6a,OsLti6b|ddOs32 Isolation of cold stress-responsive genes in the reproductive organs, and characterization of the OsLti6b gene from rice (Oryza sativa L.) 2007 Plant Cell Rep Department of Life Science, Sogang University, Seoul 121-742, Korea. During their reproductive stage, rice crops often are exposed to cold stress, which leads to sterility and reduced yields. To understand the cold response mechanism at that stage, we used an mRNA differential display method to isolate cold-responsive genes from pre-anthesis flowers. Approximately 5,000 transcripts were identified here, of which 123 were found to be displayed differentially between the control (30 degrees C) and cold-treated (12 degrees C) flowers. Among them, 26 were analyzed by northern analysis; 8 of those clones were confirmed as cold-responsive. OsLti6b, encoding a hydrophobic protein homologous to Arabidopsis RCI2, was analyzed in detail. RNA blot analysis revealed that its transcript is increased by cold, salt, drought, or ABA treatments. In situ hybridization indicated that this transcript is highly accumulated in the ovaries and stamens of cold-treated flowers, particularly in the anther walls and vascular tissues of the filaments. Over-expression of OsLti6b increased cold tolerance as revealed by seedling wilting rates and ion leakages of mature leaves, demonstrating that the extent of the tolerance correlates well with its expression level. OsLti6b|ddOs32,OsPOX1|ddOs319 Cutin monomer induces expression of the rice OsLTP5 lipid transfer protein gene 2008 J Plant Physiol Department of Biological Sciences, Inha University, Incheon 402-751, Republic of Korea. Treatment with the cutin monomer 16-hydroxypalmitic acid (HPA), a major component of cutin, elicited the synthesis of hydrogen peroxide (H2O2) in rice leaves and induced the expression of the lipid transfer protein gene OsLTP5. Treatment with HPA also induced expression of OsLTP1, OsLTP2, and the pathogen-related PR-10 genes to a lesser extent. The OsLTP5 transcript was expressed prominently in stems and flowers, but was barely detectable in leaves. Expression of OsLTP5 was induced in shoots in response to ABA and salicylic acid. It is proposed that HPA is perceived by rice as a signal, inducing defense reactions. OsLTP5 The rice OsLTP6 gene promoter directs anther-specific expression by a combination of positive and negative regulatory elements 2013 Planta National Center for Gene Research and Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 500 Caobao Road, Shanghai, 200233, People's Republic of China, xhliu@ncgr.ac.cn. Characterization of tissue-specific plant gene promoters will benefit genetic improvement in crops. Here, we isolated a novel rice anther-specific plant lipid transfer protein (OsLTP6) gene through high through-put expressional profiling. The promoter of OsLTP6 was introduced to the upstream of the uidA gene, which encodes beta-glucuronidase (GUS), and transformed into rice plants for functional analysis. Histochemical and fluorometric GUS assay showed that GUS was specifically expressed in the anthers and pollens in OsLTP6 promoter::uidA transgenic plants. Transverse section of the rice anther further indicated that the OsLTP6 promoter directed the reporter gene specifically expressed in anther tapetum. To identify regulatory elements within OsLTP6 promoter region, four progressive deletions of the OsLTP6 promoter were constructed. The results indicated that the OsLTP6 promoter achieved anther-specific expression through a combination of positive and negative regulatory elements. A 26-bp motif upstream of TATA box was a key transcriptional activator for OsLTP6 gene. CAAT box and GTGA box were the putative motifs to increase the transcription level to full expression. Two negative regulatory elements were also found in two distinct regions within this promoter. They repressed the expression in leaf and stem, respectively. These results revealed the regulating complexity of anther-specific expression. OsDIL|OsLTP6 Isolation and identification of glycosylphosphatidylinositol-anchored arabinogalactan proteins and novel beta-glucosyl Yariv-reactive proteins from seeds of rice (Oryza sativa) 2004 Plant Cell Physiol Department of Applied Biological Chemistry, University of Tokyo, Bunkyo-ku, Tokyo, 113-8657 Japan. Arabinogalactan proteins (AGPs) are highly glycosylated extracellular glycoproteins playing important roles in plant growth and development. We have previously reported the possibility that AGPs are involved in the induction of alpha-amylase by gibberellin (GA) in barley aleurone layers by using the beta-glucosyl Yariv reagent (beta-GlcY), which has been presumed to specifically bind AGPs. In this present study, we isolated beta-GlcY-reactive proteins from rice bran rich in aleurone cells. The N-terminal sequences of classical AGP and AG peptides were determined from hydrophilic fractions obtained by reversed phase HPLC. Interestingly, a novel non-specific lipid transfer protein-like protein (OsLTPL1) and a novel early nodulin-like protein (OsENODL1) were also identified in the more hydrophobic fractions from HPLC as beta-GlcY-reactive proteins. Expression analysis of the genes coding for these proteins was performed. While classical AGP, AG peptides and OsLTPL1 were expressed in various parts of rice, OsENODL1 showed temporally and spatially specific expression in the aleurone layers. This new beta-GlcY-reactive protein is a promising candidate for the extracellular signaling factors of GA action in cereal seeds. Furthermore, the possibility that proteins with the AG glycomodule might react with beta-GlcY may broaden the definition of AGPs. OsLTPL1 Lysin motif-containing proteins LYP4 and LYP6 play dual roles in peptidoglycan and chitin perception in rice innate immunity 2012 Plant Cell State Key Laboratory of Biocontrol, Key Laboratory of Gene Engineering of the Ministry of Education and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, 510275 Guangzhou, People's Republic of China. Plant innate immunity relies on successful detection of microbe-associated molecular patterns (MAMPs) of invading microbes via pattern recognition receptors (PRRs) at the plant cell surface. Here, we report two homologous rice (Oryza sativa) lysin motif-containing proteins, LYP4 and LYP6, as dual functional PRRs sensing bacterial peptidoglycan (PGN) and fungal chitin. Live cell imaging and microsomal fractionation consistently revealed the plasma membrane localization of these proteins in rice cells. Transcription of these two genes could be induced rapidly upon exposure to bacterial pathogens or diverse MAMPs. Both proteins selectively bound PGN and chitin but not lipopolysaccharide (LPS) in vitro. Accordingly, silencing of either LYP specifically impaired PGN- or chitin- but not LPS-induced defense responses in rice, including reactive oxygen species generation, defense gene activation, and callose deposition, leading to compromised resistance against bacterial pathogen Xanthomonas oryzae and fungal pathogen Magnaporthe oryzae. Interestingly, pretreatment with excess PGN dramatically attenuated the alkalinization response of rice cells to chitin but not to flagellin; vice versa, pretreatment with chitin attenuated the response to PGN, suggesting that PGN and chitin engage overlapping perception components in rice. Collectively, our data support the notion that LYP4 and LYP6 are promiscuous PRRs for PGN and chitin in rice innate immunity. LYP4|OsLYP4,LYP6|OsLYP6 Morphogenesis and molecular basis on naked seed rice, a novel homeotic mutation of OsMADS1 regulating transcript level of AP3 homologue in rice 2006 Planta College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang, 310029, People's Republic of China. The floral organs are formed from floral meristem with a regular initiation pattern in angiosperm species. Flowers of naked seed rice (nsr) were characterized by the overdeveloped lemma and palea, the transformation of lodicules to palea-/lemma-like organs, the decreased number of stamens and occasionally extra pistils. Some nsr spikelets contained additional floral organs of four whorls and/or abnormal internal florets. The floral primordium of nsr spikelet is differentiated under an irregular pattern and an incomplete determination. And molecular analysis indicated that nsr was a novel homeotic mutation in OsMADS1, suggesting that OsMADS1 played a distinct role in regulating the differentiation pattern of floral primordium and in conferring the determination of flower meristem. The gain-of-function of OsMADS1 transgenic lines presented the transformation of outer glumes to lemma-/palea-like organs and no changes in length of lemma and palea, but loss-of-function of OsMADS1 transgenic lines displayed the overdeveloped lemma and palea. Both findings revealed that OsMADS1 played a role in specifying lemma and palea and acted as a repressor of overdevelopment of lemma and palea. Moreover, it was indicated that OsMADS1 upregulated the transcript level of AP3 homologue OsMADS16, using real-time PCR analysis on gain- and loss-of-function of OsMADS1 transgenic lines. OsMADS1|LHS1|AFO,OsMADS16|SPW1 MADS-box gene family in rice: genome-wide identification, organization and expression profiling during reproductive development and stress 2007 BMC Genomics Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, India. rita_genomics@yahoo.co.in BACKGROUND: MADS-box transcription factors, besides being involved in floral organ specification, have also been implicated in several aspects of plant growth and development. In recent years, there have been reports on genomic localization, protein motif structure, phylogenetic relationships, gene structure and expression of the entire MADS-box family in the model plant system, Arabidopsis. Though there have been some studies in rice as well, an analysis of the complete MADS-box family along with a comprehensive expression profiling was still awaited after the completion of rice genome sequencing. Furthermore, owing to the role of MADS-box family in flower development, an analysis involving structure, expression and functional aspects of MADS-box genes in rice and Arabidopsis was required to understand the role of this gene family in reproductive development. RESULTS: A genome-wide molecular characterization and microarray-based expression profiling of the genes encoding MADS-box transcription factor family in rice is presented. Using a thorough annotation exercise, 75 MADS-box genes have been identified in rice and categorized into MIKCc, MIKC*, Malpha, Mbeta and Mgamma groups based on phylogeny. Chromosomal localization of these genes reveals that 16 MADS-box genes, mostly MIKCc-type, are located within the duplicated segments of the rice genome, whereas most of the M-type genes, 20 in all, seem to have resulted from tandem duplications. Nine members belonging to the Mbeta group, which was considered absent in monocots, have also been identified. The expression profiles of all the MADS-box genes have been analyzed under 11 temporal stages of panicle and seed development, three abiotic stress conditions, along with three stages of vegetative development. Transcripts for 31 genes accumulate preferentially in the reproductive phase, of which, 12 genes are specifically expressed in seeds, and six genes show expression specific to panicle development. Differential expression of seven genes under stress conditions is also evident. An attempt has been made to gain insight into plausible functions of rice MADS-box genes by collating the expression data of functionally validated genes in rice and Arabidopsis. CONCLUSION: Only a limited number of MADS genes have been functionally validated in rice. A comprehensive annotation and transcriptome profiling undertaken in this investigation adds to our understanding of the involvement of MADS-box family genes during reproductive development and stress in rice and also provides the basis for selection of candidate genes for functional validation studies. OsMADS1|LHS1|AFO,OsMADS13,OsMADS14,OsMADS2,OSMADS3,OsMADS4,OsMADS5,OsMADS51|OsMADS65,OsMADS6|MFO1,OsMADS7|OsMADS45,OsMADS8|OsMADS24 Intragenic control of expression of a rice MADS box gene OsMADS1 2008 Mol Cells Plant Metabolism Research Center and Graduate School of Biotechnology, Kyung Hee University, Yongin 446-701, Korea. OsMADS1 is a rice MADS box gene necessary for floral development. To identify the key cis-regulatory regions for its expression, we utilized transgenic rice plants expressing GUS fusion constructs. Histochemical analysis revealed that the 5.7-kb OsMADS1 intragenic sequences, encompassing exon 1, intron 1, and a part of exon 2, together with the 1.9-kb 5' upstream promoter region, are required for the GUS expression pattern that coincides with flower-preferential expression of OsMADS1. In contrast, the 5' upstream promoter sequence lacking this intragenic region caused ectopic expression of the reporter gene in both vegetative and reproductive tissues. Notably, incorporation of the intragenic region into the CaMV35S promoter directed the GUS expression pattern similar to that of the endogenous spatial expression of OsMADS1 in flowers. In addition, our transient gene expression assay revealed that the large first intron following the CaMV35S minimal promoter enhances flower-preferential expression of GUS. These results suggest that the OsMADS1 intragenic sequence, largely intron 1, contains a key regulatory region(s) essential for expression. OsMADS1|LHS1|AFO The AGL6-like gene OsMADS6 regulates floral organ and meristem identities in rice 2009 Cell Res School of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai 200240, China. Although AGAMOUS-LIKE6 (AGL6) MADS-box genes are ancient with wide distributions in gymnosperms and angiosperms, their functions remain poorly understood. Here, we show the biological role of the AGL6-like gene, OsMADS6, in specifying floral organ and meristem identities in rice (Oryza sativa L.). OsMADS6 was strongly expressed in the floral meristem at early stages. Subsequently, OsMADS6 transcripts were mainly detectable in paleas, lodicules, carpels and the integument of ovule, as well as in the receptacle. Compared to wild type plants, osmads6 mutants displayed altered palea identity, extra glume-like or mosaic organs, abnormal carpel development and loss of floral meristem determinacy. Strikingly, mutation of a SEPALLATA (SEP)-like gene, OsMADS1 (LHS1), enhanced the defect of osmads6 flowers, and no inner floral organs or glume-like structures were observed in whorls 2 and 3 of osmads1-z osmads6-1 flowers. Furthermore, the osmads1-z osmads6-1 double mutants developed severely indeterminate floral meristems. Our finding, therefore, suggests that the ancient OsMADS6 gene is able to specify "floral state" by determining floral organ and meristem identities in monocot crop rice together with OsMADS1. OsMADS1|LHS1|AFO,OsMADS6|MFO1 Functional conservation and diversification of class E floral homeotic genes in rice (Oryza sativa) 2010 Plant J Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. Mutant analyses in different eudicotyledonous flowering plants demonstrated that SEPALLATA-like MADS-box genes are required for the specification of sepals, petals, stamens and carpels, and for floral determinacy, thus defining class E floral organ identity genes. SEP-like genes encode MADS-domain transcription factors and constitute an angiosperm-specific gene clade whose members show remarkably different degrees of redundancy and sub-functionalization within eudicots. To better understand the evolutionary dynamics of SEP-like genes throughout the angiosperms we have knocked down SEP-like genes of rice (Oryza sativa), a distant relative of eudicots within the flowering plants. Plants affected in both OsMADS7 and OsMADS8 show severe phenotypes including late flowering, homeotic changes of lodicules, stamens and carpels into palea/lemma-like organs, and a loss of floral determinacy. Simultaneous knockdown of the four rice SEP-like genes OsMADS1, OsMADS5, OsMADS7 and OsMADS8, leads to homeotic transformation of all floral organs except the lemma into leaf-like organs. This mimics the phenotype observed with the sep1 sep2 sep3 sep4 quadruple mutant of Arabidopsis. Detailed analyses of the spatial and temporal mRNA expression and protein interaction patterns corresponding to the different rice SEP-like genes show strong similarities, but also gene-specific differences. These findings reveal conservation of SEP-like genes in specifying floral determinacy and organ identities since the separation of eudicots and monocots about 150 million years ago. However, they indicate also monocot-specific neo- and sub-functionalization events and hence underscore the evolutionary dynamics of SEP-like genes. Moreover, our findings corroborate the view that the lodicules of grasses are homologous to eudicot petals. OsMADS1|LHS1|AFO,OsMADS5,OsMADS7|OsMADS45,OsMADS8|OsMADS24 DEP and AFO regulate reproductive habit in rice 2010 PLoS Genet State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. Sexual reproduction is essential for the life cycle of most angiosperms. However, pseudovivipary is an important reproductive strategy in some grasses. In this mode of reproduction, asexual propagules are produced in place of sexual reproductive structures. However, the molecular mechanism of pseudovivipary still remains a mystery. In this work, we found three naturally occurring mutants in rice, namely, phoenix (pho), degenerative palea (dep), and abnormal floral organs (afo). Genetic analysis of them indicated that the stable pseudovivipary mutant pho was a double mutant containing both a Mendelian mutation in DEP and a non-Mendelian mutation in AFO. Further map-based cloning and microarray analysis revealed that dep mutant was caused by a genetic alteration in OsMADS15 while afo was caused by an epigenetic mutation in OsMADS1. Thus, OsMADS1 and OsMADS15 are both required to ensure sexual reproduction in rice and mutations of them lead to the switch of reproductive habit from sexual to asexual in rice. For the first time, our results reveal two regulators for sexual and asexual reproduction modes in flowering plants. In addition, our findings also make it possible to manipulate the reproductive strategy of plants, at least in rice. OsMADS1|LHS1|AFO,OsMADS15|DEP leafy hull sterile1 Is a Homeotic Mutation in a Rice MADS Box Gene Affecting Rice Flower Development 2000 The Plant Cell Online National Research Laboratory of Plant Functional Genomics, Division of Molecular Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea. Rice contains several MADS box genes. It has been demonstrated previously that one of these genes, OsMADS1 (for Oryza sativa MADS box gene1), is expressed preferentially in flowers and causes early flowering when ectopically expressed in tobacco plants. In this study, we demonstrated that ectopic expression of OsMADS1 in rice also results in early flowering. To further investigate the role of OsMADS1 during rice flower development, we generated transgenic rice plants expressing altered OsMADS1 genes that contain missense mutations in the MADS domain. There was no visible alteration in the transgenic plants during the vegetative stage. However, transgenic panicles typically exhibited phenotypic alterations, including spikelets consisting of elongated leafy paleae and lemmas that exhibit a feature of open hull, two pairs of leafy palea-like and lemma-like lodicules, a decrease in stamen number, and an increase in the number of carpels. In addition, some spikelets generated an additional floret from the same rachilla. These characteristics are very similar to those of leafy hull sterile1 (lhs1). The map position of OsMADS1 is closely linked to that of lhs1 on chromosome 3. Examination of lhs1 revealed that it contains two missense mutations in the OsMADS1 MADS domain. A genetic complementation experiment showed that the 11.9-kb genomic DNA fragment containing the wild-type OsMADS1 gene rescued the mutant phenotypes. In addition, ectopic expression of the OsMADS1 gene isolated from the lhs1 line resulted in lhs1-conferred phenotypes. These lines of evidence demonstrate that OsMADS1 is the lhs1 gene. OsMADS1|LHS1|AFO The SEPALLATA-like gene OsMADS34 is required for rice inflorescence and spikelet development 2010 Plant Physiol School of Life Science and Biotechnology , Shanghai Jiaotong University, Shanghai 200240, China. Grass plants develop distinct inflorescences and spikelets that determine grain yields. However, the mechanisms underlying the specification of inflorescences and spikelets in grasses remain largely unknown. Here, we report the biological role of one SEPALLATA (SEP)-like gene, OsMADS34, in controlling the development of inflorescences and spikelets in rice (Oryza sativa). OsMADS34 encodes a MADS box protein containing a short carboxyl terminus without transcriptional activation activity in yeast cells. We demonstrate the ubiquitous expression of OsMADS34 in roots, leaves, and primordia of inflorescence and spikelet organs. Compared with the wild type, osmads34 mutants developed altered inflorescence morphology, with an increased number of primary branches and a decreased number of secondary branches. In addition, osmads34 mutants displayed a decreased spikelet number and altered spikelet morphology, with lemma/leaf-like elongated sterile lemmas. Moreover, analysis of the double mutant osmads34 osmads1 suggests that OsMADS34 specifies the identities of floral organs, including the lemma/palea, lodicules, stamens, and carpel, in combination with another rice SEP-like gene, OsMADS1. Collectively, our study suggests that the origin and diversification of OsMADS34 and OsMADS1 contribute to the origin of distinct grass inflorescences and spikelets. OsMADS1|LHS1|AFO,OsMADS34|PAP2 Ectopic expression of rice OsMADS1 reveals a role in specifying the lemma and palea, grass floral organs analogous to sepals 2001 Dev Genes Evol Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India. MADS-domain-containing transcription factors play diverse roles in plant development. The prototypic members of this gene family are the floral organ identity genes of the model dicotyledonous plant, Arabidopsis thaliana. Sequence relatedness and function ascribe them to AP1/AGL9, AG, AP3 and PI gene groups. The rice MADS-box gene, OsMADS1, is a member of the AP1/ AGL9 sub-group. Tomato and Petunia members of this sub-group specify floral meristem identity and control organ development in three inner whorls. Reported here are phylogenetic analyses that show OsMADS1 to form a distinct clade within the AGL9 gene family. This sub-group currently has only three other monocot genes. We have studied the expression pattern of OsMADS1 and determined the consequences of its ectopic expression in transgenic rice plants. OsMADS1 is not expressed during panicle branching; earliest expression is in spikelet meristems where it is excluded from the outer rudimentary/sterile glumes. During organogenesis, OsMADS1 expression is confined to the lemma and palea, with weak expression in the carpel. Ectopic OsMADS1 expression results in stunted panicles with irregularly positioned branches and spikelets. Additionally, in spikelets, the outer rudimentary glumes are transformed to lemma/palea-like organs. Together, these data suggest a distinct role for OsMADS1 and its monocot relatives in assigning lemma/palea identity. OsMADS1|LHS1|AFO Early flowering and reduced apical dominance result from ectopic expression of a rice MADS box gene 1994 Plant Mol Biol Institute of Biological Chemistry, Washington State University, Pullman 99164-6340. Recent studies with dicot plants reveal that floral organ development is controlled by a group of regulatory factors containing the MADS domain. In this study, we have isolated and characterized a cDNA clone from rice, OsMADS1, which encodes a MADS-domain-containing protein. The OsMADS1 amino acid sequence shows 56.2% identity to AGL2 and 44.4% identity to AP1. The MADS box region was the most homologous to other MADS-domain-containing proteins. Northern blot analysis indicated that the rice MADS gene was preferentially expressed in floral organs. In situ localization studies showed that the transcript was uniformly present in young flower primordia and later became localized in palea, lemma, and ovary. Ectopic expression of OsMAD1 with the CaMV 35S promoter in transgenic tobacco plants dramatically alters development, resulting in short, bushy, early-flowering plants with reduced apical dominance. These results suggest that the OsMADS1 gene is involved in flower induction and that it may be used for genetic manipulation of certain plant species. OsMADS1|LHS1|AFO Conservation of the E-function for floral organ identity in rice revealed by the analysis of tissue culture-induced loss-of-function mutants of the OsMADS1 gene 2005 Plant Mol Biol Plant Functional Genomics Laboratory (PFGL), Molecular Genetics Department, National Institute of Agrobiological Sciences (NIAS), Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. Rapid progress in studies on flower development has resulted in refining the classical 'ABC model' into a new 'ABCDE model' to explain properly the regulation of floral organ identity. Conservation of E-function for flower organ identity among the dicotyledonous (dicot) plants has been revealed. However, its conservation in monocotyledonous (monocot) plants remains largely unknown. Here, we show the conservation of E-function in rice (Oryza sativaL.) by characterizing tissue culture-induced mutants of two MADS-box genes, OsMADS1and OsMADS5, which form a subclade within the well-supported clade of SEP-genes (E-function) phylogeny. Severe loss-of-function mutations of OsMADS1cause complete homeotic conversion of organs (lodicules, stamens, and carpels) of three inner whorls into lemma- and palea-like structures. Such basic deformed structure is reiterated along with the pedicel at the center of the same floret, indicating the loss of determinacy of the flower meristem. These phenotypes resemble the phenotypes caused by mutations of the dicot E-class genes, such as the Arabidopsis SEP123(SEPALLATA1/2/3) and the petunia FBP2(Floral Binding Protein 2), suggesting that OsMADS1play a very similar role in rice to that of defined E-class genes in dicot plants. In case of the loss-of-function mutation of OsMADS5, no defect in either panicles or vegetative organs was observed. These results demonstrate that OsMADS1clearly possesses E-function, and so, E-function is fundamentally conserved between dicot plants and rice, a monocot model plant. OsMADS1|LHS1|AFO,OsMADS5 OsMADS13, a novel rice MADS-box gene expressed during ovule development 1999 Dev Genet Department of Genetics and Microbiology, University of Milan, Milan, Italy. MADS-box genes have been shown to play a major role in defining plant architecture. Recently, several MADS-box genes have been reported that are highly expressed in the ovule. However, only for the Petunia genes FBP7 and FBP11 has a function in defining ovule identity been shown. We have isolated a rice MADS-box gene named OsMADS13. Expression analysis has shown that this gene is highly expressed in developing ovules. In order to facilitate a detailed characterization of rice ovule-expressed genes, a comprehensive morphological description of ovule development in rice has been performed. The predicted amino acid sequence of OsMADS13 shows significant homology with ZAG2, a maize MADS-box gene, which is also expressed mainly in the ovule. Mapping of the gene in the rice genome showed that it is located on chromosome 12, which is syntenic to two maize regions where ZAG2 and its paralogous gene ZMM1 have been mapped. Our results suggest that OsMADS13 is the ortholog of ZAG2 and ZMM1 and might play a role in rice ovule and seed development. OsMADS13 Functional analysis of all AGAMOUS subfamily members in rice reveals their roles in reproductive organ identity determination and meristem determinacy 2011 Plant Cell Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita degli Studi di Milano, 20133 Milan, Italy. Reproductive organ development is one of the most important steps in the life cycle of plants. Studies using core eudicot species like thale cress (Arabidopsis thaliana) and snapdragon (Antirrhinum majus) have shown that MADS domain transcription factors belonging to the AGAMOUS (AG) subfamily regulate the identity of stamens, carpels, and ovules and that they are important for floral meristem determinacy. Here, we investigate the genetic interactions between the four rice (Oryza sativa) AG subfamily members, MADS3, MADS13, MADS21, and MADS58. Our data show that, in contrast with previous reports, MADS3 and MADS58 determine stamen and carpel identity and, together with MADS13, are important for floral meristem determinacy. In the mads3 mads58 double mutant, we observed a complete loss of reproductive organ identity and massive accumulation of lodicules in the third and fourth floral whorls. MADS21 is an AGL11 lineage gene whose expression is not restricted to ovules. Instead, its expression profile is similar to those of class C genes. However, our genetic analysis shows that MADS21 has no function in stamen, carpel, or ovule identity determination. OsMADS13,OsMADS21,OSMADS3,OSMADS58 The D-lineage MADS-box gene OsMADS13 controls ovule identity in rice 2007 Plant J Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy. Genes that control ovule identity were first identified in Petunia. Co-suppression of both FLORAL BINDING PROTEIN 7 (FBP7) and FBP11, two D-lineage genes, resulted in the homeotic transformation of ovules into carpelloid structures. Later in Arabidopsis it was shown that three genes, SHATTERPROOF1 (SHP1), SHP2, and SEEDSTICK (STK), redundantly control ovule identity, because in the stk shp1 shp2 triple mutant ovules lose identity and are transformed into carpel and leaf-like structures. Of these three Arabidopsis genes STK is the only D-lineage gene, and its expression, like FBP7 and FBP11, is restricted to ovules. OsMADS13 is the rice ortholog of STK, FBP7, and FBP11. Its amino acid sequence is similar to the Arabidopsis and Petunia proteins, and its expression is also restricted to ovules. We show that the osmads13 mutant is female sterile and that ovules are converted into carpelloid structures. Furthermore, making carpels inside carpels, the osmads13 flower is indeterminate, showing that OsMADS13 also has a function in floral meristem determinacy. OsMADS21 is most likely to be a paralog of OsMADS13, although its expression is not restricted to ovules. Interestingly, the osmads21 mutant did not show any obvious phenotype. Furthermore, combining the osmads13 and the osmads21 mutants did not result in any additive ovule defect, indicating that osmads21 does not control ovule identity. These results suggest that during evolution the D-lineage gene OsMADS21 has lost its ability to determine ovule identity. OsMADS13,OsMADS21 Overexpression of a Transcription Factor OsMADS15 Modifies Plant Architecture and Flowering Time in Rice (Oryza sativa L.) 2012 Plant Molecular Biology Reporter Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, Yuhangtang Road 866, Hangzhou, 310058, China The MADS-box gene family encodes conserved transcription factors and functions not only in reproductive development, but also in vegetative growth. In this study, a rice MADS-box gene OsMADS15, an ortholog of Arabidopsis AP1 gene, has been functionally characterized. Rice seedlings overexpressing OsMADS15 showed precocious phenotypes of early internode elongation, shoot-borne crown root development, reduced plant height and early flowering. The axillary buds developments in OsMADS15 overexpressors were accelerated, and the buds frequently grew into effective tillers. The panicles of OsMADS15 transgenic rice plants were largely compromised for growth and branching in comparison with wild type. In the tillering stage, the OsMADS15 overexpression rice plants tillered later and less than the wild type, and in the maturity stage, the culms of the overexpression lines bore more stem nodes. Quantitative polymerase chain reaction (PCR) results showed that expression levels of WUSCHEL-related Homeobox (WOX) gene, WOX11, and some flowering regulators were promoted in the OsMADS15 overexpression transgenic plants, indicating that OsMADS15 had a wide range of regulations. These results clearly indicate that OsMADS15 plays important roles not only in transition to reproductive development, but also in crown root development. OsMADS15|DEP,WOX11 Functional analysis of the rice AP3 homologue OsMADS16 by RNA interference 2003 Plant Mol Biol Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 917 Bld, Datun Rd, Andingmen-wai, Beijing 100101, China. The rice OsMADS16 gene is phylogenetically related to the angiosperm B-function MADS-box genes. To investigate if OsMADS16 functions as an AP3/DEF orthologue to regulate the development of lodicules and stamens in rice, we isolated its genomic sequences and characterized its functions in planta by RNA interference. The genomic sequence of the OsMADS16 gene shows that it shares high similarity in genomic structure and the deduced amino acid sequence with the maize B-class gene, Si1. Transgenic lines from the introduced gene expressing double-stranded RNA with the OsMADS16 cDNA fragment were male-sterile and displayed alternations of lodicules and stamens, occasionally depressed palea and overgrown glume. The two lodicules were converted into four palea/lemma-like organs and some stamens into carpels. Further investigations of the transcription of OsMADS16 gene in these transgenic lines by RT-PCR revealed that its transcript was significantly reduced. Transcription of a rice PI homologous gene, OsMADS4, was also reduced remarkably in the transgenic plants. Our results demonstrate that OsMADS16 is an AP3/DEF orthologue to specify the identities of lodicules and stamens in rice flower and also support that OsMADS4 is a PI orthologue. In addition, these results suggest that RNA interference is a useful tool for functional genomics in rice. OsMADS16|SPW1,OsMADS4 DNA polymorphism in the SUPERWOMAN1 (SPW1) locus of the wild rice Oryza rufipogon and its related species 2008 Genes Genet Syst Laboratory of Plant Genetics, Graduate School of Agriculture, Kyoto University The level and pattern of nucleotide variation in the SUPERWOMAN1(SPW1) locus of the wild rice Oriza rufipogon and its related species were analyzed. SPW1 is orthologous to APETELA3(AP3) in Arabidopsis thaliana. The estimated level of nucleotide variation for the entire region (π = 0.0046) was intermediate among those for other genes of O. rufipogon, although the estimates varied considerably among the SPW1 domains. Complicated haplotype structure was detected, resulting in a high proportion of significant linkage disequilibrium. Deviation from the neutrality was not detected. However, purifying selection was suggested by lack of replacement variation in the MADS-box and I-domain regions, which function as DNA-binding domain. On the other hand, an excess of drastic amino acid changes was detected in the C-domain, as in the AP3 region of A. thaliana. Taken together, these results imply that different types of natural selection are acting among different domains of a single protein. In the phylogenetic tree, O. sativa strains were included in the same cluster of O. rufipogon, supporting the hypothesis that O. rufipogon is the wild ancestor of O. sativa. OsMADS16|SPW1 superwoman1-cleistogamy, a hopeful allele for gene containment in GM rice 2007 Plant Biotechnol J Hokuriku Research Center, National Agricultural Research Center, Niigata 943-0193, Japan. yocida@affrc.go.jp Cleistogamy is an efficient strategy for preventing gene flow from genetically modified (GM) crops. We identified a cleistogamous mutant of rice harbouring a missense mutation (the 45th residue isoleucine to threonine; I45T) in the class-B MADS-box gene SUPERWOMAN1 (SPW1), which specifies the identities of lodicules (equivalent to petals) and stamens. In the mutant, spw1-cls, the stamens are normal, but the lodicules are transformed homeotically to lodicule-glume mosaic organs, thereby engendering cleistogamy. Since this mutation does not affect other agronomic traits, it can be used in crosses to produce transgenic lines that do not cause environmental perturbation. Molecular analysis revealed that the reduced heterodimerization ability of SPW1(I45T) with its counterpart class-B proteins OsMADS2 and OsMADS4 caused altered lodicule identity. spw1-cls is the first useful mutant for practical gene containment in GM rice. Cleistogamy is possible in many cereals by engineering class-B floral homeotic genes and thereby inducing lodicule identity changes. OsMADS16|SPW1 Alteration of floral organ identity in rice through ectopic expression of OsMADS16 2003 Planta National Research Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology (POSTECH), 790-784 Pohang, Korea. We used a transgenic approach and yeast two-hybrid experiments to study the role of the rice ( Oryza sativa L.) B-function MADS-box gene, OsMADS16. Transgenic rice plants were generated that ectopically expressed OsMADS16 under the control of the maize ( Zea mays L.) ubiquitin1 promoter. Microscopic observations revealed that the innermost-whorl carpels had been replaced by stamen-like organs, which resembled the flowers of the previously described Arabidopsis thaliana (L.) Heynh. mutation superman as well as those ectopically expressing the AP3 gene. These results indicate that expression of OsMADS16 in the innermost whorl induces stamen development. Occasionally, carpels had completely disappeared. In addition, ectopic expression of OsMADS16 enhanced expression of OsMADS4, another B-function gene, causing superman phenotypes. In the yeast two-hybrid system, OsMADS16 did not form a homodimer but, rather, the protein interacted with OsMADS4. OsMADS16 also interacted with OsMADS6 and OSMADS8, both of which are homologous to SEPALLATA proteins required for the proper function of class-B and class-C genes in Arabidopsis. Based on the gene expression pattern and our yeast two-hybrid data, we discuss a quartet model of MADS-domain protein interactions in the lodicule and stamen whorls of rice florets. OsMADS16|SPW1,OsMADS4,OsMADS6|MFO1,OsMADS8|OsMADS24 Ternary complex formation between MADS-box transcription factors and the histone fold protein NF-YB 2002 J Biol Chem Dipartimento di Genetica e Biologia dei Microrganismi, Universita di Milano, Via Celoria 26, 20133 Milano, Italy. MADS-box proteins are transcription factors present in different eukaryotic kingdoms. In contrast to plants, for mammalian and yeast MADS-box proteins ternary complex formation with unrelated transcription factors was reported. We show here the first identification of such ternary interaction in plants. A rice seed-specific NF-YB was identified as partner of OsMADS18 by two-hybrid screening. NF-YB contains a histone fold motif, HFM,(1) and is part of the trimeric CCAAT-binding NF-Y complex. OsMADS18, alone or in combination with a natural partner, interacts with OsNF-YB1 through the MADS and I regions. The mouse NF-YB also associates with OsMADS18 in vivo and in vitro as a NF-YB-NF-YC dimer. Other rice MADS-box proteins do not interact in these assays, indicating specificity for the interaction. OsNF-YB1 is capable of heterodimerizing with NF-YC, but not trimerizing with NF-YA, thus precluding CCAAT binding. Mutation of the variant Asp at position 99 of the HFM alpha2-helix into a conserved serine recovers the capacity to interact with NF-YA, but not with DNA. This is the first indication that members of the NF-YB family work through mechanisms independent of the CCAAT box. OsMADS18,OsNF-YB1 Functional characterization of OsMADS18, a member of the AP1/SQUA subfamily of MADS box genes 2004 Plant Physiol Dipartimento di Biologia, Universita degli Studi di Milano, 20133 Milan, Italy. MADS box transcription factors controlling flower development have been isolated and studied in a wide variety of organisms. These studies have shown that homologous MADS box genes from different species often have similar functions. OsMADS18 from rice (Oryza sativa) belongs to the phylogenetically defined AP1/SQUA group. The MADS box genes of this group have functions in plant development, like controlling the transition from vegetative to reproductive growth, determination of floral organ identity, and regulation of fruit maturation. In this paper we report the functional analysis of OsMADS18. This rice MADS box gene is widely expressed in rice with its transcripts accumulated to higher levels in meristems. Overexpression of OsMADS18 in rice induced early flowering, and detailed histological analysis revealed that the formation of axillary shoot meristems was accelerated. Silencing of OsMADS18 using an RNA interference approach did not result in any visible phenotypic alteration, indicating that OsMADS18 is probably redundant with other MADS box transcription factors. Surprisingly, overexpression of OsMADS18 in Arabidopsis caused a phenotype closely resembling the ap1 mutant. We show that the ap1 phenotype is not caused by down-regulation of AP1 expression. Yeast two-hybrid experiments showed that some of the natural partners of AP1 interact with OsMADS18, suggesting that the OsMADS18 overexpression phenotype in Arabidopsis is likely to be due to the subtraction of AP1 partners from active transcription complexes. Thus, when compared to AP1, OsMADS18 during evolution seems to have conserved the mechanistic properties of protein-protein interactions, although it cannot complement the AP1 function. OsMADS18 Double-stranded RNA interference of a rice PI/GLO paralog, OsMADS2, uncovers its second-whorl-specific function in floral organ patterning 2003 Genetics Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India. Unlike many eudicot species, grasses have duplicated PI/GLO-like genes. Functional analysis of one of the rice PI/GLO paralogs, OsMADS2, is reported here. Our data demonstrate its essential role in lodicule development and implicate the second PI/GLO paralog, OsMADS4, to suffice for stamen specification. We provide the first evidence for differential contributions of grass PI/GLO paralogs in patterning second- and third-whorl floral organs. OsMADS2,OsMADS4 Divergent regulatory OsMADS2 functions control size, shape and differentiation of the highly derived rice floret second-whorl organ 2007 Genetics Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India. Functional diversification of duplicated genes can contribute to the emergence of new organ morphologies. Model eudicot plants like Arabidopsis thaliana and Antirrhinum majus have a single PI/GLO gene that together with AP3/DEF regulate petal and stamen formation. Lodicules of grass flowers are morphologically distinct reduced organs occupying the position of petals in other flowers. They serve a distinct function in partial and transient flower opening to allow stamen emergence and cross-pollination. Grasses have duplicated PI/GLO-like genes and in rice (Oryza sativa) one these genes, OsMADS2, controls lodicule formation without affecting stamen development. In this study, we investigate the mechanistic roles played by OsMADS2. We ascribe a function for OsMADS2 in controlling cell division and differentiation along the proximal-distal axis. OsMADS2 is required to trigger parenchymatous and lodicule-specific vascular development while maintaining a small organ size. Our data implicate the developmentally late spatially restricted accumulation of OsMADS2 transcripts in the differentiating lodicule to control growth of these regions. The global architecture of transcripts regulated by OsMADS2 gives insights into the regulation of cell division and vascular differentiation that together can form this highly modified grass organ with important functions in floret opening and stamen emergence independent of the paralogous gene OsMADS4. OsMADS2,OsMADS4 Unequal genetic redundancy of rice PISTILLATA orthologs, OsMADS2 and OsMADS4, in lodicule and stamen development 2008 Plant Cell Physiol Hokuriku Research Center, National Agricultural Research Center, Niigata, 943-0193 Japan. Two homologs of PISTILLATA have been identified in rice: OsMADS2 and OsMADS4. However, their roles in floral organ development are controversial. Here, we demonstrate that the genes show unequal redundancy of class B function. Although OsMADS2 plays an important role in lodicule development, OsMADS4 also supports the specification of lodicule identity. In contrast, the genes are roughly equally important in stamen development. Consistent with their redundant functions, both OsMADS2 and OsMADS4 interact with the unique rice AP3 ortholog SPW1. OsMADS2,OsMADS4 Functional conservation and diversification between rice OsMADS22/OsMADS55 and Arabidopsis SVP proteins 2012 Plant Sci Creative Research Initiatives, Division of Life Sciences, Korea University, Seoul 136-701, Republic of Korea. MADS-box transcription factors play pivotal roles in several aspects of plant growth and development. The Arabidopsis SHORT VEGETATIVE PHASE (SVP) protein mediates the integration of signals involved in the control of flowering time and flower development by interacting with MADS-box proteins. In the rice genome, three SVP-like genes (OsMADS22, OsMADS47, and OsMADS55) are present. To investigate the functional conservation of these SVP-like genes in rice and Arabidopsis, the phenotypes of transgenic Arabidopsis plants overexpressing OsMADS22 and OsMADS55 were analyzed. Overexpression of OsMADS22 and OsMADS55 led to abnormal floral morphologies including leaf-like sepals, whereas only OsMADS55 expression caused delayed flowering via downregulation of FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). Yeast two-hybrid assays revealed that OsMADS22 and OsMADS55 interacted with Arabidopsis AGL24 and AP1, but only OsMADS55 interacted with FLC. Overexpression of OsMADS55, but not OsMADS22, complemented the early flowering phenotype and ambient temperature-insensitive flowering phenotype seen in svp mutants, suggesting that OsMADS55 regulates flowering time associated with ambient temperature responses in Arabidopsis. Taken together, our data are consistent with functional conservation and diversification between Arabidopsis and rice SVP-like genes involved in controlling flowering time and flower development. OsMADS22 OsMADS22, an STMADS11-like MADS-box gene of rice, is expressed in non-vegetative tissues and its ectopic expression induces spikelet meristem indeterminacy 2005 Mol Genet Genomics National Agricultural Research Center for Hokkaido Region, Toyohira, Sapporo, 062-8555, Japan. sennao@affrc.go.jp We report the cDNA sequence and gene expression patterns of OsMADS22, a novel member of the STMADS11-like family of MADS-box genes, from rice. In contrast to previously reported STMADS11-like genes, whose expression is detected in vegetative tissues, OsMADS22 is mainly expressed during embryogenesis and flower development. In situ hybridization analysis revealed that OsMADS22 expression is localized in the L1 layer of embryos and in developing stamen primordia. Ectopic expression of OsMADS22 in transgenic rice plants resulted in aberrant floral morphogenesis, characterized by a disorganized palea, an elongated glume, and a two-floret spikelet. The results are discussed in terms of rice spikelet development and a novel non-vegetative role for a STMADS11-like gene. OsMADS22 Further characterization of a rice AGL12 group MADS-box gene, OsMADS26 2008 Plant Physiol Department of Life Science and National Research Laboratory of Plant Functional Genomics, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea. Plant MADS-box genes can be divided into 11 groups. Genetic analysis has revealed that most of them function in flowering-time control, reproductive organ development, and vegetative growth. Here, we elucidated the role of OsMADS26, a member of the AGL12 group. Transcript levels of OsMADS26 were increased in an age-dependent manner in the shoots and roots. Transgenic plants of both rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) overexpressing this gene manifested phenotypes related to stress responses, such as chlorosis, cell death, pigment accumulation, and defective root/shoot growth. In addition, apical hook development was significantly suppressed in Arabidopsis. Plants transformed with the OsMADS26-GR (glucocorticoid receptor) fusion construct displayed those stress-related phenotypes when treated with dexamethasone. Microarray analyses using this inducible system showed that biosynthesis genes for jasmonate, ethylene, and reactive oxygen species, as well as putative downstream targets involved in the stress-related process, were up-regulated in OsMADS26-overexpressing plants. These results suggest that OsMADS26 induces multiple responses that are related to various stresses. OsMADS26 Functional delineation of rice MADS29 reveals its role in embryo and endosperm development by affecting hormone homeostasis 2013 J Exp Bot Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India. Rice MADS29 has recently been reported to cause programmed cell death of maternal tissues, the nucellus, and the nucellar projection during early stages of seed development. However, analyses involving OsMADS29 protein expression domains and characterization of OsMADS29 gain-of-function and knockdown phenotypes revealed novel aspects of its function in maintaining hormone homeostasis, which may have a role in the development of embryo and plastid differentiation and starch filling in endosperm cells. The MADS29 transcripts accumulated to high levels soon after fertilization; however, protein accumulation was found to be delayed by at least 4 days. Immunolocalization studies revealed that the protein accumulated initially in the dorsal-vascular trace and the outer layers of endosperm, and subsequently in the embryo and aleurone and subaleurone layers of the endosperm. Ectopic expression of MADS29 resulted in a severely dwarfed phenotype, exhibiting elevated levels of cytokinin, thereby suggesting that cytokinin biosynthesis pathway could be one of the major targets of OsMADS29. Overexpression of OsMADS29 in heterologous BY2 cells was found to mimic the effects of exogenous application of cytokinins that causes differentiation of proplastids to starch-containing amyloplasts and activation of genes involved in the starch biosynthesis pathway. Suppression of MADS29 expression by RNAi severely affected seed set. The surviving seeds were smaller in size, with developmental abnormalities in the embryo and reduced size of endosperm cells, which also contained loosely packed starch granules. Microarray analysis of overexpression and knockdown lines exhibited altered expression of genes involved in plastid biogenesis, starch biosynthesis, cytokinin signalling and biosynthesis. OsMADS29 The MADS29 transcription factor regulates the degradation of the nucellus and the nucellar projection during rice seed development 2012 Plant Cell National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 20032 Shanghai, China. The MADS box transcription factors are critical regulators of rice (Oryza sativa) reproductive development. Here, we here report the functional characterization of a rice MADS box family member, MADS29, which is preferentially expressed in the nucellus and the nucellar projection. Suppressed expression of MADS29 resulted in abnormal seed development; the seeds were shrunken, displayed a low grain-filling rate and suppressed starch biosynthesis, and contained abnormal starch granules. Detailed analysis indicated that the abnormal seed development is due to defective programmed cell death (PCD) of the nucellus and nucellar projection, which was confirmed by a TUNEL assay and transcriptome analysis. Further studies showed that expression of MADS29 is induced by auxin and MADS29 protein binds directly to the putative promoter regions of genes that encode a Cys protease and nucleotide binding site-Leu-rich repeat proteins, thereby stimulating the PCD. This study identifies MADS29 as a key regulator of early rice seed development by regulating the PCD of maternal tissues. It provides informative clues to elucidate the regulatory mechanism of maternal tissue degradation after fertilization and to facilitate the studies of endosperm development and seed filling. OsMADS29 Live and let die - the B(sister) MADS-box gene OsMADS29 controls the degeneration of cells in maternal tissues during seed development of rice (Oryza sativa) 2012 PLoS One Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, PR China. B(sister) genes have been identified as the closest relatives of class B floral homeotic genes. Previous studies have shown that B(sister) genes from eudicots are involved in cell differentiation during ovule and seed development. However, the complete function of B(sister) genes in eudicots is masked by redundancy with other genes and little is known about the function of B(sister) genes in monocots, and about the evolution of B(sister) gene functions. Here we characterize OsMADS29, one of three MADS-box B(sister) genes in rice. Our analyses show that OsMADS29 is expressed in female reproductive organs including the ovule, ovule vasculature, and the whole seed except for the outer layer cells of the pericarp. Knock-down of OsMADS29 by double-stranded RNA-mediated interference (RNAi) results in shriveled and/or aborted seeds. Histological analyses of the abnormal seeds at 7 days after pollination (DAP) indicate that the symplastic continuity, including the ovular vascular trace and the nucellar projection, which is the nutrient source for the filial tissue at early development stages, is affected. Moreover, degeneration of all the maternal tissues in the transgenic seeds, including the pericarp, ovular vascular trace, integuments, nucellar epidermis and nucellar projection, is blocked as compared to control plants. Our results suggest that OsMADS29 has important functions in seed development of rice by regulating cell degeneration of maternal tissues. Our findings provide important insights into the ancestral function of B(sister) genes. OsMADS29 Functional diversification of the two C-class MADS box genes OSMADS3 and OSMADS58 in Oryza sativa 2006 Plant Cell Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Japan. The C-class MADS box gene AGAMOUS (AG) plays crucial roles in Arabidopsis thaliana development by regulating the organ identity of stamens and carpels, the repression of A-class genes, and floral meristem determinacy. To examine the conservation and diversification of C-class gene function in monocots, we analyzed two C-class genes in rice (Oryza sativa), OSMADS3 and OSMADS58, which may have arisen by gene duplication before divergence of rice and maize (Zea mays). A knockout line of OSMADS3, in which the gene is disrupted by T-DNA insertion, shows homeotic transformation of stamens into lodicules and ectopic development of lodicules in the second whorl near the palea where lodicules do not form in the wild type but carpels develop almost normally. By contrast, RNA-silenced lines of OSMADS58 develop astonishing flowers that reiterate a set of floral organs, including lodicules, stamens, and carpel-like organs, suggesting that determinacy of the floral meristem is severely affected. These results suggest that the two C-class genes have been partially subfunctionalized during rice evolution (i.e., the functions regulated by AG have been partially partitioned into two paralogous genes, OSMADS3 and OSMADS58, which were produced by a recent gene duplication event in plant evolution). OSMADS3,OSMADS58 Rice MADS3 regulates ROS homeostasis during late anther development 2011 Plant Cell School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. The rice (Oryza sativa) floral homeotic C-class gene, MADS3, was previously shown to be required for stamen identity determination during early flower development. Here, we describe a role for MADS3 in regulating late anther development and pollen formation. Consistent with this role, MADS3 is highly expressed in the tapetum and microspores during late anther development, and a newly identified MADS3 mutant allele, mads3-4, displays defective anther walls, aborted microspores, and complete male sterility. During late anther development, mads3-4 exhibits oxidative stress-related phenotypes. Microarray analysis revealed expression level changes in many genes in mads3-4 anthers. Some of these genes encode proteins involved in reactive oxygen species (ROS) homeostasis; among them is MT-1-4b, which encodes a type 1 small Cys-rich and metal binding protein. In vivo and in vitro assays showed that MADS3 is associated with the promoter of MT-1-4b, and recombinant MT-1-4b has superoxide anion and hydroxyl radical scavenging activity. Reducing the expression of MT-1-4b causes decreased pollen fertility and an increased level of superoxide anion in transgenic plants. Our findings suggest that MADS3 is a key transcriptional regulator that functions in rice male reproductive development, at least in part, by modulating ROS levels through MT-1-4b. OSMADS3 Ectopic Expression of OsMADS3, a Rice Ortholog of AGAMOUS, Caused a Homeotic Transformation of Lodicules to Stamens in Transgenic Rice Plants 2002 Plant and Cell Physiology Laboratory for Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, 630-0101 Japan In order to clarify the evolutionary relationship of floral organs between grasses and dicots, we expressed OsMADS3, a rice (Oryza sativa L.) AGAMOUS(AG) ortholog, in rice plants under the control of an Actin1 promoter. As a consequence of the ectopic expression of the OsMADS3, lodicules were homeotically transformed into stamens. In total, the transformation of lodicules to staminoid organs was observed in 18 out of 26 independent transgenic lines. In contrast to the almost complete transformation occurring in lodicules, none of the transgenic plants exhibited any morphological alterations in the palea or the lemma. Our results confirmed the prediction that the lodicule is an equivalent of a dicot petal and that the ABC model can be applied to rice at least for organ specification in lodicules and stamens. OSMADS3 The pleiotropic SEPALLATA-like gene OsMADS34 reveals that the 'empty glumes' of rice (Oryza sativa) spikelets are in fact rudimentary lemmas 2014 New Phytol Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China; University of Chinese Academy of Sciences, Beijing, 100039, China. The single floret of the rice (Oryza sativa) spikelet is subtended by a pair of enigmatic organs usually termed 'empty glumes' or 'sterile lemmas'. As the identity of these organs remains essentially unknown, we refer to them as 'organs of unknown identity' (OUIs). Here we present a novel mutant of the rice SEPALLATA-like gene OsMADS34 which develops, in addition to disorganized branches and sterile seeds, elongated OUIs. The function and evolution of OsMADS34 were studied. Morphological and molecular markers indicate that the elongated OUIs have adopted lemma identity. Evolutionary analyses show that the ancestral genes of the OsM34 subclade evolved under positive selection, and that three specific motifs occur in the C-terminal region of proteins in the OsM34 subclade. Yeast two-hybrid assays revealed that the C-terminal region of OsMADS34 plays a crucial role in mediating protein interactions. Sequence analyses for the wild rice Oryza grandiglumis which has elongated OUIs revealed the association of OsMADS34 functionality with OUI identity. Our findings support the hypothesis that OUIs originated from the lemmas of degenerate florets under the negative control of OsMADS34. As OUIs neither are homologues to glumes nor have the identity of lemmas any more, but originated from these organs, we suggest calling them 'rudimentary lemmas'. OsMADS34|PAP2 PANICLE PHYTOMER2 (PAP2), encoding a SEPALLATA subfamily MADS-box protein, positively controls spikelet meristem identity in rice 2010 Plant Cell Physiol Graduate School of Agriculture and Life Sciences, University of Tokyo, Tokyo, Japan. In rice panicle development, new meristems are generated sequentially in an organized manner and acquire their identity in a time- and position-dependent manner. In the panicle of the panicle phytomer2-1 (pap2-1) mutant, the pattern of meristem initiation is disorganized and newly formed meristems show reduced competency to become spikelet meristems, resulting in the transformation of early arising spikelets into rachis branches. In addition, rudimentary glumes and sterile lemmas, the outermost organs of the spikelet, elongate into a leafy morphology. We propose that PAP2 is a positive regulator of spikelet meristem identity. Map-based cloning revealed that PAP2 encodes OsMADS34, a member of the SEPALLATA (SEP) subfamily of MADS-box proteins. PAP2/OsMADS34 belongs to the LOFSEP subgroup of MADS-box genes that show grass-specific diversification caused by gene duplication events. All five SEP subfamily genes in rice are expressed exclusively during panicle development, while their spatial and temporal expression patterns vary. PAP2 expression starts the earliest among the five SEP genes, and a low but significant level of PAP2 mRNA was detected in the inflorescence meristem, in branch meristems immediately after the transition, and in glume primordia, consistent with its role in the early development of spikelet formation. Our study provides new evidence supporting the hypothesis that the genes of the LOFSEP subgroup control developmental processes that are unique to grass species. OsMADS34|PAP2 Reciprocal control of flowering time by OsSOC1 in transgenic Arabidopsis and by FLC in transgenic rice 2003 Plant Biotechnol J CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia. In a screen for MADS box genes which activate and/or repress flowering in rice, we identified a gene encoding a MADS domain protein (OsSOC1) related to the Arabidopsis gene AtSOC1. AtSOC1 and OsSOC1 show a 97% amino acid similarity in their MADS domain. The rice gene contains a large first intron of 27.6 kb compared to the 1 kb intron in Arabidopsis. OsSOC1 is located on top of the short arm of chromosome 3, tightly linked to the heading date locus, Hd9. OsSOC1 is expressed in vegetative tissues, and expression is elevated at the time of floral initiation, 40-50 days after sowing, and remains uniformly high thereafter, similar to the expression pattern of AtSOC1. The constitutive expression of OsSOC1 in Arabidopsis results in early flowering, suggesting that the rice gene is a functional equivalent of AtSOC1. We were not able to identify FLC-like sequences in the rice genome; however, we show that ectopic expression of the Arabidopsis FLC delays flowering in rice, and the up-regulation of OsSOC1 at the onset of flowering initiation is delayed in the AtFLC transgenic lines. The reciprocal recognition and flowering time effects of genes introduced into either Arabidopsis or rice suggest that some components of the flowering pathways may be shared. This points to a potential application in the manipulation of flowering time in cereals using well characterized Arabidopsis genes. OsMADS50|OsSOC1|DTH3 Distinct regulatory role for RFL, the rice LFY homolog, in determining flowering time and plant architecture 2008 Proc Natl Acad Sci U S A Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India. Activity of axillary meristems dictates the architecture of both vegetative and reproductive parts of a plant. In Arabidopsis thaliana, a model eudicot species, the transcription factor LFY confers a floral fate to new meristems arising from the periphery of the reproductive shoot apex. Diverse orthologous LFY genes regulate vegetative-to-reproductive phase transition when expressed in Arabidopsis, a property not shared by RFL, the homolog in the agronomically important grass, rice. We have characterized RFL by knockdown of its expression and by its ectopic overexpression in transgenic rice. We find that reduction in RFL expression causes a dramatic delay in transition to flowering, with the extreme phenotype being no flowering. Conversely, RFL overexpression triggers precocious flowering. In these transgenics, the expression levels of known flowering time genes reveal RFL as a regulator of OsSOC1 (OsMADS50), an activator of flowering. Aside from facilitating a transition of the main growth axis to an inflorescence meristem, RFL expression status affects vegetative axillary meristems and therefore regulates tillering. The unique spatially and temporally regulated RFL expression during the development of vegetative axillary bud (tiller) primordia and inflorescence branch primordia is therefore required to produce tillers and panicle branches, respectively. Our data provide mechanistic insights into a unique role for RFL in determining the typical rice plant architecture by regulating distinct downstream pathways. These results offer a means to alter rice flowering time and plant architecture by manipulating RFL-mediated pathways. OsMADS50|OsSOC1|DTH3,RFL|APO2 Distinct regulatory role for RFL, the rice LFY homolog, in determining flowering time and plant architecture 2008 Proceedings of the National Academy of Sciences Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India. Activity of axillary meristems dictates the architecture of both vegetative and reproductive parts of a plant. In Arabidopsis thaliana, a model eudicot species, the transcription factor LFY confers a floral fate to new meristems arising from the periphery of the reproductive shoot apex. Diverse orthologous LFY genes regulate vegetative-to-reproductive phase transition when expressed in Arabidopsis, a property not shared by RFL, the homolog in the agronomically important grass, rice. We have characterized RFL by knockdown of its expression and by its ectopic overexpression in transgenic rice. We find that reduction in RFL expression causes a dramatic delay in transition to flowering, with the extreme phenotype being no flowering. Conversely, RFL overexpression triggers precocious flowering. In these transgenics, the expression levels of known flowering time genes reveal RFL as a regulator of OsSOC1 (OsMADS50), an activator of flowering. Aside from facilitating a transition of the main growth axis to an inflorescence meristem, RFL expression status affects vegetative axillary meristems and therefore regulates tillering. The unique spatially and temporally regulated RFL expression during the development of vegetative axillary bud (tiller) primordia and inflorescence branch primordia is therefore required to produce tillers and panicle branches, respectively. Our data provide mechanistic insights into a unique role for RFL in determining the typical rice plant architecture by regulating distinct downstream pathways. These results offer a means to alter rice flowering time and plant architecture by manipulating RFL-mediated pathways. OsMADS50|OsSOC1|DTH3,RFL|APO2,RFT1 A simple method for predicting the functional differentiation of duplicate genes and its application to MIKC-type MADS-box genes 2005 Nucleic Acids Res Institute of Molecular Evolutionary Genetics and Department of Biology, Pennsylvania State University University Park, PA 16802, USA. JYN101@PSU.EDU A simple statistical method for predicting the functional differentiation of duplicate genes was developed. This method is based on the premise that the extent of functional differentiation between duplicate genes is reflected in the difference in evolutionary rate because the functional change of genes is often caused by relaxation or intensification of functional constraints. With this idea in mind, we developed a window analysis of protein sequences to identify the protein regions in which the significant rate difference exists. We applied this method to MIKC-type MADS-box proteins that control flower development in plants. We examined 23 pairs of sequences of floral MADS-box proteins from petunia and found that the rate differences for 14 pairs are significant. The significant rate differences were observed mostly in the K domain, which is important for dimerization between MADS-box proteins. These results indicate that our statistical method may be useful for predicting protein regions that are likely to be functionally differentiated. These regions may be chosen for further experimental studies. OsMADS56 Male-sterile and cleistogamous phenotypes in tall fescue induced by chimeric repressors of SUPERWOMAN1 and OsMADS58 2012 Plant Sci Forage Crop Research Division, NARO Institute of Livestock and Grassland Science, 768 Senbonmatsu, Nasushiobara, Tochigi 329-2793, Japan. s.hiroko@affrc.go.jp Since tall fescue (Festuca arundinacea Schreb.) is an anemophilous (wind-pollinated) grass species, male sterility is strongly desired for transgenic tall fescue to prevent pollen dispersal. To create male-sterile tall fescue, we applied Chimeric REpressor gene-Silencing Technology (CRES-T) based on rice APETALA3 (AP3) and AGAMOUS (AG) orthologues that specify the formation of stamens. We fused the coding regions of rice AP3 orthologue SUPERWOMAN1 (SPW1), and rice AG orthologues, Os12g0207000, Os01g0886200 and OsMADS58, respectively with the artificial sequence encoding the modified EAR-like motif repression domain (SRDX). We first introduced Os12g0207000SRDX, Os01g0886200SRDX and OsMADS58SRDX into rice for evaluation of their abilities to induce male sterility. The transgenic rice expressing OsMADS58SRDX had reiterated formation of lodicule-like organs instead of stamens and carpel, a typical phenotype of ag mutant. Thus, we found that OsMADS58SRDX was most suitable for our purpose. Next, we introduced SPW1SRDX and OsMADS58SRDX into tall fescue. Although the transgenic tall fescue did not have the stamen alterations seen in SPW1SRDX and OsMADS58SRDX rice, they either produced no pollen or produced immature pollen; thus, the anthers were not dehiscent and the plants were male-sterile. In addition to the male sterility, SPW1SRDX tall fescue showed a cleistogamous (closed) phenotype in which anthers were not observed outside the glumes, with thin, abnormally elongated lodicules. Some lines of OsMADS58SRDX tall fescue showed a cleistogamous phenotype in which the lodicules were homeotically transformed into lemma-like organs. In both cases, cleistogamous phenotype was associated with morphological changes to the lodicules. We also obtained a mild phenotype of OsMADS58SRDX tall fescue, which exhibited only the male sterility. In this study, we produced novel male-sterile phenotypes using chimeric repressors and thus suggest CRES-T as a tool for transgenic improvement of forage and turf grasses. OSMADS58 Characterization of Osmads6-5, a null allele, reveals that OsMADS6 is a critical regulator for early flower development in rice (Oryza sativa L.) 2012 Plant Mol Biol Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fujian Agricultural & Forestry University, Fuzhou, China. AGL6-clade genes are a subfamily of MADS-box genes and preferentially expressed in floral organs. OsMADS6 and OsMADS17 are two AGL6-like genes in rice. OsMADS17 has been shown to play a minor role in floral development and appears to result from a duplication of OsMADS6. OsMADS6 was initially named as MFO1 for mosaic floral organs based on its moderate mutant phenotypes. So far, four moderate or weak mutant alleles of OsMADS6 have been described, providing valuable insights into its role in flower development. Here, we report a null allele of OsMADS6 (Osmads6-5), which exhibited a strong mutant phenotype in spikelet without affecting vegetative traits, causing all floral organs except lemma homeotically transformed into lemma-like organs (LLOs) as well as an indeterminate floral meristem, thus resulting in a mutant floret consisting of reiterating whorls of lemma and LLOs. In consistently, over-expression of OsMADS6 led to additional lodicule-, stamen- and carpel-like organs. Expression analysis showed that OsMADS6 controls the formation of the incipient primordia of lodicule, stamen and carpel via regulating the expression of class B, C and SEP-like MADS-box genes. Taken together, our results revealed that OsMADS6 acts as a critical regulator for early flower development in rice and provide novel insights into the molecular mechanism of OsMADS6. OsMADS6|MFO1 OsMADS6 plays an essential role in endosperm nutrient accumulation and is subject to epigenetic regulation in rice (Oryza sativa) 2010 Plant J Department of Biochemistry and Molecular Biology, Mississippi State University, MS 39762, USA. MADS-box transcription factors are known for their roles in plant growth and development. The regulatory mechanisms of spatial and temporal specific expression of MADS-box genes and the function of MADS-box genes in other biological processes are still to be explored. Here, we report that OsMADS6 is highly expressed in flower and endosperm in Oryza sativa (rice). In addition to displaying a homeotic organ identity phenotype in all the four whorls of the flowers, the endosperm development is severely affected in its mutant. At least 32% of the seeds lacked starch filling and aborted. For seeds that have starch filling and develop to maturity, the starch content is reduced by at least 13%. In addition, the seed shape changes from elliptical to roundish, and the protein content increases from 12.1 to 15.0% (P < 0.05). Further investigation shows that ADP-glucose pyrophosphorylase genes, encoding the rate-limiting step enzyme in the starch synthesis pathway, are subject to the regulation of OsMADS6. Chromatin immunoprecipitation (ChIP)-PCR analyses on the chromatin of the OsMADS6 gene find that H3K27 is trimethylated in tissues where OsMADS6 is silenced, and that H3K36 is trimethylated in tissues where OsMADS6 is highly activated. Point mutation analysis reveals that leucine at position 83 is critical to OsMADS6 function. OsMADS6|MFO1 MOSAIC FLORAL ORGANS1, an AGL6-like MADS box gene, regulates floral organ identity and meristem fate in rice 2009 Plant Cell Rice Biotechnology Research Subteam (Hokuriku Region), National Agricultural Research Center, National Agriculture and Food Research Organization, Niigata 943-0193, Japan. Floral organ identity and meristem determinacy in plants are controlled by combinations of activities mediated by MADS box genes. AGAMOUS-LIKE6 (AGL6)-like genes are MADS box genes expressed in floral tissues, but their biological functions are mostly unknown. Here, we describe an AGL6-like gene in rice (Oryza sativa), MOSAIC FLORAL ORGANS1 (MFO1/MADS6), that regulates floral organ identity and floral meristem determinacy. In the flower of mfo1 mutants, the identities of palea and lodicule are disturbed, and mosaic organs were observed. Furthermore, the determinacy of the floral meristem was lost, and extra carpels or spikelets developed in mfo1 florets. The expression patterns of floral MADS box genes were disturbed in the mutant florets. Suppression of another rice AGL6-like gene, MADS17, caused no morphological abnormalities in the wild-type background, but it enhanced the phenotype in the mfo1 background, indicating that MADS17 has a minor but redundant function with that of MFO1. Whereas single mutants in either MFO1 or the SEPALLATA-like gene LHS1 showed moderate phenotypes, the mfo1 lhs1 double mutant showed a severe phenotype, including the loss of spikelet meristem determinacy. We propose that rice AGL6-like genes help to control floral organ identity and the establishment and determinacy of the floral meristem redundantly with LHS1. OsMADS6|MFO1 Overexpression of the mitogen-activated protein kinase gene OsMAPK33 enhances sensitivity to salt stress in rice (Oryza sativa L.) 2011 Journal of Biosciences Department of Agricultural Biotechnology, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-857, Republic of Korea. Mitogen-activated protein kinases (MAPK) signalling cascades are activated by extracellular stimuli such as environmental stresses and pathogens in higher eukaryotic plants. To know more about MAPK signalling in plants, aMAPK cDNA clone, OsMAPK33, was isolated from rice. The gene is mainly induced by drought stress. In phylogenetic analysis, OsMAPK33 (Os02g0148100) showed approximately 47-93% identity at the amino acid level with other plant MAPKs. It was found to exhibit organ-specific expression with relatively higher expression in leaves as compared with roots or stems, and to exist as a single copy in the rice genome. To investigate the biological functions of OsMAPK33 in rice MAPK signalling, transgenic rice plants that either overexpressed or suppressed OsMAPK33 were made. Under dehydration conditions, the suppressed lines showed lower osmotic potential compared with that of wild-type plants, suggesting a role of OsMAPK33 in osmotic homeostasis. Nonetheless, the suppressed lines did not display any significant difference in drought tolerance compared with their wild-type plants. With increased salinity, there was still no difference in salt tolerance between OsMAPK33-suppressed lines and their wild-type plants. However, the overexpressing lines showed greater reduction in biomass accumulation and higher sodium uptake into cells, resulting in a lower K+/Na+ ratio inside the cell than that in the wild-type plants and OsMAPK33-suppressed lines. These results suggest that OsMAPK33 could play a negative role in salt tolerance through unfavourable ion homeostasis. Gene expression profiling of OsMAPK33 transgenic lines through rice DNA chip analysis showed that OsMAPK33 altered expression of genes involved in ion transport. Further characterization of downstream components will elucidate various biological functions of this novel rice MAPK. OsMAPK33|OsMAPK3 Arsenic stress activates MAP kinase in rice roots and leaves 2011 Arch Biochem Biophys National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India. The toxic metalloid arsenite has become a potential threat to rice growing regions leading to serious contamination in food chain. In the present study effect of different physiological concentration of arsenite that is toxic and triggers the molecular events were evaluated in rice seedlings. Along with severe effect on the growth of rice seedling, production of reactive oxygen species (ROS) and nitric oxide (NO) in arsenite treated rice roots was also observed. Activation of a 42kDa mitogen activated protein kinase (MAPK/MPK) by arsenite was observed in rice leaves and 42 and 44kDa in roots in dose dependent manner. The activated MAPK could be immunoprecipitated with anti-phospho-tyrosine antibody, 4G10. The kinetic of MAPK activation by arsenite was found to be dose dependent. Transcript analysis of MAPK family and immunokinase assay in arsenite treated rice seedling revealed significant level of induction in OsMPK3 transcripts in leaves and OsMPK3, OsMPK4 transcripts in roots. Among MAPK kinase (MKKs) gene family, OsMKK4 transcripts were found to be induced in arsenite treated rice leaves and roots. In-silico homology modeling and docking analysis supported OsMPK3-OsMKK4 interaction. The data indicates that arsenite stress is transduced through MAPK signaling cascade in rice. OsMAPK33|OsMAPK3,OsMKK4,OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1 The bHLH Rac Immunity1 (RAI1) Is Activated by OsRac1 via OsMAPK3 and OsMAPK6 in Rice Immunity 2012 Plant Cell Physiol Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Japan. The Rac/Rop GTPase OsRac1 plays an essential role in rice immunity. However, the regulatory genes acting downstream of OsRac1 are largely unknown. We focused on the RAI1 gene, which is up-regulated in suspension cells expressing a constitutively active form of OsRac1. RAI1 encodes a putative basic helix-loop-helix transcription factor. A microarray analysis of cells transformed with an inducible RAI1 construct showed increased expression of PAL1 and OsWRKY19 genes after induction, suggesting that these genes are regulated by RAI1. This was confirmed using RAI1 T-DNA activation-tagged and RNA interference lines. The PAL1 and OsWRKY19 genes were also up-regulated by sphingolipid and chitin elicitors, and the RAI1 activation-tagged plants had increased resistance to a rice blast fungus. These results indicated that RAI1 is involved in defense responses in rice. RAI1 interacted with OsMAPK3 and OsMAPK6 proteins in vivo and in vitro. Also, RAI1 was phosphorylated by OsMAPK3/6 and OsMKK4-dd in vitro. Overexpression of OsMAPK6 and/or OsMAPK3 together with OsMKK4-dd increased PAL1 and OsWRKY19 expression in rice protoplasts. Therefore, the regulation of PAL1 and OsWRKY19 expression by RAI1 could be controlled via an OsMKK4-OsMAPK3/6 cascade. Co-immunoprecipitation assays indicated that OsMAPK3 and OsRac1 occur in the same complex as OsMAPK6. Taken together, our results indicate that RAI1 could be regulated by OsRac1 through an OsMAPK3/6 cascade. In this study, we have identified RAI1 as the first transcription factor acting downstream of OsRac1. This work will help us to understand the immune system regulated by OsRac1 in rice and its orthologs in other plant species. OsMAPK33|OsMAPK3,OsMKK4,OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1,OsMPK1|OsMAPK6|OsSIPK,RAI1,OsbHLH004,OsbHLH155 Disease Resistance and Abiotic Stress Tolerance in Rice Are Inversely Modulated by an Abscisic Acid-Inducible Mitogen-Activated Protein Kinase 2003 The Plant Cell Online Department of Plant Pathology and Program in Cell and Molecular Biology, University of Arkansas, Fayetteville, Arkansas 72701 Mitogen-activated protein kinase (MAPK) cascades play an important role in mediating stress responses in eukaryotic organisms. However, little is known about the role of MAPKs in modulating the interaction of defense pathways activated by biotic and abiotic factors. In this study, we have isolated and functionally characterized a stress-responsive MAPK gene (OsMAPK5) from rice. OsMAPK5 is a single-copy gene but can generate at least two differentially spliced transcripts. The OsMAPK5 gene, its protein, and kinase activity were inducible by abscisic acid as well as various biotic (pathogen infection) and abiotic (wounding, drought, salt, and cold) stresses. To determine its biological function, we generated and analyzed transgenic rice plants with overexpression (using the 35S promoter of Cauliflower mosaic virus) or suppression (using double-stranded RNA interference [dsRNAi]) of OsMAPK5. Interestingly, suppression of OsMAPK5 expression and its kinase activity resulted in the constitutive expression of pathogenesis-related (PR) genes such as PR1 and PR10 in the dsRNAi transgenic plants and significantly enhanced resistance to fungal (Magnaporthe grisea) and bacterial (Burkholderia glumae) pathogens. However, these same dsRNAi lines had significant reductions in drought, salt, and cold tolerance. By contrast, overexpression lines exhibited increased OsMAPK5 kinase activity and increased tolerance to drought, salt, and cold stresses. These results strongly suggest that OsMAPK5 can positively regulate drought, salt, and cold tolerance and negatively modulate PR gene expression and broad-spectrum disease resistance. OsMAPK33|OsMAPK3,OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1 Expression of OsMATE1 and OsMATE2 alters development, stress responses and pathogen susceptibility in Arabidopsis 2014 Sci Rep National Botanical Research Institute, Council of Scientific and Industrial Research (CSIR), Rana Pratap Marg, Lucknow-226 001, INDIA. Multidrug and Toxic compound Extrusion proteins (MATE) are a group of secondary active transporters with ubiquitous occurrences in all domains of life. This is a newly characterized transporter family with limited functional knowledge in plants. In this study, we functionally characterised two members of rice MATE gene family, OsMATE1 and OsMATE2 through expression in heterologous system, Arabidopsis. Expression of OsMATEs in Arabidopsis altered growth and morphology of transgenic plants. Genome-wide expression analysis revealed modulation of genes involved in plant growth, development and biotic stress in transgenic lines. Transgenic plants displayed sensitivity for biotic and abiotic stresses. Elevated pathogen susceptibility of transgenic lines was correlated with reduced expressions of defence related genes. Promoter and cellular localization studies suggest that both MATEs express in developing and reproductive organs and are plasma-membrane localised. Our results reveal that OsMATE1 and OsMATE2 regulate plant growth and development as well as negatively affect disease resistance. OsMATE1,OsMATE2 Plasma membrane protein OsMCA1 is involved in regulation of hypo-osmotic shock-induced Ca2+ influx and modulates generation of reactive oxygen species in cultured rice cells 2012 BMC Plant Biol Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, Japan. BACKGROUND: Mechanosensing and its downstream responses are speculated to involve sensory complexes containing Ca2+-permeable mechanosensitive channels. On recognizing osmotic signals, plant cells initiate activation of a widespread signal transduction network that induces second messengers and triggers inducible defense responses. Characteristic early signaling events include Ca2+ influx, protein phosphorylation and generation of reactive oxygen species (ROS). Pharmacological analyses show Ca2+ influx mediated by mechanosensitive Ca2+ channels to influence induction of osmotic signals, including ROS generation. However, molecular bases and regulatory mechanisms for early osmotic signaling events remain poorly elucidated. RESULTS: We here identified and investigated OsMCA1, the sole rice homolog of putative Ca2+-permeable mechanosensitive channels in Arabidopsis (MCAs). OsMCA1 was specifically localized at the plasma membrane. A promoter-reporter assay suggested that OsMCA1 mRNA is widely expressed in seed embryos, proximal and apical regions of shoots, and mesophyll cells of leaves and roots in rice. Ca2+ uptake was enhanced in OsMCA1-overexpressing suspension-cultured cells, suggesting that OsMCA1 is involved in Ca2+ influx across the plasma membrane. Hypo-osmotic shock-induced ROS generation mediated by NADPH oxidases was also enhanced in OsMCA1-overexpressing cells. We also generated and characterized OsMCA1-RNAi transgenic plants and cultured cells; OsMCA1-suppressed plants showed retarded growth and shortened rachises, while OsMCA1-suppressed cells carrying Ca2+-sensitive photoprotein aequorin showed partially impaired changes in cytosolic free Ca2+ concentration ([Ca2+]cyt) induced by hypo-osmotic shock and trinitrophenol, an activator of mechanosensitive channels. CONCLUSIONS: We have identified a sole MCA ortholog in the rice genome and developed both overexpression and suppression lines. Analyses of cultured cells with altered levels of this putative Ca2+-permeable mechanosensitive channel indicate that OsMCA1 is involved in regulation of plasma membrane Ca2+ influx and ROS generation induced by hypo-osmotic stress in cultured rice cells. These findings shed light on our understanding of mechanical sensing pathways. OsMCA1|PAD Roles of a putative mechanosensitive plasma membrane Ca2+-permeable channel OsMCA1 in generation of reactive oxygen species and hypo-osmotic signaling in rice 2012 Plant Signal Behav Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba, Japan. Mechanosensing and its downstream responses are speculated to involve sensory complexes containing Ca(2+)-permeable mechanosensitive channels. On recognizing hypo-osmotic stress, plant cells initiate activation of a widespread signal transduction network involving second messengers such as Ca(2+) to trigger inducible defense responses including the induction of transcriptional factors. However, most of the components involved in these signaling networks still remain to be identified. Recently we identified and investigated OsMCA1, the sole homolog of the MCA family putative Ca(2+)-permeable mechanosensitive channels in rice. Functional characterization of the OsMCA1-suppressed cells as well as the overexpressing cells indicated that OsMCA1 is involved in the regulation of plasma membrane Ca(2+) influx and NADPH oxidase-mediated generation of reactive oxygen species (ROS) induced by hypo-osmotic stress. Here we will discuss possible molecular mechanisms and physiological functions of the MCA protein in hypo-osmotic signaling. OsMCA1|PAD A SNP in OsMCA1 responding for a plant architecture defect by deactivation of bioactive GA in rice 2014 Plant Molecular Biology State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Luojia Hill, Wuhan, 430072, Hubei Province, China Plant architecture directly affects biomass in higher plants, especially grain yields in agricultural crops. In this study, we characterized a recessive mutant, plant architecture determinant (pad), derived from the Oryza sativa ssp. indica cultivar MH86. The mutant exhibited severe dwarf phenotypes, including shorter and stunted leaves, fewer secondary branches during both the vegetative and reproductive growth stages. Cytological studies revealed that pad mutant growth defects are primarily due to the inhibition of cell expansion. The PAD gene was isolated using a map-based cloning strategy. It encodes a plasma membrane protein OsMCA1 and a SNP responsible for a single amino acid change was found in the mutant. PAD was universally expressed in rice tissues from the vegetative to reproductive growth stages, especially in seedlings, nodes and rachillae. Quantitative real-time PCR analysis revealed that the most of the genes responding to gibberellin (GA) metabolism were up-regulated in pad mutant internodes. The endogenous GA content measurement revealed that the levels of GA1 were significantly decreased in the third internode of pad mutants. Moreover, a GA response assay suggested that OsMCA1/PAD might be involved in the regulation of GA metabolism and signal transduction. Our results revealed the pad is a loss-of-function mutant of the OsMCA1/PAD, leading to upregulation of genes related to GA deactivation, which decreased bioactive GA levels. OsMCA1|PAD Identification and characterization of a rice MCM2 homologue required for DNA replycation 2008 BMB reports Department of Biological Sciences, Myongji University, Yongin, Korea. The pre-replication complex (pre-RC), including the core hexameric MCM2-7 complex, ensures that the eukaryotic genome is replicated only once per cell division cycle. In this study, we identified a rice minichromosome maintenance (MCM) homologue (OsMCM2) that functionally complemented fission yeast MCM2 (CDC19) mutants. We found OsMCM2 transcript expression in roots, leaves, and seeds, although expression levels differed slightly among the organs. Likewise, the OsMCM2 protein was ubiquitously expressed, but it was downregulated when nutritients were limiting, indicating that MCM2 expression (and therefore cell cycle progression) requires adequate nutrition. Yeast two-hybrid and GST pull-down assays demonstrated that OsMCM2 interacted with the COP9 signalosome 5 (CSN5). Taken as a whole, our results indicated that OsMCM2 functions as a subunit of the rice MCM complex and interacts with CSN5 during developmental regulation. OsMCM2 Molecular cloning and characterization of plant genes encoding novel peroxisomal molybdoenzymes of the sulphite oxidase family 2002 J Exp Bot Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Nara 630-0101, Japan The Arabidopsis AtMCP and rice OsMCP genes which encode proteins highly homologous to molybdoenzymes of the sulphite oxidase family were isolated and characterized. Both proteins seemed to possess only a molybdenum cofactor as the redox centre, unlike all the other eukaryotic molybdoenzymes. Putative MCP orthologues were identified in 17 plant species, indicating that Mo possess only a molybdenum cofactor as the redox centre, unlike all the other eukaryotic molybdoenzymes. Putative MCP orthologues were identified in 17 plant species, indicating that MCPs are widely distributed over the plant kingdom. An analysis using a green fluorescent protein fusion showed that AtMCP possesses a peroxisomal targeting signal at its C‐terminus. Putative peroxisomal targeting signals were also found in all plant MCPs, suggesting the existence of a new redox pathway in this organelle. OsMCP A brassinolide-suppressed rice MADS-box transcription factor, OsMDP1, has a negative regulatory role in BR signaling 2006 Plant J Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, 20 Nanxincun Road, 100093 Beijing, China. The MADS-box transcription factor-encoding genes are expressed mainly during plant reproductive development, where they play important roles in controlling floral organ initiation and identity. Few previous reports have investigated the functions of MADS-box transcription factors expressed in vegetative tissues. Here we describe the functional characterization of a rice AG-like MADS-box protein, OsMDP1 (Oryza sativa MADS-domain-containing protein 1). A partial cDNA encoding a MADS-box domain was identified via high-throughput screening of rice brassinolide-regulated genes, and the full-length cDNA was subsequently isolated via screening of a cDNA library constructed from rice materials at tillering stage. Expression pattern analyses indicated that OsMDP1 is transcribed mainly in vegetative tissues, including the mature leaf, coleoptile, root-elongation zone, culm internode, and especially the joint region between the leaf blade and sheath. Further studies revealed that transcription of OsMDP1 is stimulated by darkness and suppressed by brassinolide treatment. OsMDP1 deficiency resulted in shortened primary roots, elongated coleoptiles and enhanced lamina joint inclinations. Moreover, transgenic plants showed hypersensitivities to exogenous brassinolide in terms of lamina joint inclination and coleoptile elongation. OsMDP1 deficiency resulted in enhanced expression of OsXTR1, which encodes xyloglucan endotransglycosylase, the cell-wall loosening enzyme necessary for cell elongation, and modulated expressions of multiple genes involved in cell signalling and gene transcription, indicating the key negative regulatory role of OsMDP1 in BR signalling. OsMDP1,OsXTR1|XTH2 Overexpression of constitutively active mitogen activated protein kinase kinase 6 enhances tolerance to salt stress in rice 2013 Rice (N Y) National Institute of Plant Genome Research, Aruna Asaf Ali Road, New Delhi 110067, India. alok@nipgr.ac.in. BACKGROUND: Salinity is one of the most common abiotic stresses encountered by plants in the environment and transgenic approaches offer new opportunities to improve tolerance. The mitogen activated protein kinase (MAPK) kinase (MKK) is a key component of MAPK cascade that plays important roles in intra and extra cellular signaling in plants. In the present study, a MKK from rice (Oryza sativa), OsMKK6 was functionally characterized in salt stress by transforming its constitutively active form. FINDINGS: OsMKK6 was made constitutively active by mutating serine and threonine to glutamic acid by site directed mutagenesis, and transformed in indica cultivar rice var. Pusa Basmati-1. The transgenic seedlings growing in 200 mM NaCl solution showed increased root/shoot length and weight, less chlorophyll beaching and higher MAPK activity compared to the wild types. CONCLUSION: Present work suggest role of OsMKK6 gene in salt stress signaling in rice. OsMEK1|OsMKK6 Biochemical identification of the OsMKK6-OsMPK3 signalling pathway for chilling stress tolerance in rice 2012 Biochem J Crop Breeding Research Division, Hokkaido Agricultural Research Center, National Agriculture and Food Research Organization, Hitsujigaoka 1, Toyohira-ku, Sapporo 062-8555, Japan. MAPK (mitogen-activated protein kinase) pathways have been implicated in stress signalling in plants. In the present study, we performed yeast two-hybrid screening to identify partner MAPKs for OsMKK (Oryza sativa MAPK kinase) 6, a rice MAPK kinase, and revealed specific interactions of OsMKK6 with OsMPK3 and OsMPK6. OsMPK3 and OsMPK6 each co-immunoprecipitated OsMKK6, and both were directly phosphorylated by OsMKK6 in vitro. An MBP (myelin basic protein) kinase assay of the immunoprecipitation complex indicated that OsMPK3 and OsMPK6 were activated in response to a moderately low temperature (12 degrees C), but not a severely low temperature (4 degrees C) in rice seedlings. A constitutively active form of OsMKK6, OsMKK6DD, showed elevated phosphorylation activity against OsMPK3 and OsMPK6 in vitro. OsMPK3, but not OsMPK6, was constitutively activated in transgenic plants overexpressing OsMKK6DD, indicating that OsMPK3 is an in vivo target of OsMKK6. Enhanced chilling tolerance was observed in the transgenic plants overexpressing OsMKK6DD. Taken together, our data suggest that OsMKK6 and OsMPK3 constitute a moderately low-temperature signalling pathway and regulate cold stress tolerance in rice. OsMEK1|OsMKK6,OsMPK6|OsMPK4,OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1 Two novel mitogen-activated protein signaling components, OsMEK1 and OsMAP1, are involved in a moderate low-temperature signaling pathway in rice 2002 Plant Physiol Winter Stress Laboratory, National Agricultural Research Center for Hokkaido Region, Hitsujigaoka 1, Toyohira-ku, Sapporo 062-8555, Japan. Rice (Oryza sativa) anther development is easily damaged by moderately low temperatures above 12 degrees C. Subtractive screening of cDNA that accumulated in 12 degrees C-treated anthers identified a cDNA clone, OsMEK1, encoding a protein with features characteristic of a mitogen-activated protein (MAP) kinase kinase. The putative OsMEK1 protein shows 92% identity to the maize (Zea mays) MEK homolog, ZmMEK1. OsMEK1 transcript levels were induced in rice anthers by 12 degrees C treatment for 48 h. Similar OsMEK1 induction was observed in shoots and roots of seedlings that were treated at 12 degrees C for up to 24 h. It is interesting that no induction of OsMEK1 transcripts was observed in 4 degrees C-treated seedlings. In contrast, rice lip19, encoding a bZIP protein possibly involved in low temperature signal transduction, was not induced by 12 degrees C treatment but was induced by 4 degrees C treatment. Among the three MAP kinase homologs cloned, only OsMAP1 displayed similar 12 degrees C-specific induction pattern as OsMEK1. A yeast two-hybrid system revealed that OsMEK1 interacts with OsMAP1, but not with OsMAP2 and OsMAP3, suggesting that OsMEK1 and OsMAP1 probably function in the same signaling pathway. An in-gel assay of protein kinase activity revealed that a protein kinase (approximately 43 kD), which preferentially uses myelin basic protein as a substrate, was activated by 12 degrees C treatment but not by 4 degrees C treatment. Taken together, these results lead us to conclude that at least two signaling pathways for low temperature stress exist in rice, and that a MAP kinase pathway with OsMEK1 and OsMAP1 components is possibly involved in the signaling for the higher range low-temperature stress. OsMEK1|OsMKK6,OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1 Alternative splicing of the rice OsMET1 genes encoding maintenance DNA methyltransferase 2008 J Plant Physiol National Institute for Basic Biology, Myodaiji, Okazaki 444-8585, Japan. While the Arabidopsis genome carries one copy of the methyltransferase 1 (MET1) gene for DNA methyltransferase, which is mainly responsible for maintaining CpG methylation, the rice genome bears two copies of the MET1 genes, OsMET1a and OsMET1b. The transcripts of OsMET1b accumulate more abundantly than those of OsMET1a in all of the tissues examined, and both genes actively transcribed at the callus, imbibed embryo, root, meristem, young panicle, anther, pistil, and endosperm, all of which contain actively dividing cells. The OsMET1a transcripts contain two 5'-untranslated exons and alternatively spliced 3'-terminal exons. The alternatively spliced transcripts consist of 14, 15, or 16 exons, and all of them encode a putative protein of 1527 amino acids. While the 3'-terminal exon of OsMET1b is unique, alternative splicing occurs in the 5'-terminal regions, which comprise either exons containing 5'-untranslated regions or an exon bearing the initiation codon. Depending upon alternative usage of 5' exons by alternative splicing, the OsMET1b transcripts comprise 11, 12, 13, or 14 exons, and the former two and the latter two longer transcripts encode putative proteins of 1486 and 1529 amino acids, respectively. Moreover, the 5' splicing patterns of OsMET1b can vary in different tissues. These findings are discussed with respect to the possible regulation of the OsMET1 genes. OsMet1-1|OsMET1a,OsMet1-2|OsMET1b Purification and characterization of rice DNA methyltransferase 2009 Plant Physiol Biochem Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand. Epigenetic modification is essential for normal development and plays important roles in gene regulation in higher plants. Multiple factors interact to regulate the establishment and maintenance of DNA methylation in plant genome. We had previously cloned and characterized DNA methyltransferase (DNA MTase) gene homologues (OsMET1) from rice. In this present study, determination of DNA MTase activity in different cellular compartments showed that DNA MTase was enriched in nuclei and the activity was remarkably increased during imbibing dry seeds. We had optimized the purification technique for DNA MTase enzyme from shoots of 10-day-old rice seedlings using the three successive chromatographic columns. The Econo-Pac Q, the Hitrap-Heparin and the Superdex-200 columns yielded a protein fraction of a specific activity of 29, 298 and 800 purification folds, compared to the original nuclear extract, respectively. The purified protein preferred hemi-methylated DNA substrate, suggesting the maintenance activity of methylation. The native rice DNA MTase was approximately 160-170 kDa and exhibited a broad pH optimum in the range of 7.6 and 8.0. The enzyme kinetics and inhibitory effects by methyl donor analogs, base analogs, cations, and cationic amines on rice DNA MTase were examined. Global cytosine methylation status of rice genome during development and in various tissue culture systems were monitored and the results suggested that the cytosine methylation level is not directly correlated with the DNA MTase activity. The purification and characterization of rice DNA MTase enzyme are expected to enhance our understanding of this enzyme function and their possible contributions in Gramineae plant development. OsMet1-1|OsMET1a,OsMet1-2|OsMET1b De novo DNA methylation induced by siRNA targeted to endogenous transcribed sequences is gene-specific and OsMet1-independent in rice 2008 Plant J Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma 630-0101, Japan. Small interfering RNA (siRNA) is an essential factor for epigenetic modification of the genome. Recent studies have suggested that endogenous siRNAs induce DNA methylation, chromatin modification and chromatin inactivation at homologous sequences. We have shown that siRNAs targeted to promoter regions of endogenous rice genes induce strong DNA methylation of the targeted sequences, but transcriptional gene silencing is rarely observed. Here, an analysis of epigenetic modifications induced by RNAi targeted to transcribed regions of endogenous rice genes shows that the effects of siRNA are gene-specific, but that they tend to induce higher de novo DNA methylation of CpG dinucleotides than of other cytosines. However, loss of OsMet1 expression by RNAi did not significantly affect levels and patterns of de novo DNA methylation or post-transcriptional mRNA suppression. We also showed that sequence-specific de novo DNA methylation extended both 5' and 3' of the targeted sequences, but there was no significant extension of siRNA signals either 5' or 3'. These results suggest that exogenous siRNAs are strong inducers of de novo DNA methylation in transcribed sequences of rice endogenous genes, but are insufficient to induce heterochromatin formation. OsMet1-1|OsMET1a,OsMet1-2|OsMET1b Homologous recombination-mediated knock-in targeting of the MET1a gene for a maintenance DNA methyltransferase reproducibly reveals dosage-dependent spatiotemporal gene expression in rice 2009 Plant J National Institute for Basic Biology, Okazaki 444-8585, Japan. Although homologous recombination-promoted knock-in targeting to monitor the expression of a gene by fusing a reporter gene with its promoter is routine practice in mice, gene targeting to modify endogenous genes in flowering plants remains in its infancy. In the knock-in targeting, the junction sequence between a reporter gene and an endogenous target promoter can be designed properly, and transgenic plants carrying an identical and desired knock-in allele can be repeatedly obtained. By employing a reproducible gene-targeting procedure with positive-negative selection in rice, we were able to obtain fertile transgenic knock-in plants with the promoterless GUS reporter gene encoding beta-glucuronidase fused with the endogenous promoter of MET1a, one of two rice MET1 genes encoding a maintenance DNA methyltransferase. All of the primary (T(0)) transgenic knock-in plants obtained were found to carry only one copy of GUS, with the anticipated structure in the heterozygous condition, and no ectopic events associated with gene targeting could be detected. We showed the reproducible, dosage-dependent and spatiotemporal expression of GUS in the selfed progenies of independently isolated knock-in targeted plants. The results in knock-in targeted plants contrast sharply with the results in transgenic plants with the MET1a promoter-fused GUS reporter gene integrated randomly in the genome: clear interindividual variation of GUS expression was observed among independently obtained plants bearing the randomly integrated transgenes. As our homologous recombination-mediated gene-targeting strategy with positive-negative selection is, in principle, applicable to modify any endogenous gene, knock-in targeting would facilitate basic and applied plant research. OsMet1-1|OsMET1a Cloning of a putative monogalactosyldiacylglycerol synthase gene from rice (Oryza sativa L.) plants and its expression in response to submergence and other stresses 2004 Planta Laboratory of Plant Biotechnology, Faculty of Agriculture, Tottori University, Koyama, 680-8553, Japan. Suppression subtractive hybridization was used to construct a subtractive cDNA library from plants of non-submerged and 7-day-submerged rice (Oryza sativa L., FR13A, a submergence-tolerant cultivar). One clone of the subtractive cDNA library, S23, was expressed abundantly during submergence. The full length of S23 was amplified using 5'- and 3'-rapid amplification of cDNA ends, and found to consist of 1,671 bp with an open reading frame of 1,077 bp (181-1257) encoding 358 amino acids. Its deduced amino acid sequence showed a high homology with monogalactosyldiacylglycerol synthase (UDPgalactose: 1,2-diacylglycerol 3-beta-D-galactosyl transferase; EC 2.4.1.46, MGDG synthase) from Arabidopsis thaliana; therefore, we named the gene OsMGD. Time-course studies showed that the expression of OsMGD in the rice cultivars FR13A and IR42 (submergence-susceptive cultivar) during submergence was gradually increased and that expression in FR13A was higher than in IR42. The expression of OsMGD in FR13A was influenced by benzyladenine and illumination. The accumulation of OsMGD mRNA in both FR13A and IR42 was also increased by ethephon, gibberellin, drought and salt treatment, but cold stress had no effect on the expression of the gene. These results suggest that the expression of OsMGD mRNA requires benzyladenine or illumination, and that the process is also mediated by ethephon and gibberellin. Salt and drought stress have an effect similar to that of submergence. Furthermore, the enhanced expression of OsMGD may relate to photosynthesis, and play an important role during submergence. OsMGD Up-regulation of a magnesium transporter gene OsMGT1 is required for conferring aluminum tolerance in rice 2012 Plant Physiol Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan. Magnesium (Mg)-mediated alleviation of aluminum (Al) toxicity has been observed in a number of plant species, but the mechanisms underlying the alleviation are still poorly understood. When a putative rice (Oryza sativa) Mg transporter gene, Oryza sativa MAGNESIUM TRANSPORTER1 (OsMGT1), was knocked out, the tolerance to Al, but not to cadmium and lanthanum, was decreased. However, this inhibition could be rescued by addition of 10 mum Mg, but not by the same concentration of barium or strontium. OsMGT1 was expressed in both the roots and shoots in the absence of Al, but the expression only in the roots was rapidly up-regulated by Al. Furthermore, the expression did not respond to low pH and other metals including cadmium and lanthanum, and was regulated by an Al-responsive transcription factor, AL RESISTANCE TRANSCRIPTION FACTOR1. An investigation of subcellular localization showed that OsMGT1 was localized to the plasma membrane. A short-term (30 min) uptake experiment with stable isotope (25)Mg showed that knockout of OsMGT1 resulted in decreased Mg uptake, but that the uptake in the wild type was enhanced by Al. Mg concentration in the cell sap of the root tips was also increased in the wild-type rice, but not in the knockout lines in the presence of Al. A microarray analysis showed that transcripts of genes related to stress were more up- and down-regulated in the knockout lines. Taken together, our results indicate that OsMGT1 is a transporter for Mg uptake in the roots and that up-regulation of this gene is required for conferring Al tolerance in rice by increasing Mg concentration in the cell. OsMGT1 Characterization of OsMIK in a rice mutant with reduced phytate content reveals an insertion of a rearranged retrotransposon 2013 Theor Appl Genet State Key Laboratory of Rice Biology and Key Laboratory of Nuclear-Agricultural Sciences of the Ministry of Agriculture and Zhejiang Province, Institute of Nuclear Agricultural Sciences, Zhejiang University, Hangzhou, 310029, China. The rice low phytic acid (lpa) mutant Os-lpa-XS110-1(XS-lpa) has ~45 % reduction in seed phytic acid (PA) compared with the wild-type cultivar Xiushui 110. Previously, a single recessive gene mutation was shown to be responsible for the lpa phenotype and was mapped to a region of chromosome 3 near OsMIK (LOC_Os03g52760) and OsIPK1 (LOC_Os03g51610), two genes involved in PA biosynthesis. Here, we report the identification of a large insert in the intron of OsMIK in the XS-lpa mutant. Sequencing of fragments amplified through TAIL-PCRs revealed that the insert was a derivative of the LINE retrotransposon gene LOC_Os03g56910. Further analyses revealed the following characteristics of the insert and its impacts: (1) the inserted sequence of LOC_Os03g56910 was split at its third exon and rejoined inversely, with its 5' and 3' flanking sequences inward and the split third exon segments outward; (2) the LOC_Os03g56910 remained in its original locus in XS-lpa, and the insertion probably resulted from homologous recombination repair of a DNA double strand break; (3) while the OsMIK transcripts of XS-lpa and Xiushui 110 were identical, substantial reductions of the transcript abundance (~87 %) and the protein level (~60 %) were observed in XS-lpa, probably due to increased methylation in its promoter region. The above findings are discussed in the context of plant mutagenesis, epigenetics and lpa breeding. OsMIK Isolation and characterization of a low phytic acid rice mutant reveals a mutation in the rice orthologue of maize MIK 2008 Theor Appl Genet USDA-ARS Crops Pathology and Genetics Research Unit, Department of Plant Sciences, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA. Using a forward genetics approach, we isolated two independent low phytic acid (lpa) rice mutants, N15-186 and N15-375. Both mutants are caused by single gene, recessive non-lethal mutations, which result in approximately 75% (N15-186) and 43% (N15-375) reductions in seed phytic acid (inositol hexakisphosphate). High-performance liquid chromatography and GC-MS analysis of seed extracts from N15-186 indicated that, in addition to phytic acid, inositol monophosphate was significantly reduced whereas inorganic phosphorus and myo-inositol were greatly increased when compared with wild-type. The changes observed in N15-186 resemble those previously described for the maize lpa3 mutant. Analysis of N15-375 revealed changes similar to those observed in previously characterized rice lpa1 mutants (i.e. significant reduction in phytic acid and corresponding increase in inorganic phosphorus with little or no change in inositol phosphate intermediates or myo-inositol). Further genetic analysis of the N15-186 mutant indicated that the mutation, designated lpa N15-186, was located in a region on chromosome 3 between the microsatellite markers RM15875 and RM15907. The rice orthologue of maize lpa3, which encodes a myo-inositol kinase, is in this interval. Sequence analysis of the N15-186 allele of this orthologue (Os03g52760) revealed a single base pair change (C/G to T/A) in the first exon of the gene, which results in a nonsense mutation. Our results indicate that lpa N15-186 is a mutant allele of the rice myo-inositol kinase (OsMIK) gene. Identification and characterization of lpa mutants, such as N15-186, will facilitate studies on the regulation of phytic acid biosynthesis and accumulation and help address questions concerning the contribution of the inositol lipid-dependent and independent biosynthetic pathways to the production of seed phytic acid. OsMIK OsMIOX, a myo-inositol oxygenase gene, improves drought tolerance through scavenging of reactive oxygen species in rice (Oryza sativa L.) 2012 Plant Sci Key Lab of Crop Heterosis and Utilization of Ministry of Education, Beijing Key Lab of Crop Genetic Improvement, China Agricultural University, Beijing 100193, PR China. Myo-inositol oxygenase (MIOX), a unique monooxygenase, catalyzes the oxidation of myo-inositol to d-glucuronic acid. However, the protective role of MIOX in plants against oxidative stress or drought stress remains unknown. In this study, the functional characterization of MIOX obtained from the cDNA library of upland rice (Oryza sativa L. cv. IRAT109), was performed. OsMIOX was expressed predominantly in the roots and induced by drought, H(2)O(2), salt, cold and abscisic acid. The transgenic rice lines overexpressing OsMIOX showed obviously improved growth performance in the medium containing 200 mM mannitol. Further, the survival rate of leaves from the transgenic rice lines was significantly higher than that of the wild type plants under polyethylene glycol treatment. It was discovered that the activity of ROS-scavenging enzymes and proline content, as well as the transcript levels of many ROS scavenging genes were significantly increased in transgenic plants compared to the wild type plants under drought stress conditions. Together, these data suggest that OsMIOX has a specific function in drought stress tolerance by decreasing oxidative damage. OsMIOX SMALL GRAIN 1, which encodes a mitogen-activated protein kinase kinase 4, influences grain size in rice 2014 Plant J State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100039, China. Although grain size is one of the most important components of grain yield, little information is known about the mechanisms that determine final grain size in crops. Here we characterize rice small grain1 (smg1) mutants, which exhibit small and light grains, dense and erect panicles and comparatively slightly shorter plants. The short grain and panicle phenotypes of smg1 mutants are caused by a defect in cell proliferation. The smg1 mutations were identified, using a map-based cloning approach, in mitogen-activated protein kinase kinase 4 (OsMKK4). Relatively higher expression of OsMKK4/SMG1 was detected in younger organs than in older ones, consistent with its role in cell proliferation. Green fluorescent protein (GFP)-OsMKK4/SMG1 fusion proteins appear to be distributed ubiquitously in plant cells. Further results revealed that OsMKK4 influenced brassinosteroid (BR) responses and the expression of BR-related genes. Thus, our findings have identified OsMKK4 as a factor for grain size, and suggest a possible link between the MAPK pathways and BRs in grain growth. OsMKK4 Involvement of the OsMKK4-OsMPK1 Cascade and its Downstream Transcription Factor OsWRKY53 in the Wounding Response in Rice 2014 Plant Pathol J Department of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, Korea. Plant has possessed diverse stress signals from outside and maintained its fitness. Out of such plant responses, it is well known that mitogen-activated protein kinase (MAPK) cascade plays important role in wounding and pathogen attack in most dicot plants. However, little is understood about its role in wounding response for the economically important monocot rice plant. In this study, therefore, the involvement of MAPK was investigated to understand the wounding signaling pathway in rice. The OsMPK1 was rapidly activated by wounding within 10 min, and OsMPK1 was also activated by challenge of rice blast fungus. Further analysis revealed that OsMKK4, the upstream kinase of OsMPK1, phosphorylated OsMPK1 by wounding in vivo. Furthermore, OsMPK1 directly interacted with a rice defense-related transcription factor OsWRKY53. To understand a functional link between MAPK and its target transcription factor, we showed that OsMPK1 activated by the constitutively active mutant OsMKK4(DD) phosphorylated OsWRKY53 in vitro. Taken together, components involving in the wounding signaling pathway, OsMKK4-OsMPK1-OsWRKY53, can be important players in regulating crosstalk between abiotic stress and biotic stress. OsMKK4,OsMPK1|OsMAPK6|OsSIPK,OsWRKY53 A calmodulin-binding mitogen-activated protein kinase phosphatase is induced by wounding and regulates the activities of stress-related mitogen-activated protein kinases in rice 2007 Plant Cell Physiol National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan. The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) are negative regulators of MAPKs. In dicotyledons such as Arabidopsis and tobacco, MKPs have been shown to play pivotal roles in abiotic stress responses, hormone responses and microtubule organization. However, little is known about the role of MKPs in monocotyledons such as rice. Database searches identified five putative MKPs in rice. We investigated their expression in response to wounding, and found that the expression of OsMKP1 is rapidly induced by wounding. In this study, we functionally characterized the involvement of OsMKP1 in wound responses. The deduced amino acid sequence of OsMKP1 shows strong similarity to Arabidopsis AtMKP1 and tobacco NtMKP1. Moreover, OsMKP1 bound calmodulin in a manner similar to NtMKP1. To determine the biological function of OsMKP1, we obtained osmkp1, a loss-of-function mutant, in which retrotransposon Tos17 was inserted in the second exon of OsMKP1. Unlike the Arabidopsis atmkp1 loss-of-function mutant, which shows no abnormal phenotype without stimuli, osmkp1 showed a semi-dwarf phenotype. Exogenous supply of neither gibberellin nor brassinosteroid complemented the semi-dwarf phenotype of osmkp1. Activities of two stress-responsive MAPKs, OsMPK3 and OsMPK6, in osmkp1 were higher than those in the wild type both before and after wounding. Microarray analysis identified 13 up-regulated and eight down-regulated genes in osmkp1. Among the up-regulated genes, the expression of five genes showed clear responses to wounding, indicating that wound responses are constitutively activated in osmkp1. These results suggest that OsMKP1 is involved in the negative regulation of rice wound responses. OsMKP1,OsMPK6|OsMPK4,OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1,OsDsPTP1 OsMOGS is required for N-glycan formation and auxin-mediated root development in rice (Oryza sativa L.) 2014 The Plant Journal State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, China N-glycosylation is a major modification of glycoproteins in eukaryotic cells. In Arabidopsis, great progress has been made in functional analysis of N-glycan production, however there are few studies in monocotyledons. Here, we characterized a rice (Oryza sativa L.) osmogs mutant with shortened roots and isolated a gene that coded a putative mannosyl-oligosaccharide glucosidase (OsMOGS), an ortholog of α-glucosidase I in Arabidopsis, which trims the terminal glucosyl residue of the oligosaccharide chain of nascent peptides in the endoplasmic reticulum (ER). OsMOGS is strongly expressed in rapidly cell-dividing tissues and OsMOGS protein is localized in the ER. Mutation of OsMOGS entirely blocked N-glycan maturation and inhibited high-mannose N-glycan formation. The osmogs mutant exhibited severe defects in root cell division and elongation, resulting in a short-root phenotype. In addition, osmogs plants had impaired root hair formation and elongation, and reduced root epidemic cell wall thickness due to decreased cellulose synthesis. Further analysis showed that auxin content and polar transport in osmogs roots were reduced due to incomplete N-glycosylation of the B subfamily of ATP-binding cassette transporter proteins (ABCBs). Our results demonstrate that involvement of OsMOGS in N-glycan formation is required for auxin-mediated root development in rice. OsMOGS Functional screening of cDNA library from a salt tolerant rice genotype Pokkali identifies mannose-1-phosphate guanyl transferase gene (OsMPG1) as a key member of salinity stress response 2012 Plant Mol Biol Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India. Salinity, one of the most deleterious stresses, affects growth and overall yield of crop plants. To identify new "candidate genes" having potential role in salinity tolerance, we have carried out 'functional screening' of a cDNA library (made from a salt tolerant rice-Pokkali). Based on this screening, we identified a cDNA clone that was allowing yeast cells to grow in the presence of 1.2 M NaCl. Sequencing and BLAST search identified it as mannose-1-phosphate guanyl transferase (OsMPG1) gene from rice. Analysis of rice genome sequence database indicated the presence of 3 additional genes for MPG. Out of four, three MPG genes viz. OsMPG1, 3 and 4 were able to functionally complement yeast MPG mutant -YDL055C. We have carried out detailed transcript profiling of all members of MPG family by qRT-PCR using two contrasting rice genotypes (IR64 and Pokkali) under different abiotic stresses (salinity, drought, oxidative stress, heat stress, cold or UV light). These MPG genes showed differential expression under various abiotic stresses with two genes (OsMPG1 and 3) showing high induction in response to multiple stresses. Analysis of rice microarray data indicated higher expression levels for OsMPG1 in specific tissues such as roots, leaves, shoot apical meristem and different stages of panicle and seed development, thereby indicating its developmental regulation. Functional validation of OsMPG1 carried out by overexpression in the transgenic tobacco revealed its involvement in enhancing salinity stress tolerance. OsMPG1 Molecular analysis of the rice MAP kinase gene family in relation to Magnaporthe grisea infection 2006 Mol Plant Microbe Interact Department of Plant Pathology, Program in Cell and Molecular Biology, University of Arkansas, Fayetteville, AR 72701, USA. Mitogen-activated protein kinase (MAPK) cascades play a crucial role in plant growth and development as well as biotic and abiotic stress responses. In Arabidopsis, 20 MAPKs have been identified and divided into four major groups. In rice, a monocot model and economically important cereal crop, only five MAPKs were characterized, including three related to the host defense response. In this study, we have identified 17 members of the rice MAPK gene (OsMPK) family through an in silico search of rice genome databases. Based on the phylogenetic analysis and pairwise comparison of Arabidopsis and rice MAPKs, we propose that MAPKs can be divided into six groups. Interestingly, the rice genome contains many more MAPKs with the TDY phosphorylation site (11 members) than with the TEY motif (six members). In contrast, the Arabidopsis genome contains more MAPKs with the TEY motif (12 members) than with the TDY motif (eight members). Upon inoculation with the blast fungus (Magnaporthe grisea), nine of 17 OsMPK genes were found to be induced at the mRNA level during either early, late, or both stages of infection. Four of the M. grisea-induced OsMPK genes were associated with host-cell death in the lesion-mimic rice mutant, and eight of them were differentially induced in response to defense signal molecules such as jasmonic acid, salicylic acid, abscisic acid, and ethylene. The genome-wide expression analysis suggests that about half of the rice MAPK genes are associated with pathogen infection and host defense response. OsMPK10,OsMPK11,OsWJUMK1|OsMPK12|OsBWMK1,OsMPK13|OsBIMK2,OsMPK14,OsMPK15,OsMPK16,OsMPK17,OsMPK2|OsMAPK2,OsMPK6|OsMPK4,OsMPK7,OsMPK8|OsWJUMK1,OsMPK9,OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1,OsMPK1|OsMAPK6|OsSIPK Novel rice MAP kinases OsMSRMK3 and OsWJUMK1 involved in encountering diverse environmental stresses and developmental regulation 2003 Biochem Biophys Res Commun Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), GPO Box 8207, Kathmandu, Nepal. None OsWJUMK1|OsMPK12|OsBWMK1,OsMAPK4|OsMSRMK3 Alternative splicing of the OsBWMK1 gene generates three transcript variants showing differential subcellular localizations 2007 Biochem Biophys Res Commun Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660-701, Republic of Korea. In eukaryotes, mitogen-activated protein kinases (MAPKs) play important roles in various developmental processes and in environmental stress responses. Here, we show that alternative splicing of the OsBWMK1, a member of the rice MAPK family, generates three transcript variants, OsBWMK1L, OsBWMK1M, and OsBWMK1S. The OsBWMK1L transcript variant was highly and constitutively expressed in all rice tissues tested and its expression was not altered by various stress conditions, whereas OsBWMK1M and OsBWMK1S were normally expressed at low levels but were induced by various stresses. A transient expression assay demonstrated that OsBWMK1L::GFP and OsBWMK1M::GFP were predominantly localized in the cytoplasm, whereas most OsBWMK1S::GFP was localized in the nucleus. Moreover, treatment with defense signaling related molecules, such as H(2)O(2) and SA, induced translocation of OsBWMK1 isoforms from the cytoplasm to the nucleus. Thus, our results suggest that alternative splicing of OsBWMK1 generates three different transcript variants that produce proteins with different subcellular localizations. OsWJUMK1|OsMPK12|OsBWMK1 OsBWMK1 mediates SA-dependent defense responses by activating the transcription factor OsWRKY33 2009 Biochem Biophys Res Commun Division of Applied Life Science, Gyeongsang National University, Jinju 660-701, Republic of Korea. Mitogen-activated protein kinases (MAPKs) play important roles in responses to various environmental stresses. In a previous study, we demonstrated that OsBWMK1, which localizes in the nucleus, mediates PR gene expression by activating the OsEREBP1 transcription factor, and that the constitutive expression of OsBWMK1 also enhances resistance against pathogen infections [Y.H. Cheong, B.C. Moon, J.K. Kim, C.Y. Kim, M.C. Kim, I.H. Kim, C.Y. Park, J.C. Kim, B.O. Park, S.C. Koo, H.W. Yoon, W.S. Chung, C.O. Lim, S.Y. Lee, M.J. Cho, BWMK1, rice mitogen-activated protein kinase, locates in the nucleus and mediates pathogenesis-related gene expression by activation of a transcription factor, Plant Physiol. 132 (2003) 1961--1972]. Here, we report that OsBWMK1 phosphorylates OsWRKY33, which binds to the W-box element (TTGACCA) in several PR gene promoters, thereby enhancing DNA-binding activity of the factor to its in vitro cognate binding site. Transient coexpression of OsBWMK1 and OsWRKY33 in Arabidopsis protoplasts elevates SA-dependent expression of the GUS-reporter gene driven by the W-box element and the PR1 promoter. Furthermore, the levels of SA and H(2)O(2) are elevated in 35S-OsBWMK1 transgenic plants that show HR-like cell death. Altogether, OsBWMK1 may mediate SA-dependent defense responses by activating the WRKY transcription factor in plants. OsWJUMK1|OsMPK12|OsBWMK1 Identification and characterization of alternative promoters of the rice MAP kinase gene OsBWMK1 2009 Mol Cells Division of Applied Life Science, Graduate School of Gyeongsang National University, Jinju, Korea. Our previous study suggested that OsBWMK1, a gene which encodes a member of the rice MAP kinase family, generates transcript variants which show distinct expression patterns in response to environmental stresses. The transcript variants are generated by alternative splicing and by use of alternative promoters. To test whether the two alternative promoters, pOsBWMK1L (promoter for the OsBWMK1L splice variant) and pOsBWMK1S (promoter for the OsBWMK1S splice variant), are biologically functional, we analyzed transgenic plants expressing GUS fusion constructs for each promoter. Both pOsBWMK1L and pOsBWMK1S are biologically active, although the activity of pOsBWMK1S is lower than that of pOsBWMK1L. Histochemical analysis revealed that pOsBWMK1L is constitutively active in most tissues at various developmental stages in rice and Arabidopsis, whereas pOsBWMK1S activity is spatially and temporally restricted. Furthermore, the expression of pOsBWMK1S::GUS was upregulated in response to hydrogen peroxide, a plant defense signaling molecule, in both plant species. These results suggest that the differential expression of OsBWMK1 splice variants is the result of alternative promoter usage and, moreover, that the mechanisms controlling OsBWMK1 gene expression are conserved in both monocot and dicot plants. OsWJUMK1|OsMPK12|OsBWMK1 BWMK1, a novel MAP kinase induced by fungal infection and mechanical wounding in rice 1999 Mol Plant Microbe Interact Institute of Molecular Agrobiology, National University of Singapore, Singapore. The activation of the mitogen-activated protein (MAP) kinases by different environmental stresses has been previously observed in several dicot plant species. Here, we report the isolation of a novel MAP kinase in rice that is induced during infection by the blast fungus Magnaporthe grisea or upon mechanical wounding. The gene is designated as BWMK1 for blast- and wound-induced MAP kinase. The cDNA of BWMK1 was isolated from rice leaves challenged by the blast pathogen. Transcripts of the corresponding gene accumulated in rice leaves 4 h after blast inoculation and 30 min after mechanical wounding. This gene encodes a 506 amino acid protein that contains a new dual-phosphorylation activation motif TDY and about 150 unique amino acids on its C terminus. In-gel kinase activity and immunoprecipitation assays confirmed that BWMK1 is a functional MAP kinase. These results show that BWMK1 is a new member of the plant MAP kinase family and may mediate both defense and wound signaling in rice. OsWJUMK1|OsMPK12|OsBWMK1 A novel rice MAPK gene, OsBIMK2, is involved in disease-resistance responses 2006 Plant Biol (Stuttg) Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang 310029, PR China. The mitogen-activated protein kinase (MAPK) cascades play important roles in transmission of extracellular signals to the downstream effector proteins through a mechanism of protein phosphorylation. In this study, we isolated and identified a novel rice MAPK gene, OSBIMK2 ( ORYZAE SATIVA L. BTH-Induced MAP Kinase 2). The OSBIMK2 encodes a 506 amino acid protein with molecular weight of 63 kD. The recombinant OSBIMK2 expressed in ESCHERICHIA COLI showed an autophosphorylation activity IN VITRO. OSBIMK2 is a single-copy gene in the rice genome. Expression of OSBIMK2 was activated upon treatment with benzothiadiazole (BTH), which is capable of inducing disease resistance in rice. Expression of OsBIMK2 was also up-regulated during early stage after inoculation with MAGNAPORTHE GRISEA in BTH-treated rice seedlings and during an incompatible interaction between M. GRISEA and a blast-resistant rice genotype. Over-expression of the rice OSBIMK2 gene in transgenic tobacco resulted in an enhanced disease resistance against tomato mosaic virus and a fungal pathogen, ALTERNARIA ALTERNATA. These results suggest that OSBIMK2 plays a role in disease resistance responses. OsMPK13|OsBIMK2 Cloning and Expression Analysis of a Mitogen-Activated Protein Kinase Gene OsMPK14 in Rice 2010 Rice Science College of Life Science, Henan Normal University, Xinxiang 453007, China Mitogen activated-protein kinases (MAPKs) are important components in signal transduction pathways responding to various biotic and abiotic stresses. An MAPK gene, OsMPK14 (GenBank Accession No. GQ265780) from rice (Oryza sativa L.), was cloned by RT-PCR. The full-length cDNA of OsMPK14 consists of 1660 bp in size, containing an open reading frame of 1629 bp, which encodes a 542-amino-acid polypeptide and has a typical protein kinase domain and a phosphorylation activation motif TDY. Sequence alignment and analysis revealed that OsMPK14 was located on rice chromosome 5, and composed of nine exons and eight introns in the coding region. Semi-quantitative RT-PCR was performed to detect the expression patterns of OsMPK14 in rice shoots and roots under darkness, drought, high salinity, low temperature and abscisic acid treatments. The OsMPK14 mRNA was induced by abscisic acid, low temperature and high salinity, but weakly inhibited by drought. In addition, the expression of OsMPK14 was up-regulated in roots, but down-regulated in shoots by light. The results indicate that OsMPK14 could be implicated in diverse rice stimuli-responsive signaling cascades, and its expression might be regulated by multiple factors. OsMPK14 OsWRKY30 is activated by MAP kinases to confer drought tolerance in rice 2012 Plant Mol Biol Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, People's Republic of China. Both the WRKY transcription factor (TF) and MAP kinases have been shown to regulate gene expression in response to biotic and abiotic stresses in plants. Several reports have shown that WRKY TFs may function downstream of MAPK cascades. Here, we have shown that OsWRKY30 interacted with OsMPK3, OsMPK4, OsMPK7, OsMPK14, OsMPK20-4, and OsMPK20-5, and could be phosphorylated by OsMPK3, OsMPK7, and OsMPK14. Overexpression of OsWRKY30 in rice dramatically increased drought tolerance. Overexpression of OsWRKY30AA, in which all SP (serine residue followed by proline residue) sites were replaced by AP (A, alanine), resulted in no improvement in drought tolerance. In addition, the function of transcriptional activation of OsWRKY30 was impaired after SP was replaced by AP. These results proved that the phosphorylation of OsWRKY30 by MAPKs was crucial in order for OsWRKY30 to perform its biological function. OsMPK14,OsMPK6|OsMPK4,OsMPK7,OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1,OsWRKY30 Mitogen-activated protein kinase OsMPK6 negatively regulates rice disease resistance to bacterial pathogens 2007 Planta National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Mitogen-activated protein kinase (MAPK) cascades play important roles in diverse developmental and physiological processes of plants, including pathogen-induced defense responses. Although at least 17 rice MAPKs have been identified and more than half of these MAPK genes have been shown to be pathogen or elicitor responsive, the exact role of most of the MAPKs in host-pathogen interaction is unknown. Here we report that OsMPK6 is an important regulator in rice disease resistance. Suppressing OsMPK6 or knocking out of OsMPK6 enhanced rice resistance to different races of Xanthomonas oryzae pv. oryzae, causing bacterial blight, one of the most devastating diseases of rice worldwide. The resistant plants showed increased expression of a subset of defense-responsive genes functioning in the NH1 (an Arabidopsis NPR1 orthologue)-involved defense signal transduction pathway. These results suggest that OsMPK6 functions as a repressor to regulate rice defense responses upon bacterial invasion. OsMPK6|OsMPK4,OsNPR1|NH1,OsPR5|Pir2|PR-5|PR5-1 Opposite functions of a rice mitogen-activated protein kinase during the process of resistance against Xanthomonas oryzae 2010 Plant J National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. The pathogen-induced plant defense signaling network consists of multiple components, although only some of them are characterized. Most of the known components function either as activators or repressors in host-pathogen interactions. Here we report that a mitogen-activated protein kinase, OsMPK6, functions both as an activator and a repressor in rice resistance against Xanthomonas oryzae pv. oryzae (Xoo), the causal organism of bacterial blight disease. Activation of OsMPK6 resulted in the formation of lesion mimics and local resistance to Xoo, accompanied by the accumulation of salicylic acid (SA) and jasmonic acid (JA), and the induced expression of SA- and JA-signaling genes. Nuclear localization of OsMPK6 was essential for local resistance, suggesting that modulating the expression of defense-responsive genes through transcription regulators may be the primary mechanism of OsMPK6-mediated local resistance. The knock-out of OsMPK6 resulted in enhanced Xoo resistance, increased accumulation of SA and enhanced resistance to X. oryzae pv. oryzicola, the causal organism of bacterial streak disease, in systemic tissues. Xoo infection induced the expression of PR1a, the marker gene of systemic acquired resistance (SAR), in systemic health tissues of OsMPK6-knock-out plants. These results suggest that OsMPK6 negatively regulates SAR. Thus OsMPK6 is a two-faced player in the rice-Xoo interaction. OsMPK6|OsMPK4,OsPR1a A cold-induced thioredoxin h of rice, OsTrx23, negatively regulates kinase activities of OsMPK3 and OsMPK6 in vitro 2009 FEBS Lett National Agricultural Research Center for Hokkaido Region, National Agricultural and Food Research Organization, Sapporo, Japan. Cytosolic thioredoxins are small conserved proteins that are involved in cellular redox regulation. Here, we report that a major and cold-induced thioredoxin h of rice, OsTrx23, has an inhibitory activity on stress-activated mitogen-activated protein kinases (MAPKs), OsMPK3 and OsMPK6 in vitro. This inhibition effects were redox-dependent and did not involve stable physical interaction. The data suggested a novel mechanism for redox regulation of MAPKs in plants. OsMPK6|OsMPK4,OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1,OsTrx23|OsTRXh1,OsTrx15 MRE11 is required for homologous synapsis and DSB processing in rice meiosis 2013 Chromosoma State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China. Mre11, a conserved protein found in organisms ranging from yeast to multicellular organisms, is required for normal meiotic recombination. Mre11 interacts with Rad50 and Nbs1/Xrs2 to form a complex (MRN/X) that participates in double-strand break (DSB) ends processing. In this study, we silenced the MRE11 gene in rice and detailed its function using molecular and cytological methods. The OsMRE11-deficient plants exhibited normal vegetative growth but could not set seed. Cytological analysis indicated that in the OsMRE11-deficient plants, homologous pairing was totally inhibited, and the chromosomes were completely entangled as a formation of multivalents at metaphase I, leading to the consequence of serious chromosome fragmentation during anaphase I. Immunofluorescence studies further demonstrated that OsMRE11 is required for homologous synapsis and DSB processing but is dispensable for meiotic DSB formation. We found that OsMRE11 protein was located on meiotic chromosomes from interphase to late pachytene. This protein showed normal localization in zep1, Oscom1 and Osmer3, as well as in OsSPO11-1(RNAi) plants, but not in pair2 and pair3 mutants. Taken together, our results provide evidence that OsMRE11 performs a function essential for maintaining the normal HR process and inhibiting non-homologous recombination during meiosis. OsMRE11 The MSP1 Gene Is Necessary to Restrict the Number of Cells Entering into Male and Female Sporogenesis and to Initiate Anther Wall Formation in Rice 2003 The Plant Cell Online Experimental Farm/Plant Genetics Laboratory, National Institute of Genetics, Yata1111, Mishima, Shizuoka 411-8540, Japan. The function of the novel gene MSP1 (MULTIPLE SPOROCYTE), which controls early sporogenic development, was elucidated by characterizing a retrotransposon-tagged mutation of rice. The MSP1 gene encoded a Leu-rich repeat receptor-like protein kinase. The msp1 mutation gave rise to an excessive number of both male and female sporocytes. In addition, the formation of anther wall layers was disordered and the tapetum layer was lost completely. Although the mutation never affected homologous chromosome pairing and chiasma maintenance, the development of pollen mother cells was arrested at various stages of meiotic prophase I, which resulted in complete male sterility. Meanwhile, plural megaspore mother cells in a mutant ovule generated several megaspores, underwent gametogenesis, and produced germinable seeds when fertilized with wild-type pollen despite disorganized female gametophytes. In situ expression of MSP1 was detected in surrounding cells of male and female sporocytes and some flower tissues, but never in the sporocytes themselves. These results suggest that the MSP1 product plays crucial roles in restricting the number of cells entering into male and female sporogenesis and in initiating anther wall formation in rice. OsMSP1 A novel rice calmodulin-like gene, OsMSR2, enhances drought and salt tolerance and increases ABA sensitivity in Arabidopsis 2011 Planta Laboratory for Agro-ecological Process in the Subtropical Region, Institute of Subtropical Agriculture, The Chinese Academy of Sciences, Hunan 410125, China. Many abiotic stimuli, such as drought and salt stresses, elicit changes in intracellular calcium levels that serve to convey information and activate adaptive responses. Ca(2)(+) signals are perceived by different Ca(2)(+) sensors, and calmodulin (CaM) is one of the best-characterized Ca(2)(+) sensors in eukaryotes. Calmodulin-like (CML) proteins also exist in plants, but their functions at the physiological and molecular levels are largely unknown. In this report, we present data on OsMSR2 (Oryza sativa L. Multi-Stress-Responsive gene 2), a novel calmodulin-like protein gene isolated from rice Pei'ai 64S (Oryza sativa L.). Expression of OsMSR2 was strongly up-regulated by a wide spectrum of stresses, including cold, drought, and heat in different tissues at different developmental stages of rice, as revealed by both microarray and quantitative real-time RT-PCR analyses. Analysis of the recombinant OsMSR2 protein demonstrated its potential ability to bind Ca(2)(+) in vitro. Expression of OsMSR2 conferred enhanced tolerance to high salt and drought in Arabidopsis (Arabidopsis thaliana) accompanied by altered expression of stress/ABA-responsive genes. Transgenic plants also exhibited hypersensitivity to ABA during the seed germination and post-germination stages. The results suggest that expression of OsMSR2 modulated salt and drought tolerance in Arabidopsis through ABA-mediated pathways. OsMSR2 OsMSRA4.1 and OsMSRB1.1, two rice plastidial methionine sulfoxide reductases, are involved in abiotic stress responses 2009 Planta State Key Laboratory of Plant Genomics, National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (CAS), 100101 Beijing, China. In proteins, methionine residues are especially sensitive to oxidation, leading to the formation of S- and R-methionine sulfoxide diastereoisomers, and these two methionine sulfoxides can be specifically reversed by two types of methionine sulfoxide reductases (MSRs), MSRA and MSRB. Previously, we have identified a gene encoding a putative MSR from NaCl-treated roots of Brazilian upland rice (Oryza sativa L. cv. IAPAR 9) via subtractive suppression hybridization (Wu et al. in Plant Sci 168:847-853, 2005). Blast database analysis indicated that at least four MSRA and three MSRB orthologs exist in rice, and two of them, OsMSRA4.1 and OsMSRB1.1, were selected for further functional analysis. Expression analysis showed that both OsMSRA4.1 and OsMSRB1.1 are constitutively expressed in all organs and can be induced by various stress conditions. Subcellular localization and in vitro activity assay revealed that both OsMSR proteins are targeted to the chloroplast and have MSR activity. Overexpression of either OsMSRA4.1 or OsMSRB1.1 in yeast enhanced cellular resistance to oxidative stress. In addition, OsMSRA4.1-overexpressing transgenic rice plants also showed enhanced viability under salt treatment. Our results provide genetic evidence of the involvement of OsMSRs in the plant stress responses. OsMSRA4.1,OsMSRB1.1 Isolation of novel rice (Oryza sativa L.) multiple stress responsive MAP kinase gene, OsMSRMK2, whose mRNA accumulates rapidly in response to environmental cues 2002 Biochem Biophys Res Commun Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), GPO Box 8207, Kathmandu, Nepal. gkagrawal@onebox.com In search for components of MAPK (mitogen-activated protein kinase) cascades in rice (Oryza sativa L. cv. Nipponbare), we identified a single copy gene called OsMSRMK2 from jasmonic acid (JA) treated rice seedling leaf cDNA library. This gene has a conserved protein kinase domain, including a MAPK family signature, and encodes a 369 amino acid polypeptide with a predicted molecular mass of 42995.43 and a pI of 5.48. OsMSRMK2 did not show constitutive expression in leaves and was induced within 15 min in response to wounding by cut. Using in vitro system, we show that the expression of OsMSRMK2 mRNA was potently enhanced within 15 min by signalling molecules, protein phosphatase inhibitors, ultraviolet irradiation, fungal elicitor, heavy metals, high salt and sucrose, and drought. OsMSRMK2 expression was further modulated by co-application of JA, salicylic acid, and ethylene and required de novo synthesized protein factor(s) in its transient regulation. Moreover, high (37 degrees C) and low temperatures (12 degrees C) and environmental pollutants-ozone and sulfur dioxide-differentially regulate the OsMSRMK2 mRNA accumulation in leaves of intact plants. Present results demonstrating dramatic transcriptional and transient regulation of the OsMSRMK2 expression by diverse biotic/abiotic stresses, a first report for any rice (or plant) MAPK to date, suggest a role for OsMSRMK2 in rice defense/stress response pathways. OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1 Expression of Oryza sativa MAP kinase gene is developmentally regulated and stress-responsive 2002 Physiologia Plantarum Department of Biology, National Cheng Kung University, Tainan 701, Taiwan. Mitogen-activated protein kinase (MAPK) pathways are modules involved in the transduction of extracellular signals to intracellular targets in all eukaryotes. In plants, there is evidence for MAPKs playing a role in the signalling of abiotic stresses, pathogens, plant hormones, and cell cycle cues. The large number and divergence of plant MAPKs indicates that this ancient mechanism of signal transduction is extensively used in plants. However, there have been no reports of classical MAPK module in rice. In this report, we have isolated a MAPK from rice (Oryza sativa) termed OsMAPK2. The cloned cDNA is 1457 nucleotides long and the deduced amino acid sequence comprised 369 amino acid residues. Sequence analysis revealed that the predicted amino acid sequence is 72% identical to tobacco wound-induced protein kinase (WIPK). Southern analysis suggested a single OsMAPK2 gene in rice. Analysis at the mRNA level has shown that OsMAPK2 is expressed in all plant organs and high relative amounts of OsMAPK2 were detected in the mature panicles in comparison with in the immature panicles. In suspension-cultured cells, the OsMAPK2 mRNA transcript increased markedly upon temperature downshift from 26degreesC to 4degreesC and sucrose starvation. In contrast, the OsMAPK2 mRNA level rapidly declined in rice cell challenged by high temperature. A similarly rapid response of OsMAPK2 was observed in stress-treated seedlings, demonstrating that response of the MAPK pathway occurs also in intact plants. These results suggest that this OsMAPK2 may function in the stress-signalling pathway as well as panicle development in rice. OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1 Overexpression of OsMAPK2 Enhances Low Phosphate Tolerance in Rice and Arabidopsis thaliana 2014 American Journal of Plant Sciences None The mitogen-activated protein kinase (MAPK) cascade is the most important mechanism in environmental responses and developmental processes in plants. The OsMAPK2 gene has been found to function in plant tolerance to diverse biotic/abiotic stresses. This paper presents evidence that OsMAPK2 (Oryza sativa MAP kinase gene 2) is responsive to Pi deficiency and involved in Pi homeostasis. We found that full-length expression of OsMAPK2 was up-regulated in both rice plants and cell culture in the absence of inorganic phosphate (Pi). The transgenic rice and Arabidopsis plants overexpressing OsMAPK2 showed affected root development and increased plant Pi content compared with wild-type plants. Overexpression of OsMAPK2 controlled the expression of several Pi starvation-responsive genes. Our results indicated that OsMAPK2 enables tolerance phosphate deficiency and is involved in Pi homeostasis. OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1 The C2H2-type zinc finger protein ZFP182 is involved in abscisic acid-induced antioxidant defense in rice 2012 J Integr Plant Biol College of Life Sciences, Nanjing Agricultural University, Nanjing, China. C(2) H(2) -type zinc finger proteins (ZFPs) are thought to play important roles in modulating the responses of plants to drought, salinity and oxidative stress. However, direct evidence is lacking for the involvement of these ZFPs in abscisic acid (ABA)-induced antioxidant defense in plants. In this study, the role of the rice (Oryza sativa L. sub. japonica cv. Nipponbare) C(2) H(2) -type ZFP ZFP182 in ABA-induced antioxidant defense and the relationship between ZFP182 and two rice MAPKs, OsMPK1 and OsMPK5 in ABA signaling were investigated. ABA treatment induced the increases in the expression of ZFP182, OsMPK1 and OsMPK5, and the activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in rice leaves. The transient gene expression analysis and the transient RNA interference (RNAi) analysis in protoplasts showed that ZFP182, OsMPK1 and OsMPK5 are involved in ABA-induced up-regulation in the activities of SOD and APX. Besides, OsMPK1 and OsMPK5 were shown to be required for the up-regulation in the expression of ZFP182 in ABA signaling, but ZFP182 did not mediate the ABA-induced up-regulation in the expression of OsMPK1 and OsMPK5. These results indicate that ZFP182 is required for ABA-induced antioxidant defense and the expression of ZFP182 is regulated by rice MAPKs in ABA signaling. OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1,OsMPK1|OsMAPK6|OsSIPK,ZFP182|ZOS3-21 Cadmium activates a mitogen-activated protein kinase gene and MBP kinases in rice 2004 Plant Cell Physiol Department of Life Sciences, Center for Biosciences and Biotechology, National Cheng Kung University, No. 1 University Road 701, Tainan, Taiwan. Mitogen-activated protein kinase (MAPK) pathways are modules involved in the transduction of extracellular signals to intracellular targets in all eukaryotes. In plants, it has been evidenced that MAPKs play a role in the signaling of biotic and abiotic stresses, plant hormones, and cell cycle cues. However, the effect of heavy metals on plant MAPKs has not been well examined. The Northern blot analysis of OsMAPK mRNA levels has shown that only OsMAPK2, but not OsMAPK3 and OsMAPK4, expressed in suspension-cultured cells in response to 100-400 microM Cd treatments. The OsMAPK2 transcripts increased within 12 h upon 400 microM Cd treatment. In addition, we found that 42- and 50-kDa MBP kinases were significantly activated by Cd treatment in rice suspension-cultured cells. And 40-, 42-, 50- and 64-kDa MBP kinases were activated in rice roots. Furthermore, GSH inhibits Cd-induced 40-kDa MBP kinase activation. By immunoblot analysis and immunoprecipitation followed by in-gel kinase assay, we confirmed that Cd-activated 42-kDa MBP kinase is a MAP kinase. Our results suggest that a MAP kinase cascade may function in the Cd-signalling pathway in rice. OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1 Abscisic acid-induced resistance against the brown spot pathogen Cochliobolus miyabeanus in rice involves MAP kinase-mediated repression of ethylene signaling 2010 Plant Physiol Laboratory of Phytopathology, Faculty of Bioscience Engineering, Ghent University, B-9000 Ghent, Belgium. The plant hormone abscisic acid (ABA) is involved in an array of plant processes, including the regulation of gene expression during adaptive responses to various environmental cues. Apart from its well-established role in abiotic stress adaptation, emerging evidence indicates that ABA is also prominently involved in the regulation and integration of pathogen defense responses. Here, we demonstrate that exogenously administered ABA enhances basal resistance of rice (Oryza sativa) against the brown spot-causing ascomycete Cochliobolus miyabeanus. Microscopic analysis of early infection events in control and ABA-treated plants revealed that this ABA-inducible resistance (ABA-IR) is based on restriction of fungal progression in the mesophyll. We also show that ABA-IR does not rely on boosted expression of salicylic acid-, jasmonic acid -, or callose-dependent resistance mechanisms but, instead, requires a functional Galpha-protein. In addition, several lines of evidence are presented suggesting that ABA steers its positive effect on brown spot resistance through antagonistic cross talk with the ethylene (ET) response pathway. Exogenous ethephon application enhances susceptibility, whereas genetic disruption of ET signaling renders plants less vulnerable to C. miyabeanus attack, thereby inducing a level of resistance similar to that observed on ABA-treated wild-type plants. Moreover, ABA treatment alleviates C. miyabeanus-induced activation of the ET reporter gene EBP89, while derepression of pathogen-triggered EBP89 transcription via RNA interference-mediated knockdown of OsMPK5, an ABA-primed mitogen-activated protein kinase gene, compromises ABA-IR. Collectively, these data favor a model whereby exogenous ABA enhances resistance against C. miyabeanus at least in part by suppressing pathogen-induced ET action in an OsMPK5-dependent manner. OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1 OsBIMK1, a rice MAP kinase gene involved in disease resistance responses 2002 Planta Present address: Department of Plant Protection, College of Agriculture and Biotechnology, Zhejiang University-Huajiachi Campus, Hangzhou, Zhejiang 310029, P.R. China. The activation of mitogen-activated protein kinases (MAPKs) has been previously implicated in signal transduction during plant responses to pathogen attack as well as to various environmental stresses. We have isolated from rice a new MAPK cDNA, OsBIMK1 ( O ryza s ativa L. BTH-induced MAPK 1), which encodes a 369-amino-acid protein with moderate to high nucleotide sequence similarity to previously reported plant MAPK genes. OsBIMK1 contains all 11 of the MAPK conserved subdomains and the phosphorylation-activation motif, TEY. We analyzed in detail the expression of OsBIMK1 upon treatment with various chemical and biological inducers of resistance responses in rice and in both incompatible and compatible interactions between rice and Magnaporthe grisea. Expression of OsBIMK1 was activated rapidly upon treatment with benzothiadiazole (BTH) as well as with dichloroisonicotinic acid, probenazole, jasmonic acid and its methyl ester, Pseudomonas syringae pv. syringae, or wounding. Expression of OsBIMK1 was induced rapidly during the first 36 h after inoculation with M. grisea in BTH-treated rice seedlings and in an incompatible interaction between M. grisea and a blast-resistant rice genotype. BTH treatment induced a systemic activation of OsBIMK1 expression. These results suggest that OsBIMK1 plays an important role in rice disease resistance. OsMSRMK2|OsMAP1|OsMPK5|OsMAPK2|OsMAPK5|OsBIMK1 Transcriptional Regulation of a Rice Mitogen-Activated Protein Kinase Gene, OsMAPK4, in Response to Environmental Stresses 2002 Plant and Cell Physiology Department of Biology, National Cheng-Kung University, No. 1 University Road 701, Tainan, Taiwan Mitogen-activated protein kinase (MAPK) cascades play important roles in signal transduction of extracellular stimuli in eukaryotes. However, stimulatory signals for plant MAPKs have not been well elucidated. Here, a cDNA clone, termed Oryza sativa MAPK4 (OsMAPK4), from rice encoding a protein that showed homology with the eukaryotic MAPKs was isolated. According to the phylogenetic analysis, OsMAPK4 belongs to subgroup IV MAPK in plants. OsMAPK4 transcripts were expressed strongly in mature leaves and weakly in young leaves and panicles. The gene was also differentially expressed in roots at different developmental stages. In addition, the mRNA level of OsMAPK4 was up-regulated under sugar starvation, high salinity and cold treatments. These results suggest that this OsMAPK4 functions not only in developmental programs but also in stress-signaling pathways. OsMAPK4|OsMSRMK3 Characterization and expression of monosaccharide transporters (osMSTs) in rice 2000 Plant Cell Physiol Bioscience Center and Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Japan. This study deals with the cloning and characterization of monosaccharide transporter cDNAs in rice. OsMST1-3 (Oryza sativa monosaccharide transporters 1-3) have two sets of putative six transmembrane domains separated by a central long hydrophilic region. Heterologous expression of OsMST3 in the yeast Saccharomyces cerevisiae indicated that OsMST3 has transport activity for some monosaccharides in an energy-dependent H+ co-transport manner. Northern blot and in situ hybridization analyses showed that OsMST3 mRNA is detectable in leaf blades, leaf sheaths, calli and roots, especially the xylem as well as in sclerenchyma cells in the root. These results suggested that OsMST3 is involved in the accumulation of monosaccharides required for cell wall synthesis at the stage of cell thickening. OsMST1,OsMST2,OsMST3 Molecular cloning, functional characterization and expression analysis of a novel monosaccharide transporter gene OsMST6 from rice (Oryza sativa L.) 2008 Planta State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing, China. Monosaccharides transporters play important roles in assimilate supply for sink tissue development. In this study, a new monosaccharide transporter gene OsMST6 was identified from rice (Oryza sativa L.). The predicted OsMST6 protein shows typical features of sugar transporters and shares 79.6% identity with the rice monosaccharide transporter OsMST3. Heterologous expression in yeast (Saccharomyces cerevisiae) demonstrated that OsMST6 is a broad-spectrum monosaccharide transporter, with a K (m) of 266.1 muMu for glucose. OsMST6-green fluorescent protein fusion protein is localized to the plasma membrane in plant. Semi-quantitative RT-PCR analysis exhibited that OsMST6 is expressed in all tested organs/tissues. In developing seeds, OsMST6 expression level is high at the early and middle grain filling stages and gradually declines later. Further analysis detected its expression in both maternal and filial tissues. RNA in situ hybridization analysis indicated that OsMST6 is predominantly expressed in the vascular parenchyma of the chalazal vein, cross-cells, nucellar tissue and endosperm of young seeds, in mesophyll cells of source leaf blades, and in pollens and the connective vein of anthers. In addition, OsMST6 expression is up-regulated by salt stress and sugars. The physiological role of OsMST6 for seed development and its roles in other sink and source tissues are discussed. OsMST3,OsMST6 Molecular cloning and expression analysis of a monosaccharide transporter gene OsMST4 from rice (Oryza sativa L.) 2007 Plant Mol Biol State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, China. Monosaccharide transporters mediate the membrane transport of a variable range of monosaccharides, which plays a crucial role in sugar distribution throughout the plant. To investigate the significance of monosaccharide transporters for rice (Oryza sativa L.) seed development, cDNA of a new putative monosaccharide transporter gene OsMST4 was isolated. The deduced OsMST4 protein shows typical features of monosaccharide transporters, and shares high homology with other plant homologues. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that OsMST4 is a functional monosaccharide transporter capable of transporting glucose, fructose, mannose and galactose. Transcriptional analysis revealed that OsMST4 is expressed in all tested organs/tissues. In developing caryopses, its expression is high at the early and middle grain filling stages, and declines gradually to low levels after that. Further analysis revealed that it is expressed in both the maternal tissue and the filial tissue, with its highest expression in embryo. Cellular location in young caryopses through RNA in situ hybridization showed that OsMST4 mRNA mainly accumulates in the vascular parenchyma of the chalazal vein, cross-cells, nucellar tissue and endosperm. The expression pattern of OsMST4 was further confirmed by histochemical analysis of the OsMST4-promoter-beta-glucuronidase (GUS) transgenic rice plants. These data indicate that OsMST4 is actively involved in monosaccharides supply for seed development during the course of grain filling. In addition, the cell type-specific expression patterns of OsMST4 in other sink and source tissues were also investigated, and its corresponding physiological roles were discussed. OsMST4 Sugar transporters involved in flowering and grain development of rice 2001 Journal of Plant Physiology Bioscience Center and Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan Developing seeds offer a fine experimental system for study on sugar transport mechanism(s) in sink tissue. The sugar transporters identified as being specific to the seed development may play a crucial role for flowering and grain development. The rice sucrose transporterOsSUT1 and the monosaccharide transportersOsMST1-3 have been previously characterized. To investigate sugar transport processes during flowering and in developing grains of rice, we newly isolated two genomic clonesOsSUT2(Oryzasativasucrosetransporter 2) andOsMST5(Oryzasativamonosaccharidetransporter 5) and their corresponding cDNAs.OsSUT2 andOsMST5are encoded by open reading frames of 1485 and 1557 bp encoding 495 and 519amino acids, respectively. The putative amino acid sequence ofOsSUT2showed 63.0 and 62.4 percnt; identity to that ofOsSUT1and barley transporterHvSUT1, respectively. Northern blot analysis revealed thatOsSUT2andOsMST5mRNA accumulates in panicles before pollination.In situ hybridization analysis showed thatOsSUT2transcript is specific to the developing pollen. These data presented suggest that bothOsSUT2andOsMST5 play a role during the development at the early stage of the seed development. On the other handOsSUT1 was expressed at the early stage of the grain development, suggesting a different physiological role compared to Os SUT2. OsMST5,OsSUT1,OsSUT2|OsSUT2M Characterization of Rice Functional Monosaccharide Transporter,OsMST5 2014 Bioscience, Biotechnology and Biochemistry Graduate School of Science, Hokkaido University cDNA of a monosaccharide transporter in rice, OsMST5 (Oryza sativa monosaccharide transporter 5) was cloned and its sugar transport activity was characterized by heterologous expression analysis. The amino acid sequence and topology were similar to the sequences and topology of other plant monosaccharide transporters. Yeast cells co-expressed with OsMST5 cDNA transported some monosaccharide substrates. The transport rate increased when ethanol as an electron donor was added, so the transporter was an energy-dependent active one. Most of the OsMST5 was expressed in panicles before pollination, indicating that it is associated with pollen development in rice. OsMST5 Expression patterns of the rice class I metallothionein gene family in response to lead stress in rice seedlings and functional complementation of its members in lead-sensitive yeast cells 2007 Chinese Science Bulletin Laboratory of Molecular Biology and Protein Science Key Laboratory of the Ministry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, 100084, China Metallothioneins (MTs) are a group of low molecular mass and cysteine-rich proteins that can chelate heavy-metal ions. In this paper, Northern blot analysis was used to investigate the influence of lead stress on the expression patterns of 10 rice class I MT genes (OsMT-Is) in rice seedlings. With the exception of OsMT-I-3b, the data demonstrate dynamic changes of 9 OsMT-I transcripts in response to Pb2+ treatment in rice seedling roots. Of these genes, transcription of OsMT-I-1a, OsMT-I-1b, OsMT-I-2c, OsMT-I-4a, OSMT-I-4b and OsMT-I-4c increased significantly, while transcription of OsMT-I-2a and OsMT-I-3a increased marginally. In contrast, the expression of OsMT-I-2b was inhibited. Pb2+ induced the expression of 6 OsMT-I genes in seedling shoots, but had no obvious effects on the expression of OsMT-I-1a, OsMT-I-1b, OsMT-I-4a and OsMT-I-4b. All the 10 OsMT-Is had enhanced lead tolerance when heterologously expressed in lead-sensitive yeast mutant cells. These results provide an expression profile of the rice MT gene family in response to Pb2+ stress in rice seedlings and demonstrate increased lead tolerance in sensitive yeast mutant cells expressing OsMT-Is. This study lays a foundation for further analysis of the role of the rice MT gene family in respond to Pb2+ stress. OsMT-I-4b Characterization of a novel rice metallothionein gene promoter: its tissue specificity and heavy metal responsiveness 2010 J Integr Plant Biol Tsinghua University, Beijing, China. The rice (Oryza sativa L.) metallothionein gene OsMT-I-4b has previously been identified as a type I MT gene. To elucidate the regulatory mechanism involved in its tissue specificity and abiotic induction, we isolated a 1 730 bp fragment of the OsMT-I-4b promoter region. Histochemical beta-glucuronidase (GUS) staining indicated a precise spacial and temporal expression pattern in transgenic Arabidopsis. Higher GUS activity was detected in the roots and the buds of flower stigmas, and relatively lower GUS staining in the shoots was restricted to the trichomes and hydathodes of leaves. No activity was observed in the stems and seeds. Additionally, in the root of transgenic plants, the promoter activity was highly upregulated by various environmental signals, such as abscisic acid, drought, dark, and heavy metals including Cu(2)(+) , Zn(2)(+) , Pb(2)(+) and Al(3)(+) . Slight induction was observed in transgenic seedlings under salinity stress, or when treated with Co(2)(+) and Cd(2)(+) . Promoter analysis of 5'-deletions revealed that the region -583/-1 was sufficient to drive strong GUS expression in the roots but not in the shoots. Furthermore, deletion analysis indicated important promoter regions containing different metal-responsive cis-elements that were responsible for responding to different heavy metals. Collectively, these findings provided important insight into the transcriptional regulation mechanisms of the OsMT-I-4b promoter, and the results also gave us some implications for the potential application of this promoter in plant genetic engineering. OsMT-I-4b Characterization of a rice class II metallothionein gene: tissue expression patterns and induction in response to abiotic factors 2005 J Plant Physiol Laboratory of Molecular Biology and MOE Laboratory of Protein Science, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, PR China. Data mining the complete rice genome sequences revealed a genomic fragment encoding a characteristic metallothionein (MT) protein, and its full-length cDNA was isolated from rice developing seeds by RT-PCR. This cDNA, designated OsMT-II-1a, contains an open reading frame of 264 bp encoding a protein of 87 amino acid residues. The predicted amino acid sequence was shown to have structural features characteristic of plant class II MT proteins. By sequence analysis of its 5'-flanking region, one putative TATA box, four putative CAAT boxes, and several short sequences homologous to previously reported regulatory cis-elements were identified. Northern blot analysis showed that accumulation of OsMT-II-1a mRNA is specifically abundant in developing seeds and 2-day glumes after pollination, and OsMT-II-1a transcription can markedly be induced by H2O2, paraquat, SNP, ethephon, ABA and SA, but barely by metal ions or other exogenous abiotic factors such as low temperature and PEG. These results coincide with the prediction of existing regulatory cis-elements in its 5'-flanking region. Taken together, the above results suggest that the processes of pollination and seed development might be mediated, at least in part, by expression of the OsMT-II-1a gene that is regulated by several abiotic factors. OsMT-II-1a The promoter and the 5'-untranslated region of rice metallothionein OsMT2b gene are capable of directing high-level gene expression in germinated rice embryos 2014 Plant Cell Rep Department of Bioagricultural Science, National Chiayi University, Chiayi, 60004, Taiwan, ROC. KEY MESSAGE: Critical regions within the rice metallothionein OsMT2b gene promoter are identified and the 5'-untranslated region (5'-UTR) is found essential for the high-level promoter activity in germinated transgenic rice embryos. Many metallothionein (MT) genes are highly expressed in plant tissues. A rice subfamily p2 (type 2) MT gene, OsMT2b, has been shown previously to exhibit the most abundant gene expression in young rice seedling. In the present study, transient expression assays and a transgenic approach were employed to characterize the expression of the OsMT2b gene in rice. We found that the OsMT2b gene is strongly and differentially expressed in germinated rice embryos during seed germination and seedling development. Histochemical staining analysis of transgenic rice carrying OsMT2b::GUS chimeric gene showed that high-level GUS activity was detected in germinated embryos and at the meristematic part of other tissues during germination. Deletion analysis of the OsMT2b promoter revealed that the 5'-flanking region of the OsMT2b between nucleotides -351 and -121 relative to the transcriptional initiation site is important for promoter activity in rice embryos, and this region contains the consensus sequences of G box and TA box. Our study demonstrates that the 5'-untranslated region (5'-UTR) of OsMT2b gene is not only necessary for the OsMT2b promoter activity, but also sufficient to augment the activity of a minimal promoter in both transformed cell cultures and germinated transgenic embryos in rice. We also found that addition of the maize Ubi intron 1 significantly enhanced the OsMT2b promoter activity in rice embryos. Our studies reveal that OsMT2b351-ubi(In) promoter can be applied in plant transformation and represents potential for driving high-level production of foreign proteins in transgenic rice. OsMT2b Down-regulation of metallothionein, a reactive oxygen scavenger, by the small GTPase OsRac1 in rice 2004 Plant Physiol Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Takayama 8916-5, Ikoma, Nara 630-0101, Japan. Metallothioneins are small, ubiquitous Cys-rich proteins known to be involved in reactive oxygen species (ROS) scavenging and metal homeostasis. We found that the expression of a metallothionein gene (OsMT2b) was synergically down-regulated by OsRac1 and rice (Oryza sativa) blast-derived elicitors. Transgenic plants overexpressing OsMT2b showed increased susceptibility to bacterial blight and blast fungus. OsMT2b-overexpressing cells showed reduced elicitor-induced hydrogen peroxide production. In contrast, homozygous OsMT2b::Tos17-inserted mutant and OsMT2b-RNAi-silenced transgenic cells showed significantly higher elicitor-induced hydrogen peroxide production than the wild-type cells. In vitro assay showed that recombinant OsMT2b protein possessed superoxide- and hydroxyl radical-scavenging activities. Taken together, these results showed that OsMT2b is an ROS scavenger and its expression is down-regulated by OsRac1, thus potentiating ROS, which function as signals in resistance response. The results suggest that OsRac1 plays a dual role as an inducer of ROS production and a suppressor of ROS scavenging. OsMT2b,OsRac1 Characteristic and expression analysis of a metallothionein gene, OsMT2b, down-regulated by cytokinin suggests functions in root development and seed embryo germination of rice 2008 Plant Physiol College of Life Sciences, Wuhan University, Wuhan 430072, China. Metallothioneins (MTs) are low molecular mass and cysteine-rich metal-binding proteins known to be mainly involved in maintaining metal homeostasis and stress responses. But, their functions in higher plant development are scarcely studied. Here, we characterized rice (Oryza sativa) METALLOTHIONEIN2b (OsMT2b) molecularly and found that its expression was down-regulated by cytokinins. OsMT2b was preferentially expressed in rice immature panicles, scutellum of germinating embryos, and primordium of lateral roots. In contrast with wild-type plants, OsMT2b-RNA interference (RNAi) transgenic plants had serious handicap in plant growth and root formation, whereas OsMT2b-overexpressing transformants were dwarfed and presented more adventitious roots and big lateral roots. The increased cytokinin levels in RNAi plants and decreased cytokinin levels in overexpressing plants were confirmed by high-performance liquid chromatography quantitative analysis in the roots of wild-type and transgenic plants. In RNAi plants, localization of isopentenyladenosine, a kind of endogenous cytokinin, in roots and germinating embryos expanded to the whole tissues, whereas in overexpressing plants, the isopentenyladenosine signals were very faint in the vascular tissues of roots and scutellum cells of germinating embryos. In vitro culture of embryos could largely resume the reduced germination frequency in RNAi plants but had no obvious change in overexpressing plants. Taken together, these results indicate a possible feedback regulation mechanism of OsMT2b to the level of endogenous cytokinins that is involved in root development and seed embryo germination of rice. OsMT2b Molecular characterization of a rice metal tolerance protein, OsMTP1 2012 Plant Cell Rep Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, People's Republic of China. Rice (Oryza sativa L. 'Nipponbare') cDNA subtractive suppression hybridization (SSH) libraries constructed using cadmium (Cd)-treated seedling roots were screened to isolate Cd-responsive genes. A cDNA clone, encoding the rice homolog of Metal Tolerance Protein (OsMTP1), was induced by Cd treatment. Plant MTPs belong to cation diffusion facilitator (CDF) protein family, which are widespread in bacteria, fungi, plants, and animals. OsMTP1 heterologous expression in yeast mutants showed that OsMTP1 was able to complement the mutant strains' hypersensitivity to Ni, Cd, and Zn, but not other metals including Co and Mn. OsMTP1 expression increased tolerance to Zn, Cd, and Ni in wild-type yeast BY4741 during the exponential growth phase. OsMTP1 fused to green fluorescent protein was localized in onion epidermal cell plasma membranes, consistent with an OsMTP1 function in heavy metal transporting. OsMTP1 dsRNAi mediated by transgenic assay in rice seedlings resulted in heavy metal sensitivity and changed the heavy metal accumulation in different organs of mature rice under low-concentration heavy metal stress. Taken together, our results show that OsMTP1 is a bivalent cation transporter localized in the cell membrane, which is necessary for efficient translocation of Zn, Cd and other heavy metals, and maintain ion homeostasis in plant. OZT1|OsMTP1 Functional analysis of the rice vacuolar zinc transporter OsMTP1 2013 J Exp Bot Centre for Biological Science, University of Southampton, Life Sciences Building 85, Highfield Campus, Southampton SO17 1B, UK. Heavy metal homeostasis is maintained in plant cells by specialized transporters which compartmentalize or efflux metal ions, maintaining cytosolic concentrations within a narrow range. OsMTP1 is a member of the cation diffusion facilitator (CDF)/metal tolerance protein (MTP) family of metal cation transporters in Oryza sativa, which is closely related to Arabidopsis thaliana MTP1. Functional complementation of the Arabidopsis T-DNA insertion mutant mtp1-1 demonstrates that OsMTP1 transports Zn in planta and localizes at the tonoplast. When heterologously expressed in the yeast mutant zrc1 cot1, OsMTP1 complemented its Zn hypersensitivity and was also localized to the vacuole. OsMTP1 alleviated, to some extent, the Co sensitivity of this mutant, rescued the Fe hypersensitivity of the ccc1 mutant at low Fe concentrations, and restored growth of the Cd-hypersensitive mutant ycf1 at low Cd concentrations. These results suggest that OsMTP1 transports Zn but also Co, Fe, and Cd, possibly with lower affinity. Site-directed mutagenesis studies revealed two substitutions in OsMTP1 that alter the transport function of this protein. OsMTP1 harbouring a substitution of Leu82 to a phenylalanine can still transport low levels of Zn, with an enhanced affinity for Fe and Co, and a gain of function for Mn. A substitution of His90 with an aspartic acid completely abolishes Zn transport but improves Fe transport in OsMTP1. These amino acid residues are important in determining substrate specificity and may be a starting point for refining transporter activity in possible biotechnological applications, such as biofortification and phytoremediation. OZT1|OsMTP1 Characterization of a vacuolar zinc transporter OZT1 in rice (Oryza sativa L.) 2013 Mol Biol Rep State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China. The CDF family is a ubiquitous family that has been identified in prokaryotes, eukaryotes, and archaea. Members of this family are important heavy metal transporters that transport metal ions out of the cytoplasm. In this research, a full length cDNA named Oryza sativa Zn Transporter 1 (OZT1) that closely related to rat ZnT-2 (Zn Transporter 2) gene was isolated from rice. The OZT1 encoding a CDF family protein shares 28.2 % ~ 84.3 % of identities and 49.3 % ~ 90.9 % of similarities with other zinc transporters such as RnZnT-2, HsZnT-8, RnZnT-8 and AtMTP1. OZT1 was constitutively expressed in various rice tissues. The OZT1 expression was significantly induced both in the seedlings of japonica rice Nipponbare and indica rice IR26 in response to Zn(2+) and Cd(2+) treatments. Besides, OZT1 expression was also increased when exposed to other excess metals, such as Cu(2+), Fe(2+) and Mg(2+). Subcellular localization analysis indicated that OZT1 localized to vacuole. Heterologous expression of OZT1 in yeast increased tolerance to Zn(2+) and Cd(2+) stress but not the Mg(2+) stress. Together, OZT1 is a CDF family vacuolar zinc transporter conferring tolerance to Zn(2+) and Cd(2+) stress, which is important to transporting and homeostasis of Zn, Cd or other heavy metals in plants. OZT1|OsMTP1 Mn tolerance in rice is mediated by MTP8.1, a member of the cation diffusion facilitator family 2013 J Exp Bot Faculty of Agriculture, Kochi University, Nankoku 783-8502 Japan. Manganese (Mn) is an essential micronutrient for plants, but is toxic when present in excess. The rice plant (Oryza sativa L.) accumulates high concentrations of Mn in the aerial parts; however, the molecular basis for Mn tolerance is poorly understood. In the present study, genes encoding Mn tolerance were screened for by expressing cDNAs of genes from rice shoots in Saccharomyces cerevisiae. A gene encoding a cation diffusion facilitator (CDF) family member, OsMTP8.1, was isolated, and its expression was found to enhance Mn accumulation and tolerance in S. cerevisiae. In plants, OsMTP8.1 and its transcript were mainly detected in shoots. High or low supply of Mn moderately induced an increase or decrease in the accumulation of OsMTP8.1, respectively. OsMTP8.1 was detected in all cells of leaf blades through immunohistochemistry. OsMTP8.1 fused to green fluorescent protein was localized to the tonoplast. Disruption of OsMTP8.1 resulted in decreased chlorophyll levels, growth inhibition in the presence of high concentrations of Mn, and decreased accumulation of Mn in shoots and roots. However, there was no difference in the accumulation of other metals, including Zn, Cu, Fe, Mg, Ca, and K. These results suggest that OsMTP8.1 is an Mn-specific transporter that sequesters Mn into vacuoles in rice and is required for Mn tolerance in shoots. OsMTP8.1 MTR1 encodes a secretory fasciclin glycoprotein required for male reproductive development in rice 2012 Dev Cell State Key Laboratory of Hybrid Rice, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. In flowering plants, formation of the haploid male gametophytes in anthers requires the interaction between reproductive cells and the neighboring somatic cells, yet the underlying mechanism remains poorly understood. Here, we reveal the crucial role of a fasciclin glycoprotein, MICROSPORE AND TAPETUM REGULATOR1 (MTR1), in controlling the development of sporophytic and reproductive cells in rice (Oryza sativa). MTR1 is specifically expressed in the male reproductive cells, yet its mutant exhibits defects in both tapetum and microspore development, causing complete male sterility. We also demonstrate that the fasciclin domains, N-glycolation, and N-terminal signal peptide-mediated plasma membrane localization of MTR1 are required for normal anther development and pollen fertility. Our findings show that rice male reproductive cells secrete the MTR1 protein to control the development of reproductive cells and their adjacent somatic cells, thus providing novel insights into the mechanism of plant male reproductive development. OsMTR1 Two alternatively spliced transcripts generated from OsMUS81, a rice homolog of yeast MUS81, are up-regulated by DNA-damaging treatments 2007 Plant Cell Physiol National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, 305-8602 Japan. OsMUS81, a rice homolog of the yeast MUS81 endonuclease gene, produced two alternative transcripts, OsMUS81alpha and OsMUS81beta. OsMus81alpha contained a Helix-hairpin-Helix (HhH) motif at the N- and C-termini, and a conserved XPF-like motif in the center, while the OsMus81beta isoform lacked the second HhH motif by alternative splicing of a cryptic intron generating a truncated protein. The two transcripts were induced after DNA-damaging treatments such as high intensity light, UV-C and gamma-radiation. The yeast two-hybrid assay detected a strong interaction between OsMus81 and OsRad54 recombinational repair proteins. These findings suggest that OsMus81 functions in maintaining genome integrity through homologous recombination. OsMUS81 Isolation and characterization of OsMY1, a putative partner of OsRac5 from Oryza sativa L 2014 Mol Biol Rep College of Life Science, Henan Normal University, Xinxiang, 453007, China. OsRac5 belongs to the rice Rho of plants family, and acts as the molecular switch in the signal pathway which is pivotally involved in the rice fertility control. One of its putative partners, OsMY1, was isolated by yeast two-hybrid screening from rice panicle cDNA library. Bioinformatics analysis shows that OsMY1 contains a coiled-coil domain which generally appeared in the partners of Rho GTPases. By yeast two-hybrid assay, it is confirmed that OsMY1 binds both the wild type (WT) and constitutively active (CA) OsRac5, but does not interact with dominantly negative OsRac5. In addition, the interactions between OsMY1 and WT-OsRac5 or CA-OsRac5 in vivo are demonstrated by bimolecular fluorescence complementation assay. Using PCR-mediated sequence deletion and point mutation of OsMY1, the interaction between OsMY1 and OsRac5 was identified to be mediated by the coiled-coil domain in OsMY1, and their binding was quantified by O-nitro-phenyl-beta-D-galactopyranoside assay. Real-time PCR shows that OsMY1 and OsRac5 are coordinately expressed in rice leaves and panicles with similar expression patterns. Our results suggest that OsMY1 is an important target of OsRac5 and that these two genes are involved in the same biological processes in rice growth and development. OsMY1,OsRacD|OsRac5 Cloning and expression of five myb-related genes from rice seed 1997 Gene Department of Biotechnology, National Institute of Agrobiological Resources, Tsukuba, Ibaraki, Japan. Three elements in the promoter of rice glutelin genes are important for their endosperm specific expression. One of these, an AACA motif, has been shown to be a negative regulator in non-seed tissues and has a similarity to the barley gibberellin responsive element recognized by MYB-like DNA binding proteins. A cDNA library constructed from immature rice seed was screened using two types of myb gene probes to isolate cDNA clones representing genes encoding MYB-like DNA binding proteins that may recognize the AACA motif in rice glutelin gene promoter. We obtained four cDNA clones encoding MYB-related proteins, Oryza sativa MYB (OSMYB) 1-4, using the maize C1 probe. Another myb-like clone, Osmyb5, was obtained by screening a rice seed cDNA library with probes designed to recognize the AACA-like binding domain in GAMYB and PHMYB3. RT-PCR was used to analyze Osmyb expression during rice seed development and their presence in other rice tissues, as it was not possible to detect these mRNAs by conventional Northern analysis. RT-PCR analysis showed that Osmyb2, Osmyb3 and Osmyb5 genes were expressed in all tissues examined. In seed, the mRNA levels of Osmyb1 and Osmyb4 genes reached a maximum at 14 days after flowering (DAF), suggesting that these genes may play a role in seed maturation. As Osmyb5 exhibits a high similarity to the regions in both GAMYB and PHMYB3, which can bind to the AACA motif, there is a possibility that the OSMYB5 protein may bind to the AACA motif of glutelin genes. Osmyb1,Osmyb2,Osmyb3,Osmyb4,Osmyb5 Overexpression of an R1R2R3 MYB Gene, OsMYB3R-2, Increases Tolerance to Freezing, Drought, and Salt Stress in Transgenic Arabidopsis 2007 Plant Physiol Department of Physiology and Biochemistry of Plants, Faculty of Biology, University of Bielefeld, 33615 Bielefeld, Germany We used a cDNA microarray approach to monitor the expression profile of rice (Oryza sativa) under cold stress and identified 328 cold-regulated genes. Thirteen such genes encoding MYB, homeodomain, and zinc finger proteins with unknown functions showed a significant change in expression under 72-h cold stress. Among them, OsMYB3R-2 was selected for further study. Unlike most plant R2R3 MYB transcription factors, OsMYB3R-2 has three imperfect repeats in the DNA-binding domain, the same as in animal c-MYB proteins. Expression of OsMYB3R-2 was induced by cold, drought, and salt stress. The Arabidopsis (Arabidopsis thaliana) transgenic plants overexpressing OsMYB3R-2 showed increased tolerance to cold, drought, and salt stress, and the seed germination of transgenic plants was more tolerant to abscisic acid or NaCl than that of wild type. The expression of some clod-related genes, such as dehydration-responsive element-binding protein 2A, COR15a, and RCI2A, was increased to a higher level in OsMYB3R-2-overexpressing plants than in wild type. These results suggest that OsMYB3R-2 acts as a master switch in stress tolerance. OsMYB3R-2 Evaluation of transgenic tomato plants ectopically expressing the rice Osmyb4 gene 2007 Plant Science Dipartimento Ambiente-Salute-Sicurezza, University of Insubria, 21100 Varese, Italy The rice Osmyb4 gene, coding for a MYB transcription factor, is expressed at low levels in rice coleoptiles under normal conditions and strongly induced at 4 °C. Its overexpression in Arabidopsis thaliana plants increases biotic and abiotic stress tolerance and results in the accumulation of several metabolites, essential in defence response. The heterologous expression of the Myb4 transcription factor represents a promising potential approach to improve stress tolerance in crops, avoiding endogenous mechanisms that often co-suppress the transgene of interest. In order to explore the potential of the Osmyb4 gene to enhance tolerance toward multiple stresses in different host plant genomes, we generated transgenic tomato (Solanum lycopersicum L. cv. Tondino) plants. Like Arabidopsis, tomato plants overexpressing Osmyb4 acquired a higher tolerance to drought stress and to virus disease. However, the transgenic plants did not appear to be more cold tolerant than the WT, in any tested condition. The data obtained indicate that the specificity and the degree of Osmyb4 activity depend on the host genomic background. Osmyb4 The ectopic expression of the rice Osmyb4 gene in Arabidopsis increases tolerance to abiotic, environmental and biotic stresses 2006 Physiological and Molecular Plant Pathology Dipartimento Ambiente, Salute, Sicurezza, Università degli Studi dell’Insubria, via J. H. Dunant 3, 21100 Varese, Italy The Osmyb4 rice gene encodes a Myb transcription factor involved in cold acclimation. Its constitutive expression in Arabidopsis thaliana results in improved cold and freezing tolerance. Osmyb4 up-regulated 254 genes, 22% of which encode proteins involved in gene expression regulation and signal transduction, suggesting an upstream role of Myb4 in stress response. Most of the up-regulated genes are known to be involved in tolerance not only to cold, but also to other abiotic and environmental stresses (drought, salt, oxidative stresses). Moreover, a high proportion has known functions in resistance to pathogen attacks. Therefore, we analyzed the biochemical and physiological differences between Osmyb4-expressing and wild-type plants and found increased levels of several amino acids that are involved in stress adaptation, acting as osmolytes, scavengers and/or metabolite precursors. When exposed to different adverse conditions, namely drought, salt, u.v., ozone, viruses, bacteria and fungi, transgenic plants effectively demonstrated improved tolerance/resistance to all these stress conditions, suggesting that Osmyb4 represents a crucial knot in the cross-talk of stress signalling cascades through the activation of multiple components. Osmyb4 Metabolic response to cold and freezing of Osteospermum ecklonis overexpressing Osmyb4 2010 Plant Physiol Biochem Unita di Ricerca per la Floricoltura e le Specie Ornamentali, Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Corso Degli Inglesi 508, 18038 Sanremo (IM), Italy. The constitutive expression of the rice Osmyb4 gene in Arabidopsis plants gives rise to enhanced abiotic and biotic stress tolerance, probably by activating several stress-inducible pathways. However, the effect of Osmyb4 on stress tolerance likely depends on the genetic background of the transformed species. In this study, we explored the potential of Osmyb4 to enhance the cold and freezing tolerance of Osteospermum ecklonis, an ornamental and perennial plant native to South Africa, because of an increasing interest in growing this species in Europe where winter temperatures are low. Transgenic O. ecklonis plants were obtained through transformation with the Osmyb4 rice gene under the control of the CaMV35S promoter. We examined the phenotypic adaptation of transgenic plants to cold and freezing stress. We also analysed the ability of wild-type and transgenic Osteospermum to accumulate several solutes, such as proline, amino acids and sugars. Using nuclear magnetic resonance, we outlined the metabolic profile of this species under normal growth conditions and under stress for the first time. Indeed, we found that overexpression of Osmyb4 improved the cold and freezing tolerance and produced changes in metabolite accumulation, especially of sugars and proline. Based on our data, it could be of agronomic and economic interest to use this gene to produce Osteospermum plants capable of growing in open field, even during the winter season in climatic zone Z9. Osmyb4 The rice Osmyb4 gene enhances tolerance to frost and improves germination under unfavourable conditions in transgenic barley plants 2012 J Appl Genet Agricultural Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, 2462, Martonvasar, Brunszvik u. 2, Hungary. The Osmyb4 rice gene, coding for a transcription factor, proved to be efficient against different abiotic stresses as a trans(cis)gene in several plant species, although the effectiveness was dependent on the host genomic background. Eight barley transgenic lines carrying the rice Osmyb4 gene under the control of the Arabidopsis cold inducible promoter cor15a were produced to test the efficiency of this gene in barley. After a preliminary test, the best performing lines were subjected to freezing at -11 degrees C and -12 degrees C. Frost tolerance was assessed measured the F(v)/F(m) parameter widely used to indicate the maximum quantum yield of photosystem II photochemistry in the dark adapted state. Three transgenic lines showed significantly increased tolerance. These selected lines were further studied under a complex stress applying cold and hypoxia at germinating stage. In these conditions the three selected transgenic lines outperformed the wild type barley in terms of germination vigour. The transgenic plants also showed a significant modification of their metabolism under cold/hypoxia conditions as demonstrated through the assessment of the activity of key enzymes involved in anoxic stress response. None of the transgenic lines showed dwarfism, just a slight retarded growth. These results provide evidence that the cold dependent expression of Osmyb4 can efficiently improved frost tolerance and germination vigour at low temperature without deleterious effect on plant growth. Osmyb4 Response of transgenic rape plants bearing the Osmyb4 gene from rice encoding a trans-factor to low above-zero temperature 2011 Russian Journal of Plant Physiology People’s Friedenship University, Moscow, Russia Accumulation of soluble sugars (sucrose, fructose, and glucose), proline, phenols (total phenols and flavonoids), and antocyanins during adaptation to low-temperature stress (4°C) of two lines of spring rape (Brassica napus L., cv. Westar) characterized by weak (Bn-1) and strong (Bn-3) expression of the Osmyb4 transgene was studied. Vegetatively propagated transgenic and wild-type plants were grown in the hydroponic culture at 24°C; at the stage of 5–6 leaves, plants were exposed to 4°C for 5 days and then returned to the optimum temperature of 24°C for recovery. Transgenic plants were established to manifest improved cold and frost tolerance, which was evident from more active biomass accumulation at 4°C as compared with wild-type plants and from sustaining their viability after 2-day-long exposure to −6°C. Determination of MDA content showed that one of the reasons of their improved cold tolerance was their capability of maintaining oxidative homeostasis under low-temperature stress. This suggestion is supported by intense accumulation of phenols and antocyanins, manifesting pronounced antioxidant effects, by transgenic plants during their cold adaptation. Thus, during 2–5 days of plant exposure to 4°C, in transgenic plants the total content of phenols increased by 2.6–3.7 times, flavonoids — by 3.7–4.7 times, and antocyanins — by 3.5–5.3 times as compared with control plants growing at 24°C. Transgenic Bn-3 plants with strong expression of the Osmyb4 gene accumulated phenols and antocyanins at 4°C more actively than Bn-1 plants characterized by weak expression of this gene. Transgenic rape plants subjected to cold stress accumulated more proline, manifesting stress-protection effects, and lesser accumulation of soluble sugars. Before the beginning of experiment, the content of soluble sugars was approximately similar in wild-type plants and transgenic lines; at 4°C their level in transgenic plants was substantially lower than in control plants. As distinct from the process of cold adaptation, during recovery, the content of all tested stress-protection compounds dropped sharply. The results obtained indicate that active expression of the Osmyb4 gene from rice in the rape plants was accompanied not only by accumulation of compatible osmolytes but also by biosynthesis of antioxidants of phenolic nature. Osmyb4 Supra-optimal expression of the cold-regulated OsMyb4 transcription factor in transgenic rice changes the complexity of transcriptional network with major effects on stress tolerance and panicle development 2010 Plant Cell Environ School of Biology and Ecology, University of Maine, Orono, ME 04469, USA. The R2R3-type OsMyb4 transcription factor of rice has been shown to play a role in the regulation of osmotic adjustment in heterologous overexpression studies. However, the exact composition and organization of its underlying transcriptional network has not been established to be a robust tool for stress tolerance enhancement by regulon engineering. OsMyb4 network was dissected based on commonalities between the global chilling stress transcriptome and the transcriptome configured by OsMyb4 overexpression. OsMyb4 controls a hierarchical network comprised of several regulatory sub-clusters associated with cellular defense and rescue, metabolism and development. It regulates target genes either directly or indirectly through intermediary MYB, ERF, bZIP, NAC, ARF and CCAAT-HAP transcription factors. Regulatory sub-clusters have different combinations of MYB-like, GCC-box-like, ERD1-box-like, ABRE-like, G-box-like, as1/ocs/TGA-like, AuxRE-like, gibberellic acid response element (GARE)-like and JAre-like cis-elements. Cold-dependent network activity enhanced cellular antioxidant capacity through radical scavenging mechanisms and increased activities of phenylpropanoid and isoprenoid metabolic processes involving various abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), ethylene and reactive oxygen species (ROS) responsive genes. OsMyb4 network is independent of drought response element binding protein/C-repeat binding factor (DREB/CBF) and its sub-regulons operate with possible co-regulators including nuclear factor-Y. Because of its upstream position in the network hierarchy, OsMyb4 functions quantitatively and pleiotrophically. Supra-optimal expression causes misexpression of alternative targets with costly trade-offs to panicle development. Osmyb4 Overexpression of the rice Osmyb4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants 2004 Plant J Dipartimento di Biologia Strutturale e Funzionale, Universita dell'Insubria, via J.H. Dunant 3, 21100 Varese, Italy. The expression of the gene Osmyb4, detected at low level in rice (Oryza sativa) coleoptiles grown for 3 days at 29 degrees C, is strongly induced by treatments at 4 degrees C. At sublethal temperatures of 10 and 15 degrees C, its expression in rice seedlings is already evident, but this effect cannot be vicariated by other stresses or ABA treatment. We demonstrate by transient expression that Myb4 transactivates the PAL2, ScD9 SAD and COR15a cold-inducible promoters. The Osmyb4 function in vivo is demonstrated overexpressing its cDNA in Arabidopsis thaliana plants (ecotype Wassilewskija) under the control of the constitutive CaMV 35S promoter. Myb4 overexpressing plants show a significant increased cold and freezing tolerance, measured as membrane or Photosystem II (PSII) stability and as whole plant tolerance. Finally, in Osmyb4 transgenic plants, the expression of genes participating in different cold-induced pathways is affected, suggesting that Myb4 represents a master switch in cold tolerance. Osmyb4 Transcriptional activation of secondary wall biosynthesis by rice and maize NAC and MYB transcription factors 2011 Plant Cell Physiol Department of Plant Biology, University of Georgia, Athens, GA 30602, USA. The bulk of grass biomass potentially useful for cellulose-based biofuel production is the remains of secondary wall-containing sclerenchymatous fibers. Hence, it is important to uncover the molecular mechanisms underlying the regulation of secondary wall thickening in grass species. So far, little is known about the transcriptional regulatory switches responsible for the activation of the secondary wall biosynthetic program in grass species. Here, we report the roles of a group of rice and maize NAC and MYB transcription factors in the regulation of secondary wall biosynthesis. The rice and maize secondary wall-associated NACs (namely OsSWNs and ZmSWNs) were able to complement the Arabidopsis snd1 nst1 double mutant defective in secondary wall thickening. When overexpressed in Arabidopsis, OsSWNs and ZmSWNs were sufficient to activate a number of secondary wall-associated transcription factors and secondary wall biosynthetic genes, and concomitantly result in the ectopic deposition of cellulose, xylan and lignin. It was also found that the rice and maize MYB transcription factors, OsMYB46 and ZmMYB46, are functional orthologs of Arabidopsis MYB46/MYB83 and, when overexpressed in Arabidopsis, they were able to activate the entire secondary wall biosynthetic program. Furthermore, the promoters of OsMYB46 and ZmMYB46 contain secondary wall NAC-binding elements (SNBEs), which can be bound and activated by OsSWNs and ZmSWNs. Together, our results indicate that the rice and maize SWNs and MYB46 are master transcriptional activators of the secondary wall biosynthetic program and that OsSWNs and ZmSWNs activate their direct target genes through binding to the SNBE sites. OsMYB46,OsSWN1,OsSWN2,OsSWN3,OsSWN4,OsSWN5,OsSWN6,OsSWN7 Overexpression of OsMYB4P, an R2R3-type MYB transcriptional activator, increases phosphate acquisition in rice 2014 Plant Physiol Biochem College of Life Science and Natural Resources, Dong-A University, Busan 604-714, Republic of Korea. R2R3 MYB transcription factors play regulatory roles in plant responses to various environmental stresses and nutrient deficiency. In this study, we isolated and designated OsMYB4P, an R2R3 MYB transcription factor, from rice (Oryza sativa L. 'Dongjin') under phosphate-deficient conditions. OsMYB4P was localized in the nucleus and acted as a transcriptional activator. Transcriptional levels of OsMYB4P in cell suspension, shoots, and roots of rice increased under phosphate-deficient conditions. Shoots and roots of OsMYB4P-overexpressing plants grew well in high- and phosphate-deficient conditions. In addition, root system architecture was altered considerably as a result of OsMYB4P overexpression. Under both phosphate-sufficient and -deficient conditions, more Pi accumulated in shoots and roots of OsMYB4P-overexpressing plants than in the wild type. Overexpression of OsMYB4P led to greater expression of Pi transporter-family proteins OsPT1, OsPT2, OsPT4, OsPT7, and OsPT8 in shoots, and to decreased or unchanged expression of these proteins in roots, with the exception of OsPT8. These results demonstrate that OsMYB4P may be associated with efficient utilization of Pi in rice through transcriptional activation of Pi homeostasis-related genes. OsMYB4P Rice MYB Protein OSMYB5 Specifically Binds to the AACA Motif Conserved among Promoters of Genes for Storage Protein Glutelin 1998 Plant Cell Physiol Department of Biotechnology, National Institute of Agrobiological Resources Tsukuba, Ibaraki, 305 Japan Binding analyses revealed that the AAC--A sequence in glutelin gene promoters is the target site of OSMYB5 protein and that both the distal and proximal AACA motifs are recognized by this protein. These results suggest that the OSMYB5 protein functions as a trans-acting factor for glutelin gene expression. Osmyb5 MYB80, a regulator of tapetal and pollen development, is functionally conserved in crops 2012 Plant Mol Biol Botany Department, La Trobe University, Melbourne, Australia. The Arabidopsis AtMYB80 transcription factor (formerly AtMYB103) regulate genes essential for tapetal and pollen development. One of these genes, coding for an aspartic protease (UNDEAD), may control the timing of tapetal programmed cell death (PCD). In crop plants such as rice and wheat, abiotic stresses lead to abnormal tapetal development resulting in delayed PCD. Manipulation of AtMYB80 function has been used to develop a reversible male sterility system applicable to hybrid crop production. MYB80 homologs were cloned from wheat, rice, canola and cotton. The promoters of the homologs drove temporal and spatial expression patterns of the GUS reporter gene in the tapetum and microspores of Arabidopsis anthers identical to the AtMYB80 promoter. A short region is conserved in all five MYB80 promoters. The MYB80 homolog genes, driven by the AtMYB80 or their respective promoters, rescued the atmyb80 mutant, completely restoring male fertility. The canola MYB80 was fused to the EAR (ERF-associated amphiphilic repression) repressor and canola plants transgenic for the construct exhibited premature tapetal degradation and subsequent pollen abortion. The five MYB80 homologs all shared a 44 amino acid sequence immediately adjacent to the R2R3 domain which appears to be necessary for MYB80 function. OsMYB80 Expression of StMYB1R-1, a novel potato single MYB-like domain transcription factor, increases drought tolerance 2011 Plant Physiol Bio-crop Development Division, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-857, Republic of Korea. Potato (Solanum tuberosum) is relatively vulnerable to abiotic stress conditions such as drought, but the tolerance mechanisms for such stresses in potato are largely unknown. To identify stress-related factors in potato, we previously carried out a genetic screen of potato plants exposed to abiotic environmental stress conditions using reverse northern-blot analysis. A cDNA encoding a putative R1-type MYB-like transcription factor (StMYB1R-1) was identified as a putative stress-response gene. Here, the transcript levels of StMYB1R-1 were enhanced in response to several environmental stresses in addition to drought but were unaffected by biotic stresses. The results of intracellular targeting and quadruple 9-mer protein-binding microarray analysis indicated that StMYB1R-1 localizes to the nucleus and binds to the DNA sequence (G)/(A)GATAA. Overexpression of a StMYB1R-1 transgene in potato plants improved plant tolerance to drought stress while having no significant effects on other agricultural traits. Transgenic plants exhibited reduced rates of water loss and more rapid stomatal closing than wild-type plants under drought stress conditions. In addition, overexpression of StMYB1R-1 enhanced the expression of drought-regulated genes such as AtHB-7, RD28, ALDH22a1, and ERD1-like. Thus, the expression of StMYB1R-1 in potato enhanced drought tolerance via regulation of water loss. These results indicated that StMYB1R-1 functions as a transcription factor involved in the activation of drought-related genes. OsMYBS1 Three Novel MYB Proteins with One DNA Binding Repeat Mediate Sugar and Hormone Regulation of alpha-Amylase Gene Expression 2002 The Plant Cell Online Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan 115, Republic of China. The expression of alpha-amylase genes in cereals is induced by both gibberellin (GA) and sugar starvation. All alpha-amylase genes isolated from cereals contain a TATCCA element or its variants at positions approximately 90 to 150 bp upstream of the transcription start sites. The TATCCA element was shown previously to be an important component of the GA response complex and the sugar response complex of alpha-amylase gene promoters. In the present study, three cDNA clones encoding novel MYB proteins with single DNA binding domains were isolated from a rice suspension cell cDNA library and designated OsMYBS1, OsMYBS2, and OsMYBS3. Gel mobility shift experiments with OsMYBSs showed that they bind specifically to the TATCCA element in vitro. Yeast one-hybrid experiments demonstrated that OsMYBS1 and OsMYBS2 bind to the TATCCA element and transactivate a promoter containing the TATCCA element in vivo. Transient expression assays with barley half-seeds showed that OsMYBS1 and OsMYBS2 transactivate a promoter containing the TATCCA element when sugar is provided, whereas OsMYBS3 represses transcription of the same promoter under sugar starvation. Transient expression assays also showed that these three OsMYBSs cooperate with a GA-regulated transcription factor, HvMYBGa, in the transactivation of a low-pI barley alpha-amylase gene promoter in the absence of GA. Two-hybrid experiments with barley half-seeds showed that OsMYBS1 is able to form a homodimer. The present study demonstrates that differential DNA binding affinity, promoter transactivation ability, dimerization, and interactions with other protein factors determine the biological function of OsMYBSs. This study also suggests that common transcription factors are involved in the sugar and hormonal regulation of alpha-amylase gene expression in cereals. OsMYBS1,OsMYBS2,MYBS3|OsMYBS3 A novel MYBS3-dependent pathway confers cold tolerance in rice 2010 Plant Physiol Institute of Biotechnology, National Cheng Kung University, Tainan 701, Taiwan, Republic of China. Rice (Oryza sativa) seedlings are particularly sensitive to chilling in early spring in temperate and subtropical zones and in high-elevation areas. Improvement of chilling tolerance in rice may significantly increase rice production. MYBS3 is a single DNA-binding repeat MYB transcription factor previously shown to mediate sugar signaling in rice. In this study, we observed that MYBS3 also plays a critical role in cold adaptation in rice. Gain- and loss-of-function analyses indicated that MYBS3 was sufficient and necessary for enhancing cold tolerance in rice. Transgenic rice constitutively overexpressing MYBS3 tolerated 4 degrees C for at least 1 week and exhibited no yield penalty in normal field conditions. Transcription profiling of transgenic rice overexpressing or underexpressing MYBS3 led to the identification of many genes in the MYBS3-mediated cold signaling pathway. Several genes activated by MYBS3 as well as inducible by cold have previously been implicated in various abiotic stress responses and/or tolerance in rice and other plant species. Surprisingly, MYBS3 repressed the well-known DREB1/CBF-dependent cold signaling pathway in rice, and the repression appears to act at the transcriptional level. DREB1 responded quickly and transiently while MYBS3 responded slowly to cold stress, which suggests that distinct pathways act sequentially and complementarily for adapting short- and long-term cold stress in rice. Our studies thus reveal a hitherto undiscovered novel pathway that controls cold adaptation in rice. MYBS3|OsMYBS3 Jasmonic acid regulates spikelet development in rice 2014 Nature Communications State Key Laboratory of Hybrid Rice, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China The spikelet is the basal unit of inflorescence in grasses, and its formation is crucial for reproductive success and cereal yield. Here, we report a previously unknown role of the plant hormone jasmonic acid (JA) in determining rice (Oryza sativa) spikelet morphogenesis. The extra glume 1 (eg1) and eg2 mutants exhibit altered spikelet morphology with changed floral organ identity and number, as well as defective floral meristem determinacy. We show that EG1 is a plastid-targeted lipase that participates in JA biosynthesis, and EG2/OsJAZ1 is a JA signalling repressor that interacts with a putative JA receptor, OsCOI1b, to trigger OsJAZ1’s degradation during spikelet development. OsJAZ1 also interacts with OsMYC2, a transcription factor in the JA signalling pathway, and represses OsMYC2’s role in activating OsMADS1, an E-class gene crucial to the spikelet development. This work discovers a key regulatory mechanism of grass spikelet development and suggests that the role of JA in reproduction has diversified during the flowering plant evolution. OsMYC2 ORYZA SATIVA MYOSIN XI B controls pollen development by photoperiod-sensitive protein localizations 2007 Dev Biol Rice Functional Genomics Group, Temasek Life Sciences Laboratory, 1 Research Link, the National University of Singapore 117604, Singapore. Myosins are actin-based motor proteins responsible for various motility and signal transduction. Only a small set of myosin classes is present inplants, and little is known about their functions. Here we showed how a rice myosin gene controlled pollen development by sensing changed environmental factors. The analysis is based on a gene-trapped Ds insertion mutant Oryza sativa myosin XI B (osmyoXIB). This mutant showed male sterility under short day length (SD) conditions and fertility under long day length (LD) conditions. Under both SD and LD conditions, the OSMYOXIB transcript was detected in whole anthers. However, under SD conditions, the OSMYOXIB-GUS fusion protein was localized only in the epidermal layer of anthers due to the lack of 3'-untranslated region (3'-UTR) and to dilute (DIL) domain sequences following the Ds insertion. As a result, mutant pollen development was affected, leading to male sterility. By contrast, under LD conditions, the fusion protein was localized normally in anthers. Despite normal localization, the protein was only partially functional due to the lack of DIL domain sequences, resulting in limited recovery of pollen fertility. This study also provides a case for a novel molecular aspect of gene expression, i.e., cell layer-specific translation in anthers. osmyoXIB Root-specific expression of OsNAC10 improves drought tolerance and grain yield in rice under field drought conditions 2010 Plant Physiol School of Biotechnology and Environmental Engineering, Myongji University, Yongin 449-728, Korea. Drought poses a serious threat to the sustainability of rice (Oryza sativa) yields in rain-fed agriculture. Here, we report the results of a functional genomics approach that identified a rice NAC (an acronym for NAM [No Apical Meristem], ATAF1-2, and CUC2 [Cup-Shaped Cotyledon]) domain gene, OsNAC10, which improved performance of transgenic rice plants under field drought conditions. Of the 140 OsNAC genes predicted in rice, 18 were identified to be induced by stress conditions. Phylogenic analysis of the 18 OsNAC genes revealed the presence of three subgroups with distinct signature motifs. A group of OsNAC genes were prescreened for enhanced stress tolerance when overexpressed in rice. OsNAC10, one of the effective members selected from prescreening, is expressed predominantly in roots and panicles and induced by drought, high salinity, and abscisic acid. Overexpression of OsNAC10 in rice under the control of the constitutive promoter GOS2 and the root-specific promoter RCc3 increased the plant tolerance to drought, high salinity, and low temperature at the vegetative stage. More importantly, the RCc3:OsNAC10 plants showed significantly enhanced drought tolerance at the reproductive stage, increasing grain yield by 25% to 42% and by 5% to 14% over controls in the field under drought and normal conditions, respectively. Grain yield of GOS2:OsNAC10 plants in the field, in contrast, remained similar to that of controls under both normal and drought conditions. These differences in performance under field drought conditions reflect the differences in expression of OsNAC10-dependent target genes in roots as well as in leaves of the two transgenic plants, as revealed by microarray analyses. Root diameter of the RCc3:OsNAC10 plants was thicker by 1.25-fold than that of the GOS2:OsNAC10 and nontransgenic plants due to the enlarged stele, cortex, and epidermis. Overall, our results demonstrated that root-specific overexpression of OsNAC10 enlarges roots, enhancing drought tolerance of transgenic plants, which increases grain yield significantly under field drought conditions. ONAC122|OsNAC10 Overexpression of a NAC-domain protein promotes shoot branching in rice 2007 New Phytol State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, 310058, PR China. For a better understanding of shoot branching in rice (Oryza sativa), a rice activation-tagging library was screened for mutations in tiller development. Here, an activation-tagging mutant Ostil1 (Oryza sativa tillering1) was characterized, which showed increased tillers, enlarged tiller angle and semidwarf phenotype. Flanking sequence was obtained by plasmid rescue. RNA-interfering and overexpression transgenic rice plants were produced using Agrobacterium-mediated transformation. The mutant phenotype was cosegregated with the reallocation of Ds element, and the flanking region of the reallocated Ds element was identified as part of the OsNAC2 gene. Northern analysis showed that expression of OsNAC2 was greatly induced in the mutant plants. Transgenic rice overexpressing the OsNAC2 resulted in recapture of the mutant phenotype, while downregulation of OsNAC2 in the Ostil1 mutant through RNA interfering (RNAi) complemented the mutant phenotype, confirming that the Ostil1 was caused by overexpression of OsNAC2. Overexpression of OsNAC2 regulates shoot branching in rice. Overexpression of OsNAC2 contributes tiller bud outgrowth, but does not affect tiller bud initiation. This suggests that OsNAC2 has potential utility for improving plant structure for higher light-use efficiency and higher yield potential in rice. OsNAC2|OsTIL1 Rice gene OsNAC19 encodes a novel NAC-domain transcription factor and responds to infection by Magnaporthe grisea 2007 Plant Science Department of Plant Pathology, China Agricultural University, Beijing 100094, PR China The plant-specific NAC-domain proteins have been identified to play important roles in plant responses to stresses or in plant development regulation. In this research, a full-length cDNA clone OsNAC19 (Oryza sativa NAC19), encoding a novel NAC-domain protein, was isolated from a cDNA library prepared with rice leaves infected by incompatible race 131 of blast fungus (Magnaporthe grisea). The similarity between OsNAC19 and the members of OsNAC3 subgroup is from 65.87 to 51.53%. One plant-specific NAC domain is located in the N-terminus of OsNAC19. The protein OsNAC19 was localized in the nucleus of onion epidemical cells. The fusion protein of OsNAC19 with GAL4 DNA-binding domain (BD) activated the reporter genes LacZ and HIS3 in a yeast assay system, and the activation domain was located within amino acid 181-240 of its C-terminal region. The expression of gene OsNAC19 was high in rice seedling roots, culms and blade sheathes, but its expression in rice leaves was low. The expression of OsNAC19 in rice leaves could be induced by the infection of blast fungus, and by application of exogenous methyl jasmonate (MeJA), ABA and ethylene but ethylene had a relatively weak induction effect. These data suggest that OsNAC19 protein is a transcriptional activator involved in rice response to infection by M. grisea and may play a role in the MeJA-mediated signaling pathway. OsNAC3,OsNAC19|SNAC1|OsNAC9 The transcription factor OsNAC4 is a key positive regulator of plant hypersensitive cell death 2009 EMBO J Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama Ikoma, Nara, Japan. The hypersensitive response (HR) is a common feature of plant immune responses and a type of programmed cell death. However, little is known about the induction mechanism of HR cell death. We report that overexpression of OsNAC4, which encodes a plant-specific transcription factor, leads to HR cell death accompanied by the loss of plasma membrane integrity, nuclear DNA fragmentation and typical morphological changes. In OsNAC4 knock-down lines, HR cell death is markedly decreased in response to avirulent bacterial strains. After induction by an avirulent pathogen recognition signal, OsNAC4 is translocated into the nucleus in a phosphorylation-dependent manner. A microarray analysis showed that the expression of 139 genes including OsHSP90 and IREN, encoding a Ca(2+)-dependent nuclease, were different between the OsNAC4 knock-down line and control line during HR cell death. During the induction of HR cell death, OsHSP90 is involved in the loss of plasma membrane integrity, whereas IREN causes nuclear DNA fragmentation. Overall, our results indicate that two important events occurring during HR cell death are regulated by independent pathways. OsNAC4 Role of OsHSP90 and IREN, Ca2+ dependent nuclease, in plant hypersensitive cell death induced by transcription factor OsNAC4 2009 Plant Signal Behav Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Shiga, Japan. The hypersensitive response (HR) is a form of programmed cell death (PCD) commonly associated with the immune response in plants. HR cell death is often characterized by DNA fragmentation, loss of plasma membrane integrity, protein degradation and typical morphological changes such as plasma membrane shrinkage and nuclear condensation. Initiation of HR cell death requires de novo protein synthesis, suggesting that HR cell death induction involves a transcriptional network regulated by a key factor. We recently identified the OsNAC4 gene, which encodes a plant-specific transcription factor that exhibited rapid but transient transcriptional activation during the early stages of HR cell death. Overexpression of OsNAC4 in rice plants induced cell death accompanied by all characteristics of HR cell death: DNA fragmentation, loss of plasma membrane integrity, and protein degradation. In OsNAC4 RNAi knock-down lines exposed to an avirulent bacterial strain, the cellular response was characterized by a marked decrease in HR cell death compared to wild-type rice cells. Gene expression profiling, which compared rice cells and OsNAC4 knock-down transformants using a rice cDNA microarray, demonstrated that OsNAC4 controls the transcription of at least 139 genes including OsHSP90, involved in loss of plasma membrane integrity, and IREN, which encodes novel plant nuclease involved in cleavage of nuclear DNA. Here we report that although OsNAC4 overexpression caused rapid protein degradation during HR cell death, neither IREN nor OsHSP90 were involved. Thus, three important processes that accompany HR cell death are regulated by independent signaling pathways that are collectively induced by OsNAC4. OsNAC4 Physiological mechanisms underlying OsNAC5-dependent tolerance of rice plants to abiotic stress 2011 Planta State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, The Chinese Academy of Sciences, Beijing, 100093, People's Republic of China. To understand the functions of transcription factor OsNAC5 in response to abiotic stress, we generated transgenic rice plants with knockdown OsNAC5 by RNA-interfered (RNAi) and overexpressing OsNAC5, and investigated the effects of cold, drought and salt stress on wild-type (WT), RNAi and overexpression rice lines. Our results demonstrated that RNAi lines became less tolerant to these stresses than WT plants, while overexpression of OsNAC5 in Arabidopsis and rice enhanced tolerance to these stresses. The mechanisms underlying the changes in tolerance of the transgenic rice plants to abiotic stresses were explored by measuring free proline (Pro) and soluble sugar contents in WT and transgenic plants. Accumulation of Pro and soluble sugars was positively correlated with OsNAC5 expression levels. The less accumulation of Pro in RNAi lines may be accounted for by inhibition of Pro synthesis and transport at transcriptional levels. In addition, knockdown and overexpression of OsNAC5 enhanced and reduced accumulation of malondialdehyde and H(2)O(2), suggesting that knockdown of OsNAC5 renders RNAi plants more susceptible to oxidative damage. The RNAi lines displayed higher Na(+)/K(+) ratio due to greater accumulation of Na(+) ions than WT under salt stress conditions, and expression of genes encoding tonoplast Na(+)/H(+) antiporter was lower in RNAi lines than in WT under both control and salt-stressed conditions. Seed germination of RNAi and overexpression plants was more and less inhibited by salt and mannitol than that of WT, respectively. Seed germination of overexpression and RNAi plants was more and less sensitive than that of WT to ABA. These findings highlight the important role of OsNAC5 played in the tolerance of rice plants to abiotic stress by regulating downstream targets associated with accumulation of compatible solutes, Na(+) ions, H(2)O(2) and malondialdehyde. OsNAC5 OsNAC5 overexpression enlarges root diameter in rice plants leading to enhanced drought tolerance and increased grain yield in the field 2013 Plant Biotechnol J School of Biotechnology and Environmental Engineering, Myongji University, Yongin, Korea. Drought conditions are among the most serious challenges to crop production worldwide. Here, we report the results of field evaluations of transgenic rice plants overexpressing OsNAC5, under the control of either the root-specific (RCc3) or constitutive (GOS2) promoters. Field evaluations over three growing seasons revealed that the grain yield of the RCc3:OsNAC5 and GOS2:OsNAC5 plants were increased by 9%-23% and 9%-26% under normal conditions, respectively. Under drought conditions, however, RCc3:OsNAC5 plants showed a significantly higher grain yield of 22%-63%, whilst the GOS2:OsNAC5 plants showed a reduced or similar yield to the nontransgenic (NT) controls. Both the RCc3:OsNAC5 and GOS2:OsNAC5 plants were found to have larger roots due to an enlarged stele and aerenchyma at flowering stage. Cell numbers per cortex layer and stele of developing roots were higher in both transgenic plants than NT controls, contributing to the increase in root diameter. The root diameter was enlarged to a greater extent in the RCc3:OsNAC5, suggesting the importance of this phenotype for enhanced drought tolerance. Microarray experiments identified 25 up-regulated genes by more than three-fold (P < 0.01) in the roots of both transgenic lines. Also identified were 19 and 18 up-regulated genes that are specific to the RCc3:OsNAC5 and GOS2:OsNAC5 roots, respectively. Of the genes specifically up-regulated in the RCc3:OsNAC5 roots, GLP, PDX, MERI5 and O-methyltransferase were implicated in root growth and development. Our present findings demonstrate that the root-specific overexpression of OsNAC5 enlarges roots significantly and thereby enhances drought tolerance and grain yield under field conditions. OsNAC5 Molecular analysis of the NAC gene family in rice 2000 Mol Gen Genet Laboratory of Plant Breeding and Genetics, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan. Genes that encode products containing a NAC domain, such as NO APICAL MERISTEM (NAM) in petunia, CUP-SHAPED COTYLEDON2 (CUC2) and NAP in Arabidopsis thaliana, have crucial functions in plant development. We describe here molecular aspects of the OsNAC genes that encode proteins with NAC domains in rice (Oryza sativa L.). Sequence analysis revealed that the NAC genes in plants can be divided into several subfamilies, such as the NAM, ATAF, and OsNAC3 subfamilies. In rice, OsNAC1 and OsNAC2 are classified in the NAM subfamily, which includes NAM and CUC2, while OsNAC5 and OsNAC6 fall into the ATAF subfamily. In addition to the members of these subfamilies, the rice genome contains the NAC genes OsNAC3, OsNAC4 (both in the OsNAC3 subfamily), OsNAC7, and OsNAC8. These results and Southern analysis indicate that the OsNAC genes constitute a large gene family in the rice genome. Each OsNAC gene is expressed in a specific pattern in different organs, suggesting that this family has diverse and important roles in rice development. OsNAC5 Identification of up-regulated genes in flag leaves during rice grain filling and characterization of OsNAC5, a new ABA-dependent transcription factor 2009 Planta Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, 91501-970 Porto Alegre, RS, Brazil. Rice is a poor source of micronutrients such as iron and zinc. To help clarify the molecular mechanisms that regulate metal mobilization from leaves to developing seeds, we conducted suppression subtractive hybridization analysis in flag leaves of two rice cultivars. Flag leaves are the major source of remobilized metals for developing seeds. We isolated 78 sequences up-regulated in flag leaves at the grain filling stage relative to the panicle exertion stage. Differential expression of selected genes (encoding 7 transport proteins, the OsNAS3 enzyme and the OsNAC5 transcription factor) was confirmed by quantitative RT-PCR. We show that OsNAC5 expression is up-regulated by natural (aging) and induced senescence processes (dark, ABA application, high salinity, cold and Fe-deficiency) and its expression is not affected in the presence of 6-benzylaminopurine (a senescence inhibitor) under dark-induced senescence. Salt induction of OsNAC5 expression is abolished by nicotinamide, an inhibitor of ABA effects. This result and the presence of cis-acting elements in the promoter region of the OsNAC5 gene suggest an ABA-dependent regulation. Using four different rice cultivars, we show that OsNAC5 up-regulation is higher and earlier in flag leaves and panicles of IR75862 plants, which have higher seed concentrations of Fe, Zn and protein. We suggest that OsNAC5 is a novel senescence-associated ABA-dependent NAC transcription factor and its function could be related to Fe, Zn and amino acids remobilization from green tissues to seeds. OsNAC5,OsNAS3 Disruption of a Novel NADH-Glutamate Synthase2 Gene Caused Marked Reduction in Spikelet Number of Rice 2011 Front Plant Sci Graduate School of Agricultural Science, Tohoku University Sendai, Japan. Inorganic ammonium ions are assimilated by a coupled reaction of glutamine synthetase and glutamate synthase (GOGAT). In rice, three genes encoding either ferredoxin (Fd)-GOGAT, NADH-GOGAT1, or NADH-GOGAT2, have been identified. OsNADH-GOGAT2, a newly identified gene, was expressed mainly in fully expanded leaf blades and leaf sheaths. Although the distinct expression profile to OsNADH-GOGAT1, which is mainly detected in root tips, developing leaf blades, and grains, was shown in our previous studies, physiological role of NADH-GOGAT2 is not yet known. Here, we isolated retrotransposon mediated-knockout mutants lacking OsNADH-GOGAT2. In rice grown under paddy field conditions, disruption of the OsNADH-GOGAT2 gene caused a remarkable decrease in spikelet number per panicle associated with a reductions in yield and whole plant biomass, when compared with wild-type (WT) plants. The total nitrogen contents in the senescing leaf blade of the mutants were approximately a half of the WT plants. Expression of this gene was mainly detected in phloem companion cells and phloem parenchyma cells associated with large vascular bundles in fully expanded leaf blades, when the promoter region fused with a beta-glucuronidase gene was introduced into the WT rice. These results suggest that the NADH-GOGAT2 is important in the process of glutamine generation in senescing leaves for the remobilization of leaf nitrogen through phloem to the panicle during natural senescence. These results also indicate that other GOGATs, i.e., NADH-GOGAT1 and ferredoxin-GOGAT are not able to compensate the function of NADH-GOGAT2. OsNADH-GOGAT2 Identification and functional characterization of a rice NAC gene involved in the regulation of leaf senescence 2013 BMC Plant Biol National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan, 430070, China. yongjunlin@mail.hzau.edu.cn. BACKGROUND: As the final stage of leaf development, leaf senescence may cause the decline of photosynthesis and gradual reduction of carbon assimilation, which makes it a possible limiting factor for crop yield. NACs are plant-specific transcription factors and some NACs have been confirmed to play important roles in regulating leaf senescence. RESULTS: In this study, we reported a member of the NAC transcription factor family named OsNAP whose expression is associated with leaf senescence, and investigated its preliminary function during the process of leaf senescence. The results of qRT-PCR showed that the OsNAP transcripts were accumulated gradually in response to leaf senescence and treatment with methyl jasmonic acid (MeJA). A subcellular localization assay indicated that OsNAP is a nuclear-localized protein. Yeast one-hybrid experiments indicated that OsNAP can bind the NAC recognition site (NACRS)-like sequence. OsNAP-overexpressing transgenic plants displayed an accelerated leaf senescence phenotype at the grain-filling stage, which might be caused by the elevated JA levels and the increased expression of the JA biosynthesis-related genes LOX2 and AOC1, and showed enhanced tolerance ability to MeJA treatment at the seedling stage. Nevertheless, the leaf senescence process was delayed in OsNAP RNAi transgenic plants with a dramatic drop in JA levels and with decreased expression levels of the JA biosynthesis-related genes AOS2, AOC1 and OPR7. CONCLUSIONS: These results suggest that OsNAP acts as a positive regulator of leaf senescence and this regulation may occur via the JA pathway. OsNAP OsNAP connects abscisic acid and leaf senescence by fine-tuning abscisic acid biosynthesis and directly targeting senescence-associated genes in rice 2014 Proceedings of the National Academy of Sciences State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China It has long been established that premature leaf senescence negatively impacts the yield stability of rice, but the underlying molecular mechanism driving this relationship remains largely unknown. Here, we identified a dominant premature leaf senescence mutant, prematurely senile 1 (ps1-D). PS1 encodes a plant-specific NAC (no apical meristem, Arabidopsis ATAF1/2, and cup-shaped cotyledon2) transcriptional activator, Oryza sativa NAC-like, activated by apetala3/pistillata (OsNAP). Overexpression of OsNAP significantly promoted senescence, whereas knockdown of OsNAP produced a marked delay of senescence, confirming the role of this gene in the development of rice senescence. OsNAP expression was tightly linked with the onset of leaf senescence in an age-dependent manner. Similarly, ChIP-PCR and yeast one-hybrid assays demonstrated that OsNAP positively regulates leaf senescence by directly targeting genes related to chlorophyll degradation and nutrient transport and other genes associated with senescence, suggesting that OsNAP is an ideal marker of senescence onset in rice. Further analysis determined that OsNAP is induced specifically by abscisic acid (ABA), whereas its expression is repressed in both aba1 and aba2, two ABA biosynthetic mutants. Moreover, ABA content is reduced significantly in ps1-D mutants, indicating a feedback repression of OsNAP on ABA biosynthesis. Our data suggest that OsNAP serves as an important link between ABA and leaf senescence. Additionally, reduced OsNAP expression leads to delayed leaf senescence and an extended grain-filling period, resulting in a 6.3% and 10.3% increase in the grain yield of two independent representative RNAi lines, respectively. Thus, fine-tuning OsNAP expression should be a useful strategy for improving rice yield in the future. OsNAP Three rice nicotianamine synthase genes, OsNAS1, OsNAS2, and OsNAS3 are expressed in cells involved in long-distance transport of iron and differentially regulated by iron 2003 The Plant Journal Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Nicotianamine (NA), a chelator of metals, is ubiquitously present in higher plants. In graminaceous plants, NA is a biosynthetic precursor of phytosiderophores and is thus a crucial component for iron (Fe) acquisition. Here, we show that three rice NA synthase (NAS) genes, OsNAS1, OsNAS2, and OsNAS3 are expressed in cells involved in long-distance transport of Fe and that the three genes are differentially regulated by Fe. OsNAS1 and OsNAS2 transcripts were detected in Fe-sufficient roots but not in leaves, and levels of both increased markedly in both roots and leaves in response to Fe deficiency. In contrast, the OsNAS3 transcript was present in leaves but was very low in roots of Fe-sufficient plants. Further, OsNAS3 expression was induced in roots but was suppressed in leaves in response to Fe deficiency. Promoter-GUS analysis revealed that OsNAS1 and OsNAS2 were expressed in Fe-sufficient roots in companion cells and pericycle cells adjacent to the protoxylem. With Fe deficiency, OsNAS1 and OsNAS2 expression extended to all root cells along with an increase in phytosiderophore secretion. In Fe-deficient plants, OsNAS1 and OsNAS2 were expressed in the vascular bundles of green leaves and in all cells of leaves showing severe chlorosis. OsNAS3 expression was restricted to the pericycle and companion cells of the roots, and in companion cells of leaves irrespective of Fe status. These results strongly suggested that NAS and NA play an important role in long-distance transport of Fe in rice plants, in addition to their roles in phytosiderophore secretion from roots. OsNAS1,OsNAS2,OsNAS3 Bio-available zinc in rice seeds is increased by activation tagging of nicotianamine synthase 2011 Plant Biotechnol J Department of Plant Molecular Systems Biotechnology and Crop Biotech Center, Kyung Hee University, Yongin, Republic of Korea. We generated rice lines with increased content of nicotianamine (NA), a key ligand for metal transport and homeostasis. This was accomplished by activation tagging of rice nicotianamine synthase 2 (OsNAS2). Enhanced expression of the gene resulted in elevated NA levels, greater Zn accumulations and improved plant tolerance to a Zn deficiency. Expression of Zn-uptake genes and those for the biosynthesis of phytosiderophores (PS) were increased in transgenic plants. This suggests that the higher amount of NA led to greater exudation of PS from the roots, as well as stimulated Zn uptake, translocation and seed-loading. In the endosperm, the OsNAS2 activation-tagged line contained up to 20-fold more NA and 2.7-fold more zinc. Liquid chromatography combined with inductively coupled plasma mass spectrometry revealed that the total content of zinc complexed with NA and 2'-deoxymugineic acid was increased 16-fold. Mice fed with OsNAS2-D1 seeds recovered more rapidly from a zinc deficiency than did control mice receiving WT seeds. These results demonstrate that the level of bio-available zinc in rice grains can be enhanced significantly by activation tagging of OsNAS2. OsNAS2 Activation of Rice nicotianamine synthase 2 (OsNAS2) enhances iron availability for biofortification 2012 Mol Cells Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755, USA. Because micronutrients in human diets ultimately come from plant sources, malnutrition of essential minerals is a significant public health concern. By increasing the expression of nicotianamine synthase (NAS), we fortified the level of bioavailable iron in rice seeds. Activation of iron deficiency-inducible OsNAS2 resulted in a rise in Fe content (3.0-fold) in mature seeds. Its ectopic expression also increased that content. Enhanced expression led to higher tolerance of Fe deficiency and better growth under elevated pH. Mice fed with OsNAS2-D1 seeds recovered more rapidly from anemia, indicating that bioavailable Fe contents were improved by this increase in OsNAS2 expression. OsNAS2 Iron fortification of rice seeds through activation of the nicotianamine synthase gene 2009 Proc Natl Acad Sci U S A Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea. The most widespread dietary problem in the world is mineral deficiency. We used the nicotianamine synthase (NAS) gene to increase mineral contents in rice grains. Nicotianamine (NA) is a chelator of metals and a key component of metal homeostasis. We isolated activation-tagged mutant lines in which expression of a rice NAS gene, OsNAS3, was increased by introducing 35S enhancer elements. Shoots and roots of the OsNAS3 activation-tagged plants (OsNAS3-D1) accumulated more Fe and Zn. Seeds from our OsNAS3-D1 plants grown on a paddy field contained elevated amounts of Fe (2.9-fold), Zn (2.2-fold), and Cu (1.7-fold). The NA level was increased 9.6-fold in OsNAS3-D1 seeds. Analysis by size exclusion chromatography coupled with inductively coupled plasma mass spectroscopy showed that WT and OsNAS3-D1 seeds contained equal amounts of Fe bound to IP6, whereas OsNAS3-D1 had 7-fold more Fe bound to a low molecular mass, which was likely NA. Furthermore, this activation led to increased tolerance to Fe and Zn deficiencies and to excess metal (Zn, Cu, and Ni) toxicities. In contrast, disruption of OsNAS3 caused an opposite phenotype. To test the bioavailability of Fe, we fed anemic mice with either engineered or WT seeds for 4 weeks and measured their concentrations of hemoglobin and hematocrit. Mice fed with engineered seeds recovered to normal levels of hemoglobin and hematocrit within 2 weeks, whereas those that ate WT seeds remained anemic. Our results suggest that an increase in bioavailable mineral content in rice grains can be achieved by enhancing NAS expression. OsNAS3 Ectopic expression of rice OsNCED3 in Arabidopsis increases ABA level and alters leaf morphology 2010 Plant Science Institute of Plant and Microbial Biology, Academia Sinica, 128, Sect 2, Academia Rd, Nankang, Taipei 115, Taiwan, ROC In higher plants, many genes are involved in the abscisic acid (ABA) biosynthetic pathway. Of these, 9-cis-epoxycarotenoid dioxygenase (NCED) encodes what is considered to be the key enzyme. In the present study, we investigated the effects of ectopically expressing a monocot rice NCED gene, OsNCED3, in dicot Arabidopsis (Arabidopsis thaliana) plants. We showed that OsNCED3 is functionally active in dicot Arabidopsis plants since the ectopic expression of OsNCED3 successfully complements the 129B08/nced3 mutant phenotype. Furthermore, overexpression of OsNCED3 in wild-type Arabidopsis plants results in increased accumulation of ABA, reduced relative water loss, delayed seed germination, and greater drought tolerance relative to that of wild-type. Additionally, we observed that seed ABA content and germination patterns are similar between wild-type and the 129B08/nced3 mutant, suggesting functional redundancy or differential spatial expression of other NCED gene family members. Transgenic Arabidopsis lines overexpressing the monocot OsNCED3 gene in a wild-type background result in a smaller and rounder leaf shape and midvein. The data suggest that, in addition to ABA biosynthesis, the monocot OsNCED3 gene may have additional functions in shaping leaf morphology and vascular bundle development in Arabidopsis. OsNCED3 Physiological and Biochemical Characterization of Three Nucleoside Diphosphate Kinase Isozymes from Rice (Oryza sativaL.) 2014 Bioscience, Biotechnology and Biochemistry Laboratory of Biochemistry, Research Faculty of Agriculture, Hokkaido University. Nucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme that catalyzes the transfer of the γ-phosphoryl group from a nucleoside triphosphate to a nucleoside diphosphate. In this study, we examined the subcellular localization, tissue-specific gene expression, and enzymatic characteristics of three rice NDPK isozymes (OsNDPK1-OsNDPK3). Sequence comparison of the three OsNDPKs suggested differential subcellular localization. Transient expression of green fluorescence protein-fused proteins in onion cells indicated that OsNDPK2 and OsNDPK3 are localized to plastid and mitochondria respectively, while OsNDPK1 is localized to the cytosol. Expression analysis indicated that all the OsNDPKs are expressed in the leaf, leaf sheath, and immature seeds, except for OsNDPK1, in the leaf sheath. Recombinant OsNDPK2 and OsNDPK3 showed lower optimum pH and higher stability under acidic pH than OsNDPK1. In ATP formation, all the OsNDPKs displayed lower K(m) values for the second substrate, ADP, than for the first substrate, NTP, and showed lowest and highest K(m) values for GTP and CTP respectively. OsNDPK1,OsNDPK2,OsNDPK3 Enhanced expression of a gene encoding a nucleoside diphosphate kinase 1 (OsNDPK1) in rice plants upon infection with bacterial pathogens 2004 Mol Cells Agricultural Plant Stress Research Center, Applied Plant Science Division, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, Korea. A cDNA library was constructed using mRNA extracted from rice leaves infected with Xanthomonas oryzae pv. oryzae (Xoo), a bacterial leaf blight pathogen, to isolate rice genes induced by Xoo infection. Subtractive hybridization and differential screening of the cDNA library led to the isolation of many induced genes including a nucleotide diphosphate kinase 1 (OsNDPK1) and a pathogenesis-related protein 1 (OSPR1) cDNA. Nucleoside diphosphate kinases (NDPKs) are key metabolic enzymes that maintain the balance between cellular ATP and other nucleoside triphosphates (NTPs). Three other OsNDPK genes (NP922751, OsNDPK2 and OsNDPK3) found in databases were obtained by RTPCR. Three different programs for predicting subcellular targeting indicated that OsNDPK1 and NP922751 were non-organellar, OsNDPK2 plastidic, and OsNDPK3 mitochondrial. Only transcripts of OsNDPK1 accumulated strongly after infection with Xoo. When rice plants were infected with Burkholderia glumae, a bacterial grain/seedling rot pathogen, the pattern of expression of the rice NDPK genes was similar to that following infection with Xoo. OsNDPK1 gene expression was also strongly induced in response to exposure to salicylic acid, jasmonic acid, and abscisic acid, although the level of transcripts and their pattern of expression depended on the inducer. OsNDPK1,OsNDPK2,OsNDPK3 Molecular cloning and expression of the Na+/H+ exchanger gene in Oryza sativa 1999 Biochim Biophys Acta Department of Plant Physiology, National Institute of Agrobiological Resources, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. fukuda@abr.affrc.go.jp Na+/H+ exchanger catalyzes the countertransport of Na+ and H+ across membranes. We isolated a rice cDNA clone the deduced amino acid sequence of which had homology with a putative Na+/H+ exchanger in Saccharomyces cerevisiae, NHX1. The sequence contains 2330 bp with an open reading frame of 1608 bp. The deduced amino acid sequence is similar to that of NHX1 and NHE isoforms in mammals, and shares high similarity with the sequences within predicted transmembrane segments and an amiloride-binding domain. The expression of the gene was increased by salt stress. These results suggest that the product of the novel gene, OsNHX1, functions as a Na+/H+ exchanger, and plays important roles in salt tolerance of rice. OsNHX1 Molecular and functional analyses of rice NHX-type Na+/H+ antiporter genes 2011 Planta Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, Japan. fukuda@affrc.go.jp We previously cloned a vacuolar Na+/H+ antiporter gene (OsNHX1) from rice (Oryza sativa). Here we identified four additional NHX-type antiporter genes in rice (OsNHX2 through OsNHX5) and performed molecular and functional analyses of those genes. The exon-intron structure of the OsNHX genes and the phylogenetic tree of the OsNHX proteins suggest that the OsNHX proteins are categorized into two subgroups (OsNHX1 through OsNHX4 and OsNHX5). OsNHX1, OsNHX2, OsNHX3, and OsNHX5 can suppress the Na+, Li+, and hygromycin sensitivity of yeast nhx1 mutants and their sensitivity to a high K+ concentration. The expression of OsNHX1, OsNHX2, OsNHX3, and OsNHX5 is regulated differently in rice tissues and is increased by salt stress, hyperosmotic stress, and ABA. When we studied the expression of beta-glucuronidase (GUS) driven by either the OsNHX1 or the OsNHX5 promoter, we observed activity in the stele, the emerging part of lateral roots, the vascular bundle, the water pore, and the basal part of seedling shoots with both promoters. In addition, each promoter had a unique expression pattern. OsNHX1 promoter-GUS activity only was localized to the guard cells and trichome, whereas OsNHX5 promoter-GUS activity only was localized to the root tip and pollen grains. Our results suggest that the members of this gene family play important roles in the compartmentalization into vacuoles of the Na+ and K+ that accumulate in the cytoplasm and that the differential regulation of antiporter gene expression in different rice tissues may be an important factor determining salt tolerance in rice. OsNHX1,OsNHX2|OsNHX3,OsNHX4,OsNHX5 Function, intracellular localization and the importance in salt tolerance of a vacuolar Na(+)/H(+) antiporter from rice 2004 Plant Cell Physiol National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602 Japan. fukuda@affrc.go.jp We examined the function and intracellular localization of the product of the Na(+)/H(+) antiporter gene (OsNHX1) cloned from rice (Oryza sativa). OsNHX1 has the ability to suppress Na(+), Li(+) and hygromycin sensitivity of yeast nhx1 mutants and sensitivity to a high K(+) concentration, a novel phenotype of the nhx1 mutants. Analysis using rice cells expressing a fusion protein of OsNHX1 and green fluorescent protein and Western blot analysis using antibodies specific for OsNHX1 confirmed the localization of OsNHX1 on the tonoplasts. These results indicate that the OsNHX1 gene encodes a vacuolar (Na(+), K(+))/H(+) antiporter. Treatment with high concentrations of NaCl and KCl increased the transcript levels of OsNHX1 in rice roots and shoots. In addition, overexpression of OsNHX1 improved the salt tolerance of transgenic rice cells and plants. These results suggest that OsNHX1 on the tonoplasts plays important roles in the compartmentation of Na(+) and K(+) highly accumulated in the cytoplasm into the vacuoles, and the amount of the antiporter is one of the most important factors determining salt tolerance in rice. OsNHX1 Genes for alkaline/neutral invertase in rice: alkaline/neutral invertases are located in plant mitochondria and also in plastids 2007 Planta Research Team for Crop Cold Tolerance, National Agricultural Research Center for Hokkaido Region, 1 Hitsuji-ga-oka, Sapporo 062-8555, Japan. Two cDNA clones (OsNIN1 and OsNIN3) encoding an alkaline/neutral invertase localized in organelles were identified from rice. The deduced amino acid sequences of these cDNA clones showed high homology to other plant alkaline/neutral invertases. Semi-quantitative reverse transcription polymerase chain reaction revealed that the expression of OsNIN1 was constitutive and independent of organ difference, although its expression level was low. Analyses using five types of web software for the prediction of protein localization in the cell, Predotar, PSORT, Mitoprot, TargetP, and ChloroP, strongly supported the possibility that OsNIN1 is transported into the mitochondria and that OsNIN3 is transported into plastids. Transient expression of fusion proteins combining the amino terminal region of these two proteins with sGFP demonstrated that N-OsNIN1::GFP and N-OsNIN3::GFP fusion proteins were transported into the mitochondria and plastids, respectively. We expressed the OsNIN1 protein in vitro and revealed that the translated protein had an invertase activity. These results clearly indicate that some of alkaline/neutral invertases are located in plant organelles, mitochondria and plastids, and that they might have a novel physiological function in plant organelles. OsNIN1,OsNIN3 OsNIP3;1, a rice boric acid channel, regulates boron distribution and is essential for growth under boron-deficient conditions 2014 The Plant Journal Biotechnology Research Center, University of Tokyo, Tokyo, Japan Boron is an essential micronutrient for higher plants. Boron deficiency is an important agricultural issue because it results in loss of yield quality and/or quantity in cereals and other crops. To understand boron transport mechanisms in cereals, we characterized OsNIP3;1, a member of the major intrinsic protein family in rice (Oryza sativa L.), because OsNIP3;1 is the most similar rice gene to the Arabidopsis thaliana boric acid channel genes AtNIP5;1 and AtNIP6;1. Yeast cells expressing OsNIP3;1 imported more boric acid than control cells. GFP-tagged OsNIP3;1 expressed in tobacco BY2 cells was localized to the plasma membrane. The accumulation of OsNIP3;1 transcript increased fivefold in roots within 6 h of the onset of boron starvation, but not in shoots. Promoter-GUS analysis suggested that OsNIP3;1 is expressed mainly in exodermal cells and steles in roots, as well as in cells around the vascular bundles in leaf sheaths and pericycle cells around the xylem in leaf blades. The growth of OsNIP3;1 RNAi plants was impaired under boron limitation. These results indicate that OsNIP3;1 functions as a boric acid channel, and is required for acclimation to boron limitation. Boron distribution among shoot tissues was altered in OsNIP3;1 knockdown plants, especially under boron-deficient conditions. This result demonstrates that OsNIP3;1 regulates boron distribution among shoot tissues, and that the correct boron distribution is crucial for plant growth. OsNIP3;1 Expression of a rice gene OsNOA1 re-establishes nitric oxide synthesis and stress-related gene expression for salt tolerance in Arabidopsis nitric oxide-associated 1 mutant Atnoa1 2009 Environmental and Experimental Botany Peking-Yale Joint Center for Plant Molecular Genetics and Agro-Biotechnology, National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China Nitric oxide (NO) has been indicated in regulating a wide-spectrum of plant developmental events and stress responses. An Arabidopsis gene AtNOA1 encodes a NO-associated protein, which plays a role in salt tolerance. We employed the knockout mutant for AtNOA1, Atnoa1 that is sensitive to salinity, as a tool to evaluate the functions of a rice homologous gene, OsNOA1. OsNOA1 transgenic expression rescued Atnoa1 in seedling development and vegetative growth under normal conditions, enhanced the salt tolerance of Atnoa1 in seed germination, seedling root growth and chlorophyll synthesis, and reduced Na+/K+ ratio in Atnoa1; NO relative content assay implicates that NO synthesis was re-established via OsNOA1 expression in Atnoa1; Northern blot and Semi-Q RT-PCR assays demonstrate that salt tolerance-related gene expression was re-established as well via OsNOA1 expression in Atnoa1. Our data indicate that the re-establishment of NO synthesis and salt tolerance-related gene expression by OsNOA1 expression may account for the restoration of Atnoa1 in terms of developmental and salt tolerance phenotypes. All the above results point to a notion that OsNOA1 may play similar roles as AtNOA1, and NO involvement in salt tolerance may be ascribed to its regulation of salt tolerance-related gene expression. OsNOA1 OsNOA1/RIF1 is a functional homolog of AtNOA1/RIF1: implication for a highly conserved plant cGTPase essential for chloroplast function 2010 New Phytol Crop Molecular Breeding Center, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang, China. *The bacterial protein YqeH is a circularly permuted GTPase with homologs encoded by plant nuclear genomes. The rice homolog OsNOA1/RIF1 is encoded by the single-copy gene Os02g01440. OsNOA1/RIF1 is expressed in different tissues and is light-inducible. The OsNOA1/RIF1-EYFP fusion protein was targeted to chloroplasts in transgenic Arabidopsis plants. In addition, the rice homolog was able to rescue most of the growth phenotypes in an Arabidopsis rif1 mutant. *Rice (Oryza sativa) OsNOA1/RIF1 RNAi mutant seedlings were chlorotic with reduced pigment contents and lower photosystem II (PSII) efficiency. However, the expressions of the chloroplast-encoded genes rbcL, atpB, psaA and psbA were not affected. By contrast, reduced abundance of the chloroplast 16S rRNA was observed in the mutant. *Quantitative iTRAQ-LC-MS/MS proteomics investigations revealed proteome changes in the rice mutant consistent with the expected functional role of OsNOA1/RIF1 in chloroplast translation. The RNAi mutant showed significantly decreased expression levels of chloroplast-encoded proteins as well as nuclear-encoded components of chloroplast enzyme complexes. Conversely, upregulation of some classes of nonchloroplastic proteins, such as glycolytic and phenylpropanoid pathway enzymes, was detected. *Our work provides independent indications that a highly conserved nuclear-encoded cGTPase of likely prokaryotic origin is essential for proper chloroplast ribosome assembly and/or translation in plants. OsNOA1 NOA1 functions in a temperature-dependent manner to regulate chlorophyll biosynthesis and Rubisco formation in rice 2011 PLoS One Key Laboratory of Plant Functional Genomics and Biotechnology, Education Department of Guangdong Province, South China Agricultural University, Guangzhou, China. NITRIC OXIDE-ASSOCIATED1 (NOA1) encodes a circularly permuted GTPase (cGTPase) known to be essential for ribosome assembly in plants. While the reduced chlorophyll and Rubisco phenotypes were formerly noticed in both NOA1-suppressed rice and Arabidopsis, a detailed insight is still necessary. In this study, by using RNAi transgenic rice, we further demonstrate that NOA1 functions in a temperature-dependent manner to regulate chlorophyll and Rubisco levels. When plants were grown at 30 degrees C, the chlorophyll and Rubisco levels in OsNOA1-silenced plants were only slightly lower than those in WT. However, at 22 degrees C, the silenced plants accumulated far less chlorophyll and Rubisco than WT. It was further revealed that the regulation of chlorophyll and Rubisco occurs at the anabolic level. Etiolated WT seedlings restored chlorophyll and Rubisco accumulations readily once returned to light, at either 30 degrees C or 15 degrees C. Etiolated OsNOA1-silenced plants accumulated chlorophyll and Rubisco to normal levels only at 30 degrees C, and lost this ability at low temperature. On the other hand, de-etiolated OsNOA1-silenced seedlings maintained similar levels of chlorophyll and Rubisco as WT, even after being shifted to 15 degrees C for various times. Further expression analyses identified several candidate genes, including OsPorA (NADPH: protochlorophyllide oxidoreductase A), OsrbcL (Rubisco large subunit), OsRALyase (Ribosomal RNA apurinic site specific lyase) and OsPuf4 (RNA-binding protein of the Puf family), which may be involved in OsNOA1-regulated chlorophyll biosynthesis and Rubisco formation. Overall, our results suggest OsNOA1 functions in a temperature-dependent manner to regulate chlorophyll biosynthesis, Rubisco formation and plastid development in rice. OsNOA1,OsPORA,OsPuf4,OsRALyase Naringenin 7-O-methyltransferase involved in the biosynthesis of the flavanone phytoalexin sakuranetin from rice (Oryza sativa L.) 2000 Plant Sci United Graduate School, Tokyo University of Agriculture and Technology, Tokyo, Japan An inducible S-adenosyl-L-methionine:naringenin 7-O-methyltransferase (NOMT) catalyzing the methylation of naringenin to sakuranetin, a major rice phytoalexin was purified approximately 985-fold from ultraviolet (UV)-irradiated rice leaves. The enzyme is not found in healthy tissues and was purified to a nearly homogeneous preparation in one step using adenosine-agarose affinity chromatography, with 1 g rice leaves (UV-irradiated) as starting material. Gel filtration chromatography resulted in an almost pure enzyme, as evidenced by a major band migrating to a position corresponding to a molecular mass of approximately 41 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified NOMT was strongly inhibited by Mn(2+), Ni(2+), Cu(2+), Zn(2+), Hg(2+), and Cd(2+), and to a low degree by Co(2+), Mg(2+), Ba(2+), Ca(2+) and ethylenediamine tetraacetic acid. The amino acid sequence of a NOMT cyanogen bromide (CNBr)-cleavage peptide was highly homologous to that of a caffeic acid 3-O-methyltransferase from maize, and about 70% of the amino acid sequence was obtained after sequencing the peptides generated by CNBr and/or formic acid hydrolysis. NOMT was also shown to be induced in a time-dependent manner, and purified from rice leaves treated with jasmonic acid and copper chloride. OsNOMT The potential bioproduction of the pharmaceutical agent sakuranetin, a flavonoid phytoalexin in rice 2012 Bioengineered Biotechnology Research Center; The University of Tokyo, Bunkyo-ku, Tokyo, Japan. Sakuranetin, the major flavonoid phytoalexin in rice, can be induced by ultraviolet (UV) irradiation, treatment with CuCl 2 or jasmonic acid (JA), or phytopathogenic infection. In addition to sakuranetin's biological significance on disease resistance in rice, its broad bioactivities have recently been described. Results from these studies have shown that sakuranetin is a useful compound as a plant antibiotic and a potential pharmaceutical agent. Sakuranetin is biosynthesized from naringenin, a precursor of sakuranetin, by naringenin 7-O-methyltransferase (NOMT), but the relevant gene has not yet been identified in rice. Recently, we identified the OsNOMT gene, which is involved in the final step of sakuranetin biosynthesis in rice. In previous studies, OsNOMT was purified to apparent homogeneity from UV-treated wild-type rice leaves; however, the purified protein, termed OsCOMT1, exhibited caffeic acid 3-O-methyltransferase (COMT) activity, but not NOMT activity. Based on the analysis of an oscomt1 T-DNA tagged mutant, we determined that OsCOMT1 did not contribute to sakuranetin production in rice in vivo. Therefore, we took advantage of the oscomt1 mutant to purify OsNOMT. A crude protein preparation from UV-treated oscomt1 leaves was subjected to three sequential purification steps resulting in a 400-fold purification from the crude enzyme preparation with a minor band at an apparent molecular mass of 40 kDa in the purest enzyme preparation. Matrix-assisted laser desorption/ionization time of flight/time of flight analysis showed that the 40 kDa protein band included two O-methyltransferase-like proteins, but one of the proteins encoded by Os12g0240900 exhibited clear NOMT activity; thus, this gene was designated OsNOMT. Gene expression was induced by treatment with jasmonic acid in rice leaves prior to sakuranetin accumulation, and the recombinant protein showed reasonable kinetic properties to NOMT. Identification of the OsNOMT gene enables the production of large amounts of sakuranetin through transgenic rice and microorganisms. This finding also allows for the generation of disease-resistant and sakuranetin biofortified rice in the future. OsNOMT Purification and identification of naringenin 7-O-methyltransferase, a key enzyme in biosynthesis of flavonoid phytoalexin sakuranetin in rice 2012 J Biol Chem Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, Japan. Sakuranetin, the major flavonoid phytoalexin in rice, is induced by ultraviolet (UV) irradiation, CuCl(2) treatment, jasmonic acid treatment, and infection by phytopathogens. It was recently demonstrated that sakuranetin has anti-inflammatory activity, anti-mutagenic activity, anti-pathogenic activities against Helicobacter pylori, Leishmania, and Trypanosoma and contributes to the maintenance of glucose homeostasis in animals. Thus, sakuranetin is a useful compound as a plant antibiotic and a potential pharmaceutical agent. Sakuranetin is biosynthesized from naringenin by naringenin 7-O-methyltransferase (NOMT). In previous research, rice NOMT (OsNOMT) was purified to apparent homogeneity from UV-treated wild-type rice leaves, but the purified protein, named OsCOMT1, exhibited caffeic acid O-methyltransferase (COMT) activity and not NOMT activity. In this study, we found that OsCOMT1 does not contribute to sakuranetin production in rice in vivo, and we purified OsNOMT using the oscomt1 mutant. A crude protein preparation from UV-treated oscomt1 leaves was subjected to three sequential purification steps, resulting in a 400-fold purification from the crude enzyme preparation. Using SDS-PAGE, the purest enzyme preparation showed a minor band at an apparent molecular mass of 40 kDa. Two O-methyltransferase-like proteins, encoded by Os04g0175900 and Os12g0240900, were identified from the 40-kDa band by MALDI-TOF/TOF analysis. Recombinant Os12g0240900 protein showed NOMT activity, but the recombinant Os04g0175900 protein did not. Os12g0240900 expression was induced by jasmonic acid treatment in rice leaves prior to sakuranetin accumulation, and the Os12g0240900 protein showed reasonable kinetic properties to OsNOMT. On the basis of these results, we conclude that Os12g0240900 encodes an OsNOMT. OsNOMT The Oryza sativa no pollen (Osnop) gene plays a role in male gametophyte development and most likely encodes a C2-GRAM domain-containing protein 2005 Plant Mol Biol Rice Functional Genomics Group, Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, 117604, Singapore. Phenotype screens of Ds insertional lines identified a male sterile Orysa sativa no pollen (Osnop) mutant with a pollen-less phenotype at the flowering stage. The mutant phenotype showed linkage to Ds insertion into Osnop gene region. This mutant contained a deletion of 65 kb chromosomal region at the site of Ds insertion containing 14 predicted genes. Out of these deleted genes, Delegen 5-7, 9-10 were redundant, as two or three copies were present with 100% homology in other regions of rice genome. RT-PCR analysis showed that Delegen 5-7 were expressed not only in wild type plants but also in the mutant plants. In addition to this, Delegen 8-10 transcripts could not be detected under normal growth conditions, and Delegen 12 was expressed only in roots, thus deletion of these genes may not affect the pollen development. Our data and analysis also ruled out the possibility of delegen 1-4, 11, and 13 as candidates contributing to the pollen-less phenotype. Further investigation showed that the delegen 14 was expressed only in late stage of pollen development with the highest expression at the stage of pollen release and germination by RT-PCR, Northern blotting, in situ hybridization, and promoter-GUS transgenic plants. Thus, the delegen 14 gene is the best candidate for Osnop, corresponding to the pollen-less phenotype in the mutant. Our data suggest that delegen 14 may play an important role during late stage of pollen development and its germination. Since the delegen 14 gene has both C(2) and GRAM domains, it can be assumed that this gene cross-links both calcium and phosphoinositide signaling pathways. This is the first report to suggest possible functions for this gene in plant development. Osnop Rice nitrate transporter OsNPF2.4 functions in low-affinity acquisition and long-distance transport 2014 J Exp Bot State Key Laboratory of Crop Genetics and Germplasm Enhancement and MOA Key Laboratory of Plant Nutrition and Fertilization in Lower-Middle Reaches of the Yangtze River, Nanjing Agricultural University, Nanjing 210095, PR China. Plant proteins belonging to the NPF (formerly NRT1/PTR) family are well represented in every genome and function in transporting a wide variety of substrates. In this study, we showed that rice OsNPF2.4 is located in the plasma membrane and is expressed mainly in the epidermis, xylem parenchyma, and phloem companion cells. Functional analysis in oocytes showed that OsNPF2.4 is a pH-dependent, low-affinity NO3- transporter. Short-term 15NO3- influx rate, long-term NO3- acquisition by root, and upward transfer from root to shoot were decreased by disruption of OsNPF2.4 and increased by OsNPF2.4 overexpression under high NO3- supply. Moreover, the redistribution of NO3- in the mutants in comparison with the wild type from the oldest leaf to other organs, particularly to N-starved roots, was dramatically changed. Knockout of OsNPF2.4 decreased rice growth and potassium (K) concentration in xylem sap, root, culm, and sheath, but increased the shoot:root ratio of tissue K under higher NO3-. We conclude that OsNPF2.4 functions in acquisition and long-distance transport of NO3-, and that altering its expression has an indirect effect on K recycling between the root and shoot. OsNPF2.4 Involvement of OsNPR1/NH1 in rice basal resistance to blast fungus Magnaporthe oryzae 2011 European Journal of Plant Pathology State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering, and College of Life Science and Technology, Guangxi University, 100 Daxue Road, Nanning, Guangxi, 530004, China Rice blast disease, caused by the fungus Magnaporthe oryzae, is a major threat to worldwide rice production. Plant basal resistance is activated by virulent pathogens in susceptible host plants. OsNPR1/NH1, a rice homolog of NPR1 that is the key regulator of systemic acquired resistance in Arabidopsis thaliana, was shown to be involved in the resistance of rice to bacterial blight disease caused by Xanthomonas oryzae pv. oryzae and benzothiadiazole (BTH)-induced blast resistance. However, the role of OsNPR1/NH1 in rice basal resistance to blast fungus M. oryzae remains uncertain. In this study, the OsNPR1 gene was isolated and identified from rice cultivar Gui99. Transgenic Gui99 rice plants harbouring OsNPR1-RNAi were generated, and the OsNPR1-RNAi plants were significantly more susceptible to M. oryzae infection. Northern hybridization analysis showed that the expression of pathogenesis-related (PR) genes, such as PR-1a, PBZ1, CHI, GLU, and PAL, was significantly suppressed in the OsNPR1-RNAi plants. Consistently, overexpression of OsNPR1 in rice cultivars Gui99 and TP309 conferred significantly enhanced resistance to M. oryzae and increased expression of the above-mentioned PR genes. These results revealed that OsNPR1 is involved in rice basal resistance to the blast pathogen M. oryzae, thus providing new insights into the role of OsNPR1 in rice disease resistance. OsNPR1|NH1,OsPR10a|PBZ1,OsPR1a Isolation and characterization of the rice NPR1 promoter 2009 Plant Biotechnology Reports National Academy of Agricultural Sciences, Rural Development Administration, Suwon, 441-707, Republic of Korea NPR1 is a positive regulator of systemic acquired resistance in Arabidopsis and rice. Expression of the rice gene OsNPR1 is induced by salicylic acid (SA). To identify the region of the OsNPR1 promoter involved in response to SA, we carried out deletion mutagenesis of the region 1005 bp upstream of the OsNPR1 start codon. Cis-element analysis revealed that the OsNPR1 promoter contains W-boxes and ASF1 motifs, both of which are known to be functional cis-elements of the WRKY and bZIP proteins, respectively. The deletion constructs 1005:LUC and 752:LUC, were induced by up to 4.3- and 3.8-fold, respectively, following SA treatment, suggesting that W-boxes and ASF1 motifs may play an important role in the strong induction of these constructs by SA. Using mutation analysis, we also showed that both the W-box and ASF1 motif are necessary for SA-induced expression of OsNPR1. OsNPR1|NH1 OsWRKY71, a rice transcription factor, is involved in rice defense response 2007 J Plant Physiol National Key Laboratory of Plant Genomics and Plant Gene Research Center (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, China. WRKY proteins are a large family of transcription factors that mainly participate in plant biotic stress responses. So far, one hundred and five OsWRKY genes have been predicted in the rice genome. To identify OsWRKY genes that might function in inducible defense responses, a phylogenetic tree including 184 WRKY proteins from Arabidopsis thaliana, rice, and other species was constructed. Based on the phylogenetic analysis, ten candidate OsWRKY genes that may be involved in defense responses were isolated from salicylic acid (SA)-treated rice seedlings. One of them, OsWRKY71, was up-regulated by several defense signaling molecules, such as SA, methyl jasmonate (MeJA), 1-aminocyclo-propane-1-carboxylic acid (ACC), as well as wounding and pathogen infection, suggesting that OsWRKY71 might function in rice biotic stress response. Transient expression of OsWRKY71:GFP fusion protein in onion epidermis cells revealed that OsWRKY71 was localized in the nucleus. Overexpression of OsWRKY71 gene in rice resulted in enhanced resistance to virulent bacterial pathogens Xanthomonas oryzae pv. oryzae (Xoo) 13751. Furthermore, two marker genes in defense signaling pathway, OsNPR1 and OsPR1b, were constitutively expressed in OsWRKY71-overexpressing transgenic plants. These results suggest that OsWRKY71 might function as a transcriptional regulator upstream of OsNPR1 and OsPR1b in rice defense signaling pathways. OsNPR1|NH1,OsPR1b,OsWRKY71 Building a mutant resource for the study of disease resistance in rice reveals the pivotal role of several genes involved in defence 2012 Mol Plant Pathol UMR BGPI INRA/CIRAD/SupAgro, Campus International de Baillarguet, TA A 54/K, 34398 Montpellier, France. In Arabidopsis, gene expression studies and analysis of knock-out (KO) mutants have been instrumental in building an integrated view of disease resistance pathways. Such an integrated view is missing in rice where shared tools, including genes and mutants, must be assembled. This work provides a tool kit consisting of informative genes for the molecular characterization of the interaction of rice with the major fungal pathogen Magnaporthe oryzae. It also provides for a set of eight KO mutants, all in the same genotypic background, in genes involved in key steps of the rice disease resistance pathway. This study demonstrates the involvement of three genes, OsWRKY28, rTGA2.1 and NH1, in the establishment of full basal resistance to rice blast. The transcription factor OsWRKY28 acts as a negative regulator of basal resistance, like the orthologous barley gene. Finally, the up-regulation of the negative regulator OsWRKY28 and the down-regulation of PR gene expression early during M. oryzae infection suggest that the fungus possesses infection mechanisms that enable it to block host defences. OsNPR1|NH1,OsWRKY28,rTGA2.1|OsbZIP63|OsNIF1 Functional analysis and expressional characterization of rice ankyrin repeat-containing protein, OsPIANK1, in basal defense against Magnaporthe oryzae attack 2013 PLoS One College of Life Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China. The ankyrin repeat-containing protein gene OsPIANK1 (AK068021) in rice (Oryza sativa L.) was previously shown to be upregulated following infection with the rice leaf blight pathogen Xanthomonas oryzae pv oryzae (Xoo). In this study, we further characterized the role of OsPIANK1 in basal defense against Magnaporthe oryzae (M.oryzae) by 5' deletion analysis of its promoter and overexpression of the gene. The promoter of OsPIANK1 with 1,985 bps in length was sufficient to induce the OsPIANK1 response to inoculation with M.oryzae and to exogenous application of methyl jasmonate (MeJA) or salicylic acid (SA), but not to exogenous application of abscisic acid (ABA). A TCA-element present in the region between -563 bp and -249 bp may be responsible for the OsPIANK1 response to both M.oryzae infection and exogenous SA application. The JERE box, CGTCA-box, and two MYB binding sites locating in the region between -1985 bp and -907 bp may be responsible for the response of OsPIANK1 to exogenous MeJA. OsPIANK1 expression was upregulated after inoculation with M.oryzae and after treatment with exogenous SA and MeJA. Overexpression of OsPIANK1 enhanced resistance of rice to M.oryzae, although it did not confer complete resistance. The enhanced resistance to M.oryzae was accompanied by enhanced transcriptional expression of SA- and JA-dependent genes such as NH1, WKRY13, PAL, AOS2, PR1b, and PR5. This evidence suggests that OsPIANK1 acted as a positive regulator in rice basal defense mediated by SA- and JA-signaling pathways. OsNPR1|NH1,OsPIANK1,OsPR1b,OsWRKY13,OsPR5|Pir2|PR-5|PR5-1 Cytokinins act synergistically with salicylic acid to activate defense gene expression in rice 2013 Mol Plant Microbe Interact National Institute of Agrobiological Sciences, Tsukuba, Japan. cjjiang@affrc.go.jp Hormone crosstalk is pivotal in plant-pathogen interactions. Here, we report on the accumulation of cytokinins (CK) in rice seedlings after infection of blast fungus Magnaporthe oryzae and its potential significance in rice-M. oryzae interaction. Blast infection to rice seedlings increased levels of N(6)-(Delta(2)-isopentenyl) adenine (iP), iP riboside (iPR), and iPR 5'-phosphates (iPRP) in leaf blades. Consistent with this, CK signaling was activated around the infection sites, as shown by histochemical staining for beta-glucuronidase activity driven by a CK-responsive OsRR6 promoter. Diverse CK species were also detected in the hyphae (mycelium), conidia, and culture filtrates of blast fungus, indicating that M. oryzae is capable of production as well as hyphal secretion of CK. Co-treatment of leaf blades with CK and salicylic acid (SA), but not with either one alone, markedly induced pathogenesis-related genes OsPR1b and probenazole-induced protein 1 (PBZ1). These effects were diminished by RNAi-knockdown of OsNPR1 or WRKY45, the key regulators of the SA signaling pathway in rice, indicating that the effects of CK depend on these two regulators. Taken together, our data imply a coevolutionary rice-M. oryzae interaction, wherein M. oryzae probably elevates rice CK levels for its own benefits such as nutrient translocation. Rice plants, on the other hand, sense it as an infection signal and activate defense reactions through the synergistic action with SA. OsNPR1|NH1,OsPR10a|PBZ1,OsPR1b,OsRR6,OsWRKY45 Overexpression of a rice NPR1 homolog leads to constitutive activation of defense response and hypersensitivity to light 2005 Mol Plant Microbe Interact Department of Plant Pathology, University of California, Davis 95616, USA. Arabidopsis NPR1/NIM1 is a key regulator of systemic acquired resistance (SAR), which confers lasting broad-spectrum resistance. Previous reports indicate that rice has a disease-resistance pathway similar to the Arabidopsis SAR pathway. Here we report the isolation and characterization of a rice NPR1 homologue (NH1). Transgenic rice plants overexpressing NH1 (NH1ox) acquire high levels of resistance to Xanthomonas oryzae pv. oryzae. The resistance phenotype is heritable and correlates with the presence of the transgene and reduced bacterial growth. Northern analysis shows that NH1ox rice spontaneously activates defense genes, contrasting with NPR1-overexpressing Arabidopsis, where defense genes are not activated until induction. Wild-type NH1, but not a point mutant corresponding to npr1-1, interacts strongly with the rice transcription factor rTGA2.2 in yeast two-hybrid. Greenhouse-grown NH1ox plants develop lesion-mimic spots on leaves at preflowering stage although no other developmental effects are observed. However, when grown in growth chambers (GCs) under low light, NH1ox plants are dwarfed, indicating elevated sensitivity to light. The GC-grown NH1ox plants show much higher salicylic acid (SA) levels than the wild type, whereas greenhouse-grown NH1ox plants contain lower SA. These results indicate that NH1 may be involved in the regulation of SA in response to environmental changes. OsNPR1|NH1 The nuclear ubiquitin proteasome degradation affects WRKY45 function in the rice defense program 2012 Plant J Disease Resistant Crops Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. The transcriptional activator WRKY45 plays a major role in the salicylic acid (SA)/benzothiadiazole-induced defense program in rice. Here, we show that the nuclear ubiquitin proteasome system (UPS) plays a role in regulating the function of WRKY45. Proteasome inhibitors induced accumulation of polyubiquitinated WRKY45 and transient upregulation of WRKY45 target genes in rice cells, suggesting that WRKY45 is constantly degraded by the UPS to suppress defense responses in the absence of defense signals. Mutational analysis of the nuclear localization signal (NLS) indicated that UPS-dependent WRKY45 degradation occurs in the nuclei. Interestingly, transcriptional activity of WRKY45 after SA treatment was impaired by proteasome inhibition. The same C-terminal region in WRKY45 was essential for both transcriptional activity and UPS-dependent degradation. These results suggest that UPS regulation also plays a role in transcriptional activity of WRKY45. It has been reported that AtNPR1, the central regulator of the SA pathway in Arabidopsis, is regulated by the UPS. We found that OsNPR1/NH1, the rice counterpart of NPR1, was not stabilized by proteasome inhibition under an uninfected condition. We discuss the differences in the post-translational regulation of the SA-pathway components between rice and Arabidopsis. (c) 2012 The Authors. The Plant Journal (c) 2012 Blackwell Publishing Ltd. OsNPR1|NH1,OsWRKY45 Cloning and characterization of three genes encoding Qb-SNARE proteins in rice 2008 Mol Genet Genomics State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, 210095, China. Qb-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and function as important components of the vesicle trafficking machinery in eukaryotic cells. Here, we report three novel plant SNARE (NPSN) genes isolated from rice and named OsNPSN11, OsNPSN12 and OsNPSN13. They have about 70% nucleotide identity over their entire coding regions and similar genomic organization with ten exons and nine introns in each gene. Multiple alignment of deduced amino acid sequences indicate that the OsNPSNs proteins are homologous to AtNPSNs from Arabidopsis, containing a Qb-SNARE domain and a membrane-spanning domain in the C-terminal region. Semi-quantitative RT-PCR assays showed that the OsNPSNs were ubiquitously and differentially expressed in roots, culms, leaves, immature spikes and flowering spikes. The expression of OsNPSNs was significantly activated in rice seedlings treated with H(2)O(2), but down-regulated under NaCl and PEG6000 stresses. Transient expression method in onion epidermal cells revealed that OsNPSNs were located in the plasma membrane. Transformed yeast cells with OsNPSNs had better growth rates than empty-vector transformants when cultured on either solid or liquid selective media containing various concentrations of H(2)O(2), but more sensitive to NaCl and mannitol stresses. The 35S:OsNPSN11 transgenic tobacco also showed more tolerance to H(2)O(2) and sensitivity to NaCl and mannitol than non-transgenic tobacco. These results indicate that OsNPSNs may be involved in different aspects of the signal transduction in plant and yeast responses to abiotic stresses. OsNPSN11,OsNPSN12,OsNPSN13 The OsNRAMP1 iron transporter is involved in Cd accumulation in rice 2011 J Exp Bot Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Cadmium (Cd) is a heavy metal toxic to humans and the accumulation of Cd in the rice grain is a major agricultural problem, particularly in Asia. The role of the iron transporter OsNRAMP1 in Cd uptake and transport in rice was investigated here. An OsNRAMP1:GFP fusion protein was localized to the plasma membrane in onion epidermal cells. The growth of yeast expressing OsNRAMP1 was impaired in the presence of Cd compared with yeast transformed with an empty vector. Moreover, the Cd content of OsNRAMP1-expressing yeast exceeded that of the vector control. The expression of OsNRAMP1 in the roots was higher in a high Cd-accumulating cultivar (Habataki) than a low Cd-accumulating cultivar (Sasanishiki) regardless of the presence of Cd, and the amino acid sequence of OsNRAMP1 showed 100% identity between Sasanishiki and Habataki. Over-expression of OsNRAMP1 in rice increased Cd accumulation in the leaves. These results suggest that OsNRAMP1 participates in cellular Cd uptake and Cd transport within plants, and the higher expression of OsNRAMP1 in the roots could lead to an increase in Cd accumulation in the shoots. Our results indicated that OsNRAMP1 is an important protein in high-level Cd accumulation in rice. OsNRAMP1 Role of the iron transporter OsNRAMP1 in cadmium uptake and accumulation in rice 2011 Plant Signal Behav Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan. The heavy metal cadmium (Cd) is toxic to humans, and its accumulation in rice grains is a major agricultural problem. Rice has seven putative metal transporter NRAMP genes, but microarray analysis showed that only OsNRAMP1 is highly up-regulated by iron (Fe) deficiency. OsNRAMP1 localized to the plasma membrane and transported Cd as well as Fe. OsNRAMP1 expression was observed mainly in roots and was higher in the roots of a high-Cd-accumulating cultivar (Habataki) than in those of a low-Cd-accumulating cultivar (Sasanishiki). The amino acid sequence of OsNRAMP1 in the Sasanishiki and Habataki cultivars was found to be 100% identical. These results suggest that OsNRAMP1 participates in cellular Cd uptake and that the differences observed in Cd accumulation among cultivars are because of differences in OsNRAMP1 expression levels in roots. OsNRAMP1 OsNRAMP3 is a vascular bundles-specific manganese transporter that is responsible for manganese distribution in rice 2013 PLoS One National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China. Manganese (Mn) is an essential trace element for plants. Recently, the genes responsible for uptake of Mn in plants were identified in Arabidopsis and rice. However, the mechanism of Mn distribution in plants has not been clarified. In the present study we identified a natural resistance-associated macrophage protein (NRAMP) family gene in rice, OsNRAMP3, involved in Mn distribution. OsNRAMP3 encodes a plasma membrane-localized protein and was specifically expressed in vascular bundles, especially in phloem cells. Yeast complementation assay showed that OsNRAMP3 is a functional Mn-influx transporter. When OsNRAMP3 was absent, rice plants showed high sensitivity to Mn deficiency. Serious necrosis appeared on young leaves and root tips of the OsNRAMP3 knockout line cultivated under low Mn conditions, and high Mn supplies could rescue this phenotype. However, the necrotic young leaves of the knockout line possessed similar levels of Mn to the wild type, suggesting that the necrotic appearance was caused by disturbed distribution of Mn but not a general Mn shortage. Additionally, compared with wild type, leaf Mn content in osnramp3 plants was mostly in older leaves. We conclude that OsNRAMP3 is a vascular bundle-localized Mn-influx transporter involved in Mn distribution and contributes to remobilization of Mn from old to young leaves. OsNRAMP3 Characterizing the role of rice NRAMP5 in Manganese, Iron and Cadmium Transport 2012 Sci Rep Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Metals like manganese (Mn) and iron (Fe) are essential for metabolism, while cadmium (Cd) is toxic for virtually all living organisms. Understanding the transport of these metals is important for breeding better crops. We have identified that OsNRAMP5 contributes to Mn, Fe and Cd transport in rice. OsNRAMP5 expression was restricted to roots epidermis, exodermis, and outer layers of the cortex as well as in tissues around the xylem. OsNRAMP5 localized to the plasma membrane, and complemented the growth of yeast strains defective in Mn, Fe, and Cd transport. OsNRAMP5 RNAi (OsNRAMP5i) plants accumulated less Mn in the roots, and less Mn and Fe in shoots, and xylem sap. The suppression of OsNRAMP5 promoted Cd translocation to shoots, highlighting the importance of this gene for Cd phytoremediation. These data reveal that OsNRAMP5 contributes to Mn, Cd, and Fe transport in rice and is important for plant growth and development. OsNRAMP5,OsZIP3 Ion-beam irradiation, gene identification, and marker-assisted breeding in the development of low-cadmium rice 2012 Proc Natl Acad Sci U S A Soil Environment Division, National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki 305-8604, Japan. isatoru@affrc.go.jp Rice (Oryza sativa L.) grain is a major dietary source of cadmium (Cd), which is toxic to humans, but no practical technique exists to substantially reduce Cd contamination. Carbon ion-beam irradiation produced three rice mutants with <0.05 mg Cdkg(-1) in the grain compared with a mean of 1.73 mg Cdkg(-1) in the parent, Koshihikari. We identified the gene responsible for reduced Cd uptake and developed a strategy for marker-assisted selection of low-Cd cultivars. Sequence analysis revealed that these mutants have different mutations of the same gene (OsNRAMP5), which encodes a natural resistance-associated macrophage protein. Functional analysis revealed that the defective transporter protein encoded by the mutant osnramp5 greatly decreases Cd uptake by roots, resulting in decreased Cd in the straw and grain. In addition, we developed DNA markers to facilitate marker-assisted selection of cultivars carrying osnramp5. When grown in Cd-contaminated paddy fields, the mutants have nearly undetectable Cd in their grains and exhibit no agriculturally or economically adverse traits. Because mutants produced by ion-beam radiation are not transgenic plants, they are likely to be accepted by consumers and thus represent a practical choice for rice production worldwide. OsNRAMP5 Functional Characterization of a Putative Nitrate Transporter Gene Promoter from Rice 2006 Acta Biochimica et Biophysica Sinica Department of Biochemistry, School of Life Science, Fudan University, Shanghai 200433, China. Drought is one of the most significant abiotic stresses that influence plant growth and development. Expression analysis revealed that OsNRT1.3, a putative nitrate transporter gene in rice, was induced by drought. To confirm if the OsNRT1.3 promoter can respond to drought stress, a 2019 bp upstream sequence of OsNRT1.3 was cloned. Three OsNRT1.3 promoter fragments were generated by 5'-deletion, and fused to the beta-glucuronidase (GUS) gene. The chimeric genes were introduced into rice plants. NRT2019::GUS, NRT1196::GUS and NRT719::GUS showed similar expression patterns in seeds, roots, leaves and flowers in all transgenic rice, and GUS activity conferred by different OsNRT1.3 promoter fragments was significantly upregulated by drought stress, indicating that OsNRT1.3 promoter responds to drought stress and the 719 bp upstream sequence of OsNRT1.3 contains the drought response elements. OsNRT1.3 Function of GRAS proteins in root nodule symbiosis is retained in homologs of a non-legume, rice 2010 Plant Cell Physiol National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan. Root nodule (RN) symbiosis in legumes shares genes involved in the early signaling pathway with more ancient arbuscular mycorrhiza (AM) symbiosis, which is widespread in higher plants. The non-legume homologs of such genes have been well documented to be not only essential for the AM symbiosis in non-legume mycorrhizal plants but also functional in the RN symbiosis in legume plants. In contrast, it has not been investigated in detail whether RN symbiosis-specific genes, which are not essential for AM symbiosis, are functionally conserved in non-legumes. Two GRAS-domain transcription factors, NSP1 and NSP2, have been shown to be required for RN symbiosis, but not for AM symbiosis. In this study, we demonstrated that their homologs, OsNSP1 and OsNSP2, from rice are able to fully rescue the RN symbiosis-defective phenotypes of the mutants of corresponding genes in the model legume, Lotus japonicus. Our results indicate that some of the genes essential for RN symbiosis conserve their functions in homologs from non-legumes, which do not nodulate. OsNSP1,OsNSP2 Golgi nucleotide sugar transporter modulates cell wall biosynthesis and plant growth in rice 2011 Proc Natl Acad Sci U S A State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Golgi-localized nucleotide sugar transporters (NSTs) are considered essential for the biosynthesis of wall polysaccharides and glycoproteins based on their characteristic transport of a large number of nucleotide sugars to the Golgi lumen. The lack of NST mutants in plants has prevented evaluation of this hypothesis in plants. A previously undescribed Golgi NST mutant, brittle culm14 (bc14), displays reduced mechanical strength caused by decreased cellulose content and altered wall structure, and exhibits abnormalities in plant development. Map-based cloning revealed that all of the observed mutant phenotypes result from a missense mutation in a putative NST gene, Oryza sativa Nucleotide Sugar Transporter1 (OsNST1). OsNST1 was identified as a Golgi-localized transporter by analysis of a fluorescence-tagged OsNST1 expressed in rice protoplast cells and demonstration of UDP-glucose transport activity via uptake assays in yeast. Compositional sugar analyses in total and fractionated wall residues of wild-type and bc14 culms showed a deficiency in the synthesis of glucoconjugated polysaccharides in bc14, indicating that OsNST1 supplies the glucosyl substrate for the formation of matrix polysaccharides, and thereby modulates cellulose biosynthesis. OsNST1 is ubiquitously expressed, with high expression in mechanical tissues. The inferior mechanical strength and abnormal development of bc14 plants suggest that OsNST1 has pleiotropic effects on cell wall biosynthesis and plant growth. Identification of OsNST1 has improved our understanding of how cell wall polysaccharide synthesis is regulated by Golgi NSTs in plants. OsNST1|BC14 Golgi-localized UDP-glucose transporter is required for cell wall integrity in rice 2011 Plant Signal Behav State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. Cell wall-related nucleotide sugar transporters (NSTs) theoretically supply the cytosolic nucleotide sugars for glycosyltransferases (GTs) to carry out ploysaccharide synthesis and modification in the Golgi apparatus. However, the regulation of cell wall synthesis by NSTs remains undescribed. Recently, we have reported the functional characterization of Oryza sativa nucleotide sugar transport (Osnst1) mutant and its corresponding gene. OsNST1/BC14 is localized in the Golgi apparatus and transports UDP-glucose. This mutant provides us with a unique opportunity for evaluation of its abroad impacts on cell wall structure and components. We previously examined cell wall composition of bc14 and wild type plants. Here, the spatial distribution of these cell wall alterations was analyzed by immunolabeling approach. Analysis of the sugar yield in different cell wall fractions indicated that this mutation improves the extractability of cell wall components. Field emission scanning electron microscopy further showed that the orientation of microfibrils in bc14 is irregular when compared to that in wild type. Therefore, this UDP-glucose transporter, making substrates available for polysaccharide biosynthesis, plays a critical role in maintaining cell wall integrity. OsNST1|BC14 Overexpression of a partial fragment of the salt-responsive gene OsNUC1 enhances salt adaptation in transgenic Arabidopsis thaliana and rice (Oryza sativa L.) during salt stress 2013 Plant Sci Biological Sciences Program, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand; Environmental and Plant Physiology Research Unit, Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. The rice (Oryza sativa L.) nucleolin gene, OsNUC1, transcripts were expressed in rice leaves, flowers, seeds and roots but differentially expressed within and between two pairs of salt-sensitive and salt-resistant rice lines when subjected to salt stress. Salt-resistant lines exhibited higher OsNUC1 transcript expression levels than salt-sensitive lines during 0.5% (w/v) NaCl salt stress for 6d. Two sizes of OsNUC1 full-length cDNA were found in the rice genome database and northern blot analysis confirmed their existence in rice tissues. The longer transcript (OsNUC1-L) putatively encodes for a protein with a serine rich N-terminal, RNA recognition motifs in the central domain and a glycine- and arginine-rich repeat in the C-terminal domain, while the shorter one (OsNUC1-S) putatively encodes for the similar protein without the N-terminus. Without salt stress, OsNUC1-L expressing Arabidopsis thaliana Atnuc1-L1 plants displayed a substantial but incomplete revertant phenotype, whereas OsNUC1-S expression only induced a weak effect. However, under 0.5% (w/v) NaCl salt stress they displayed a higher relative growth rate, longer root length and a lower H2O2 level than the wild type plants, suggesting a higher salt resistance. Moreover, they displayed elevated AtSOS1 and AtP5CS1 transcript levels. We propose that OsNUC1-S plays an important role in salt resistance during salt stress, a new role for nucleolin in plants. OsNUC1 Contribution of chloroplast biogenesis to carbon-nitrogen balance during early leaf development in rice 2010 J Plant Res Department of Biology, Faculty of Science, Kyushu University, Fukuoka, 812-8581, Japan. kkususcb@kyushu-u.org Chloroplast biogenesis is most significant during the changes in cellular organization associated with leaf development in higher plants. To examine the physiological relationship between developing chloroplasts and host leaf cells during early leaf development, we investigated changes in the carbon and nitrogen contents in leaves at the P4 developmental stage of rice, during which leaf blade structure is established and early events of chloroplast differentiation occur. During the P4 stage, carbon content on a dry mass basis remained constant, whereas the nitrogen content decreased by 30%. Among carbohydrates, sucrose and starch accumulated to high levels early in the P4 stage, and glucose, fructose and cellulose degradation increased during the mid-to-late P4 stage. In the chloroplast-deficient leaves of the virescent-1 mutant of rice, however, the carbon and nitrogen contents, as well as the C/N ratio during the P4 stage, were largely unaffected. These observations suggest that developing rice leaves function as sink organs at the P4 stage, and that chloroplast biogenesis and carbon and nitrogen metabolism in the leaf cell is regulated independently at this stage. OsNUS1|V1 A virescent gene V1 determines the expression timing of plastid genes for transcription/translation apparatus during early leaf development in rice 1997 The Plant Journal Department of Biology, Faculty of Science, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-81, Japan Differentiation of proplastids into functionally active chloroplasts is one of the most significant changes in cellular organization associated with leaf development in higher plants. This process involves activation of a large number of nuclear and chloroplast genes. A central question, therefore, concerns the nature and origin of the signals that initiate and control this process. The rice nuclear mutant, virescent-1 (v1), is temperature-conditional and develops chlorotic leaves when grown at restrictive temperatures. We report here the effects of v1 mutation on the expressions of plastid and nuclear genes during leaf development. In the wild-type rice seedlings, the transcripts of the plastid RNA polymerase gene (rpoB) and ribosomal protein genes (rps7, rps15) accumulated during a strictly limited period of early leaf development, prior to the accumulation of the transcripts of photosynthetic genes (rbcL, RbcS, psbA, Lhc). This period coincides very closely with the leaf developmental stage (late P4) at which the V1 gene gives the signal that determines the virescent phenotype. On the contrary, in the v1 seedlings grown at a restrictive temperature (20°C), this stage-specific accumulation of the rpo and rps transcripts was missing. Instead, the accumulation of these transcripts occurred during a later stage of leaf maturation. In such mutant seedlings, the expression of other plastid genes (psbA, rbcL, 16S rDNA) was strongly suppressed and the normal chloroplast development was disturbed. These data indicate that the V1 gene controls the timing of expression of the key plastid genes for the transcription/ translation apparatus that are essential for the subsequent activation of other plastid genes. OsNUS1|V1 A plastid protein NUS1 is essential for build-up of the genetic system for early chloroplast development under cold stress conditions 2011 Plant J Department of Biology, Kyushu University, Fukuoka 812-8581, Japan. kkususcb@kyushu-u.org During early chloroplast differentiation, the regulation of the plastid genetic system including transcription and translation differs greatly from that in the mature chloroplast, suggesting the existence of a stage-dependent mechanism that regulates the chloroplast genetic system during this period. The virescent-1 (v(1)) mutant of rice (Oryza sativa) is temperature-conditional and develops chlorotic leaves under low-temperature conditions. We reported previously that leaf chlorosis in the v(1) mutant is caused by blockage of the activation of the chloroplast genetic system during early leaf development. Here we identify the V(1) gene, which encodes a chloroplast-localized protein NUS1. Accumulation of NUS1 specifically occurred in the pre-emerged immature leaves, and is enhanced by low-temperature treatment. The C-terminus of NUS1 shows structural similarity to the bacterial antitermination factor NusB, which is known to play roles in the regulation of ribosomal RNA transcription. The RNA-immunoprecipitation and gel mobility shift assays indicated that NUS1 binds to several regions of chloroplast RNA including the upstream leader region of the 16S rRNA precursor. In the leaves of the NUS1-deficient mutant, accumulation of chloroplast rRNA during early leaf development was impaired and chloroplast translation/transcription capacity was severely suppressed under low temperature. Our results suggest that NUS1 is involved in the regulation of chloroplast RNA metabolism and promotes the establishment of the plastid genetic system during early chloroplast development under cold stress conditions. OsNUS1|V1 An ornithine delta-aminotransferase gene OsOAT confers drought and oxidative stress tolerance in rice 2012 Plant Sci National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Ornithine delta-aminotransferase (delta-OAT) is a pyridoxal-5'-phosphate-dependent enzyme that has been proposed to be involved in proline (Pro) and arginine (Arg) metabolism. However, the actual role of delta-OAT in abiotic responses in plants remains to be clarified. Here we characterized an ornithine delta-aminotransferase gene OsOAT that confers multi-stress tolerance in rice (Oryza sativa). We confirmed that OsOAT is a direct target of the stress-responsive NAC transcription factor SNAC2. OsOAT is responsive to multiple stresses and phytohormone treatments. Both ABA-dependent and ABA-independent pathways contributed to the drought-induced expression of OsOAT. Overexpression of the OsOAT gene in rice resulted in significantly enhanced drought and osmotic stress tolerance. Overexpression of OsOAT caused significantly increased delta-OAT activity and Pro accumulation under normal growth conditions. In addition, OsOAT-overexpressing plants showed significantly increased tolerance to oxidative stress. The glutathione (GSH) content and activity of reactive oxygen species (ROS)-scavenging enzymes, such as glutathione peroxidase, were also increased in OsOAT-overexpressing plants. We conclude that OsOAT is a target gene of SNAC2 and confers stress tolerance mainly through enhancing ROS-scavenging capacity and Pro pre-accumulation. OsOAT Cation dependent O-methyltransferases from rice 2008 Planta Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, South Korea. Two lower molecular mass OMT genes (ROMT-15 and -17) were cloned from rice and expressed in Escherichia coli as glutathione S-transferase fusion proteins. ROMT-15 and -17 metabolized caffeoyl-CoA, flavones and flavonols containing two vicinal hydroxyl groups, although they exhibited different substrate specificities. The position of methylation in both luteolin and quercetin was determined to be the 3' hydroxyl group and myricetin and tricetin were methylated not only at 3' but also at 5' hydroxyl groups. ROMT-15 and -17 are cation-dependent and mutation of the predicted metal binding sites resulted in the loss of the enzyme activity, indicating that the metal ion has a critical role in the enzymatic methylation. OsOMT26|ROMT-15|ROMT15 Diverse environmental cues transiently regulate OsOPR1 of the “octadecanoid pathway" revealing its importance in rice defense/stress and development 2003 Biochem Biophys Res Commun Bio-Resource Research and Development Company Pvt. Ltd. (BIRD), G.P.O. Box 8207, Kathmandu, Nepal. None OsOPR1 Identification of a Jasmonic Acid-Responsive Region in the Promoter of the Rice 12-Oxophytodienoic Acid Reductase 1 GeneOsOPR1 2014 Bioscience, Biotechnology and Biochemistry Biotechnology Research Center, The University of Tokyo, Tokyo, Japan. The rice 12-oxophytodienoic acid reductase 1 gene (OsOPR1), isolated as a jasmonic acid (JA)-responsive gene, has been suggested to be involved in defense responses in rice. We identified a 19-base pair region that is essential to the JA-responsiveness of OsOPR1 by deletion and mutation analysis of the promoter by dual luciferase assay. This region contains possible recognition sites for basic leucine zipper transcription factors. OsOPR1 Identification of the OsOPR7 gene encoding 12-oxophytodienoate reductase involved in the biosynthesis of jasmonic acid in rice 2008 Planta Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Enzyme 12-oxophytodienoate (OPDA) reductase (EC1.3.1.42), which is involved in the biosynthesis of jasmonic acid (JA), catalyses the reduction of 10, 11-double bonds of OPDA to yield 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0). The rice OsOPR1 gene encodes OPDA reductase (OPR) converting (-)-cis-OPDA preferentially, rather than (+)-cis-OPDA, a natural precursor of JA. Here, we provide evidence that an OPR family gene in rice chromosome 8, designated OsOPR7, encodes the enzyme involved in the JA biosynthesis. Recombinant OsOPR7-His protein efficiently catalysed the reduction of both enantiomers of cis-OPDA, similar to the OPR3 protein in Arabidopsis thaliana (L.) Heynh. The expression of OsOPR7 mRNA was induced and reached maximum levels within 0.5 h of mechanical wounding and drought stress, and the endogenous JA level started to increase in accordance with the increase in OsOPR7 expression. The GFP-OsOPR7 fusion protein was detected exclusively in peroxisomes in onion epidermal cells. Furthermore, complementation analysis using an Arabidopsis opr3 mutant indicated that the OsOPR7 gene, but not OsOPR1, was able to complement the phenotypes of male sterility in the mutant caused by JA deficiency, and that JA production in the opr3 mutant was also restored by the expression of the OsOPR7 gene. We conclude that the OsOPR7 gene encodes the enzyme catalysing the reduction of natural (+)-cis-OPDA for the JA biosynthesis in rice. OsOPR7 Molecular cloning and characterization of a plant homologue of the origin recognition complex 1 (ORC1) 2000 Plant Sci Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda-shi, 278-8510, Chiba-ken, Japan By using the rice EST database, we have isolated a 2.8 kb cDNA, termed Oryza sativa ORC1 (OsORC1), from rice (O. sativa) encoding a protein that shows homology with the eukaryotic ORC1 proteins. Alignment of the OsORC1 protein sequence with the sequence of ORC1 from human and yeasts S. cerevisiae and S. pombe showed a high degree of sequence homology (38.7, 32.9 and 35.0% identity, respectively), particularly around the C-terminal region containing the CDC-NTP domain. Interestingly, the OsORC1 protein had an A+T hook-like motif, which was not present in the human or yeast genes. Genomic analysis indicated that OsORC1 existed as a single copy per genome. OsORC1 transcripts were expressed strongly in root tips and weakly in young leaves containing root apical meristem and marginal meristem, respectively. No expression was detected in the mature leaves. The level of OsORC1 expression was significantly reduced when cell proliferation was temporarily halted by the removal of sucrose from the growth medium. When the growth-halted cells began to re-grow following addition of sucrose to the medium, OsORC1 was again expressed at high levels. These results suggested that OsORC1 is required for cell proliferation. The role of OsORC1 in plant DNA replication will be discussed. OsORC1 Cloning and characterization of OsORC2, a new member of rice origin recognition complex 2005 Biotechnol Lett Institute of Genetics, State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, People's Republic of China. In eukaryotic cells, the origin recognition complex (ORC) governs the initiation site of DNA replication and formation of the prereplication complex. The isolation, characterization and tissue-specific expression of a putative ORC subunit 2 (OsORC2) in Oryza sativa is described here. A novel cDNA fragment encoding rice ORC2 was isolated by screening the subtractive library, which had a higher expression level in inflorescence meristem than in shoot apical meristem. The full-length cDNA of rice ORC2 was obtained by the method of rapid amplification of cDNA ends, which contained an 1140 bp open reading frame encoding a 379 amino acid polypeptide. Sequence alignment shows that there is a high homology between the deduced amino sequence of OsORC2 and maize ORC2 (85%). The tissue-specific expression pattern of OsORC2 reveals that it is abundant in roots, seedling and inflorescence meristem, while its expression level is much lower in mature leaves and shoot. OsORC2 OsORC3 is required for lateral root development in rice 2013 Plant J The State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, 310058, Hangzhou, China. The origin recognition complex (ORC) is a pivotal element in DNA replication, heterochromatin assembly, checkpoint regulation and chromosome assembly. Although the functions of the ORC have been determined in yeast and model animals, they remain largely unknown in the plant kingdom. In this study, Oryza sativa Origin Recognition Complex subunit 3 (OsORC3) was cloned using map-based cloning procedures, and functionally characterized using a rice (Oryza sativa) orc3 mutant. The mutant showed a temperature-dependent defect in lateral root (LR) development. Map-based cloning showed that a G-->A mutation in the 9th exon of OsORC3 was responsible for the mutant phenotype. OsORC3 was strongly expressed in regions of active cell proliferation, including the primary root tip, stem base, lateral root primordium, emerged lateral root primordium, lateral root tip, young shoot, anther and ovary. OsORC3 knockdown plants lacked lateral roots and had a dwarf phenotype. The root meristematic zone of ORC3 knockdown plants exhibited increased cell death and reduced vital activity compared to the wild-type. CYCB1;1::GUS activity and methylene blue staining showed that lateral root primordia initiated normally in the orc3 mutant, but stopped growing before formation of the stele and ground tissue. Our results indicate that OsORC3 plays a crucial role in the emergence of lateral root primordia. OsORC3 Divergent evolution of oxidosqualene cyclases in plants 2012 New Phytol Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Nanxincun 20, Fragrant Hill, Beijing 100093, China. Triterpenes are one of the largest classes of plant metabolites and have important functions. A diverse array of triterpenoid skeletons are synthesized via the isoprenoid pathway by enzymatic cyclization of 2,3-oxidosqualene. The genomes of the lower plants Chlamydomonas reinhardtii and moss (Physcomitrella patens) contain just one oxidosqualene cyclase (OSC) gene (for sterol biosynthesis), whereas the genomes of higher plants contain nine to 16 OSC genes. Here we carry out functional analysis of rice OSCs and rigorous phylogenetic analysis of 96 OSCs from higher plants, including Arabidopsis thaliana, Oryza sativa, Sorghum bicolor and Brachypodium distachyon. The functional analysis identified an amino acid sequence for isoarborinol synthase (OsIAS) (encoded by Os11g35710/OsOSC11) in rice. Our phylogenetic analysis suggests that expansion of OSC members in higher plants has occurred mainly through tandem duplication followed by positive selection and diversifying evolution, and consolidated the previous suggestion that dicot triterpene synthases have been derived from an ancestral lanosterol synthase instead of directly from their cycloartenol synthases. The phylogenetic trees are consistent with the reaction mechanisms of the protosteryl and dammarenyl cations which parent a wide variety of triterpene skeletal types, allowing us to predict the functions of the uncharacterized OSCs. OsOSC11|OsIAS1,OsPS1|OsOSC7 A mutation of OSOTP 51 leads to impairment of photosystem I complex assembly and serious photo-damage in rice 2012 J Integr Plant Biol National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Gene expression in chloroplasts is regulated by many nuclear-encoded proteins. In this study, we isolated a rice (Oryza sativa subsp. japonica) mutant osotp51 with significant reduction in photosystem I (PSI). The osotp51 is extremely sensitive to light and accumulates a higher level of reactive oxygen species. Its leaves are almost albino when grown at 40 mumol photons/m(2) per s. However, grown at 4 mumol photons/m(2) per s, osotp51 has a similar phenotype to the wild-type. 77K chlorophyll fluorescence analysis showed a blue shift in the highest peak emission from PSI in osotp51. In addition, the level of PSI and PSII dimer is dramatically reduced in osotp51. OSOTP 51 encodes a pentatricopeptide repeats protein, homologous to organelle transcript processing 51 in Arabidopsis. Loss-of-function OSOTP51 affects intron splicing of a number of plastid genes, particularly the ycf3 coding a protein involved in the assembly of PSI complex. OSOTP51 is functionally conserved in higher plants. The mutation of osotp51 indirectly leads to a widespread change in the structure and functions of PSI, results in severe photoinhibition, and finally dies, even when grown under very low light intensity. OSOTP51 AGC kinase OsOxi1 positively regulates basal resistance through suppression of OsPti1a-mediated negative regulation 2010 Plant Cell Physiol Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. OsPti1a, a functional ortholog of tomato SlPti1, negatively regulates both basal resistance and R-gene-mediated resistance in rice. To investigate the molecular function of OsPti1a in defense responses, we searched for components interacting with OsPti1a using a yeast two-hybrid system. One of the interacting proteins is a Ser/Thr kinase that directly phosphorylates OsPti1a in vitro. This protein belongs to the AGC kinase family and is highly similar to AtOxi1, which is induced in response to a wide range of reactive oxygen species (ROS)-generating stimuli in Arabidopsis. Thus, it was designated OsOxi1. OsOxi1 was transiently phosphorylated in response to ROS and chitin elicitor. Both OsOxi1-overexpressing transgenic lines and the ospti1a mutant were highly sensitive to ROS treatment, indicating that OsOxi1 and OsPti1a are involved in ROS-mediated signaling in opposing ways. OsOxi1 is specifically expressed at infection sites where ROS are produced after inoculation with a blast fungus, Magnaporthe oryzae. Overexpression of OsOxi1 enhanced basal resistance to the blast fungus, indicating that OsOxi1 positively regulates disease resistance. OsOxi1 phosphorylates Thr-233 of OsPti1a and a point mutation of Thr-233 enhanced disease susceptibility to a bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), suggesting that the phosphorylation of OsPti1a by OsOxi1 is essential for basal resistance to Xoo. Taken together, our data suggest that OsOxi1 positively regulates defense responses through the phosphorylation of OsPti1a, causing the release from an OsPti1a-dependent inhibition of the responses. OsOxi1,OsPti1a Pdk1 Kinase Regulates Basal Disease Resistance Through the OsOxi1-OsPti1a Phosphorylation Cascade in Rice 2010 Plant and Cell Physiology Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8602 Japan. The AGC kinase OsOxi1, which has been isolated as an interactor with OsPti1a, positively regulates basal disease resistance in rice. In eukaryotes, AGC kinase family proteins are regulated by 3-phosphoinositide-dependent protein kinase 1 (Pdk1). In Arabidopsis, AtPdk1 directly interacts with phosphatidic acid, which functions as a second messenger in both biotic and abiotic stress responses. However, the functions of Pdk1 are poorly understood in plants. We show here that OsPdk1 acts upstream of the OsOxi1-OsPti1a signal cascade in disease resistance in rice. OsPdk1 interacts with OsOxi1 and phosphorylates the Ser283 residue of OsOxi1 in vitro. To investigate whether OsPdk1 is involved in immunity that is triggered by microbial-associated molecular patterns, we analyzed the phosphorylation status of OsPdk1 in response to chitin elicitor. Like OsOxi1, OsPdk1 is rapidly phosphorylated in response to chitin elicitor, suggesting that OsPdk1 participates in signal transduction through pathogen recognition. The overexpression of OsPdk1 enhanced basal resistance against a blast fungus, Magnaporthe oryzae, and a bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo). Taken together, these results suggest that OsPdk1 positively regulates basal disease resistance through the OsOxi1-OsPti1a phosphorylation cascade in rice. OsOxi1,OsPti1a,PDK1 The evolution of CONSTANS-like gene families in barley, rice, and Arabidopsis 2003 Plant Physiol John Innes Centre, Norwich Research Park, Colney, United Kingdom. The CO (CONSTANS) gene of Arabidopsis has an important role in the regulation of flowering by photoperiod. CO is part of a gene family with 17 members that are subdivided into three classes, termed Group I to III here. All members of the family have a CCT (CO, CO-like, TOC1) domain near the carboxy terminus. Group I genes, which include CO, have two zinc finger B-boxes near the amino terminus. Group II genes have one B-box, and Group III genes have one B-box and a second diverged zinc finger. Analysis of rice (Oryza sativa) genomic sequence identified 16 genes (OsA-OsP) that were also divided into these three groups, showing that their evolution predates monocot/dicot divergence. Eight Group I genes (HvCO1-HvCO8) were isolated from barley (Hordeum vulgare), of which two (HvCO1 and HvCO2) were highly CO like. HvCO3 and its rice counterpart (OsB) had one B-box that was distantly related to Group II genes and was probably derived by internal deletion of a two B-box Group I gene. Sequence homology and comparative mapping showed that HvCO1 was the counterpart of OsA (Hd1), a major determinant of photoperiod sensitivity in rice. Major genes determining photoperiod response have been mapped in barley and wheat (Triticum aestivum), but none corresponded to CO-like genes. Thus, selection for variation in photoperiod response has affected different genes in rice and temperate cereals. The peptides of HvCO1, HvCO2 (barley), and Hd1 (rice) show significant structural differences from CO, particularly amino acid changes that are predicted to abolish B-box2 function, suggesting an evolutionary trend toward a one-B-box structure in the most CO-like cereal genes. OsP|OsBBX9 The rice B-box zinc finger gene family: genomic identification, characterization, expression profiling and diurnal analysis 2012 PLoS One National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China. BACKGROUND: The B-box (BBX) -containing proteins are a class of zinc finger proteins that contain one or two B-box domains and play important roles in plant growth and development. The Arabidopsis BBX gene family has recently been re-identified and renamed. However, there has not been a genome-wide survey of the rice BBX (OsBBX) gene family until now. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we identified 30 rice BBX genes through a comprehensive bioinformatics analysis. Each gene was assigned a uniform nomenclature. We described the chromosome localizations, gene structures, protein domains, phylogenetic relationship, whole life-cycle expression profile and diurnal expression patterns of the OsBBX family members. Based on the phylogeny and domain constitution, the OsBBX gene family was classified into five subfamilies. The gene duplication analysis revealed that only chromosomal segmental duplication contributed to the expansion of the OsBBX gene family. The expression profile of the OsBBX genes was analyzed by Affymetrix GeneChip microarrays throughout the entire life-cycle of rice cultivar Zhenshan 97 (ZS97). In addition, microarray analysis was performed to obtain the expression patterns of these genes under light/dark conditions and after three phytohormone treatments. This analysis revealed that the expression patterns of the OsBBX genes could be classified into eight groups. Eight genes were regulated under the light/dark treatments, and eleven genes showed differential expression under at least one phytohormone treatment. Moreover, we verified the diurnal expression of the OsBBX genes using the data obtained from the Diurnal Project and qPCR analysis, and the results indicated that many of these genes had a diurnal expression pattern. CONCLUSIONS/SIGNIFICANCE: The combination of the genome-wide identification and the expression and diurnal analysis of the OsBBX gene family should facilitate additional functional studies of the OsBBX genes. OsP|OsBBX9 Isolation and mapping of a family of putative zinc-finger protein cDNAs from rice 1998 DNA Res Rice Genome Research Program, Institute of the Society for Techno-Innovation of Agriculture, Forestry and Fisheries, Ibaraki, Japan. To understand the functions of rice homologues of the Arabidopsis flowering-time gene CONSTANS (CO) and salt-tolerance gene STO, we performed a similarity search of the single-run sequence data of cDNA clones accumulated by the Rice Genome Research Program, and isolated seven rice cDNA clones (S3574, C60910, S12569, R2931, R1479, R1577, and E10707) coding for proteins containing one of two zinc-finger-like motifs. Comparison of the deduced amino acid sequences between these cDNAs and the CO gene revealed significant similarities (46%-61%) in the region of zinc-finger motifs. A domain having a high content of basic amino acids at the C-terminus of the CO protein was found in the corresponding region of proteins predicted by from cDNAs S3574, C60910, and S12569. Two amino acid sequences, "CCADEAAL" and "FCV(L)EDRA," which were present inside each zinc-finger in the Arabidposis regulatory protein STO, were also found in each of the two zinc-finger regions of proteins predicted from cDNAs R2931, R1479, R1577, and E10707. Using restriction fragment length polymorphism (RFLP) linkage analysis, we determined the chromosomal location of the seven cDNA clones. The position of R2931 on the RFLP linkage map was closely linked to Hd-3, one of the putative quantitative trait loci (QTL) controlling heading date in rice. OsP|OsBBX9 Proline accumulation and Δ1-pyrroline-5-carboxylate synthetase gene properties in three rice cultivars differing in salinity and drought tolerance 2003 Plant Science Laboratory of Plant Genetics, Vrije Universiteit Brussel (VUB), Campus Etterbeek, Gebouw E Pleinlaan 2, B-1050 Brussels, Belgium Three indica rice cultivars (Oryza sativa) differing in their tolerance to salt and drought stress in field conditions in Vietnam were analyzed at the molecular and biochemical levels with the goal to reveal the basis for their differential behavior and in particular their ability to accumulate proline. An in vitro growth test showed that after a 7-day period of stress, the fresh weight of plantlet roots appears to be a relevant parameter for differentiating drought and salt tolerance of the concerned cultivars. Sodium level was lower in the salt tolerant cultivar than in the other rice cultivars. Proline accumulation in roots of tolerant cultivars starts earlier after the initiation of the stress treatment than that of the osmotic stress sensitive cultivar and also reaches a higher level. Proline accumulation was not related to proteolysis and so could be the result from induction of proline biosynthesis by osmotic stress. However, neither the sequence of amino acids involved in the proline feedback inhibition of the key regulatory enzyme Δ1-pyrroline-5-carboxylate synthetase (P5CS; EC not assigned), nor the expression of the p5cs genes were modified in the tolerant cultivars. These observations suggest that proline accumulation in roots is a possible indicator of the osmotic tolerance in these rice cultivars. However, other mechanisms than those related to a change in P5CS regulation are responsible for the increased proline content. OsP5CS|OsP5CS1 Overexpression of the receptor-like protein kinase genes AtRPK1 and OsRPK1 reduces the salt tolerance of Arabidopsis thaliana 2014 Plant Sci College of Life Science, Hebei Normal University, Shijiazhuang 050024, PR China. AtRPK1 (AT1G69270) is a leucine-rich repeat receptor-like protein kinase (LRR-RLK) gene in Arabidopsis thaliana. The rice gene Os07g0602700 (OsRPK1) is the homolog of AtRPK1. AtRPK1 and OsRPK1 were overexpressed and the expression of AtRPK1 was inhibited by RNAi in A. thaliana. The functional results showed that the degrees of salt tolerance of the 35S:RPK1 A. thaliana plants were significantly lower than that of the control plants. The AtRPK1-RNAi A. thaliana plants exhibited higher salt tolerance than the wild-type plants (Col). The subcellular localisation results showed that the RPK1 proteins were mainly distributed on the cell membrane and that the overexpressed AtRPK1 proteins exhibited a significantly clustered distribution. The physiological analyses revealed that the overexpression of the RPK1 genes increased the membrane permeability in the transgenic A. thaliana plants. In response to salt stress, these plants exhibited an increased Na(+) flux into the cell, which caused greater damage to the cell. The real-time quantitative PCR analysis showed that the expression of the P5CS1 gene was inhibited and the SOS signalling pathway was blocked in the 35S:AtRPK1 A. thaliana plants. These effects at least partially contribute to the salt-sensitive phenotype of the 35S:RPK1 plants. OsP5CS|OsP5CS1,OsRPK1 Characterization of the gene for delta1-pyrroline-5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L. 1997 Plant Mol Biol Advanced Research Laboratory, Hitachi Ltd., Hatoyama, Saitama, Japan. A cDNA for delta1-pyrroline-5-carboxylate (P5C) synthetase (cOsP5CS), an enzyme involved in the biosynthesis of proline, was isolated and characterized from a cDNA library prepared from 14-day-old seedlings of Oryza sativa cv. Akibare. The deduced amino acid sequence of the P5CS protein (OsP5CS) from O. sativa exhibited 74.2% and 75.5% homology to that of the P5CS from Arabidopsis thaliana and Vigna aconitifolia, respectively. Northern blot analysis revealed that the gene for P5CS (OsP5CS) was induced by high salt, dehydration, treatment of ABA and cold treatment, while it was not induced by heat treatment. Simultaneously, accumulation of proline was observed as a result of high salt treatment in O. sativa. Moreover, the levels of expression of OsP5CS mRNA and content of proline under salt stress condition were compared between a salt-tolerant cultivar, Dee-gee-woo-gen (DGWG) and a salt-sensitive breeding line, IR28. It was observed that the expression of the P5CS gene and the accumulation of proline in DGWG steadily increased, whereas those in IR28 increased slightly. OsP5CS|OsP5CS1 OsPAA2, a distinct α1 subunit gene for the 20S proteasome in rice (Oryza sativa L.) 2001 Gene Kihara Institute for Biological Research and Graduate School of Integrated Science, Yokohama City University, Maioka 641-12, Totsuka-ku, Yokohama 244-0813, Japan The 20S proteasome is the proteolytic complex that is involved in removing abnormal proteins and other diverse biological functions. The 20S proteasome is constituted of 28 subunits arranged in four rings of seven subunits, and exists as a hollow cylinder. The two outer rings and the two inner rings are composed of seven different α and beta type subunits, respectively, giving an α7beta7beta7α7 structure. We previously reported the primary structures of the 14 proteasomal subunit subfamilies in rice (Oryza sativa), representing the first set for all the subfamilies from monocot. In this study, a distinct cDNA sequence encoding the α1 subunit, OsPAA2, was identified. The amino acid sequence similarity between the two rice α1 subunits was as low as 59.6%, contrasting with those between paralogs of Arabidopsis proteasome subunit genes. The expression pattern of the OsPAA2 gene was different from that of another α1 gene, OsPAA1. These data suggest that OsPAA2 might play a distinct role from that of OsPAA1 in the 20S proteasome complex. OsPAA1,OsPAA2 Overexpression of OgPAE1 from wild rice confers fungal resistance against Botrytis cinerea 2008 J Plant Res Life Science and Natural Resources, Dong-A University, 840 Hadan-dong, Busan 604-714, Korea. A full-length cDNA of the OgPAE1 gene encoding the alpha5 subunit of the 20S proteasome was isolated from wild rice (Oryza grandiglumis) treated by wounding or with a fungal elicitor. The deduced amino acid sequence of OgPAE1 comprises 237 amino acids (25.99 kDa), and shows 94.5% homology with Arabidopsis thaliana AtPAE1. Expression of OgPAE1 is regulated by defense-related signaling chemicals such as cantharidin, endothall and jasmonic acid. Overexpression of OgPAE1 in A. thaliana leads to resistance to the fungal pathogen Botrytis cinerea by lowering disease rate and size of necrotic lesions, and by less penetration and colonization of fungal hyphae. The results indicate that the 20S proteasome from wild rice is involved in the B. cinerea defense pathway via an as yet undetermined mechanism. OgPAE1 Knockdown of OsPAO and OsRCCR1 cause different plant death phenotypes in rice 2011 J Plant Physiol Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, PR China. Pheophorbide a oxygenase (PAO) and red chlorophyll catabolite reductase (RCCR) catalyze key steps in chlorophyll degradation by opening the porphyrin macrocycle of pheophorbide a and forming the primary non-photoreactive fluorescent chlorophyll catabolite. These genes strongly participate in senescence and reportedly involved in plants' responses to physical wounding and pathogens. In this report, a single PAO gene (OsPAO) and two RCCR genes (OsRCCR1 and OsRCCR2) have been isolated from rice. Expression analysis by semi-quantitative PCR or quantitative real-time PCR showed that OsRCCR1 transcripts were much more abundant than OsRCCR2, and all of these genes were upregulated during senescence and following wound treatment. RNA interference knockdown of OsPAO led to pheophorbide a accumulation in leaves (especially dark-induced senescent leaves) and leaf death from regeneration stage onwards, even transgenic plants inviability after transplantation. While, knockdown of OsRCCR1 resulted in lesion-mimic spots generation in older leaves which died off early in the transgenic plants. These results suggest that OsPAO and OsRCCR1 play key roles in senescence and are involved in wound responses. OsPAO,OsRCCR1 Molecular cloning and function analysis of the stay green gene in rice 2007 Plant J South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China. Chloroplasts undergo drastic morphological and physiological changes during senescence with a visible symptom of chlorophyll (Chl) degradation. A stay green mutant was identified and then isolated from the japonica rice (Oryza sativa) cv. Huazhiwu by gamma-ray irradiation. The stay green mutant was characterized by Chl retention, stable Chl-protein complexes, and stable thylakoid membrane structures, but lost its photosynthetic competence during senescence. The gene, designated Stay Green Rice (SGR), was cloned by a positional cloning strategy encoding an ancient protein containing a putative chloroplast transit peptide. SGR protein was found in both soluble and thylakoid membranes in rice. SGR, like the gene for pheophorbide a oxygenase (PaO), was constitutively expressed, but was upregulated by dark-induced senescence in rice leaves. Senescence-induced expression of SGR and PaO was enhanced by ABA, but inhibited by cytokinin. Overexpression of SGR reduced the number of lamellae in the grana thylakoids and reduced the Chl content of normally growing leaves. This indicates that upregulation of SGR increases Chl breakdown during senescence in rice. A small quantity of chlorophyllide a accumulated in sgr leaves, but this also accumulated in wild-type rice leaves during senescence. Some pheophorbide a was detected in sgr leaves in the dark. According to these observations, we propose that SGR may be involved in regulating or taking part in the activity of PaO, and then may influence Chl breakdown and degradation of pigment-protein complex. OsPAO,SGR Overexpression of OsPAP10a, a root-associated acid phosphatase, increased extracellular organic phosphorus utilization in rice 2012 J Integr Plant Biol State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China. Phosphorus (P) deficiency is a major limitation for plant growth and development. Among the wide set of responses to cope with low soil P, plants increase their level of intracellular and secreted acid phosphatases (APases), which helps to catalyze inorganic phosphate (Pi) hydrolysis from organo-phosphates. In this study we characterized the rice (Oryza sativa) purple acid phosphatase 10a (OsPAP10a). OsPAP10a belongs to group Ia of purple acid phosphatases (PAPs), and clusters with the principal secreted PAPs in a variety of plant species including Arabidopsis. The transcript abundance of OsPAP10a is specifically induced by Pi deficiency and is controlled by OsPHR2, the central transcription factor controlling Pi homeostasis. In gel activity assays of root and shoot protein extracts, it was revealed that OsPAP10a is a major acid phosphatase isoform induced by Pi starvation. Constitutive overexpression of OsPAP10a results in a significant increase of phosphatase activity in both shoot and root protein extracts. In vivo root 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) assays and activity measurements on external media showed that OsPAP10a is a root-associated APase. Furthermore, overexpression of OsPAP10a significantly improved ATP hydrolysis and utilization compared with wild type plants. These results indicate that OsPAP10a can potentially be used for crop breeding to improve the efficiency of P use. OsPAP10a,OsPHR2 Molecular characterization of OsPAP2: transgenic expression of a purple acid phosphatase up-regulated in phosphate-deprived rice suspension cells 2010 Biotechnol Lett College of Natural Resources and Life Science, Dong-A University, Busan, 604-714, Korea. A phosphate starvation-induced, purple, acid phosphatase cDNA was cloned from rice, Oryza sativa. The cDNA encoding the phosphatase (OsPAP2) has 1,893 bp with an open reading frame of 630 amino acid residues. The deduced amino acid sequence of OsPAP2 shows identities of 60-63% with other plant purple acid phosphatases and appears to have five conserved motifs containing the residues involved in metal binding. OsPAP2 expression is up-regulated in the rice plant and in cell cultures in the absence of phosphate (P( i )). The induced expression of OsPAP2 is a specific response to P( i ) starvation, and is not affected by the deprivation of other nutrients. OsPAP2 expression was responsive to the level of P( i )-supply, and transcripts of OsPAP2 were abundant in P( i )-deprived roots. The OsPAP2 cDNA was expressed as a 69 kDa polypeptide in baculovirus-infected insect Sf9 cells. In addition, the OsPAP2 gene was introduced into Arabidopsis via an Agrobacterium-mediated transformation. Functional expression of the OsPAP2 gene in the transgenic Arabidopsis line was confirmed by northern and western blot analyses, as well as by phosphatase activity assays. These results suggest that the OsPAP2 gene can be used to develop new transgenic dicotyledonous plants that are able to adapt to P( i )-deficient conditions. OsPAP2 Rice OsERG3 encodes an unusual small C2-domain protein containing a Ca(2+)-binding module but lacking phospholipid-binding properties 2011 Biochim Biophys Acta Division of Applied Life Sciences (BK21 Program), Gyeongsang National University, Jinju 660701, Republic of Korea. BACKGROUND: The C2 domain is a Ca(2+)/phospholipid-binding motif found in many proteins involved in signal transduction or membrane trafficking. OsERG3 is a homolog of OsERG1, a gene encoding a small C2-domain protein in rice. METHODS: OsERG3 Ca(2+)-binding and phospholipid-binding assays were carried out using (3)H-labeled phospholipid liposomes and a (45)Ca(2+) overlay assay, respectively. Cytosolic expression of OsERG3 was investigated by Western blot analysis and the OsERG3::smGFP transient expression assay. RESULTS: OsERG3 transcript levels were greatly enhanced by treatment with a fungal elicitor and Ca(2+)-ionophore. OsERG3 protein proved unable to interact with phospholipids regardless of the presence or absence of Ca(2+) ions. Nonetheless, OsERG3 displayed calcium-binding activity in an in vitro(45)Ca(2+)-binding assay, a property not observed with OsERG1. The cytosolic location of OsERG3 was not altered by the presence of fungal elicitor or Ca(2+)-ionophore. CONCLUSIONS: OsERG3 encodes a small C2-domain protein consisting of a single C2 domain. OsERG3 binds Ca(2+) ions but not phospholipids. OsERG3 is a cytosolic soluble protein. The OsERG3 gene may play a role in signaling pathway involving Ca(2+) ions. GENERAL SIGNIFICANCE: The data demonstrate that OsERG3 is an unusual small C2-domain protein containing a Ca(2+)-binding module but lacking phospholipid-binding properties. Rpp16|OsPBP1 Rpp16 and Rpp17, from a Common Origin, have Different Protein Characteristics but Both Genes are Predominantly Expressed in Rice Phloem Tissues 2002 Plant and Cell Physiology Genetic Diversity Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602 Japan The genes for two types of rice phloem protein (RPP16 and RPP17) were isolated and characterized. Conservation of five exon sizes as well as splicing positions between the two genes suggest that either RPP16 or RPP17 is a resultant of gene duplication. By protein blot analysis, RPP16 and RPP17 proteins were specifically detected in soluble and insoluble fractions of a crude extract of rice plants, respectively, suggesting that these proteins play different roles in individual cells. The expression of Rpp16 and Rpp17 was monitored by the beta-glucuronidase (gusA) reporter-gene method. Rpp16-gusA and Rpp17-gusA were expressed preferentially in the phloem tissues from different parts of the plant, but almost no GUS staining was observed in the rest of the tissues. In roots of both constructs, interestingly, stronger GUS-accumulation was detected in younger vascular tissues than in aged vascular tissues. In situ hybridization also showed that Rpp17 was more strongly expressed in vascular tissues of tiller buds. These results suggest that transcript of these genes was more abundant in young tissues. The presence of two copies of the gene in higher plants, from a common origin, which have different protein characteristics, indicates that evolutionary diversification might have occurred in the gene function. Rpp16|OsPBP1,Rpp17|OsERG1 A novel C2-domain phospholipid-binding protein, OsPBP1, is required for pollen fertility in rice 2008 Mol Plant National Centre for Plant Gene Research, Beijing 100190, China. Pollen fertility is a crucial factor for successful pollination and essential for seed formation. Recent studies have suggested that a diverse range of internal and external factors, signaling components and their related pathways are likely involved in pollen fertility. Here, we report a single C2-domain containing protein, OsPBP1, initially identified through cDNA microarray analysis. OsPBP1 is a single copy gene and preferentially expressed in pistil and pollen but down-regulated by pollination. OsPBP1 had a calcium concentration-dependent phospholipid-binding activity and was localized mainly in cytoplasm and nucleus, but translocated onto the plasma membrane in response to an intracellular Ca(2+) increase. Pollen grains of antisense OsPBP1 transgenic lines were largely nonviable, germinated poorly in vitro and of low fertility. OsPBP1 protein was localized in a region peripheral to pollen wall and vesicles of elongating pollen tube, and its repressed expression reduced substantially this association and led to alteration of microfilament polymerization during pollen germination. Taken together, these results indicate that OsPBP1 is a novel functional C2-domain phospholipids-binding protein that is required for pollen fertility likely by regulating Ca(2+) and phospholipid signaling pathways. Rpp16|OsPBP1 Characterization of plant proliferating cell nuclear antigen (PCNA) and flap endonuclease-1 (FEN-1), and their distribution in mitotic and meiotic cell cycles 2002 The Plant Journal Department of Applied Biological Science, Faculty of Science and Technology, Tokyo The biochemical and cell cycle-dependent properties of proliferating cell nuclear antigen (OsPCNA) and flap endonuclease-1 (OsFEN-1) were characterized from rice (Oryza sativa). OsPCNA was physically associated with OsFEN-1 and increased the flap-endonuclease activity of OsFEN-1 by 2.5-fold. Northern and Western blotting analysis revealed that OsPCNA and OsFEN-1 were present in meristematic tissues such as cultured cells, shoot apical meristem and root apical meristem. No expression was detected in the mature leaves, although they were exposed to UV. Both of these proteins were localized in the nuclei of the interphase cells including G1, S and G2, and in the nuclear region at telophase. The distribution patterns of plant PCNA and FEN-1 in meiotic cell progression were investigated using microsporocytes of lily (Lilium longiflorum cv. Hinomoto). During the leptotene to pachytene stages, PCNA and FEN-1 were localized in the nuclear region. The florescence gradually disappeared from diplotene to metaphase I. Interestingly, signals for PCNA formed 10-20 intense spots at leptotene. The number of spots decreased to 1-5 at zygotene and finally to 1 at pachytene. The roles of OsPCNA and OsFEN-1 in mitotic and meiotic cell cycles are discussed. OsPCNA|PCNA Gene for proliferating-cell nuclear antigen (DNA polymerase 6 auxiliary protein) is present in both mammalian and higher plant genomes 1989 Proc Natl Acad Sci U S A Department of Molecular Biology, National Institute of Agrobiological Resources, Tsukuba Science City, Japan. Proliferating-cell nuclear antigen (PCNA; also called cyclin) was originally described in proliferating mammalian cells as a nuclear protein with an apparent Mr of 33,000-36,000 and recently was found to be a DNA polymerase delta auxiliary protein. To elucidate whether PCNA/cyclin is a universal protein necessary for proliferation of eukaryotes, a search was conducted for PCNA/cyclin homologues in higher plants. In Southern blot-hybridization analysis, a rat PCNA/cyclin cDNA probe hybridized with homologous sequences in genomic DNAs from rice, soybean, and tobacco. A PCNA/cyclin-related molecular clone (pCJ-1) was isolated from rice DNA and was partially sequenced. The pCJ-1 probe hybridized with a 1.2-kilobase transcript in RNA from rice root tips and shoots. Immunoblot analysis of the soluble extract of soybean root tips with monospecific anti-PCNA/cyclin identified an immunoreactive protein with an apparent Mr of 34,000. Immunohistochemical analysis revealed the presence of an immunoreactive PCNA/cyclin protein in the nuclei of cells in the meristem of soybean root tips. The highly homologous nature of the gene for PCNA/cyclin throughout the animal and plant kingdoms suggests that the product of the gene plays an essential role in DNA replication in eukaryotes. OsPCNA|PCNA The OsGEN-L protein from Oryza sativa possesses Holliday junction resolvase activity as well as 5'-flap endonuclease activity 2012 J Biochem Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, Fukuoka 812-8581, Japan. OsGEN-L has a 5'-flap endonuclease activity and plays an essential role in rice microspore development. The Class 4 RAD2/XPG family nucleases, including OsGEN-L, were recently found to have resolving activity for the Holliday junction (HJ), the intermediate of DNA strand recombination. In this study, we performed a detailed characterization of OsGEN-L, as a structure-specific endonuclease. Highly purified OsGEN-L was prepared as the full-length protein for in vitro endonuclease assays using various structured DNAs, and the 5'-flap endonuclease activity, which is stimulated in a PCNA-dependent manner, was demonstrated. In addition, the in vitro HJ resolving activity of OsGEN-L represents the first such activity originating from plant cells. OsGEN-L cleaved HJ at symmetrically related sites of the branch point. However, the two branched strands seemed to be cleaved individually, and not cooperatively, by each OsGEN-L monomer protein. The substrate specificity suggests that OsGEN-L functions in multiple processes of DNA metabolism in rice cells. OsPCNA|PCNA Production and phenotypic analysis of rice transgenics with altered levels of pyruvate decarboxylase and alcohol dehydrogenase proteins 2007 Plant Physiol Biochem Department of Plant Molecular Biology, University of Delhi, South Campus, Benito Juarez Road, Dhaula Kuan, New Delhi 110021, India. Pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) enzymes are responsible for the operation of ethanolic fermentation pathway that appears to correlate to an extent with anoxia tolerance in plants. This study was undertaken with the objective of (a) analysing the rice pdc gene family and (b) altering the efficacy of the ethanolic fermentation process, through production of transgenic rice plants over- and under-expressing pyruvate decarboxylase (employing Ospdc1 gene from rice) as well as over-expressing alcohol dehydrogenase (employing Ghadh2 gene from cotton) proteins. Correlations noted in this study between the pattern of expression of the Pdc alpha-subunit and Ospdc2 transcript as well as between the Pdc beta-subunit and Ospdc1 transcript suggest the possibility that alpha-subunit is encoded by Ospdc2 and that beta-subunit is encoded by Ospdc1. The fact that levels of Pdc beta-subunit were particularly high in pUH-sPdc1 (plasmid construct designed for over-expression of Ospdc1) seedlings while levels of beta-subunit levels were negligible or lower in pUH-asPdc1 (plasmid construct designed for under-expression of Ospdc1) seedlings also support these observations. Transgenics raised for over-expression of Pdc and Adh and under-expression of Pdc were confirmed for the transgene presence and effects by PCR, Southern blotting, Northern blotting, Western blotting and isozyme assays. Pdc and Adh over-expressing rice transgenics at early seedling stage under unstressed control growth conditions showed slight, consistent advantage in root vigour as compared to that of wild-type seedlings. OsPDC1,OsPDC2 Characterization of pyruvate decarboxylase genes from rice 1996 Plant Mol Biol Department of Botany & Plant Pathology, Purdue University, West Lafayette, IN 47907, USA. The pdc1 gene encoding pyruvate decarboxylase has been isolated and sequenced from an IR54 rice genomic library. In contrast to a previously isolated intron-less rice genomic pdc, pRgpdc3, this gene contains five intervening introns in the coding region and corresponds to a cDNA clone, pRcpdc1, isolated from an IR54-cDNA library constructed from anaerobically-induced mRNAs. Comparison of the deduced amino acid sequence of this gene with that of the rice pdc2 and pdc3 showed 88% and 89% similarity, and 78% and 79% identity, respectively. Southern blots indicated that more than three genes constitute the pdc gene family in rice. pdc1 is highly inducible under anaerobic conditions. Rice pdc2 is also inducible by anoxia but to a much lesser extent than pdc1. OsPDC1,OsPDC2 Down-regulation of the OsPDCD5 gene induced photoperiod-sensitive male sterility in rice 2010 Plant Science State Key Laboratory of Genetic Engineering, Institute of Genetics School of Life Sciences, Fudan University, Shanghai 200433, PR China Programmed cell death (PCD) is a crucial process for plants during development and environmental stress. OsPDCD5, is an ortholog to mammalian programmed cell death 5 gene. Here we report that decreased expression of OsPDCD5 caused by antisense technology could induce pollen sterility in photoperiod-sensitive rice. The male sterility of transgenic plants is reversible, determined mainly by photoperiod. Transgenic plants are sterile under long-day photoperiod (>13.5 h sunlight) with postponed heading time, while they can restore fertility under short-day photoperiod (below 13 h sunlight). Analysis of tissue sections of anthers displays the tapetum cells of the anther wall retard PCD process in transgenic plants under long-day photoperiod. The retarded PCD was also confirmed by DNA fragmentation. In F1 hybrids made from transgenic plants with antisense-OsPDCD5 and japonica rice varieties, lower transcript inhibition could restore fertility, under certain photoperiod and temperatures. The surprising discovery of the photosensitive male fertility suggested that by using specific photoperiods, the male sterility (PGMS) could be used for commercial production of hybrid rice using a two-line breeding system. The transgenic hybrid strategy was easy to apply, reduced costs and shortened the breeding period. It provides great advantages for commercial applications in rice and other crops. OsPDCD5 Overexpression of the OsPDCD5 gene induces programmed cell death in rice 2005 J Integr Plant Biol Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China The primary aim of the present study was to investigate the overexpression of the rice (Oryza sativa L.) programmed cell death 5 (OsPDCD5) gene in rice plant. Constitutive expression of OsPDCD5 from the cauliflower mosaic virus (CaMV) 35S promoter induced programmed cell death (PCD) in transgenic rice. Programmed cell death was accompanied by typical features, including inhibition of developmental growth, a reduction of fresh weight, degradation of total protein content, and fragmentation of genomic DNA. These results suggest that OsPDCD5 plays an essential role in the regulation of PCD in rice plants. OsPDCD5 Rice peptide deformylase PDF1B is crucial for development of chloroplasts 2008 Plant Cell Physiol National Research Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea. Because protein synthesis begins with N-formylmethionine in plant endosymbiotic organelles, removal of the formyl group by peptide deformylase (PDF) is essential to allowing the excision of the first methionine. Rice contains three copies (OsPDF1A, OsPDF1B and OsPDF1B2) of the PDF genes. Unlike OsPDF1A and OsPDF1B, OsPDF1B2 is apparently non-functional, with several deleterious substitutions and deletions. OsPDF1A is more strongly expressed in the roots, while OsPDF1B is expressed at higher levels in mature leaves. Transient expression of PDF-green fluorescent protein (GFP) fusion proteins in the protoplasts demonstrates that, unlike OsPDF1A, OsPDF1B is localized in both the chloroplasts and the mitochondria. We used T-DNA insertional alleles to elucidate functional roles associated with OsPDF1B. Homozygous plants of pdf1b/pdf1b exhibited the phenotypes of chlorina and growth retardation. Histochemical analysis showed that the length of their mesophyll cells was increased 4- to 5-fold, resulting in a reduction in the total number of cells. Transmission electron microscopy analyses revealed that chloroplasts were severely damaged and mitochondria appeared to be mildly altered in the pdf1b mutants. Expression of genes encoded in the chloroplasts and mitochondria was altered in the mutants. Based on these results, we conclude that OsPDF1B is essential for the development of chloroplast and perhaps mitochondria. OsPDF1A,OsPDF1B Gibberellin regulates mitochondrial pyruvate dehydrogenase activity in rice 2006 Plant Cell Physiol National Institute of Agrobiological Sciences, Tsukuba, 305-8602 Japan. Pyruvate dehydrogenase kinase (PDK) is a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC) that plays a key role in intermediary metabolism. OsPDK1 was identified as a gibberellin-up-regulated gene using a cDNA microarray. The full-length cDNA for OsPDK1 was 1498 bp and encoded a predicted polypeptide of 363 amino acids. Genomic DNA analysis showed the presence of another isoform of PDK, OsPDK2, in rice. Reverse transcriptase-PCR analysis revealed differential expression of the two isoforms. OsPDK1 was expressed in leaf blade and leaf sheath but not in callus and root, while OsPDK2 was expressed constitutively in all tissues examined. Maximum expression of OsPDK1 in leaf sheath was detected by Northern blot analysis when seedlings were treated with 5 microM GA3 for 24 h. OsPDK1 expression was up-regulated by GA3, and there was little effect of other plant hormones. Mitochondrial pyruvate dehydrogenase (PDH) activity was reduced compared with control plants in 2-week-old seedlings treated with GA3. The beta-glucuronidase (GUS) reporter gene, driven by a 2,067 bp OsPDK1 promoter region fragment, was mainly expressed in the aleurone layer of germinating seed and leaf sheath. Transgenic rice expressing PDK1 RNAi had altered vegetative growth with reduced accumulation of vegetative tissues. These results suggest that gibberellin modulates the activity of mtPDC by regulating OsPDK1 expression and subsequently controlling plant growth and development. OsPDK1 Ospdr9, which encodes a PDR-type ABC transporter, is induced by heavy metals, hypoxic stress and redox perturbations in rice roots 2003 FEBS Letters Department of Biology, Faculty of Science, University of Ottawa, 30 Marie Curie, Ottawa, ON K1N 6N5, Canada. Little is known about the role of pleiotropic drug resistance (PDR)-type ATP-binding (ABC) proteins in plant responses to environmental stresses. We characterised ospdr9, which encodes a rice ABC protein with a reverse (ABC-TMS(6))(2) configuration. Polyethylene glycol and the heavy metals Cd (20 microM) and Zn (30 microM) rapidly and markedly induced ospdr9 in roots of rice seedlings. Hypoxic stress also induced ospdr9 in rice roots, salt stress induced ospdr9 at low levels but cold and heat shock had no effect. The plant growth regulator jasmonic acid, the auxin alpha-naphthalene acetic acid and the cytokinin 6-benzylaminopurine triggered ospdr9 expression. The antioxidants dithiothreitol and ascorbic acid rapidly and markedly induced ospdr9 in rice roots; the strong oxidant hydrogen peroxide also induced ospdr9 but at three times lower levels. The results suggested that redox changes may be involved in the abiotic stress response regulation of ospdr9 in rice roots. Ospdr9 The polyembryo gene (OsPE) in rice 2010 Funct Integr Genomics Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247667, Uttarakhand, India. T-DNA insertional mutagenesis is one of the most important approaches for gene discovery and cloning. A fertile polyembryo mutant generated by T-DNA/Ds insertion in Oryza sativa, cv. Basmati 370 showed twin or triple seedlings at a frequency of 15-20%. T-DNA insertion was confirmed by 950 bp hpt gene amplification in the promoter region of the candidate gene. The annotated protein corresponding to the OsPE candidate gene has been reported as a hypothetical protein in O. sativa. OsPE gene lacked functional homologs in other species. No OsPE paralog was found in rice. No conserved domains were found in the protein coded by OsPE. RT-PCR showed the expression of OsPE gene in Basmati 370 shoots. Full-length OsPE gene was cloned in Basmati 370. The combined use of Southern blot, genome walking, TAIL-PCR, RT-PCR techniques, and bioinformatics led to the identification of a candidate gene controlling the multiple embryos in rice. There is gain of function, i.e., multiple embryos in the seeds in the knockout mutant OsPE whereas its wild-type allele strictly controls single embryo per seed. The seeds with multiple embryos are distributed at random in the rice mutant panicle. The origin of multiple embryos, whether apomictic, zygotic or both is under investigation. OsPE Development of multiple embryos in polyembryonic insertional mutant OsPE of rice 2012 Plant Cell Rep Department of Biotechnology, Indian Institute of Technology, Roorkee, 247 667, Uttaranchal, India. priyaincal@gmail.com A T-DNA insertional mutant OsPE of rice gives twin and triplet seedlings in up to 20 % of the seeds. Detailed cytological and histological analysis of OsPE indicated normal male and female gametogenesis in the OsPE mutant. Confocal laser scanning microscopic (CLSM) analysis of the developing seeds of OsPE showed multiple embryo development in up to 60 % of the ovules. The multiple embryos, mostly twins and triplets, and rarely quadruplets, developed through sequential cleavage from a single zygotic embryo in each ovule. The reduced number of multiple seedlings compared with multiple embryos observed in CLSM study may be attributed to their inability to develop further due to competition in a single embryo sac. Key message Multiple seedlings in the OsPE mutant are due to sequential proliferation and cleavage of the zygotic embryos. The nucellar tissue was not involved in multiple embryo development. OsPE Cloning of two splice variants of the rice PTS1 receptor, OsPex5pL and OsPex5pS, and their functional characterization using pex5-deficient yeast and Arabidopsis 2006 Plant J Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Korea. Using the rice PEX14 cDNA as a bait in a yeast two-hybrid assay, two splice variants of the type I peroxisomal targeting signal (PTS1) receptor, OsPex5pL and OsPex5pS, were cloned from a pathogen-treated rice leaf cDNA library. The proteins were produced from a single gene by alternative splicing, which generated a full-length variant, OsPEX5L, and a variant that lacked exon 7, OsPEX5S. OsPex5pL contained 11 copies of the pentapeptide motif WXXXF/Y in its N-terminus, and seven tetratricopeptide repeats in its C-terminus. Expression of OsPEX5L and OsPEX5S predominantly occurred in leaf tissues, and was induced by various stresses, such as exposure to the pathogen Magnaporthe grisea, and treatment with fungal elicitor, methyl viologen, NaCl or hydrogen peroxide. The Arabidopsis T-DNA insertional pex5 mutant, Atpex5, which does not germinate in the absence of sucrose and was resistant to indole-3-butyric acid (IBA), was perfectly rescued by over-expression of OsPex5pL, but not by OsPex5pS. Using transient expression of OsPex5pL and OsPex5pS in the Atpex5 mutant, we show that OsPex5pL translocates both PTS1- and PTS2-containing proteins into the peroxisome by interacting with OsPex7p, whereas OsPex5pS is involved only in PTS1-dependent import in Arabidopsis. OsPEX5 OsPFA-DSP1, a rice protein tyrosine phosphatase, negatively regulates drought stress responses in transgenic tobacco and rice plants 2012 Plant Cell Rep Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, 510275 Guangzhou, People's Republic of China. Dephosphorylation plays a pivotal role in regulating plant growth, development and abiotic/biotic stress responses. Here, we characterized a plant and fungi atypical dual-specificity phosphatase (PFA-DSP) subfamily member, OsPFA-DSP1, from rice. OsPFA-DSP1 was determined to be a functional protein tyrosine phosphatase (PTP) in vitro using phosphatase activity assays. Quantitative real-time PCR and GENEVESTIGATOR analysis showed that OsPFA-DSP1 mRNA was induced by drought stress. Transfection of rice protoplasts showed that OsPFA-DSP1 accumulated in both the cytoplasm and nucleus. Ectopic overexpression of OsPFA-DSP1 in tobacco increased sensitivity to drought stress and insensitivity to ABA-induced stomatal closure and inhibition of stomatal opening. Furthermore, overexpression of OsPFA-DSP1 in rice also increased sensitivity to drought stress. These results indicated that OsPFA-DSP1 is a functional PTP and may act as a negative regulator in drought stress responses. OsPFA-DSP1 Two homologous putative protein tyrosine phosphatases, OsPFA-DSP2 and AtPFA-DSP4, negatively regulate the pathogen response in transgenic plants 2012 PLoS One Key Laboratory of Gene Engineering of Ministry of Education, State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, Guangzhou, People's Republic of China. Protein phosphatases, together with protein kinases, regulate protein phosphorylation and dephosphorylation, and play critical roles in plant growth and biotic stress responses. However, little is known about the biological functions of plant protein tyrosine dual-specificity phosphatase (PFA-DSP) in biotic stresses. Here, we found that OsPFA-DSP2 was mainly expressed in calli, seedlings, roots, and young panicles, and localized in cytoplasm and nucleus. Ectopic overexpression of OsPFA-DSP2 in rice increased sensitivity to Magnaporthe grisea (M. grisea Z1 strain), inhibited the accumulation of hydrogen peroxide (H(2)O(2)) and suppressed the expression of pathogenesis-related (PR) genes after fungal infection. Interestingly, transgenic Arabidopsis plants overexpressing AtPFA-DSP4, which is homologous to OsPFA-DSP2, also exhibited sensitivity to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), reduced accumulation of H(2)O(2) and decreased photosynthesic capacity after infection compared with Col-0. These results indicate that OsPFA-DSP2 and AtPFA-DSP4 act as negative regulators of the pathogen response in transgenic plants. OsPFA-DSP2,OsPR5|Pir2|PR-5|PR5-1 Candidacy of a chitin-inducible gibberellin-responsive gene for a major locus affecting plant height in rice that is closely linked to Green Revolution gene sd1 2011 Theor Appl Genet National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, 430070, China. Appropriate plant height is crucial for lodging resistance to improve the rice crop yield. The application of semi-dwarf 1 led to the green revolution in the 1960s, by predominantly increasing the rice yield. However, the frequent use of single sd1 gene sources may cause genetic vulnerability to pests and diseases. Identifying useful novel semi-dwarf genes is important for the genetic manipulation of plant architecture in practical rice breeding. In this study, introgression lines derived from two parents contrasting in plant height, Zhenshan 97 and Pokkali were employed to locate a gene with a large effect on plant height by the bulk segregant analysis method. A major gene, ph1, was mapped to a region closely linked to sd1 on chromosome 1; the additive effects of ph1 were more than 50 cm on the plant height and 2 days on the heading date in a BC(4)F(2) population and its progeny. ph1 was then fine mapped to BAC AP003227. Gene annotation indicated that LOC_OS01g65990 encoding a chitin-inducible gibberellin-responsive protein (CIGR), which belongs to the GRAS family, might be the right candidate gene of ph1. Co-segregation analysis of the candidate gene-derived marker finally confirmed its identity as the candidate gene. A higher expression level of the CIGR was detected in all the tested tissues in tall plants compared to those of short plants, especially in the young leaf sheath containing elongating tissues, which indicated its importance role in regulating plant height. ph1 showed a tremendous genetic effect on plant height, which is distinct from sd1 and could be a new resource for breeding semi-dwarf varieties. OsPH1 OsPHF1 regulates the plasma membrane localization of low- and high-affinity inorganic phosphate transporters and determines inorganic phosphate uptake and translocation in rice 2011 Plant Physiol State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, People's Republic of China. PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (PHF1) is known to regulate the plasma membrane localization of PHT1;1, a high-affinity inorganic phosphate (Pi) transporter in Arabidopsis (Arabidopsis thaliana). OsPHF1, a rice (Oryza sativa) gene homologous to AtPHF1, was isolated and found to regulate the localization of both low- and high-affinity Pi transporters to the plasma membrane. Three OsPHF1 allelic mutants carrying one-point mutations at the fifth WD-repeat motif and two at the transmembrane helix, respectively, showed arsenate resistance and severely reduced Pi accumulation. The data indicate that mutation of OsPHF1 results in the endoplasmic reticulum retention of the low-affinity Pi transporter OsPT2 and high-affinity Pi transporter OsPT8. Mutation of OsPHF1 also reduced Pi accumulation in plants exhibiting excessive shoot Pi accumulation due to the overexpression of OsPHR2. However, the transcript level of OsPHF1 itself is not controlled by OsPHR2. Overexpression of OsPHF1 increased Pi accumulation in both roots and shoots in a solution culture with Pi-supplied condition. These results indicate that the role of OsPHF1 is unique in the localization of both low- and high-affinity Pi transporters on the plasma membrane in rice and determines Pi uptake and translocation in rice. The similar function of PHF1 required to facilitate PHT1 transit through the endoplasmic reticulum between Arabidopsis and rice provides an example of expectations from what one would deduce from sequence comparisons to extend knowledge from Arabidopsis to crops. OsPHF1,OsPHR2,OsPht1;2|OsPT2,OsPht1;8|OsPT8,OsSQD2,OsPHF1L Investigating the contribution of the phosphate transport pathway to arsenic accumulation in rice 2011 Plant Physiol State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, China. Arsenic (As) accumulation in rice (Oryza sativa) may pose a significant health risk to consumers. Plants take up different As species using various pathways. Here, we investigated the contribution of the phosphate (Pi) transport pathway to As accumulation in rice grown hydroponically or under flooded soil conditions. In hydroponic experiments, a rice mutant defective in OsPHF1 (for phosphate transporter traffic facilitator1) lost much of the ability to take up Pi and arsenate and to transport them from roots to shoots, whereas transgenic rice overexpressing either the Pi transporter OsPht1;8 (OsPT8) or the transcription factor OsPHR2 (for phosphate starvation response2) had enhanced abilities of Pi and arsenate uptake and translocation. OsPT8 was found to have a high affinity for both Pi and arsenate, and its overexpression increased the maximum influx by 3- to 5-fold. In arsenate-treated plants, both arsenate and arsenite were detected in the xylem sap, with the proportion of the latter increasing with the exposure time. Under the flooded soil conditions, the phf1 mutant took up less Pi whereas the overexpression lines took up more Pi. But there were no similar effects on As accumulation and distribution. Rice grain contained predominantly dimethylarsinic acid and arsenite, with arsenate being a minor species. These results suggest that the Pi transport pathway contributed little to As uptake and transport to grain in rice plants grown in flooded soil. Transgenic approaches to enhance Pi acquisition from paddy soil through the overexpression of Pi transporters may not increase As accumulation in rice grain. OsPHF1,OsPHR2,OsPht1;8|OsPT8 Effects of signaling molecules, protein phosphatase inhibitors and blast pathogen (Magnaporthe grisea) on the mRNA level of a rice (Oryza sativa L.) phospholipid hydroperoxide glutathione peroxidase (OsPHGPX) gene in seedling leaves 2002 Gene Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), GPO 8207, Kathmandu, Nepal. None OsPHGPX A novel AP2-type transcription factor, SMALL ORGAN SIZE1, controls organ size downstream of an auxin signaling pathway 2014 Plant Cell Physiol Bioscience and Biotechnology Center, Nagoya University, Nagoya, 464-8601 Japan. The organ size of flowering plants is determined by two post-embryonic developmental events: cell proliferation and cell expansion. In this study, we identified a new rice loss-of-function mutant, small organ size1 (smos1), that decreases the final size of various organs due to decreased cell size and abnormal microtubule orientation. SMOS1 encodes an unusual APETALA2 (AP2)-type transcription factor with an imperfect AP2 domain, and its product belongs to the basal AINTEGUMENTA (ANT) lineage, including WRINKLED1 (WRI1) and ADAP. SMOS1 expression was induced by exogenous auxin treatment, and the auxin response element (AuxRE) of the SMOS1 promoter acts as a cis-motif through interaction with auxin response factor (ARF). Furthermore, a functional fluorophore-tagged SMOS1 was localized to the nucleus, supporting the role of SMOS1 as a transcriptional regulator for organ size control. Microarray analysis showed that the smos1 mutation represses expression of several genes involved in microtubule-based movement and DNA replication. Among the down-regulated genes, we demonstrated by gel-shift and chromatin immunoprecipitation (ChIP) experiments that OsPHI-1, which is involved in cell expansion, is a target of SMOS1. SMOS1 homologs in early-diverged land plants partially rescued the smos1 phenotype of rice. We propose that SMOS1 acts as an auxin-dependent regulator for cell expansion during organ size control, and that its function is conserved among land plants. OsPHI-1,SMOS1 Characterization of the rice PHO1 gene family reveals a key role for OsPHO1;2 in phosphate homeostasis and the evolution of a distinct clade in dicotyledons 2010 Plant Physiol Departement de Biologie Moleculaire Vegetale, Biophore, Universite de Lausanne, CH-1015 Lausanne, Switzerland. Phosphate homeostasis was studied in a monocotyledonous model plant through the characterization of the PHO1 gene family in rice (Oryza sativa). Bioinformatics and phylogenetic analysis showed that the rice genome has three PHO1 homologs, which cluster with the Arabidopsis (Arabidopsis thaliana) AtPHO1 and AtPHO1;H1, the only two genes known to be involved in root-to-shoot transfer of phosphate. In contrast to the Arabidopsis PHO1 gene family, all three rice PHO1 genes have a cis-natural antisense transcript located at the 5 ' end of the genes. Strand-specific quantitative reverse transcription-PCR analyses revealed distinct patterns of expression for sense and antisense transcripts for all three genes, both at the level of tissue expression and in response to nutrient stress. The most abundantly expressed gene was OsPHO1;2 in the roots, for both sense and antisense transcripts. However, while the OsPHO1;2 sense transcript was relatively stable under various nutrient deficiencies, the antisense transcript was highly induced by inorganic phosphate (Pi) deficiency. Characterization of Ospho1;1 and Ospho1;2 insertion mutants revealed that only Ospho1;2 mutants had defects in Pi homeostasis, namely strong reduction in Pi transfer from root to shoot, which was accompanied by low-shoot and high-root Pi. Our data identify OsPHO1;2 as playing a key role in the transfer of Pi from roots to shoots in rice, and indicate that this gene could be regulated by its cis-natural antisense transcripts. Furthermore, phylogenetic analysis of PHO1 homologs in monocotyledons and dicotyledons revealed the emergence of a distinct clade of PHO1 genes in dicotyledons, which include members having roles other than long-distance Pi transport. OsPHO1;2 Fine characterization of OsPHO2 knockout mutants reveals its key role in Pi utilization in rice 2014 J Plant Physiol State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China. Previous research using forward genetics approaches demonstrated that OsPHO2 regulates multiple phosphate-starvation responses in rice. In this work, we finely characterized two independent OsPHO2 knockout rice mutants under inorganic phosphate (Pi)-sufficient conditions. The ospho2 mutants exhibited defects in growth and reproductive development in the whole growing period. The cells in the elongation zone of ospho2 seedling roots were much shorter than those of the wild type. The phosphorus concentration in the blades of ospho2 mutants was 5.8-fold higher than those of wild-type plants, whereas it was only slightly higher in the sheaths, culms, spikelets, and seeds. Furthermore, Pi levels in the ospho2 mutants were highest in the oldest leaf and lowest in the youngest leaf, whereas there was no significant difference in the corresponding leaves of wild-type plants. These results suggest that ospho2 mutant phenotype results from a partial defect in Pi translocation and remobilization in the shoot of rice. This study thus provides evidence that OsPHO2, which functions at the downstream of OsPHF1, modulates Pi utilization by regulating the expression of Pht1 transporters in rice. LTN1|OsPHO2 Molecular mechanisms regulating Pi-signaling and Pi homeostasis under OsPHR2, a central Pi-signaling regulator, in rice 2011 Frontiers in Biology State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, 310058, China Phosphorus (P) is one of the most important major mineral elements for plant growth and metabolism. Plants have evolved adaptive regulatory mechanisms to maintain phosphate (Pi) homeostasis by improving phosphorus uptake, translocation, remobilization and efficiency of use. Here we review recent advances in our understanding of the OsPHR2-mediated phosphate-signaling pathway in rice. OsPHR2 positively regulates the low-affinity Pi transporter OsPT2 through physical interaction and reciprocal regulation of OsPHO2 in roots. OsPT2 is responsible for most of the OsPHR2-mediated accumulation of excess Pi in shoots. OsSPX1 acts as a repressor in the OsPHR2-mediated phosphate-signaling pathway. Some mutants screened from ethyl methanesulfonate (EMS)-mutagenized M2 population of OsPHR2 overexpression transgenic line removed the growth inhibition, indicating that some unknown factors are crucial for Pi utilization or plant growth under the regulation of OsPHR2. LTN1|OsPHO2,OsPHR2,OsPht1;2|OsPT2,OsSPX1 OsSPX1 suppresses the function of OsPHR2 in the regulation of expression of OsPT2 and phosphate homeostasis in shoots of rice 2010 Plant J State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, China. Phosphate (Pi) homeostasis in plants is required for plant growth and development, and is achieved by the coordination of Pi acquisition, translocation from roots to shoots, and remobilization within plants. Previous reports have demonstrated that over-expression of OsPHR2 (the homolog of AtPHR1) and knockdown of OsSPX1 result in accumulation of excessive shoot Pi in rice. Here we report that OsPHR2 positively regulates the low-affinity Pi transporter gene OsPT2 by physical interaction and upstream regulation of OsPHO2 in roots. OsPT2 is responsible for most of the OsPHR2-mediated accumulation of excess shoot Pi. OsSPX1 suppresses the regulation on expression of OsPT2 by OsPHR2 and the accumulation of excess shoot Pi, but it does not suppress induction of OsPT2 or the accumulation of excessive shoot Pi in the Ospho2 mutant. Our data also show that OsSPX1 is a negative regulator of OsPHR2 and is involved in the feedback of Pi-signaling network in roots that is defined by OsPHR2 and OsPHO2. This finding provides new insight into the regulatory mechanism of Pi uptake, translocation, allocation and homeostasis in plants. LTN1|OsPHO2,OsPHR2,OsPht1;2|OsPT2,OsSPX1 Involvement of OsSPX1 in phosphate homeostasis in rice 2009 Plant J State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, China. Arabidopsis thaliana SPX (SYG/PHO81/XPR1) domain genes have recently been shown to be involved in the phosphate (Pi) signaling pathway. We show here that a rice (Oryza sativa) SPX gene, OsSPX1, is specifically induced by Pi starvation in roots. Suppression of OsSPX1 by RNA interference resulted in severe signs of toxicity caused by the over-accumulation of Pi, similar to that found in OsPHR2 (phosphate starvation response transcription factor 2) overexpressors and pho2 (phosphate-responsive mutant 2). Quantitative RT-PCR showed that expression of OsSPX1 was strongly induced in OsPHR2 overexpression and pho2 mutant plants, indicating that OsSPX1 occurs downstream of OsPHR2 and PHO2. The expression of 10 genes associated with the phosphate-starvation signal pathways was analyzed. Expression of OsPT2 (phosphate transporter 2) and OsPT8 was significantly induced in OsSPX1-RNAi (OsSPX1-Ri) plants, suggesting that over-accumulation of Pi in OsSPX1-Ri plants results from an increase in Pi transport. In contrast, overexpression of OsSPX1 suppressed the induction of expression by Pi starvation of all 10 phosphate starvation-induced genes tested: IPS1 (induced by phosphate starvation 1), IPS2, OsPAP10 (purple acid phosphatase 10), OsSQD2 (sulfoquinovosyldiacylglycerol 2), miR399d and miR399j (microRNA 399), OsPT2, OsPT3, OsPT6 and OsPT8. This suggests that OsSPX1 acts via a negative feedback loop to optimize growth under phosphate-limited conditions. LTN1|OsPHO2,OsPHR2,OsPht1;2|OsPT2,OsPht1;3|OsPT3,OsLPT1|OsPht1;6|OsPT6,OsPht1;8|OsPT8,OsSPX1,OsSQD2,Pi1-5,Pi1-6 Regulation of OsSPX1 and OsSPX3 on expression of OsSPX domain genes and Pi-starvation signaling in rice 2009 J Integr Plant Biol State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China. The rice (Oryza sativa L.) genome contains at least six genes exclusively with an SPX (SYG1/PHO81/XPR1) domain at the N-terminal, designated as OsSPX1-6. Here we report the diverse expression patterns of the OsSPX genes in different tissues and their responses to Pi-starvation. Among them, five genes, OsSPX1, 2, 3, 5 and 6 are responsive to Pi-starvation in shoots and/or in roots. The subcellular localization analysis indicates that OsSPX1 and OsSPX2 is exclusively located in nucleus, OsSPX3 in the cytoplasm, and OsSPX4 is a membrane localization protein. OsSPX1 regulates OsSPX2, 3 and 5 at the transcription level and is positively involved in the responses of the genes to Pi-starvation. Overexpression of OsSPX3 downregulates OsSPX5 in shoots under Pi-sufficiency. OsSPX3 negatively regulates the PSI (Pi-starvation induced) gene, OsIPS1 and is involved in the responses of miR399 and OsPHO2 to Pi-starvation. Our results suggest that OsSPX1 may be a regulator involved in the transcriptions of OsSPX2, 3 and 5. OsSPX3 plays a role in OsIPS1/miR399 mediated long distance regulation on OsPHO2. Our results also indicate that OsSPX3 is involved in plant tolerance to Pi-starvation stress. LTN1|OsPHO2,OsSPX1,OsSPX3,OsSPX6 Down-regulation of OsSPX1 causes high sensitivity to cold and oxidative stresses in rice seedlings 2013 PLoS One State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China. Rice SPX domain gene, OsSPX1, plays an important role in the phosphate (Pi) signaling network. Our previous work showed that constitutive overexpression of OsSPX1 in tobacco and Arabidopsis plants improved cold tolerance while also decreasing total leaf Pi. In the present study, we generated rice antisense and sense transgenic lines of OsSPX1 and found that down-regulation of OsSPX1 caused high sensitivity to cold and oxidative stresses in rice seedlings. Compared to wild-type and OsSPX1-sense transgenic lines, more hydrogen peroxide accumulated in seedling leaves of OsSPX1-antisense transgenic lines for controls, cold and methyl viologen (MV) treatments. Glutathione as a ROS scavenger could protect the antisense transgenic lines from cold and MV stress. Rice whole genome GeneChip analysis showed that some oxidative-stress marker genes (e.g. glutathione S-transferase and P450s) and Pi-signaling pathway related genes (e.g. OsPHO2) were significantly down-regulated by the antisense of OsSPX1. The microarray results were validated by real-time RT-PCR. Our study indicated that OsSPX1 may be involved in cross-talks between oxidative stress, cold stress and phosphate homeostasis in rice seedling leaves. LTN1|OsPHO2,OsSPX1 LEAF TIP NECROSIS1 plays a pivotal role in the regulation of multiple phosphate starvation responses in rice 2011 Plant Physiol State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Although phosphate (Pi) starvation signaling is well studied in Arabidopsis (Arabidopsis thaliana), it is still largely unknown in rice (Oryza sativa). In this work, a rice leaf tip necrosis1 (ltn1) mutant was identified and characterized. Map-based cloning identified LTN1 as LOC_Os05g48390, the putative ortholog of Arabidopsis PHO2, which plays important roles in Pi starvation signaling. Analysis of transgenic plants harboring a LTN1 promoter::beta-glucuronidase construct revealed that LTN1 was preferentially expressed in vascular tissues. The ltn1 mutant exhibited increased Pi uptake and translocation, which led to Pi overaccumulation in shoots. In association with enhanced Pi uptake and transport, some Pi transporters were up-regulated in the ltn1 mutant in the presence of sufficient Pi. Furthermore, the elongation of primary and adventitious roots was enhanced in the ltn1 mutant under Pi starvation, suggesting that LTN1 is involved in Pi-dependent root architecture alteration. Under Pi-sufficient conditions, typical Pi starvation responses such as stimulation of phosphatase and RNase activities, lipid composition alteration, nitrogen assimilation repression, and increased metal uptake were also activated in ltn1. Moreover, analysis of OsmiR399-overexpressing plants showed that LTN1 was down-regulated by OsmiR399. Our results strongly indicate that LTN1 is a crucial Pi starvation signaling component downstream of miR399 involved in the regulation of multiple Pi starvation responses in rice. LTN1|OsPHO2 A constitutive expressed phosphate transporter, OsPht1;1, modulates phosphate uptake and translocation in phosphate-replete rice 2012 Plant Physiol State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China. A number of phosphate (Pi) starvation- or mycorrhiza-regulated Pi transporters belonging to the Pht1 family have been functionally characterized in several plant species, whereas functions of the Pi transporters that are not regulated by changes in Pi supply are lacking. In this study, we show that rice (Oryza sativa) Pht1;1 (OsPT1), one of the 13 Pht1 Pi transporters in rice, was expressed abundantly and constitutively in various cell types of both roots and shoots. OsPT1 was able to complement the proton-coupled Pi transporter activities in a yeast mutant defective in Pi uptake. Transgenic plants of OsPT1 overexpression lines and RNA interference knockdown lines contained significantly higher and lower phosphorus concentrations, respectively, compared with the wild-type control in Pi-sufficient shoots. These responses of the transgenic plants to Pi supply were further confirmed by the changes in depolarization of root cell membrane potential, root hair occurrence, (33)P uptake rate and transportation, as well as phosphorus accumulation in young leaves at Pi-sufficient levels. Furthermore, OsPT1 expression was strongly enhanced by the mutation of Phosphate Overaccumulator2 (OsPHO2) but not by Phosphate Starvation Response2, indicating that OsPT1 is involved in the OsPHO2-regulated Pi pathway. These results indicate that OsPT1 is a key member of the Pht1 family involved in Pi uptake and translocation in rice under Pi-replete conditions. LTN1|OsPHO2,OsPht1;1|OsPT1 OsPHR2 is involved in phosphate-starvation signaling and excessive phosphate accumulation in shoots of plants 2008 Plant Physiol State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, China. Previous research has demonstrated that AtPHR1 plays a central role in phosphate (Pi)-starvation signaling in Arabidopsis thaliana. In this work, two OsPHR genes from rice (Oryza sativa) were isolated and designated as OsPHR1 and OsPHR2 based on amino acid sequence homology to AtPHR1. Their functions in Pi signaling in rice were investigated using transgenic plants. Our results showed that both OsPHR1 and OsPHR2 are involved in Pi-starvation signaling pathway by regulation of the expression of Pi-starvation-induced genes, whereas only OsPHR2 overexpression results in the excessive accumulation of Pi in shoots under Pi-sufficient conditions. Under Pi-sufficient conditions, overexpression of OsPHR2 mimics Pi-starvation stress in rice with enhanced root elongation and proliferated root hair growth, suggesting the involvement of OsPHR2 in Pi-dependent root architecture alteration by both systematic and local pathways. In OsPHR2-overexpression plants, some Pi transporters were up-regulated under Pi-sufficient conditions, which correlates with the strongly increased content of Pi. The mechanism behind the OsPHR2 regulated Pi accumulation will provide useful approaches to develop smart plants with high Pi efficiency. OsPHR1,OsPHR2 Role of OsPHR2 on phosphorus homeostasis and root hairs development in rice (Oryza sativa L.) 2008 Plant Signal Behav State Key Laboratory of Plant Physiology and Biochemistry; College of Life Science; Zhejiang University; Hangzhou, P. R. China. AtPHR1 plays a central role in Pi-starvation signaling in Arabidopsis (Arabidopsis thaliana). Two PHR genes were isolated from rice and designated as OsPHR1 and OsPHR2 based on amino acid sequence homology to AtPHR1. Transgenic plants with overexpression and repression of OsPHR1 and OsPHR2, respectively, were used for investigation of roles of the genes in Pi-signaling pathway and Pi homeostasis under Pi-sufficient and deficient conditions. The results showed that both of the genes are involved in the Pi-signaling pathway, while overexpression of OsPHR2 mimics Pi-starvation stress with enhanced root elongation and proliferated root hairs, and results in the excessive accumulation of Pi in shoots under Pi-sufficient conditions. OsPHR2 regulated proliferation of root hair growth and root elongation suggests that OsPHR2 is involved in both systematic and local Pi-signaling pathways. OsPHR1,OsPHR2 Novel Phr1 mutations and the evolution of phenol reaction variation in US weedy rice (Oryza sativa) 2009 New Phytol Department of Biology, Washington University, St Louis, MO 63130-4899, USA. *Red rice, a major agricultural weed, is phenotypically diverse and possesses traits that are similar to both wild and cultivated rice. The genetic resources available for rice make it possible to examine the molecular basis and evolution of traits characterizing this weed. Here, we assess the phenol reaction - a classical trait for distinguishing among cultivated rice varieties - in red rice at the phenotypic and molecular levels. *We phenotyped more than 100 US weed samples for the phenol reaction and sequenced the underlying Phr1 locus in a subset of samples. Data were analyzed in combination with previously published Phr1 data for cultivated rice. *Most weed accessions (96.3%) are positive for the phenol reaction, and samples with a negative response carry loss-of-function alleles that are rare or heretofore undocumented. One such allele may have evolved through mutational convergence of a 1-bp frameshift insertion. Haplotype sharing between red rice and US cultivars suggests occasional crop-weed hybridization. *Our discovery of previously undocumented nonfunctional phr1 alleles suggests that there are likely to be other loss-of-function mutations segregating in Oryza sativa around the world. Red rice may provide a useful study system for understanding the adaptive significance of Phr1 variation in agricultural settings. OsPHR1 Independent losses of function in a polyphenol oxidase in rice: differentiation in grain discoloration between subspecies and the role of positive selection under domestication 2008 Plant Cell State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Asian rice (Oryza sativa) cultivars originated from wild rice and can be divided into two subspecies by several criteria, one of which is the phenol reaction (PHR) phenotype. Grains of indica cultivars turn brown in a phenol solution that accelerates a similar process that occurs during prolonged storage. By contrast, the grains of japonica do not discolor. This distinction may reflect the divergent domestication of these two subspecies. The PHR is controlled by a single gene, Phr1; here, we report the cloning of Phr1, which encodes a polyphenol oxidase. The Phr1 gene is indeed responsible for the PHR phenotype, as transformation with a functional Phr1 can complement a PHR negative cultivar. Phr1 is defective in all japonica lines but functional in nearly all indica and wild strains. Phylogenetic analysis showed that the defects in Phr1 arose independently three times. The multiple recent origins and rapid spread of phr1 in japonica suggest the action of positive selection, which is further supported by several population genetic tests. This case may hence represent an example of artificial selection driving the differentiation among domesticated varieties. OsPHR1 Rice SPX1 and SPX2 inhibit phosphate starvation responses through interacting with PHR2 in a phosphate-dependent manner 2014 Proc Natl Acad Sci U S A State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China In plants, sensing the levels of external and internal nutrients is essential for reprogramming the transcriptome and adapting to the fluctuating environment. Phosphate (Pi) is a key plant nutrient, and a large proportion of Pi starvation-responsive genes are under the control of PHOSPHATE STARVATION RESPONSE REGULATOR 1 (PHR1) in Arabidopsis (AtPHR1) and its homologs, such as Oryza sativa (Os)PHR2 in rice. AtPHR1 and OsPHR2 expression is not very responsive to Pi starvation, raising the question as to how plants sense changes in cellular Pi levels to activate the central regulator. SPX [named after SYG1 (suppressor of yeast gpa1), Pho81 (CDK inhibitor in yeast PHO pathway), and XPR1 (xenotropic and polytropic retrovirus receptor)] proteins that harbor only the SPX domain are reported to be involved in the negative regulation of Pi starvation responses. Here, we show that the nuclear localized SPX proteins SPX1 and SPX2 are Pi-dependent inhibitors of the activity of OsPHR2 in rice. Indeed, SPX1 and SPX2 proteins interact with PHR2 through their SPX domain, inhibiting its binding to P1BS (the PHR1-binding sequence: GNATATNC). In vivo data, as well as results from in vitro experiments using purified SPX1, SPX2, and OsPHR2 proteins, showed that SPX1 and SPX2 inhibition of OsPHR2 activity is Pi-dependent. These data provide evidence to support the involvement of SPX1 and SPX2 in the Pi-sensing mechanism in plants. OsPHR2,OsSPX1,OsSPX2 Rice phosphate transporters include an evolutionarily divergent gene specifically activated in arbuscular mycorrhizal symbiosis 2002 Proc Natl Acad Sci U S A Torrey Mesa Research Institute, San Diego, CA 92121, USA. uta.paszkowski@syngenta.com Using a genome-wide approach, we asked how many transporter genes contribute to symbiotic phosphate uptake and analyzed their evolutionary conservation. Considering the sequenced rice genome at hand, only the Oryza sativa phosphate transporter (OsPT) gene OsPT11 was specifically induced during the arbuscular mycorrhizal symbiosis. This induction was confined to the root system and was tightly correlated with the degree of root colonization by Glomus intraradices. OsPT11 activation was independent of the nutritional status of the plant and phosphate availability in the rhizosphere. Moreover, infection of roots with the fungal pathogens Rhizoctonia solani and Fusarium moniliforme did not activate OsPT11, corroborating the high signal specificity for OsPT11 activation in the arbuscular mycorrhizal symbiosis. OsPT11 expression complemented a defect in phosphate uptake in a yeast strain mutated in its high-affinity P(i) transporter (pho84), thereby confirming its function. Recently, a phosphate transporter gene in potato was shown to be induced during arbuscular mycorrhizal symbiosis. Assessment of the phylogenetic relationship of the rice and potato protein revealed that the rice is nonorthologous to the potato protein. Further, there are no structural commonalities in the promoter regions. Thus, although cytological and physiological features of the arbuscular mycorrhizal symbiosis seem to be conserved, the molecular components may differ significantly between distantly related plant species. OsPht1;1|OsPT1,OsPht1;10|OsPT10,OsPht1;11|OsPT11,OsPht1;12|OsPT12,OsPht1;13|OsPT13,OsPht1;2|OsPT2,OsPht1;3|OsPT3,OsPht1;4|OsPT4,OsPht1;5|OsPT5,OsLPT1|OsPht1;6|OsPT6,OsPht1;7|OsPT7,OsPht1;8|OsPT8,OsPht1;9|OsPT9 Increased expression of OsPT1, a high-affinity phosphate transporter, enhances phosphate acquisition in rice 2008 Biotechnol Lett Department of Molecular Biotechnology, Dong-A University, Busan 604-714, Korea. Most high-affinity phosphate transporter genes (OsPTs) in rice were highly induced in roots when phosphate was depleted. OsPT1, however, was highly expressed in primary roots and leaves regardless of external phosphate concentrations. This finding was confirmed histochemically using transgenic rice plants that express the GUS reporter gene under the control of the OsPT1 promoter, which exhibited high GUS activity even in the phosphate sufficient condition. Furthermore, transgenic rice plants overexpressing the OsPT1 gene accumulated almost twice as much phosphate in the shoots as did wild-type plants. As a result, transgenic plants had more tillers than did wild-type plants, which is a typical physiological indicator for phosphate status in rice. OsPht1;1|OsPT1 Phosphate transporters OsPHT1;9 and OsPHT1;10 are involved in phosphate uptake in rice 2014 Plant Cell Environ State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou, 310058, China; The Key Laboratory for Quality Improvement of Agricultural Products of Zhejiang Province, College of Agriculture and Food Science, Zhejiang A & F University, Lin'an, 311300, China. We characterized the function of two rice phosphate (Pi) transporters: OsPHT1;9 (OsPT9) and OsPHT1;10 (OsPT10). OsPT9 and OsPT10 were expressed in the root epidermis, root hairs and lateral roots, with their expression being specifically induced by Pi starvation. In leaves, expression of the two genes was observed in both mesophyll and vasculature. High-affinity Km values for Pi transport of OsPT9 and OsPT10 were determined by yeast experiments and two-electrode voltage clamp analysis of anion transport in Xenopus oocytes expressing OsPT9 and OsPT10. Pi uptake and Pi concentrations in transgenic plants harbouring overexpressed OsPT9 and OsPT10 were determined by Pi concentration analysis and (33) P-labelled Pi uptake rate analysis. Significantly higher Pi uptake rates in transgenic plants compared with wild-type plants were observed under both high-Pi and low-Pi solution culture conditions. Conversely, although no alterations in Pi concentration were found in OsPT9 or OsPT10 knockdown plants, a significant reduction in Pi concentration in both shoots and roots was observed in double-knockdown plants grown under both high- and low-Pi conditions. Taken together, our results suggest that OsPT9 and OsPT10 redundantly function in Pi uptake. OsPht1;10|OsPT10,OsPht1;9|OsPT9 Nonredundant regulation of rice arbuscular mycorrhizal symbiosis by two members of the phosphate transporter1 gene family 2012 Plant Cell Department of Plant Molecular Biology, University of Lausane, CH-1015 Lausane, Switzerland. Pi acquisition of crops via arbuscular mycorrhizal (AM) symbiosis is becoming increasingly important due to limited high-grade rock Pi reserves and a demand for environmentally sustainable agriculture. Here, we show that 70% of the overall Pi acquired by rice (Oryza sativa) is delivered via the symbiotic route. To better understand this pathway, we combined genetic, molecular, and physiological approaches to determine the specific functions of two symbiosis-specific members of the PHOSPHATE TRANSPORTER1 (PHT1) gene family from rice, ORYsa;PHT1;11 (PT11) and ORYsa;PHT1;13 (PT13). The PT11 lineage of proteins from mono- and dicotyledons is most closely related to homologs from the ancient moss, indicating an early evolutionary origin. By contrast, PT13 arose in the Poaceae, suggesting that grasses acquired a particular strategy for the acquisition of symbiotic Pi. Surprisingly, mutations in either PT11 or PT13 affected the development of the symbiosis, demonstrating that both genes are important for AM symbiosis. For symbiotic Pi uptake, however, only PT11 is necessary and sufficient. Consequently, our results demonstrate that mycorrhizal rice depends on the AM symbiosis to satisfy its Pi demands, which is mediated by a single functional Pi transporter, PT11. OsPht1;11|OsPT11,OsPht1;13|OsPT13 Two rice phosphate transporters, OsPht1;2 and OsPht1;6, have different functions and kinetic properties in uptake and translocation 2009 Plant J State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, China. Plant phosphate (Pi) transporters mediate the uptake and translocation of this nutrient within plants. A total of 13 sequences in the rice (Oryza sativa) genome can be identified as belonging to the Pi transporter (Pht1) family. Here, we report on the expression patterns, biological properties and the physiological roles of two members of the family: OsPht1;2 (OsPT2) and OsPht1;6 (OsPT6). Expression of both genes increased significantly under Pi deprivation in roots and shoots. By using transgenic rice plants expressing the GUS reporter gene, driven by their promoters, we detected that OsPT2 was localized exclusively in the stele of primary and lateral roots, whereas OsPT6 was expressed in both epidermal and cortical cells of the younger primary and lateral roots. OsPT6, but not OsPT2, was able to complement a yeast Pi uptake mutant in the high-affinity concentration range. Xenopus oocytes injected with OsPT2 mRNA showed increased Pi accumulation and a Pi-elicited depolarization of the cell membrane electrical potential, when supplied with mM external concentrations. Both results show that OsPT2 mediated the uptake of Pi in oocytes. In transgenic rice, the knock-down of either OsPT2 or OsPT6 expression by RNA interference significantly decreased both the uptake and the long-distance transport of Pi from roots to shoots. Taken together, these data suggest OsPT6 plays a broad role in Pi uptake and translocation throughout the plant, whereas OsPT2 is a low-affinity Pi transporter, and functions in translocation of the stored Pi in the plant. OsPht1;2|OsPT2,OsLPT1|OsPht1;6|OsPT6 OsPT2, a phosphate transporter, is involved in the active uptake of selenite in rice 2014 New Phytol Henan University of Science and Technology, Luoyang, 471003, China. * Selenite is a predominant form of selenium (Se) available to plants, especially in anaerobic soils, but the molecular mechanism of selenite uptake by plants is not well understood. * ltn1, a rice mutant previously shown to have increased phosphate (Pi) uptake, was found to exhibit higher selenite uptake than the wild-type in both concentration- and time-dependent selenite uptake assays. Respiratory inhibitors significantly inhibited selenite uptake in the wildtype and the ltn1 mutant, indicating that selenite uptake was coupled with H(+) and energy-dependent. Selenite uptake was greatly enhanced under Pi-starvation conditions, suggesting that Pi transporters are involved in selenite uptake. * OsPT2, the most abundantly expressed Pi transporter in the roots, is also significantly up-regulated in ltn1 and dramatically induced by Pi starvation. OsPT2-overexpressing and knockdown plants displayed significantly increased and decreased rates of selenite uptake, respectively, suggesting that OsPT2 plays a crucial role in selenite uptake. Se content in rice grains also increased significantly in OsPT2-overexpressing plants. * These data strongly demonstrate that selenite and Pi share similar uptake mechanisms and that OsPT2 is involved in selenite uptake, which provides a potential strategy for breeding Se-enriched rice varieties. OsPht1;2|OsPT2 Cloning and characterization of cDNA for the Oryza sativa phosphate transporter 2005 Cell Mol Biol Lett Institute of Genetics, State Key Laboratory of Genetic Engineering, School of Life Science, Fudan University 220 Handan Road, Shanghai 200433, PR China. fming@fudan.edu.cn A putative high-affinity phosphate (Pi) transporter gene in rice (Oryza sativa), OsLPT1, was isolated by RT-PCR from the leaves of the plants. The 1635-bp nucleotide sequence of OsLPT1 spans an open reading frame encoding a polypeptide of 535 amino acids with sequence similarity to phosphate transporters from other plant species. Southern blot analysis showed that the OsLPT1 gene might be present in three transcripts in the rice genome. RT-PCR analysis demonstrated the expression of OsLPT1 in both leaves and roots. The expression of OsLPT1 in the roots was enhanced by Pi deprivation. In situ hybridization revealed OsLPT1 expression in mesophyll cells, xylem parenchyma and phloem cells in the leaves, and in the epidermis, exodermis, and in the vasculature surrounding metaxylem vessels in the roots. The data suggests that the OsLPT1 protein may be involved in enhancing phosphate uptake under conditions of Pi starvation, and in the translocation of Pi among cells in shoots to increase the efficiency of internal Pi use. OsLPT1|OsPht1;6|OsPT6 The phosphate transporter gene OsPht1;8 is involved in phosphate homeostasis in rice 2011 Plant Physiol State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, China. Plant phosphate transporters (PTs) are active in the uptake of inorganic phosphate (Pi) from the soil and its translocation within the plant. Here, we report on the biological properties and physiological roles of OsPht1;8 (OsPT8), one of the PTs belonging to the Pht1 family in rice (Oryza sativa). Expression of a beta-glucuronidase and green fluorescent protein reporter gene driven by the OsPT8 promoter showed that OsPT8 is expressed in various tissue organs from roots to seeds independent of Pi supply. OsPT8 was able to complement a yeast Pi-uptake mutant and increase Pi accumulation of Xenopus laevis oocytes when supplied with micromolar (33)Pi concentrations at their external solution, indicating that it has a high affinity for Pi transport. Overexpression of OsPT8 resulted in excessive Pi in both roots and shoots and Pi toxic symptoms under the high-Pi supply condition. In contrast, knockdown of OsPT8 by RNA interference decreased Pi uptake and plant growth under both high- and low-Pi conditions. Moreover, OsPT8 suppression resulted in an increase of phosphorus content in the panicle axis and in a decrease of phosphorus content in unfilled grain hulls, accompanied by lower seed-setting rate. Altogether, our data suggest that OsPT8 is involved in Pi homeostasis in rice and is critical for plant growth and development. OsPht1;8|OsPT8 Transcriptomic analysis indicates putative metabolic changes caused by manipulation of phosphorus availability in rice leaves 2006 J Exp Bot Creative Research Initiative Sousei (CRIS), Hokkaido University, Kita-ku, Sapporo, Japan. Plants have developed several strategies for coping with phosphorus (P) deficiency. However, the details of the regulation of gene expression of adaptations to low P are still unclear. Using a cDNA microarray, transcriptomic analyses were carried out of the rice genes regulated by P deficiency and P re-supply to P-deficient plants. The OsPI1 gene, which was isolated as the most significant up-regulated gene under -P conditions, was also the most significant down-regulated gene following P re-supply. Many starch metabolism-related genes, as well as several genes for P(i)-liberating enzymes, were up-regulated by -P treatment, suggesting a homeostatic contribution to the P(i) concentration in leaf tissues. mRNAs for glucanases were also induced by P re-supply: these are suspected to play a role in loosening the cell wall compounds. Most of the genes up-regulated by -P treatment were down-regulated by P re-supply, suggesting that their responses were specific to -P conditions. Conversely, the number of genes up-regulated by P re-supply was also larger following P re-supply than in the -P condition. It is proposed that the genes up-regulated by P re-supply play an important role in P acquisition by P-deficient plants. OsPI1 Expression of the OsPI1 gene, cloned from rice roots using cDNA microarray, rapidly responds to phosphorus status 2003 New Phytologist Graduate School of Agriculture, Hokkaido University, N9W9, Kita-ku, Sapporo, 060-8589, Japan; Plants have developed several methods of adapting to conditions of low phosphorus (P). However, details of the regulation of the gene expression system that responds to P status of plants is unknown. Here, a phosphorus limitation inducible novel gene was isolated and characterized to provide further information of plant adaptation to low P. Rice plants (Oryza sativa) were grown hydroponically with or without P. A novel gene was isolated by cDNA microarray analysis and designated as OsPI1 (Oryza sativaPhosphate-limitation Inducible Gene 1). mRNA accumulation was examined by Northern blot and quantitative real time PCR. The OsPI1 gene was rapidly induced by phosphate starvation in both shoots and roots. When phosphate was supplied to phosphate-deficient plants, the OsPI1 transcripts rapidly disappeared. OsPI1 cDNA consisted of 375 bp and contained several small open reading frames (ORFs). The OsPI1 gene shows the same characteristics as the TPSI1/Mt4 family (the phosphate starvation inducible novel gene family). It is suggested that OsPI1 acts as riboregulator, that is, it binds with other molecules under phosphate starvation and regulates their function. OsPI1 Isolation and functional characterization of the C-terminus of rice phosphatidylinositol 4-kinase in vitro 2003 Cell Res National Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Science, The Chinese Academy of Sciences, Shanghai, China. A partial rice (Oryza sativa L.) cDNA clone, OsPI4K1c, was isolated through screening of a cDNA library constructed from tillering materials. OsPI4K1c encoded a peptide of 608 amino acids with a calculated molecular mass of 68.4 kDa. The OsPI4K1c peptide shared high homology and possessed the highly conserved domains present in most isolated cloned PI4-kinases, i.e. a lipid kinase unique (LKU) domain and a catalytic (CAT) domain. A region with similarity to pleckstrin homology (PH) domain was present in OsPI4K1c as well. Further comparison with genomic sequences in databases revealed that OsPI4K1c is located at the 3'-end of a putative rice PI 4-kinase coding gene OsPI4K1, and its coding region corresponded to the C-terminal half of OsPI4K1 protein. Twelve exons (49-562 bp in size) and 11 introns (77-974 bp in size) were identified in OsPI4K1c. The recombinant protein expressed in Escherichia coli phosphorylates phosphatidylinositol at the D4 position of the inositol ring. OsPI4K1 transcript levels were detected in a low but constitutive manner in shoot, stem, leaf, spike and root tissues and did not change upon treatment with different hormones, calcium and jasmonic acid (JA). However, treatment with salicylic acid (SA) elevated the mRNA level of the OsPI4K1 gene, which suggested the involvement of OsPI4K1 in wounding responses. OsPI4K1 The highly charged region of plant beta-type phosphatidylinositol 4-kinase is involved in membrane targeting and phospholipid binding 2006 Plant Mol Biol National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Science (SiBS), Chinese Academy of Sciences, Shanghai 200032, P.R. China. In Arabidopsis thaliana and Oryza sativa, two types of PI 4-kinase (PI4Ks) have been isolated and functionally characterized. The alpha-type PI4Ks (approximately 220 kDa) contain a PH domain, which is lacking in beta-type PI4Ks (approximately 120 kDa). Beta-type PI4Ks, exemplified by Arabidopsis AtPI4Kbeta and rice OsPI4K2, contain a highly charged repetitive segment designated PPC (Plant PI4K Charged) region, which is an unique domain only found in plant beta-type PI4Ks at present. The PPC region has a length of approximately 300 amino acids and harboring 11 (AtPI4Kbeta) and 14 (OsPI4K2) repeats, respectively, of a 20-aa motif. Studies employing a modified yeast-based "Sequence of Membrane-Targeting Detection" system demonstrate that the PPC(OsPI4K2) region, as well as the former 8 and latter 6 repetitive motifs within the PPC region, are able to target fusion proteins to the plasma membrane. Further detection on the transiently expressed GFP fusion proteins in onion epidermal cells showed that the PPC(OsPI4K2) region alone, as well as the region containing repetitive motifs 1-8, was able to direct GFP to the plasma membrane, while the regions containing less repetitive motifs, i.e. 6, 4, 2 or single motif(s) led to predominantly intracellular localization. Agrobacterium-mediated transient expression of PPC-GFP fusion protein further confirms the membrane-targeting capacities of PPC region. In addition, the predominant plasma membrane localization of AtPI4Kbeta was mediated by the PPC region. Recombinant PPC peptide, expressed in E. coli, strongly binds phosphatidic acid, PI and PI4P, but not phosphatidylcholine, PI5P, or PI(4,5)P2 in vitro, providing insights into potential mechanisms for regulating sub-cellular localization and lipid binding for the plant beta-type PI4Ks. OsPI4K2 Characterization of OsPID, the rice ortholog of PINOID, and its possible involvement in the control of polar auxin transport 2007 Plant Cell Physiol Graduate School of Agriculture and Life Sciences, University of Tokyo, Yayoi, Bunkyo, Tokyo, 113-0032, Japan. PINOID, a serine threonine protein kinase in Arabidopsis, controls auxin distribution through a positive control of subcellular localization of PIN auxin efflux carriers. Compared with the rapid progress in understanding mechanisms of auxin action in dicot species, little is known about auxin action in monocot species. Here, we describe the identification and characterization of OsPID, the PINOID ortholog of rice. Phylogenetic analysis showed that the rice genome contains a single PID ortholog, OsPID. Constitutive overexpression of OsPID caused a variety of abnormalities, such as delay of adventitious root development, curled growth of shoots and agravitropism. Abnormalities observed in the plants that overexpress OsPID could be phenocopied by treatment with an inhibitor of active polar transport of auxin, indicating that OsPID could be involved in the control of polar auxin transport in rice. Analysis of OsPID mRNA distribution showed a complex pattern in shoot meristems, indicating that it probably plays a role in the pattern formation and organogenesis in the rice shoot. OsPID,OsPIDlike Expression Patterns of OsPIL11, a Phytochrome-Interacting Factor in Rice, and Preliminary Analysis of Its Roles in Light Signal Transduction 2012 Rice Science High-Tech Research Center, Shandong Academy of Agricultural Sciences, Ji'nan 250100, China The expression patterns of OsPIL11, one of six putative phytochrome-interacting factors, were analyzed in different organs of transgenic tobacco (Nicotiana tabacum). The expression of OsPIL11 was organ-specific and was regulated by leaf development, abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA). To further explore the role of OsPIL11 in plant light signal transduction, a plant expression vector of OsPIL11 was constructed and introduced into tobacco. When grown under continuous red light, OsPIL11-overexpressed transgenic tobacco exhibited shorter hypocotyls and larger cotyledons and leaves compared to wild-type seedlings. When grown under continuous far-red light, however, transgenic and wild-type seedlings showed similar phenotypes. These results indicate that OsPIL11 is involved in red light induced de-etiolation, but not in far-red light induced de-etiolation in transgenic tobacco, which lays the foundation for dissecting the function of OsPIL11 in phytochrome-mediated light signal transduction in rice. OsPIL11 Characterization of a Set of Phytochrome-Interacting Factor-Like bHLH Proteins inOryza sativa 2014 Bioscience, Biotechnology and Biochemistry Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Nagoya, Japan. The model dicotyledon Arabidopsis thaliana has a characteristic small sub-family of phytochrome-interacting bHLH (basic Helix-Loop-Helix) factors, which are collectively designated the PIL (or PIF) (PHYTOCHROME INTERACTING FACTOR-LIKE) family proteins. In this study, we identified and characterized a set of highly homologous members (designated OsPIL11 to OsPIL16) in the model monocotyledon rice (Oryza sativa). Some of them (OsPIL11, OsPIL12, and OsPIL13) showed the ability to interact with the putative OsPRR1 (PSEUDO-RESPONSE REGULATOR 1) clock component, as far as the results of yeast two-hybrid assays were concerned. It was found that the expression of OsPIL13 is under the control of circadian rhythms (clock), while the expression of OsPIL15 is negatively regulated by light upon the onset to light exposure of etiolated seedlings. When the rice genes (OsPIL11 to OsPIL15) were over-expressed in A. thaliana, the resulting transgenic seedlings displayed anomalous morphologies with very long hypocotyls during early photomorphogenesis. These results suggest the view that the identified OsPILs are functional counterparts (or orthologs) of AtPILs, which are known to play important roles in red light-mediated (phyA and/or phyB-dependent) signal transduction pathways at immediate positions downstream of the photoreceptor in A. thaliana. OsPIL11,OsPIL12,OsPIL13|OsPIL1,OsPIL14,OsPIL15,APG|OsPIL16,OsPRR1 Phytochrome-interacting factors have both shared and distinct biological roles 2013 Mol Cells Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Korea. Phytochromes are plant photoreceptors that perceive red and far-red light. Upon the perception of light in Arabidopsis, light-activated phytochromes enter the nucleus and act on a set of interacting proteins, modulating their activities and thereby altering the expression levels of approximately 10% of the organism's entire gene complement. Phytochromeinteracting factors (PIFs) belonging to Arabidopsis basic helix-loop-helix (bHLH) subgroup 15 are key interacting proteins that play negative roles in light responses. Their activities are post-translationally countered by light-activated phytochromes, which promote the degradation of PIFs and directly or indirectly inhibit their binding to DNA. The PIFs share a high degree of similarity, but examinations of pif single and multiple mutants have indicated that they have shared and distinct functions in various developmental and physiological processes. These are believed to stem from differences in both intrinsic protein properties and their gene expression patterns. In an effort to clarify the basis of these shared and distinct functions, we compared recently published genome-wide ChIP data, developmental gene expression maps, and responses to various stimuli for the various PIFs. Based on our observations, we propose that the biological roles of PIFs stem from their shared and distinct DNA binding targets and specific gene expression patterns. OsPIL11 Rice phytochrome-interacting factor-like protein OsPIL1 functions as a key regulator of internode elongation and induces a morphological response to drought stress 2012 Proc Natl Acad Sci U S A Biological Resources and Post-harvest Division, Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki, Japan. The mechanisms for plant growth restriction during stress conditions remains unclear. Here, we demonstrate that a phytochrome-interacting factor-like protein, OsPIL1/OsPIL13, acts as a key regulator of reduced internode elongation in rice under drought conditions. The level of OsPIL1 mRNA in rice seedlings grown under nonstressed conditions with light/dark cycles oscillated in a circadian manner with peaks in the middle of the light period. Under drought stress conditions, OsPIL1 expression was inhibited during the light period. We found that OsPIL1 was highly expressed in the node portions of the stem using promoter-glucuronidase analysis. Overexpression of OsPIL1 in transgenic rice plants promoted internode elongation. In contrast, transgenic rice plants with a chimeric repressor resulted in short internode sections. Alteration of internode cell size was observed in OsPIL1 transgenic plants, indicating that differences in cell size cause the change in internode length. Oligoarray analysis revealed OsPIL1 downstream genes, which were enriched for cell wall-related genes responsible for cell elongation. These data suggest that OsPIL1 functions as a key regulatory factor of reduced plant height via cell wall-related genes in response to drought stress. This regulatory system may be important for morphological stress adaptation in rice under drought conditions. OsPIL13|OsPIL1 Overexpression of OsPIL15, a phytochrome-interacting factor-like protein gene, represses etiolated seedling growth in rice 2014 J Integr Plant Biol Shandong Rice Research Institute, Shandong Academy of Agricultural Sciences, Jinan, 250100, China; Shandong Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Jinan, 250100, China. Phytochrome-interacting factors (PIFs) regulate an array of developmental responses ranging from seed germination to vegetational architecture in Arabidopsis. However, information regarding the functions of the PIF family in monocots has not been widely reported. Here, we investigate the roles of OsPIL15, a member of the rice (Oryza sativa L. cv. Nipponbare) PIF family, in regulating seedling growth. OsPIL15 encodes a basic helix-loop-helix factor localized in the nucleus. OsPIL15-OX seedlings exhibit an exaggerated shorter aboveground part and undeveloped root system relative to wild-type seedlings, suggesting that OsPIL15 represses seedling growth in the dark. Microarray analysis combined with gene ontology analysis revealed that OsPIL15 represses a set of genes involved in auxin pathways and cell wall organization or biogenesis. Given the important roles of the auxin pathway and cell wall properties in controlling plant growth, we speculate that OsPIL15 represses seedling growth likely by regulating the auxin pathway and suppressing cell wall organization in etiolated rice seedlings. Additionally, exposure to red light or far-red light relieved growth retardation and promoted seedling elongation in the OsPIL15-OX lines, despite higher levels of OsPIL15 transcripts under red light and far-red light than in the dark. These results suggest that light regulation of OsPIL15 expression is probably involved in photomorphogenesis in rice. OsPIL15 An atypical bHLH protein encoded by POSITIVE REGULATOR OF GRAIN LENGTH 2 is involved in controlling grain length and weight of rice through interaction with a typical bHLH protein APG 2012 Breed Sci Graduate School of Horticulture, Chiba University , 648 Matsudo, Matsudo, Chiba 271-8510, Japan. Grain size is an important yield component in rice, however, genes controlling the trait remain poorly understood. Previously, we have shown that an antagonistic pair of basic helix-loop-helix (bHLH) proteins, POSITIVE REGULATOR OF GRAIN LENGTH 1 (PGL1) and ANTAGONIST OF PGL1 (APG), is involved in controlling rice grain length. Here, we report the involvement of another atypical bHLH protein gene, POSITIVE REGULATOR OF GRAIN LENGTH 2 (PGL2), in the regulation of rice grain length. Over-expression of PGL2 in the lemma/palea increased grain length and weight in correlation with the level of transgene expression. Observation of the inner epidermal cells of lemma of PGL2-overexpressing lines revealed that the long grain size is caused by an increase in cell length. PGL2 interacts with a typical bHLH protein APG, a negative regulator of rice grain length and weight, in vitro and in vivo. It was reported that overexpression of BU1 (BRASSINOSTEROID UPREGULATED 1), the closest homolog of PGL2, caused an increase in grain length. However, we detected no interaction between BU1 and APG. These findings suggest that PGL2 and PGL1 redundantly suppress the function of APG by forming heterodimers to positively regulate the rice grain length, while the pathway through which BU1, the closest homolog of PGL2, controls grain length is independent of APG. APG|OsPIL16,PGL1,PGL2 OsRPK1, a novel leucine-rich repeat receptor-like kinase, negatively regulates polar auxin transport and root development in rice 2014 Biochim Biophys Acta Dept. of Biochemistry & Molecular Biology, College of Life Science, Nanjing Agricultural University, Nanjing 210095, China. BACKGROUND: Leucine-rich-repeat receptor-like kinases (LRR-RLKs) represent the largest subfamily of putative RLKs in plants. Although several members in this subfamily have been identified, the studies about the relationships between LRR-RLKs and root development are still few. We previously identified a novel LRR-RLK in rice roots, and named it OsRPK1. METHODS: In this study, we first detected OsRPK1 kinase activity in vitro, and assessed its expression profile. We then investigated its biological function using transgenic rice plants over- and under-expressing OsRPK1. RESULTS: The OsRPK1 gene, which encodes a Ca(2+)-independent Ser/Thr kinase, was predominantly expressed in root tips, leaf blades, and undifferentiated suspension cells, and was markedly induced by treatment with auxin or ABA. Knockdown of OsRPK1 promoted the growth of transgenic rice plants, and increased plant height and tiller numbers. In contrast, over-expressing plants showed undeveloped adventitious roots, lateral roots, and a reduced root apical meristem. OsRPK1 over-expression also inhibited the expression of most auxin efflux carrier OsPIN genes, which was accompanied by changes in PAT and endogenous free IAA distribution in the leaves and roots. CONCLUSIONS: The data indicated that OsRPK1, a novel leucine-rich-repeat receptor-like kinase, affects the root system architecture by negatively regulating polar auxin transport in rice. GENERAL SIGNIFICANCE: This study demonstrated a common regulatory pathway of root system development in higher plants, which might be initiated by external stimuli via upstream receptor-like kinases and downstream carriers for polar auxin transport. OsPIN1|REH1,OsRPK1 A PIN1 family gene, OsPIN1, involved in auxin-dependent adventitious root emergence and tillering in rice 2005 Plant Cell Physiol State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, PR China. Auxin transport affects a variety of important growth and developmental processes in plants, including the regulation of shoot and root branching. The asymmetrical localization of auxin influx and efflux carriers within the plasma membrane establishes the auxin gradient and facilitates its transport. REH1, a rice EIR1 (Arabidopsis ethylene insensitive root 1)-like gene, is a putative auxin efflux carrier. Phylogenetic analysis of 32 members of the PIN family, taken from across different species, showed that in terms of evolutionary relationship, OsPIN1 is closer to the PIN1 family than to the PIN2 family. It is, therefore, renamed as OsPIN1 in this study. OsPIN1 was expressed in the vascular tissues and root primordial in a manner similar to AtPIN1. Adventitious root emergence and development were significantly inhibited in the OsPIN1 RNA interference (RNAi) transgenic plants, which was similar to the phenotype of NPA (N-1-naphthylphalamic acid, an auxin-transport inhibitor)-treated wild-type plants. alpha-naphthylacetic acid (alpha-NAA) treatment was able to rescue the mutated phenotypes occurring in the RNAi plants. Overexpression or suppression of the OsPIN1 expression through a transgenic approach resulted in changes of tiller numbers and shoot/root ratio. Taken together, these data suggest that OsPIN1 plays an important role in auxin-dependent adventitious root emergence and tillering. OsPIN1|REH1 The putative auxin efflux carrier OsPIN3t is involved in the drought stress response and drought tolerance 2012 Plant J Hebei Key Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hebei Normal University, Shijiazhuang, Hebei 050024, China. The phytohormone auxin plays a critical role in plant growth and development, and its spatial distribution largely depends on the polar localization of the PIN-FORMED (PIN) auxin efflux carrier family members. In this study, we identify a putative auxin efflux carrier gene in rice, OsPIN3t, which acts in auxin polar transport but is also involved in the drought stress response in rice. We show that OsPIN3t-GFP fusion proteins are localized in plasma membranes, and this subcellular localization changes under 1-N-naphthylphthalamic acid (NPA) treatment. The tissue-specific expression patterns of OsPIN3t were also investigated using a beta-glucuronidase (GUS) reporter, which showed that OsPIN3t was mainly expressed in vascular tissue. The GUS activity in OsPIN3tpro::GUS plants increased by NAA treatment and decreased by NPA treatment. Moreover, knockdown of OsPIN3t caused crown root abnormalities in the seedling stage that could be phenocopied by treatment of wild-type plants with NPA, which indicated that OsPIN3t is involved in the control of polar auxin transport. Overexpression of OsPIN3t led to improved drought tolerance, and GUS activity significantly increased when OsPIN3tpro::GUS plants were subjected to 20% polyethylene glycol stress. Taken together, these results suggest that OsPIN3t is involved in auxin transport and the drought stress response, which suggests that a polar auxin transport pathway is involved in the regulation of the response to water stress in plants. OsPIN3t The role of water channel proteins and nitric oxide signaling in rice seed germination 2007 Cell Res Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. Previous studies have demonstrated the possible role of several aquaporins in seed germination. But systematic investigation of the role of aquaporin family members in this process is lacking. Here, the developmental regulation of plasma membrane intrinsic protein (PIP) expression throughout germination and post-germination processes in rice embryos was analyzed. The expression patterns of the PIPs suggest these aquaporins play different roles in seed germination and seedling growth. Partial silencing of the water channel genes, OsPIP1;1 and OsPIP1;3, reduced seed germination while over-expression of OsPIP1;3 promoted seed germination under water-stress conditions. Moreover, spatial expression analysis indicates that OsPIP1;3 is expressed predominantly in embryo during seed germination. Our data also revealed that the nitric oxide (NO) donors, sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO), promoted seed germination; furthermore, the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, inhibited germination and reduced the stimulative effects of SNP and GSNO on rice germination. Exogenous NO stimulated the transcription of OsPIP1;1, OsPIP1;2, OsPIP1;3 and OsPIP2;8 in germinating seeds. These results suggest that water channels play an important role in seed germination, acting, at least partly, in response to the NO signaling pathway. OsPIP1;2,OsPIP2;4,OsPIP1;1|RWC1,RWC3|OsPIP1;3 Identification of 33 rice aquaporin genes and analysis of their expression and function 2005 Plant Cell Physiol CO2 and Temperature Research Laboratory, National Agricultural Research Center for Tohoku Region, Morioka, 020-0198 Japan. junkoi@affrc.go.jp Plant aquaporins form a large protein family including plasma membrane-type (PIPs) and tonoplast-type aquaporins (TIPs), and facilitate osmotic water transport across membranes as a key physiological function. We identified 33 genes for aquaporins in the genome sequence of rice (Oryza sativa L. cv. Nipponbare). We investigated their expression levels in leaf blades, roots and anthers of rice (cv. Akitakomachi) using semi-quantitative reverse transcription-PCR (RT-PCR). At both early tillering (21 d after germination) and panicle formation (56 d) stages, six genes, including OsPIP2;4 and OsPIP2;5, were expressed predominantly in roots, while 14 genes, including OsPIP2;7 and OsTIP1;2, were found in leaf blades. Eight genes, such as OsPIP1;1 and OsTIP4;1, were evenly expressed in leaf blades, roots and anthers. Analysis by stopped-flow spectrophotometry revealed high water channel activity when OsPIP2;4 or OsPIP2;5 were expressed in yeast but not when OsPIP1;1 or OsPIP1;2 were expressed. Furthermore, the mRNA levels of OsPIP2;4 and OsPIP2;5 showed a clear diurnal fluctuation in roots; they showed a peak 3 h after the onset of light and dropped to a minimum 3 h after the onset of darkness. The mRNA levels of 10 genes including OsPIP2;4 and OsPIP2;5 markedly decreased in roots during chilling treatment and recovered after warming. The changes in mRNA levels during and after the chilling treatment were comparable with that of the bleeding sap volume. These results suggested the relationship between the root water uptake and mRNA levels of several aquaporins with high water channel activity, such as OsPIP2;4 and OsPIP2;5. OsPIP1;2 Members of rice plasma membrane intrinsic proteins subfamily are involved in arsenite permeability and tolerance in plants 2012 Transgenic Res Department of Plant, Soil and Insect Sciences, University of Massachusetts, 202 French Hall, Amherst, MA 01003, USA. Rice accumulates high level of arsenic (As) in its edible parts and thus plays an important role in the transfer of As into the food chain. However, the mechanisms of As uptake and its detoxification in rice are not well understood. Recently, members of the Nodulin 26-like intrinsic protein (NIP) subfamily of plant aquaporins were shown to transport arsenite in rice and Arabidopsis. Here we report that members of the rice plasma membrane intrinsic protein (PIP) subfamily are also involved in As tolerance and transport. Based on the homology search with the mammalian AQP9 and yeast Fps1 arsenite transporters, we identified and cloned five rice PIP gene subfamily members. qRT-PCR analysis of PIPs in rice root and shoot tissues revealed a significant down regulation of transcripts encoding OsPIP1;2, OsPIP1;3, OsPIP2;4, OsPIP2;6, and OsPIP2;7 in response to arsenite treatment. Heterologous expression of OsPIP2;4, OsPIP2;6, and OsPIP2;7 in Xenopus laevis oocytes significantly increased the uptake of arsenite. Overexpression of OsPIP2;4, OsPIP2;6, and OsPIP2;7 in Arabidopsis yielded enhanced arsenite tolerance and higher biomass accumulation. Further, these transgenic plants showed no significant accumulation of As in shoot and root tissues in long term uptake assays. Whereas, short duration exposure to arsenite caused both active influx and efflux of As in the roots. The data suggests a bidirectional arsenite permeability of rice PIPs in plants. These rice PIPs genes will be highly useful for engineering important food and biofuel crops for enhanced crop productivity on contaminated soils without increasing the accumulation of toxic As in the biomass or edible tissues. OsPIP1;2,OsPIP2;4,OsPIP2;6,OsPIP2;7 Two rice plasma membrane intrinsic proteins, OsPIP2;4 and OsPIP2;7, are involved in transport and providing tolerance to boron toxicity 2014 Planta Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA, 01003, USA. Boron (B) toxicity is responsible for low cereal crop production in a number of regions worldwide. In this report, we characterized two rice genes, OsPIP2;4 and OsPIP2;7, for their involvement in B permeability and tolerance. Transcript analysis demonstrated that the expression of OsPIP2;4 and OsPIP2;7 were downregulated in shoots and strongly upregulated in rice roots by high B treatment. Expression of both OsPIP2;4 and OsPIP2;7 in yeast HD9 strain lacking Fps1, ACR3, and Ycf1 resulted in an increased B sensitivity. Furthermore, yeast HD9 strain expressing OsPIP2;4 and OsPIP2;7 accumulated significantly higher B as compared to empty vector control, which suggests their involvement in B transport. Overexpression of OsPIP2;4 and OsPIP2;7 in Arabidopsis imparted higher tolerance under B toxicity. Arabidopsis lines overexpressing OsPIP2;4 and OsPIP2;7 showed significantly higher biomass production and greater root length, however there was no difference in B accumulation in long term uptake assay. Short-term uptake assay using tracer B ((1)(0)B) in shoots and roots demonstrated increased (1)(0)B accumulation in Arabidopsis lines expressing OsPIP2;4 and OsPIP2;7, compare to wild type control plants. Efflux assay of B in the roots showed that (1)(0)B was effluxed from the Arabidopsis transgenic plants overexpressing OsPIP2;4 or OsPIP2;7 during the initial 1-h of assay. These data indicate that OsPIP2;4 and OsPIP2;7 are involved in mediating B transport in rice and provide tolerance via efflux of excess B from roots and shoot tissues. These genes will be highly useful in developing B tolerant crops for enhanced yield in the areas affected by high B toxicity. OsPIP2;4,OsPIP2;7 Influence of low air humidity and low root temperature on water uptake, growth and aquaporin expression in rice plants 2012 Plant Cell Physiol National Institute for Agro-environmental Sciences, Agro-Meteorology Division, Tsukuba, 305-8604 Japan. The effects of low air humidity and low root temperature (LRT) on water uptake, growth and aquaporin gene expression were investigated in rice plants. The daily transpiration of the plants grown at low humidity was 1.5- to 2-fold higher than that at high humidity. LRT at 13 degrees C reduced transpiration, and the extent was larger at lower humidity. LRT also reduced total dry matter production and leaf area expansion, and the extent was again larger at lower humidity. These observations suggest that the suppression of plant growth by LRT is associated with water stress due to decreased water uptake ability of the root. On the other hand, the net assimilation rate was not affected by low humidity and LRT, and water use efficiency was larger for LRT. We found that low humidity induced coordinated up-regulation of many PIP and TIP aquaporin genes in both the leaves and the roots. Expression levels of two root-specific aquaporin genes, OsPIP2;4 and OsPIP2;5, were increased significantly after 6 and 13 d of LRT exposure. Taken together, we discuss the possibility that aquaporins are part of an integrated response of this crop to low air humidity and LRT. OsPIP2;4,OsPIP2;5 Characterization of OsPIP2;7, a water channel protein in rice 2008 Plant Cell Physiol Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, PR China. Aquaporins are water channel proteins that facilitate passage of water and other small neutral molecules across biological membranes. There are usually a large number of members of this family in higher plants, which exhibit various physiological functions and are regulated in a time-specific and particular mode. We have previously shown that a rice gene, OsPIP2;7, was generally up-regulated in roots but down-regulated in shoots at the early stage of chilling stress. Here, OsPIP2;7 was cloned and proved to be an aquaporin with high activity in Xenopus oocytes. OsPIP2;7 was localized mainly in mesophyll cells of leaves. In roots it was detected in the vascular tissues, epidermis cells and exodermis cells at the elongation zone, as well as in the epidermis cells, exodermis cells and root hair at the maturation zone. Yeast cells overexpressing OsPIP2;7 showed a higher survival rate after freeze-thaw stress. Furthermore, OsPIP2;7 enhanced the transpiration rate and tolerance to low temperature when overexpressed in rice. These results indicated that OsPIP2;7 was involved in rapid water transport and maintenance of the water balance in cells, and ultimately improves the tolerance of yeast and rice to low temperature stress. OsPIP2;7 OsPIPK1, a rice phosphatidylinositol monophosphate kinase, regulates rice heading by modifying the expression of floral induction genes 2004 Plant Mol Biol National Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences (SiBS), Chinese Academy of Sciences, 200032 Shanghai, China. A rice gene, OsPIPK1, encoding a 792-aa putative phosphatidylinositol 4-phosphate 5-kinase (PIPK), was identified and characterized. Comparison between the cDNA and genomic sequences revealed the presence of 10 exons ( 39 - 1050 bp) and 9 introns ( 88 - 745 bp) in OsPIPK1 gene. The deduced amino acid sequence of OsPIPK1 contains a lipid kinase domain that is highly homologous to those of previously isolated PIPKs, and structural analysis revealed the intriguing presence of multiple MORN motifs at the N-terminus. The MORN motifs have also been detected in PIPKs from Arabidopsis thaliana and Oryza sativa, but not in the well-characterized PIPKs from animal and yeast cells. RT-PCR analysis indicated that OsPIPK1 was expressed almost constitutively in roots, shoots, stems, leaves and flowers, and upregulated following treatment with plant hormones or application of various stresses. An antisense transgenic strategy was used to suppress the expression of OsPIPK1, and homozygous transgenic plants showed earlier heading ( 7 - 14 days earlier) than control plants, suggesting that OsPIPK1 negatively regulates. oral initiation. This was further confirmed by morphologic observation showing earlier. oral development in antisense plants, as well as leaf emergence measurement indicating delayed leaf development under OsPIPK1 deficiency, a common phenotype observed with earlier flowering. RT-PCR analysis and cDNA chip technology were used to examine transcripts of various genes in the transgenic plants and the results showed altered transcriptions of several flowering-time or - identity related genes, suggesting that OsPIPK1 is involved in rice heading through regulation of. oral induction genes, signaling and metabolic pathways. OsPIPK1 Downregulation of OsPK1, a cytosolic pyruvate kinase, by T-DNA insertion causes dwarfism and panicle enclosure in rice 2012 Planta School of Life Sciences, Tsinghua University, Beijing, 100084, China. Pyruvate kinase (PK) catalyzes the final step of glycolysis. There are few reports on the role of PK in rice. Here, we identified a novel rice dwarf mutant, designated as ospk1, showing dwarfism, panicle enclosure, reduced seed set, and outgrowth of axillary buds from culm nodes. Sequence analyses of 5'-RACE indicated that a single T-DNA was inserted in the transcriptional regulatory region of OsPK1 in ospk1. Quantitative RT-PCR result showed that OsPK1 expression was decreased by approximately 90% in ospk1 compared with that in WT. Enzyme assay and transient expression in protoplasts indicated that OsPK1 encodes a cytosolic PK (PK(c)). Complementation with OsPK1 demonstrated that OsPK1 is responsible for the phenotype of ospk1. Quantitative RT-PCR and GUS staining analyses exhibited that OsPK1 was expressed mainly in leaf mesophyll cells, phloem companion cells in stems, and cortical parenchyma cells in roots. The transcriptions of four other putative enzymes involved in the glycolysis/gluconeogenesis pathway were altered in ospk1. The amount of pyruvate is decreased in ospk1. We propose that OsPK1 plays an important role through affecting the glycolytic pathway. The contents of glucose and fructose were markedly accumulated in flag leaf blade and panicle of ospk1. The sucrose level in panicle of ospk1 was decreased by approximately 84%. These findings indicated that both monosaccharide metabolism and sugar transport are altered due to the decreased expression of OsPK1. Together, these results provide new insights into the role of PK(c) in plant morphological development, especially plant height. OsPK1,ALT-2,PEPC-1,PEPCK Isolation and characterization of two cDNA clones for mRNAs that are abundantly expressed in immature anthers of rice (Oryza sativa L.) 1996 Plant Mol Biol Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi 1-1-1, 113, Bunkyo-ku, Tokyo, Japan The relationship between the length of anthers and the stage of development of microspores was examined in rice (Oryza sativa L. cv. Hayayuki). Anthers of >2 mm and 2.1-2.2 mm in length and those ready to dehiscence were determined to be at the uninucleate, binucleate and trinucleate microspore stage, respectively. Two cDNAs (YY1 and YY2), representing genes that are specifically expressed in anthers at the uninucleate microspore stage, were isolated and characterized. YY1 cDNA encoded an open reading frame of 95 amino acids. Eight cysteine residues with the potential to form disulfide bridges were present in the amino acid sequence. There was a hydrophobic region at the N-terminus of the putative protein, suggesting that the YY1 protein might be secreted. This cysteine motif and the hydrophobic N-terminus are conserved among products of several anther-specific genes or cDNAs isolated from various plant species. These proteins are thought to form a superfamily of proteins that are confined to anthers. The YY1 transcript was localized in the tapetal cells and the peripheral cells of the vascular bundle. YY2 cDNA encoded an open reading frame of 389 amino acids and the deduced amino acid sequence exhibited substantial homology to that of chalcone synthase. Expression of YY2 mRNA was confined to the tapetal cells. The genes correspond to YY1 and YY2 cDNAs were shown to exist as single copies in the rice genome. OsPKS1|YY2,YY1|OsLTPc2|LTPL45 Conserved metabolic steps for sporopollenin precursor formation in tobacco and rice 2013 Physiol Plant School of Biological Sciences, The University of Hong Kong, Hong Kong, China. The development of pollen wall with proper sporopollenin deposition is essential for pollen viability and male fertility in flowering plants. Sporopollenin is a complex biopolymer synthesized from fatty acid and phenolic derivatives. Recent investigations in Arabidopsis have identified a number of anther-specific genes involved in the production of fatty-acyl monomers potentially required for exine formation. The existence of ancient biochemical pathways for sporopollenin biosynthesis has been widely proposed but experimental evidence from plant species other than Arabidopsis is not extensively available. Here, we investigated the metabolic steps catalyzed by the anther-specific acyl-CoA synthetase (ACOS), polyketide synthase (PKS) and tetraketide alpha-pyrone reductase (TKPR). Using fatty acids as starting substrates, sequential activities of heterologously expressed tobacco enzymes NtACOS1, NtPKS1 and NtTKPR1 resulted in the production of reduced tetraketide alpha-pyrones. Transgenic RNA interference lines were then generated for the different tobacco genes which were demonstrated to be indispensable for normal pollen development and male fertility. Similarly, recombinant rice OsPKS1 and OsTKPR1 were shown to function as downstream enzymes of NtACOS1. In addition, insertion mutant lines for these rice genes displayed different levels of impaired pollen and seed formation. Taken together, reduced tetraketide alpha-pyrones appear to represent common sporopollenin fatty-acyl precursors essential for male fertility in taxonomically distinct plant species. OsPKS1|YY2,OsTKPR1|OsDFR2|OS-DFR2 Suppression of a phospholipase D gene, OsPLDbeta1, activates defense responses and increases disease resistance in rice 2009 Plant Physiol National Agricultural Research Center, Joetsu, Niigata 943-0193, Japan. tkyama@affrc.go.jp Phospholipase D (PLD) plays an important role in plants, including responses to abiotic as well as biotic stresses. A survey of the rice (Oryza sativa) genome database indicated the presence of 17 PLD genes in the genome, among which OsPLDalpha1, OsPLDalpha5, and OsPLDbeta1 were highly expressed in most tissues studied. To examine the physiological function of PLD in rice, we made knockdown plants for each PLD isoform by introducing gene-specific RNA interference constructs. One of them, OsPLDbeta1-knockdown plants, showed the accumulation of reactive oxygen species in the absence of pathogen infection. Reverse transcription-polymerase chain reaction and DNA microarray analyses revealed that the knockdown of OsPLDbeta1 resulted in the up-/down-regulation of more than 1,400 genes, including the induction of defense-related genes such as pathogenesis-related protein genes and WRKY/ERF family transcription factor genes. Hypersensitive response-like cell death and phytoalexin production were also observed at a later phase of growth in the OsPLDbeta1-knockdown plants. These results indicated that the OsPLDbeta1-knockdown plants spontaneously activated the defense responses in the absence of pathogen infection. Furthermore, the OsPLDbeta1-knockdown plants exhibited increased resistance to the infection of major pathogens of rice, Pyricularia grisea and Xanthomonas oryzae pv oryzae. These results suggested that OsPLDbeta1 functions as a negative regulator of defense responses and disease resistance in rice. OsPLDalpha5,OsPLDbeta1 Isolation and Expression analysis of OsPME1, encoding for a putative Pectin Methyl Esterase from Oryza sativa (subsp. indica) 2009 Physiol Mol Biol Plants Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, 625021 Tamil Nadu India. Pectin Methyl Esterases (PMEs) play an essential role during plant development by affecting the mechanical properties of the plant cell walls. Recent studies indicated that PMEs play important role in pollen tube development. In this study, we isolated a 1.3 kb cDNA clone from rice panicle cDNA library. It contained a 1038 bp of open reading frame (ORF) encoding for a putative pectin methyl esterase of 345 aminoacids with a 20 aminoacid signal peptide and was hence designated as OsPME1 (Oryza sativaPectin Methyl Esterase 1). It contained the structural arrangement GXYXE and GXXDFIF, found in the active groups of all PMEs. OsPME1 gene product shared varying identities, ranging from 52 % to 33 % with PMEs from other plant species belonging to Brassicaceae, Fabaceae, Amaranthaceae and Funariaceae. Southern blot analysis indicated that PME1 exists as a single copy in the rice genome. Expression pattern analysis revealed that OsPME1 is expressed only in pollen grains, during the later stages of their development and was also regulated by various abiotic stress treatments and phytohormones. Functional characterization of this pollen specific PME from rice would enable us to understand its role in pollen development. OsPME1 Rapid generation of rice mutants via the dominant negative suppression of the mismatch repair protein OsPMS1 2012 Theor Appl Genet Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, South China Normal University, Guangzhou 510631, People's Republic of China. Mismatch repair (MMR) is a conservative pathway for maintaining the genome integrity of different organisms. Although suppression of MMR has resulted in various mutation phenotypes in Arabidopsis, the use of this strategy for mutation breeding in major crops has not been reported. Here, we overexpressed a truncated version of the OsPMS1 protein in rice; this approach is expected to suppress the rice MMR system through a dominant negative mechanism. We observed a wide spectrum of mutation phenotypes in the progeny of the transgenic plants during seed germination and the plant growth stages. Genomic variations were detected with inter-simple sequence repeat (ISSR), and sequencing of the differential ISSR bands revealed that the mutation occurred as a point mutation or as microsatellite instability at high frequencies. Plant lines with agronomically important traits, such as salt and drought tolerance, various tiller number, and early flowering, were obtained. Furthermore, we obtained mutants with important traits that are free of the transgene. Together, these results demonstrate that MMR suppression can be used as an efficient strategy for mutation breeding in rice. OsPMS1,OsMLH1 OsPNH1regulates leaf development and maintenance of the shoot apical meristem in rice 2002 The Plant Journal BioScience Centre, Nagoya University, Nagoya 464-8601, Japan. The Arabidopsis PINHEAD/ZWILLE (PNH/ZLL) gene is thought to play an important role in the formation of the shoot apical meristem (SAM) and in leaf adaxial cell specification. To investigate the molecular mechanisms of rice development, we have isolated a rice homologue of PNH/ZLL, called OsPNH1. Around the SAM, OsPNH1 was strongly expressed in developing leaf primordia, specifically in the presumptive vascular domains, developing vascular tissues, a few cell-layers of the adaxial region, and future bundle sheath extension cells. In the SAM, only weak expression was observed in the central region, whereas strong expression was detected in the mid-vein region of leaf founder cells in the peripheral SAM domain. We produced transgenic rice plants containing the antisense OsPNH1 strand. The antisense OsPNH1 plants developed malformed leaves with an altered vascular arrangement and abnormal internal structure. These plants also formed an aberrant SAM with reduced KNOX gene expression. We examined the subcellular localization of the OsPNH1-GFP fusion protein and found that it was localized in the cytoplasm. On the basis of these observations, we propose that OsPNH1 functions not only in SAM maintenance as previously thought, but also in leaf formation through vascular development. OsPNH1 A novel DNA polymerase homologous to Escherichia coli DNA polymerase I from a higher plant, rice (Oryza sativa L.) 2002 Nucleic Acids Res Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan A novel DNA polymerase, designated as OsPolI-like, has been identified from the higher plant, rice (Oryza sativa L. cv. Nipponbare). The OsPolI-like cDNA was 3765 bp in length, and the open reading frame encoded a predicted product of 977 amino acid residues with a molecular weight of 100 kDa. The OsPolI-like gene has been mapped to chromosome 8 and contains 12 exons and 11 introns. The encoded protein showed a high degree of sequence and structural homology to Escherichia coli pol I protein, but differed from DNA polymerase γ and θ. The DNA polymerase domain of OsPolI-like showed DNA polymerase activity. Subcellular fractionation analysis suggested that the protein is localized in the plastid. Northern and western blotting, and in situ hybridization analyses demonstrated preferential expression of OsPolI-like in meristematic tissues such as shoot apical meristem, root apical meristem, leaf primordia and the marginal meristem. Interestingly, no expression was detected in mature leaves, although they have a high chloroplast content. These properties indicated that OsPolI-like is a novel plant DNA polymerase. The function of OsPolI-like is discussed in relation to plastid maturation. OsPOLP1 FtsZ Ring Formation at the Chloroplast Division Site in Plants 2001 The Journal of Cell Biology Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824 Among the events that accompanied the evolution of chloroplasts from their endosymbiotic ancestors was the host cell recruitment of the prokaryotic cell division protein FtsZ to function in chloroplast division. FtsZ, a structural homologue of tubulin, mediates cell division in bacteria by assembling into a ring at the midcell division site. In higher plants, two nuclear-encoded forms of FtsZ, FtsZ1 and FtsZ2, play essential and functionally distinct roles in chloroplast division, but whether this involves ring formation at the division site has not been determined previously. Using immunofluorescence microscopy and expression of green fluorescent protein fusion proteins in Arabidopsis thaliana, we demonstrate here that FtsZ1 and FtsZ2 localize to coaligned rings at the chloroplast midpoint. Antibodies specific for recognition of FtsZ1 or FtsZ2 proteins in Arabidopsis also recognize related polypeptides and detect midplastid rings in pea and tobacco, suggesting that midplastid ring formation by FtsZ1 and FtsZ2 is universal among flowering plants. Perturbation in the level of either protein in transgenic plants is accompanied by plastid division defects and assembly of FtsZ1 and FtsZ2 into filaments and filament networks not observed in wild-type, suggesting that previously described FtsZ-containing cytoskeletal-like networks in chloroplasts may be artifacts of FtsZ overexpression. OsPOLP1 Biochemical properties of a plastidial DNA polymerase of rice 2007 Plant Mol Biol Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba-ken, 278-8510, Japan. j6498055@yahoo.co.jp Plastids are organelles unique to plant cells and are responsible for photosynthesis and other metabolic functions. Despite their important cellular roles, relatively little is known about the mechanism of plastidial DNA replication and repair. Recently, we identified a novel DNA polymerase in Oryza Sativa L. (OsPOLP1, formerly termed OsPolI-like) that is homologous to prokaryotic DNA polymerase Is (PolIs), and suggested that this polymerase might be involved in plastidial DNA replication and repair. Here, we propose to rename the plant PolI homologs as DNA polymerase pi (POLP), and investigate the biochemical properties of full-length OsPOLP1. The purified OsPOLP1 elongated both DNA and RNA primer hybridized to a DNA template, and possessed a 3' exonuclease activity. Moreover, OsPOLP1 displayed high processivity and fidelity, indicating that this polymerase has the biochemical characteristics appropriate for DNA replication. We found that POLPs have two extra sequences in the polymerase domain that are absent in prokaryotic PolIs. Deletion of either insert from OsPOLP1 caused a decrease in DNA synthetic activity, processivity, and DNA binding activity. In addition, OsPOLP1 efficiently catalyzed strand displacement on nicked DNA with a 5'-deoxyribose phosphate, suggesting that this enzyme might be involved in a repair pathway similar to long-patch base excision repair. These results provide insights into the possible role of POLPs in plastidial DNA replication and repair. OsPOLP1 The rice faded green leaf locus encodes protochlorophyllide oxidoreductase B and is essential for chlorophyll synthesis under high light conditions 2013 Plant J Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-921, Korea. NADPH:protochlorophyllide oxidoreductase (POR) catalyzes photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in chlorophyll (Chl) synthesis, and is required for prolamellar body (PLB) formation in etioplasts. Rice faded green leaf (fgl) mutants develop yellow/white leaf variegation and necrotic lesions during leaf elongation in field-grown plants. Map-based cloning revealed that FGL encodes OsPORB, one of two rice POR isoforms. In fgl, etiolated seedlings contained smaller PLBs in etioplasts, and lower levels of total and photoactive Pchlide. Under constant or high light (HL) conditions, newly emerging green leaves rapidly turned yellow and formed lesions. Increased levels of non-photoactive Pchlide, which acts as a photosensitizer, may cause reactive oxygen accumulation and lesion formation. OsPORA expression is repressed by light and OsPORB expression is regulated in a circadian rhythm in short-day conditions. OsPORA was expressed at high levels in developing leaves and decreased dramatically in fully mature leaves, whereas OsPORB expression was relatively constant throughout leaf development, similar to expression patterns of AtPORA and AtPORB in Arabidopsis. However, OsPORB expression is rapidly upregulated by HL treatment, similar to the fluence rate-dependent regulation of AtPORC. This suggests that OsPORB function is equivalent to both AtPORB and AtPORC functions. Our results demonstrate that OsPORB is essential for maintaining light-dependent Chl synthesis throughout leaf development, especially under HL conditions, whereas OsPORA mainly functions in the early stages of leaf development. Developmentally and physiologically distinct roles of monocot OsPORs are discussed by comparing with those of dicot AtPORs. OsPORA,OsPORB|FGL Cold-Responsive Regulation of a Flower-Preferential Class III Peroxidase Gene, OsPOX1, in Rice (Oryza sativa L.) 2011 Journal of Plant Biology Department of Life Science, Sogang University, Seoul, 121-742, South Korea A full-length cDNA corresponding to ddOs319, previously isolated as a cold-responsive gene in the flowers by mRNA differential display (Plant Cell Rep 26:1097-1110, 2007), was obtained from the cold-treated flowers by reverse transcription and nested PCR. The cDNA encodes a putative class III peroxidase of 335 amino acids with 77-98% identity with rice peroxidases and named OsPOX1. To understand the regulation of OsPOX1 expression, a 1.8 kb promoter region of OsPOX1 was isolated and fused to beta-glucuronidase (GUS) reporter gene. Transgenic rice plants expressing P OsPOX1 -GUS showed minimal GUS activity in both shoots and roots at the vegetative stage. In the flowers at early young microspore stage, GUS activity was detected in the veins and anthers. Interestingly, at the later vacuolated pollen stage, the GUS activity was highly induced by cold stress, suggesting that OsPOX1 is a flower-preferential cold-responsive gene in rice. OsPOX1|ddOs319 A SNAC1-regulated protein phosphatase gene OsPP18 modulates drought and oxidative stress tolerance through ABA-independent reactive oxygen species scavenging in rice 2014 Plant Physiol Huazhong Agricultural University Plants respond to abiotic stresses through a complexity of signaling pathways, and the dephosphorylation mediated by protein phosphatase is an important event in this process. We identified a rice protein phosphatase 2C (PP2C) gene, OsPP18, as a STRESS-RESPOSIVE NAC1 (SNAC1)-regulated downstream gene. The ospp18 mutant was more sensitive than wild-type plants to drought stress at both the seedling and panicle development stages. Rice plants with OsPP18-suppresed via artificial microRNA were also hypersensitive to drought stress. Microarray analysis of the mutant revealed that genes encoding reactive oxygen species (ROS) scavenging enzymes were down-regulated in the ospp18 mutant, and the mutant exhibited reduced activities of ROS-scavenging enzymes and increased sensitivity to oxidative stresses. Over-expression of OsPP18 in rice led to enhanced osmotic and oxidative stress tolerance. The expression of OsPP18 was induced by drought stress, but not induced by abscisic acid. Although OsPP18 is a typical PP2C with enzymatic activity, it did not interact with SnRK2 protein kinases which function in ABA signaling. Meanwhile, the expression of ABA-responsive genes was not affected in the ospp18 mutant, and the ABA sensitivity of the ospp18 mutant and OsPP18-over-expressing plants was not altered either. Together, these findings suggest that OsPP18 is a novel PP2C gene which is regulated by SNAC1 and confers drought and oxidative stress resistance by regulating ROS homeostasis through ABA-independent pathways. OsPP18|OsPP2C10 Molecular characterization of catalytic-subunit cDNA sequences encoding protein phosphatases 1 and 2A and study of their roles in the gibberellin-dependent Osamy-cexpression in rice 1999 Plant Mol Biol Department of Plant Biology, Cornell University, Ithaca, NY, 14853, USA To understand the molecular mechanism of gibberellin-dependent gene regulation, the effect of three phosphatase inhibitors on the germination of rice seeds and the expression of a target gene, the α-amylase gene, Osamy-c, were measured. We found that okadaic acid, microcystin-LR, and calyculin A, which are known to specifically inhibit Ser/Thr phosphatases 1 and 2A, strongly inhibit the expression of the Osamy-c and may be involved in the germination of rice seeds. The protein phosphatase enzyme activity assays showed that there is no obvious effect of GA3 on total PP1/PP2A activities. To further understand the possible role of protein phosphatases 1 and 2A in the GA-dependent expression of Osamy-c, we isolated cDNA clones encoding protein phosphatase 1 and protein phosphatase 2A from a rice aleurone cDNA library. These were designated OsPP1c and OsPP2Ac, respectively. Comparison of the deduced amino acid sequences of OsPP1c and OsPP2Ac with the catalytic subunits of PP1 or PP2A of rabbit skeletal muscle, Arabidopsis thaliana, maize and Brassica napus showed that the catalytic subunit sequences of PP1 or PP2A among these organisms are highly conserved (73% to 90% similarity). Genomic Southern blot analysis indicated that there are only one or two copies of OsPP1c genes and more than two copies of OsPP2Ac genes in the rice genome. Northern blot analysis showed that OsPP1c and OsPP2Ac genes are expressed in several organs of rice, including seed, shoot and root. We also showed by using 3 gene-specific probes of OsPP1c and OsPP2Ac cDNA, that the expression of neither gene is regulated by GA. Taken together, our results suggest that protein phosphatases PP1 or PP2A are involved in the GA-dependent expression of the rice Osamy-c gene, though the PP1 or/and PP2A enzymatic activities as well as mRNA levels do not increase upon GA3 treatment. OsPP1c,OsPP2A-1,OsPP2A-3 Structure, evolution and expression of a second subfamily of protein phosphatase 2A catalytic subunit genes in the rice plant (Oryza sativa L.) 2005 Planta Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong. Protein phosphatase 2A (PP2A) is one of the major serine/threonine protein phosphatases in the cell and plays a variety of regulatory roles in metabolism and signal transduction. Previously, we described the structure and expression of two genes encoding PP2A catalytic subunits (PP2Ac)--OsPP2A-1 and OsPP2A-3--in the rice plant (Yu et al. 2003). Here, we report the isolation and characterisation of a second structurally distinguishable PP2Ac subfamily comprised of three additional isogenes, OsPP2A-2, OsPP2A-4 (each containing ten introns) and OsPP2A-5 (which contains nine introns). Northern blot analysis demonstrated that the three isogenes are ubiquitously expressed in all rice tissues during plant development, and differentially expressed in response to high salinity and the combined stresses of drought and heat. Phylogenetic analyses indicated that the two PP2Ac subfamilies are descended from two ancient lineages, which derived from gene duplications that occurred after the monocotyledon-dicotyledon split. In the second subfamily, it is proposed that two duplication events were involved; in which, the initial duplication of a ten-intron primordial gene yielded OsPP2A-2 and the progenitor of OsPP2A-4 and OsPP2A-5. The OsPP2A-4/OsPP2A-5 progenitor, in turn, underwent a second duplication event, resulting in the present day OsPP2A-4 and OsPP2A-5. It is proposed that loss of the 5'-most intron from OsPP2A-5 occurred after these two duplication events. OsPP2A-2 Phosphoenolpyruvate carboxylase intrinsically located in the chloroplast of rice plays a crucial role in ammonium assimilation 2010 Proc Natl Acad Sci U S A Photobiology and Photosynthesis Research Unit, National Institute of Agrobiological Sciences, Kannondai, Tsukuba 305-8602, Japan. Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of primary metabolism in bacteria, algae, and vascular plants, and is believed to be cytosolic. Here we show that rice (Oryza sativa L.) has a plant-type PEPC, Osppc4, that is targeted to the chloroplast. Osppc4 was expressed in all organs tested and showed high expression in the leaves. Its expression in the leaves was confined to mesophyll cells, and Osppc4 accounted for approximately one-third of total PEPC protein in the leaf blade. Recombinant Osppc4 was active in the PEPC reaction, showing V(max) comparable to cytosolic isozymes. Knockdown of Osppc4 expression by the RNAi technique resulted in stunting at the vegetative stage, which was much more marked when rice plants were grown with ammonium than with nitrate as the nitrogen source. Comparison of leaf metabolomes of ammonium-grown plants suggested that the knockdown suppressed ammonium assimilation and subsequent amino acid synthesis by reducing levels of organic acids, which are carbon skeleton donors for these processes. We also identified the chloroplastic PEPC gene in other Oryza species, all of which are adapted to waterlogged soil where the major nitrogen source is ammonium. This suggests that, in addition to glycolysis, the genus Oryza has a unique route to provide organic acids for ammonium assimilation that involves a chloroplastic PEPC, and that this route is crucial for growth with ammonium. This work provides evidence for diversity of primary ammonium assimilation in the leaves of vascular plants. Osppc4 Low-oxygen stress and water deficit induce cytosolic pyruvate orthophosphate dikinase (PPDK) expression in roots of rice, a C3 plant 1998 The Plant Journal Department of Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), Universiteit Gent, Belgium. Pyruvate orthophosphate dikinase (PPDK) is known for its role in C4 photosynthesis but has no established function in C3 plants. Abscisic acid, PEG and submergence were found to markedly induce a protein of about 97 kDa, identified by microsequencing as PPDK, in rice roots (C3). The rice genome was found to contain two ppdk loci, osppdka and osppdkb. We isolated osppdka cDNA, which encodes a cytosolic rice PPDK isoform of 96.6 kDa, that corresponded to the ABA-induced protein from roots. Western blot analysis showed a PPDK induction in roots of rice seedlings during gradual drying, cold, high salt and mannitol treatment, indicating a water deficit response. PPDK was also induced in the roots and sheath of submerged rice seedlings, and in etiolated rice seedlings exposed to an oxygen-free N2 atmosphere, which indicated a low-oxygen stress response. None of the stress treatments induced PPDK protein accumulation in the lamina of green rice seedlings. Ppdk transcripts were found to accumulate in roots of submerged seedlings, concomitant with the induction of alcohol dehydrogenase 1. Low-oxygen stress triggered an increase in PPDK activity in roots and etiolated rice seedlings, accompanied by increases in phosphoenolpyruvate carboxylase and malate dehydrogenase activities. The results indicate that cytosolic PPDK is involved in a metabolic response to water deficit and low-oxygen stress in rice, an anoxia-tolerant species. OsPPDKA OsPPR1, a pentatricopeptide repeat protein of rice is essential for the chloroplast biogenesis 2005 Plant Mol Biol School of Life Sciences and Biotechnology, Korea University, Sungbuk-ku, 136-701 Seoul, Anam-Dong, Korea. In this paper, we report a novel pentatricopeptide repeat (PPR) protein gene in rice. PPR, a characteristic repeat motif consisted of tandem 35 amino acids, has been found in various biological systems including plant. Sequence analysis revealed that the gene designated OsPPR1 consisted of an open reading frame of 2433 nucleotides encoding 810 amino acids that include 11 PPR motifs. Blast search result indicated that the gene did not align with any of the characterized PPR genes in plant. The OsPPR1 gene was found to contain a putative chloroplast transit peptide in the N-terminal region, suggesting that the gene product targets to the chloroplast. Southern blot hybridization indicated that the OsPPR1 is the member of a gene family within the rice genome. Expression analysis and immunoblot analysis suggested that the OsPPR1 was accumulated mainly in rice leaf. Antisense transgenic strategy was used to suppress the expression of OsPPR1 and the resulted transgenic rice showed the typical phenotypes of chlorophyll-deficient mutants; albinism and lethality. Cytological observation using microscopy revealed that the antisense transgenic plant contained a significant defect in the chloroplast development. Taken together, the results suggest that the OsPPR1 is a nuclear gene of rice, encoding the PPR protein that might play a role in the chloroplast biogenesis. This is the first report on the PPR protein required for the chloroplast biogenesis in rice. OsPPR1 Functional characterization of OsPPT1, which encodes p-hydroxybenzoate polyprenyltransferase involved in ubiquinone biosynthesis in Oryza sativa 2006 Plant Cell Physiol Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Uji, Kyoto, 611-0011 Japan. Prenylation of the aromatic intermediate p-hydroxybenzoate (PHB) is a critical step in ubiquinone (UQ) biosynthesis. The enzyme that catalyzes this prenylation reaction is p-hydroxybenzoate polyprenyltransferase (PPT), which substitutes an aromatic proton at the m-position of PHB with a prenyl chain provided by polyprenyl diphosphate synthase. The rice genome contains three PPT candidates that share significant similarity with the yeast PPT (COQ2 gene), and the rice gene showing the highest similarity to COQ2 was isolated by reverse transcription-PCR and designated OsPPT1a. The deduced amino acid sequence of OsPPT1a contained a putative mitochondrial sorting signal at the N-terminus and conserved domains for putative substrate-binding sites typical of PPT protein family members. The subcellular localization of OsPPT1a protein was shown to be mainly in mitochondria based on studies using a green fluorescent protein-PPT fusion. A yeast complementation study revealed that OsPPT1a expression successfully recovered the growth defect of the coq2 mutant. A prenyltransferase assay using recombinant protein showed that OsPPT1a accepted prenyl diphosphates of various chain lengths as prenyl donors, whereas it showed strict substrate specificity for the aromatic substrate PHB as a prenyl acceptor. The apparent K (m) values for geranyl diphosphate and PHB were 59.7 and 6.04 microM, respectively. The requirement by OsPPT1a and COQ2 for divalent cations was also studied, with Mg2+ found to produce the highest enzyme activity. Northern analysis showed that OsPPT1a mRNA was accumulated in all tissues of O. sativa. These results suggest that OsPPT1a is a functional PPT involved in UQ biosynthesis in O. sativa. OsPPT1 Light-dependent induction of OsPR10 in rice (Oryza sativa L.) seedlings by the global stress signaling molecule jasmonic acid and protein phosphatase 2A inhibitors 2001 Plant Science United Graduate School, Tokyo University of Agriculture and Technology, Tokyo, Japan The OsPR10 gene was first shown to be weakly induced only 3 days after treatment with a herbicide, probenazole (N. Midoh, M. Iwata, Cloning and characterization of a probenazole-inducible gene for an intracellular pathogenesis-related protein in rice, Plant Cell Physiol. 37 (1996) 918), and remains uncharacterized till date. We provide first evidence for the induction of OsPR10 by the global signaling molecule, jasmonic acid (JA), and two protein phosphatase (PP) 2A inhibitors, cantharidin (CN) and endothall (EN). These inductions were light- and time-dependent, and also specific to the dose of applied JA, CN and EN. OsPR10 was not responsive to cut, and was expressed mainly in leaves and leaf sheaths, but not in roots. Furthermore, OsPR10 induction was blocked completely by cycloheximide, indicating requirement of de novo protein synthesis in its induction. Two signal transducers, salicylic acid (SA) and abscisic acid (ABA) induced OsPR10, and a cytokinin, kinetin (KN) also strongly up-regulated its expression, under light. Simultaneously applied staurosporine, a serine/threonine protein kinase inhibitor, strongly enhanced the JA inducible OsPR10 expression. The inducible nature of OsPR10 was affected significantly by a combination of applied JA, with varying concentrations of SA, ABA and KN. Induction of OsPR10 was followed by the appearance of necrotic lesions/coalescing necrotic regions clearly visible at 48 h under light, with CN and EN, but not with JA at a similar time period. Finally, using two-dimensional polyacrylamide gel electrophoresis, we identified a new member of the OsPR10 protein family, the OsPR10-3 protein. This represents the first systematic report in rice demonstrating that the inducible OsPR10 expression is light dependent, and may be regulated via a kinase-signaling cascade. OsPR10a|PBZ1 Identification of an OsPR10a promoter region responsive to salicylic acid 2008 Planta National Institute of Agricultural Biotechnology, Rural Development Administration, Suwon, South Korea. Orysa sativa pathogenesis-related protein 10a (OsPR10a) was induced by pathogens, salicylic acid (SA), jasmonic acid (JA), ethephon, abscisic acid (ABA), and NaCl. We tried to analyze the OsPR10a promoter to investigate the transcriptional regulation of OsPR10a by SA. We demonstrated the inducibility of OsPR10a promoter by SA using transgenic Arabidopsis carrying OsPR10a:GFP as well as by transient expression assays in rice. To further identify the promoter region responsible for its induction by SA, four different deletions of the OsPR10a promoter were made, and their activities were measured by transient assays. The construct containing 687-bp OsPR10a promoter from its start codon exhibited a six-fold increase of induction compared to the control in response to SA. Mutation in the W-box like element 1 (WLE 1) between 687 and 637-bp from TGACA to TGAAA completely abolished induction of the OsPR10a promoter by SA, indicating that the WLE 1 between -687 and -637 of OsPR10a promoter is important in SA-mediated OsPR10a expression. We show for the first time that the W-box like element plays a role in SA mediated PR gene expression. OsPR10a|PBZ1 Characterization of PBZ1, a Probenazole-inducible Gene, in Suspension-Cultured Rice Cells 2014 Bioscience, Biotechnology and Biochemistry RIKEN (The Institute of Physical and Chemical Research) Probenazole (PBZ) induces non-race specific resistance in rice plants against rice blast fungus and PBZ1 was identified as a PBZ-inducible gene from rice. The induction of PBZ1 expression in suspension-cultured rice cells was investigated. Northern blot analysis indicated that PBZ1 was induced by PBZ in a dosedependent manner. Enzyme-linked immunosorbent assay (ELISA) showed a dose and time-dependent accumulation of PBZ1 protein. Both mRNA and protein analysis showed that PBZ1 was not induced by salicylic acid or an active metabolite, 1,2-benzisothiazole-1,1-dioxide. OsPR10a|PBZ1 A novel rice PR10 protein, RSOsPR10, specifically induced in roots by biotic and abiotic stresses, possibly via the jasmonic acid signaling pathway 2004 Plant Cell Physiol Department of Biological Sciences, Tokyo Metropolitan University, Minami-Osawa, Hachioji-shi, Tokyo, 192-0397 Japan. Plant roots have important roles not only in absorption of water and nutrients, but also in stress tolerance such as desiccation, salt, and low temperature. We have investigated stress-response proteins from rice roots using 2-dimensional polyacrylamide-gel electrophoresis and found a rice protein, RO-292, which was induced specifically in roots when 2-week-old rice seedlings were subjected to salt and drought stress. The full-length RO-292 cDNA was cloned, and was determined to encode a protein of 160 amino acid residues (16.9 kDa, pI 4.74). The deduced amino acid sequence showed high similarity to known rice PR10 proteins, OsPR10a/PBZ1 and OsPR10b. RO-292 mRNA accumulated rapidly upon drought, NaCl, jasmonic acid and probenazole, but not by exposure to low temperature or by abscisic acid and salicylic acid. The RO-292 gene was also up-regulated by infection with rice blast fungus. Interestingly, induction was observed almost exclusively in roots, thus we named the gene RSOsPR10 (root specific rice PR10). The present results indicate that RSOsPR10 is a novel rice PR10 protein, which is rapidly induced in roots by salt, drought stresses and blast fungus infection possibly through activation of the jasmonic acid signaling pathway, but not the abscisic acid and salicylic acid signaling pathway. OsPR10a|PBZ1,OsPR10b,RSOsPR10 Gene editing a constitutively active OsRac1 by homologous recombination-based gene targeting induces immune responses in rice 2013 Plant Cell Physiol Laboratory of Plant Molecular Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, 630-0192 Japan. OsRac1 is a member of the plant small GTPase Rac/Rop family and plays a key role in rice immunity. The constitutively active (CA) G19V mutation of OsRac1 was previously shown to induce reactive oxygen species production, phytoalexin synthesis and defense gene activation, leading to resistance to rice blast infection. To study further the effect of the G19V mutation in disease resistance, we introduced a single base substitution by gene targeting and removed the selectable marker using Cre-loxP site-specific recombination. The CA-OsRac1 gene generated by gene targeting was termed CA-gOsRac1. The G19V mutation was transferred from a targeting vector to the OsRac1 locus and stably transmitted to the next generation. In the leaf blade of homozygous CA-gOsRac1 plants, mutant transcript levels were much lower than in those of wild-type plants. In contrast, mutant transcripts in roots, leaf sheaths and panicles were more abundant than those in leaf blades. However, upon chitin treatment, the expression of defense-related genes PAL1 and PBZ1 in the cell culture was greater in the mutants compared with wild-type plants. Furthermore, induction of hypersensitive response (HR)-like cell death was observed in the leaf sheaths of mutant plants infected with a compatible race of rice blast fungus. In the CA-gOsRac1 plants, a number of genes previously shown to be induced by Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae (Xoo) infection were induced in the leaf sheath without pathogen infection. These results suggest that gene targeting will provide mutations useful for gene function studies and crop improvement. OsPR10a|PBZ1,OsRac1 OsRap2.6 transcription factor contributes to rice innate immunity through its interaction with Receptor for Activated Kinase-C 1 (RACK1) 2012 Rice (N Y) Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. BACKGROUND: The rice small GTPase OsRac1 is a molecular switch in rice innate immunity. The Receptor for Activated Kinase C-1 (RACK1) interacts with OsRac1 to suppress the growth of the rice blast fungus, Magnaporthe oryzae. RACK1 has two homologs in rice, RACK1A and RACK1B. Overexpressing RACK1A enhances resistance to the rice blast fungus. However, RACK1A downstream signals are largely unknown. RESULTS: Here, we report the identification of OsRap2.6, a transcription factor that interacts with RACK1A. We found a 94% similarity between the OsRap2.6 AP2 domain and Arabidopsis Rap2.6 (AtRap2.6). Bimolecular fluorescence complementation (BiFC) assays in rice protoplasts using tagged OsRap2.6 and RACK1A with the C-terminal and N-terminal fragments of Venus (Vc/Vn) indicated that OsRap2.6 and RACK1A interacted and localized in the nucleus and the cytoplasm. Moreover, OsRap2.6 and OsMAPK3/6 interacted in the nucleus and the cytoplasm. Expression of defense genes PAL1 and PBZ1 as well as OsRap2.6 was induced after chitin treatment. Disease resistance analysis using OsRap2.6 RNAi and overexpressing (Ox) plants infected with the rice blast fungus indicated that OsRap2.6 RNAi plants were highly susceptible, whereas OsRap2.6 Ox plants had an increased resistance to the compatible blast fungus. CONCLUSIONS: OsRap2.6 contributes to rice innate immunity through its interaction with RACK1A in compatible interactions. OsPR10a|PBZ1,OsRap2.6,OsRACK1A A novel rice (Oryza sativa L.) acidic PR1 gene highly responsive to cut, phytohormones, and protein phosphatase inhibitors 2000 Biochem Biophys Res Commun Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal. gkanitu@abr.affrc.go.jp A novel rice acidic pathogenesis-related (PR) class 1 cDNA (OsPR1a) was isolated from jasmonic acid (JA)-treated rice seedling leaf. The OsPR1a cDNA is 830 bp long and contains an open reading frame of 507 nucleotides encoding 168 amino acid residues with a predicted molecular mass of 17,560 and pI of 4.4. The deduced amino acid sequence of OsPR1a has a high level of identity with acidic and basic PR1 proteins from plants. Southern analysis revealed that OsPR1a is a member of a multigene family. The OsPR1a gene was found to be cut-inducible, whereas the phytohormones JA, salicylic acid (SA), 3-indoleacetic acid, gibberellin, and ethylene (using ethylene generator ethephon, ET) enhanced accumulation of OsPR1a transcript, as well as the protein phosphatase inhibitors cantharidin (CN) and endothall (EN). Induced expression of OsPR1a gene by JA, CN or EN, and ET was light/dark- and dose-dependent and was almost completely inhibited by cycloheximide. Dark downregulated CN-, EN-, and ET-induced OsPR1a gene expression, whereas it was further enhanced with JA. SA and abscisic acid blocked JA-induced OsPR1a transcript. Simultaneous application of staurosporine (ST) enhances CH- or EN-induced OsPR1a transcript, but not with JA. This is the first report on cloning of a rice acidic PR1 gene (OsPR1a), which is regulated by phytohormones, phosphorylation/dephosphorylation event(s), and light. OsPR1a Rice (Oryza sativa L.) OsPR1b gene is phytohormonally regulated in close interaction with light signals 2000 Biochem Biophys Res Commun Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal. gkanitu@abr.affrc.go.jp Strategies evolved by plants to counteract a variety of biotic/abiotic stresses include induction of genes encoding pathogenesis-related (PR) protein, in particular the PR class 1 (PR1) gene family, widely used in stress response studies. In spite of its immense importance as a PR family member, and an accepted gene marker in plant disease/defense in dicots, little is known about rice PR1 genes. Recently, we cloned and characterized the first OsPR1a (rice acidic PR1) gene (Agrawal et al. (2000) Biochem. Biophys. Res. Commun. 274, 157-165). Here, we report characterization of a rice basic PR1 (OsPR1b) gene, identified from screening a cDNA library prepared from jasmonic acid (JA)-treated rice seedling leaf, providing detailed and valuable insights into rice PR1 gene expression. The deduced amino acid sequence of OsPR1b reveals only 63.1% homology with the OsPR1a protein, whereas Southern blot analyses indicate that OsPR1b is a multigene family. The JA-inducible OsPR1b gene was also up-regulated by salicylic acid (SA), abscisic acid (ABA), and kinetin (KN). Furthermore, protein phosphatase inhibitors, cantharidin (CN) and endothall (EN) strongly induced the OsPR1b transcript. However, OsPR1b was not cut-responsive, diagrammatically opposite to cut inducibility of OsPR1a. This induction was light-, time-, and dose-dependent, as demonstrated by using, JA, CN, and EN, and completely inhibited by cycloheximide, but not by tetracycline. The simultaneous application of SA, and ABA, with JA, respectively, showed almost complete inhibition of the JA-induced OsPR1b transcript by 200 microM SA or ABA, but not by 100 microM concentrated solutions, suggesting a potential interaction among JA, SA, and ABA, whereas KN dramatically enhanced JA-induced OsPR1b transcript upon simultaneous application. Moreover, a simultaneous application of staurosporine enhances JA-, CN-, and EN-induced OsPR1b transcript, in particular with CN. Finally, a comparative analysis with the OsPR1a gene gives us insight into the differential regulation of the PR1 gene family, while proposing OsPR1 genes as important gene markers in rice, with potential use(s) in analyzing plant defense responses. OsPR1b The jasmonate pathway is a key player in systemically induced defense against root knot nematodes in rice 2011 Plant Physiol Department of Molecular Biotechnology, Ghent University, B-9000 Ghent, Belgium. Complex defense signaling pathways, controlled by different hormones, are involved in the reaction of plants to a wide range of biotic and abiotic stress factors. We studied the ability of salicylic acid, jasmonate (JA), and ethylene (ET) to induce systemic defense in rice (Oryza sativa) against the root knot nematode Meloidogyne graminicola. Exogenous ET (ethephon) and JA (methyl jasmonate) supply on the shoots induced a strong systemic defense response in the roots, exemplified by a major up-regulation of pathogenesis-related genes OsPR1a and OsPR1b, while the salicylic acid analog BTH (benzo-1,2,3-thiadiazole-7-carbothioic acid S-methyl ester) was a less potent systemic defense inducer from shoot to root. Experiments with JA biosynthesis mutants and ET-insensitive transgenics showed that ET-induced defense requires an intact JA pathway, while JA-induced defense was still functional when ET signaling was impaired. Pharmacological inhibition of JA and ET biosynthesis confirmed that JA biosynthesis is needed for ET-induced systemic defense, and quantitative real-time reverse transcription-polymerase chain reaction data revealed that ET application onto the shoots strongly activates JA biosynthesis and signaling genes in the roots. All data provided in this study point to the JA pathway to play a pivotal role in rice defense against root knot nematodes. The expression of defense-related genes was monitored in root galls caused by M. graminicola. Different analyzed defense genes were attenuated in root galls caused by the nematode at early time points after infection. However, when the exogenous defense inducers ethephon and methyl jasmonate were supplied to the plant, the nematode was less effective in counteracting root defense pathways, hence making the plant more resistant to nematode infection. OsPR1b Characteristic expression of twelve rice PR1 family genes in response to pathogen infection, wounding, and defense-related signal compounds (121/180) 2008 Mol Genet Genomics National Institute of Agrobiological Sciences (NIAS), Kannon-dai 2-1-2, Tsukuba, Ibaraki, 305-8602, Japan. mituhara@affrc.go.jp Pathogenesis-related (PR) proteins have been used as markers of plant defense responses, and are classified into 17 families. However, precise information on the majority members in specific PR families is still limited. We were interested in the individual characteristics of rice PR1 family genes, and selected 12 putatively active genes using rice genome databases for expressed genes. All were upregulated upon compatible and/or incompatible rice-blast fungus interactions; three were upregulated in the early infection period and four in the late infection period. Upon compatible rice-bacterial blight interaction, four genes were upregulated, six were not affected, and one was downregulated. These results are in striking contrast to those among 22 Arabidopsis PR1 genes where only one gene was pathogen-inducible. The responses of individual genes to salicylic acid, jasmonic acid, and ethylene induced defense signaling pathways in rice are likely to be different from those in dicot plants. Transcript levels in healthy leaves, roots, and flowers varied according to each gene. Analysis of the partially overlapping expression patterns of rice PR1 genes in healthy tissues and in response to pathogens and other stresses would be useful to understand their possible functions and for use as characteristic markers for defense-related studies in rice. OsPR1b Molecular cloning and expression analysis of rice phosphoribulokinase gene that is regulated by environmental stresses 2004 Mol Biol Rep Key Laboratory of Ministry of Education for Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, P R China. Phosphoribulokinase (PRKase, EC 2.7.1.19) plays an important role in regulating the flow of sugar through the Calvin cycle. To investigate its regulatory character and expression pattern, the gene encoding PRKase in rice was cloned by RACE. A full-length cDNA with an open reading frame of 1212 bp encoding 403 amino acids residues was obtained from an indica rice variety, 9311. The OsPrk is a single locus gene in the rice genome. It is localized in the region of 28.32 similar to 28.33 Mb on the Chromosome 2, flanked by the genetic markers RM450 and MRG0168. RT-PCR analysis revealed that OsPrk gene was expressed in all the tissues we tested and OsPrk transcript level could be dramatically boosted by light illumination. Its expression was down-regulated by externally applied NaCl, ABA, MeJA and glucose over 24 hr, whereas it was up-regulated by GA after 24 hr treatments. These results also indicated that OsPrk gene expression is modulated by these factors at multiple levels. OsPrk Overexpression of a TFIIIA-type zinc finger protein gene ZFP252 enhances drought and salt tolerance in rice (Oryza sativa L.) 2008 FEBS Lett State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, PR China. We previously identified a salt and drought stress-responsive TFIIIA-type zinc finger protein gene ZFP252 from rice. Here we report the functional analysis of ZFP252 using gain- and loss-of-function strategies. We found that overexpression of ZFP252 in rice increased the amount of free proline and soluble sugars, elevated the expression of stress defense genes and enhanced rice tolerance to salt and drought stresses, as compared with ZFP252 antisense and non-transgenic plants. Our findings suggest that ZFP252 plays an important role in rice response to salt and drought stresses and is useful in engineering crop plants with enhanced tolerance to salt and drought stresses. OsProT|ProT,ZFP252|RZF71 OsPRP3, a flower specific proline-rich protein of rice, determines extracellular matrix structure of floral organs and its overexpression confers cold-tolerance 2010 Plant Mol Biol School of Bio Sciences & Technology, VIT University, Vellore, 632 014, Tamil Nadu, India. gothandam@yahoo.com Proline-rich protein (PRP), a cell wall protein of plant, has been studied in many plant species. Yet, none of the PRPs has been functionally elucidated. Here we report a novel flower-specific PRP designated OsPRP3 from rice. Expression analysis showed that the OsPRP3 transcript was mainly present in rice flower and accumulated abundantly during the late stage of the flower development. To study the function of OsPRP3, we constructed and transformed a binary vector containing a full clone of OsPRP3 in sense orientation and also an RNAi vector to achieve overexpression and knockout of the gene, respectively. Our overexpression plants showed a significant increase in cold tolerance than the WT plants which is conferred by the accumulation of OsPRP3 protein during cold treatment. Further the microscopic analysis revealed that OsPRP3 enhances the cell wall integrity in the cold tolerant plant and confers cold-tolerance in rice. Microscopic analysis of the RNAi mutant flower revealed that blocking OsPRP3 function caused significant defects in floral organogenesis. Taken together, the results suggested that OsPRP3 is a cell wall protein, playing a crucial role in determining extracellular matrix structure of floral organs. OsPRP3 A Rapid and Efficient System of Agrobacterium Infection-Mediated Transient Gene Expression in Rice Oc Cells and Its Application for Analysis of the Expression and Antisense Suppression of Preprophytosulfokine, a Precursor of Phytosulfokine-alpha, Encoded by OsPSK Gene 2000 Plant Cell Physiol Laboratory of Bioactive Natural Product Chemistry, Graduate School of Bio-Agricultural Sciences, Nagoya University, Chikusa, Japan. A rapid and efficient system for Agrobacterium infection-mediated transient gene expression in rice has been developed. Using this system, transient expression of preprophytosulfokine, a precursor of phytosulfokine-a, encoded by OsPSK gene was analyzed. The results suggest that the Agrobacterium infection-mediated transient gene expression system is as efficient in rice Oc cells as in tobacco BY-2 cells and might be useful for rapid analysis not only of foreign gene expression, but also of antisense gene suppression. OsPSK Molecular cloning and characterization of OsPSK, a gene encoding a precursor for phytosulfokine-alpha, required for rice cell proliferation 2000 Plant Mol Biol Laboratory of Bioactive Natural Product Chemistry, Graduate School of Bio-Agricultural Sciences, Nagoya University, Chikusa, Japan. hyang@nagoya-u.ac.jp We previously characterized an OsPSK cDNA encoding a precursor of phytosulfokine-alpha (PSK-alpha), a peptide plant growth factor. Southern blot analysis suggested that OsPSK is a single-copy gene in rice, which we have isolated and characterized. The OsPSK gene consists of one large intron and two exons. The 5-amino acid PSK-alpha sequence located close to the COOH-terminus of the precursor is encoded in the second exon. A putative TATA box was found at position -68 with respect to the transcription initiation site. Upstream of this sequence, several potential regulatory elements, including one CAAT-box, three CCAAT-boxes, one enhancer core-like sequence, and three E-boxes could be identified. By constructing plasmids with various lengths of the 5'-upstream regions of the OsPSK gene fused to the coding sequence for bacterial beta-glucuronidase (GUS), we demonstrated a region 1.9 kb upstream of the transcription initiation point, which contains most of the putative 5'-regulatory elements, to be sufficient for maximal-level GUS expression in transformed rice Oc cells. The promoter of the OsPSK gene gave significantly higher levels of GUS expression than the CaMV 35S promoter. These results suggest that the OsPSK promoter could be useful for the constitutive expression of a foreign gene at high levels in transformed rice culture cells. Northern blot analyses suggest that the expression of OsPSK is reinforced by auxin and cytokinin. OsPSK Oryza sativa PSK gene encodes a precursor of phytosulfokine-alpha , a sulfated peptide growth factor found in plants 1999 Proceedings of the National Academy of Sciences Laboratories of Bioactive Natural Product Chemistry and Biochemistry, Graduate School of Bio-Agricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan Phytosulfokine-α [PSK-α, Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln], a sulfated mitogenic peptide found in plants, strongly promotes proliferation of plant cells in culture at very low concentrations. Oryza sativa PSK (OsPSK) cDNA encoding a PSK-α precursor has been isolated. The cDNA is 725 base pairs long, and the 89-aa product, preprophytosulfokine, has a 22-aa hydrophobic region that resembles a cleavable leader peptide at its NH2 terminus. The PSK-α sequence occurs only once within the precursor, close to the COOH terminus. [Ser4]PSK-α was secreted by transgenic rice Oc cells harboring a mutated OsPSK cDNA, suggesting proteolytic processing from the larger precursor, a feature commonly found in animal systems. Whereas PSK-α in conditioned medium with sense transgenic Oc cells was 1.6 times as concentrated as in the control case, antisense transgenic Oc cells produced less than 60% of the control level. Preprophytosulfokine mRNA was detected at an elevated constitutive level in rice Oc culture cells on RNA blot analysis. Although PSK-α molecules have never been identified in any intact plant, reverse transcription–PCR analysis demonstrated that OsPSK is expressed in rice seedlings, indicating that PSK-α may be important for plant cell proliferation both in vitro and in vivo. DNA blot analysis demonstrated that OsPSK homologs may occur in dicot as well as monocot plants. OsPSK PSY3, a new member of the phytoene synthase gene family conserved in the Poaceae and regulator of abiotic stress-induced root carotenogenesis 2008 Plant Physiol Department of Biological Sciences, Lehman College, The City University of New York, Bronx, NY 10468, USA. Abscisic acid (ABA) plays a vital role in mediating abiotic stress responses in plants. De novo ABA biosynthesis involves cleavage of carotenoid precursors by 9-cis-epoxycarotenoid dioxygenase (NCED), which is rate controlling in leaves and roots; however, additional bottlenecks in roots must be overcome, such as biosynthesis of upstream carotenoid precursors. Phytoene synthase (PSY) mediates the first committed step in carotenoid biosynthesis; with PSY3 described here, maize (Zea mays) and other members of the Poaceae have three paralogous genes, in contrast to only one in Arabidopsis thaliana. PSY gene duplication has led to subfunctionalization, with each paralog exhibiting differential gene expression. We showed that PSY3 encodes a functional enzyme for which maize transcript levels are regulated in response to abiotic stresses, drought, salt, and ABA. Drought-stressed roots showed elevated PSY3 transcripts and ABA, responses reversed by rehydration. By blocking root carotenoid biosynthesis with the maize y9 mutation, we demonstrated that PSY3 mRNA elevation correlates with carotenoid accumulation and that blocking carotenoid biosynthesis interferes with stress-induced ABA accumulation. In parallel, we observed elevated NCED transcripts and showed that, in contrast to dicots, root zeaxanthin epoxidase transcripts were unchanged. PSY3 was the only paralog for which transcripts were induced in roots and abiotic stress also affected leaf PSY2 transcript levels; PSY1 mRNA was not elevated in any tissues tested. Our results suggest that PSY3 expression influences root carotenogenesis and defines a potential bottleneck upstream of NCED; further examination of PSY3 in the grasses is of value for better understanding root-specific stress responses that impact plant yield. OsPSY|OsPSY3,PSY1,PSY2 A third phytoene synthase is devoted to abiotic stress-induced abscisic acid formation in rice and defines functional diversification of phytoene synthase genes 2008 Plant Physiol Faculty of Biology, Center for Applied Biosciences, University of Freiburg, 79104 Freiburg, Germany. We here report on the characterization of a novel third phytoene synthase gene (PSY) in rice (Oryza sativa), OsPSY3, and on the differences among all three PSY genes with respect to the tissue-specific expression and regulation upon various environmental stimuli. The two already known PSYs are under phytochrome control and involved in carotenoid biosynthesis in photosynthetically active tissues and exhibit different expression patterns during chloroplast development. In contrast, OsPSY3 transcript levels are not affected by light and show almost no tissue-specific differences. Rather, OsPSY3 transcripts are up-regulated during increased abscisic acid (ABA) formation upon salt treatment and drought, especially in roots. The simultaneous induction of genes encoding 9-cis-epoxycarotenoid dioxygenases (NCEDs), involved in the initial steps of ABA biosynthesis, indicate that decreased xanthophyll levels are compensated by the induction of the third PSY gene. Furthermore, OsPSY3 and the OsNCEDs investigated were also induced by the application of ABA, indicating positive feedback regulation. The regulatory differences are mirrored by cis-acting elements in the corresponding promoter regions, with light-responsive elements for OsPSY1 and OsPSY2 and an ABA-response element as well as a coupling element for OsPSY3. The investigation of the gene structures and 5' untranslated regions revealed that OsPSY1 represents a descendant of an ancient PSY gene present in the common ancestor of monocots and dicots. Since the genomic structures of OsPSY2 and OsPSY3 are comparable, we conclude that they originated from the most recent common ancestor, OsPSY1. OsPSY|OsPSY3,PSY1,PSY2 OsPTF1, a novel transcription factor involved in tolerance to phosphate starvation in rice 2005 Plant Physiol State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310029, People's Republic of China. We report here on a novel transcription factor with a basic helix-loop-helix domain for tolerance to inorganic phosphate (Pi) starvation in rice (Oryza sativa). The gene is designated OsPTF1. The expression of OsPTF1 is Pi starvation induced in roots while constitutively expressed in shoots, as shown by northern-blot analysis. Overexpression of OsPTF1 enhanced tolerance to Pi starvation in transgenic rice. Tillering ability, root and shoot biomass, and phosphorus content of transgenic rice plants were about 30% higher than those of the wild-type plants in Pi-deficient conditions in hydroponic experiments. In soil pot and field experiments, more than 20% increase in tiller number, panicle weight, and phosphorus content was observed in transgenic plants compared to wild-type plants at low-Pi levels. In Pi-deficient conditions, transgenic rice plants showed significantly higher total root length and root surface area, which results in a higher instantaneous Pi uptake rate over their wild-type counterparts. Microarray analysis for transgenic plants overexpressing OsPTF1 has been performed to investigate the downstream regulation of OsPTF1. OsPTF1 Rice Pti1a negatively regulates RAR1-dependent defense responses 2007 Plant Cell Department of Molecular Genetics, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan. Tomato (Solanum lycopersicum) Pto encodes a protein kinase that confers resistance to bacterial speck disease. A second protein kinase, Pti1, physically interacts with Pto and is involved in Pto-mediated defense signaling. Pti1-related sequences are highly conserved among diverse plant species, including rice (Oryza sativa), but their functions are largely unknown. Here, we report the identification of a null mutant for the Pti1 homolog in rice and the functional characterization of Os Pti1a. The rice pti1a mutant was characterized by spontaneous necrotic lesions on leaves, which was accompanied by a series of defense responses and resistance against a compatible race of Magnaporthe grisea. Overexpression of Pti1a in rice reduced resistance against an incompatible race of the fungus recognized by a resistance (R) protein, Pish. Plants overexpressing Pti1a were also more susceptible to a compatible race of the bacterial pathogen Xanthomonas oryzae pv oryzae. These results suggest that Os Pti1a negatively regulates defense signaling for both R gene-mediated and basal resistance. We also demonstrated that repression of the rice RAR1 gene suppressed defense responses induced in the pti1a mutant, indicating that Pti1a negatively regulates RAR1-dependent defense responses. Expression of a tomato Pti1 cDNA in the rice pti1a mutant suppressed the mutant phenotypes. This contrasts strikingly with the previous finding that Sl Pti1 enhances Pto-mediated hypersensitive response (HR) induction when expressed in tobacco (Nicotiana tabacum), suggesting that the molecular switch controlling HR downstream of pathogen recognition has evolved differently in rice and tomato. OsPti1a,PISH Altered expression of the PTR/NRT1 homologue OsPTR9 affects nitrogen utilization efficiency, growth and grain yield in rice 2013 Plant Biotechnol J Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China. The plant PTR/NRT1 (peptide transporter/nitrate transporter 1) gene family comprises di/tripeptide and low-affinity nitrate transporters; some members also recognize other substrates such as carboxylates, phytohormones (auxin and abscisic acid), or defence compounds (glucosinolates). Little is known about the members of this gene family in rice (Oryza sativa L.). Here, we report the influence of altered OsPTR9 expression on nitrogen utilization efficiency, growth, and grain yield. OsPTR9 expression is regulated by exogenous nitrogen and by the day-night cycle. Elevated expression of OsPTR9 in transgenic rice plants resulted in enhanced ammonium uptake, promotion of lateral root formation and increased grain yield. On the other hand, down-regulation of OsPTR9 in a T-DNA insertion line (osptr9) and in OsPTR9-RNAi rice plants had the opposite effect. These results suggest that OsPTR9 might hold potential for improving nitrogen utilization efficiency and grain yield in rice breeding. OsPTR9 OsPUB15, an E3 ubiquitin ligase, functions to reduce cellular oxidative stress during seedling establishment 2011 Plant J Department of Life Science, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Korea. The plant U-box (PUB) protein functions as an E3 ligase to poly-ubiquitinate a target protein for its degradation or post-translational modification. Here, we report functional roles for OsPUB15, which encodes a cytosolic U-box protein in the class-II PUB family. Self-ubiquitination assays showed that bacterially expressed MBP-OsPUB15 protein has E3 ubiquitin ligase activity. A T-DNA insertional mutation in OsPUB15 caused severe growth retardation and a seedling-lethal phenotype. Mutant seeds did not produce primary roots, and their shoot development was significantly delayed. Transgenic plants expressing the OsPUB15 antisense transcript phenocopied these mutant characters. The abnormal phenotypes were partially rescued by two antioxidants, catechin and ascorbic acid. Germinating seeds in the dark also recovered the rootless defect. Levels of H2O2 and oxidized proteins were higher in the knock-out mutant compared with the wild type. OsPUB15 transcript levels were increased upon H2O2, salt and drought stresses; plants overexpressing the gene grew better than the wild type under high salinity. These results indicate that PUB15 is a regulator that reduces reactive oxygen species (ROS) stress and cell death. OsPUB15 Characterization of a purine permease family gene OsPUP7 involved in growth and development control in rice 2013 J Integr Plant Biol National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, 430070, China. In this study, PUP-type cytokinin transporter genes were identified in rice (Oryza sativa L.). The Oryza sativa purine permease (OsPUP) family has 12 members that show similar predicted protein sequences with AtPUPs. To reveal the functions of OsPUP genes, we searched the T-DNA mutant library of rice and found one mutant for the member OsPUP7. The T-DNA insertion caused a new transcript that encodes a protein with 26 amino acids different from the native OsPUP7 at the C-terminus. The mutant showed multiple phenotypic changes including increased plant height, big seeds, and delayed flowering. The mutant also showed increased sensitivity to drought and salt stresses and treatments with kinetin and abscisic acid. OsPUP7 is expressed mainly in the vascular bundle, pistil, and stamens. The measurement of cytokinins (CKs) showed that CK content in the mutant spikelets accumulated higher than that in the wild type. Moreover, uptake experiment in the yeast fcy2 mutant suggested that OsPUP7 has the ability to transport caffeine, a CK derivative. Our results indicate that the PUP transport system also exists in rice, and OsPUP7 has an important role in the transport of CK, thus affecting developmental process and stress responses. OsPUP7 Functional analysis of OsPUT1, a rice polyamine uptake transporter 2012 Planta Department of Biological Sciences, Bowling Green State University, 442, Life Sciences Building, Bowling Green, OH, 43403-09, USA. Polyamines are nitrogenous compounds found in all eukaryotic and prokaryotic cells and absolutely essential for cell viability. In plants, they regulate several growth and developmental processes and the levels of polyamines are also correlated with the plant responses to various biotic and abiotic stresses. In plant cells, polyamines are synthesized in plastids and cytosol. This biosynthetic compartmentation indicates that the specific transporters are essential to transport polyamines between the cellular compartments. In the present study, a phylogenetic analysis was used to identify candidate polyamine transporters in rice. A full-length cDNA rice clone AK068055 was heterologously expressed in the Saccharomyces cerevisiae spermidine uptake mutant, agp2. Radiological uptake and competitive inhibition studies with putrescine indicated that rice gene encodes a protein that functioned as a spermidine-preferential transporter. In competition experiments with several amino acids at 25-fold higher levels than spermidine, only methionine, asparagine, and glutamine were effective in reducing uptake of spermidine to 60% of control rates. Based on those observations, this rice gene was named polyamine uptake transporter 1 (OsPUT1). Tissue-specific expression of OsPUT1 by semiquantitative RT-PCR showed that the gene was expressed in all tissues except seeds and roots. Transient expression assays in onion epidermal cells and rice protoplasts failed to localize to a cellular compartment. The characterization of the first plant polyamine transporter sets the stage for a systems approach that can be used to build a model to fully define how the biosynthesis, degradation, and transport of polyamines in plants mediate developmental and biotic responses. OsPUT1 Kinetic and phylogenetic analysis of plant polyamine uptake transporters 2012 Planta Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403, USA. The rice gene Polyamine Uptake Transporter1 (PUT1) was originally identified based on its homology to the polyamine uptake transporters LmPOT1 and TcPAT12 in Leishmania major and Trypanosoma cruzi, respectively. Here we show that five additional transporters from rice and Arabidopsis that cluster in the same clade as PUT1 all function as high affinity spermidine uptake transporters. Yeast expression assays of these genes confirmed that uptake of spermidine was minimally affected by 166 fold or greater concentrations of amino acids. Characterized polyamine transporters from both Arabidopsis thaliana and Oryza sativa along with the two polyamine transporters from L. major and T. cruzi were aligned and used to generate a hidden Markov model. This model was used to identify significant matches to proteins in other angiosperms, bryophytes, chlorophyta, discicristates, excavates, stramenopiles and amoebozoa. No significant matches were identified in fungal or metazoan genomes. Phylogenic analysis showed that some sequences from the haptophyte, Emiliania huxleyi, as well as sequences from oomycetes and diatoms clustered closer to sequences from plant genomes than from a homologous sequence in the red algal genome Galdieria sulphuraria, consistent with the hypothesis that these polyamine transporters were acquired by horizontal transfer from green algae. Leishmania and Trypansosoma formed a separate cluster with genes from other Discicristates and two Entamoeba species. We surmise that the genes in Entamoeba species were acquired by phagotrophy of Discicristates. In summary, phylogenetic and functional analysis has identified two clades of genes that are predictive of polyamine transport activity. OsPUT1,OsPUT2 A rice orthologue of the ABA receptor, OsPYL/RCAR5, is a positive regulator of the ABA signal transduction pathway in seed germination and early seedling growth 2012 J Exp Bot Department of Bio-crop development, National Academy of Agricultural Science, Rural Development Administration, Suwon, 441-707, Korea. Abscisic acid (ABA) is a phytohormone that positively regulates seed dormancy and stress tolerance. PYL/RCARs were identified an intracellular ABA receptors regulating ABA-dependent gene expression in Arabidopsis thaliana. However, their function in monocot species has not been characterized yet. Herein, it is demonstrated that PYL/RCAR orthologues in Oryza sativa function as a positive regulator of the ABA signal transduction pathway. Transgenic rice plants expressing OsPYL/RCAR5, a PYL/RCAR orthologue of rice, were found to be hypersensitive to ABA during seed germination and early seedling growth. A rice ABA signalling unit composed of OsPYL/RCAR5, OsPP2C30, SAPK2, and OREB1 for ABA-dependent gene regulation was further identified, via interaction assays and a transient gene expression assay. Thus, a core signalling unit for ABA-responsive gene expression modulating seed germination and early seedling growth in rice has been unravelled. This study provides substantial contributions toward understanding the ABA signal transduction pathway in rice. OsPYL|RCAR5 Overexpression of PYL5 in rice enhances drought tolerance, inhibits growth, and modulates gene expression 2014 J Exp Bot Molecular Breeding Division, National Academy of Agricultural Science, RDA, Suwon 441-707, Republic of Korea. Abscisic acid (ABA) is a phytohormone that plays important roles in the regulation of seed dormancy and adaptation to abiotic stresses. Previous work identified OsPYL/RCARs as functional ABA receptors regulating ABA-dependent gene expression in Oryza sativa. OsPYL/RCARs thus are considered to be good candidate genes for improvement of abiotic stress tolerance in crops. This work demonstrates that the cytosolic ABA receptor OsPYL/RCAR5 in O. sativa functions as a positive regulator of abiotic stress-responsive gene expression. The constitutive expression of OsPYL/RCAR5 in rice driven by the Zea mays ubiquitin promoter induced the expression of many stress-responsive genes even under normal growth conditions and resulted in improved drought and salt stress tolerance in rice. However, it slightly reduced plant height under paddy field conditions and severely reduced total seed yield. This suggests that, although exogenous expression of OsPYL/RCAR5 is able to improve abiotic stress tolerance in rice, fine regulation of its expression will be required to avoid deleterious effects on agricultural traits. OsPYL|RCAR5 Overexpression of OsRAA1 causes pleiotropic phenotypes in transgenic rice plants, including altered leaf, flower, and root development and root response to gravity 2004 Plant Physiol Research Center for Molecular and Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China. There are very few root genes that have been described in rice as a monocotyledonous model plant so far. Here, the OsRAA1 (Oryza sativa Root Architecture Associated 1) gene has been characterized molecularly. OsRAA1 encodes a 12.0-kD protein that has 58% homology to the AtFPF1 (Flowering Promoting Factor 1) in Arabidopsis, which has not been reported as modulating root development yet. Data of in situ hybridization and OsRAA1::GUS transgenic plant showed that OsRAA1 expressed specifically in the apical meristem, the elongation zone of root tip, steles of the branch zone, and the young lateral root. Constitutive expression of OsRAA1 under the control of maize (Zea mays) ubiquitin promoter resulted in phenotypes of reduced growth of primary root, increased number of adventitious roots and helix primary root, and delayed gravitropic response of roots in seedlings of rice (Oryza sativa), which are similar to the phenotypes of the wild-type plant treated with auxin. With overexpression of OsRAA1, initiation and growth of adventitious root were more sensitive to treatment of auxin than those of the control plants, while their responses to 9-hydroxyfluorene-9-carboxylic acid in both transgenic line and wild type showed similar results. OsRAA1 constitutive expression also caused longer leaves and sterile florets at the last stage of plant development. Analysis of northern blot and GUS activity staining of OsRAA1::GUS transgenic plants demonstrated that the OsRAA1 expression was induced by auxin. At the same time, overexpression of OsRAA1 also caused endogenous indole-3-acetic acid to increase. These data suggested that OsRAA1 as a new gene functions in the development of rice root systems, which are mediated by auxin. A positive feedback regulation mechanism of OsRAA1 to indole-3-acetic acid metabolism may be involved in rice root development in nature. OsRAA1 Rice ROOT ARCHITECTURE ASSOCIATED1 binds the proteasome subunit RPT4 and is degraded in a D-box and proteasome-dependent manner 2008 Plant Physiol Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China. Root growth is mainly determined by cell division and subsequent elongation in the root apical area. Components regulating cell division in root meristematic cells are largely unknown. Previous studies have identified rice (Oryza sativa) ROOT ARCHITECTURE ASSOCIATED1 (OsRAA1) as a regulator in root development. Yet, the function of OsRAA1 at the cellular and molecular levels is unclear. Here, we show that OsRAA1-overexpressed transgenic rice showed reduced primary root growth, increased numbers of cells in metaphase, and reduced numbers of cells in anaphase, which suggests that OsRAA1 is responsible for limiting root growth by inhibiting the onset of anaphase. The expression of OsRAA1 in fission yeast also induced metaphase arrest, which is consistent with the fact that OsRAA1 functions through a conserved mechanism of cell cycle regulation. Moreover, a colocalization assay has shown that OsRAA1 is expressed predominantly at spindles during cell division. Yeast two-hybrid and pull-down assays, as well as a bimolecular fluorescence complementation assay, all have revealed that OsRAA1 interacts with a rice homolog of REGULATORY PARTICLE TRIPLE-A ATPASE4, a component that is involved in the ubiquitin pathway. Treating transgenic rice with specific inhibitors of the 26S proteasome blocked the degradation of OsRAA1 and increased the number of cells in metaphase. Mutation of a putative ubiquitination-targeting D-box (RGSLDLISL) in OsRAA1 interrupted the destruction of OsRAA1 in transgenic yeast. These results suggest that ubiquitination and proteasomic proteolysis are involved in OsRAA1 degradation, which is essential for the onset of anaphase, and that OsRAA1 may modulate root development mediated by the ubiquitin-proteasome pathway as a novel regulatory factor of the cell cycle. OsRAA1,OsRPT4 OsRab5a regulates endomembrane organization and storage protein trafficking in rice endosperm cells 2010 Plant J State Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, China. Rice glutelins are synthesized at the endoplasmic reticulum (ER) as precursors (pro-glutelins), and are transported to protein storage vacuoles, where they are processed into mature proteins. The molecular basis of this process is largely unknown. Here, we report the isolation of a rice mutant, gpa1, that accumulates 57 kDa pro-glutelins in seeds and whose endosperm has a floury appearance. Transmission electron microscopy analysis showed that the gpa1 endosperm cells have an enlarged ER lumen and a smaller protein body II (PBII), and accumulated three types of newly generated subcellular structures. Moreover, a proportion of glutelins in the gpa1 endosperm cells were not delivered to PBII, and instead were mis-targeted to two of the newly generated structures or secreted. The gene corresponding to the gpa1 mutation was found to be OsRab5a, which encodes a small GTPase. In Arabidopsis protoplasts, OsRab5a protein was found to co-localize predominantly with AtVSR2, a molecular marker for the pre-vacuolar compartments (PVC). We conclude that OsRab5a plays an essential role in trafficking of storage protein to PBII, possibly as part of its function in organizing the endomembrane system in developing endosperm cells of rice. OsRab5a|gpa1|glup4 The small GTPase Rab5a is essential for intracellular transport of proglutelin from the Golgi apparatus to the protein storage vacuole and endosomal membrane organization in developing rice endosperm 2011 Plant Physiol Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan. Rice (Oryza sativa) glutelins are synthesized on the endoplasmic reticulum as larger precursors, which are then transported via the Golgi to the protein storage vacuole (PSV), where they are processed into acidic and basic subunits. Three independent glutelin precursor mutant4 (glup4) rice lines, which accumulated elevated levels of proglutelin over the wild type, were identified as loss-of-function mutants of Rab5a, the small GTPase involved in vesicular membrane transport. In addition to the plasma membrane, Rab5a colocalizes with glutelins on the Golgi apparatus, Golgi-derived dense vesicles, and the PSV, suggesting that Rab5a participates in the transport of the proglutelin from the Golgi to the PSV. This spatial distribution pattern was dramatically altered in the glup4 mutants. Numerous smaller protein bodies containing glutelin and alpha-globulin were evident, and the proteins were secreted extracellularly. Moreover, all three independent glup4 allelic lines displayed the novel appearance of a large dilated, structurally complex paramural body containing proglutelins, alpha-globulins, membrane biomarkers for the Golgi apparatus, prevacuolar compartment, PSV, and the endoplasmic reticulum luminal chaperones BiP and protein disulfide isomerase as well as beta-glucan. These results indicate that the formation of the paramural bodies in glup4 endosperm was due to a significant disruption of endocytosis and membrane vesicular transport by Rab5a loss of function. Overall, Rab5a is required not only for the intracellular transport of proglutelins from the Golgi to the PSV in rice endosperm but also in the maintenance of the general structural organization of the endomembrane system in developing rice seeds. OsRab5a|gpa1|glup4,VSR3|OsVSR3 Overexpression ofOsRab7B3, a Small GTP-Binding Protein Gene, Enhances Leaf Senescence in Transgenic Rice 2014 Bioscience, Biotechnology and Biochemistry Department of Biology, Faculty of Science, Chiang Mai University, Muang, Chiang Mai, Thailand. Rab family proteins are small GTP-binding proteins involved in intracellular trafficking. They play critical roles in several plant development processes. Different expression patterns of 46 Rabs in the rice genome were examined in various rice tissues and in leaves treated with plant growth regulators and under senescence conditions. One of the OsRab genes, OsRab7B3, closely associated with senescence in expression pattern, was chosen for functional analysis. Expression of sGFP under the control of the OsRab7B3 promoter increased in leaves when ABA and NaCl were applied or when kept in dark. In transgenic rice overexpressing OsRab7B3, the senescence-related genes were upregulated and leaf senescence was significantly enhanced under dark conditions. Moreover, leaf yellowing occurred earlier in the transgenic plants than in the wild type at the ripening stage. Hence it is suggested that OsRab7B3 act as a stress-inducible gene that plays an important role in the leaf senescence process. OsRab7|OsRab7B3 Molecular and biochemical analyses of OsRab7, a rice Rab7 homolog 2003 Plant Cell Physiol Division of Applied Life Science (BK21), Gyeongsang National University, Jinju, 660-701 Korea. Rab7 is a small GTP-binding protein important in early to late endosome/lysosome vesicular transport in mammalian cells. We have isolated a Rab7 cDNA clone, OsRab7, from a cold-treated rice cDNA library by the subtraction screening method. The cDNA encodes a polypeptide of 206 amino acids with a calculated molecular mass of about 23 kDa. Its predicted amino acid sequence shows significantly high identity with the sequences of other Rab7 proteins. His-tagged OsRab7 bound to radiolabeled GTPgammaS in a specific and stoichiometric manner. Biochemical and structural properties of the Rab7 wild type (WT) protein were compared to those of Q67L and T22N mutants. The detergent 3-([3-cholamidopropyl]dimethylammonio)-1-propane sulfonate (CHAPS) increased the guanine nucleotide binding and hydrolysis activities of Rab7WT. The OsRab7Q67L mutant showed much lower GTPase activity compared to the WT protein untreated with CHAPS, and the T22N mutant showed no GTP binding activity at all. The OsRab7Q67L mutant was constitutively active for guanine nucleotide binding while the T22N mutant (dominant negative) showed no guanine nucleotide binding activity. When bound to GTP, the Rab7WT and the Q67L mutants were protected from tryptic proteolysis. The cleavage pattern of the Rab7T22N mutant, however, was not affected by GTP addition. Northern and Western blot analyses suggested that OsRab7 is distributed in various tissues of rice. Furthermore, expression of a rice Rab7 gene was differentially regulated by various environmental stimuli such as cold, NaCl, dehydration, and ABA. In addition, subcellular localization of OsRab7 was investigated in the Arabidopsis protoplasts by a double-labeling experiment using GFP-fused OsRab7 and FM4-64. GFP-OsRab7 is localized to the vacuolar membrane, suggesting that OsRab7 is implicated in a vesicular transport to the vacuole in plant cells. OsRab7|OsRab7B3 The rice bright green leaf (bgl) locus encodes OsRopGEF10, which activates the development of small cuticular papillae on leaf surfaces 2011 Plant Mol Biol Department of Plant Science, Plant Genomics and Breeding Institute, Seoul National University, Seoul 151-921, Republic of Korea. Development of specialized epidermal cells and structures plays a key role in plant tolerance to biotic and abiotic stresses. In the paddy field, the bright green leaf (bgl) mutants of rice (Oryza sativa) exhibit a luminous green color that is clearly distinguishable from the normal green of wild-type plants. Transmission and scanning electron microscopy revealed that small cuticular papillae (or small papillae; SP), nipple-like structures, are absent on the adaxial and abaxial leaf surfaces of bgl mutants, leading to more direct reflection and less diffusion of green light. Map-based cloning revealed that the bgl locus encodes OsRopGEF10, one of eleven OsRopGEFs in rice. RopGEFs (guanine nucleotide exchange factors for Rop) activate Rop/Rac GTPases, acting as molecular switches in eukaryotic signal transduction by replacing the bound GDP (inactive form) with GTP (active form) in response to external or internal cues. In agreement with the timing of SP initiation on the leaf epidermis, OsRopGEF10 is most strongly expressed in newly developing leaves before emergence from the leaf sheath. In yeast two-hybrid assays, OsRopGEF10 interacts with OsRac1, one of seven OsRac proteins; consistent with this, both proteins are localized in the plasma membrane. These results suggest that OsRopGEF10 activates OsRac1 to turn on the molecular signaling pathway for SP development. Together, our findings provide new insights into the molecular genetic mechanism of SP formation during early leaf morphogenesis. OsRac1,OsRopGEF10|BGL SWAP70 functions as a Rac/Rop guanine nucleotide-exchange factor in rice 2012 Plant J Department of Advanced Bioscience, Graduate School of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan. Rho family small GTPases are involved in diverse signaling processes including immunity, growth, and development. The activity of Rho GTPases is regulated by cycling between guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active forms, in which guanine nucleotide exchange factors (GEFs) predominantly function to promote activation of the GTPases. In animals, most Rho GEFs possess a Dbl (diffuse B-cell lymphoma) homology (DH) domain which functions as a GEF-catalytic domain. However, no proteins with the DH domain have been identified in plants so far. Instead, plant-specific Rho GEFs with the PRONE domain responsible for GEF activity have been found to constitute a large family in plants. In this study, we found rice homologs of human SWAP70, Oryza sativa (Os) SWAP70A and SWAP70B, containing the DH domain. OsSWAP70A interacted with rice Rho GTPase OsRac1, an important signaling factor for immune responses. The DH domain of OsSWAP70A exhibited the GEF-catalytic activity toward OsRac1 as found in animal Rho GEFs, indicating that plants have the functional DH domains. Transient expression of OsSWAP70A enhanced OsRac1-mediated production of reactive oxygen species in planta. Reduction of OsSWAP70A and OsSWAP70B mRNA levels by RNA interference resulted in the suppression of chitin elicitor-induced defense gene expression and ROS production. Thus, it is likely that OsSWAP70 regulates immune responses through activation of OsRac1. OsRac1,OsSWAP70A,OsSWAP70B Proteome analysis of detergent-resistant membranes (DRMs) associated with OsRac1-mediated innate immunity in rice 2009 Plant Cell Physiol Laboratory of Plant Protein Analysis, Plant Education Unit, Nara Institute of Science and Technology, Takayama, Ikoma, Japan. OsRac1, a member of the Rac/Rop GTPase family, plays important roles as a molecular switch in rice innate immunity, and the active form of OsRac1 functions in the plasma membrane (PM). To study the precise localization of OsRac1 in the PM and its possible association with other signaling components, we performed proteomic analysis of DRMs (detergent-resistant membranes) isolated from rice suspension-cultured cells transformed with myc-tagged constitutively active (CA) OsRac1. DRMs are regions of the PM that are insoluble after Triton X-100 treatment under cold conditions and are thought to be involved in various signaling processes in animal, yeast and plant cells. We identified 192 proteins in DRMs that included receptor-like kinases (RLKs) such as Xa21, nucleotide-binding leucine-rich repeat (NB-LRR)-type disease resistance proteins, a glycosylphosphatidylinositol (GPI)-anchored protein, syntaxin, NADPH oxidase, a WD-40 repeat family protein and various GTP-binding proteins. Many of these proteins have been previously identified in the DRMs isolated from other plant species, and animal and yeast cells, validating the methods used in our study. To examine the possible association of DRMs and OsRac1-mediated innate immunity, we used rice suspension-cultured cells transformed with myc-tagged wild-type (WT) OsRac1 and found that OsRac1 and RACK1A, an effector of OsRac1, shifted to the DRMs after chitin elicitor treatment. These results suggest that OsRac1-mediated innate immunity is associated with DRMs in the PM. OsRac1,OsRACK1A,xa21 RACK1 functions in rice innate immunity by interacting with the Rac1 immune complex 2008 Plant Cell Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0192, Japan. A small GTPase, Rac1, plays a key role in rice (Oryza sativa) innate immunity as part of a complex of regulatory proteins. Here, we used affinity column chromatography to identify rice RACK1 (for Receptor for Activated C-Kinase 1) as an interactor with Rac1. RACK1 functions in various mammalian signaling pathways and is involved in hormone signaling and development in plants. Rice contains two RACK1 genes, RACK1A and RACK1B, and the RACK1A protein interacts with the GTP form of Rac1. Rac1 positively regulates RACK1A at both the transcriptional and posttranscriptional levels. RACK1A transcription was also induced by a fungal elicitor and by abscisic acid, jasmonate, and auxin. Analysis of transgenic rice plants and cell cultures indicates that RACK1A plays a role in the production of reactive oxygen species (ROS) and in resistance against rice blast infection. Overexpression of RACK1A enhances ROS production in rice seedlings. RACK1A was shown to interact with the N terminus of NADPH oxidase, RAR1, and SGT1, key regulators of plant disease resistance. These results suggest that RACK1A functions in rice innate immunity by interacting with multiple proteins in the Rac1 immune complex. OsRac1,OsRAR1,OsRACK1A,RACK1B|OsWD40-122 Proteomics of Rac GTPase signaling reveals its predominant role in elicitor-induced defense response of cultured rice cells 2006 Plant Physiol Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Ikoma 630-0101, Japan. We have previously shown that a human small GTPase Rac homolog, OsRac1, from rice (Oryza sativa) induces cascades of defense responses in rice plants and cultured cells. Sphingolipid elicitors (SEs) have been similarly shown to activate defense responses in rice. Therefore, to systematically analyze proteins whose expression levels are altered by OsRac1 and/or SE treatment, we performed a differential display analysis of proteins by the use of two-dimensional gel electrophoresis and mass spectrometry. A total of 271 proteins whose expression levels were altered by constitutively active (CA)-OsRac1 or SE were identified. Interestingly, of 100 proteins that were up-regulated by a SE, 87 were also induced by CA-OsRac1, suggesting that OsRac1 plays a pivotal role in defense responses induced by SE in cultured rice cells. In addition, CA-OsRac1 induces the expression of 119 proteins. Many proteins, such as pathogenesis-related proteins, SGT1, and prohibitin, which are known to be involved in the defense response, were found among these proteins. Proteins involved in redox regulation, chaperones such as heat shock proteins, BiP, and chaperonin 60, proteases and protease inhibitors, cytoskeletal proteins, subunits of proteasomes, and enzymes involved in the phenylpropanoid and ethylene biosynthesis pathways were found to be induced by CA-OsRac1 or SE. Results of our proteomic analysis revealed that OsRac1 is able to induce many proteins in various signaling and metabolic pathways and plays a predominant role in the defense response in cultured rice cells. OsRac1,OsSGT1 The small GTP-binding protein Rac is a regulator of cell death in plants 1999 Proc Natl Acad Sci U S A Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan. Cell death plays important roles in the development and defense of plants as in other multicellular organisms. Rapid production of reactive oxygen species often is associated with plant defense against pathogens, but their molecular mechanisms are not known. We introduced the constitutively active and the dominant negative forms of the small GTP-binding protein OsRac1, a rice homolog of human Rac, into the wild type and a lesion mimic mutant of rice and analyzed H(2)O(2) production and cell death in transformed cell cultures and plants. The results indicate that Rac is a regulator of reactive oxygen species production as well as cell death in rice. OsRac1 Essential role of the small GTPase Rac in disease resistance of rice 2001 Proc Natl Acad Sci U S A Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan. Production of reactive oxygen intermediates (ROI) and a form of programmed cell death called hypersensitive response (HR) are often associated with disease resistance of plants. We have previously shown that the Rac homolog of rice, OsRac1, is a regulator of ROI production and cell death in rice. Here we show that the constitutively active OsRac1 (i) causes HR-like responses and greatly reduces disease lesions against a virulent race of the rice blast fungus; (ii) causes resistance against a virulent race of bacterial blight; and (iii) causes enhanced production of a phytoalexin and alters expression of defense-related genes. The dominant-negative OsRac1 suppresses elicitor-induced ROI production in transgenic cell cultures, and in plants suppresses the HR induced by the avirulent race of the fungus. Taken together, our findings strongly suggest that OsRac1 has a general role in disease resistance of rice. OsRac1 Phosphatidylinositol 3-kinase plays a vital role in regulation of rice seed vigor via altering NADPH oxidase activity 2012 PLoS One MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, China. Phosphatidylinositol 3-kinase (PI3K) has been reported to be important in normal plant growth and stress responses. In this study, it was verified that PI3K played a vital role in rice seed germination through regulating NADPH oxidase activity. Suppression of PI3K activity by inhibitors wortmannin or LY294002 could abate the reactive oxygen species (ROS) formation, which resulted in disturbance to the seed germination. And then, the signal cascades that PI3K promoted the ROS liberation was also evaluated. Diphenylene iodonium (DPI), an NADPH oxidase inhibitor, suppressed most of ROS generation in rice seed germination, which suggested that NADPH oxidase was the main source of ROS in this process. Pharmacological experiment and RT-PCR demonstrated that PI3K promoted the expression of Os rboh9. Moreover, functional analysis by native PAGE and the measurement of the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-lium-5- carboxanilide (XTT) formazan concentration both showed that PI3K promoted the activity of NADPH oxidase. Furthermore, the western blot analysis of OsRac-1 demonstrated that the translocation of Rac-1 from cytoplasm to plasma membrane, which was known as a key factor in the assembly of NADPH oxidase, was suppressed by treatment with PI3K inhibitors, resulting in the decreased activity of NADPH oxidase. Taken together, these data favored the novel conclusion that PI3K regulated NADPH oxidase activity through modulating the recruitment of Rac-1 to plasma membrane and accelerated the process of rice seed germination. OsRac1,Osrboh9,OsrbohA|Osrboh2,OsrbohB|Osrboh7,OsrbohD|Osrboh5,OsrbohE|Osrboh6,PI3K Purification, crystallization and preliminary X-ray crystallographic analysis of a rice Rac/Rop GTPase, OsRac1 2014 Acta Crystallogr F Struct Biol Commun Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Small GTPases regulate a large variety of key cellular processes. Plant small Rac/Rop GTPases have recently received broad attention as it is becoming clear that these enzymes regulate various plant cellular processes. OsRac1, a rice Rac/Rop protein, is a key regulator of reactive oxygen species (ROS) production and induces immune responses. Although four structures of plant small GTPases have been reported, all of these were of the inactive form. Here, OsRac1 was purified and co-crystallized with the GTP analogue 5'-guanylyl imidodiphosphate (GMPPNP). The crystal belonged to space group P2(1)2(1)2(1) and a complete data set was collected to 1.9 A resolution. OsRac1 RAR1 and HSP90 form a complex with Rac/Rop GTPase and function in innate-immune responses in rice 2007 Plant Cell Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Ikoma 630-0101, Japan. A rice (Oryza sativa) Rac/Rop GTPase, Os Rac1, is involved in innate immunity, but its molecular function is largely unknown. RAR1 (for required for Mla12 resistance) and HSP90 (a heat shock protein 90 kD) are important components of R gene-mediated disease resistance, and their function is conserved in several plant species. HSP90 has also recently been shown to be important in mammalian innate immunity. However, their functions at the molecular level are not well understood. In this study, we examined the functional relationships between Os Rac1, RAR1, and HSP90. Os RAR1-RNA interference (RNAi) rice plants had impaired basal resistance to a compatible race of the blast fungus Magnaporthe grisea and the virulent bacterial blight pathogen Xanthomonas oryzae. Constitutively active Os Rac1 complemented the loss of resistance, suggesting that Os Rac1 and RAR1 are functionally linked. Coimmunoprecipitation experiments with rice cell culture extracts indicate that Rac1 forms a complex with RAR1, HSP90, and HSP70 in vivo. Studies with Os RAR1-RNAi and treatment with geldanamycin, an HSP90-specific inhibitor, showed that RAR1 and HSP90 are essential for the Rac1-mediated enhancement of pathogen-associated molecular pattern-triggered immune responses in rice cell cultures. Furthermore, the function of HSP90, but not RAR1, may be essential for their association with the Rac1 complex. Os Rac1 also regulates RAR1 expression at both the mRNA and protein levels. Together, our results indicate that Rac1, RAR1, HSP90, and HSP70 form one or more protein complexes in rice cells and suggest that these proteins play important roles in innate immunity in rice. OsRac1,OsRAR1,Hsp90|rHsp90 Functional characterization of OsRacB GTPase--a potentially negative regulator of basal disease resistance in rice 2006 Plant Physiol Biochem Department of Molecular Biology, College of Natural Science, Sejong University, Seoul 143-747, Korea. The rice genome contains at least seven expressed Rop small GTPase genes. Of these Rops, OsRac1 is the only characterized gene that has been implicated in disease resistance as a positive regulator. To our interest in finding a negative ROP regulator of disease resistance in rice, we applied a "phylogeny of function" approach to rice Rops, and identified OsRacB based on its close genetic orthologous relationship with the barley HvRacB gene, a known negative regulator of disease resistance. To determine the function of OsRacB, we isolated the OsRacB cDNA and conducted gene expression and transgenic studies. OsRacB, a single copy gene in the genome of rice, shared 98% identity with HvRacB at the amino acid level. Its mRNA was strongly expressed in leaf sheath (LS) and in panicles, but was very weakly expressed in young and mature leaves. The basal mRNA level of OsRacB in LS of two-week-old seedlings was strongly down-regulated upon wounding by cut and treatment with jasmonic acid. A dramatic down-regulation in the OsRacB transcripts was also found in plants inoculated with the blast pathogen, Magnaporthe grisea. Interestingly, transgenic rice plants over-expressing OsRacB showed increased symptom development in response to rice blast pathogens. Additionally, fluorescence microscopy of green fluorescent protein (GFP):OsRacB-transformed onion cells and Arabidopsis protoplasts revealed OsRacB association with plasma membrane (PM), suggesting that PM localization is required for proper function of OsRacB. Based on these results, we suggest that OsRacB functions as a potential regulator for a basal disease resistance pathway in rice. OsRacB Rice GTPase OsRacB: Potential Accessory Factor in Plant Salt-stress Signaling 2006 Acta Biochimica et Biophysica Sinica Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080, China. As the sole ubiquitous signal small guanosine triphosphate-binding protein in plants, Rop gene plays an important role in plant growth and development. In this study, we focus on the relationship between the novel rice Rop gene OsRacB and plant salt tolerance. Results show that OsRacB transcription is highly accumulated in roots after treatment with salinity, but only slightly accumulated in stems and leaves under the same treatment. Promoter analysis showed that OsRacB promoter is induced by salinity and exogenous salicylic acid, not abscisic acid. To elucidate its physiological function, we generated OsRacB sense and antisense transgenic tobacco and rice. Under proper salinity treatment, sense transgenic plants grew much better than the control. This suggests that overexpression of OsRacB in tobacco and rice can improve plant salt tolerance. But under the same treatment, no difference could be observed between OsRacB antisense plants and the control. The results indicated that OsRacB is only an accessory factor in plant salt tolerance. OsRacB Structural analysis and identification of cis-elements of rice osRACD gene 2004 Acta Biochim Biophys Sin (Shanghai) Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080, China. The osRACD gene correlated with fertility transformation in the photoperiod sensitive genic male sterile rice (PGMR), Nongken 58S, encoded a rice (Oryza sativa L. ssp. japonica) small GTPase belonging to the Rac/Rho family. Inverse PCR was performed to amplify a fragment about 1.4 kb in 5' upstream region of the osRACD promoter. Deletion mutation and gel mobility shift assay characterized two fragments (-799 to -686 nt, and -686 to -431 nt) in the osRACD promoter that could be involved in its transcriptional regulation. When these two deletion fragments were used as probe respectively, a retarded band appeared in the nuclear extracts of fertile 58S rice under short day (58S-SD). Whereas no retarded band was shown in the nuclear extracts of sterile 58S rice under long day (58S-LD). Competition assay indicated that the factors in the retarded bands binding to these two fragments were the same trans-acting factor (termed rice factor, RF). The binding affinity of RF was affected by phosphorylation and was higher in SD-growth rice than that of LD-growth rice. OsRacD|OsRac5 Isolation of osRACD gene encoding a small GTP-binding protein from rice 2002 Chinese Science Bulletin Chinese Acad Sci, Inst Dev Biol, Beijing 100080, Peoples R China Using an improved version of mRNA differential display technology, we have obtained a differentially displayed fragment RDP-8. Homologous comparison indicated that the fragment RDP-8 has high homology with the gene encoding maize small GTP-binding protein. By screening cDNA library of the rice Nongken 58N panicle using the newly obtained fragment RDP-8 as probe, we further found the full-length cDNA of osRACD gene that encodes a rice small GTP-binding protein. As compared with maize RACD gene, the osRACD of rice shows remarkable homology in both nucleotide sequence and amino acid sequence, 88% and 97% respectively. Evidence from RT-PCR study indicates that osRACD gene is related to photoperiod fertility conversion of photoperiod sensitive genic male sterility (PSGMS) rice. OsRacD|OsRac5 Molecular characterization of OsRAD21-1, a rice homologue of yeast RAD21 essential for mitotic chromosome cohesion 2004 J Exp Bot Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Research Center for Molecular and Developmental Biology, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China. Rad21/Rec8 is an important component and key regulator of cohesins. A RAD21-like gene from rice (Oryza sativa L. ssp. japonica) has been cloned and termed OsRAD21-1. OsRAD21-1 is a single-copy gene in the rice genome and is expressed in the entire plant. OsRad21-1 consists of 1055 amino acid residues and is the largest of the Rad21/Rec8 family identified to date. Based on sequence similarity comparison with other members of this family and gene expression patterns, it is concluded that OsRad21 is a rice orthologue of yeast Rad21. OsRAD21-1 Rice OsRAD21-2 is expressed in actively dividing tissues and its ectopic expression in yeast results in aberrant cell division and growth 2011 J Integr Plant Biol Research Center for Molecular & Development Biology, Key Laborartory of Photosynthesis & Environmental Molecular Physiology, Insitute of Botany, Chinese Academy of Sciences, National Center for Plant Gene Research, Beijing , China. Rad21 and its meiotic counterpart Rec8, the key components of the cohesin complex, are essential for sister chromatid cohesion and chromosome segregation in mitosis and meiosis, respectively. In contrast to yeast and vertebrates, which have only two RAD21/REC8 genes, the rice genome encodes four Rad21/Rec8 proteins. Here, we report on the cloning and characterization of OsRAD21-2 from rice (Oryza sativa L.). Phylogenetic analysis of the full-length amino acids showed that OsRad21-2 was grouped into the plant-specific Rad21 subfamily. Semi-quantitative reverse transcription-polymerase chain reaction revealed OsRAD21-2 preferentially expressed in premeiotic flowers. Further RNA in situ hybridization analysis and promoter::beta-glucuronidase staining indicated that OsRAD21-2 was mainly expressed in actively dividing tissues including premeiotic stamen, stem intercalary meristem, leaf meristem, and root pericycle. Ectopic expression of OsRAD21-2 in fission yeast resulted in cell growth delay and morphological abnormality. Flow cytometric analysis revealed that the OsRAD21-2-expressed cells were arrested in G2 phase. Our results suggest that OsRad21-2 functions in regulation of cell division and growth. OsRAD21-2 OsRAD21-3, an orthologue of yeast RAD21, is required for pollen development in Oryza sativa 2007 Plant J Research Center for Molecular and Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, 20 Nanxincun, Xiangshan, Haidianqu, Beijing, China. In contrast to animals, in which products of meiosis differentiate directly into sperm, flowering plants employ a specific mechanism to give rise to functional sperm cells, the specifics of which remain largely unknown. A previous study revealed that, compared to yeast and vertebrates, which have two proteins (Rad21 and its meiosis-specific variant Rec8) that play a vital role in sister chromatid cohesion and segregation for mitosis and meiosis, respectively, the rice genome encodes four Rad21/Rec8 proteins (OsRad21s). In this paper, phylogenetic and immunostaining analyses reveal that OsRad21-3 is an orthologue of yeast Rad21. OsRAD21-3 transcript and protein accumulated preferentially in flowers, with low levels in vegetative tissues. In flowers, they persisted from the stamen and carpel primordia stages until the mature pollen stage. OsRAD21-3-deficient RNAi lines showed arrested pollen mitosis, aberrant pollen chromosome segregation and aborted pollen grains, which led to disrupted pollen viability. However, male meiosis in these RNAi lines did not appear to be severely disrupted, which suggests that the main involvement of OsRAD21-3 is in post-meiotic pollen development by affecting pollen mitosis. Furthermore, of the four OsRAD21 genes in the rice genome, only OsRAD21-3 was expressed in pollen grains. Given that the mechanism involving generation of sperm cells differs between flowering plants and metozoans, this study shows, in part, why flowering plants of rice and Arabidopsis have four Rad21/Rec8 proteins, as compared with two in yeast and metozoans, and gives some clues to the functional differentiation of Rad21/Rec8 proteins during evolution. OsRAD21-3 Characterization and expression of a rice RAD23 gene 1997 Plant Mol Biol Department of Biology, University of North Carolina, Chapel Hill 27599-3280, USA. In order to identify proteins that interact with plant transcriptional complexes, we performed a two-hybrid screen in yeast using a cDNA library from embryogenic rice suspension cultures and the plant transcriptional activator viviparous-1 (vp1) as 'bait'. In this screen, we detected an interaction between VP1 and a rice homologue of the Saccharomyces cerevisiae RAD23 gene (osRAD23). The RAD23 protein is associated with the general transcriptional machinery in yeast, and is believed to play a role in the processes of nucleotide excision repair in yeast and mammalian cells. This report is the first identification of a RAD23 homolog in plants. The osRAD23 amino acid sequence shares 50-60% similarity throughout its length with RAD23 sequences from yeast, mice, and man. osRAD23 contains a characteristic ubiquitin-like domain at its N-terminus, which is similar to other RAD23 genes. Analysis of the expressed sequence tag database identifies two different classes of RAD23 genes in both Arabidopsis and rice. Southern analysis of rice genomic DNA indicated the presence of at least two RAD23-like genes. A single transcript (1.5 kb) of osRAD23 was detected in total RNA from rice embryonic tissue, while three transcripts (1.8, 1.5 and 1.0 kb) were observed in total RNA from vegetative tissues of rice. osRAD23 Homologous recombination properties of OsRad51, a recombinase from rice 2008 Plant Mol Biol Plant Biochemistry Section, Molecular Biology Division, Bhabha Atomic Research Center, Mumbai 400085, India. cDNA corresponding to OsRad51 protein was isolated from cDNA library of rice flowers (Oryza sativa, Indica cultivar group) and cloned in to pET28a expression vector. The protein was over expressed in E. coli BL21 (DE3) and purified. Purified OsRad51 could bind single and double stranded DNA, however it showed higher affinity for single stranded DNA. Transmission Electron Microscopy (TEM) studies of OsRad51-DNA complexes showed that this protein formed ring like structures and bound DNA forming filaments. OsRad51 protein promoted renaturation of complementary single strands in to duplex DNA molecules and also showed ATPase activity, which was stimulated by single strand DNA. Fluorescence resonance energy transfer (FRET) assays revealed that OsRad51 promoted homology dependent renaturation as well as strand exchange reactions. Renaturation activity was ATP dependent; however strand exchange activity was ATP independent. This is the first report on in vitro characterization of Rad51 protein from crop plants. OsRad51 Suppression of OsRAD51D results in defects in reproductive development in rice (Oryza sativa L.) 2014 Plant J Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul, 120-749, Korea. The cellular roles of RAD51 paralogs in somatic and reproductive growth have been extensively described in a wide range of animal systems and, to a lesser extent, in Arabidopsis, a dicot model plant. Here, the OsRAD51D gene was identified and characterized in rice (Oryza sativa L.), a monocot model crop. In the rice genome, three alternative OsRAD51D mRNA splicing variants, OsRAD51D.1, OsRAD51D.2, and OsRAD51D.3, were predicted. Yeast two-hybrid studies, however, showed that only OsRAD51D.1 interacted with OsRAD51B and OsRAD51C paralogs, suggesting that OsRAD51D.1 is a functional OsRAD51D protein in rice. Loss-of-function osrad51d mutant rice plants displayed normal vegetative growth. However, the mutant plants were defective in reproductive growth, resulting in sterile flowers. Homozygous osrad51d mutant flowers exhibited impaired development of lemma and palea and contained unusual numbers of stamens and stigmas. During early meiosis, osrad51d pollen mother cells (PMCs) failed to form normal homologous chromosome pairings. In subsequent meiotic progression, mutant PMCs represented fragmented chromosomes. The osrad51d pollen cells contained numerous abnormal micro-nuclei that resulted in malfunctioning pollen. The abnormalities of heterozygous mutant and T2 Ubi:RNAi-OsRAD51D RNAi-knock-down transgenic plants were intermediate between those of wild type and homozygous mutant plants. The osrad51d and Ubi:RNAi-OsRAD51D plants contained longer telomeres compared with wild type plants, indicating that OsRAD51D is a negative factor for telomere lengthening. Overall, these results suggest that OsRAD51D plays a critical role in reproductive growth in rice. This essential function of OsRAD51D is distinct from Arabidopsis, in which AtRAD51D is not an essential factor for meiosis or reproductive development. OsRAD51D Characterization of Rad6 from a higher plant, rice (Oryza sativa L.) and its interaction with Sgt1, a subunit of the SCF ubiquitin ligase complex 2004 Biochem Biophys Res Commun Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda-shi, 278-8510 Chiba-ken, Japan. We report here the existence of interactions between a ubiquitin-conjugating enzyme, Rad6, from rice, Oryza sativa L. cv. Nipponbare (OsRad6), and Sgt1 (OsSgt1), a novel subunit of the SCF ubiquitin ligase complex. Rad6 is not only related to post-replicational repair but also to the proteasome system, while Sgt1 has a function in kinetochore assembly. The relationship between the two is unexpected, but of great interest. The open reading frames of OsRad6 and OsSgt1 encode predicted products of 152 and 367 amino acid residues, respectively, with molecular weights of 17.3 and 40.9kDa. Two-hybrid and pull-down analyses indicated that OsRad6 binds to OsSgt1, and transcripts of both OsRad6 and OsSgt1 were found to be strongly expressed only in the proliferating tissues such as the shoot apical meristem, suggesting that their expression is cell cycle-dependent. The amount of the Rad6 mRNA in cultured cells increased rapidly after division was halted, and mRNA levels of Rad6 and Sgt1 were induced by UV- and DNA-damaging agents such as MMS or H(2)O(2). The Rad6 pathway for repair or the proteasome system may thus require Sgt1 as ubiquitin-conjugating enzyme. OsRad6,OsSGT1 OsRAF is an ethylene responsive and root abundant factor gene of rice 2007 Plant Growth Regulation Research Center for Molecular & Developmental Biology, Key Laboratory of Photosynthesis & Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, 20 Nanxincun, Xiangshan, Haidianqu, Beijing, 100093, China ERFs, the largest group of AP2/EREBP transcription factors, are involved in diverse processes in plants. However, their importance in growth and development is not fully understood. Here, we report OsRAF (a Root Abundant Factor gene in Oryza sativa), a new member of the rice ERF group, expressed more abundantly in roots than in other organs of rice at the transcriptional level. Moreover, the expression could be up-regulated by ethylene or low temperature. Transient expression of OsRAF::GFP fusion in onion epidermis cells and its overexpression in Arabidopsis revealed that OsRAF was a nucleus localized protein. Down-regulation of OsRAF’s expression by RNAi method failed to produce aberrance in transgenic rice, which suggests existence of functionally redundant gene(s). OsRAF Overexpression of OsRAN2 in rice and Arabidopsis renders transgenic plants hypersensitive to salinity and osmotic stress 2010 J Exp Bot Institute of Plant Physiology and Ecology,Graduate School of Chinese Academy of Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China. Nucleo-cytoplasmic partitioning of regulatory proteins is increasingly being recognized as a major control mechanism for the regulation of signalling in plants. Ras-related nuclear protein (Ran) GTPase is required for regulating transport of proteins and RNA across the nuclear envelope and also has roles in mitotic spindle assembly and nuclear envelope (NE) assembly. However, thus far little is known of any Ran functions in the signalling pathways in plants in response to changing environmental stimuli. The OsRAN2 gene, which has high homology (77% at the amino acid level) with its human counterpart, was isolated here. Subcellular localization results showed that OsRan2 is mainly localized in the nucleus, with some in the cytoplasm. Transcription of OsRAN2 was reduced by salt, osmotic, and exogenous abscisic acid (ABA) treatments, as determined by real-time PCR. Overexpression of OsRAN2 in rice resulted in enhanced sensitivity to salinity, osmotic stress, and ABA. Seedlings of transgenic Arabidopsis thaliana plants overexpressing OsRAN2 were overly sensitive to salinity stress and exogenous ABA treatment. Furthermore, three ABA- or stress-responsive genes, AtNCED3, AtPLC1, and AtMYB2, encoding a key enzyme in ABA synthesis, a phospholipase C homologue, and a putative transcriptional factor, respectively, were shown to have differentially induced expression under salinity and ABA treatments in transgenic and wild-type Arabidopsis plants. OsRAN2 overexpression in tobacco epidermal leaf cells disturbed the nuclear import of a maize (Zea mays L.) leaf colour transcription factor (Lc). In addition, gene-silenced rice plants generated via RNA interference (RNAi) displayed pleiotropic developmental abnormalities and were male sterile. OsRAN2 OsRAN2, essential for mitosis, enhances cold tolerance in rice by promoting export of intranuclear tubulin and maintaining cell division under cold stress 2011 Plant Cell Environ Research Center for Molecular and Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. With global climate change, abnormally low temperatures have affected the world's rice production. Many genes have been shown to be essential for molecular improvement of rice cold-tolerance traits. However, less is known about the molecular cellular mechanism of their response to cold stress. Here, we investigated OsRAN2 involved in regulation of cell division during cold stress in rice. Expression of OsRAN2 was increased under cold treatment, but not during salt and drought stress. The mean root mitotic index was closely related to the expression level of OsRAN2. Knockdown transgenic rice lines showed an aberrant organization of spindles during mitosis and stunted growth during development. Overexpression of OsRAN2 enhanced cold tolerance in rice. The transgenic rice overexpressing OsRAN2 showed maintained cell division, decreased proportion of cells with intranuclear tubulin and formation of a normal nuclear envelope under the cold condition. Our study suggests a mechanism for OsRAN2 in regulating cold resistance in rice by maintaining cell division through promoting the normal export of intranuclear tubulin at the end of mitosis. This insight could help improve the cold-tolerance trait in rice. OsRAN2 OsRAR1 and OsSGT1 physically interact and function in rice basal disease resistance 2008 Mol Plant Microbe Interact Institute of Plant Physiology & Ecology, Chinese Academy of Sciences, Shangai, China. The RAR1 and SGT1 proteins function synergistically or antagonistically in plant innate immune responses. Here, we show that the rice orthologs OsRAR1 and OsSGT1 physically interact in vivo and in yeast. They displayed conserved roles in Arabidopsis disease resistance through ectopic expression in the Arabidopsis rar1 and sgt1 mutants. Overexpression of OsRar1 and OsSGT1 in rice significantly increased basal resistance to a virulent bacterial blight Xanthomonas oryzae pv. oryzae PXO99 but not to another virulent strain DY89031, suggesting race-specific-like basal resistance conferred by OsRar1 and OsSGT1. OsRar1-OE and OsSGT1-OE plants also enhanced resistance to all four virulent blast fungal Magnaporthe oryzae races. Overexpression of the OsSGT1-green fluorescent protein (GFP) fusion most likely caused a dominant negative phenotype which led to race-specific-like basal resistance. Transgenic plants overexpressing OsSGT1-GFP show enhanced resistance to DY89031 but decreased resistance to PXO99, implying that OsSGT1 might be the target of a component required for DY89031 virulence or OsSGT1-GFP might stabilize weak resistance proteins against DY89031. Consistent with the hypothesis of the dominant negative regulation, we observed the reduced sensitivity to auxin of OsSGT1-GFP plants compared with the wild-type ones, and the curling-root phenotype in OsSGT1-OE plants. These results collectively suggest that OsRar1 and OsSGT1 might be differentially required for rice basal disease resistance. Our current study also provides new insight into the roles of OsSGT1 in basal disease resistance. OsRAR1,OsSGT1 Differential requirement of Oryza sativa RAR1 in immune receptor-mediated resistance of rice to Magnaporthe oryzae 2013 Mol Cells Graduate School of Biotechnology and Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Korea. The required for Mla12 resistance (RAR1) protein is essential for the plant immune response. In rice, a model monocot species, the function of Oryza sativa RAR1 (OsRAR1) has been little explored. In our current study, we characterized the response of a rice osrar1 T-DNA insertion mutant to infection by Magnaporthe oryzae, the causal agent of rice blast disease. osrar1 mutants displayed reduced resistance compared with wild type rice when inoculated with the normally virulent M. oryzae isolate PO6-6, indicating that OsRAR1 is required for an immune response to this pathogen. We also investigated the function of OsRAR1 in the resistance mechanism mediated by the immune receptor genes Pib and Pi5 that encode nucleotide binding-leucine rich repeat (NB-LRR) proteins. We inoculated progeny from Pib/osrar1 and Pi5/osrar1 heterozygous plants with the avirulent M. oryzae isolates, race 007 and PO6-6, respectively. We found that only Pib-mediated resistance was compromised by the osrar1 mutation and that the introduction of the OsRAR1 cDNA into Pib/osrar1 rescued Pib-mediated resistance. These results indicate that OsRAR1 is required for Pib-mediated resistance but not Pi5-mediated resistance to M. oryzae. OsRAR1 Effect of individual suppression of RBCS multigene family on Rubisco contents in rice leaves 2012 Plant Cell Environ Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Sendai, Japan. In higher plants, a small subunit of Rubisco is encoded for by an RBCS multigene family in the nuclear genome. However, it is unknown how each multigene member contributes to the accumulation of Rubisco holoenzyme. Here, four RBCS genes that are highly expressed in leaf blaedes of rice (Oryza sativa L.) were individually suppressed by RNAi, and the effects on leaf Rubisco content were examined at seedling, vegetative and reproductive stages. Rubisco contents in each transgenic line declined irrespective of growth stage, and the ratios of Rubisco-N to total N were 66-96% of wild-type levels. The mRNA levels of the suppressed RBCS genes declined significantly, whereas those of the unsuppressed ones did not change drastically. These results indicate that four RBCS genes all contribute to accumulation of Rubisco holoenzyme irrespective of growth stage and that suppression of one RBCS gene is not fully compensated by other RBCS genes. Additionally, the mRNA levels of the large subunit of Rubisco showed a change similar to that of total RBCS mRNA level irrespective of genotype and growth stage. These results suggest that gene expression of RBCS and RBCL is regulated in a coordinated manner at the transcript level in rice. OsRBCS1,OsRBCS2,OsRBCS4,OsRBCS3|OsRBCS5 Increased Rubisco content in transgenic rice transformed with the 'sense' rbcS gene 2007 Plant Cell Physiol Department of Applied Plant Science, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Sendai 981-8555, Japan. Rice (Oryza sativa L.) plants with substantially increased Rubisco content were obtained by Agrobacterium-mediated transformation with the rice rbcS sense gene under the control of the rice rbcS promoter. The primary transformants were screened for the ratio of Rubisco to leaf-N content, and the transformants with >120% wild-type levels of Rubisco were selected. In the progeny of the selected lines of the transformants, the mRNA levels of one member of the rbcS gene family were increased from 3.9- to 6.2-fold, whereas those of other members of the rbcS gene family were unchanged. The total levels of rbcS mRNA were increased from 2.1- to 2.8-fold. The levels of rbcL mRNA were increased from 1.2- to 1.9-fold. Rubisco protein content was significantly increased by 30% on a leaf area basis. The ratio of Rubisco-N to leaf-N was also increased by 10-20%, irrespective of N treatment. The specific activity of Rubisco per unit of enzyme protein was not different. However, light-saturated photosynthesis was not enhanced even when the rate was measured at low [CO2] where Rubisco becomes limiting for photosynthesis. Some lines showed lower photosynthesis at high [CO2] (>60 Pa). We conclude that introduction of additional sense rbcS leads to overexpression of rbcS and that this overexpression slightly up-regulates the gene expression of rbcL at the transcript level and enhances the amount of Rubisco holoenzyme. However, overproduction of Rubisco protein does not improve photosynthesis. OsRBCS1,OsRBCS2,OsRBCS4,OsRBCS3|OsRBCS5 Availability of Rubisco small subunit up-regulates the transcript levels of large subunit for stoichiometric assembly of its holoenzyme in rice 2012 Plant Physiol Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan. ysuzuki@biochem.tohoku.ac.jp Rubisco is composed of eight small subunits coded for by the nuclear RBCS multigene family and eight large subunits coded for by the rbcL gene in the plastome. For synthesis of the Rubisco holoenzyme, both genes need to be expressed coordinately. To investigate this molecular mechanism, the protein synthesis of two subunits of Rubisco was characterized in transgenic rice (Oryza sativa) plants with overexpression or antisense suppression of the RBCS gene. Total RBCS and rbcL messenger RNA (mRNA) levels and RBCS and RbcL synthesis simultaneously increased in RBCS-sense plants, although the increase in total RBCS mRNA level was greater. In RBCS-antisense plants, the levels of these mRNAs and the synthesis of the corresponding proteins declined to a similar extent. The amount of RBCS synthesized was tightly correlated with rbcL mRNA level among genotypes but not associated with changes in mRNA levels of other major chloroplast-encoded photosynthetic genes. The level of rbcL mRNA, in turn, was tightly correlated with the amount of RbcL synthesized, the molar ratio of RBCS synthesis to RbcL synthesis being identical irrespective of genotype. Polysome loading of rbcL mRNA was not changed. These results demonstrate that the availability of RBCS protein up-regulates the gene expression of rbcL primarily at the transcript level in a quantitative manner for stoichiometric assembly of Rubisco holoenzyme. OsRBCS2,OsRBCS4,OsRBCS3|OsRBCS5 Rubisco content and photosynthesis of leaves at different positions in transgenic rice with an overexpression of RBCS 2009 Plant Cell Environ Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Sendai 981-8555, Japan. ysuzuki@biochem.tohoku.ac.jp As ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco) activity limits light-saturated photosynthesis under present atmospheric condition, the effects of an overexpression of RBCSon Rubisco content and photosynthesis were examined in the leaves at different positions in rice (Oryza sativa L.). Rubisco content in the transformant was significantly greater in the uppermost, fully expanded leaves but decreased to levels similar to those in wild-type plants in the lower leaves. The mRNA levels of total RBCS and rbcL in these leaves were much less than those in the expanding leaves, where Rubisco synthesis is active, suggesting commensurately low level of synthesis. Although the activation state of Rubisco was lower in the uppermost, fully expanded leaves of the transformant, it recovered to its full level in the lower leaves. As a result, the photosynthetic rate did not differ in leaves at the same position between the transformant and the wild type. Similarly, whole plant biomass did not differ between these genotypes. Thus, we conclude that although the overexpression of RBCS led to an enhancement of Rubisco protein content in the uppermost, fully expanded leaves, it does not result in increased photosynthetic rates or plant biomass, because of an apparent down-regulation in its activation state. OsRBCS2 rbohA, a rice homologue of the mammalian gp91phox respiratory burst oxidase gene 1996 The Plant Journal Department of Biochemistry and Genetics, University of Newcastle, UK. It has been hypothesized that plants contain respiratory burst oxidase which, upon activation, oxidize NADPH and generate extracellular superoxide, O2.-. These proteins are proposed to play a central role in defence against pathogens. However, plant DNA sequences that encode proteins with similarity to components of respiratory burst oxidase have not previously been reported. This paper describes the complete cDNA and genomic DNA sequence of the rice rbohA (for respiratory burst oxidase homologue) gene. The predicted RbohA product is most similar to the main catalytic subunit, gp91phox, of the respiratory burst oxidase of neutrophils. Reverse transcriptase PCR detects rbohA transcripts in both roots and shoots of healthy rice plants. OsrbohA|Osrboh2 Reactive oxygen species production and activation mechanism of the rice NADPH oxidase OsRbohB 2012 J Biochem Department of Applied Biological Science, Tokyo University of Science, Noda, 278-8510 Chiba, Japan. Reactive oxygen species (ROS) produced by plant NADPH oxidases (NOXes) are important in plant innate immunity. The Oryza sativa respiratory burst oxidase homologue B (OsRbohB) gene encodes a NOX the regulatory mechanisms of which are largely unknown. Here, we used a heterologous expression system to demonstrate that OsRbohB shows ROS-producing activity. Treatment with ionomycin, a Ca(2+) ionophore, and calyculin A, a protein phosphatase inhibitor, activated ROS-producing activity; it was thus OsRbohB activated by both Ca(2+) and protein phosphorylation. Mutation analyses revealed that not only the first EF-hand motif but also the upstream amino-terminal region were necessary for Ca(2+)-dependent activation, while these regions are not required for phosphorylation-induced ROS production. OsrbohB|Osrboh7 Involvement of a Ca(2+)-dependent protein kinase component downstream to the gibberellin-binding phosphoprotein, RuBisCO activase, in rice 2002 Biochem Biophys Res Commun Japan Science and Technology Corporation, Japan. Previously, we reported the identification of a gibberellin (GA)-binding protein in rice using ligand binding assay that was homologous to RuBisCO activase (Komatsu et al., FEBS Lett. 384, 167-171, 1996). Here, we provide an evidence for the involvement of protein kinases components downstream to the GA-binding phosphoprotein, RuBisCO activase in rice. Ca(2+)-dependent protein kinase activity was studied in subcellular fractions of leaf sheath from transgenic rice containing sense and antisense constructs of RuBisCO activase. In-gel kinase assay using histone III-S as a substrate showed constitutive induction of a 46- and 48-kDa Ca(2+)-dependent protein kinase activity in the sense transgenic plants. Kinase activities of these proteins were significantly reduced in the presence of uniconazole, a potent GA biosynthesis inhibitor, but one of them was strongly promoted by GA(3) treatment in transgenic plants carrying a smaller subunit of RuBisCO activase (OsrcaA1) compared to the larger subunit OsrcaA2. Also, in vitro phosphorylation studies using two-dimensional polyacrylamide gel showed changes in the degree of phosphorylation of several proteins in OsrcaA1- and OsrcaA2-sense transgenic rice. These studies suggest the presence of two independent cytosolic Ca(2+)-dependent protein kinase signaling components downstream to the GA-binding protein in rice suggesting their role in GA signaling. OsrcaA1|OsrcaA2 Overexpression of OsRDCP1, a rice RING domain-containing E3 ubiquitin ligase, increased tolerance to drought stress in rice (Oryza sativa L.) 2011 Plant Sci Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea. CaRma1H1 was previously identified as a hot pepper drought-induced RING E3 Ub ligase. We have identified five putative proteins that display a significant sequence identity with CaRma1H1 in the rice genome database (http://signal.salk.edu/cgi-bin/RiceGE). These five rice paralogs possess a single RING motif in their N-terminal regions, consistent with the notion that RING proteins are encoded by a multi-gene family. Therefore, these proteins were named OsRDCPs (Oryza sativa RING domain-containing proteins). Among these paralogs, OsRDCP1 was induced by drought stress, whereas the other OsRDCP members were constitutively expressed, with OsRDCP4 transcripts expressed at the highest level in rice seedlings. osrdcp1 loss-of-function knockout mutant and OsRDCP1-overexpressing transgenic rice plants were developed. Phenotypic analysis showed that wild-type plants and the homozygous osrdcp1 G2 mutant line displayed similar phenotypes under normal growth conditions and in response to drought stress. This may be due to complementation by other OsRDCP paralogs. In contrast, 35S:OsRDCP1 T2 transgenic rice plants exhibited improved tolerance to severe water deficits. Although the physiological function of OsRDCP1 remains unclear, there are several possible mechanisms for its involvement in a subset of physiological responses to counteract dehydration stress in rice plants. OsRDCP1 Both OsRecQ1 and OsRDR1 Are Required for the Production of Small RNA in Response to DNA-Damage in Rice 2013 PLoS One Faculty of Agriculture, Ehime University, Matsuyama, Ehime, Japan Small RNA-mediated gene silencing pathways play important roles in the regulation of development, genome stability and various stress responses in many eukaryotes. Recently, a new type of small interfering RNAs (qiRNAs) approximately 20-21 nucleotides long in Neurospora crassa have been shown to mediate gene silencing in the DNA damage response (DDR) pathway. However, the mechanism for RNA silencing in the DDR pathway is largely unknown in plants. Here, we report that a class of small RNAs (qiRNAs) derived from rDNA was markedly induced after treatment by DNA-damaging agents [ ethyl methanesulphonate (EMS and UV-C)], and that aberrant RNAs (aRNAs) as precursors were also highly induced after the DNA damage treatment in rice. However, these RNAs were completely abolished in OsRecQ1 (RecQ DNA helicase homologue) and OsRDR1 (RNA-dependent RNA polymerase homologue) mutant lines where either gene was disrupted by the insertion of rice retrotransposon Tos17 after the same treatment. DNA damage resulted in a more significant increase in cell death and a more severe inhibition of root growth in both mutant lines than in the WT. Together, these results strongly suggest that both OsRecQ1 and OsRDR1 play a pivotal role in the aRNA and qiRNA biogenesis required for the DDR and repair pathway in rice, and it may be a novel mechanism of regulation to the DDR through the production of qiRNA in plants. OsRDR1,OsRecQ1 RNA-dependent RNA polymerase 6 of rice (Oryza sativa) plays role in host defense against negative-strand RNA virus, Rice stripe virus 2012 Virus Res State Key Laboratory of Protein and Plant Gene Research, Peking-Yale Joint Center for Plant Molecular Genetics and Agrobiotechnology, College of Life Sciences, Peking University, Beijing 100871, People's Republic of China. RNA-dependent RNA polymerases (RDRs) from fungi, plants and some invertebrate animals play fundamental roles in antiviral defense. Here, we investigated the role of RDR6 in the defense of economically important rice plants against a negative-strand RNA virus (Rice stripe virus, RSV) that causes enormous crop damage. In three independent transgenic lines (OsRDR6AS line A, B and C) in which OsRDR6 transcription levels were reduced by 70-80% through antisense silencing, the infection and disease symptoms of RSV were shown to be significantly enhanced. The hypersusceptibilities of the OsRDR6AS plants were attributed not to enhanced insect infestation but to enhanced virus infection. The rise in symptoms was associated with the increased accumulation of RSV genomic RNA in the OsRDR6AS plants. The deep sequencing data showed reduced RSV-derived siRNA accumulation in the OsRDR6AS plants compared with the wild type plants. This is the first report of the antiviral role of a RDR in a monocot crop plant in the defense against a negative-strand RNA virus and significantly expands upon the current knowledge of the antiviral roles of RDRs in the defense against different types of viral genomes in numerous groups of plants. OsRDR6|shl-2 The SHOOTLESS2 and SHOOTLESS1 genes are involved in both initiation and maintenance of the shoot apical meristem through regulating the number of indeterminate cells 2003 Genetics Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. To characterize the SHL2 and SHL1 genes in detail, we analyzed three strains carrying weak alleles of SHL2, shl2-6, shl2-7, and shl2-8, and one weak allele of SHL1, shl1-3. In contrast to strong alleles, which result in lack of shoot meristem, strains bearing these weak alleles formed shoot meristem frequently, during embryogenesis. In shl2-6 and shl2-7 mutants, the meristem vas lost during seed development. Only the shl2-8 mutant could survive after germination, but it showed abnormal initiation pattern and morphology of leaves. In strains bearing the weak alleles, the shoot meristem was composed of a small number of indeterminate cells and ultimately converted into leaf primordium. The shl1-3 mutant showed phenotypes similar to those of shl2-8. Thus SHL2 and SHL1 are required for both initiation and maintenance of shoot meristem. In shl2 mutants, there was a positive correlation between the size of the expression domain of OSH1 representing the number of indeterminate cells, the frequency of shoot meristem initiation, and the duration of meristem survival. Thus the shoot meristem will not initiate in an "all-or-nothing" fashion, but is formed in various degrees depending on the strength of the alleles. Double-mutant analyses indicate that SHL2 functions upstream of SHO to establish proper organization of the shoot meristem. OsRDR6|shl-2 Rice RNA-dependent RNA polymerase 6 acts in small RNA biogenesis and spikelet development 2012 Plant J State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Higher plants have evolved multiple RNA-dependent RNA polymerases (RDRs), which work with Dicer-like (DCL) proteins to produce different classes of small RNAs with specialized molecular functions. Here we report that OsRDR6, the rice (Oryza sativa L.) homolog of Arabidopsis RDR6, acts in the biogenesis of various types and sizes of small RNAs. We isolated a rice osrdr6-1 mutant, which was temperature sensitive and showed spikelet defects. This mutant displays reduced accumulation of tasiR-ARFs, the conserved trans-acting siRNAs (tasiRNAs) derived from the TAS3 locus, and ectopic expression of tasiR-ARF target genes, the Auxin Response Factors (including ARF2 and ARF3/ETTIN). The loss of tasiR-mediated repression of ARFs in osrdr6-1 can explain its morphological defects, as expression of two non-targeted ARF3 gene constructs (ARF3muts) in a wild-type background mimics the osrdr6 and osdcl4-1 mutant phenotypes. Small RNA high-throughput sequencing also reveals that besides tasiRNAs, 21-nucleotide (nt) phased small RNAs are also largely dependent on OsRDR6. Unexpectedly, we found that osrdr6-1 has a strong impact on the accumulation of 24-nt phased small RNAs, but not on unphased ones. Our work uncovers the key roles of OsRDR6 in small RNA biogenesis and directly illustrates the crucial functions of tasiR-ARFs in rice development. OsRDR6|shl-2 Initiation of shoot apical meristem in rice: characterization of four SHOOTLESS genes 1999 Development Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. The regulatory mechanism of shoot apical meristem (SAM) initiation is an important subject in developmental plant biology. We characterized nine recessive mutations derived from four independent loci (SHL1-SHL4) causing the deletion of the SAM. Radicles were produced in these mutant embryos. Concomitant with the loss of SAM, two embryo-specific organs, coleoptile and epiblast, were lost, but the scutellum was formed normally. Therefore, differentiation of radicle and scutellum is regulated independently of SAM, but that of coleoptile and epiblast may depend on SAM. Regeneration experiments using adventitious shoots from the scutellum-derived calli showed that no adventitious shoots were regenerated in any shl mutant. However, small adventitious leaves were observed in both mutant and wild-type calli, but they soon became necrotic and showed no extensive growth. Thus, leaf primordia can initiate in the absence of SAM, but their extensive growth requires the SAM. An in situ hybridization experiment using a rice homeobox gene, OSH1, as a probe revealed that shl1 and shl2 modified the expression domain of OSH1, but normal expression of OSH1 was observed in shl3 and shl4 embryos. Accordingly, SHL1 and SHL2 function upstream of OSH1, and SHL3 and SHL4 downstream or independently of OSH1. These shl mutants are useful for elucidating the genetic program driving SAM initiation and for unraveling the interrelationships among various organs in grass embryos. OsRDR6|shl-2,OsAGO7|shl4|SHO2 p2 of rice stripe virus (RSV) interacts with OsSGS3 and is a silencing suppressor 2011 Mol Plant Pathol Institute of Plant Virology, Fujian Agriculture and Forestry University, Key Laboratory of Plant Virology of Fujian Province, Fuzhou, Fujian, 350002, China. A rice cDNA library was screened by a galactosidase 4 (Gal4)-based yeast two-hybrid system with Rice stripe virus (RSV) p2 as bait. The results revealed that RSV p2 interacted with a rice protein exhibiting a high degree of identity with Arabidopsis thaliana suppressor of gene silencing 3 (AtSGS3). The interaction was confirmed by bimolecular fluorescence complementation assay. SGS3 has been shown to be involved in sense transgene-induced RNA silencing and in the biogenesis of trans-acting small interfering RNAs (ta-siRNAs), possibly functioning as a cofactor of RNA-dependent RNA polymerase 6 (RDR6). Given the intimate relationships between virus and RNA silencing, further experiments were conducted to show that p2 was a silencing suppressor. In addition, p2 enhanced the accumulation and pathogenicity of Potato virus X in Nicotiana benthamiana. Five genes that have been demonstrated to be targets of TAS3-derived ta-siRNAs were up-regulated in RSV-infected rice. The implications of these findings are discussed. OsRDR6|shl-2,OsSGS3 Phytohormone abscisic acid control RNA-dependent RNA polymerase 6 gene expression and post-transcriptional gene silencing in rice cells 2008 Nucleic Acids Res The Basic Science Research Institute, Department of Biological Science, Sungkyunkwan University, Suwon 440-746, Korea. RNA-dependent RNA polymerase 6 (RDR6) catalyses dsRNA synthesis for post-transcriptional gene silencing (PTGS)-associated amplification and the generation of endogeneous siRNAs involved in developmental determinations or stress responses. The functional importance of RDR6 in PTGS led us to examine its connection to the cellular regulatory network by analyzing the hormonal responses of RDR6 gene expression in a cultured cell system. Delivery of dsRNA, prepared in vitro, into cultured rice (Oryza sativa cv. Japonica Dongjin) cells successfully silenced the target isocitrate lyase (ICL) transcripts. Silencing was transient in the absence of abscisic acid (ABA), while it became persistent in the presence of ABA in growth medium. A transcription assay of the OsRDR6 promoter showed that it was positively regulated by ABA. OsRDR6-dependent siRNA(ICL) generation was also significantly up-regulated by ABA. The results showed that, among the five rice OsRDR isogenes, only OsRDR6 was responsible for the observed ABA-mediated amplification and silencing of ICL transcripts. We propose that ABA modulates PTGS through the transcriptional control of the OsRDR6 gene. OsRDR6|shl-2 Distinct regulation of adaxial-abaxial polarity in anther patterning in rice 2010 Plant Cell Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan. Establishment of adaxial-abaxial polarity is essential for lateral organ development. The mechanisms underlying the polarity establishment in the stamen remain unclear, whereas those in the leaf are well understood. Here, we investigated a rod-like lemma (rol) mutant of rice (Oryza sativa), in which the development of the stamen and lemma is severely compromised. We found that the rod-like structure of the lemma and disturbed anther patterning resulted from defects in the regulation of adaxial-abaxial polarity. Gene isolation indicated that the rol phenotype was caused by a weak mutation in SHOOTLESS2 (SHL2), which encodes an RNA-dependent RNA polymerase and functions in trans-acting small interfering RNA (ta-siRNA) production. Thus, ta-siRNA likely plays an important role in regulating the adaxial-abaxial polarity of floral organs in rice. Furthermore, we found that the spatial expression patterns of marker genes for adaxial-abaxial polarity are rearranged during anther development in the wild type. After this rearrangement, a newly formed polarity is likely to be established in a new developmental unit, the theca primordium. This idea is supported by observations of abnormal stamen development in the shl2-rol mutant. By contrast, the stamen filament is likely formed by abaxialization. Thus, a unique regulatory mechanism may be involved in regulating adaxial-abaxial polarity in stamen development. OsRDR6|shl-2 MER3 is required for normal meiotic crossover formation, but not for presynaptic alignment in rice 2009 J Cell Sci State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. MER3, a ZMM protein, is required for the formation of crossovers in Saccharomyces cerevisiae and Arabidopsis. Here, MER3, the first identified ZMM gene in a monocot, is characterized by map-based cloning in rice (Oryza sativa). The null mutation of MER3 results in complete sterility without any vegetative defects. Cytological analyses show that chiasma frequency is reduced dramatically in mer3 mutants and the remaining chiasmata distribute randomly among different pollen mother cells, implying possible coexistence of two kinds of crossover in rice. Immunocytological analyses reveal that MER3 only exists as foci in prophase I meiocytes. In addition, MER3 does not colocalize with PAIR2 at the beginning of prophase I, but locates on one end of PAIR2 fragments at later stages, whereas MER3 foci merely locate on one end of REC8 fragments when signals start to be seen in early prophase I. The normal loading of PAIR2 and REC8 in mer3 implies that their loading is independent of MER3. On the contrary, the absence of MER3 signal in pair2 mutants indicates that PAIR2 is essential for the loading and further function of MER3. OsRad21-4|OsREC8,PAIR2,MER3|RCK OsREC8 is essential for chromatid cohesion and metaphase I monopolar orientation in rice meiosis 2011 Plant Physiol State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. The successful transmission of chromosomes during mitosis and meiosis relies on the establishment and subsequent release of cohesion between replicated chromatids. Cohesion is mediated by a four-subunit structural maintenance of chromosome complex, called cohesins. REC8 is a key component of this meiotic cohesion complex in most model organisms studied to date. Here, we isolated and dissected the functions of OsREC8, a rice (Oryza sativa) REC8 homolog, using two null Osrec8 mutants. We showed that OsREC8 encodes a protein that localized to meiotic chromosomes from approximately meiotic interphase to metaphase I. Homologous pairing and telomere bouquet formation were abnormal in Osrec8 meiocytes. Furthermore, fluorescent in situ hybridization experiments on Osrec8 meiocytes demonstrated that the mutation eliminated meiotic centromeric cohesion completely during prophase I and also led to the bipolar orientation of the kinetochores during the first meiotic division and accordingly resulted in premature separation of sister chromatid during meiosis I. Immunolocalization analyses revealed that the loading of PAIR2, PAIR3, OsMER3, and ZEP1 all depended on OsREC8. By contrast, the presence of the OsREC8 signal in pair2, pair3, Osmer3, and zep1 mutants indicated that the loading of OsREC8 did not rely on these four proteins. These results suggest that OsREC8 has several essential roles in the meiotic processes. OsRad21-4|OsREC8,PAIR2,MER3|RCK,ZEP1 OsSPO11-1 is essential for both homologous chromosome pairing and crossover formation in rice 2010 Chromosoma Key Laboratory of Crop Genetics and Physiology of Jiangsu Province/Key Laboratory of Plant Functional Genomics of Ministry of Education, Yangzhou University, Yangzhou, 225009, China. Spo11 is a homolog of a subunit of archaebacterial topoisomerase, which catalyzes DNA double-strand breaks and initiates homologous chromosome recombination. In the present study, we silenced the SPO11-1 gene in rice (Oryza sativa) using RNAi. Rice plants with loss-of-function of OsSPO11-1 have no apparent growth defects during vegetative development, but homologous chromosome pairing and recombination are significantly obstructed. Telomeres can be assembled as bouquet during the zygotene stage of the OsSPO11-1-deficient plants, just as that in wild type. Although the two axial-associated proteins, REC8 and PAIR2, are loaded onto the chromosomes, the depletion of PAIR2 from the chromosomes is much later than in wild type. The central element of the synaptonemal complex (SC), ZEP1, does not load onto the chromosomes normally, implying that SC formation is disturbed severely. The crossover protein, MER3, isn't efficiently assembled onto chromosomes and the lack of bivalent suggests that crossovers are also affected in the absence of OsSPO11-1. Thus, OsSPO11-1 is essential for both homologous chromosomes pairing and crossover formation during meiosis in rice. OsRad21-4|OsREC8,OsSPO11-1|OsTOP6A1,PAIR2,MER3|RCK,ZEP1 The rice OsRad21-4, an orthologue of yeast Rec8 protein, is required for efficient meiosis 2006 Plant Mol Biol Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Research Center of Molecular & Developmental Biology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. In yeast, Rad21/Scc1 and its meiotic variant Rec8 are key players in the establishment and subsequent dissolution of sister chromatid cohesion for mitosis and meiosis, respectively, which are essential for chromosome segregation. Unlike yeast, our identification revealed that the rice genome has 4 RAD21-like genes that share lower than 21% identity at polypeptide levels, and each is present as a single copy in this genome. Here we describe our analysis of the function of OsRAD21-4 by RNAi. Western blot analyses indicated that the protein was most abundant in young flowers and less in leaves and buds but absent in roots. In flowers, the expression was further defined to premeiotic pollen mother cells (PMCs) and meiotic PMCs of anthers. Meiotic chromosome behaviors were monitored from male meiocytes of OsRAD21-4-deficient lines mediated by RNAi. The male meiocytes showed multiple aberrant events at meiotic prophase I, including over-condensation of chromosomes, precocious segregation of homologues and chromosome fragmentation. Fluorescence in situ hybridization experiments revealed that the deficient lines were defective in homologous pairing and cohesion at sister chromatid arms. These defects resulted in unequal chromosome segregation and aberrant spore generation. These observations suggest that OsRad21-4 is essential for efficient meiosis. OsRad21-4|OsREC8 Characterization of four RecQ homologues from rice (Oryza sativa L. cv. Nipponbare) 2006 Biochem Biophys Res Commun Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda-shi, Chiba-ken, Japan. The RecQ family of DNA helicases is conserved throughout the biological kingdoms. In this report, we have characterized four RecQ homologues clearly expressed in rice. OsRecQ1, OsRecQ886, and OsRecQsim expressions were strongly detected in meristematic tissues. Transcription of the OsRecQ homologues was differentially induced by several types of DNA-damaging agents. The expression of four OsRecQ homologues was induced by MMS and bleomycin. OsRecQ1 and OsRecQ886 were induced by H(2)O(2), and MitomycinC strongly induced the expression of OsRecQ1. Transient expression of OsRecQ/GFP fusion proteins demonstrated that OsRecQ2 and OsRecQ886 are found in nuclei, whereas OsRecQ1 and OsRecQsim are found in plastids. Neither OsRecQ1 nor OsRecQsim are induced by light. These results indicate that four of the RecQ homologues have different and specific functions in DNA repair pathways, and that OsRecQ1 and OsRecQsim may not involve in plastid differentiation but different aspects of a plastid-specific DNA repair system. OsRecQ1 OsRecQ1, a QDE-3 homologue in rice, is required for RNA silencing induced by particle bombardment for inverted repeat DNA, but not for double-stranded RNA 2008 Plant J Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan. Based on the nucleotide sequence of QDE-3 in Neurospora crassa, which is involved in RNA silencing, rice (Oryza sativa) mutant lines disrupted by the insertion of the rice retrotransposon Tos17 were selected. Homozygous individuals from the M(1) and M(2) generations were screened and used for further analyses. The expression of the gene was not detected in leaves or calli of the mutant lines, in contrast to the wild type (WT). Induction of RNA silencing by particle bombardment was performed to investigate any effects of the OsRecQ1 gene on RNA silencing with silencing inducers of the GFP (green fluorescence protein)/GUS (beta-glucuronidase) gene in the mutant lines. The results showed that OsRecQ1 is required for RNA silencing induced by particle bombardment for inverted-repeat DNA, but not for double-stranded RNA (dsRNA). The levels of transcripts from inverted-repeat DNA were much lower in the mutant lines than those in the WT. Furthermore, no effects were observed in the accumulation of endogenous microRNAs (miR171 and miR156) and the production of the short interspersed nuclear element retroelement by small interfering RNA. On the basis of these results, we propose that OsRecQ1 may participate in the process that allows inverted repeat DNA to be transcribed into dsRNA, which can trigger RNA silencing. OsRecQ1 OsRFPH2-10, a RING-H2 Finger E3 Ubiquitin Ligase, Is Involved in Rice Antiviral Defense in the Early Stages of Rice dwarf virus Infection 2014 Mol Plant The State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, People's Republic of China None OsRFPH2-10 A rice DEAD-box protein, OsRH36, can complement an Arabidopsis atrh36 mutant, but cannot functionally replace its yeast homolog Dbp8p 2010 Plant Mol Biol Department of Life Science, National Central University, Zhongli, Taoyuan County, Taiwan, ROC. DEAD-box proteins comprise a large family and function in RNA metabolism. Although DEAD-box proteins are highly conserved among eukaryotes, little is known about the role of DEAD-box proteins in rice. In this study, we identified a rice DEAD-box protein, OsRH36, and demonstrated that OsRH36 and AtRH36 are functional orthologs. OsRH36 was expressed ubiquitously throughout the plant and the OsRH36-GFP fusion protein was localized to the nucleus and nucleolus. Furthermore, functional complementation tests among three homologous DEAD-box protein genes indicated that OsRH36 can restore segregation distortion in the Arabidopsis atrh36-1 mutant, but cannot complement the lethal phenotype in the yeast dbp8p mutant. Moreover, mutation of osrh36 also led to defective transmission of the osrh36 allele, as shown for the atrh36 mutants of Arabidopsis. Previously, we have shown that AtRH36 is involved at an early stage of rRNA maturation and is required for synchronous development of female gametophytes in Arabidopsis. Therefore, we suggest that OsRH36 is required for rRNA biogenesis and megagametogenesis in rice, consistent with the function of AtRH36 in Arabidopsis. OsRH36 A transcription factor with a bHLH domain regulates root hair development in rice 2009 Cell Res Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, People's Republic of China. A subtractive cDNA library was constructed using rice (Oryza sativa L.) callus cDNA as driver and differentiating callus cDNA as tester. A novel cDNA fragment encoding RNase L inhibitor (RLI) was isolated by screening the subtractive library, which had a higher expression level in differentiating callus than in callus. The full-length cDNA of rice-RLI was obtained by the method of rapid amplification of cDNA ends, which contained a 1812-bp open reading frame encoding a 604 amino acid polypeptide. Homologous analysis showed that rice-RLI contained the conserved motifs (two repeated P-loops, two ATP-binding boxes and an iron-sulfur binding motif). The fluorescence quantitative PCR analysis showed that it was a constitutive expressed gene but up-regulated in abiotic stress (low temperature and NaCl treatment) and down-regulated under the treatments of NAA and IAA. OsRHL1 Molecular cloning and characterization of a rice blast-inducible RING-H2 type zinc finger gene 2006 DNA Seq Department of Plant Pathology, China Agricultural University, Beijing 100094, People's Republic of China. A novel blast-inducible RING-H2 type zinc finger protein gene OsRING-1 was cloned from rice by cDNA library screening. OsRING-1 is 1670 bp in length and encodes a 46.6 kDa basic protein with two transmembrane (TM) domains, a basic domain (BD), a conserved domain (CD), a RING finger domain and a serine rich (S-rich) domain. By database search, OsRING-1 was mapped on chromosome 2 and clustered together with other six zinc finger genes. The promoter sequence analysis of OsRING-1 gene revealed that some ABA, GA, ethylene, wound, drought, heat stress and pathogen infection responsive elements were found within the OsRING-1 promoter region. Northern analysis showed that OsRING-1 was induced in different degree by pathogen infections, SA, ABA, JA and ethephon (ET) treatments. Tissue expression analysis showed that OsRING-1 was constitutively strongly expressed in roots, but faintly in stems, leaves and sheaths. Taken together, OsRING-1, as a novel C3H2C3-type zinc finger protein involved in many stress responses in rice might plays a role as a transcription regulator in plant stress response signal transduction pathways. OsRING-1 Molecular characterization and concerted evolution of two genes encoding RING-C2 type proteins in rice 2012 Gene Plant Genomics Lab, Department of Applied Plant Sciences, Kangwon National University, Chuncheon 200-713, Korea. RING (Really Interesting New Gene) finger proteins are believed to play a critical role in mediating the transfer of ubiquitin to heterogeneous substrate(s). While the two canonical types, RING-H2 and RING-HC, have been well-characterized, the molecular functions of the modified types, particularly the RING-C2 types, remain elusive. We isolated two rice genes harboring the RING-C2 domain on the distal parts of rice chromosomes 11 and 12, termed OsRINGC2-1 and OsRINGC2-2, respectively. A comparison of sequence divergences between 10 duplicate pairs on the distal parts of rice chromosomes 11 and 12 and randomly selected duplicate pairs suggested that OsRINGC2-1 and OsRINGC2-2 have evolved in concert via gene conversion. An in vitro ubiquitination assay revealed that both proteins possess E3 ligase activity, suggesting that the innate functions of these RING domains have not been affected by their modifications during evolution. Subcellular localizations were strikingly different; OsRINGC2-1 was found only in the cytoplasm with many punctate complexes, whereas OsRINGC2-2 was observed in both the nucleus and cytoplasm. The expression patterns of both genes showed striking differences in response to salt stress, whereas plants heterogeneous for both genes mediated salt tolerance in Arabidopsis, supporting the notion of concerted evolution. These results shed light on the molecular functions of OsRINGC2-1 and OsRINGC2-2 and provide insight into their molecular evolution. OsRINGC2-1,OsRINGC2-2 Over-expression of OSRIP18 increases drought and salt tolerance in transgenic rice plants 2012 Transgenic Res Rice Functional Genomics Group, Temasek Life Sciences Laboratory, 1 Research Link, The National University of Singapore, Singapore 117604, Singapore. Both drought and high salinity stresses are major abiotic factors that limit the yield of agricultural crops. Transgenic techniques have been regarded as effective ways to improve crops in their tolerance to these abiotic stresses. Functional characterization of genes is the prerequisite to identify candidates for such improvement. Here, we have investigated the biological functions of an Oryza sativa Ribosome-inactivating protein gene 18 (OSRIP18) by ectopically expressing this gene under the control of CaMV 35S promoter in the rice genome. We have generated 11 independent transgenic rice plants and all of them showed significantly increased tolerance to drought and high salinity stresses. Global gene expression changes by Microarray analysis showed that more than 100 probe sets were detected with up-regulated expression abundance while signals from only three probe sets were down-regulated after over-expression of OSRIP18. Most of them were not regulated by drought or high salinity stresses. Our data suggested that the increased tolerance to these abiotic stresses in transgenic plants might be due to up-regulation of some stress-dependent/independent genes and OSRIP18 may be potentially useful in further improving plant tolerance to various abiotic stresses by over-expression. OSRIP18 Early signaling network in rice PRR-mediated and R-mediated immunity 2013 Curr Opin Plant Biol Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. Recent studies on plant immunity and pathogen infection have revealed sophisticated forms of plant-pathogen interaction. Considerable progress has been made recently in our understanding of the molecular mechanism underlying chitin signaling in rice. The identification and characterization of two direct substrates, OsRacGEF1 and OsRLCK185, as components in the chitin receptor complex of OsCERK1 have revealed how pattern recognition receptors transduce pathogen signals to downstream molecules in rice. In this review, we highlight these and other recent studies that have contributed to our current understanding of the signaling network in rice immunity, especially with regard to pattern recognition receptors, disease resistance (R) proteins, and their downstream targets. OsRLCK185 A receptor-like cytoplasmic kinase targeted by a plant pathogen effector is directly phosphorylated by the chitin receptor and mediates rice immunity 2013 Cell Host Microbe Department of Advanced Bioscience, Graduate School of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan. CERK1 is a lysine motif-containing plant pattern recognition receptor for chitin and peptidoglycan. Chitin recognition by OsCERK1 triggers rapid engagement of a rice MAP kinase cascade, which leads to defense response activation. How the MAP kinase cascades are engaged downstream of OsCERK1 remains obscure. Searching for host proteins that interact with Xoo1488, an effector of the rice pathogen Xanthomonas oryzae, we identified the rice receptor-like cytoplasmic kinase, OsRLCK185. Silencing OsRLCK185 suppressed peptidoglycan- and chitin-induced immune responses, including MAP kinase activation and defense-gene expression. In response to chitin, OsRLCK185 associates with, and is directly phosphorylated by, OsCERK1 at the plasma membrane. Xoo1488 inhibits peptidoglycan- and chitin-induced immunity and pathogen resistance. Additionally, OsCERK1-mediated phosphorylation of OsRLCK185 is suppressed by Xoo1488, resulting in the inhibition of chitin-induced MAP kinase activation. These data support a role for OsRLCK185 as an essential immediate downstream signaling partner of OsCERK1 in mediating chitin- and peptidoglycan-induced plant immunity. OsRLCK185,OsRLCK55 Rice A20/AN1 zinc-finger containing stress-associated proteins (SAP1/11) and a receptor-like cytoplasmic kinase (OsRLCK253) interact via A20 zinc-finger and confer abiotic stress tolerance in transgenic Arabidopsis plants 2011 New Phytol Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi 110021, India. * The inbuilt mechanisms of plant survival have been exploited for improving tolerance to abiotic stresses. Stress-associated proteins (SAPs), containing A20/AN1 zinc-finger domains, confer abiotic stress tolerance in different plants, however, their interacting partners and downstream targets remain to be identified. * In this study, we have investigated the subcellular interactions of rice SAPs and their interacting partner using yeast two-hybrid and fluorescence resonance energy transfer (FRET) approaches. Their efficacy in improving abiotic stress tolerance was analysed in transgenic Arabidopsis plants. Regulation of gene expression by genome-wide microarray in transgenics was used to identify downstream targets. * It was found that the A20 domain mediates the interaction of OsSAP1 with self, its close homolog OsSAP11 and a rice receptor-like cytoplasmic kinase, OsRLCK253. Such interactions between OsSAP1/11 and with OsRLCK253 occur at nuclear membrane, plasma membrane and in nucleus. Functionally, both OsSAP11 and OsRLCK253 could improve the water-deficit and salt stress tolerance in transgenic Arabidopsis plants via a signaling pathway affecting the expression of several common endogenous genes. * Components of a novel stress-responsive pathway have been identified. Their stress-inducible expression provided the protection against yield loss in transgenic plants, indicating the agronomic relevance of OsSAP11 and OsRLCK253 in conferring abiotic stress tolerance. OsRLCK253,OsSAP1,OsDOG|OsSAP11 RNAi knockdown of Oryza sativa root meander curling gene led to altered root development and coiling which were mediated by jasmonic acid signalling in rice 2007 Plant Cell Environ Research Center for Molecular Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, and Graduate School of the Chinese Academy of Sciences, Beijing, China. Jasmonic acid (JA) is a well-known defence hormone, but its biological function and mechanism in rice root development are less understood. Here, we describe a JA-induced putative receptor-like protein (OsRLK, AAL87185) functioning in root development in rice. RNA in situ hybridization revealed that the gene was expressed largely in roots, and a fusion protein showed its localization on the plasma membrane. The primary roots in RNAi transgenic rice plants meandered and curled more easily than wild-type (WT) roots under JA treatment. Thus, this gene was renamed Oryza sativa root meander curling (OsRMC). The transgenic primary roots were shorter, the number of adventitious roots increased and the number of lateral roots decreased as compared to the WT. As well, the second sheath was reduced in length. Growth of both primary roots and second sheaths was sensitive to JA treatment. No significant change of JA level appeared in the roots between the transgenic rice line and WT. Expression of RSOsPR10, involved in the JA signalling pathway, was induced in transgenic rice. Western blotting revealed OsRMC induced by JA. Our results suggest that OsRMC of the DUF26 subfamily involved in JA signal transduction mediates root development and negatively regulates root curling in rice. OsRMC|OsRLK,RSOsPR10 Identification of an apoplastic protein involved in the initial phase of salt stress response in rice root by two-dimensional electrophoresis 2009 Plant Physiol Institute of Molecular and Cell Biology, Hebei Normal University, Shijiazhuang 050016, China. The apoplast of plant cells, which carries out multiple functions in plant metabolism and signaling, is not only a barrier but also the linker between the environment and the protoplast. To investigate the role of apoplastic proteins in the salt stress response, 10-d-old rice (Oryza sativa) plants were treated with 200 mM NaCl for 1, 3, or 6 h, and the soluble apoplast proteins were extracted for differential analysis compared with untreated controls using two-dimensional electrophoresis. Ten protein spots that increased or decreased significantly in abundance were identified by mass spectrometry. These proteins included some well-known biotic and abiotic stress-related proteins. Among them, an apoplastic protein, with extracellular domain-like cysteine-rich motifs (DUF26), O. sativa root meander curling (OsRMC), has shown drastically increased abundance in response to salt stress during the initial phase. OsRMC RNA interference transgenic rice has been generated to assess the function of OsRMC in the salt stress response. The results show that knocking down the expression level of OsRMC in transgenic rice led to insensitive seed germination, enhanced growth inhibition, and improved salt stress tolerance to NaCl than in untransgenic plants. These results indicate that plant apoplastic proteins may have important roles in the plant salt stress response. OsRMC|OsRLK The rice RMR1 associates with a distinct prevacuolar compartment for the protein storage vacuole pathway 2011 Mol Plant School of Life Sciences, Centre for Cell and Developmental Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China. Transport of vacuolar proteins from Golgi apparatus or trans-Golgi network (TGN) to vacuoles is a receptor-mediated process via an intermediate membrane-bound prevacuolar compartment (PVC) in plant cells. Both vacuolar sorting receptor (VSR) and receptor homology region-transmembrane domain-RING-H2 (RMR) proteins have been shown to function in transporting storage proteins to protein storage vacuole (PSV), but little is known about the nature of the PVC for the PSV pathway. Here, we use the rice RMR1 (OsRMR1) as a probe to study the PSV pathway in plants. Immunogold electron microscopy (EM) with specific OsRMR1 antibodies showed that OsRMR1 proteins were found in the Golgi apparatus, TGN, and a distinct organelle with characteristics of PVC in both rice culture cells and developing rice seeds, as well as the protein body type II (PBII) or PSV in developing rice seeds. This organelle, also found in both tobacco BY-2 and Arabidopsis suspension cultured cells, is morphologically distinct from the VSR-positive multivesicular lytic PVC or multivesicular body (MVB) and thus represent a PVC for the PSV pathway that we name storage PVC (sPVC). Further in vivo and in vitro interaction studies using truncated OsRMR1 proteins secreted into the culture media of transgenic BY-2 suspension cells demonstrated that OsRMR1 functions as a sorting receptor in transporting vicilin-like storage proteins. OsRMR1 Replication protein A (RPA1a) is required for meiotic and somatic DNA repair but is dispensable for DNA replication and homologous recombination in rice 2009 Plant Physiol National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China. Replication protein A (RPA), a highly conserved single-stranded DNA-binding protein in eukaryotes, is a stable complex comprising three subunits termed RPA1, RPA2, and RPA3. RPA is required for multiple processes in DNA metabolism such as replication, repair, and homologous recombination in yeast (Saccharomyces cerevisiae) and human. Most eukaryotic organisms, including fungi, insects, and vertebrates, have only a single RPA gene that encodes each RPA subunit. Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), however, possess multiple copies of an RPA gene. Rice has three paralogs each of RPA1 and RPA2, and one for RPA3. Previous studies have established their biochemical interactions in vitro and in vivo, but little is known about their exact function in rice. We examined the function of OsRPA1a in rice using a T-DNA insertional mutant. The osrpa1a mutants had a normal phenotype during vegetative growth but were sterile at the reproductive stage. Cytological examination confirmed that no embryo sac formed in female meiocytes and that abnormal chromosomal fragmentation occurred in male meiocytes after anaphase I. Compared with wild type, the osrpa1a mutant showed no visible defects in mitosis and chromosome pairing and synapsis during meiosis. In addition, the osrpa1a mutant was hypersensitive to ultraviolet-C irradiation and the DNA-damaging agents mitomycin C and methyl methanesulfonate. Thus, our data suggest that OsRPA1a plays an essential role in DNA repair but may not participate in, or at least is dispensable for, DNA replication and homologous recombination in rice. OsRPA1a Plant-specific regulation of replication protein A2 (OsRPA2) from rice during the cell cycle and in response to ultraviolet light exposure 2003 Planta Institut fur Allgemeine Botanik, Universitat Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany. DNA replication is a process that is highly conserved among eukaryotes. Nonetheless, little is known about the proteins involved in it in plants. Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding protein with several functions in DNA metabolism in humans and yeast and supposedly also in plants. Here we report on the regulation of OsRPA2, the 32-kDa subunit of RPA from rice ( Oryza sativa L.). We found conserved regulation mechanisms at the level of gene expression between animal and plant RPA2 genes and distinct features of OsRPA2 regulation at the level of protein expression. Unlike in animals or in yeast, protein abundance in rice was regulated in a cell cycle phase-specific manner and was altered after UV-C light exposure. On the other hand, posttranslational modification through phosphorylation did not appear to play a pivotal role in rice as it does in animal cells. Our results indicate that plant-specific mechanisms of regulation have evolved for RPA2 within the generally well-conserved process of DNA replication, suggesting specific requirements for regulation of DNA metabolism in plants as compared to other eukaryotes. OsRPA2|OsRPA32-1 Two types of replication protein A 70 kDa subunit in rice, Oryza sativa: molecular cloning, characterization, and cellular & tissue distribution 2001 Gene Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda-shi, Chiba-ken 278-8510, Japan. Replication protein A (RPA), which is comprised of three subunits, is an important factor involved in DNA replication, repair, and transcription. We isolated and characterized 70 and 32 kDa subunits of RPA from rice (Oryza sativa cv. Nipponbare) termed OsRPA70a and OsRPA32. OsRPA70a shows a low level of homology with OsRPA1 which was isolated from deepwater rice (Oryza sativa cv. Pin Gaew 56), previously. We also succeeded to isolate OsRPA70b which is homologue to OsRPA1 from Oryza sativa cv. Nipponbare. OsRPA70a shows only 33.8% sequence identity with OsRPA70b, indicating that two different types of 70 kDa RPA subunits are present in Oryza sativa cv. Nipponbare. These subunits showed differences in their expression patterns among tissues. The transcripts of OsRPA70a and OsRPA32 were expressed strongly in proliferating tissues such as root tips and young leaves that contain root apical meristem and marginal meristem, respectively, and weakly in the mature leaves which have no proliferating tissues. On the other hand, OsRPA70b was expressed mostly in the proliferating tissues. The roles of these molecules in plant DNA replication and DNA repair are discussed. OsRPA1|OsRPA70b A higher plant has three different types of RPA heterotrimeric complex 2006 J Biochem Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510. Replication protein A (RPA) is a protein complex composed of three subunits known as RPA70, RPA32, and RPA14. Generally, only one version of each of the three RPA genes is present in animals and yeast (with the exception of the human RPA32 ortholog). In rice (Oryza sativa L.), however, two paralogs of RPA70 have been reported. We screened the rice genome for RPA subunit genes, and identified three OsRPA70 (OsRPA70a, OsRPA70b and OsRPA70c), three OsRPA32 (OsRPA32-1, OsRPA32-2 and OsRPA32-3), and one OsRPA14. Through two-hybrid assays and pull down analyses, we showed that OsRPA70a interacted preferentially with OsRPA32-2, OsRPA70b with OsRPA32-1, and OsRPA70c with OsRPA32-3. OsRPA14 interacted with all OsRPA32 paralogs. Thus, rice has three types of RPA complex: OsRPA70a-OsRPA32-2-OsRPA14 (type A), OsRPA70b-OsRPA32-1-OsRPA14 (type B), and OsRPA70c-OsRPA32-3-OsRPA14 (type C). Subcellular localization analysis suggested that the type-A RPA complex is required for chloroplast DNA metabolism, whereas types B and C function in nuclear DNA metabolism. OsRPA1|OsRPA70b New changes in the plasma-membrane-associated proteome of rice roots under salt stress 2009 Proteomics Department of Biochemistry and Molecular Biology, College of Life Science, Nanjing Agricultural University, Nanjing, Jiangsu, PR China. To gain a better understanding of salt stress responses in plants, we used a proteomic approach to investigate changes in rice (Oryza sativa) root plasma-membrane-associated proteins following treatment with 150 mmol/L NaCl. With or without a 48 h salt treatment, plasma membrane fractions from root tip cells of a salt-sensitive rice cultivar, Wuyunjing 8, were purified by PEG aqueous two-phase partitioning, and plasma-membrane-associated proteins were separated by IEF/SDS-PAGE using an optimized rehydration buffer. Comparative analysis of three independent biological replicates revealed that the expressions of 18 proteins changed by more than 1.5-fold in response to salt stress. Of these proteins, nine were up-regulated and nine were down-regulated. MS analysis indicated that most of these membrane-associated proteins are involved in important physiological processes such as membrane stabilization, ion homeostasis, and signal transduction. In addition, a new leucine-rich-repeat type receptor-like protein kinase, OsRPK1, was identified as a salt-responding protein. Immuno-blots indicated that OsRPK1 is also induced by cold, drought, and abscisic acid. Using immuno-histochemical techniques, we determined that the expression of OsRPK1 was localized in the plasma membrane of cortex cells in roots. The results suggest that different rice cultivars might have different salt stress response mechanisms. OsRPK1 Transfer of rice mitochondrial ribosomal protein L6 gene to the nucleus: acquisition of the 5'-untranslated region via a transposable element 2008 BMC Evol Biol Graduate School of Agriculture, Kyoto Prefectural University, Seika, Kyoto 619-0244, Japan. nk0103@kab.seika.kyoto.jp BACKGROUND: The mitochondria of contemporary organisms contain fewer genes than the ancestral bacteria are predicted to have contained. Because most of the mitochondrial proteins are encoded in the nucleus, the genes would have been transferred from the mitochondrion to the nucleus at some stage of evolution and they must have acquired cis-regulatory elements compatible with eukaryotic gene expression. However, most of such processes remain unknown. RESULTS: The ribosomal protein L6 gene (rpl6) has been lost in presently-known angiosperm mitochondrial genomes. We found that each of the two rice rpl6 genes (OsRpl6-1 and OsRpl6-2) has an intron in an identical position within the 5'-untranslated region (UTR), which suggests a duplication of the rpl6 gene after its transfer to the nucleus. Each of the predicted RPL6 proteins lacks an N-terminal extension as a mitochondrial targeting signal. Transient assays using green fluorescent protein indicated that their mature N-terminal coding regions contain the mitochondrial targeting information. Reverse transcription-PCR analysis showed that OsRpl6-2 expresses considerably fewer transcripts than OsRpl6-1. This might be the result of differences in promoter regions because the 5'-noncoding regions of the two rpl6 genes differ at a point close to the center of the intron. There are several sequences homologous to the region around the 5'-UTR of OsRpl6-1 in the rice genome. These sequences have characteristics similar to those of the transposable elements (TE) belonging to the PIF/Harbinger superfamily. CONCLUSION: The above evidences suggest a novel mechanism in which the 5'-UTR of the transferred mitochondrial gene was acquired via a TE. Since the 5'-UTRs and introns within the 5'-UTRs often contain transcriptional and posttranscriptional cis-elements, the transferred rice mitochondrial rpl6 gene may have acquired its cis-element from a TE. OsRpl6-1 Characterization of a rice nuclear-encoded plastid RNA polymerase gene OsRpoTp 2004 Plant Cell Physiol Department of Biology, Faculty of Science, Kyushu University, Hakozaki, Higashi-ku, Fukuoka, 812-81 Japan. kkususcb@mbox.nc.kyushu-u.ac.jp We isolated and characterized two rice genes, OsRpoTp and OsRpoTm, that encode putative phage-type RNA polymerases. Predicted amino acid sequences showed high homology of these genes to known RpoT genes. A transient expression assay using green fluorescent protein indicated that the encoded proteins were localized to plastids and mitochondria, respectively. We demonstrated by reverse transcription-PCR experiments and immunoblot analysis that OsRpoTp expression occurred at an early stage of leaf development, prior to the transcript accumulation of the genes that were transcribed by the nuclear-encoded plastid RNA polymerase (NEP). Expression analyses of the chloroplast-deficient rice mutant, virescent-1, showed a discrepancy between OsRpoTp protein accumulation and the level of transcripts of NEP-transcribed genes. Our results suggest that NEP activation is regulated by a process after transcription, and is affected by the developmental state of chloroplast biogenesis. OsRpoTm,OsRpoTp The virescent-2 mutation inhibits translation of plastid transcripts for the plastid genetic system at an early stage of chloroplast differentiation 2004 Plant and Cell Physiology Department of Biology, Faculty of Science, Kyushu University, Fukuoka, 812-8581 Japan. The rice virescent-2 mutant (v(2)) is temperature conditional and develops chlorotic, chloroplast-deficient leaves at the restrictive temperature. In the v(2) mutant, plastid-encoded proteins involved in photosynthesis and plastid transcriptional regulation were not detectable at any time during chloroplast differentiation. However, the plastid transcripts for these two classes of proteins behaved differently in the mutant, with those for the plastid transcription/translation apparatus accumulating to wild-type levels and those for photosynthetic apparatus being suppressed. Polysome analysis showed that translation of the plastid transcripts encoding the plastid transcription/translation apparatus was blocked at an early stage of chloroplast differentiation. Accumulation of transcripts of nuclear-encoded photosynthetic genes, such as cab and rbcS, was strongly suppressed in the mutant at later stages of chloroplast differentiation, whereas transcripts of genes for the plastid transcription apparatus, such as OsRpoTp and OsSIG2A, accumulated to abnormally high levels at these stages. These results suggest that activation of the plastid translation machinery at an early stage of chloroplast differentiation is important for triggering the transmission of information about plastid developmental state to the nucleus, which in turn is required for the induction of nuclear-encoded chloroplast proteins at later stages of chloroplast differentiation. OsRpoTp,OsSIG2A,v2 Identification of the 19S regulatory particle subunits from the rice 26S proteasome 2002 Eur J Biochem Yokohama City University, Kihara Institute for Biological Research/Graduate School of Integrated Science, Japan. The 26S proteasome, a protein complex consisting of a 20S proteasome and a pair of 19S regulatory particles (RP), is involved in ATP-dependent proteolysis in eukaryotes. In yeast, the RP contains six different ATPase subunits and, at least, 11 non-ATPase subunits. In this study, we identified the rice homologs of yeast RP subunit genes from the rice expressed sequence tag (EST) library. The complete nucleotide sequences of the homologs for five ATPase subunits, OsRpt1, OsRpt2, OsRpt4, OsRpt5 and OsRpt6, and five non-ATPase subunits, OsRpn7, OsRpn8, OsRpn10, OsRpn11 and OsRpn12, and the partial sequences of one ATPase subunit, OsRpt3, and six non-ATPase subunits, OsRpn1, OsRpn2, OsRpn3, OsRpn5, OsRpn6 and OsRpn9, were determined. Gene homologs of four ATPase subunits, OsRpt1, OsRpt2, OsRpt4 and OsRpt5, and three non- ATPase subunits, OsRpn1, OsRpn2 and OsRpn9, were found to be encoded by duplicated genes. The rice RP was purified by immunoaffinity chromatography with a Protein A column immobilized antibody against rice 20S proteasome, and the subunit composition was determined. The homologs obtained from the rice EST library were identified as genes encoding subunits of RP purified from rice, including the both products of duplicated genes by using electrospray ionization quadrupole time-of-flight mass spectrometry. Post-translational modifications and processing in rice RP subunits were also identified. Various types of RP complex with different subunit compositions are present in rice cells, suggesting the multiple functions of rice proteasome. OsRPT4 Molecular characterization and differential expression of cytokinin-responsive type-A response regulators in rice (Oryza sativa) 2006 BMC Plant Biol Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India. mjainanid@gmail.com BACKGROUND: The response regulators represent the elements of bacterial two-component system and have been characterized from dicot plants like Arabidopsis but little information is available on the monocots, including the cereal crops. The aim of this study was to characterize type-A response regulator genes from rice, and to investigate their expression in various organs as well as in response to different hormones, including cytokinin, and environmental stimuli. RESULTS: By analysis of the whole genome sequence of rice, we have identified ten genes encoding type-A response regulators based upon their high sequence identity within the receiver domain. The exon-intron organization, intron-phasing as well as chromosomal location of all the RT-PCR amplified rice (Oryza sativa) response regulator (OsRR) genes have been analyzed. The transcripts of OsRR genes could be detected by real-time PCR in all organs of the light- and dark-grown rice seedlings/plants, although there were quantitative differences. The steady-state transcript levels of most of the OsRR genes increased rapidly (within 15 min) on exogenous cytokinin application even in the presence of cycloheximide. Moreover, the expression of the OsRR6 gene was enhanced in rice seedlings exposed to salinity, dehydration and low temperature stress. CONCLUSION: Ten type-A response regulator genes identified in rice, the model monocot plant, show overlapping/differential expression patterns in various organs and in response to light. The induction of OsRR genes by cytokinin even in the absence of de novo protein synthesis qualifies them to be primary cytokinin response genes. The induction of OsRR6 in response to different environmental stimuli indicates its role in cross-talk between abiotic stress and cytokinin signaling. These results provide a foundation for further investigations on specific as well as overlapping cellular functions of type-A response regulators in rice. OsRR1,OsRR2,OsRR3,OsRR4,OsRR5,OsRR6,OsRR7,OsRR8,OsRR9 OsRRM, a Spen-like rice gene expressed specifically in the endosperm 2007 Cell Res National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. We used the promoter trap technique to identify a rice plant, named 107#, in which the beta-glucuronidase (GUS) reporter gene was expressed specifically in the endosperm. A single copy of the T-DNA was inserted into the plant genome, and a candidate gene OsRRM was identified by the insertion. The OsRRM promoter directed GUS expression specifically in rice endosperm, analogous to the GUS expression pattern observed in 107#. OsRRM is a single-copy gene in rice and encodes a nuclear protein containing 1005 amino-acid residues with two RNA recognition motifs and one Spen paralog and ortholog C-terminal domain. Western blot analysis confirmed that the OsRRM protein was specifically expressed in rice endosperm. Ectopic expression of OsRRM in transgenic plants led to abnormalities, such as short stature, retarded growth and low fructification rates. Our data, in conjunction with the reported function of Spen genes, implicated OsRRM in the regulation of cell development in rice endosperm. OsRRM OsRRMh, a Spen-like gene, plays an important role during the vegetative to reproductive transition in rice 2013 J Integr Plant Biol National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, the Chinese Academy of Sciences, Shanghai, 200032, China; University of Chinese Academy of Sciences, the Chinese Academy of Sciences, Beijing, 100049, China. OsRRMh, a homologue of OsRRM, encodes a Spen-like protein, and is composed of two N-terminal RNA recognition motifs (RRM) and one C-terminal Spen paralogue and an orthologue C-terminal domain (SPOC). The gene has been found to be constitutively expressed in the root, stem, leaf, spikelet, and immature seed, and alternative splicing patterns were confirmed in different tissues, which may indicate diverse functions for OsRRMh. The OsRRMh dsRNAi lines exhibited late-flowering and a larger panicle phenotype. When full-length OsRRMh and/or its SPOC domain were overexpressed, the fertility rate and number of spikelets per panicle were both markedly reduced. Also, overexpression of OsRRMh in the Arabidopsis fpa mutant did not restore the normal flowering time, and it delayed flowering in Col plants. Therefore, we propose that OsRRMh may confer one of its functions in the vegetative-to-reproductive transition in rice (Oryza sativa L. subsp. japonica cv. Zhonghua No. 11 (ZH11)). OsRRMh Overexpression of an S-like ribonuclease gene, OsRNS4, confers enhanced tolerance to high salinity and hyposensitivity to phytochrome-mediated light signals in rice 2014 Plant Sci Shandong Rice Research Institute, Shandong Academy of Agricultural Sciences, Jinan 250100, China; Shandong Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, 250100 Jinan, China. Electronic address: xiaofeijuner@126.com. S-like ribonucleases (S-like RNases) are homologous to S-ribonucleases (S-RNases), but are not involved in self-incompatibility. In dicotyledonous plants, S-like RNases play an important role in phosphate recycling during senescence and are induced by inorganic phosphate-starvation and in response to defense and mechanical wounding. However, little information about the functions of the S-like RNase in monocots has been reported. Here, we investigated the expression patterns and roles of an S-like RNase gene, OsRNS4, in abscisic acid (ABA)-mediated responses and phytochrome-mediated light responses as well as salinity tolerance in rice. The OsRNS4 gene was expressed at relatively high levels in leaves although its transcripts were detected in various organs. OsRNS4 expression was regulated by salt, PEG and ABA. The seedlings overexpressing OsRNS4 had longer coleoptiles and first leaves than wild-type seedlings under red light (R) and far-red light (FR), suggesting negative regulation of OsRNS4 in photomorphogenesis in rice seedlings. Moreover, ABA-induced growth inhibition of rice seedlings was significantly increased in the OsRNS4-overexpression (OsRNS4-OX) lines compared with that in WT, suggesting that OsRNS4 probably acts as a positive regulator in ABA responses in rice seedlings. In addition, our results demonstrate that OsRNS4-OX lines have enhanced tolerance to high salinity compared to WT. Our findings supply new evidence on the functions of monocot S-like RNase in regulating photosensitivity and abiotic stress responses. OsRNS4|OsRRP Cloning and characterization of an RNase-related protein gene preferentially expressed in rice stems 2006 Biosci Biotechnol Biochem State Key Laboratory of Biocontrol and Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou, China. RNase-related proteins (RRPs) are S- and S-like RNase homologs lacking the active site required for RNase activity. Here we describe the cloning and characterization of the rice (Oryza sativa) RRP gene (OsRRP). A single copy of OsRRP occurs in the rice genome. OsRRP contains three introns and an open reading frame encoding 252 amino acids, with the replacement of two histidines involved in the active site of RNase by lysine and tyrosine respectively. OsRRP is preferentially expressed in stems of wild-type rice and is significantly down-regulated in an increased tillering dwarf mutant ext37. OsRNS4|OsRRP Modulation of ethylene responses by OsRTH1 overexpression reveals the biological significance of ethylene in rice seedling growth and development 2012 J Exp Bot National Key Laboratory of Plant Molecular Genetics and National Center for Plant Gene Research (Shanghai), Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Overexpression of Arabidopsis Reversion-To-ethylene Sensitivity1 (RTE1) results in whole-plant ethylene insensitivity dependent on the ethylene receptor gene Ethylene Response1 (ETR1). However, overexpression of the tomato RTE1 homologue Green Ripe (GR) delays fruit ripening but does not confer whole-plant ethylene insensitivity. It was decided to investigate whether aspects of ethylene-induced growth and development of the monocotyledonous model plant rice could be modulated by rice RTE1 homologues (OsRTH genes). Results from a cross-species complementation test in Arabidopsis showed that OsRTH1 overexpression complemented the rte1-2 loss-of-function mutation and conferred whole-plant ethylene insensitivity in an ETR1-dependent manner. In contrast, OsRTH2 and OsRTH3 overexpression did not complement rte1-2 or confer ethylene insensitivity. In rice, OsRTH1 overexpression substantially prevented ethylene-induced alterations in growth and development, including leaf senescence, seedling leaf elongation and development, coleoptile elongation or curvature, and adventitious root development. Results of subcellular localizations of OsRTHs, each fused with the green fluorescent protein, in onion epidermal cells suggested that the three OsRTHs were predominantly localized to the Golgi. OsRTH1 may be an RTE1 orthologue of rice and modulate rice ethylene responses. The possible roles of auxins and gibberellins in the ethylene-induced alterations in growth were evaluated and the biological significance of ethylene in the early stage of rice seedling growth is discussed. OsRTH1,OsRTH2,OsRTH3 Zinc finger-containing glycine-rich RNA-binding protein in Oryza sativa has an RNA chaperone activity under cold stress conditions 2010 Plant Cell Environ Department of Plant Biotechnology and Agricultural Plant Stress Research Center, College of Agriculture and Life Sciences, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, Korea. The rice (Oryza sativa) genome harbours three genes encoding CysCysHisCys (CCHC)-type zinc finger-containing glycine-rich RNA-binding proteins, designated OsRZ proteins, but their importance and physiological functions remain largely unknown. Here, the stress-responsive expression patterns of OsRZs were assessed, and the biological and cellular functions of OsRZs were evaluated under low temperature conditions. The expression levels of the three OsRZs were up-regulated by cold stress, whereas drought or high salt stress did not significantly alter its transcript level. OsRZ2 complemented the cold sensitivity of BX04 Escherichia coli cells under low temperatures, and had DNA-melting activity and transcription anti-termination activity, thereby indicating that OsRZ2 possesses an RNA chaperone activity. By contrast, neither OsRZ1 nor OsRZ3 harboured these activities. Ectopic expression of OsRZ2, but not OsRZ3, in cold-sensitive Arabidopsis grp7 knockout plants rescued the grp7 plants from cold and freezing damage, and OsRZ2 complemented the defect in mRNA export from the nucleus to the cytoplasm in grp7 mutant during cold stress. The present findings support the emerging idea that the regulation of mRNA export is one of the adaptive processes in plants under stress conditions, and RNA chaperone functions as a regulator in mRNA export under cold stress conditions. OsRZ1,OsRZ2,OsRZ3 Structural features important for the RNA chaperone activity of zinc finger-containing glycine-rich RNA-binding proteins from wheat (Triticum avestivum) and rice (Oryza sativa) 2013 Phytochemistry Department of Plant Biotechnology and Kumho Life Science Laboratory, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, Republic of Korea. Despite the increase in understanding of RNA chaperone activity of zinc finger-containing glycine-rich RNA-binding proteins (RZs) during the cold adaptation process, the structural features relevant to the RNA chaperone activity of RZs still largely remain to be established. To investigate the structural determinants important for the RNA chaperone activity of RZs, domain-swapping and deletion analyses was carried out to assess the contribution of the N-terminal zinc finger RNA-recognition motif (RRM) domain and the C-terminal glycine-rich region of wheat (Triticum avestivum) and rice (Oryza sativa) RZs to RNA chaperone activity. Although the amino acid sequence similarity among wheat TaRZ2, wheat TaRZ3, and rice OsRZ1 was high, only TaRZ2 had RNA chaperone activity as evidenced by complementation ability in cold-sensitive Escherichia coli mutant cell under cold stress and in vivo and in vitro nucleic acid-melting activity. Domain-swapping and deletion analysis demonstrated that the overall folding of RZs governed by the N-terminal RRM domain and the C-terminal glycine-rich region, as well as the size of the disordered C-terminal glycine-rich region, are crucial for the RNA chaperone activity of RZs. Collectively, these results indicate that a specific modular arrangement of RRM domain and the disordered C-terminal region determines the RNA chaperone activity of RZs in cells. OsRZ1,OsRZ2 Differential expression of an S-adenosyl-L-methionine decarboxylase gene involved in polyamine biosynthesis under low temperature stress in japonica and indica rice genotypes 2004 Mol Genet Genomics Department of Low Temperature Science, National Agricultural Research Center for Hokkaido Region, 1 Hitsujigaoka, Toyohira, 062-8555 Sapporo, Hokkaido, Japan. mapillai1@hotmail.com We have investigated the regulation of the rice (Oryza sativa) gene OsSAMDC, which encodes an S-adenosyl-L-methionine decarboxylase (SAMDC) involved in polyamine biosynthesis. Clones representing genes differentially expressed at 5 degrees C and 20 degrees C were isolated from a cDNA library prepared from the chilling-tolerant japonica-type cultivar Yukihikari. The full-length OsSAMDC cDNA consists of 1560 bp, with the longest ORF encoding a polypeptide of 398 amino acids. Southern analysis indicated that there are two types of gene for SAMDC in the Yukihikari genome. Analysis of the expression of OsSAMDC by Northern hybridization revealed relatively high levels of mRNA in the leaves, nodes and internodes. We also analyzed the response of OsSAMDC to various abiotic stress treatments and plant hormones. Upon exposure to cold stress (5 degrees C) the level of OsSAMDC transcripts in the cold-resistant Yukihikari genotype continued to increase for up to 72 h. In contrast, there was no change in OsSAMDC transcription in the susceptible indica cultivar TKM9 under the same conditions. Ethephon induced the accumulation of OsSAMDC transcripts to similar extents in both genotypes. Examination of polyamine levels in the cold-resistant Yukihikari genotype revealed that spermidine levels were elevated during the course of cold treatment. These results suggest that the induction of the OsSAMDC gene in response to cold may be used as a molecular marker for the ability of rice seedlings to withstand exposure to low temperatures. OsSAMDC Identification of a drought-induced rice gene, OsSAP, that suppresses Bax-induced cell death in yeast 2013 Mol Biol Rep Division of Plant Biosciences, School of Applied BioSciences, College of Agriculture and Life Science, Kyungpook National University, Taegu, 702-701, Korea. We identified rice genes that might be involved in drought stress tolerance by virtue of their anti-apoptotic activity. Potential anti-apoptosis related genes were identified by screening an Oryza sativa cDNA library derived from drought stressed tissues in a yeast functional assay. About 28 O. sativa cDNAs promoted yeast survival following engagement of Bax-induced apoptosis. An O. sativa cDNA encoding R12H780 was a highly conserved putative senescence-associated-protein (OsSAP). OsSAP was both highly and rapidly expressed in response to drought stress. Additionally, OsSAP was found to be localized to the mitochondria. Overall, OsSAP represents a new type of Bax suppressor related gene and endows multiple stress tolerance in yeast. OsSAP Small GTPase Sar1 is crucial for proglutelin and alpha-globulin export from the endoplasmic reticulum in rice endosperm 2013 J Exp Bot Key Laboratory of Plant Molecular Physiology, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China. Rice seed storage proteins glutelin and alpha-globulin are synthesized in the endoplasmic reticulum (ER) and deposited in protein storage vacuoles (PSVs). Sar1, a small GTPase, acts as a molecular switch to regulate the assembly of coat protein complex II, which exports secretory protein from the ER to the Golgi apparatus. To reveal the route by which glutelin and alpha-globulin exit the ER, four putative Sar1 genes (OsSar1a/b/c/d) were cloned from rice, and transgenic rice were generated with Sar1 overexpressed or suppressed by RNA interference (RNAi) specifically in the endosperm under the control of the rice glutelin promoter. Overexpression or suppression of any OsSar1 did not alter the phenotype. However, simultaneous knockdown of OsSar1a/b/c resulted in floury and shrunken seeds, with an increased level of glutelin precursor and decreased level of the mature alpha- and beta-subunit. OsSar1abc RNAi endosperm generated numerous, spherical, novel protein bodies with highly electron-dense matrixes containing both glutelin and alpha-globulin. Notably, the novel protein bodies were surrounded by ribosomes, showing that they were derived from the ER. Some of the ER-derived dense protein bodies were attached to a blebbing structure containing prolamin. These results indicated that OsSar1a/b/c play a crucial role in storage proteins exiting from the ER, with functional redundancy in rice endosperm, and glutelin and alpha-globulin transported together from the ER to the Golgi apparatus by a pathway mediated by coat protein complex II. OsSar1a,OsSar1b,OsSar1c,OsSar1d The novel rice (Oryza sativa L.) gene OsSbf1 encodes a putative member of the Na+/bile acid symporter family 2002 J Exp Bot Institut für Allgemeine Botanik, Universität Hamburg, Ohnhorststrasse 18, D-22609 Hamburg, Germany PCR‐based differential screening was used to identify ethylene‐induced genes in deep‐water rice (Oryza sativa L.). One of the isolated cDNAs represented a novel protein, OsSBF1, with high homology to mammalian Na+/bile acid transporters and to sodium‐dependent transporters from bacteria. One highly homologous protein and three less conserved homologues were identified in Arabidopsis thaliana indicating that Sbf proteins exist in monocot and dicot plant species. Expression of OsSbf1 in deep‐water rice was shown to be elevated by growth‐inducing treatments. Since bile acids have not been found in plants to date a possible function of SBF proteins may be in the transport of structurally related sulphonated brassinosteroids. Ossbf1 Enhanced Resistance to Blast Fungus and Bacterial Blight in Transgenic Rice Constitutively ExpressingOsSBP, a Rice Homologue of Mammalian Selenium-binding Proteins 2014 Bioscience, Biotechnology and Biochemistry Research Association for Biotechnology The rice Oryza sativa selenium-binding protein homologue (OsSBP) gene encodes a homologue of mammalian selenium-binding proteins, and it has been isolated as one of the genes induced by treating a plant with a cerebroside elicitor from rice blast fungus. The possible role of OsSBP in plant defense was evaluated by using a transgenic approach. Plants overexpressing OsSBP showed enhanced resistance to a virulent strain of rice blast fungus as well as to rice bacterial blight. The expression of defense-related genes and the accumulation of phytoalexin after infection by rice blast fungus were accelerated in the OsSBP overexpressors. A higher level of H2O2 accumulation and reduced activity of such scavenging enzymes as ascorbate peroxidase and catalase were seen when the OsSBP-overexpressing plants were treated with the protein phosphatase 1 inhibitor, calyculin A. These results suggest that the upregulation of OsSBP expression conferred enhanced tolerance to different pathogens, possibly by increasing plant sensitivity to endogenous defense responses. Additionally, the OsSBP protein might have a role in modulating the defense mechanism to biotic stress in rice. OsSBP Rice SCAMP1 Defines Clathrin-Coated, trans-Golgi-Located Tubular-Vesicular Structures as an Early Endosome in Tobacco BY-2 Cells 2007 The Plant Cell Online Department of Biology and Molecular Biotechnology Program, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China. We recently identified multivesicular bodies (MVBs) as prevacuolar compartments (PVCs) in the secretory and endocytic pathways to the lytic vacuole in tobacco (Nicotiana tabacum) BY-2 cells. Secretory carrier membrane proteins (SCAMPs) are post-Golgi, integral membrane proteins mediating endocytosis in animal cells. To define the endocytic pathway in plants, we cloned the rice (Oryza sativa) homolog of animal SCAMP1 and generated transgenic tobacco BY-2 cells expressing yellow fluorescent protein (YFP)-SCAMP1 or SCAMP1-YFP fusions. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that YFP-SCAMP1 fusions and native SCAMP1 localize to the plasma membrane and mobile structures in the cytoplasm of transgenic BY-2 cells. Drug treatments and confocal immunofluorescence studies demonstrated that the punctate cytosolic organelles labeled by YFP-SCAMP1 or SCAMP1 were distinct from the Golgi apparatus and PVCs. SCAMP1-labeled organelles may represent an early endosome because the internalized endocytic markers FM4-64 and AM4-64 reached these organelles before PVCs. In addition, wortmannin caused the redistribution of SCAMP1 from the early endosomes to PVCs, probably as a result of fusions between the two compartments. Immunogold electron microscopy with high-pressure frozen/freeze-substituted samples identified the SCAMP1-positive organelles as tubular-vesicular structures at the trans-Golgi with clathrin coats. These early endosomal compartments resemble the previously described partially coated reticulum and trans-Golgi network in plant cells. OsSCAMP1 The SCARECROW gene's role in asymmetric cell divisions in rice plants 2003 The Plant Journal BioScience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan. Asymmetric cell division is one of the most important mechanisms in the diversification of cell function and fate. In Arabidopsis, SCARECROW (SCR) is essential for the asymmetric division of the cortex/endodermis progenitor cell in the root. To learn more about how SCR is involved in asymmetric division, we analyzed the rice SCR (OsSCR) expression. In the root tip, OsSCR expression was observed in the endodermal cell layer and downregulated in the daughter cortex cell after asymmetric division, just as with Arabidopsis SCR. In leaf primordia, expression of OsSCR was observed in stomatal and ligule formation. In stomatal development, OsSCR was specifically expressed in the stomatal cell files before formation of guard mother cells (GMCs), and then, its expression was localized in GMCs, when the first asymmetric division occurred to generate the GMCs. Before the second asymmetric division of subsidiary mother cells (SMCs), localized OsSCR expression was observed in SMCs in the area close to the GMCs. Before these asymmetric divisions, the localization of OsSCR mRNA in GMC-forming cells and SMCs was observed in the area of the daughter GMC and subsidiary cells. OsSCR expression was also observed in the initiation area of ligule formation, and its downregulation occurred in the inner L2 cells generated by asymmetric division. Based on these observations, we proposed that OsSCR is involved not only in the asymmetric division of the cortex/endodermis progenitor cell but also during stomata and ligule formation by establishing the polarization of cytoplasm. OsSCR OsSDIR1 overexpression greatly improves drought tolerance in transgenic rice 2011 Plant Mol Biol State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing, China. Recent genomic and genetic analyses based on Arabidopsis suggest that ubiquitination plays crucial roles in the plant response to abiotic stress and the phytohormone abscisic acid (ABA). However, few such studies have been reported in rice as a monocotyledonous model plant. Taking advantage of strategies in biochemistry, molecular cell biology and genetics, the RING-finger containing E3 ligase OsSDIR1 (Oryza sativa SALT-AND DROUGHT-INDUCED RING FINGER 1) was found to be a candidate drought tolerance gene for engineering of crop plants. The expression of OsSDIR1 was detected in all tissues of rice and up-regulated by drought and NaCl, but not by ABA. In vitro ubiquitination assays demonstrated that OsSDIR1 is a functional E3 ubiquitin ligase and that the RING finger region is required for its activity. OsSDIR1 could complement the drought sensitive phenotype of the sdir1 mutant and overexpressing transgenic Arabidopsis were more sensitive to ABA, indicating that the OsSDIR1 gene is a functional ortholog of SDIR1. Upon drought treatment, the OsSDIR1-transgenic rice showed strong drought tolerance compared to control plants. Analysis of the stomata aperture revealed that there were more closed stomatal pores in transgenic plants than those of control plants. This result was also confirmed by the water loss assay and leaf related water content (RWC) measurements during drought treatment. Thus, we demonstrated that monocot- and dicot- SDIR1s are conserved yet have diverse functions. OsSDIR1 OsSEND-1: a new RAD2 nuclease family member in higher plants 2003 Plant Mol Biol Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan. A novel endonuclease, a new member of the RAD2 nuclease family, has been identified from the higher plant, rice (Oryza sativa L. cv. Nipponbare), and designated as OsSEND-1. The open reading frame of the OsSEND-1 cDNA encoded a predicted product of 641 amino acid residues with a molecular weight of 69.9 kDa. The encoded protein showed a relatively high degree of sequence homology with the RAD2 nuclease family proteins, especially RAD2 nuclease, but it differed markedly from FEN-1, XPG or HEX1/EXO1. The N- and I-domains in the family were highly conserved in the OsSEND-1 sequence. The protein was much smaller than XPG, but larger than HEX1/EXO1 and FEN-1. The genome sequence was composed of 14 exons, and was localized at the almost terminal region of the short arm of chromosome 8. Northern blotting and in situ hybridization analyses demonstrated preferential expression of OsSEND-1 mRNA in proliferating tissues such as meristem. The mRNA level of OsSEND-1 was induced by UV and DNA-damaging agent such as MMS or H2O2, indicating that OsSEND-1 has some roles in the repair of many types of damaged DNA. The recombinant peptide showed endonuclease activity. OsSEND-1 Expression of SERK family receptor-like protein kinase genes in rice 2005 Biochim Biophys Acta Plant Genetics Laboratory, National Institute of Genetics, Yata 1111, Mishima, Shizuoka-ken 411-8540, Japan. Some SERK-family receptor-like protein kinase genes have been shown to confer embryonic competence to cells. In this study, we isolated two novel rice genes, OsSERK1 and OsSERK2, belonging to the SERK-family. OsSERK2 showed constitutive expression. The OsSERK1 promoter showed reporter gene activities in some specific tissues in a germinating seed, leaf and root, but not in a developing embryo. This promoter activity suggests that OsSERK1 may have roles in non-embryonic tissues rather than in the embryo. OsBISERK1|OsSERK1|OsBAK1,OsSERK2,ORK1 Rice SERK1 gene positively regulates somatic embryogenesis of cultured cell and host defense response against fungal infection 2005 Planta National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Here we report on the isolation and characterization of a somatic embryogenesis receptor-like kinase (OsSERK1) gene in rice (Oryza sativa). The OsSERK1 gene belongs to a small subfamily of receptor-like kinase genes in rice and shares a highly conserved gene structure and extensive sequence homology with previously reported plant SERK genes. Though it has a basal level of expression in various rice organs/tissues, as high expression level was detected in rice callus during somatic embryogenesis. Suppression of OsSERK1 expression in transgenic calli by RNA interference resulted in a significant reduction of shoot regeneration rate (from 72% to 14% in the japonica rice Zhonghua11). Overexpression of OsSERK1, however, increased the shoot regeneration rate (from 72% to 86%). Interestingly, OsSERK1 is significantly activated by the rice blast fungus, particularly during the incompatible interaction, and is associated with host cell death in Sekigushi lesion mimic mutants. This gene is also inducible by defense signaling molecules such as salicylic acid, jasmonic acid, and abscisic acid. Furthermore, constitutive overexpression of OsSERK1 in two rice cultivars led to an increase in host resistance to the blast fungus. Our data suggest that OsSERK1 may partially mediate defense signal transduction in addition to its basic role in somatic embryogenesis. OsBISERK1|OsSERK1|OsBAK1 Engineering OsBAK1 gene as a molecular tool to improve rice architecture for high yield 2009 Plant Biotechnol J Research Center for Molecular & Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China. Generating a new variety of plant with erect-leaf is a critical strategy to improve rice grain yield, as plants with this trait can be dense-planted. The erect-leaf is a significant morphological trait partially regulated by Brassinosteroids (BRs) in rice plants. So far, only a few genes can be used for molecular breeding in rice. Here, we identified OsBAK1 as a potential gene to alter rice architecture. Based on rice genome sequences, four closely related homologs of Arabidopsis BAK1 (AtBAK1) gene were amplified. Phylogenetic analysis and suppression of a weak Arabidopsis mutant bri1-5 indicated that OsBAK1 (Os08g0174700) is the closest relative of AtBAK1. Genetic, physiological, and biochemical analyses all suggest that the function of OsBAK1 is conserved with AtBAK1. Overexpression of a truncated intracellular domain of OsBAK1, but not the extracellular domain of OsBAK1, resulted in a dwarfed phenotype, similar to the rice BR-insensitive mutant plants. The expression of OsBAK1 changed important agricultural traits of rice such as plant height, leaf erectness, grain morphologic features, and disease resistance responses. Our results suggested that a new rice variety with erect-leaf and normal reproduction can be generated simply by suppressing the expression level of OsBAK1. Therefore, OsBAK1 is a potential molecular breeding tool for improving rice grain yield by modifying rice architecture. OsBISERK1|OsSERK1|OsBAK1,OsSERK2,OsSERK3,OsSERK4 A subset of OsSERK genes, including OsBAK1, affects normal growth and leaf development of rice 2011 Mol Cells Department of Biological Science, Sookmyung Women's University, Seoul 140-742, Korea. Since the identification of BRI1-Associated receptor Kinase 1 (BAK1), a member of the Somatic Embryogenesis Receptor Kinase (SERK) family, the dual functions of BAK1 in BR signaling and innate immunity in Arabidopsis have attracted considerable attention as clues for understanding developmental processes that must be balanced between growth and defense over the life of plants. Here, we extended our research to study cellular functions of OsSERKs in rice. As it was difficult to identify an authentic ortholog of AtBAK1 in rice, we generated transgenic rice in which the expression of multiple OsSERK genes, including OsBAK1, was reduced by OsBAK1 RNA interference. Resulting transgenic rice showed reduced levels of Os-BAK1 and decreased sensitivity to BL, leading to semidwarfism in overall growth. Moreover, they resulted in abnormal growth patterns, especially in leaf development. Most of the OsBAK1RNAi transgenic rice plants were defective in the development of bulliform cells in the leaf epidermal layer. They also showed increased expression level of pathogenesis-related gene and enhanced susceptibility to a rice blast-causing fungal pathogen, Magnaporthe oryzae. These results indicate that OsSERK genes, such as OsBAK1, play versatile roles in rice growth and development. OsBISERK1|OsSERK1|OsBAK1,OsSERK2,OsSERK3,OsSERK4 Molecular characterization and expression analysis of OsBISERK1, a gene encoding a leucine-rich repeat receptor-like kinase, during disease resistance responses in rice 2008 Mol Biol Rep State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang, 310029, P.R. China. A rice gene, OsBISERK1, encoding a protein belonging to SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) type of leucine-rich repeat receptor-like kinases (LRR-RLKs) was identified. The OsBISERK1 encodes a 624 aa protein with high level of identity to known plant SERKs. OsBISERK1 contains a hydrophobic signal peptide, a leucine zipper, and five leucine-rich repeat motifs in the extracellular domain; the cytoplasmic region carries a proline-rich region and a single transmembrane domain, as well as a conserved intracellular serine/threonine protein kinase domain. OsBISERK1 has a low level of basal expression in leaf tissue. However, expression of OsBISERK1 was induced by treatment with benzothiadiazole (BTH), which is capable of inducing disease resistance in rice, and also up-regulated after inoculation with Magnaporthe grisea in BTH-treated rice seedlings and during incompatible interaction between a blast-resistant rice genotype and M. grisea. The results suggest that OsBISERK1 may be involved in disease resistance responses in rice. OsBISERK1|OsSERK1|OsBAK1 OsSERK1 regulates rice development but not immunity to Xanthomonas oryzae pv. oryzae or Magnaporthe oryzae 2014 J Integr Plant Biol Department of Plant Pathology and the Genome Center, University of California, Davis, California, 95616, USA; Joint Bioenergy Institute, Emeryville, California, 94710, USA; Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, College of Agriculture, Yangzhou University/Key Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou, 225009, China. Somatic embryogenesis receptor kinase (SERK) proteins play pivotal roles in regulation of plant development and immunity. The rice genome contains two SERK genes, OsSerk1 and OsSerk2. We previously demonstrated that OsSerk2 is required for rice Xa21-mediated resistance to Xanthomonas oryzae pv. oryzae (Xoo) and for normal development. Here we report the molecular characterization of OsSerk1. Overexpression of OsSerk1 results in a semi-dwarf phenotype whereas silencing of OsSerk1 results in a reduced angle of the lamina joint. OsSerk1 is not required for rice resistance to Xoo or Magnaporthe oryzae (M. oryzae). Overexpression of OsSerk1 in OsSerk2-silenced lines complements phenotypes associated with brassinosteroid (BR) signaling defects, but not the disease resistance phenotype mediated by Xa21. In yeast, OsSERK1 interacts with itself forming homodimers, and also interacts with the kinase domains of OsSERK2 and BRI1, respectively. OsSERK1 is a functional protein kinase capable of auto-phosphorylation in vitro. We conclude that, whereas OsSERK2 regulates both rice development and immunity, OsSERK1 functions in rice development but not immunity to Xoo and M. oryzae. OsBISERK1|OsSERK1|OsBAK1 Characterisation of the legume SERK-NIK gene superfamily including splice variants: implications for development and defence 2011 BMC Plant Biol Australian Research Council Centre of Excellence for Integrative Legume Research, School of Environmental and Life Sciences, The University of Newcastle, University Dr, Callaghan, NSW, 2308, Australia. BACKGROUND: SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) genes are part of the regulation of diverse signalling events in plants. Current evidence shows SERK proteins function both in developmental and defence signalling pathways, which occur in response to both peptide and steroid ligands. SERKs are generally present as small gene families in plants, with five SERK genes in Arabidopsis. Knowledge gained primarily through work on Arabidopsis SERKs indicates that these proteins probably interact with a wide range of other receptor kinases and form a fundamental part of many essential signalling pathways. The SERK1 gene of the model legume, Medicago truncatula functions in somatic and zygotic embryogenesis, and during many phases of plant development, including nodule and lateral root formation. However, other SERK genes in M. truncatula and other legumes are largely unidentified and their functions unknown. RESULTS: To aid the understanding of signalling pathways in M. truncatula, we have identified and annotated the SERK genes in this species. Using degenerate PCR and database mining, eight more SERK-like genes have been identified and these have been shown to be expressed. The amplification and sequencing of several different PCR products from one of these genes is consistent with the presence of splice variants. Four of the eight additional genes identified are upregulated in cultured leaf tissue grown on embryogenic medium. The sequence information obtained from M. truncatula was used to identify SERK family genes in the recently sequenced soybean (Glycine max) genome. CONCLUSIONS: A total of nine SERK or SERK-like genes have been identified in M. truncatula and potentially 17 in soybean. Five M. truncatula SERK genes arose from duplication events not evident in soybean and Lotus. The presence of splice variants has not been previously reported in a SERK gene. Upregulation of four newly identified SERK genes (in addition to the previously described MtSERK1) in embryogenic tissue cultures suggests these genes also play a role in the process of somatic embryogenesis. The phylogenetic relationship of members of the SERK gene family to closely related genes, and to development and defence function is discussed. OsSERK3 OsSFR6 is a functional rice orthologue of SENSITIVE TO FREEZING-6 and can act as a regulator of COR gene expression, osmotic stress and freezing tolerance in Arabidopsis 2011 New Phytol School of Biological and Biomedical Sciences, Durham University, Durham, UK. The Arabidopsis protein SENSITIVE TO FREEZING-6 (AtSFR6) is required for cold- and drought-inducible expression of COLD-ON REGULATED (COR) genes and, as a consequence, AtSFR6 is essential for osmotic stress and freezing tolerance in Arabidopsis. Therefore, orthologues of AtSFR6 in crop species represent important candidate targets for future manipulation of stress tolerance. We identified and cloned a homologue of AtSFR6 from rice (Oryza sativa), OsSFR6, and confirmed its orthology in Arabidopsis. OsSFR6 was identified by homology searches, and a full-length coding region isolated using reverse transcription polymerase chain reaction (RT-PCR) from Oryza sativa cDNA. To test for orthology, OsSFR6 was expressed in an Arabidopsis sfr6 loss-of-function mutant background, and restoration of wild-type phenotypes was assessed. Searching the rice genome revealed a single homologue of AtSFR6. Cloning and sequencing the OsSFR6 coding region showed OsSFR6 to have 61.7% identity and 71.1% similarity to AtSFR6 at the predicted protein sequence level. Expression of OsSFR6 in the atsfr6 mutant background restored the wild-type visible phenotype, as well as restoring wild-type levels of COR gene expression and tolerance of osmotic and freezing stresses. OsSFR6 is an orthologue of AtSFR6, and thus a target for future manipulation to improve tolerance to osmotic and other abiotic stresses. OsSFR6 contribution of salicylic acid glucosyltransferase, ossgt1, to chemically induced disease resistance in rice plants 2008 Plant J Agricultural & Veterinary Research Laboratories, Meiji Seika Kaisha Ltd, Yokohama 222-8567, Japan. kenji_umemura@meiji.co.jp Systemic acquired resistance (SAR), a natural disease response in plants, can be induced chemically. Salicylic acid (SA) acts as a key endogenous signaling molecule that mediates SAR in dicotyledonous plants. However, the role of SA in monocotyledonous plants has yet to be elucidated. In this study, the mode of action of the agrochemical protectant chemical probenazole was assessed by microarray-based determination of gene expression. Cloning and characterization of the most highly activated probenazole-responsive gene revealed that it encodes UDP-glucose:SA glucosyltransferase (OsSGT1), which catalyzes the conversion of free SA into SA O-beta-glucoside (SAG). We found that SAG accumulated in rice leaf tissue following treatment with probenazole or 2,6-dichloroisonicotinic acid. A putative OsSGT1 gene from the rice cultivar Akitakomachi was cloned and the gene product expressed in Escherichia coli was characterized, and the results suggested that probenazole-responsive OsSGT1 is involved in the production of SAG. Furthermore, RNAi-mediated silencing of the OsSGT1 gene significantly reduced the probenazole-dependent development of resistance against blast disease, further supporting the suggestion that OsSGT1 is a key mediator of development of chemically induced disease resistance. The OsSGT1 gene may contribute to the SA signaling mechanism by inducing up-regulation of SAG in rice plants. OsSGT1 Parallel domestication of the Shattering1 genes in cereals 2012 Nat Genet Department of Agronomy, Kansas State University, Manhattan, Kansas, USA. A key step during crop domestication is the loss of seed shattering. Here, we show that seed shattering in sorghum is controlled by a single gene, Shattering1 (Sh1), which encodes a YABBY transcription factor. Domesticated sorghums harbor three different mutations at the Sh1 locus. Variants at regulatory sites in the promoter and intronic regions lead to a low level of expression, a 2.2-kb deletion causes a truncated transcript that lacks exons 2 and 3, and a GT-to-GG splice-site variant in the intron 4 results in removal of the exon 4. The distributions of these non-shattering haplotypes among sorghum landraces suggest three independent origins. The function of the rice ortholog (OsSh1) was subsequently validated with a shattering-resistant mutant, and two maize orthologs (ZmSh1-1 and ZmSh1-5.1+ZmSh1-5.2) were verified with a large mapping population. Our results indicate that Sh1 genes for seed shattering were under parallel selection during sorghum, rice and maize domestication. OsSh1 Differential Expression of Three Plastidial Sigma Factors,OsSIG1,OsSIG2A, andOsSIG2B, during Leaf Development in Rice 2014 Bioscience, Biotechnology and Biochemistry National Food Research Institute We isolated and characterized two rice nuclear genes, OsSIG2A and OsSIG2B, encoding the putative σ-factor of the plastid RNA polymerase. Deduced protein sequences predicted a plastid-localizing signal in the N-terminus and subsequent polypeptides similar to known SIG2 proteins. Gene expression analysis revealed that the OsSIG2A transcript is more abundant than the OsSIG2B transcript in all tissues tested and that both rice SIG2s are expressed from earlier stages of leaf development than that in the case of OsSIG1. These results indicate differential expression of SIG genes in leaf morphogenesis, suggesting the existence of tissue- and stage-specific functions of SIG proteins for transcriptional regulation of chloroplast genes in plant development. OsSIG1,OsSIG2A The plastid sigma factor SIG1 maintains photosystem I activity via regulated expression of the psaA operon in rice chloroplasts 2007 Plant J Cell-Free Science and Technology Research Center, Ehime University, Matsuyama 790-8577, Japan. tozaway@ccr.ehime-u.ac.jp Sigma factors encoded by the nucleus of plants confer promoter specificity on the bacterial-type RNA polymerase in chloroplasts. We previously showed that transcripts of OsSIG1, which encodes one such sigma factor in rice, accumulate relatively late during leaf development. We have now isolated and characterized two allelic mutants of OsSIG1, in which OsSIG1 is disrupted by insertion of the retrotransposon Tos17, in order to characterize the functions of OsSIG1. The OsSIG1-/- plants were found to be fertile but they manifested an approximately one-third reduction in the chlorophyll content of mature leaves. Quantitative RT-PCR and northern blot analyses of chloroplast gene expression revealed that the abundance of transcripts derived from the psaA operon was markedly reduced in OsSIG1-/- plants compared with that in wild-type homozygotes. This effect was accompanied by a reduction in the abundance of the core protein complex (PsaA-PsaB) of photosystem I. Analysis of chlorophyll fluorescence also revealed a substantial reduction in the rate of electron transfer from photosystem II to photosystem I in the OsSIG1 mutants. Our results thus indicate that OsSIG1 plays an important role in the maintenance of photosynthetic activity in mature chloroplasts of rice by regulating expression of chloroplast genes for components of photosystem I. OsSIG1 Two novel nuclear genes, OsSIG5 and OsSIG6, encoding potential plastid sigma factors of RNA polymerase in rice: tissue-specific and light-responsive gene expression 2007 Plant Cell Physiol Laboratory of Molecular Genetics, Collage of Agriculture, Ibaraki University, Ami, Inashiki, Ibaraki, 300-0393 Japan. Two novel nuclear genes, OsSIG5 and OsSIG6, encoding potential plastid sigma factors of RNA polymerase (RNAP) were identified in Oryza sativa. The deduced amino acid sequences contain conserved regions, regions 1.2-4.2, and a novel region A/B at the N-terminus. Tissue-specific and light-responsive transcripts of OsSIG5 and OsSIG6 were observed. The N-terminal region of OsSig5 conferred import of green fluorescent protein into the chloroplast. Specific transcripts of rice psbA were synthesized in vitro by reconstituted OsSig5-RNAP holoenzymes. These results indicated that OsSig5 is a plastid sigma factor. This is the first report of the Sig5-type sigma factor in crops. OsSIG5,OsSIG6 Receptor-like kinase OsSIK1 improves drought and salt stress tolerance in rice (Oryza sativa) plants 2010 Plant J Plant Gene Research Center, National Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Receptor-like kinases (RLKs) play essential roles in plant growth, development and responses to environmental stresses. A putative RLK gene, OsSIK1, with extracellular leucine-rich repeats was cloned and characterized in rice (Oryza sativa). OsSIK1 exhibits kinase activity in the presence of Mn(2+), and the OsSIK1 kinase domain has the ability to autophosphorylate and phosphorylate myelin basic protein (MBP). OsSIK1 promoter-GUS analysis revealed that OsSIK1 is expressed mainly in the stem and spikelet in rice. The expression of OsSIK1 is mainly induced by salt, drought and H(2)O(2) treatments. Transgenic rice plants with overexpression of OsSIK1 show higher tolerance to salt and drought stresses than control plants. On the contrary, the knock-out mutants sik1-1 and sik1-2, as well as RNA interference (RNAi) plants, are sensitive to drought and salt stresses. The activities of peroxidase, superoxide dismutase and catalase are enhanced significantly in OsSIK1-overexpressing plants. Also, the accumulation of H(2)O(2) in leaves of OsSIK1-overexpressing plants is much less than that of the mutants, RNAi plants and control plants, as measured by 3,3'-diamino benzidine (DAB) staining. We also show that OsSIK1 affects stomatal density in the abaxial and adaxial leaf epidermis of rice. These results indicate that OsSIK1 plays important roles in salt and drought stress tolerance in rice, through the activation of the antioxidative system. OsSIK1 An S-domain receptor-like kinase, OsSIK2, confers abiotic stress tolerance and delays dark-induced leaf senescence in rice 2013 Plant Physiol State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Receptor-like kinases play important roles in plant development and defense responses; however, their functions in other processes remain unclear. Here, we report that OsSIK2, an S-domain receptor-like kinase from rice (Oryza sativa), is involved in abiotic stress and the senescence process. OsSIK2 is a plasma membrane-localized protein with kinase activity in the presence of Mn(2+). OsSIK2 is expressed mainly in rice leaf and sheath and can be induced by NaCl, drought, cold, dark, and abscisic acid treatment. Transgenic plants overexpressing OsSIK2 and mutant sik2 exhibit enhanced and reduced tolerance to salt and drought stress, respectively, compared with the controls. Interestingly, a truncated version of OsSIK2 without most of the extracellular region confers higher salt tolerance than the full-length OsSIK2, likely through the activation of different sets of downstream genes. Moreover, seedlings of OsSIK2-overexpressing transgenic plants exhibit early leaf development and a delayed dark-induced senescence phenotype, while mutant sik2 shows the opposite phenotype. The downstream PR-related genes specifically up-regulated by full-length OsSIK2 or the DREB-like genes solely enhanced by truncated OsSIK2 are all induced by salt, drought, and dark treatments. These results indicate that OsSIK2 may integrate stress signals into a developmental program for better adaptive growth under unfavorable conditions. Manipulation of OsSIK2 should facilitate the improvement of production in rice and other crops. OsSIK2,OsPR5|Pir2|PR-5|PR5-1 Overexpression of OsSIN, encoding a novel small protein, causes short internodes in Oryza sativa 2005 Plant Science Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China We report here the characterization of function of a novel gene, OsSIN (Orayza sativa SHORT INTERNODES). OsSIN with no intron, encodes a 106 amino acid polypeptide and has no homology proteins in GenBank. The gene shows predominant expression in shoots and is localized in the nucleus. Constitutive expression of OsSIN resulted in dwarfism and reduced number of inferior spikelets. The functional strength was correlated with the level of transgene expression. Transgenic plants were more insensitive to endogenous gibberellic acid (GA) levels than the wild type. The rice with OsSIN overexpression was less sensitive to GA than the wild type in terms of the second leaf sheath length. Expression of OsGAI at protein level was increased in OsSIN-overexpressed rice compared with the wild type. Our results suggest that OsSIN is a new member in the GA pathway regulating rice stem development. OsSIN,SLR1|OsGAI Rice OsSIPK and its orthologs: a "central master switch" for stress responses 2009 Biochem Biophys Res Commun Environmental Biology Division, National Institute for Environmental Studies, Tsukuba, Japan. Mitogen-activated protein kinase (MAPK) plays a central role in controlling a vast array of plant biochemical and physiological processes. It is regulated by a characteristic phosphorelay system in which a series of three kinases phosphorylate and activate each other. Over the past years, several plants MAPKs have been identified and characterized. Of these, rice OsSIPK (Salicylic acid (SA)-Induced Protein Kinase) and its orthologs in other plants are of particular interest. A large body of evidence demonstrates the involvement of SIPKs in fine-tuned regulation of the plant responses to ozone, wounding, SA, and jasmonic acid (JA). Interestingly, their function appears to be conserved across reference plants, such as rice, tobacco, and Arabidopsis. In this minireview, we discuss the recent progress on rice OsSIPK and its orthologs as a "central master switch" for mediating plant responses against ozone, wounding, and JA as examples. OsMPK1|OsMAPK6|OsSIPK Novel rice OsSIPK is a multiple stress responsive MAPK family member showing rhythmic expression at mRNA level 2007 Planta Department of Molecular Biology, College of Natural Science, Sejong University, Gwangjin-Gu, Seoul, South Korea. We report isolation and transcriptional profiling of rice (Oryza sativa L.) mitogen-activated protein kinase (MAPK), OsSIPK (salicylic acid-induced protein kinase). OsSIPK gene is located on chromosome 6 most probably existing as a single copy in the rice genome, and encodes 398 amino acid polypeptide having the MAPK family signature and phosphorylation activation motif TEY. Steady state mRNA analyses of OsSIPK showed weak constitutive expression in leaves of 2-week-old rice seedlings. A time course (30-120 min) experiment using a variety of elicitors and stresses revealed that the OsSIPK mRNA is strongly induced by jasmonic acid (JA), salicylic acid (SA), ethephon, abscisic acid, cycloheximide (CHX), JA/SA + CHX, cantharidin, okadaic acid, hydrogen peroxide, chitosan, sodium chloride, and cold stress (12 degrees C), but not with wounding by cut, gaseous pollutants ozone, and sulfur dioxide, high temperature, ultraviolet C irradiation, sucrose, and drought. Its transcription was also found to be tissue-specifically regulated, and followed a rhythmic dark induction in leaves. Finally, we showed that the OsSIPK protein is localized to the nucleus. From these results, OsSIPK can be implicated in diverse stimuli-responsive signaling cascades and transcription of certain genes. OsMPK1|OsMAPK6|OsSIPK OsSIZ1 Regulates the Vegetative Growth and Reproductive Development in Rice 2010 Plant Molecular Biology Reporter State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, 210095, China SAP and MIZ (SIZ) is a small ubiquitin-related modifier (SUMO) E3 ligase that facilitates conjugation of SUMO to protein substrates. Although there have been a number of reports about the functions of SIZ1 in Arabidopsis in the regulation of diverse life processes, no information regarding the role of SIZ in other plants is available yet. In this work, two homologous genes from rice (Oryza sativa) were isolated and designated as OsSIZ1 and OsSIZ2 based on amino acid sequence homology to AtSIZ1 and their phylogenetic relationship. The function in the vegetative growth and reproductive development in rice was investigated using OsSIZ1 mutants containing a T-DNA insertion. The results showed that the mutant Ossiz1 exhibited the significant changes in several growth and developmental parameters, including primary root length, adventitious root number, plant height, leaf and panicle length, flower formation, and seed-setting rate compared with wild type. Taking together these results indicate that OsSIZ1 plays an important role in regulating growth and development in rice. OsSIZ1,OsSIZ2 Functional characterization of the SIZ/PIAS-type SUMO E3 ligases, OsSIZ1 and OsSIZ2 in rice 2010 Plant Cell Environ Division of Applied Life Science (BK21 program), Plant Molecular Biology and Biotechnology Research Center and Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Korea. hcpark@gnu.ac.kr Sumoylation is a post-translational regulatory process in diverse cellular processes in eukaryotes, involving conjugation/deconjugation of small ubiquitin-like modifier (SUMO) proteins to other proteins thus modifying their function. The PIAS [protein inhibitor of activated signal transducers and activators of transcription (STAT)] and SAP (scaffold attachment factor A/B/acinus/PIAS)/MIZ (SIZ) proteins exhibit SUMO E3-ligase activity that facilitates the conjugation of SUMO proteins to target substrates. Here, we report the isolation and molecular characterization of Oryza sativa SIZ1 (OsSIZ1) and SIZ2 (OsSIZ2), rice homologs of Arabidopsis SIZ1. The rice SIZ proteins are localized to the nucleus and showed sumoylation activities in a tobacco system. Our analysis showed increased amounts of SUMO conjugates associated with environmental stresses such as high and low temperature, NaCl and abscisic acid (ABA) in rice plants. The expression of OsSIZ1 and OsSIZ2 in siz1-2 Arabidopsis plants partially complemented the morphological mutant phenotype and enhanced levels of SUMO conjugates under heat shock conditions. In addition, ABA-hypersensitivity of siz1-2 seed germination was partially suppressed by OsSIZ1 and OsSIZ2. The results suggest that rice SIZ1 and SIZ2 are able to functionally complement Arabidopsis SIZ1 in the SUMO conjugation pathway. Their effects on the Arabidopsis mutant suggest a function for these genes related to stress responses and stress adaptation. OsSIZ1,OsSIZ2 Rice SIZ1, a SUMO E3 ligase, controls spikelet fertility through regulation of anther dehiscence 2011 New Phytol Molecular and Biological Agricultural Sciences, Taiwan International Graduate Program, National Chung-Hsing University-Academia Sinica, Taipei, Taiwan. * Sumoylation, a post-translational modification, has important functions in both animals and plants. However, the biological function of the SUMO E3 ligase, SIZ1, in rice (Oryza sativa) is still under investigation. * In this study, we employed two different genetic approaches, the use of siz1 T-DNA mutant and SIZ1-RNAi transgenic plants, to characterize the function of rice SIZ1. * Genetic results revealed the co-segregation of single T-DNA insertional recessive mutation with the observed phenotypes in siz1. In addition to showing reduced plant height, tiller number and seed set percentage, both the siz1 mutant and SIZ1-RNAi transgenic plants showed obvious defects in anther dehiscence, but not pollen viability. The anther indehiscence in siz1 was probably a result of defects in endothecium development before anthesis. Interestingly, rice orthologs of AtIRX and ZmMADS2, which are essential for endothecium development during anther dehiscence, were significantly down-regulated in siz1. Compared with the wild-type, the sumoylation profile of high-molecular-weight proteins in mature spikelets was reduced significantly in siz1 and the SIZ1-RNAi line with notably reduced SIZ1 expression. The nuclear localization signal located in the SIZ1 C-terminus was sufficient for its nuclear targeting in bombarded onion epidermis. * The results suggest the functional role of SIZ1, a SUMO E3 ligase, in regulating rice anther dehiscence. OsSIZ1 Identification of three shikimate kinase genes in rice: characterization of their differential expression during panicle development and of the enzymatic activities of the encoded proteins 2005 Planta Japan Science and Technology Agency for Core Research for Evolutional Science and Technology, Kawaguchi 332-0012, Japan. The shikimate pathway is common to the biosynthesis of the three aromatic amino acids and that of various secondary metabolites in land plants. Shikimate kinase (SK; EC 2.7.1.71) catalyzes the phosphorylation of shikimate to yield shikimate 3-phosphate. In an attempt to elucidate the functional roles of enzymes that participate in the shikimate pathway in rice (Oryza sativa), we have now identified and characterized cDNAs corresponding to three SK genes--OsSK1, OsSK2, and OsSK3--in this monocotyledenous plant. These SK cDNAs encode proteins with different NH(2)-terminal regions and with putative mature regions that share sequence similarity with other plant and microbial SK proteins. An in vitro assay of protein import into intact chloroplasts isolated from pea (Pisum sativum) seedlings revealed that the full-length forms of the three rice SK proteins are translocated into chloroplasts and processed, consistent with the assumption that the different NH(2)-terminal sequences function as chloroplast transit peptides. The processed forms of all three rice proteins synthesized in vitro manifested SK catalytic activity. Northern blot analysis revealed that the expression of OsSK1 and OsSK2 was induced in rice calli by treatment with the elicitor N-acetylchitoheptaose, and that expression of OsSK1 and OsSK3 was up-regulated specifically during the heading stage of panicle development. These results suggest that differential expression of the three rice SK genes and the accompanying changes in the production of shikimate 3-phosphate may contribute to the defense response and to panicle development in rice. OsSK1,OsSK2,OsSK3 A homolog of human ski-interacting protein in rice positively regulates cell viability and stress tolerance 2009 Proc Natl Acad Sci U S A National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China. Abiotic stresses are major limiting factors for growth, development, and productivity of crop plants. Here, we report on OsSKIPa, a rice homolog of human Ski-interacting protein (SKIP) that can complement the lethal defect of the knockout mutant of SKIP homolog in yeast and positively modulate cell viability and stress tolerance of rice. Suppression of OsSKIPa in rice resulted in growth arrest and reduced cell viability. The expression OsSKIPa is induced by various abiotic stresses and phytohormone treatments. Transgenic rice overexpressing OsSKIPa exhibited significantly improved growth performance in the medium containing stress agents (abscisic acid, salt, or mannitol) and drought resistance at both the seedling and reproductive stages. The OsSKIPa-overexpressing rice showed significantly increased reactive oxygen species-scavenging ability and transcript levels of many stress-related genes, including SNAC1 and rice homologs of CBF2, PP2C, and RD22, under drought stress conditions. More than 30 OsSKIPa-interacting proteins were identified, but most of these proteins have no matches with the reported SKIP-interacting proteins in animals and yeast. Together, these data suggest that OsSKIPa has evolved a specific function in positive modulation of stress resistance through transcriptional regulation of diverse stress-related genes in rice. OsSKIPa,OsNAC19|SNAC1|OsNAC9 Sekiguchi lesion gene encodes a cytochrome P450 monooxygenase that catalyzes conversion of tryptamine to serotonin in rice 2010 J Biol Chem Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0192, Japan. Serotonin is a well known neurotransmitter in mammals and plays an important role in various mental functions in humans. In plants, the serotonin biosynthesis pathway and its function are not well understood. The rice sekiguchi lesion (sl) mutants accumulate tryptamine, a candidate substrate for serotonin biosynthesis. We isolated the SL gene by map-based cloning and found that it encodes CYP71P1 in a cytochrome P450 monooxygenase family. A recombinant SL protein exhibited tryptamine 5-hydroxylase enzyme activity and catalyzed the conversion of tryptamine to serotonin. This pathway is novel and has not been reported in mammals. Expression of SL was induced by the N-acetylchitooligosaccharide (chitin) elicitor and by infection with Magnaporthe grisea, a causal agent for rice blast disease. Exogenously applied serotonin induced defense gene expression and cell death in rice suspension cultures and increased resistance to rice blast infection in plants. We also found that serotonin-induced defense gene expression is mediated by the RacGTPase pathway and by the G alpha subunit of the heterotrimeric G protein. These results suggest that serotonin plays an important role in rice innate immunity. OsSL Overproduction of OsSLRL2 alters the development of transgenic Arabidopsis plants 2007 Biochem Biophys Res Commun The National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, PR China. SLR1 (SLENDER RICE 1) was thought to be the sole DELLA protein in rice considering the constitutive GA response phenotype of slr1 mutants. There were two other SLR1 homologous SLRL1 and SLRL2 (SLR1 like 1 and 2) which did not have DELLA domain but still shared high level similarity to the C-terminal region of SLR1 found after searching the whole rice genome. SLRL2 specially expressed in the embryo of immature rice seeds and the expression of SLRL2 was increased when treated with GA(3). The SLRL2 over-expressed transgenic Arabidopsis plants were semi-dwarfed, late flowering, and insensitive to GA. Moreover, the expression of AtGA20ox1 and AtGA3ox1 was increased and the expression of AtGA2ox1 decreased, indicating SLRL2 was a repressor of GA signaling. We suggested SLRL2 might function to overcome too strong GA responses and maintained a basic repression. Furthermore, a different form of DELLA family in monocots against dicots was discussed. OsSLRL2,OsSLRL1 Overexpression of a GRAS protein lacking the DELLA domain confers altered gibberellin responses in rice 2005 Plant J Bioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya, Aichi 464-8601, Japan. The rice SLR1 (SLENDER RICE 1) gene encodes a DELLA protein that belongs to a subfamily of the GRAS protein superfamily and that functions as a repressor of gibberellin (GA) signaling. Based on the constitutive GA response phenotype of slr1 mutants, SLR1 has been thought to be the sole DELLA-type protein suppressing GA signals in rice. However, in rice genome databases we identified two sequences homologous to SLR1: SLR1-like1 and -2 (SLRL1 and -2). SLRL1 and SLRL2 contain regions with high similarity to the C-terminal conserved domains in SLR1, but lack the N-terminal conserved region of the DELLA proteins. The expression of SLRL1 was positively regulated by GA at the mRNA level and occurred preferentially in reproductive organs, whereas SLRL2 was moderately expressed in mature leaf organs and was not affected by GA. Transformation of SLRL1 into the slr1 mutant rescued the slender phenotype of this mutant. Moreover, overexpression of SLRL1 in normal rice plants induced a dwarf phenotype with an increased level of OsGA20ox2 gene expression and diminished the GA-induced shoot elongation, suggesting that SLRL1 acts as a repressor of GA signaling. Consistent with the fact that SLRL1 does not have a DELLA domain, which is essential for degradation of DELLA proteins, a level of SLRL1 protein was not degraded by application of gibberellic acid. However, the repressive activity of SLRL1 against GA signaling was much weaker than a truncated SLR1 lacking the DELLA domain. Based on these characteristics of SLRL1, the functional roles of SLRL1 in GA signaling in rice are discussed. OsSLRL2,sd1|GA20ox2,SLR1|OsGAI,OsSLRL1 Overexpression of a rice gene encoding a small C2 domain protein OsSMCP1 increases tolerance to abiotic and biotic stresses in transgenic Arabidopsis 2009 Plant Mol Biol Research Institute for Biological Sciences Okayama, 7549-1 Yoshikawa, Kibichuo, Okayama 716-1241, Japan. Plant growth and crop production are limited by environmental stress. We used a large population of transgenic Arabidopsis expressing rice full-length cDNAs to isolate the rice genes that improve the tolerance of plants to environmental stress. By sowing T2 seeds of the transgenic lines under conditions of salinity stress, the salt-tolerant line R07047 was isolated. It expressed a rice gene, OsSMCP1, which encodes a small protein with a single C2 domain, a Ca(2+)-dependent membrane-targeting domain. Retransformation of wild-type Arabidopsis revealed that OsSMCP1 is responsible for conferring the salt tolerance. It is particularly interesting that R07047 and newly constructed OsSMCP1-overexpressing Arabidopsis showed enhanced tolerance not only to high salinity but also to osmotic, dehydrative, and oxidative stresses. Furthermore, R07047 showed improved resistance to Pseudomonas syringae. The OsSMCP1 expression in rice is constitutive. Particle-bombardment-mediated transient expression analysis revealed that OsSMCP1 is targeted to plastids in rice epidermal cells. It induced overexpression of several nuclear encoded genes, including the stress-associated genes, in transgenic Arabidopsis. No marked morphological change or growth retardation was observed in R07047 or retransformants. For molecular breeding to improve the tolerance of crops against environmental stress, OsSMCP1 is a promising candidate. OsSMCP1 Molecular cloning and characterization of a novel SNAP25-type protein gene OsSNAP32 in rice (Oryza sativa L.) 2008 Mol Biol Rep State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, 210095, China. The SNAP25-type proteins belong to the superfamily of the SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), and function as important components of the vesical trafficking machinery in eukaryotic cells. In this paper, we report the cloning and expression characterization of OsSNAP32 gene, and the subcellular localization of its encoded protein. The OsSNAP32 gene contains five exons and four introns, and is located between RFLP markers C12276S and S1917 on chromosome 2 in rice. The OsSNAP32 has a molecular weight of 31.3 kD, comprises 283 amino acid residues, and contains Qb-SNARE and Qc-SNARE domains in the N- and C-terminal, respectively. Multiple sequence alignment of the SNARE domains indicates that OsSNAP32 protein is homologous to HvSNAP34 and HvSNAP28 (63% and 55% of amino acid identity respectively) from barley. The transient expression method in onion epidermal cells, revealed that OsSNAP32 is located in the plasma membrane, like other SNAP25-type proteins. Semi-quantitative RT-PCR assay showed that the OsSNAP32 is highly expressed in leaves and culms, and low in roots of rice, while hardly detected in immature spikes and flowering spikes. The expression of OsSNAP32 was significantly activated in rice seedlings treated with H2O2, PEG6000, and low temperature or after inoculation with rice blast (Magnaporthe grisea strain Hoku 1). The results suggest that this gene belongs to a novel member of this gene family encoding SNAP25-type proteins, involved in the rice responses to biotic and abiotic stresses. OsSNAP32 OsSNDP1, a Sec14-nodulin domain-containing protein, plays a critical role in root hair elongation in rice 2013 Plant Mol Biol Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center (PMBBRC), Gyeongsang National University, Jinju 660-701, Korea. Rice is cultivated in water-logged paddy lands. Thus, rice root hairs on the epidermal layers are exposed to a different redox status of nitrogen species, organic acids, and metal ions than root hairs growing in drained soil. To identify genes that play an important role in root hair growth, a forward genetics approach was used to screen for short-root-hair mutants. A short-root-hair mutant was identified and isolated by using map-based cloning and sequencing. The mutation arose from a single amino acid substitution of OsSNDP1 (Oryza sativa Sec14-nodulin domain protein), which shows high sequence homology with Arabidopsis COW1/AtSFH1 and encodes a phosphatidylinositol transfer protein (PITP). By performing complementation assays with Atsfh1 mutants, we demonstrated that OsSNDP1 is involved in growth of root hairs. Cryo-scanning electron microscopy was utilized to further characterize the effect of the Ossndp1 mutation on root hair morphology. Aberrant morphogenesis was detected in root hair elongation and maturation zones. Many root hairs were branched and showed irregular shapes due to bulged nodes. Many epidermal cells also produced dome-shaped root hairs, which indicated that root hair elongation ceased at an early stage. These studies showed that PITP-mediated phospholipid signaling and metabolism is critical for root hair elongation in rice. OsSNDP1 The expression of the rice (Oryza sativa L.) homologue of Snm1 is induced by DNA damages 2005 Biochem Biophys Res Commun Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan. We isolated and characterized the rice homologue of the DNA repair gene Snm1 (OsSnm1). The length of the cDNA was 1862bp; the open reading frame encoded a predicted product of 485 amino acid residues with a molecular mass of 53.2kDa. The OsSnm1 protein contained the conserved beta-lactamase domain in its internal region. OsSnm1 was expressed in all rice organs. The expression was induced by MMS, H(2)O(2), and mitomycin C, but not by UV. Transient expression of an OsSnm1/GFP fusion protein in onion epidermal cells revealed the localization of OsSnm1 to the nucleus. These results suggest that OsSnm1 is involved not only in the repair of DNA interstrand crosslinks, but also in various other DNA repair pathways. OsSnm1 Isolation and characterization of a new Na+/H+ antiporter gene OsNHA1 from rice (Oryza sativa L.) 2006 DNA Seq State key laboratory of crop genetics and germplasm enhancement, Nanjing Agricultural University, Nanjing 210095, China. The full-length cDNA (3612 bp) of OsNHA1 was cloned by RT-PCR approach from rice (Oryza sativa L.), which encodes a putative plasma membrane Na+/H+ antiporter. Its deduced protein, OsNHA1, has 11 transmembrane domains and a significant similarity to a plasma membrane Na+/H+ antiporter AtNHA1 from Arabidopsis thaliana. Phylogenetic analysis showed that the OsNHA1 clusters with the plasma membrane Na+/H+ antiporters from various organisms. The semi-quantitative RT-PCR assay revealed that the expression of OsNHA1 was up-regulated in both shoots and roots of rice seedlings under salt stress, whereas it was not induced in the rice seedlings treated by drought stress. OsNHA1|OsSOS1 Conservation of the salt overly sensitive pathway in rice 2007 Plant Physiol Instituto de Recursos Naturales y Agrobiologia, Consejo Superior de Investigaciones Cientificas, Sevilla 41012, Spain. The salt tolerance of rice (Oryza sativa) correlates with the ability to exclude Na+ from the shoot and to maintain a low cellular Na+/K+ ratio. We have identified a rice plasma membrane Na+/H+ exchanger that, on the basis of genetic and biochemical criteria, is the functional homolog of the Arabidopsis (Arabidopsis thaliana) salt overly sensitive 1 (SOS1) protein. The rice transporter, denoted by OsSOS1, demonstrated a capacity for Na+/H+ exchange in plasma membrane vesicles of yeast (Saccharomyces cerevisiae) cells and reduced their net cellular Na+ content. The Arabidopsis protein kinase complex SOS2/SOS3, which positively controls the activity of AtSOS1, phosphorylated OsSOS1 and stimulated its activity in vivo and in vitro. Moreover, OsSOS1 suppressed the salt sensitivity of a sos1-1 mutant of Arabidopsis. These results represent the first molecular and biochemical characterization of a Na+ efflux protein from monocots. Putative rice homologs of the Arabidopsis protein kinase SOS2 and its Ca2+-dependent activator SOS3 were identified also. OsCIPK24 and OsCBL4 acted coordinately to activate OsSOS1 in yeast cells and they could be exchanged with their Arabidopsis counterpart to form heterologous protein kinase modules that activated both OsSOS1 and AtSOS1 and suppressed the salt sensitivity of sos2 and sos3 mutants of Arabidopsis. These results demonstrate that the SOS salt tolerance pathway operates in cereals and evidences a high degree of structural conservation among the SOS proteins from dicots and monocots. OsNHA1|OsSOS1 STV11 encodes a sulphotransferase and confers durable resistance to rice stripe virus 2014 Nat Commun National key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, China Rice stripe virus (RSV) causes one of the most serious viral diseases of rice (Oryza sativa L.), but the molecular basis of RSV resistance has remained elusive. Here we show that the resistant allele of rice STV11 (STV11-R) encodes a sulfotransferase (OsSOT1) catalysing the conversion of salicylic acid (SA) into sulphonated SA (SSA), whereas the gene product encoded by the susceptible allele STV11-S loses this activity. Sequence analyses suggest that the STV11-R and STV11-S alleles were predifferentiated in different geographic populations of wild rice, Oryza rufipogon, and remained prevalent in cultivated indica and japonica rice varieties, respectively. Introgression of the STV11-R allele into susceptible cultivars or heterologous transfer of STV11-R into tobacco plants confers effective resistance against RSV. Our results shed new insights into plant viral defense mechanisms and suggest effective means of breeding RSV-resistant crops using molecular marker-assisted selection or genetic engineering. OsSOT1|STV11 A distinctive class of spermidine synthase is involved in chilling response in rice 2004 Journal of Plant Physiology Winter Stress Laboratory, Department of Low Temperature Sciences, National Agricultural Research Center for Hokkaido Region, Hitsujigaoka, Toyohira-ku, Sapporo, Japan. A cDNA for a putative 42 kD spermidine synthase (OsSPDS2) was cloned from rice. The deduced OsSPDS2 sequence showed highest similarity with Arabidopsis AtSPDS3. Phylogenetic analysis revealed that OsSPDS2 and AtSPDS3 form a distinctive subclass in the spermidine synthase family in plants. OsSPDS2 mRNA accumulated in roots during long term exposure to chilling temperature (12 degrees C). In contrast, no such induction of the paralogous OsSPDS1 was observed during the chilling treatment. ABA treatment up-regulated OsSPDS2, whereas salt stress did not change OsSPDS2 levels significantly. Data suggested a distinct function of OsSPDS2 in chilling response in rice. OsSPDS1,OsSPDS2 Overexpression of rice sphingosine-1-phoshpate lyase gene OsSPL1 in transgenic tobacco reduces salt and oxidative stress tolerance 2012 J Integr Plant Biol State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang 310058, China. Sphingolipids, including sphingosine-1-phosphate (S1P), have been shown to function as signaling mediators to regulate diverse aspects of plant growth, development, and stress response. In this study, we performed functional analysis of a rice (Oryza sativa) S1P lyase gene OsSPL1 in transgenic tobacco plants and explored its possible involvement in abiotic stress response. Overexpression of OsSPL1 in transgenic tobacco resulted in enhanced sensitivity to exogenous abscisic acid (ABA), and decreased tolerance to salt and oxidative stress, when compared with the wild type. Furthermore, the expression levels of some selected stress-related genes in OsSPL1-overexpressing plants were reduced after application of salt or oxidative stress, indicating that the altered responsiveness of stress-related genes may be responsible for the reduced tolerance in OsSPL1-overexpressing tobacco plants under salt and oxidative stress. Our results suggest that rice OsSPL1 plays an important role in abiotic stress responses. OsSPL1 Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice 2010 Nat Genet State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. Increasing crop yield is a major challenge for modern agriculture. The development of new plant types, which is known as ideal plant architecture (IPA), has been proposed as a means to enhance rice yield potential over that of existing high-yield varieties. Here, we report the cloning and characterization of a semidominant quantitative trait locus, IPA1 (Ideal Plant Architecture 1), which profoundly changes rice plant architecture and substantially enhances rice grain yield. The IPA1 quantitative trait locus encodes OsSPL14 (SOUAMOSA PROMOTER BINDING PROTEIN-LIKE 14) and is regulated by microRNA (miRNA) OsmiR156 in vivo. We demonstrate that a point mutation in OsSPL14 perturbs OsmiR156-directed regulation of OsSPL14, generating an 'ideal' rice plant with a reduced tiller number, increased lodging resistance and enhanced grain yield. Our study suggests that OsSPL14 may help improve rice grain yield by facilitating the breeding of new elite rice varieties. OsSPL14|IPA1|WFP OsSPL14 promotes panicle branching and higher grain productivity in rice 2010 Nat Genet Bioscience and Biotechnology Center, Nagoya University, Nagoya, Japan. Identification of alleles that improve crop production and lead to higher-yielding varieties are needed for food security. Here we show that the quantitative trait locus WFP (WEALTHY FARMER'S PANICLE) encodes OsSPL14 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 14, also known as IPA1). Higher expression of OsSPL14 in the reproductive stage promotes panicle branching and higher grain yield in rice. OsSPL14 controls shoot branching in the vegetative stage and is affected by microRNA excision. We also demonstrate the feasibility of using the OsSLP14(WFP) allele to increase rice crop yield. Introduction of the high-yielding OsSPL14(WFP) allele into the standard rice variety Nipponbare resulted in increased rice production. OsSPL14|IPA1|WFP Shaping a better rice plant 2010 Nat Genet None Two studies describe how regulatory variation at the rice gene OsSPL14 can lead to altered plant morphology and improve grain yield. These studies support the possibility of improving rice yield through changing plant architecture. OsSPL14|IPA1|WFP The double-stranded break-forming activity of plant SPO11s and a novel rice SPO11 revealed by a Drosophila bioassay 2012 BMC Mol Biol Cellular & Molecular Biology Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. BACKGROUND: SPO11 is a key protein for promoting meiotic recombination, by generating chromatin locus- and timing-specific DNA double-strand breaks (DSBs). The DSB activity of SPO11 was shown by genetic analyses, but whether SPO11 exerts DSB-forming activity by itself is still an unanswered question. DSB formation by SPO11 has not been detected by biochemical means, probably because of a lack of proper protein-folding, posttranslational modifications, and/or specific SPO11-interacting proteins required for this activity. In addition, plants have multiple SPO11-homologues. RESULTS: To determine whether SPO11 can cleave DNA by itself, and to identify which plant SPO11 homologue cleaves DNA, we developed a Drosophila bioassay system that detects the DSB signals generated by a plant SPO11 homologue expressed ectopically. We cytologically and genetically demonstrated the DSB activities of Arabidopsis AtSPO11-1 and AtSPO11-2, which are required for meiosis, in the absence of other plant proteins. Using this bioassay, we further found that a novel SPO11-homologue, OsSPO11D, which has no counterpart in Arabidopsis, displays prominent DSB-forming activity. Quantitative analyses of the rice SPO11 transcripts revealed the specific increase in OsSPO11D mRNA in the anthers containing meiotic pollen mother cells. CONCLUSIONS: The Drosophila bioassay system successfully demonstrated that some plant SPO11 orthologues have intrinsic DSB activities. Furthermore, we identified a novel SPO11 homologue, OsSPO11D, with robust DSB activity and a possible meiotic function. OsSpo11-4|OsSPO11D,TOP6A2 OsSpo11-4, a rice homologue of the archaeal TopVIA protein, mediates double-strand DNA cleavage and interacts with OsTopVIB 2011 PLoS One Research Center of Molecular and Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China. DNA topoisomerase VI from Archaea, a heterotetrameric complex composed of two TopVIA and two TopVIB subunits, is involved in altering DNA topology during replication, transcription and chromosome segregation by catalyzing DNA strand transfer through transient double-strand breaks. The sequenced yeast and animal genomes encode only one homologue of the archaeal TopVIA subunit, namely Spo11, and no homologue of the archaeal TopVIB subunit. In yeast, Spo11 is essential for initiating meiotic recombination and this function appears conserved among other eukaryotes. In contrast to yeast and animals, studies in Arabidopsis and rice have identified three Spo11/TopVIA homologues and one TopVIB homologue in plants. Here, we further identified two novel Spo11/TopVIA homologues (named OsSpo11-4 and OsSpo11-5, respectively) that exist just in the monocot model plant Oryza sativa, indicating that at least five Spo11/TopVIA homologues are present in the rice genome. To reveal the biochemical function of the two novel Spo11/TopVIA homologues, we first examined the interactions among OsSpo11-1, OsSpo11-4, OsSpo11-5, and OsTopVIB by yeast two-hybrid assay. The results showed that OsSpo11-4 and OsTopVIB can self-interact strongly and among the 3 examined OsSpo11 proteins, only OsSpo11-4 interacted with OsTopVIB. Pull-down assay confirmed the interaction between OsSpo11-4 and OsTopVIB, which indicates that OsSpo11-4 may interact with OsTopVIB in vivo. Further in vitro enzymatic analysis revealed that among the above 4 proteins, only OsSpo11-4 exhibited double-strand DNA cleavage activity and its enzymatic activity appears dependent on Mg(2+) and independent of OsTopVIB, despite its interaction with OsTopVIB. We further analyzed the biological function of OsSpo11-4 by RNA interference and found that down-regulated expression of OsSpo11-4 led to defects in male meiosis, indicating OsSpo11-4 is required for meiosis. OsSpo11-4|OsSPO11D,OsSpo11-5,TOP6A2 Signal peptide peptidases are expressed in the shoot apex of rice, localized to the endoplasmic reticulum 2009 Plant Cell Rep Department of Applied Biological Chemistry, The University of Tokyo, Japan. Signal peptide peptidase (SPP) is a multi-transmembrane aspartic proteinase involved in regulated intramembrane proteolysis, which is implicated in fundamental life processes such as immunological response, cell signaling, tissue differentiation, and embryogenesis. In this study, we identified two rice SPPs: OsSPP1 and OsSPP2. Green fluorescent protein-fused OsSPP1 and OsSPP2 were localized to the ER in cultured plant cells. In situ hybridization showed that OsSPPs were strongly expressed in vegetative shoot apex, young panicle, developing panicle, and the early developing florets. Undifferentiated cells, which have the potential to differentiate into all of the aerial parts of the plant are presented in the shoot apex. OsSPPs are located in both the undifferentiated cells, and the early differentiated cells at the shoot apex. These results suggest that rice SPPs have an important function in differentiation and development at the shoot apex. The expression of the shoot apex and ER localization is equal to dicot Arabidopsis thaliana, and will have common crucial roles in plant. OsSPP,OsSPP1,OsSPP2 Purification, molecular cloning, and sequence analysis of sucrose-6F-phosphate phosphohydrolase from plants 2000 Proc Natl Acad Sci U S A Commonwealth Scientific and Industrial Research Organization Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia. john.lunn@pi.csiro.au Sucrose-6(F)-phosphate phosphohydrolase (SPP; EC ) catalyzes the final step in the pathway of sucrose biosynthesis and is the only enzyme of photosynthetic carbon assimilation for which the gene has not been identified. The enzyme was purified to homogeneity from rice (Oryza sativa L.) leaves and partially sequenced. The rice leaf enzyme is a dimer with a native molecular mass of 100 kDa and a subunit molecular mass of 50 kDa. The enzyme is highly specific for sucrose 6(F)-phosphate with a K(m) of 65 microM and a specific activity of 1250 micromol min(-1) mg(-1) protein. The activity is dependent on Mg(2+) with a remarkably low K(a) of 8-9 microM and is weakly inhibited by sucrose. Three peptides from cleavage of the purified rice SPP with endoproteinase Lys-C showed similarity to the deduced amino acid sequences of three predicted open reading frames (ORF) in the Arabidopsis thaliana genome and one in the genome of the cyanobacterium Synechocystis sp. PCC6803, as well as cDNA clones from Arabidopsis, maize, and other species in the GenBank database of expressed sequence tags. The putative maize SPP cDNA clone contained an ORF encoding a 420-amino acid polypeptide. Heterologous expression in Escherichia coli showed that this cDNA clone encoded a functional SPP enzyme. The 260-amino acid N-terminal catalytic domain of the maize SPP is homologous to the C-terminal region of sucrose-phosphate synthase. A PSI-BLAST search of the GenBank database indicated that the maize SPP is a member of the haloacid dehalogenase hydrolase/phosphatase superfamily. OsSPP Identification of a novel mitochondrial protein, short postembryonic roots 1 (SPR1), involved in root development and iron homeostasis in Oryza sativa 2011 New Phytol State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou, China. * A rice mutant, Oryza sativa short postembryonic roots 1 (Osspr1), has been characterized. It has short postembryonic roots, including adventitious and lateral roots, and a lower iron content in its leaves. * OsSPR1 was identified by map-based cloning. It encodes a novel mitochondrial protein with the Armadillo-like repeat domain. * Osspr1 mutants exhibited decreased root cell elongation. The iron content of the mutant shoots was significantly altered compared with that of wild-type shoots. A similar pattern of alteration of manganese and zinc concentrations in shoots was also observed. Complementation of the mutant confirmed that OsSPR1 is involved in post-embryonic root elongation and iron homeostasis in rice. OsSPR1 was found to be ubiquitously expressed in various tissues throughout the plant. The transcript abundance of various genes involved in iron uptake and signaling via both strategies I and II was similar in roots of wild-type and mutant plants, but was higher in the leaves of mutant plants. * Thus, a novel mitochondrial protein that is involved in root elongation and plays a role in metal ion homeostasis has been identified. Osspr1 Is Reactive Oxygen Species (ROS) the underlying factor for inhibited root growth in Osspr1? 2011 Plant Signal Behav Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou, China. lqjiazaas@gmail.com Reactive oxygen species (ROS), like hydrogen peroxide (H2O2) and superoxide anion (O2(.-)), are important plant cell signaling molecules involved in diverse physiological processes, such as programmed cell death, development, cell elongation and hormonal signaling. Recently, much attention has been paid to the role of ROS in regulating plant root development. Two ROS, superoxide and hydrogen peroxide, were shown to exhibit a typical accumulation pattern in the Arabidopsis root apex and play distinct roles in root development. The latest study showed that UPBEAT1 (UPB1), a bHLH transcription factor, modulates the ROS balance by directly regulating the expression of a set of peroxidases, therefore, regulates the root cell proliferation and differentiation. In this addendum, we proposed a possible hypothesis that OsSPR1 maintained the mitochondria function to restrict H2O2 production in root apex for normal root development. Osspr1 The promoter activities of sucrose phosphate synthase genes in rice, OsSPS1 and OsSPS11, are controlled by light and circadian clock, but not by sucrose 2013 Front Plant Sci Bio Research Laboratory, Toyota Motor Corporation Toyota, Aichi, Japan. Although sucrose plays a role in sugar sensing and its signaling pathway, little is known about the regulatory mechanisms of the expressions of plant sucrose-related genes. Our previous study on the expression of the sucrose phosphate synthase gene family in rice (OsSPSs) suggested the involvement of sucrose sensing and/or circadian rhythm in the transcriptional regulation of OsSPS. To examine whether the promoters of OsSPSs can be controlled by sugars and circadian clock, we produced transgenic rice plants harboring a promoter-luciferase construct for OsSPS1 or OsSPS11 and analyzed the changes in the promoter activities by monitoring bioluminescence from intact transgenic plants in real-time. Transgenic plants fed sucrose, glucose, or mannitol under continuous light conditions showed no changes in bioluminescence intensity; meanwhile, the addition of sucrose increased the concentration of sucrose in the plants, and the mRNA levels of OsSPS remained constant. These results suggest that these OsSPS promoters may not be regulated by sucrose levels in the tissues. Next, we investigated the changes in the promoter activities under 12-h light/12-h dark cycles and continuous light conditions. Under the light-dark cycle, both OsSPS1 and OsSPS11 promoter activities were low in the dark and increased rapidly after the beginning of the light period. When the transgenic rice plants were moved to the continuous light condition, both P OsSPS1 ::LUC and P OsSPS11 ::LUC reporter plants exhibited circadian bioluminescence rhythms; bioluminescence peaked during the subjective day with a 27-h period: in the early morning as for OsSPS1 promoter and midday for OsSPS11 promoter. These results indicate that these OsSPS promoters are controlled by both light illumination and circadian clock and that the regulatory mechanism of promoter activity differs between the two OsSPS genes. OsSPS11,sps1|SPS Functional characterization of the rice SPX-MFS family reveals a key role of OsSPX-MFS1 in controlling phosphate homeostasis in leaves 2012 New Phytol State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, China. * Proteins possessing the SPX domain are found in several proteins involved in inorganic phosphate (Pi) transport and signalling in yeast and plants. Although the functions of several SPX-domain protein subfamilies have recently been uncovered, the role of the SPX-MFS subfamily is still unclear. * Using quantitative RT-PCR analysis, we studied the regulation of SPX-MFS gene expression by the central regulator, OsPHR2 and Pi starvation. The function of OsSPX-MFS1 in Pi homeostasis was analysed using an OsSPX-MFS1 mutant (mfs1) and osa-miR827 overexpression line (miR827-Oe). Finally, heterologous complementation of a yeast mutant impaired in Pi transporter was used to assess the capacity of OsSPX-MFS1 to transport Pi. * Transcript analyses revealed that members of the SPX-MFS family were mainly expressed in the shoots, with OsSPX-MFS1 and OsSPX-MFS3 being suppressed by Pi deficiency, while OsSPX-MFS2 was induced. Mutation in OsSPX-MFS1 (mfs1) and overexpression of the upstream miR827 (miR827-Oe) plants impaired Pi homeostasis in the leaves. In addition, studies in yeast revealed that OsSPX-MFS1 may be involved in Pi transport. * The results suggest that OsSPX-MFS1 is a key player in maintaining Pi homeostasis in the leaves, potentially acting as a Pi transporter. OsSPX-MFS1|OsPSS1,OsSPX-MFS3|OsPSS3,OsSPX-MFS4|OSPSS4 Complex regulation of two target genes encoding SPX-MFS proteins by rice miR827 in response to phosphate starvation 2010 Plant Cell Physiol Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan. Here we report on the characterization of rice osa-miR827 and its two target genes, OsSPX-MFS1 and OsSPX-MFS2, which encode SPX-MFS proteins predicted to be implicated in phosphate (Pi) sensing or transport. We first show by Northern blot analysis that osa-miR827 is strongly induced by Pi starvation in both shoots and roots. Hybridization of osa-miR827 in situ confirms its strong induction by Pi starvation, with signals concentrated in mesophyll, epidermis and ground tissues of roots. In parallel, we analyzed the responses of the two OsSPX-MFS1 and OsSPX-MFS2 gene targets to Pi starvation. OsSPX-MFS1 mRNA is mainly expressed in shoots under sufficient Pi supply while its expression is reduced on Pi starvation, revealing a direct relationship between induction of osa-miR827 and down-regulation of OsSPX-MFS1. In contrast, OsSPX-MFS2 responds in a diametrically opposed manner to Pi starvation. The accumulation of OsSPX-MFS2 mRNA is dramatically enhanced under Pi starvation, suggesting the involvement of complex regulation of osa-miR827 and its two target genes. We further produced transgenic rice lines overexpressing osa-miR827 and T-DNA knockout mutant lines in which the expression of osa-miR827 is abolished. Compared with wild-type controls, both target mRNAs exhibit similar changes, their expression being reduced and increased in overexpressing and knockout lines, respectively. This suggests that OsSPX-MFS1 and OsSPX-MFS2 are both negatively regulated by osa-miR827 abundance although they respond differently to external Pi conditions. We propose that this is a complex mechanism comprising fine tuning of spatial or temporal regulation of both targets by osa-miR827. OsSPX-MFS1|OsPSS1,OsSPX-MFS2 The emerging importance of the SPX domain-containing proteins in phosphate homeostasis 2012 New Phytologist Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley, WA 6009, Plant growth and development are strongly influenced by the availability of nutrients in the soil solution. Among them, phosphorus (P) is one of the most essential and most limiting macro-elements for plants. In the environment, plants are often confronted with P starvation as a result of extremely low concentrations of soluble inorganic phosphate (Pi) in the soil. To cope with these conditions, plants have developed a wide spectrum of mechanisms aimed at increasing P use efficiency. At the molecular level, recent studies have shown that several proteins carrying the SPX domain are essential for maintaining Pi homeostasis in plants. The SPX domain is found in numerous eukaryotic proteins, including several proteins from the yeast PHO regulon, involved in maintaining Pi homeostasis. In plants, proteins harboring the SPX domain are classified into four families based on the presence of additional domains in their structure, namely the SPX, SPX-EXS, SPX-MFS and SPX-RING families. In this review, we highlight the recent findings regarding the key roles of the proteins containing the SPX domain in phosphate signaling, as well as providing further research directions in order to improve our knowledge on P nutrition in plants, thus enabling the generation of plants with better P use efficiency. OsSPX-MFS4|OSPSS4 Increased expression of OsSPX1 enhances cold/subfreezing tolerance in tobacco and Arabidopsis thaliana 2009 Plant Biotechnol J State Key Laboratory for Agricultural Biotechnology, College of Biological Sciences, China Agricultural University, Beijing, China. Low temperature is a major environmental stress for plants. Many important cultivated crops have limited capacity to survive below freezing/subfreezing temperatures. Low inorganic phosphate (Pi) is reportedly important in triggering cold acclimatization. SPX (SYG1/Pho81/XPR1: SYG1, suppressor of yeast gpal; Pho81, CDK inhibitor in yeast PHO pathway; XPR1, xenotropic and polytropic retrovirus receptor) domain proteins have been shown to be involved in the phosphate-related signal transduction and regulation pathways. Recently, Arabidopsis AtSPX family genes have been found to possess diverse functions in plant tolerance to phosphorus starvation, and OsSPX1 is involved in phosphate homeostasis in rice and optimizes growth under phosphate-limited conditions through a negative feedback loop. In this study, our phylogenetic and gene expression profiling approaches identified six rice OsSPX genes up-regulated during cold stress. Transgenic tobacco plants with constitutive expression of OsSPX1 were more tolerant to cold stress than were wild-type plants, and showed better seedling survival and reduced cellular electrolyte leakage. In addition, there was decreased total leaf Pi content and accumulation of free proline and sucrose in transgenic tobacco plants during cold stress. To further establish a cause-and-effect relationship between intracellular Pi level and cold acclimatization in transgenic plants, we generated transgenic Arabidopsis plants with constitutive expression of OsSPX1. Cold stress resulted in reduced leaf Pi levels in Arabidopsis transgenic relative to wild-type plants. From real-time reverse transcriptase-polymerase chain reaction analysis, several Pi starvation-related genes, such as AtSPX1 (orthologue of OsSPX1), PHO2, PLDZ2 and ATSIZ1, showed differential expression between wild-type and transgenic plants during cold stress. Our results indicate that OsSPX1 may play an important role in linking cold stress and Pi starvation signal transduction pathways. OsSPX1 The paralogous SPX3 and SPX5 genes redundantly modulate Pi homeostasis in rice 2014 J Exp Bot State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China. The importance of SPX-domain-containing proteins to phosphate (Pi) homeostasis and signalling transduction has been established in plants. In this study, phylogenetic analysis revealed that OsSPX3 and OsSPX5 (SPX3/5) are paralogous SPX genes ( SYG1/Pho81/XPR1) in cereal crops. SPX3/5 are specifically responsive to Pi starvation at both the transcriptional and post-transcriptional levels. Similar tissue expression patterns of the two genes and proteins were identified by in situ hybridization and the transgenic plants harbouring SPX3pro-SPX3-GUS or SPX5pro-SPX5-GUS fusions, respectively. Both SPX3/5 are localized in the nucleus and cytoplasm in rice protoplasts and plants. SPX3/5 negatively regulate root-to-shoot Pi translocation with redundant function. The data showed that the Pi-starvation-accumulated SPX3/5 proteins are players in restoring phosphate balance following phosphate starvation. In vitro and in vivo protein-protein interaction analyses indicated that these two proteins can form homodimers and heterodimers, also implying their functional redundancy. Genetic interaction analysis indicated that SPX3/5 are functional repressors of OsPHR2 (PHR2), the rice orthologue of the central regulator AtPHR1 for Pi homeostasis and Pi signalling. These results suggest that the evolution of the additional redundant paralogous SPX genes is beneficial to plants recovering Pi homeostasis after Pi starvation by PHR2 pathway. OsSPX3,OsSPX5 SPX4 Negatively Regulates Phosphate Signaling and Homeostasis through Its Interaction with PHR2 in Rice 2014 Plant Cell State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, People's Republic of China PHR2, a central regulator of phosphate signaling in rice, enhanced the expression of phosphate starvation-induced (PSI) genes and resulted in the enhancement of Pi acquisition under Pi deficiency stress. This occurred via PHR2 binding to a cis-element named the PHR1 binding sequences. However, the transcription level of PHR2 was not responsive to Pi starvation. So how is activity of transcription factor PHR2 adjusted to adapt diverse Pi status? Here, we identify an SPX family protein, Os-SPX4 (SPX4 hereafter), involving in Pi starvation signaling and acting as a negative regulator of PHR2. SPX4 is shown to be a fast turnover protein. When Pi is sufficient, through its interaction with PHR2, SPX4 inhibits the binding of PHR2 to its cis-element and reduces the targeting of PHR2 to the nucleus. However, when plants grow under Pi deficiency, the degradation of SPX4 is accelerated through the 26S proteasome pathway, thereby releasing PHR2 into the nucleus and activating the expression of PSI genes. Because the level of SPX4 is responsive to Pi concentration and SPX4 interacts with PHR2 and regulates its activity, this suggests that SPX4 senses the internal Pi concentration under diverse Pi conditions and regulates appropriate responses to maintain Pi homeostasis in plants. OsSPX4 Expression of OsSPY and 14-3-3 genes involved in plant height variations of ion-beam-induced KDML 105 rice mutants 2012 Mutat Res Science and Technology Research Institute, Chiang Mai University, Chiang Mai 50200, Thailand. The culm length of two semidwarf rice mutants (PKOS1, HyKOS1) obtained from low-energy N-ion beam bombardments of dehusked Thai jasmine rice (Oryza sativa L. cv. KDML 105) seeds showed 25.7% and 21.5% height reductions and one spindly rice mutant (TKOS4) showed 21.4% increase in comparison with that of the KDML 105 control. A cDNA-RAPD analysis identified differential gene expression in internode tissues of the rice mutants. Two genes identified from the cDNA-RAPD were OsSPY and 14-3-3, possibly associated with stem height variations of the semidwarf and spindly mutants, respectively. The OsSPY gene encoded the SPY protein which is considered to be a negative regulator of gibberellin (GA). On the other hand, the 14-3-3 encoded a signaling protein which can bind and prevent the RSG (repression of shoot growth) protein function as a transcriptional repressor of the kaurene oxidase (KO) gene in the GA biosynthetic pathway. Expression analysis of OsSPY, 14-3-3, RSG, KO, and SLR1 was confirmed in rice internode tissues during the reproductive stage of the plants by semi-quantitative RT-PCR technique. The expression analysis showed a clear increase of the levels of OsSPY transcripts in PKOS1 and HyKOS1 tissue samples compared to that of the KDML 105 and TKOS4 samples at the age of 50-60 days which were at the ages of internode elongation. The 14-3-3 expression had the highest increase in the TKOS4 samples compared to those in KDML 105, PKOS1 and HyKOS1 samples. The expression analysis of RSG and KO showed an increase in TKOS4 samples compared to that of the KDML 105 and that of the two semidwarf mutants. These results indicate that changes of OsSPY and 14-3-3 expression could affect internode elongation and cause the phenotypic changes of semidwarf and spindly rice mutants, respectively. OsSPY,RSG The rice SPINDLY gene functions as a negative regulator of gibberellin signaling by controlling the suppressive function of the DELLA protein, SLR1, and modulating brassinosteroid synthesis 2006 Plant J Bioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya, Aichi 464-8601, Japan. SPINDLY (SPY) encodes an O-linked N-acetylglucosamine transferase that is considered to be a negative regulator of gibberellin (GA) signaling through an unknown mechanism. To understand the function of SPY in GA signaling in rice, we isolated a rice SPINDLY homolog (OsSPY) and produced knockdown transgenic plants in which OsSPY expression was reduced by introducing its antisense or RNAi construct. In knockdown plants, the enhanced elongation of lower internodes was correlated with decreased levels of OsSPY expression, similar to the spindly phenotype of Arabidopsis spy mutants, suggesting that OsSPY also functions as a negative factor in GA signaling in rice. The suppressive function of OsSPY in GA signaling was supported by the findings that the dwarfism was partially rescued and OsGA20ox2 (GA20 oxidase) expression was reduced in GA-deficient and GA-insensitive mutants by the knockdown of OsSPY function. The suppression of OsSPY function in a GA-insensitive mutant, gid2, also caused an increase in the phosphorylation of a rice DELLA protein, SLR1, but did not change the amount of SLR1. This indicates that the function of OsSPY in GA signaling is not via changes in the amount or stability of SLR1, but probably involves control of the suppressive function of SLR1. In addition to the GA-related phenotypes, OsSPY antisense and RNAi plants showed increased lamina joint bending, which is a brassinosteroid-related phenotype, indicating that OsSPY may play roles both in GA signaling and in the brassinosteroid pathway. OsSPY,sd1|GA20ox2,SLR1|OsGAI A special member of the rice SRO family, OsSRO1c, mediates responses to multiple abiotic stresses through interaction with various transcription factors 2014 Plant Mol Biol National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, 430070, China. SIMILAR TO RCD ONE (SRO) is a plant-specific gene family involved in development and abiotic stress responses. SRO proteins are characterized by containing poly (ADP-ribose) polymerase catalytic (PARP) and C-terminal RCD1-SRO-TAF4 domains, and can be classified into two groups and five subgroups on the basis of their PARP domain. Expression analysis of rice SRO genes in response to various abiotic stresses showed that OsSRO1c, a rice SRO gene which functions downstream of the stress-responsive transcription factor SNAC1, is the major stress-responsive gene in the rice SRO family. The ossro1c-1 mutant showed resistance not only to chloroplastic oxidative stress, but also to apoplastic oxidative stress. However, the ossro1c-1 mutant and artificial microRNA-OsSRO1c transgenic rice were significantly impaired in cold tolerance. When compared with the well-characterized Arabidopsis SRO protein radical-induced cell death 1 (RCD1), OsSRO1c has considerable variation in the protein sequence, and the two genes exhibit different expression profiles under abiotic stresses. Furthermore, ossro1c-1 and rcd1 showed different responses to multiple abiotic stresses. By screening an Arabidopsis transcription factor library, 29 transcription factors interacted with OsSRO1c in yeast, but only two of these transcription factors were reported to interact with RCD1, which may partly explain the different responses of the two mutants under various stresses. The data presented in this report provide important clues for further elucidating the molecular and biochemical mechanisms of OsSRO1c in mediating responses to multiple abiotic stresses. OsSRO1c Down-regulation of a SILENT INFORMATION REGULATOR2-related histone deacetylase gene, OsSRT1, induces DNA fragmentation and cell death in rice 2007 Plant Physiol National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China. The SILENT INFORMATION REGULATOR2 (SIR2) family proteins are NAD(+)-dependent histone deacetylases. Sir2 is involved in chromatin silencing at the mating-type loci, rDNA, and telomeres in yeast and is associated with lifespan extension in yeast, worms, and flies, but also in a broader range of additional functions. In this work, we investigated the role of OsSRT1, one of the two SIR2-related genes found in rice (Oryza sativa). We show that OsSRT1 is a widely expressed nuclear protein with higher levels in rapidly dividing tissues. OsSRT1 RNA interference induced an increase of histone H3K9 (lysine-9 of H3) acetylation and a decrease of H3K9 dimethylation, leading to H(2)O(2) production, DNA fragmentation, cell death, and lesions mimicking plant hypersensitive responses during incompatible interactions with pathogens, whereas overexpression of OsSRT1 enhanced tolerance to oxidative stress. Transcript microarray analysis revealed that the transcription of many transposons and retrotransposons in addition to genes related to hypersensitive response and/or programmed cell death was activated. Chromatin immunoprecipitation assays showed that OsSRT1 down-regulation induced histone H3K9 acetylation on the transposable elements and some of the hypersensitive response-related genes, suggesting that these genes may be among the primary targets of deacetylation regulated by OsSRT1. Our data together suggest that the rice SIR2-like gene is required for safeguard against genome instability and cell damage to ensure plant cell growth, but likely implicates different molecular mechanisms than yeast and animal homologs. OsSRT1,HSR201,HSR203J The rice NAD(+)-dependent histone deacetylase OsSRT1 targets preferentially to stress- and metabolism-related genes and transposable elements 2013 PLoS One National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China. Histone acetylation/deacetylation is an important chromatin modification for epigenetic regulation of gene expression. Silent information regulation2 (Sir2)-related sirtuins are nicotinamide-adenine dinucleotide (NAD(+))-dependent histone deacetylases (HDAC). The mammalian sirtuin family comprises 7 members (SIRT1-7) that act in different cellular compartments to regulate metabolism and aging. The rice genome contains only two Sir2-related genes: OsSRT1 (or SRT701) and OsSRT2 (orSRT702). OsSRT1 is closely related to the mammalian SIRT6, while OsSRT2 is homologous to SIRT4. Previous work has shown that OsSRT1 is required for the safeguard against genome instability and cell damage in rice plant. In this work we investigated the role of OsSRT1 on genome-wide acetylation of histone H3 lysine 9 (H3K9ac) and studied the genome-wide binding targets of OsSRT1. The study reveals that OsSRT1 binds to loci with relatively low levels of H3K9ac and directly regulates H3K9ac and expression of many genes that are related to stress and metabolism, indicating that OsSRT1 is an important site-specific histone deacetylase for gene regulation in rice. In addition, OsSRT1 is found to also target to several families of transposable elements, suggesting that OsSRT1 is directly involved in transposable element repression. OsSRT1 Molecular and functional characterization of sulfiredoxin homologs from higher plants 2006 Cell Res The State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing 100101, China. By reducing cysteine-sulfinic acid in oxidized peroxiredoxin, sulfiredoxin (Srx) plays an important role in oxidation stress resistance in yeast and human cells. Here, we report the first molecular and functional characterization of Srx homolog from higher plants. Bioinformatic analysis revealed the presence of potential Srx encoding sequences in both monocot and dicot plant species. Putative plant Srx proteins exhibited significant identities to their orthologs from yeast and human, and contained the conserved signature sequence and residues essential for catalysis. However, unlike yeast and human orthologs, plant Srxs were all predicted to possess chloroplast transit peptide in their primary structure. The Srx proteins from Arabidopsis and rice (designated as AtSrx and OsSrx, respectively) complemented functional deficiency of Srx in the SRX1 deletion yeast cells. A GFP fusion protein of AtSrx was targeted to chloroplast in Arabidopsis mesophyll protoplast. AtSrx transcription occurred in both vegetative and reproductive organs, and the highest transcript level was detected in leaves. Under oxidation stress, AtSrx transcript level was substantially increased, which paralleled with enhanced transcription of 2-Cys peroxiredoxins that have been found essential in maintaining chloroplast redox balance. In addition to oxidation stress, osmotic/water deficit or cold treatments also raised AtSrx transcript level. Consistent with above findings, the knock-out mutant of AtSrx was significantly more susceptible to oxidation stress than wild type Arabidopsis plant. Taken together, the results of this work indicate the existence of functional Srx homolog in higher plants that is essential for plants to cope with oxidation stress. OsSrx Structure, organization, and chromosomal location of the gene encoding a form of rice soluble starch synthase 1995 Plant Physiol Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan. A rice (Oryza sativa L.) genomic clone encoding the gene for a form of soluble starch synthase (SSS1) and its 5'- and 3'-flanking regions has been isolated and sequenced. The SSS1 gene contained 15 exons interrupted by 14 introns. The exon/intron organization of the SSS1 gene was divergent from that of the rice Waxy gene coding for granule-bound starch synthase, thus suggesting that the SSS1 and granule-bound starch synthase genes have evolved from an ancestral gene in a different way or that the two genes are products of different ancestral genes that have converged during evolution. However, these two genes were closely located to each other on rice chromosome 6 at an approximate map distance of 5 centimorgans. The nucleotide sequence of the 5'-end region of the gene is unique because of the presence of some repetitive sequences. OsSSI|SSS1 Function and characterization of starch synthase I using mutants in rice 2006 Plant Physiol Department of Biological Production, Akita Prefectural University, Akita City, Japan. naokof@akita-pu.ac.jp Four starch synthase I (SSI)-deficient rice (Oryza sativa) mutant lines were generated using retrotransposon Tos17 insertion. The mutants exhibited different levels of SSI activities and produced significantly lower amounts of SSI protein ranging from 0% to 20% of the wild type. The mutant endosperm amylopectin showed a decrease in chains with degree of polymerization (DP) 8 to 12 and an increase in chains with DP 6 to 7 and DP 16 to 19. The degree of change in amylopectin chain-length distribution was positively correlated with the extent of decrease in SSI activity in the mutants. The structural changes in the amylopectin increased the gelatinization temperature of endosperm starch. Chain-length analysis of amylopectin in the SSI band excised from native-polyacrylamide gel electrophoresis/SS activity staining gel showed that SSI preferentially synthesized DP 7 to 11 chains by elongating DP 4 to 7 short chains of glycogen or amylopectin. These results show that SSI distinctly generates DP 8 to 12 chains from short DP 6 to 7 chains emerging from the branch point in the A or B(1) chain of amylopectin. SSI seemingly functions from the very early through the late stage of endosperm development. Yet, the complete absence of SSI, despite being a major SS isozyme in the developing endosperm, had no effect on the size and shape of seeds and starch granules and the crystallinity of endosperm starch, suggesting that other SS enzymes are probably capable of partly compensating SSI function. In summary, this study strongly suggested that amylopectin chains are synthesized by the coordinated actions of SSI, SSIIa, and SSIIIa isoforms. OsSSI|SSS1 A comprehensive expression analysis of the starch synthase gene family in rice (Oryza sativa L.) 2004 Planta Department of Rice Research, National Agricultural Research Center, Joetsu, 943-0193 Niigata, Japan. dragon@affrc.go.jp To elucidate the roles of the isogenes encoding starch synthase (EC 2.4.1.21) in rice (Oryza sativa L.), a comprehensive expression analysis of the gene family was conducted. Extensive searches for starch synthase genes were done in the databases of both the whole genome and full-length cDNAs of rice, and ten genes were revealed to comprise the starch synthase gene family. Multi-sequence alignment analysis of the starch synthase proteins from rice and other plant species suggested that they were grouped into five classes, soluble starch synthase I (SSI), SSII, SSIII, SSIV and granule-bound starch synthase (GBSS). In rice, there was one gene for SSI, three for SSII and two each for SSIII, IV and GBSS. The expression pattern of the ten genes in the developing caryopsis was examined by semi-quantitative RT-PCR analysis. Based on the temporal expression patterns, the ten genes could be divided into three groups: (i) early expressers ( SSII-2, III-1, GBSSII), which are expressed in the early stage of grain filling; (ii) late expressers ( SSII-3, III-2, GBSSI), which are expressed in the mid to later stage of grain filling; and (iii) steady expressers ( SSI, II-1, IV-1, IV-2), which are expressed relatively constantly during grain filling. Within a caryopsis, the three gene groups spatially share their expression, i.e. "early expressers" in the pericarp, the "late expressers" in the endosperm" and the "steady expressers" in both tissues. In addition, this grouping was reflected in the expression pattern of various rice tissues: expression in non-endosperm, endosperm or all tissues examined. The implications in this spatio-temporal work sharing of starch synthesis isogenes are discussed. OsSSI|SSS1 Suppression of the rice fatty-acid desaturase gene OsSSI2 enhances resistance to blast and leaf blight diseases in rice 2009 Mol Plant Microbe Interact Plant Disease Resistance Research Unit, Division of Plant Science, National Institute of Agrobiological Sciences, Tsukuba, Japan. Fatty acids and their derivatives play important signaling roles in plant defense responses. It has been shown that suppressing a gene for stearoyl acyl carrier protein fatty-acid desaturase (SACPD) enhances the resistance of Arabidopsis (SSI2) and soybean to multiple pathogens. In this study, we present functional analyses of a rice homolog of SSI2 (OsSSI2) in disease resistance of rice plants. A transposon insertion mutation (Osssi2-Tos17) and RNAi-mediated knockdown of OsSSI2 (OsSSI2-kd) reduced the oleic acid (18:1) level and increased that of stearic acid (18:0), indicating that OsSSI2 is responsible for fatty-acid desaturase activity. These plants displayed spontaneous lesion formation in leaf blades, retarded growth, slight increase in endogenous free salicylic acid (SA) levels, and SA/benzothiadiazole (BTH)-specific inducible genes, including WRKY45, a key regulator of SA/BTH-induced resistance, in rice. Moreover, the OsSSI2-kd plants showed markedly enhanced resistance to the blast fungus Magnaporthe grisea and leaf-blight bacteria Xanthomonas oryzae pv. oryzae. These results suggest that OsSSI2 is involved in the negative regulation of defense responses in rice, as are its Arabidopsis and soybean counterparts. Microarray analyses identified 406 genes that were differentially expressed (>or=2-fold) in OsSSI2-kd rice plants compared with wild-type rice and, of these, approximately 39% were BTH responsive. Taken together, our results suggest that induction of SA-responsive genes, including WRKY45, is likely responsible for enhanced disease resistance in OsSSI2-kd rice plants. OsSSI2,OsWRKY45 Knockout of a starch synthase gene OsSSIIIa/Flo5 causes white-core floury endosperm in rice (Oryza sativa L.) 2007 Plant Cell Rep Plant Metabolism Research Center and Graduate School of Biotechnology, Kyung Hee University, Yongin 446-701, Korea. ryoo1009@khu.ac.kr To elucidate the role of SSIIIa during starch synthesis in rice (Oryza sativa L.) endosperm, we characterized null mutants of this gene, generated by T-DNA insertions. Scanning electron microscope (SEM) analysis revealed that the starch granules in these mutants are smaller and rounder compared with the wild type controls, and that the mutant endosperm is characterized by a loosely packed central portion exhibiting a floury-like phenotype. Hence, the OsSSIIIa (Oryza sativa SSIIIa) mutations are referred to as white-core floury endosperm 5-1 (flo5-1) and flo5-2. Based upon their X-ray diffraction patterns, the crystallinity of the starch in the flo5 mutant endosperm is decreased compared with wild type. Through determination of the chain-length distribution of the mutant endosperm starch, we found that flo5-1 and flo5-2 mutants have reduced the content of long chains with degree of polymerization (DP) 30 or greater compared with the controls. This suggests that OsSSIIIa/Flo5 plays an important role in generating relatively long chains in rice endosperm. In addition, DP 6 to 8 and DP 16 to 20 appeared to be reduced in endosperm starch of flo5-1 and flo5-2, whereas DP 9 to 15 and DP 22 to 29 were increased in these mutants. By the use of differential scanning calorimetry (DSC), the gelatinization temperatures of endosperm starch were found to be 1-5 degrees C lower than those of the control. We propose a distinct role for OsSSIIIa/Flo5 and the coordinated action of other SS isoforms during starch synthesis in the seed endosperm of rice. OsSSIIIa|Flo5,SSIIB,SSIIC Deciphering starch quality of rice kernels using metabolite profiling and pedigree network analysis 2012 Mol Plant RIKEN Plant Science Center, Tsurumi, Yokohama 230-0045, Japan. mkusano005@psc.riken.jp The physiological properties of rice grains are immediately obvious to consumers. High-coverage metabolomic characterization of the rice diversity research set predicted a negative correlation between fatty acid and lipid levels and amylose/total starch ratio (amylose ratio), but the reason for this is unclear. To obtain new insight into the relationships among the visual phenotypes of rice kernels, starch granule structures, amylose ratios, and metabolite changes, we investigated the metabolite changes of five Japonica cultivars with various amylose ratios and two knockout mutants (e1, a Starch synthase IIIa (SSIIIa)-deficient mutant and the SSIIIa/starch branching enzyme (BE) double-knockout mutant 4019) by using mass spectrometry-based metabolomics techniques. Scanning electron microscopy clearly showed that the two mutants had unusual starch granule structures. The metabolomic compositions of two cultivars with high amylose ratios (Hoshiyutaka and Yumetoiro) exhibited similar patterns, while that of the double-knockout mutant, which has an extremely high amylose ratio, differed. Rice pedigree network analysis of the cultivars and the mutants provided insight into the association between metabolic-trait properties and their underlying genetic basis in rice breeding in Japan. Multidimensional scaling analysis revealed that the Hoshiyutaka and Yumetoiro cultivars were Indica-like, yet they are classified as Japonica subpopulations. Exploring metabolomic traits is a powerful way to follow rice genetic traces and breeding history. OsSSIIIa|Flo5 Characterization of SSIIIa-deficient mutants of rice: the function of SSIIIa and pleiotropic effects by SSIIIa deficiency in the rice endosperm 2007 Plant Physiol Department of Biological Production, Akita Prefectural University, Akita City, Akita 010-0195, Japan. naokof@akita-pu.ac.jp Starch synthase IIIa (SSIIIa)-deficient rice (Oryza sativa) mutants were generated using retrotransposon insertion and chemical mutagenesis. The lowest migrating SS activity bands on glycogen-containing native polyacrylamide gel, which were identified to be those for SSIIIa, were completely absent in these mutants, indicating that they are SSIIIa null mutants. The amylopectin B(2) to B(4) chains with degree of polymerization (DP) >/= 30 and the M(r) of amylopectin in the mutant were reduced to about 60% and 70% of the wild-type values, respectively, suggesting that SSIIIa plays an important part in the elongation of amylopectin B(2) to B(4) chains. Chains with DP 6 to 9 and DP 16 to 19 decreased while chains with DP 10 to 15 and DP 20 to 25 increased in the mutants amylopectin. These changes in the SSIIIa mutants are almost opposite images of those of SSI-deficient rice mutant and were caused by 1.3- to 1.7-fold increase of the amount of SSI in the mutants endosperm. Furthermore, the amylose content and the extralong chains (DP >/= 500) of amylopectin were increased by 1.3- and 12-fold, respectively. These changes in the composition in the mutants starch were caused by 1.4- to 1.7-fold increase in amounts of granules-bound starch synthase (GBSSI). The starch granules of the mutants were smaller with round shape, and were less crystalline. Thus, deficiency in SSIIIa, the second major SS isozyme in developing rice endosperm affected the structure of amylopectin, amylase content, and physicochemical properties of starch granules in two ways: directly by the SSIIIa deficiency itself and indirectly by the enhancement of both SSI and GBSSI gene transcripts. OsSSIIIa|Flo5 Loss-of-function of OsSTN8 suppresses the photosystem II core protein phosphorylation and interferes with the photosystem II repair mechanism in rice (Oryza sativa) 2013 Plant J Department of Molecular Biology, Pusan National University, Busan, 609-735, Korea; Department of New Biology, Daegu Gyeongbuk Institute of Science and Technology, Daegu, 711-873, Korea. STN8 kinase is involved in photosystem II (PSII) core protein phosphorylation (PCPP). To examine the role of PCPP in PSII repair during high light (HL) illumination, we characterized a T-DNA insertional knockout mutant of the rice (Oryza sativa) STN8 gene. In this osstn8 mutant, PCPP was significantly suppressed, and the grana were thin and elongated. Upon HL illumination, PSII was strongly inactivated in the mutants, but the D1 protein was degraded more slowly than in wild-type, and mobilization of the PSII supercomplexes from the grana to the stromal lamellae for repair was also suppressed. In addition, higher accumulation of reactive oxygen species and preferential oxidation of PSII reaction center core proteins in thylakoid membranes were observed in the mutants during HL illumination. Taken together, our current data show that the absence of STN8 is sufficient to abolish PCPP in osstn8 mutants and to produce all of the phenotypes observed in the double mutant of Arabidopsis, indicating the essential role of STN8-mediated PCPP in PSII repair. OsSTN8 Expression dynamics of metabolic and regulatory components across stages of panicle and seed development in indica rice 2012 Funct Integr Genomics Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India. Carefully analyzed expression profiles can serve as a valuable reference for deciphering gene functions. We exploited the potential of whole genome microarrays to measure the spatial and temporal expression profiles of rice genes in 19 stages of vegetative and reproductive development. We could verify expression of 22,980 genes in at least one of the tissues. Differential expression analysis with respect to five vegetative tissues and preceding stages of development revealed reproductive stage-preferential/-specific genes. By using subtractive logic, we identified 354 and 456 genes expressing specifically during panicle and seed development, respectively. The metabolic/hormonal pathways and transcription factor families playing key role in reproductive development were elucidated after overlaying the expression data on the public databases and manually curated list of transcription factors, respectively. During floral meristem differentiation (P1) and male meiosis (P3), the genes involved in jasmonic acid and phenylpropanoid biosynthesis were significantly upregulated. P6 stage of panicle, containing mature gametophytes, exhibited enrichment of transcripts involved in homogalacturonon degradation. Genes regulating auxin biosynthesis were induced during early seed development. We validated the stage-specificity of regulatory regions of three panicle-specific genes, OsAGO3, OsSub42, and RTS, and an early seed-specific gene, XYH, in transgenic rice. The data generated here provides a snapshot of the underlying complexity of the gene networks regulating rice reproductive development. OsSub42|OsSP2,XYH Identification and characterization of the duplicate rice sucrose synthase genes OsSUS5 and OsSUS7 which are associated with the plasma membrane 2011 Mol Cells Graduate School of Biotechnology, Kyung Hee University, Yongin, 446-701, Korea. Systematic searches using the complete genome sequence of rice (Oryza sativa) identified OsSUS7, a new member of the rice sucrose synthase (OsSUS) gene family, which shows only nine single nucleotide substitutions in the OsSUS5 coding sequence. Comparative genomic analysis revealed that the synteny between OsSUS5 and OsSUS7 is conserved, and that significant numbers of transposable elements are scattered at both loci. In particular, a 17.6-kb genomic region containing transposable elements was identified in the 5' upstream sequence of the OsSUS7 gene. GFP fusion experiments indicated that OsSUS5 and OsSUS7 are largely associated with the plasma membrane and partly with the cytosol in maize mesophyll protoplasts. RT-PCR analysis and transient expression assays revealed that OsSUS5 and OsSUS7 exhibit similar expression patterns in rice tissues, with the highest expression evident in roots. These results suggest that two redundant genes, OsSUS5 and OsSUS7, evolved via duplication of a chromosome region and through the transposition of transposable elements. OsSUS5,OsSUS7 Modification of OsSUT1 gene expression modulates the salt response of rice Oryza sativa cv. Taipei 309 2012 Plant Sci Max Planck Institute of Molecular Plant Physiology (MPIMP), Wissenschaftspark Golm, Am Muehlenberg 1, Potsdam-Golm D-14476, Germany. siahpoosh@scu.ac.ir A metabolic depletion syndrome was discovered at early vegetative stages in roots of salt sensitive rice cultivars after prolonged exposure to 100mM NaCl. Metabolite profiling analyses demonstrate that this syndrome is part of the terminal stages of the rice salt response. The phenotype encompasses depletion of at least 30 primary metabolites including sucrose, glucose, fructose, glucose-6-P, fructose-6P, organic- and amino-acids. Based on these observations we reason that sucrose allocation to the root may modify the rice response to high salt. This hypothesis was tested using antisense lines of the salt responsive OsSUT1 gene in the salt sensitive Taipei 309 cultivar. Contrary to our expectations of a plant system impaired in one component of sucrose transport, we find improved gas exchange and photosynthetic performance as well as maintenance of sucrose levels in the root under high salinity. Two independent OsSUT1 lines with an antisense inhibition similar to the naturally occurring salt induced reduction of OsSUT1 gene expression showed these phenomena but not a more extreme antisense inhibition line. We investigated the metabolic depletion syndrome by metabolomic and physiological approaches and discuss our results with regard to the potential role of sucrose transporters and sucrose transport for rice salt acclimation. OsSUT1 Assimilate translocation and expression of sucrose transporter, OsSUT1, contribute to high-performance ripening under heat stress in the heat-tolerant rice cultivar Genkitsukushi 2013 J Plant Physiol Fukuoka Agricultural Research Center, Chikushino, Fukuoka 818-8549, Japan; Crop Science Laboratory, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. High temperature reduces the grain quality of rice, a situation likely to become more frequent because of global warming. We studied the effects of high-temperature stress on grain quality of heat-tolerant cultivar 'Genkitsukushi' and heat-sensitive cultivar 'Tsukushiroman'. When day/night temperatures were 31/26 degrees C from heading until maturity, the grain quality of 'Genkitsukushi' was rated at the first inspection grade (high quality), whereas 'Tsukushiroman' showed a remarkable increase in the percentage of white immature kernels (low quality). Nonstructural carbohydrate content in the stem of 'Genkitsukushi' the early maturation was significantly higher than in 'Tsukushiroman' and greatly decreased under high temperature. From 14 to 21 days after heading, the expression of the sucrose transporter gene, OsSUT1, was higher in the stem of 'Genkitsukushi' grown under high temperature than in 'Tsukushiroman'. In addition, the expression of OsSUT1 in the grains of 'Genkitsukushi' was significantly higher than in 'Tsukushiroman' during the ripening period. These results indicate that sugar transport functions more effectively in 'Genkitsukushi' than in 'Tsukushiroman', and that the effectiveness of sugar transport contributes to maintaining high grain quality in 'Genkitsukushi' under high-temperature conditions. OsSUT1 Arg188 in rice sucrose transporter OsSUT1 is crucial for substrate transport 2012 BMC Biochem Department of Plant Biology, University of Minnesota Twin Cities, St, Paul, MN 55108, USA. BACKGROUND: Plant sucrose uptake transporters (SUTs) are H+/sucrose symporters related to the major facilitator superfamily (MFS). SUTs are essential for plant growth but little is known about their transport mechanism. Recent work identified several conserved, charged amino acids within transmembrane spans (TMS) in SUTs that are essential for transport activity. Here we further evaluated the role of one of these positions, R188 in the fourth TMS of OsSUT1, a type II SUT. RESULTS: The OsSUT1(R188K) mutant, studied by expression in plants, yeast, and Xenopus oocytes, did not transport sucrose but showed a H+ leak that was blocked by sucrose. The H+ leak was also blocked by beta-phenyl glucoside which is not translocated by OsSUT1. Replacing the corresponding Arg in type I and type III SUTs, AtSUC1(R163K) and LjSUT4(R169K), respectively, also resulted in loss of sucrose transport activity. Fluorination at the glucosyl 3 and 4 positions of alpha-phenyl glucoside greatly decreased transport by wild type OsSUT1 but did not affect the ability to block H+ leak in the R188K mutant. CONCLUSION: OsSUT1 R188 appears to be essential for sucrose translocation but not for substrate interaction that blocks H+ leak. Therefore, we propose that an additional binding site functions in the initial recognition of substrates. The corresponding Arg in type I and III SUTs are equally important. We propose that R188 interacts with glucosyl 3-OH and 4-OH during translocation. OsSUT1 Functionally important amino acids in rice sucrose transporter OsSUT1 2012 Biochemistry Department of Plant Biology, University of Minnesota-Twin Cities, St. Paul, Minnesota 55108, United States. Six conserved, charged amino acids within membrane spans in rice sucrose transporter OsSUT1 were identified using a three-dimensional structural model based on the crystal structures of three major facilitator superfamily (MFS) proteins: LacY, GlpT, and EmrD. These positions in OsSUT1 were selected for mutagenesis and biochemical assays. Among the six mutants, D177N completely lost transport function, D331N retained only a small fraction of sucrose uptake activity (2.3% of that of the wild type), and R335H and E336Q also displayed a substantial decrease in transport activity. D329N functioned as well as wild-type OsSUT1. R188K did not transport sucrose but showed a H(+) leak that was inhibited by sucrose, indicating that R188K had uncoupled sucrose and H(+) translocation. This demonstrates that charged amino acids within membrane spans are important for the transport mechanism of OsSUT1 as they are in lactose permease. OsSUT1 The role of the sucrose transporter, OsSUT1, in germination and early seedling growth and development of rice plants 2007 J Exp Bot CSIRO Plant Industry, Canberra, ACT 2601, Australia. Using expression analysis, the role of the sucrose transporter OsSUT1 during germination and early growth of rice seedlings has been examined in detail, over a time-course ranging from 1 d to 7 d post-imbibition. Unlike the wheat orthologue, TaSUT1, which is thought to be directly involved in sugar transfer across the scutellar epithelium, OsSUT1 is not expressed in the scutellar epithelial cell layer of germinating rice and is, therefore, not involved in transport of sugars across the symplastic discontinuity between the endosperm and the embryo. OsSUT1 expression was also absent from the aleurone cells, indicating it is not involved in the transport of sucrose in this cell layer during germination. However, by 3 d post-imbibition, OsSUT1 was present in the companion cells and sieve elements of the scutellar vascular bundle, where it may play a role in phloem loading of sucrose for transport to the developing shoot and roots. This sucrose is most likely sourced from hexoses imported from the endosperm. In addition, sucrose may be remobilized from starch granules which are present at a high density in the scutellar ground tissues surrounding the vasculature and at the base of the shoot. OsSUT1 was also present in the coleoptile and the first and second leaf blades, where it was localized to the phloem along the entire length of these tissues, and was also present within the phloem of the primary roots. OsSUT1 may be involved in retrieval of sugars from the apoplasm in these tissues. OsSUT1 Involvement of the sucrose transporter, OsSUT1, in the long-distance pathway for assimilate transport in rice 2007 J Exp Bot CSIRO Plant Industry, Canberra, ACT 2601, Australia. The roles of the rice sucrose transporter, OsSUT1, have previously been examined in filling grain, germination, and early seedling growth. In the current work, the role that OsSUT1 plays in the transport of assimilate along the entire long-distance pathway, from the flag leaf blade to the base of the filling grain, was investigated. OsSUT1 promoter::GUS (beta-glucuronidase) reporter gene analysis and immunolocalization revealed that both OsSUT1 promoter::GUS activity and OsSUT protein were present in the mature phloem of all the vegetative tissues involved in the long-distance assimilate transport pathway during grain filling. In addition, expression was observed in the flag leaf blade and sheath prior to heading. The OsSUT1 promoter::GUS activity appeared to be largely confined to the companion cells within the phloem, whereas the protein localized to both the sieve tubes and the companion cells. RT-PCR analysis confirmed that the OsSUT1 transcript is expressed in the uppermost internode of the rice plant (internode-1). These OsSUT localization data were related to measurements of starch and soluble sugar content of these tissues, and localization of the carbohydrate reserves stored in the stem. Results from dye feeding experiments, to examine cellular connections, revealed a symplastic continuity between the phloem and surrounding parenchyma in the flag leaf blade, sheath, and internode-1 tissues. It is proposed that OsSUT1 may primarily play a role in phloem loading of sucrose retrieved from the apoplasm along the transport pathway. OsSUT1 Disruption of a gene for rice sucrose transporter, OsSUT1, impairs pollen function but pollen maturation is unaffected 2010 J Exp Bot Hokuriku Research Center, National Agricultural Research Center, National Agriculture and Food Research Organization, Joetsu, Niigata 943-0193, Japan. dragon@affrc.go.jp Sucrose transporters (SUTs) are known to play critical roles in the uptake of sucrose from the apoplast in various steps of sugar translocation. Because developing pollen is symplastically isolated from anther tissues, it is hypothesized that SUTs are active in the uptake of apoplastic sucrose into pollen. To investigate this possibility, a comprehensive expression analysis was performed for members of the SUT gene family in the developing pollen of rice (Oryza sativa L.) using real-time RT-PCR combined with a laser microdissection technique. Among the five SUT genes, OsSUT1 and OsSUT3 were found to be preferentially expressed and had temporal expression patterns that were distinct from each other. Expression of OsSUT1 in pollen was confirmed by a promoter-GUS fusion assay. The physiological function of OsSUT1 in pollen was further investigated using retrotransposon insertion mutant lines. While the homozygote of disrupted OsSUT1 (SUT1-/-) could not be obtained, heterozygote plants (SUT1+/-) showed normal grain filling. Their progeny segregated into SUT1+/- and SUT1+/+ with the ratio of 1:1, suggesting that the pollen disrupted for OsSUT1 is dysfunctional. This hypothesis was reinforced in vivo by a backcross of SUT1+/- plants with wild-type plants and also by in vitro pollen germination on the artificial media. However, starch accumulation during pollen development was not affected by disruption of OsSUT1, suggesting that the sugar(s) required for starch biosynthesis is supplied by other sugar transporters. OsSUT1 Antisense Expression of a Rice Sucrose Transporter OsSUT1 in Rice (Oryza sativa L.) 2001 Plant and Cell Physiology National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602 Japan We analyzed the function of a rice sucrose transporter, OsSUT1, by using antisense rice. There was no difference between antisense and wild-type plants in carbohydrate content and photosynthetic ability of the flag leaves in the vegetative growth stage, suggesting that OsSUT1 may not play an important role in carbon metabolism, at least in these materials. OsSUT1 The sucrose transporter gene family in rice 2003 Plant Cell Physiol CSIRO Plant Industry, Canberra, ACT, 2601 Australia. In this paper we report the identification, cloning and expression analysis of four putative sucrose transporter (SUT) genes from rice, designated OsSUT2, 3, 4 and 5. Three of the four genes were identified through extensive searches of the recently published draft sequence of the rice genome. Along with the previously reported OsSUT1 we propose that these five genes comprise the rice SUT gene family. Complementary DNA clones were isolated for the four newly identified genes. The deduced proteins of all five SUT genes were predicted to contain 12 membrane-spanning helices and a domain highly conserved throughout all known plant SUTs, suggesting the four additional OsSUT genes encode functional SUTs. Reverse transcription-PCR analysis was performed in order to investigate the expression pattern of each member of the SUT family in rice. A differing but overlapping expression pattern was observed for each member of the SUT family at different stages through plant development. These results, together with the structural variations apparent from the deduced protein sequences, suggest that the five SUTs possess diverse roles in both sink and source tissues. We also discuss the classification and evolution of the rice SUT gene family, using a comparison of the gene structures and deduced amino acid sequences with other known plant SUT genes. OsSUT1,OsSUT2|OsSUT2M,OsSUT3,OsSUT4,OsSUT5|OsSUT5Z Transport activity of rice sucrose transporters OsSUT1 and OsSUT5 2010 Plant Cell Physiol Department of Plant Biology, University of Minnesota, 250 Biological Sciences Center, St. Paul, MN 55108 USA. Expression in Xenopus oocytes and electrophysiology was used to test for transport activity of the five sucrose transporter (SUT) homologs from rice. Expression of OsSUT1 and OsSUT5 resulted in sucrose-dependent currents that were analyzed by two-electrode voltage clamping. We examined the transport kinetics, substrate specificity and pH dependence of sucrose transport and K(0.5) for sucrose. OsSUT1 showed similar features to those of other type II SUTs from monocots examined previously, with a K(0.5) value of 7.50 mM at pH 5.6. In contrast, OsSUT5 had a higher substrate affinity (K(0.5) = 2.32 mM at pH 5.6), less substrate specificity and less pH dependence compared with all type II SUTs tested to date. Regulation of the rice SUTs, as well as ZmSUT1 from maize and HvSUT1 from barley, by reduced (GSH) and oxidized (GSSG) forms of glutathione was tested. GSSG and GSH were found to have no significant effect on the activity of sucrose transporters when expressed in Xenopus oocytes. In conclusion, differences in transport activity between OsSUT1 and OsSUT5 indicate that type II SUTs have a range of transport activities that are tuned to their function in the plant. OsSUT1,OsSUT5|OsSUT5Z Rice sucrose transporter1 (OsSUT1) up-regulation in xylem parenchyma is caused by aphid feeding on rice leaf blade vascular bundles 2014 Plant Biol (Stuttg) Department of Botany, Rhodes University, Grahamstown, South Africa; Plant Stress Response Group, Department of Biochemistry & Microbiology, University of Fort Hare, Alice, South Africa. The role of the sucrose transporter OsSUT1 in assimilate retrieval via the xylem, as a result of damage to and leakage from punctured phloem was examined after rusty plum aphid (Hysteroneura setariae, Thomas) infestation on leaves from 3-week-old rice (Oryza sativa L. cv Nipponbare) plants. Leaves were examined over a 1- to 10-day infestation time course, using a combination of gene expression and beta-glucuronidase (GUS) reporter gene analyses. qPCR and Western blot analyses revealed differential expression of OsSUT1 during aphid infestation. Wide-field fluorescence microscopy was used to confirm the expression of OsSUT1-promoter::GUS reporter gene in vascular parenchyma associated with xylem elements, as well as in companion cells associated with phloem sieve tubes of large, intermediate and small vascular bundles within the leaf blade, in regions where the aphids had settled and were feeding. Of great interest was up-regulation of OsSUT1 expression associated with the xylem parenchyma cells, abutting the metaxylem vessels, which confirmed that OsSUT1 was not only involved in loading of sugars into the phloem under normal physiological conditions, but was apparently involved in the retrieval of sucrose leaked into the xylem conduits, which occurred as a direct result of aphid feeding, probing and puncturing of vascular bundles. The up-regulation of OsSUT1 in xylem vascular parenchyma thus provides evidence in support of the location within the xylem parenchyma cells of an efficient mechanism to ensure sucrose recovery after loss to the apoplast (xylem) after aphid-related feeding damage and its transfer back to the symplast (phloem) in O. sativa leaves. OsSUT1 Antisense suppression of the rice transporter gene, OsSUT1, leads to impaired grain filling and germination but does not affect photosynthesis 2002 Functional Plant Biology None OsSUT1 encodes a rice sucrose transport protein that is highly expressed in developing grain, leaf sheath and stem after heading, and in germinating seedlings, but only at very low levels in source leaves. In this study, we have used antisense gene suppression to elucidate the in vivo function of OsSUT1. Rice was transformed with an antisense construct containing a portion of the 3'-coding and non-coding regions of OsSUT1 driven by the maize ubiquitin-1 promoter. Twenty-six independent stably transformed lines were obtained. T0 and selfed T1 progeny were analysed for suppression of OsSUT1 expression and function. Many of the plants showed a significant reduction in their ability to produce filled grain, and final grain weight was reduced. Severe phenotypes correlated with a reduction in OsSUT1 transcript level in filling grain. Unlike SUT1 antisense suppression in dicots, source supply of photosynthate was unaffected in these transformants. This provides the first direct evidence for the requirement of a sucrose transporter for grain filling in a cereal species. Furthermore, seed from some of the T0 population showed a reduction in the rate of germination and growth, supporting the hypothesis that OsSUT1 may also play a role in transporting sucrose remobilized from starch reserves in germinating seeds. OsSUT1 Impaired function of the tonoplast-localized sucrose transporter in rice, OsSUT2, limits the transport of vacuolar reserve sucrose and affects plant growth 2011 Plant Physiol Graduate School of Biotechnology and Plant Metabolism Research Center, Kyung Hee University, Yongin 446-701, Korea. Physiological functions of sucrose (Suc) transporters (SUTs) localized to the tonoplast in higher plants are poorly understood. We here report the isolation and characterization of a mutation in the rice (Oryza sativa) OsSUT2 gene. Expression of OsSUT2-green fluorescent protein in rice revealed that OsSUT2 localizes to the tonoplast. Analysis of the OsSUT2 promoter::beta-glucuronidase transgenic rice indicated that this gene is highly expressed in leaf mesophyll cells, emerging lateral roots, pedicels of fertilized spikelets, and cross cell layers of seed coats. Results of Suc transport assays in yeast were consistent with a H(+)-Suc symport mechanism, suggesting that OsSUT2 functions in Suc uptake from the vacuole. The ossut2 mutant exhibited a growth retardation phenotype with a significant reduction in tiller number, plant height, 1,000-grain weight, and root dry weight compared with the controls, the wild type, and complemented transgenic lines. Analysis of primary carbon metabolites revealed that ossut2 accumulated more Suc, glucose, and fructose in the leaves than the controls. Further sugar export analysis of detached leaves indicated that ossut2 had a significantly decreased sugar export ability compared with the controls. These results suggest that OsSUT2 is involved in Suc transport across the tonoplast from the vacuole lumen to the cytosol in rice, playing an essential role in sugar export from the source leaves to sink organs. OsSUT2|OsSUT2M Cloning and expression analysis of rice sucrose transporter genes OsSUT2M and OsSUT5Z 2008 J Integr Plant Biol State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, the Chinese Academy of Sciences, Beijing 100101, China. Two sucrose transporter (SUT) cDNAs, OsSUT2M and OsSUT5Z, were isolated from rice (Oryza sativa L.) by reverse transcription polymerase chain reaction (RT-PCR). Sequencing results indicate they are 1,531 bp and 1,635 bp in length including complete open reading frame 1,506 bp and 1,608 bp, which encode 502 amino acids and 536 amino acids, respectively. The TopPred program suggested that both sucrose transporter proteins, OsSUT2M and OsSUT5Z, consist of potentially 12 transmembrane domains. Semi-quantitative RT-PCR was carried out to investigate the gene expression patterns of OsSUT2M and OsSUT5Z. In vegetative organs, transcripts of OsSUT2M were higher in source leaf blades than in other organs at the same development stage, whereas transcripts of OsSUT5Z were less traceable in all organs investigated. In reproductive organs, both transcripts of these two genes were high in panicles from the booting stage to 7 days after flowering (DAF) and then sharply declined. The potential physiology functions of these two sucrose transporters are discussed. OsSUT2|OsSUT2M,OsSUT5|OsSUT5Z OsSUV3 dual helicase functions in salinity stress tolerance by maintaining photosynthesis and antioxidant machinery in rice (Oryza sativa L. cv. IR64) 2013 Plant J International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India. To overcome the salinity-induced loss of crop yield, a salinity-tolerant trait is required. The SUV3 helicase is involved in the regulation of RNA surveillance and turnover in mitochondria, but the helicase activity of plant SUV3 and its role in abiotic stress tolerance have not been reported so far. Here we report that the Oryza sativa (rice) SUV3 protein exhibits DNA and RNA helicase, and ATPase activities. Furthermore, we report that SUV3 is induced in rice seedlings in response to high levels of salt. Its expression, driven by a constitutive cauliflower mosaic virus 35S promoter in IR64 transgenic rice plants, confers salinity tolerance. The T1 and T2 sense transgenic lines showed tolerance to high salinity and fully matured without any loss in yields. The T2 transgenic lines also showed tolerance to drought stress. These results suggest that the introduced trait is functional and stable in transgenic rice plants. The rice SUV3 sense transgenic lines showed lesser lipid peroxidation, electrolyte leakage and H2 O2 production, along with higher activities of antioxidant enzymes under salinity stress, as compared with wild type, vector control and antisense transgenic lines. These results suggest the existence of an efficient antioxidant defence system to cope with salinity-induced oxidative damage. Overall, this study reports that plant SUV3 exhibits DNA and RNA helicase and ATPase activities, and provides direct evidence of its function in imparting salinity stress tolerance without yield loss. The possible mechanism could be that OsSUV3 helicase functions in salinity stress tolerance by improving photosynthesis and antioxidant machinery in transgenic rice. OsSUV3 Rice SUV3 is a bidirectional helicase that binds both DNA and RNA 2014 BMC Plant Biol International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India BackgroundHelicases play crucial role in almost all the nucleic acid metabolism including replication, repair, recombination, transcription, translation, ribosome biogenesis and splicing and these processes regulate plant growth and development. It is suggested that helicases play essential roles in stabilizing growth in plants under stress because their presence in the stress-induced ORFs has been identified. Moreover in a recent study we have reported that SUV3 helicase from Oryza sativa (OsSUV3) functions in salinity stress tolerance in transgenic rice by improving the antioxidant machinery. SUV3 helicase has been identified and characterized from yeast and human systems but the properties and functions of plant SUV3 are poorly understood.ResultsIn this study, the purification and extensive characterization of recombinant OsSUV3 protein (67 kDa) is presented. OsSUV3 binds to DNA and RNA and exhibits DNA as well as RNA-dependent ATPase activities. It also contains the characteristic DNA and RNA helicase activity. OsSUV3 can use mainly ATP or dATP as energy source for the unwinding activity and it cannot unwind the blunt-end duplex DNA substrate. It is interesting to note that OsSUV3 unwinds DNA in both the 5'-3' and 3'-5' directions and thus its activity is bipolar in vitro. The Km values of OsSUV3 are 0.51 nM and 0.95 nM for DNA helicase and RNA helicase, respectively.ConclusionsThis study is the first direct evidence to show the bipolar DNA helicase activity of OsSUV3 protein. The unique properties of OsSUV3 including its dual helicase activity imply that it could be a multifunctional protein involved in biologically significant process of DNA and RNA metabolisms. These results should make significant contribution towards better understanding of SUV3 protein in plants. OsSUV3 Overexpression of the Qc-SNARE gene OsSYP71 enhances tolerance to oxidative stress and resistance to rice blast in rice (Oryza sativa L.) 2012 Gene State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Agriculture, Nanjing Agricultural University, Nanjing, China OsSYP71 is an oxidative stress and rice blast response gene that encodes a Qc-SNARE protein in rice. Qc-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), which function as important components of the vesicle trafficking machinery in eukaryotic cells. In this paper, 12 Qc-SNARE genes were isolated from rice, and expression patterns of 9 genes were detected in various tissues and in seedlings challenged with oxidative stresses and inoculated with rice blast. The expression of OsSYP71 was clearly up-regulated under these stresses. Overexpression of OsSYP71 in rice showed more tolerance to oxidative stress and resistance to rice blast than wild-type plants. These results indicate that Qc-SNAREs play an important role in rice response to environmental stresses, and OsSYP71 is useful in engineering crop plants with enhanced tolerance to oxidative stress and resistance to rice blast. OsSYP71 Identification of a beta-glucosidase hydrolyzing tuberonic acid glucoside in rice (Oryza sativa L.) 2010 Phytochemistry Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, N-9, W-9, Kita-Ku, Sapporo 060-8589, Japan. Tuberonic acid (TA) and its glucoside (TAG) have been isolated from potato (Solanum tuberosum L.) leaflets and shown to exhibit tuber-inducing properties. These compounds were reported to be biosynthesized from jasmonic acid (JA) by hydroxylation and subsequent glycosylation, and to be contained in various plant species. Here we describe the in vivo hydrolytic activity of TAG in rice. In this study, the TA resulting from TAG was not converted into JA. Tuberonic acid glucoside (TAG)-hydrolyzing beta-glucosidase, designated OsTAGG1, was purified from rice by six purification steps with an approximately 4300-fold purification. The purified enzyme migrated as a single band on native PAGE, but as two bands with molecular masses of 42 and 26 kDa on SDS-PAGE. Results from N-terminal sequencing and peptide mass fingerprinting of both polypeptides suggested that both bands were derived from a single polypeptide, which is a member of the glycosyl hydrolase family 1. In the native enzyme, the K(m) and V(max) values of TAG were 31.7 microM and 0.25 microkatal/mg protein, OsTAGG1 preferentially hydrolyzed TAG and methyl TAG. Here we report that OsTAGG1 is a specific beta-glucosidase hydrolyzing TAG, which releases the physiologically active TA. OsTAGG1 A large increase in IAA during development of rice grains correlates with the expression of tryptophan aminotransferase OsTAR1 and a grain-specific YUCCA 2012 Physiol Plant Molecular and Cellular Biology, University of New England, Armidale, New South Wales, Australia. The indole-3-acetic acid (IAA) content of developing grains of Oryza sativa subsp. japonica was measured by combined liquid chromatography, tandem mass spectrometry in multiple-reaction-monitoring mode. The increase from 50 ng g(-1) fresh weight to 2.9 microg g(-1) fresh weight from 1 to 14 days after pollination was much larger than that previously reported by enzyme-linked immunoassay methods. The largest increase in IAA content coincided with the start of the major starch deposition phase of grain-fill. The increase in IAA content was strongly correlated with the expression of putative IAA biosynthesis genes, OsYUC9, OsYUC11 and OsTAR1, measured by quantitative reverse transcriptase polymerase chain reaction. These results confirm the importance of the tryptophan aminotransferase/YUCCA pathway in this system. All three genes were expressed in endosperm; expression of OsYUC11 appeared to be confined to endosperm tissue. Phylogenetic analysis indicated that OsYUC11 and AtYUC10 belong to a separate clade of YUCCAs, which do not have orthologues outside the Angiosperms. This clade may have evolved with a specific role in endosperm. Expression of tryptophan decarboxylase in developing rice grains did not correlate with IAA levels, indicating that tryptamine is unlikely to be important for IAA synthesis in this system. In light of these observations, we hypothesize that IAA production in developing rice grains is controlled via expression of OsTAR1, OsYUC9, OsYUC11 and that IAA may be important during starch deposition in addition to its previously suggested role early in grain development. OsTAR1,OsYUC11,OsYUC9 Screening of the rice viviparous mutants generated by endogenous retrotransposon Tos17 insertion. Tagging of a zeaxanthin epoxidase gene and a novel ostatc gene 2001 Plant Physiol Department of Molecular Genetics, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan. The rice (Oryza sativa) retrotransposon Tos17 is one of a few active retrotransposons in plants and its transposition is activated by tissue culture. Here, we present the characterization of viviparous mutants of rice induced by tissue culture to demonstrate the feasibility of the use of retrotransposon Tos17 as an endogenous insertional mutagen and cloning of the tagged gene for forward genetics in unraveling the gene function. Two mutants were shown to be caused by the insertion of Tos17. Osaba1, a strong viviparous mutant with wilty phenotype, displayed low abscisic acid level and almost no further increase in its levels upon drought. The mutant is shown to be impaired in the epoxidation of zeaxanthin. On the other hand, Ostatc, a mutant with weak phenotype, exhibited the pale green phenotype and slight increase in abscisic acid levels upon drought. Deduced amino acids of the causative genes of Osaba1 and Ostatc manifested a significantly high homology with zeaxanthin epoxidase isolated from other plant species and with bacterial Sec-independent translocase TATC protein, respectively. This is the first example of transposon tagging in rice. OsTATC,OsABA1|OSZEP1 The OsTB1 gene negatively regulates lateral branching in rice 2003 The Plant Journal Bioscience Center, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan. Although the shoot apical meristem (SAM) is ultimately responsible for post-embryonic development in higher plants, lateral meristems also play an important role in determining the final morphology of the above-ground part. Axillary buds developing at the axils of leaves produce additional shoot systems, lateral branches. The rice TB1 gene (OsTB1) was first identified based on its sequence similarity with maize TEOSINTE BRANCHED 1 (TB1), which is involved in lateral branching in maize. Both genes encode putative transcription factors carrying a basic helix-loop-helix type of DNA-binding motif, named the TCP domain. The genetic locus of OsTB1 suggested that OsTB1 is a real counterpart of maize TB1. Transgenic rice plants overexpressing OsTB1 exhibited markedly reduced lateral branching without the propagation of axillary buds being affected. We also demonstrated that a rice strain carrying a classical morphological marker mutation, fine culm 1 (fc1), contain the loss-of-function mutation of OsTB1 and exhibits enhanced lateral branching. Expression of OsTB1, as examined with a putative promoter-glucuronidase (GUS) gene fusion, was observed throughout the axillary bud, as well as the basal part of the shoot apical meristem, vascular tissues in the pith and the lamina joint. Taking these data together, we concluded that OsTB1 functions as a negative regulator for lateral branching in rice, presumably through expression in axillary buds. OsTB1|FC1 Rice TATA Binding Protein Interacts Functionally with Transcription Factor IIB and the RF2a bZIP Transcriptional Activator in an Enhanced Plant in Vitro Transcription System 2002 The Plant Cell Online Plant Biology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA. TATA binding protein (TBP) and transcription factor IIB (TFIIB) are key factors for the assembly of eukaryotic transcription initiation complexes. We used a rice whole-cell extract in vitro transcription system to characterize the functional interactions of recombinant plant TBP and TFIIB. Bacterially expressed rice TBP (OsTBP2) bound to the TATA box of the rice pal gene encoding phenylalanine ammonia-lyase, caused DNA bending, and enhanced basal transcription from the pal promoter in a TATA box-dependent manner. Recombinant rice TFIIB (OsTFIIB) stimulated the DNA binding and bending activities of OsTBP2 and synergistically enhanced OsTBP2-mediated transcription from the pal promoter and the promoter of Rice tungro bacilliform virus but not from the barley pr1 promoter. We also demonstrate a physical interaction between OsTBP2 and RF2a, a rice bZIP transcription factor that bound to the box II cis element of the promoter of Rice tungro bacilliform virus, resulting in enhanced transcription from the viral promoter. Enhancement of rice whole-cell extracts with recombinant transcription factors thus provides a powerful tool for the in vitro determination of plant gene regulation mechanisms. We conclude that OsTBP2 undergoes promoter-specific functional interactions with both the basal transcription factor OsTFIIB and the accessory transcription factor RF2a. OsTBP2,OsTFIIB,RF2a|OsbZIP75 MIL2 (MICROSPORELESS2) regulates early cell differentiation in the rice anther 2012 New Phytol State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China. The formation of diverse, appropriately patterned cell types is critical in the development of all complex multicellular organisms. In flowering plants, anther patterning is a complex process essential for successful sexual reproduction. However, few genes regulating this process have been characterized to date. We report here that the gene MICROSPORELESS2 (MIL2) regulates early anther cell differentiation in rice (Oryza sativa). The anthers of mil2 mutants were characterized using molecular markers and cytological examination. The MIL2 gene was cloned and its expression pattern was analyzed through RNA in situ hybridization. The localization of the MIL2 protein was observed by immunostaining. MIL2 encodes the rice homolog of the Arabidopsis TAPETUM DETERMINANT1 (TPD1) protein. However, mil2 anthers display phenotypes different from those of tpd1 mutants, with only two layers of anther wall cells formed. MIL2 has an expression pattern distinct from that of TPD1. Its transcripts and proteins predominate in inner parietal cells, but show little accumulation in reproductive cells. Our results demonstrate that MIL2 is responsible for the differentiation of primary parietal cells into secondary parietal cells in rice anthers, and suggest that rice and Arabidopsis anthers might share similar regulators in anther patterning, but divergent mechanisms are employed in these processes. OsTDL1A|MIL2 OsTDL1A binds to the LRR domain of rice receptor kinase MSP1, and is required to limit sporocyte numbers 2008 Plant J College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China. Hybrids lose heterotic yield advantage when multiplied sexually via meiosis. A potential alternative breeding system for hybrids is apospory, where female gametes develop without meiosis. Common among grasses, apospory begins in the nucellus, where aposporous initials (AIs) appear near the sexual megaspore mother cell (MeMC). The cellular origin of AIs is obscure, but one possibility, suggested by the mac1 and msp1 mutants of maize and rice, is that AIs are apomeiotic derivatives of the additional MeMCs that appear when genetic control over sporocyte numbers is relaxed. MULTIPLE SPOROCYTES1 (MSP1) encodes a leucine-rich-repeat receptor kinase, which is orthologous to EXS/EMS1 in Arabidopsis. Like mac1 and msp1, exs/ems1 mutants produce extra sporocytes in the anther instead of a tapetum, causing male sterility. This phenotype is copied in mutants of TAPETUM DETERMINANT1 (TPD1), which encodes a small protein hypothesized to be an extracellular ligand of EXS/EMS1. Here we show that rice contains two TPD1-like genes, OsTDL1A and OsTDL1B. Both are co-expressed with MSP1 in anthers during meiosis, but only OsTDL1A and MSP1 are co-expressed in the ovule. OsTDL1A binds to the leucine-rich-repeat domain of MSP1 in yeast two-hybrid assays and bimolecular fluorescence complementation in onion cells; OsTDL1B lacks this capacity. When driven by the maize Ubiquitin1 promoter, RNA interference against OsTDL1A phenocopies msp1 in the ovule but not in the anther. Thus, RNAi produces multiple MeMCs without causing male sterility. We conclude that OsTDL1A binds MSP1 in order to limit sporocyte numbers. OsTDL1A-RNAi lines may be suitable starting points for achieving synthetic apospory in rice. OsTDL1A|MIL2,OsTDL1B Reduced tillering in Basmati rice T-DNA insertional mutant OsTEF1 associates with differential expression of stress related genes and transcription factors 2012 Funct Integr Genomics Department of Biotechnology, Indian Institute of Technology, Roorkee 247667, India. A T-DNA insertional mutant OsTEF1 of rice gives 60-80% reduced tillering, retarded growth of seminal roots, and sensitivity to salt stress compared to wild type Basmati 370. The insertion occurred in a gene encoding a transcription elongation factor homologous to yeast elf1, on chromosome 2 of rice. Detailed transcriptomic profiling of OsTEF1 revealed that mutation in the transcription elongation factor differentially regulates the expression of more than 100 genes with known function and finely regulates tillering process in rice by inducing the expression of cytochrome P450. Along with different transcription factors, several stress associated genes were also affected due to a single insertion. In silico analysis of the TEF1 protein showed high conservation among different organisms. This transcription elongation factor predicted to interact with other proteins that directly or indirectly positively regulate tillering in rice. OsTEF1 Cloning and characterization of rice (Oryza sativaL) telomerase reverse transcriptase, which reveals complex splicing patterns 2002 The Plant Journal CAMBIA: Center for the Application of Molecular Biology to International Agriculture, GPO Box 3200, Canberra, ACT 2601, Australia. Plant chromosomes terminate in telomeres as in other eukaryotes. Telomeres are vital to genome stability and their malfunctioning is lethal. One of the core components of the telomere complex is telomerase. The enzyme activity depends on RNA (TER) and reverse transcriptase (TERT) subunits. We describe here the isolation, sequencing and characterization of the telomerase reverse transcriptase catalytic subunit from the monocot plant Oryza sativa L. (OsTERT). A single copy of this gene is present in the rice genome. The protein predicted from the OsTERT sequence has all the signature motifs of the TERT family members. Our data indicate that rice telomerase activity is developmentally regulated and is high in in vitro tissue and cell culture. However, steady-state transcript levels of the TERT gene do not seem to correlate with enzyme activity. Northern and RT-PCR analyses of the OsTERT gene transcript profile show multiple differentially spliced transcripts in both telomerase-positive and telomerase-negative tissues. Based on quantitative analysis of these transcripts, we speculate that the overall balance between the quantities of particular alternatively spliced transcripts may determine whether the TERT protein(s) is active or not. The diversity of splicing variants detected suggests that, as recently discovered for mammalian TERT proteins, rice TERT protein variants may perform functions other than telomere maintenance. OsTERT OSTF1: A HD-GL2 Family Homeobox Gene is Developmentally Regulated During Early Embryogenesis in Rice 2002 Plant and Cell Physiology Graduate Institute of Agricultural Biotechnology, National Chung Hsing University, Taichung, Taiwan In many eukaryotic organisms, homeobox genes are important regulators that specify the cell fate and body plan in early embryogenesis. In this study, a gene designated OSTF1 (Oryza sativa transcription factor 1) encoding a homeodomain protein in rice was isolated and characterized. The encoded OSTF1, although sharing only approximately 51% sequence identity with other HD-GL2 members, contains four characteristic motifs (an N-terminal acidic region, a homeodomain, a truncated leucine zipper, and a START domain). OSTF1 was detected as a single copy gene in rice. The transcripts were absent in young panicle or mature spikelet before anthesis, but appeared very early in the pollinated grain with a transient profile. In vegetative tissues examined, expression was only detectable in root. In situ hybridization analysis on developing grains revealed that OSTF1 was strongly and uniformly expressed in the embryo at the globular stage and preferentially localized to the protoderm at 3-6 d after pollination. Expression was also detectable in the integument and throughout the endosperm. Although OSTF1 is not closely related to the remaining HD-GL2 members in sequences, this gene exhibits an analogous epidermis-preferential expression pattern. OSTF1 Two type III effector genes of Xanthomonas oryzae pv. oryzae control the induction of the host genes OsTFIIA 1 and OsTFX1 during bacterial blight of rice 2007 Proceedings of the National Academy of Sciences Department of Plant Pathology, Kansas State University, Manhattan, KS 66506, USA. Xanthomonas oryzae pv. oryzae strain PXO99(A) induces the expression of the host gene Os8N3, which results in increased host susceptibility to bacterial blight of rice. Here, we show that PXO99(A) affects the expression of two additional genes in a type III secretion system-dependent manner, one encoding a bZIP transcription factor (OsTFX1) and the other the small subunit of the transcription factor IIA located on chromosome 1 (OsTFIIAgamma1). Induction of OsTFX1 and OsTFIIAgamma1 depended on the type III effector genes pthXo6 and pthXo7, respectively, both encoding two previously undescribed members of the transcription activator-like (TAL) effector family. pthXo7 is strain-specific and may reflect adaptation to the resistance mediated by xa5, an allele of OsTFIIAgamma5 encoding a second form of the TFIIA small subunit on chromosome 5 of rice. The loss of pthXo6 resulted in reduced pathogen virulence, and ectopic expression of OsTFX1 abrogated the requirement for pthXo6 for full virulence. X. oryzae pv. oryzae therefore modulates the expression of multiple host genes using multiple TAL effectors from a single strain, and evidence supports the hypothesis that expression of the associated host genes contributes to host susceptibility to disease. OsTFX1 Overexpression of OsTLP27 in rice improves chloroplast function and photochemical efficiency 2012 Plant Sci College of Biological Engineering, Chongqing University, Chongqing 400030, China. The thylakoid lumen proteins are highly associated with photosynthesis functionally. In this study, we characterized the OsTLP27 gene from rice (Oryza sativa), which encodes a 27-kDa of 257-amino acid with 53% homology to the AtTLP gene from Arabidopsis thaliana. OsTLP27 was predicted to encode a thylakoid lumen protein of unknown function in chloroplast, and chloroplast targeting of OsTLP27 was confirmed by transient expression of a fusion protein with green fluorescent protein (GFP). OsTLP27 transcripts accumulated specifically in green tissues such as the leaf blade and leaf sheath, and the levels of its transcripts followed a circadian rhythm. Constitutive expression of OsTLP27 under the control of CaMV 35S promoter resulted in increased pigment content and enhanced photochemical efficiency in terms of the values of maximal photochemical efficiency of photosystem II (PSII) (F(v)/F(m)), effective quantum yield of PSII (PhiPSII), electron transport rate (ETR) and photochemical quenching (qP). Overexpression of OsTLP27 also enhanced transcript levels of genes related to chloroplast function and caused changes in the grana size and number. Further study showed that the structure and polypeptide composition of the photosynthetic apparatus were altered in transgenic lines overexpressing OsTLP27. These data suggested that OsTLP27 encodes a protein with a novel function in photosynthesis and chloroplast development in rice. OsTLP27 Expression analysis and functional characterization of the monosaccharide transporters, OsTMTs, involving vacuolar sugar transport in rice (Oryza sativa) 2010 New Phytol Graduate School of Biotechnology and Plant Metabolism Research Center, Kyung Hee University, Yongin 446-701, Korea. In Arabidopsis, the compartmentation of sugars into vacuoles is known to be facilitated by sugar transporters. However, vacuolar sugar transporters have not been studied in detail in other plant species. To characterize the rice (Oryza sativa) tonoplast monosaccharide transporters, OsTMT1 and OsTMT2, we analysed their subcellular localization using green fluorescent protein (GFP) and expression patterns using reverse-transcription polymerase chain reaction (RT-PCR), performed histochemical beta-glucuronidase (GUS) assay and in situ hybridization analysis, and assessed sugar transport ability using isolated vacuoles. Expression of OsTMT-GFP fusion protein in rice and Arabidopsis revealed that the OsTMTs localize at the tonoplast. Analyses of OsTMT promoter-GUS transgenic rice indicated that OsTMT1 and OsTMT2 are highly expressed in bundle sheath cells, and in vascular parenchyma and companion cells in leaves, respectively. Both genes were found to be preferentially expressed in the vascular tissues of roots, the palea/lemma of spikelets, and in the main vascular tissues and nucellar projections on the dorsal side of the seed coats. Glucose uptake studies using vacuoles isolated from transgenic mutant Arabidopsis (tmt1-2-3) expressing OsTMT1 demonstrated that OsTMTs are capable of transporting glucose into vacuoles. Based on expression analysis and functional characterization, our present findings suggest that the OsTMTs play a role in vacuolar glucose storage in rice. OsTMT1,OsTMT2 Overexpression of putative topoisomerase 6 genes from rice confers stress tolerance in transgenic Arabidopsis plants 2006 FEBS J Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, India. DNA topoisomerase 6 (TOP6) belongs to a novel family of type II DNA topoisomerases present, other than in archaebacteria, only in plants. Here we report the isolation of full-length cDNAs encoding putative TOP6 subunits A and B from rice (Oryza sativa ssp. indica), preserving all the structural domains conserved among archaebacterial TOP6 homologs and eukaryotic meiotic recombination factor SPO11. OsTOP6A1 was predominantly expressed in prepollinated flowers. The transcript abundance of OsTOP6A2, OsTOP6A3 and OsTOP6B was also higher in prepollinated flowers and callus. The expression of OsTOP6A2, OsTOP6A3 and OsTOP6B was differentially regulated by the plant hormones, auxin, cytokinin, and abscisic acid. Yeast two-hybrid analysis revealed that the full-length OsTOP6B protein interacts with both OsTOP6A2 and OsTOP6A3, but not with OsTOP6A1. The nuclear localization of OsTOP6A3 and OsTOP6B was established by the transient expression of their beta-glucuronidase fusion proteins in onion epidermal cells. Overexpression of OsTOP6A3 and OsTOP6B in transgenic Arabidopsis plants conferred reduced sensitivity to the stress hormone, abscisic acid, and tolerance to high salinity and dehydration. Moreover, the stress tolerance coincided with enhanced induction of many stress-responsive genes in transgenic Arabidopsis plants. In addition, microarray analysis revealed that a large number of genes are expressed differentially in transgenic plants. Taken together, our results demonstrate that TOP6 genes play a crucial role in stress adaptation of plants by altering gene expression. OsSPO11-1|OsTOP6A1,OsTOP6A3,OsTOP6B,TOP6A2 Constitutive expression of a meiotic recombination protein gene homolog, OsTOP6A1, from rice confers abiotic stress tolerance in transgenic Arabidopsis plants 2008 Plant Cell Rep Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, India. Plant productivity is greatly influenced by various environmental stresses, such as high salinity and drought. Earlier, we reported the isolation of topoisomerase 6 homologs from rice and showed that over expression of OsTOP6A3 and OsTOP6B confers abiotic stress tolerance in transgenic Arabidopsis plants. In this study, we have assessed the function of nuclear-localized topoisomerase 6 subunit A homolog, OsTOP6A1, in transgenic Arabidopsis plants. The over expression of OsTOP6A1 in transgenic Arabidopsis plants driven by cauliflower mosaic virus-35S promoter resulted in pleiotropic effects on plant growth and development. The transgenic Arabidopsis plants showed reduced sensitivity to stress hormone, abscisic acid (ABA), and tolerance to high salinity and dehydration at the seed germination; seedling and adult stages as reflected by the percentage of germination, fresh weight of seedlings and leaf senescence assay, respectively. Concomitantly, the expression of many stress-responsive genes was enhanced under various stress conditions in transgenic Arabidopsis plants. Moreover, microarray analysis revealed that the expression of a large number of genes involved in various processes of plant growth and development and stress responses was altered in transgenic plants. Although AtSPO11-1, the homolog of OsTOP6A1 in Arabidopsis, has been implicated in meiotic recombination; the present study demonstrates possible additional role of OsTOP6A1 and provides an effective tool for engineering crop plants for tolerance to different environmental stresses. OsSPO11-1|OsTOP6A1 Regulation of a Proteinaceous Elicitor-induced Ca2+ Influx and Production of Phytoalexins by a Putative Voltage-gated Cation Channel, OsTPC1, in Cultured Rice Cells 2012 Journal of Biological Chemistry Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan. Pathogen/microbe- or plant-derived signaling molecules (PAMPs/MAMPs/DAMPs) or elicitors induce increases in the cytosolic concentration of free Ca(2+) followed by a series of defense responses including biosynthesis of antimicrobial secondary metabolites called phytoalexins; however, the molecular links and regulatory mechanisms of the phytoalexin biosynthesis remains largely unknown. A putative voltage-gated cation channel, OsTPC1 has been shown to play a critical role in hypersensitive cell death induced by a fungal xylanase protein (TvX) in suspension-cultured rice cells. Here we show that TvX induced a prolonged increase in cytosolic Ca(2+), mainly due to a Ca(2+) influx through the plasma membrane. Membrane fractionation by two-phase partitioning and immunoblot analyses revealed that OsTPC1 is localized predominantly at the plasma membrane. In retrotransposon-insertional Ostpc1 knock-out cell lines harboring a Ca(2+)-sensitive photoprotein, aequorin, TvX-induced Ca(2+) elevation was significantly impaired, which was restored by expression of OsTPC1. TvX-induced production of major diterpenoid phytoalexins and the expression of a series of diterpene cyclase genes involved in phytoalexin biosynthesis were also impaired in the Ostpc1 cells. Whole cell patch clamp analyses of OsTPC1 heterologously expressed in HEK293T cells showed its voltage-dependent Ca(2+)-permeability. These results suggest that OsTPC1 plays a crucial role in TvX-induced Ca(2+) influx as a plasma membrane Ca(2+)-permeable channel consequently required for the regulation of phytoalexin biosynthesis in cultured rice cells. OsTPC1 Functional analysis of a rice putative voltage-dependent Ca2+ channel, OsTPC1, expressed in yeast cells lacking its homologous gene CCH1 2004 Plant Cell Physiol Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo, 184-8588 Japan. We isolated a cDNA (OsTPC1) from rice that was homologous to AtTPC1, a putative voltage-dependent Ca(2+) channel (VDCC) gene of Arabidopsis thaliana. The hydropathy profile of its deduced amino acid sequence showed significant structural features of the alpha 1-subunit of animal VDCCs. Functional analysis using a heterologous yeast expression system showed that OsTPC1 facilitates Ca(2+) permeation. The K(m) value for Ca(2+) of OsTPC1, 47.5 micro M, was comparable to that of intrinsic CCH1, a candidate VDCC in yeast. Ca(2+) permeation by OsTPC1 was inhibited by verapamil, a VDCC blocker. These findings indicate for the first time that OsTPC1 is a putative VDCC in rice. OsTPC1 Identification of a putative voltage-gated Ca2+ -permeable channel (OsTPC1) involved in Ca2+ influx and regulation of growth and development in rice 2004 Plant Cell Physiol Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510 Japan. Cytosolic free Ca2+ serves as an important second messenger participating in signal transduction of various environmental stresses. However, molecular bases for the plasma membrane Ca2+ influx and its regulation remain largely unknown. We here identified a gene (OsTPC1) encoding a putative voltage-gated Ca2+ channel from rice, ubiquitously expressed in mature leaves, shoots and roots as well as in cultured cells. OsTPC1 rescued the Ca2+ uptake activity and growth rate of a yeast mutant cch1. To elucidate its physiological roles, we generated transgenic rice plants and cultured cells overexpressing OsTPC1 mRNA. Furthermore, a retrotransposon (Tos17) insertional knockout mutant of OsTPC1 was isolated. OsTPC1-overexpressing cells showed hypersensitivity to excess Ca2+ but higher growth rate under Ca2+ limitation, while growth of the OsTPC1-knockout cultured cells was less sensitive to extracellular free Ca2+ concentration, suggesting that OsTPC1 has Ca2+ transport activity across the plasma membrane. OsTPC1-overexpressing plants showed reduced growth and abnormal greening of roots. Growth of Ostpc1 seedlings was comparable to the control on agar plates, while significantly reduced in adult plants. These results suggest that OsTPC1 functions as a Ca2+ -permeable channel involved in the regulation of growth and development. OsTPC1 Intracellular localization and physiological function of a rice Ca(2)(+)-permeable channel OsTPC1 2012 Plant Signal Behav Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, Japan. Two-pore channels (TPCs) are cation channels with a voltage-sensor domain conserved in plants and animals. Rice OsTPC1 is predominantly localized to the plasma membrane (PM), and assumed to play an important role as a Ca(2)(+)-permeable cation channel in the regulation of cytosolic Ca(2)(+) rise and innate immune responses including hypersensitive cell death and phytoalexin biosynthesis in cultured rice cells triggered by a fungal elicitor, xylanase from Trichoderma viride. In contrast, Arabidopsis AtTPC1 is localized to the vacuolar membrane (VM). To gain further insights into the intracellular localization of OsTPC1, we stably expressed OsTPC1-GFP in tobacco BY-2 cells. Confocal imaging and membrane fractionation revealed that, unlike in rice cells, the majority of OsTPC1-GFP fusion protein was targeted to the VM in tobacco BY-2 cells. Intracellular localization and functions of the plant TPC family is discussed. OsTPC1 Rice two-pore K+ channels are expressed in different types of vacuoles 2011 Plant Cell Biology Department/Area 9, University of York, York YO105DD, United Kingdom. Potassium (K+) is a major nutrient for plant growth and development. Vacuolar K+ ion channels of the two-pore K+ (TPK) family play an important role in maintaining K+ homeostasis. Several TPK channels were previously shown to be expressed in the lytic vacuole (LV) tonoplast. Plants also contain smaller protein storage vacuoles (PSVs) that contain membrane transporters. However, the mechanisms that define how membrane proteins reach different vacuolar destinations are largely unknown. The Oryza sativa genome encodes two TPK isoforms (TPKa and TPKb) that have very similar sequences and are ubiquitously expressed. The electrophysiological properties of both TPKs were comparable, showing inward rectification and voltage independence. In spite of high levels of similarity in sequence and transport properties, the cellular localization of TPKa and TPKb channels was different, with TPKa localization predominantly at the large LV and TPKb primarily in smaller PSV-type compartments. Trafficking of TPKa was sensitive to brefeldin A, while that of TPKb was not. The use of TPKa:TPKb chimeras showed that C-terminal domains are crucial for the differential targeting of TPKa and TPKb. Site-directed mutagenesis of C-terminal residues that were different between TPKa and TPKb identified three amino acids that are important in determining ultimate vacuolar destination. OsTPKa,OsTPKb Overexpression of the trehalose-6-phosphate phosphatase gene OsTPP1 confers stress tolerance in rice and results in the activation of stress responsive genes 2008 Planta National Key Laboratory of Plant Molecular Genetics, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032, Shanghai, China. Trehalose plays a protective role in yeast and microorganisms under abiotic stresses. However, little is known about its role in higher plants when subjected to environmental challenges. A systematic search of rice databases discovered a large TPS/TPP gene family in the rice genome, which is similar to that found in Arabidopsis thaliana, especially in the gene family structure. Expression analysis demonstrated that OsTPP1 was initially and transiently up-regulated after salt, osmotic and abscisic acid (ABA) treatments but slowly up-regulated under cold stress. OsTPP1 overexpression in rice enhanced tolerance to salt and cold stress. Analysis of the overexpression lines revealed that OsTPP1 triggered abiotic stress response genes, which suggests a possible transcriptional regulation pathway in stress induced reprogramming initiated by OsTPP1. The current study revealed the mechanism of an OsTPP gene involved in stress tolerance in rice and also suggested the use of OsTPP1 in abiotic stress engineering of crops. OsTPP1 Functional identification of a trehalose 6-phosphate phosphatase gene that is involved in transient induction of trehalose biosynthesis during chilling stress in rice 2005 Plant Mol Biol Winter Stress Laboratory, National Agricultural Research Center for Hokkaido Region, National Agricultural and Bio-oriented Research Organization, Hitsujigaoka 1, 062-8555 Toyohira-ku, Sapporo, Japan. Trehalose serves as a stress protectant and/or reserve carbohydrate in a variety of organisms including bacteria, yeast, and invertebrates. Recently, trace amounts of trehalose have been detected in higher plants, although the function of trehalose in plants remains unknown. A cDNA clone (OsTPP1) encoding a putative trehalose-6-phosphate phosphatase (TPP) for trehalose biosynthesis was isolated from rice. Functionality of the clone was demonstrated by complementation of a yeast mutant and enzymatic activity of the recombinant protein. Northern blots revealed that the OsTPP1 transcript levels were fairly low or under detectable limits in most of the tissues under ambient conditions but were highly induced within 1-2 h of chilling stress (12 degrees C) in both root and shoot tissues of seedlings. This induction was transient and disappeared after 6 h of the chilling stress. Transient expression of OsTPP1 was also induced under severe chilling stress (4 degrees C) as well as salinity and drought stresses at ambient temperatures. Application of exogenous ABA (50 microM) resulted in a transient increase of OsTPP1 expression within 20 min of the treatment, thereby suggesting involvement of ABA in OsTPP1 gene regulation. Measurements of total cellular TPP activity and trehalose content in roots indicated that both TPP activity and trehalose levels were transiently increased after chilling (12 degrees C) stress. Collectively, the data indicate that transient activation of trehalose biosynthesis is involved in early chilling stress response in rice. Possible functions of trehalose in the early stages of chilling stress response are discussed. OsTPP1 Biochemical characterization of rice trehalose-6-phosphate phosphatases supports distinctive functions of these plant enzymes 2007 FEBS J Crop Cold Tolerance Research Team, National Agricultural Research Center for Hokkaido Region, National Agriculture and Food Research Organization, Hitsujigaoka 1, Toyohira-ku, Sapporo 062-8555, Japan. Substantial levels of trehalose accumulate in bacteria, fungi, and invertebrates, where it serves as a storage carbohydrate or as a protectant against environmental stresses. In higher plants, trehalose is detected at fairly low levels; therefore, a regulatory or signaling function has been proposed for this molecule. In many organisms, trehalose-6-phosphate phosphatase is the enzyme governing the final step of trehalose biosynthesis. Here we report that OsTPP1 and OsTPP2 are the two major trehalose-6-phosphate phosphatase genes expressed in vegetative tissues of rice. Similar to results obtained from our previous OsTPP1 study, complementation analysis of a yeast trehalose-6-phosphate phosphatase mutant and activity measurement of the recombinant protein demonstrated that OsTPP2 encodes a functional trehalose-6-phosphate phosphatase enzyme. OsTPP2 expression is transiently induced in response to chilling and other abiotic stresses. Enzymatic characterization of recombinant OsTPP1 and OsTPP2 revealed stringent substrate specificity for trehalose 6-phosphate and about 10 times lower K(m) values for trehalose 6-phosphate as compared with trehalose-6-phosphate phosphatase enzymes from microorganisms. OsTPP1 and OsTPP2 also clearly contrasted with microbial enzymes, in that they are generally unstable, almost completely losing activity when subjected to heat treatment at 50 degrees C for 4 min. These characteristics of rice trehalose-6-phosphate phosphatase enzymes are consistent with very low cellular substrate concentration and tightly regulated gene expression. These data also support a plant-specific function of trehalose biosynthesis in response to environmental stresses. OsTPP1,OsTPP2,SALT Characterization of a drought-responsive gene, OsTPS1, identified by the T-DNA gene-trap system in rice 2005 Journal of Plant Biology Department of Life Science, Sogang University, 121-742, Seoul, Korea Trehalose-6-phosphate synthase (TPS) is a key enzyme for trehalose biosynthesis. To understand the function of the TPS gene, we identified the T-DNA insertion in a TPS gene of rice. Line 1C-071-05 had T-DNA inserted at the second intron of OsTPS1. its deduced OsTPS1 protein shared 52% homology with Arabidopsis TPS. Reverse transcriptase-PCR analysis of OsTPS1 showed that OsTPS1 is inducible by drought, salt, cold, and ABA. Leaves of OsTPS1 knockout (KO) plants were more sensitive to drought or cold stress than were the wild types. Furthermore, transgenic rice of UBI::CBF1 had high expression OsTPS1 mRNA, suggesting that OsTPS1 is regulated by the CBF/DREB transcription factor. Therefore, we propose that OsTPS1 plays an important role during the abiotic stress response. OsTPS1 An NADPH-dependent genetic switch regulates plant infection by the rice blast fungus 2010 Proc Natl Acad Sci U S A School of Biosciences, University of Exeter, Exeter EX4 4QD, United Kingdom. To cause rice blast disease, the fungus Magnaporthe oryzae breaches the tough outer cuticle of the rice leaf by using specialized infection structures called appressoria. These cells allow the fungus to invade the host plant and proliferate rapidly within leaf tissue. Here, we show that a unique NADPH-dependent genetic switch regulates plant infection in response to the changing nutritional and redox conditions encountered by the pathogen. The biosynthetic enzyme trehalose-6-phosphate synthase (Tps1) integrates control of glucose-6-phosphate metabolism and nitrogen source utilization by regulating the oxidative pentose phosphate pathway, the generation of NADPH, and the activity of nitrate reductase. We report that Tps1 directly binds to NADPH and, thereby, regulates a set of related transcriptional corepressors, comprising three proteins, Nmr1, Nmr2, and Nmr3, which can each bind NADP. Targeted deletion of any of the Nmr-encoding genes partially suppresses the nonpathogenic phenotype of a Deltatps1 mutant. Tps1-dependent Nmr corepressors control the expression of a set of virulence-associated genes that are derepressed during appressorium-mediated plant infection. When considered together, these results suggest that initiation of rice blast disease by M. oryzae requires a regulatory mechanism involving an NADPH sensor protein, Tps1, a set of NADP-dependent transcriptional corepressors, and the nonconsuming interconversion of NADPH and NADP acting as signal transducer. OsTPS1 Analysis of trehalose-6-phosphate synthase (TPS) gene family suggests the formation of TPS complexes in rice 2011 Plant Mol Biol Peking-Yale Joint Center for Plant Molecular Genetics and Agrobiotechnology, National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing, 100871, People's Republic of China. Trehalose-6-phosphate (T6P), an intermediate in the trehalose biosynthesis pathway, is emerging as an important regulator of plant metabolism and development. T6P levels are potentially modulated by a group of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) homologues. In this study, we have isolated 11 TPS genes encoding proteins with both TPS and TPP domains, from rice. Functional complement assays performed in yeast tps1 and tps2 mutants, revealed that only OsTPS1 encodes an active TPS enzyme and no OsTPS protein possesses TPP activity. By using a yeast two-hybrid analysis, a complicated interaction network occurred among OsTPS proteins, and the TPS domain might be essential for this interaction to occur. The interaction between OsTPS1 and OsTPS8 in vivo was confirmed by bimolecular fluorescence complementation and coimmunoprecipitation assays. Furthermore, our gel filtration assay showed that there may exist two forms of OsTPS1 (OsTPS1a and OsTPS1b) with different elution profiles in rice. OsTPS1b was particularly cofractionated with OsTPS5 and OsTPS8 in the 360 kDa complex, while OsTPS1a was predominantly incorporated into the complexes larger than 360 kDa. Collectively, these results suggest that OsTPS family members may form trehalose-6-phosphate synthase complexes and therefore potentially modify T6P levels to regulate plant development. OsTPS1,OsTPS3 The rice (E)-beta-caryophyllene synthase (OsTPS3) accounts for the major inducible volatile sesquiterpenes 2007 Phytochemistry National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Terpenoids serve as both constitutive and inducible defense chemicals in many plant species, and volatile terpenes participate in plant a indirect defense by attracting natural enemies of the herbivores. The rice (Oryza sativa L.) genome contains about 50 genes encoding putative terpene synthases (TPSs). Here we report that two of the rice sesquiterpene synthase genes, OsTPS3 and OsTPS13, encode (E)-beta-caryophyllene synthase and (E,E)-farnesol synthase, respectively. In vitro, the recombinant protein of OsTPS3 catalyzed formation of (E)-beta-caryophyllene and several other sesquiterpenes, including beta-elemene and alpha-humulene, all being components of inducible volatiles of rice plants. The transcript levels of OsTPS3 exhibit a circadian rhythm of fluctuation, and its expression was also greatly induced by methyl jasmonate (MeJA). In addition, expression of OsTPS3 in transgenic plants of Arabidopsis thaliana resulted in emitting high quantities of OsTPS3 products. We also overexpressed OsTPS3 in rice plants which then produced more (E)-beta-caryophyllene after MeJA treatment. Finally, we found that the MeJA-treated transgenic rice plants attracted more parasitoid wasps of Anagrus nilaparvatae than the wild-type. These results demonstrate that OsTPS3, an enzyme catalyzing the formation of volatile sesquiterpenes, plays a role in indirect defense of rice plants. OsTPS3 An apoplastic h-type thioredoxin is involved in the stress response through regulation of the apoplastic reactive oxygen species in rice 2011 Plant Physiol Institute of Molecular and Cell Biology, Hebei Normal University, Shijiazhuang 050016, China. Thioredoxins (Trxs) are a multigenic family of proteins in plants that play a critical role in redox balance regulation through thiol-disulfide exchange reactions. There are 10 members of the h-type Trxs in rice (Oryza sativa), and none of them has been clearly characterized. Here, we demonstrate that OsTRXh1, a subgroup I h-type Trx in rice, possesses reduction activity in vitro and complements the hydrogen peroxide sensitivity of Trx-deficient yeast mutants. OsTRXh1 is ubiquitously expressed in rice, and its expression is induced by salt and abscisic acid treatments. Intriguingly, OsTRXh1 is secreted into the extracellular space, and salt stress in the apoplast of rice induces its expression at the protein level. The knockdown of OsTRXh1 results in dwarf plants with fewer tillers, whereas the overexpression of OsTRXh1 leads to a salt-sensitive phenotype in rice. In addition, both the knockdown and overexpression of OsTRXh1 decrease abscisic acid sensitivity during seed germination and seedling growth. We also analyzed the levels of hydrogen peroxide produced in transgenic plants, and the results show that more hydrogen peroxide is produced in the extracellular space of OsTRXh1 knockdown plants than in wild-type plants, whereas the OsTRXh1 overexpression plants produce less hydrogen peroxide under salt stress. These results show that OsTRXh1 regulates the redox state of the apoplast and influences plant development and stress responses. OsTrx23|OsTRXh1 OsTRXh1 regulates the redox state of the apoplast and influences stress responses in rice 2012 Plant Signal Behav Institute of Molecular and Cell Biology, Hebei Normal University, Shijiazhuang, China. The plant cell apoplast is the compartment beyond the cell plasmalemma, including the cell wall and intercellular space. Many environmental elements can trigger reactive oxygen species (ROS) burst at the plasma membrane which then alters the redox state of the apoplast. Recently, h-type thioredoxin (Trx), OsTRXh1, was identified to be involved in apoplastic redox state regulation in rice. OsTRXh1 is conserved redox-active Trx and can be secreted into the extracellular regions. Through transgenic rice plant, we found that OsTRXh1 regulated ROS accumulation in apoplast and influenced plant development and stress responses. This provides new insights into apoplastic redox state regulation pathway and expands our understanding of h-type Trxs function. OsTrx23|OsTRXh1 Abnormal chloroplast development and growth inhibition in rice thioredoxin m knock-down plants 2008 Plant Physiol Division of Applied Life Science (BK21 Program), EB-NCRC and Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Korea. Plant cells contain several thioredoxin isoforms that are characterized by subcellular localization and substrate specificity. Here, we describe the functional characterization of a rice (Oryza sativa) thioredoxin m isoform (Ostrxm) using a reverse genetics technique. Ostrxm showed green tissue-specific and light-responsive mRNA expression. Ostrxm was localized in chloroplasts of rice mesophyll cells, and the recombinant protein showed dithiothreitol-dependent insulin beta-chain reduction activity in vitro. RNA interference (RNAi) of Ostrxm resulted in rice plants with developmental defects, including semidwarfism, pale-green leaves, abnormal chloroplast structure, and reduced carotenoid and chlorophyll content. Ostrxm RNAi plants showed remarkably decreased F(v)/F(m) values under high irradiance conditions (1,000 micromol m(-2) s(-1)) with delayed recovery. Two-dimensional electrophoresis and matrix-assisted laser-desorption/ionization time-of-flight analysis showed that the levels of several chloroplast proteins critical for photosynthesis and biogenesis were significantly decreased in Ostrxm RNAi plants. Furthermore, 2-Cys peroxiredoxin, a known target of thioredoxin, was present in oxidized forms, and hydrogen peroxide levels were increased in Ostrxm RNAi plants. The pleiotropic effects of Ostrxm RNAi suggest that Ostrxm plays an important role in the redox regulation of chloroplast target proteins involved in diverse physiological functions. Ostrxm A long leader intron of the Ostub16 rice beta-tubulin gene is required for high-level gene expression and can autonomously promote transcription both in vivo and in vitro 2002 The Plant Journal Istituto Biosintesi Vegetali, CNR, Via Bassini 15, 20133 Milano A 2 kb DNA fragment, upstream of the rice beta-tubulin isotype 16 (Ostub16) coding sequence, was isolated using inverse PCR and screening of a tubulin-enriched lambda library. An intron (863 bp) present in the 5' untranslated region (5' UTR) is spliced out to produce the most abundant mRNA species which corresponds to the previously cloned Ostub16 cDNA. Transient expression assays performed on rice embryogenic calluses with chimeric Ostub16::GUS constructs demonstrated that the entire 2 kb upstream sequence has a strong promoter activity, and that the 863 bp intron is required for high-level GUS expression. In addition, the intron sequence is capable per se of sustaining a weak but consistent GUS expression. Two rare Ostub16 transcripts, with a start site mapping within this intron sequence, were detected in rice coleoptile cells. The transcription start site mapped at position -290 with respect to the ATG codon, and the shorter molecule originated from splicing of the same precursor mRNA. Therefore transcriptional expression of rice beta-tubulin isotype 16 results in the synthesis of two premRNA molecules (I and II) encoding for three different mRNA species. We discuss these findings in terms of function and molecular evolution of the mechanisms that control plant beta-tubulin gene expression. Ostub16 Functional analysis of OsTUB8, an anther-specific beta-tubulin in rice 2007 Plant Science National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan Microtubules play important roles in many cellular processes, such as cell division and cell elongation in plants. beta-tubulins (TUB), which are one of the basic components of microtubules, are encoded by multigene family in eukaryotes and their nucleotide sequences are highly conserved in protein coding regions. OsTUB8 that was expressed in rice anthers was characterized with a multi-level approach. At the protein level, OsTUB8 was expressed mainly in anthers compared to callus, root, leaf sheath and leaf blade. In situ hybridization and GUS fusion analysis revealed that OsTUB8 was expressed in vascular bundles of anther filaments and in pollen. OsTUB8 expression was lower in the anthers of GA-deficient mutants, 'Tanginbozu' and 'Akibrarewaisei', compared to those of their respective wild types. Transgenic rice expressing OsTUB8 in an antisense orientation were suppressed in the amount of seed set upon maturity. Antisense-transgenic rice plants were 20-60% shorter compared to the vector-only control. These results suggest that OsTUB8 might be differentially expressed in rice anthers due to the action of GA, and involved in the processes of vegetative growth and seed set in rice. OsTUB8 Tissue-preferential expression of a rice alpha-tubulin gene, OsTubA1, mediated by the first intron 2000 Plant Physiol Department of Life Science and National Research Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea. The genomic clone encoding an alpha-tubulin, OsTubA1, has been isolated from rice (Oryza sativa L.). The gene consists of four exons and three introns. RNA-blot analysis showed that the gene is strongly expressed in actively dividing tissues, including root tips, young leaves, and young flowers. Analysis of chimeric fusions between OsTubA1 and beta-glucuronidase (GUS) revealed that the intron 1 was required for high-level GUS expression in actively dividing tissues, corresponding with normal expression pattern of OsTubA1. Fusion constructs lacking the intron 1 showed more GUS staining in mature tissues rather than young tissues. When the intron 1 was placed at the distal region from 5'-upstream region or at the 3'-untranslated region, no enhancement of GUS expression was observed. Sequential deletions of the OsTubA1 intron 1 brought about a gradual reduction of GUS activity in calli. These results suggest that tissue-preferential expression of the OsTubA1 gene is mediated by the intron 1 and that it may be involved in a mechanism for an efficient RNA splicing that is position dependent. OsTubA1 The cytoplasmic-localized, cytoskeletal-associated RNA binding protein OsTudor-SN: evidence for an essential role in storage protein RNA transport and localization 2008 Plant J Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA. Previous studies have demonstrated that the major storage protein RNAs found in the rice endosperm are transported as particles via actomyosin to specific subdomains of the cortical endoplasmic reticulum. In this study, we examined the potential role of OsTudor-SN, a major cytoskeletal-associated RNA binding protein, in RNA transport and localization. OsTudor-SN molecules occur as high-molecular-weight forms, the integrity of which are sensitive to RNase. Immunoprecipitation followed by RT-PCR showed that OsTudor-SN binds prolamine and glutelin RNAs. Immunofluorescence studies using affinity-purified antibodies show that OsTudor-SNs exists as particles in the cytoplasm, and are distributed to both the protein body endoplasmic reticulum (ER) and cisternal ER. Examination of OsTudor-SN particles in transgenic rice plants expressing GFP-tagged prolamine RNA transport particles showed co-localization of OsTudor-SN and GFP, suggesting a role in RNA transport. Consistent with this view, GFP-tagged OsTudor-SN is observed in living endosperm sections as moving particles, a property inhibited by microfilament inhibitors. Downregulation of OsTudor-SN by antisense and RNAi resulted in a decrease in steady state prolamine RNA and protein levels, and a reduction in the number of prolamine protein bodies. Collectively, these results show that OsTudor-SN is a component of the RNA transport particle, and may control storage protein biosynthesis by regulating one or more processes leading to the transport, localization and anchoring of their RNAs to the cortical ER. OsTudor-SN Molecular cloning and expression analysis of rice OsTVLP1, encoding a protein with similarity to TGF-beta receptor interacting proteins and vacuolar assembly Vam6p/Vps39p proteins 2006 DNA Sequence Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang, 310029, People's Republic of China We describe the cloning and identification of a rice cDNA, OsTVLP1, encoding a protein with similarity to TGF-beta receptor interacting proteins and vacuolar assembly Vam6p/Vps39p proteins. OsTVLP1 has an open reading frame of 2955 bp, which encodes a 984 amino acid protein, containing a citron homology (CNH) domain at its N-terminal and a clathrin heavy-chain repeat homology (CLH) domain at its C-terminal. The expression of OsTVLP1 was induced by treatments with benzothiadiazole (BTH), a chemical activator of plant disease resistance responses, and by infection of the blast fungus, Magnaporthe grisea. Importantly, the expression of OsTVLP1 was activated specifically in disease resistance response induced by BTH and in an incompatible interaction between rice and the blast fungus. Our observations suggest that OsTVLP1 may play a role in rice disease resistance response against pathogen infection. OsTVLP1 Overexpression of a phytochrome-regulated tandem zinc finger protein gene, OsTZF1, confers hypersensitivity to ABA and hyposensitivity to red light and far-red light in rice seedlings 2012 Plant Cell Rep High-Tech Research Center, Shandong Academy of Agricultural Sciences, Jinan 250100, People's Republic of China. Tandem zinc finger proteins (TZFs) in plants are involved in gene regulation, developmental responses, and hormone-mediated environmental responses in Arabidopsis. However, little information about the functions of the TZF family in monocots has been reported. Here, we investigated a cytoplasmic TZF protein, OsTZF1, which is involved in photomorphogenesis and ABA responses in rice seedlings. The OsTZF1 gene was expressed at relatively high levels in leaves and shoots, although its transcripts were detected in various organs. Red light (R)- and far-red light (FR)-mediated repression of OsTZF1 gene expression was attributed to phytochrome B (phyB) and phytochrome C (phyC), respectively. In addition, OsTZF1 expression was regulated by salt, PEG, and ABA. Overexpression of OsTZF1 caused a long leaf sheath relative to wild type (WT) under R and FR, suggesting that OsTZF1 probably acts as a negative regulator of photomorphogenesis in rice seedlings. Moreover, ABA-induced growth inhibition of rice seedlings was marked in the OsTZF1-overexpression lines compared with WT, suggesting the positive regulation of OsTZF1 to ABA responses. Genome-wide expression analysis further revealed that OsTZF1 also functions in other hormone or stress responses. Our findings supply new evidence on the functions of monocot TZF proteins in phytochrome-mediated light and hormone responses. KEY MESSAGE: OsTZF1 encodes a cytoplasm-localized tandem zinc finger protein and is regulated by both ABA and phytochrome-mediated light signaling. OsTZF1 functions in phytochrome-mediated light and ABA responses in rice. OsTZF1 The Minor Spliceosomal Protein U11/U12-31K Is an RNA Chaperone Crucial for U12 Intron Splicing and the Development of Dicot and Monocot Plants 2012 PLoS One Department of Plant Biotechnology and Kumho Life Science Laboratory, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Korea U12 intron-specific spliceosomes contain U11 and U12 small nuclear ribonucleoproteins and mediate the removal of U12 introns from precursor-mRNAs. Among the several proteins unique to the U12-type spliceosomes, an Arabidopsis thaliana AtU11/U12-31K protein has been shown to be indispensible for proper U12 intron splicing and for normal growth and development of Arabidopsis plants. Here, we assessed the functional roles of the rice (Oryza sativa) OsU11/U12-31K protein in U12 intron splicing and development of plants. The U11/U12-31K transcripts were abundantly expressed in the shoot apical meristems (SAMs) of Arabidopsis and rice. Ectopic expression of OsU11/U12-31K in AtU11/U12-31K-defecient Arabidopsis mutant complemented the incorrect U12 intron splicing and abnormal development phenotypes of the Arabidopsis mutant plants. Impaired cell division activity in the SAMs and inflorescence stems observed in the AtU11/U12-31K-deficient mutant was completely recovered to normal by the expression of OsU11/U12-31K. Similar to Arabidopsis AtU11/U12-31K, rice OsU11/U12-31K was determined to harbor RNA chaperone activity. Collectively, the present findings provide evidence for the emerging idea that the U11/U12-31K protein is an indispensible RNA chaperone that functions in U12 intron splicing and is necessary for normal development of monocotyledonous plants as well as dicotyledonous plants. OsU11|U12-31K Rice UBC13, a candidate housekeeping gene, is required for K63-linked polyubiquitination and tolerance to DNA damage 2012 Rice College of Life Sciences, Capital Normal University, Beijing, 100048, China While plant growth and reproduction is dependent on sunlight, UV irradiation from sunlight is one of the major genotoxic stresses that threaten plant survival and genome stability. In addition, many environmental chemicals can also damage the plant genome. In yeast and mammalian cells protection against the above genome instability is provided by an error-free DNA-damage tolerance (DDT) pathway, which is dependent on Ubc13-mediated K63-linked polyubiquitination of the proliferating cell nuclear antigen (PCNA). In this study, we isolated the UBC13 gene from rice and characterized its functions. Expression of OsUBC13 can protect a yeast ubc13 null mutant against spontaneous and environmental DNA damage. Furthermore, OsUbc13 physically interacts with human Ubc13 partners Mms2 and Uev1A, and catalyzes K63 polyubiquitination in vitro. These observations collectively suggest that the K63 polyubiquitination is conserved in rice, and that OsUBC13 may be involved in DDT and other cellular processes. In addition, OsUBC13 is constitutively expressed at a high level even under various stress conditions, suggesting that it is a housekeeping gene. OsUBC13 Active site residues and amino acid specificity of the ubiquitin carrier protein-binding RING-H2 finger domain 2005 J Biol Chem Biochemistry Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. EL5 is a rice ubiquitin-protein isopeptide ligase (E3) containing a RING-H2 finger domain that interacts with Oryza sativa (Os) UBC5b, a rice ubiquitin carrier protein. We introduced point mutations into the EL5 RING-H2 finger so that residues that functionally interact with OsUBC5b could be identified when assayed for ubiquitination activity in vitro. The residue positions were selected based on the results of an EL5 RING-H2 finger/OsUBC5b NMR titration experiment. These RING-H2 finger residues form or are adjacent to a shallow groove that is recognized by OsUBC5b. The E3 activity of EL5 is shown to be dependent on a Trp located at the center of the groove. We classified rice RING fingers according to the type of metal-chelating motif, i.e. RING-H2 or RING-HC, and according to the presence or absence of a conserved EL5-like Trp. We discuss the probable relationship between E3 activity and the conserved Trp. OsUBC5b Structure and expression of OsUBP6, an ubiquitin-specific protease 6 homolog in rice (Oryza sativa L.) 2009 Mol Cells Department of Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea. Although the possible cellular roles of several ubiquitin-specific proteases (UBPs) were identified in Arabidopsis, almost nothing is known about UBP homologs in rice, a monocot model plant. In this report, we searched the rice genome database ( http://signal.salk.edu/cgi-bin/RiceGE ) and identified 21 putative UBP family members (OsUBPs) in the rice genome. These OsUBP genes each contain a ubiquitin carboxyl-terminal hydrolase (UCH) domain with highly conserved Cys and His boxes and were subdivided into 9 groups based on their sequence identities and domain structures. RT-PCR analysis indicated that rice OsUBP genes are expressed at varying degrees in different rice tissues. We isolated a full-length cDNA clone for OsUBP6, which possesses not only a UCH domain, but also an N-terminal ubiquitin motif. Bacterially expressed OsUBP6 was capable of dismantling K48-linked tetraubiquitin chains in vitro. Quantitative real-time RT-PCR indicated that OsUBP6 is constitutively expressed in different tissues of rice plants. An in vivo targeting experiment showed that OsUBP6 is predominantly localized to the nucleus in onion epidermal cells. We also examined how knock-out of OsUBP6 affects developmental growth of rice plants. Although homozygous T3 osubp6 T-DNA insertion mutant seedlings displayed slower growth relative to wild type seedlings, mature mutant plants appeared to be normal. These results raise the possibility that loss of OsUBP6 is functionally compensated for by an as-yet unknown OsUBP homolog during later stages of development in rice plants. OsUBP6 Two uncoupling protein genes of rice (Oryza sativa L.): molecular study reveals the defects in the pre-mRNA processing for the heat-generating proteins of the subtropical cereal 2002 Planta Labvoratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. akiowat@akita-pu.ac.jp The recent finding of uncoupling proteins (UCPs) in plants has demonstrated that plant mitochondria contain at least two distinct types of potential heat-generating protein, i.e. uncoupling proteins (UCPs) and alternative oxidases. In this study, we isolated and characterized two rice genes encoding UCPs with the aim of improving cold tolerance of rice through the modification of UCP expression. The two rice nuclear genes, OsUCP1 and OsUCP2, appeared to encode functional UCPs, as far as the amino acid sequences of the gene products showed. However, our study revealed that the processing of the pre-mRNAs of both genes was defective. The defects in the pre-mRNA processing resulted in multiple abnormal forms of the transcripts, whose translation products seemed not to retain normal UCP activities. No OsUCP cDNA clones corresponding to the normal transcripts were detected in our study. A northern analysis, on the other hand, revealed that neither of the OsUCP genes exhibited cold-enhanced expression, whereas the gene expression for an alternative oxidase was strikingly enhanced in cold-treated leaves of rice. Our present data thus indicate that rice leaves, placed in cold environments, do not express functional UCPs. OsUCP2 Functional analysis of a rice late pollen-abundant UDP-glucose pyrophosphorylase (OsUgp2) promoter 2011 Mol Biol Rep Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Province, College of Life Science, South China Agricultural University, 510642 Guangzhou, China. OsUgp2, a rice UDP-glucose pyrophosphorylase gene, has previously been shown to preferentially express in maturing pollens and plays an important role in pollen starch accumulation. Here, a 1943 bp promoter fragment (P1943) of OsUgp2 was characterized by 5' deletion and gain-of-function experiments. P1943 and its 5' deletion derivatives (P1495, P1005, P665 and P159) were fused to GUS reporter gene and stably introduced into rice plants. Histochemical analyses of different tissues and pollens at different developmental stages of the transgenic plants showed that P1943 could only direct GUS expression in binucleate pollens. P1495 and P1005 could still drive GUS expression in binucleate pollens but at a lower level. On the other hand, neither P665 nor P159 transformant exhibited any GUS activity in pollens. Gain-of-function analyses showed that the region (-1005 to -665 relative to translation start site) combined with a minimal CaMV 35S promoter could direct GUS expression in pollens. Further analysis of 5' deletion truncated at -952, -847 and -740 delimited a 53 bp region (-1005 to -952) essential for pollen-specific expression. The 53 bp sequence contains two motifs of TTTCT and TTTC, which were known to be pollen-specific cis-elements. In addition, the same P1943-GUS fusion construct was introduced into tobacco to analyze its specificity in dicotyledon. Interestingly, the GUS expression pattern in transgenic tobacco was quite different from that in rice. High level of GUS expression was detected in mature pollens as well as leaves, roots, sepals and stigmas. These findings suggested a complicated transcriptional regulation of OsUgp2. OsUgp2|Ugp2 UDP-glucose pyrophosphorylase2 (OsUgp2), a pollen-preferential gene in rice, plays a critical role in starch accumulation during pollen maturation 2009 Chinese Science Bulletin Department of Botany, the University of Hong Kong, Hong Kong, China UDP-glucose pyrophosphorylase (UGPase) is predominantly present and plays significant role in carbohydrate metabolism in plants. Two homologous UGPase genes, OsUgp1 and OsUgp2, exist in rice genome. OsUgp1 has recently been reported to be essential for callose deposition during pollen mother cell and meiosis stages as well as for seed carbohydrate metabolism. In this study, a full-length cDNA of OsUgp2 was isolated from rice anther. Northern blot and RNA in situ hybridization indicated that the expression of OsUgp2 was preferentially in pollen and developmentally regulated. No transcripts were found in leaf, stem, lemma/palea, ripening grain and florets before the uninucleate microspore developmental stage, but a large quantity of OsUgp2 mRNA was found in pollen at the binucleate and mature stages. The immunolocalization of OsUgp2 showed a similar expression pattern to that by RNA in situ hybridization. The function of OsUgp2 was investigated by dsRNA-mediated transcriptional gene silencing. The pollen fertility of 16 independent transgenic rice plants was found between 25% and 90%, which was correlated with the amount of OsUgp2 mRNA. The results of morphological changes and starch variation during pollen development in transgenic rice showed that the abnormal feature of pollen development appeared after the uninucleate microspore stage. Starch failed to accumulate in pollen and thus led to sterile pollens. These results demonstrated that OsUgp2 is a pollen-preferential “late gene" and plays a key role during pollen maturation, especially for starch accumulation. OsUgp2 complements OsUgp1 to fulfill the UGPase’s functions necessary for the full process of pollen development. OsUgp2|Ugp2,OsUgp1|Ugp1|UGPase1 Rice UDP-Glucose Pyrophosphorylase1 Is Essential for Pollen Callose Deposition and Its Cosuppression Results in a New Type of Thermosensitive Genic Male Sterility 2007 The Plant Cell Online Key Laboratory of Ministry of Education for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China UDP-glucose pyrophosphorylase (UGPase) catalyzes the reversible production of glucose-1-phosphate and UTP to UDP-glucose and pyrophosphate. The rice (Oryza sativa) genome contains two homologous UGPase genes, Ugp1 and Ugp2. We report a functional characterization of rice Ugp1, which is expressed throughout the plant, with highest expression in florets, especially in pollen during anther development. Ugp1 silencing by RNA interference or cosuppression results in male sterility. Expressing a double-stranded RNA interference construct in Ugp1-RI plants resulted in complete suppression of both Ugp1 and Ugp2, together with various pleiotropic developmental abnormalities, suggesting that UGPase plays critical roles in plant growth and development. More importantly, Ugp1-cosuppressing plants contained unprocessed intron-containing primary transcripts derived from transcription of the overexpression construct. These aberrant transcripts undergo temperature-sensitive splicing in florets, leading to a novel thermosensitive genic male sterility. Pollen mother cells (PMCs) of Ugp1-silenced plants appeared normal before meiosis, but during meiosis, normal callose deposition was disrupted. Consequently, the PMCs began to degenerate at the early meiosis stage, eventually resulting in complete pollen collapse. In addition, the degeneration of the tapetum and middle layer was inhibited. These results demonstrate that rice Ugp1 is required for callose deposition during PMC meiosis and bridges the apoplastic unloading pathway and pollen development. OsUgp2|Ugp2,OsUgp1|Ugp1|UGPase1 Characterization of rice nucleotide sugar transporters capable of transporting UDP-galactose and UDP-glucose 2010 J Biochem Glyco-chain Functions Laboratory, RIKEN-FRS, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Using the basic local alignment search tool (BLAST) algorithm to search the Oryza sativa (Japanese rice) nucleotide sequence databases with the Arabidopsis thaliana UDP-galactose transporter sequences as queries, we found a number of sequences encoding putative O. sativa UDP-galactose transporters. From these, we cloned four putative UDP-galactose transporters, designated OsUGT1, 2, 3 and 4, which exhibited high sequence similarity with Arabidopsis thaliana UDP-galactose transporters. OsUGT1, 2, 3 and 4 consisted of 350, 337, 345 and 358 amino acids, respectively, and all of these proteins were predicted to have multiple transmembrane domains. To examine the UDP-galactose transporter activity of the OsUGTs, we introduced the OsUGTs' expression vectors into UDP-galactose transporter activity-deficient Lec8 cells. Our results showed that transfection with OsUGT1, 2 and 3 resulted in recovery of the deficit phenotype of Lec8 cells, but transfection with OsUGT4 did not. The results of an in vitro nucleotide sugar transport assay of OsUGTs, carried out with a yeast expression system, suggested that OsUGT4 is a UDP-glucose transporter rather than a UDP-galactose transporter. Although plants have multiple UDP-galactose transporter genes, phylogenic analysis indicates that plant UDP-galactose transporter genes are not necessarily evolutionary related to each other. OsUGT1,OsUGT2,OsUGT3,OsUGT4 Molecular cloning and characterization of OsUPS, a U-box containing E3 ligase gene that respond to phosphate starvation in rice (Oryza sativa) 2012 Mol Biol Rep College of Life Science and Natural Resources, Dong-A University, Busan, 604-714, Korea. The ubiquitin-26S proteasome system is important in the quality control of intracellular proteins. The ubiquitin-26S proteasome system includes the E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligase) enzymes. U-box proteins are a derived version of RING-finger domains, which have E3 enzyme activity. Here, we present the isolation of a novel U-box protein, U-box containing E3 ligase induced by phosphate starvation (OsUPS), from rice (Oryza sativa). The cDNA encoding the O. sativa U-box protein (OsUPS) comprises 1338 bp, with an open reading frame of 445 amino acids. The amino acid sequence of OsUPS cDNA shows 41-79% identity with other plant U-box homologous genes. The open reading frame of the OsUPS protein is comprised of notable domains: a single ~70-amino acid domain and a GKL domain that contains conserved glycine, lysine/arginine residues and leucine-rich feature. We found that full-length expression of OsUPS was up-regulated in both rice plants and cell culture in the absence of inorganic phosphate (P(i)). A self-ubiquitination assay indicated that the bacterially expressed OsUPS protein had E3 ligase activity, and subcellular localization results showed that OsUPS was located in the chloroplast. These results support the notion that OsUPS plays an important role in the P(i) signaling pathway through the ubiquitin-26S proteasome system. OsUPS The novel ethylene-regulated gene OsUsp1 from rice encodes a member of a plant protein family related to prokaryotic universal stress proteins 2002 J Exp Bot Institut fur Allgemeine Botanik, Universitat Hamburg, Ohnhorststrasse 18, D-22609 Hamburg, Germany. msauter@botanik.uni-hamburg.de Using subtractive hybridization a submergence-induced gene was identified from deepwater rice, OsUsp1, that encodes a homologue of the bacterial universal stress protein family. Sequence analysis revealed that OsUSP1 is most closely related to the bacterial MJ0577-type of ATP-binding USP proteins which have been suggested to act as a molecular switch. USP protein homologues appear to be ubiquitous in plants and are encoded by gene families, but are absent in animal species. In the youngest internode of deepwater rice plants, OsUsp1 expression was very strongly induced within 1 h of submergence. Elevated transcript levels were observed in dividing cells, in expanding cells and in differentiated tissue indicating that USP1 mediates a general process. Gene induction was shown to be regulated by ethylene with a highly similar expression pattern to that observed with submergence treatment. Based on sequence information and on expression data it is hypothesized that OsUSP1 plays a role in ethylene-mediated stress adaptation in rice. OsUsp1 The rice OsV4 encoding a novel pentatricopeptide repeat protein is required for chloroplast development during the early leaf stage under cold stress 2014 J Integr Plant Biol Development Center of Plant Germplasm Resources, College of Life and Environmental Sciences, Shanghai Normal University, Shanghai, 200234, China. Pentatricopeptide repeat (PPR) proteins, characterized by tandem arrays of a 35 amino acid motif, have been suggested to play central and broad roles in modulating the expression of organelle genes in plants. However, the molecular mechanisms of most rice PPR genes remains unclear. In this paper, we isolated and characterized a temperature-conditional virescent mutant, OsV4, in rice (Oryza sativa cultivar Jiahua1 (WT, japonica rice variety)). The mutant displays albino phenotype and abnormal chloroplasts at the three leaf stage, which gradually turns green after the four leaf stage at a low temperature (20 degrees C). But the mutant always develops green leaves and well-developed chloroplasts at a high temperature (32 degrees C). Genetic and molecular analyses uncovered that OsV4 encodes a novel chloroplast-targeted PPR protein including four PPR motifs. Further investigations show that the mutant phenotype is associated with changes in chlorophyll content and chloroplast development. The OsV4 transcripts only accumulate to high levels in young leaves, indicating that its expression is tissue-specific. In addition, transcript levels of some ribosomal components and plastid-encoded polymerase-dependent genes are dramatically reduced in the albino mutants grown at 20 degrees C. These findings suggest that OsV4 plays an important role during early chloroplast development under cold stress in rice. OsV4 Vacuolar membrane transporters OsVIT1 and OsVIT2 modulate iron translocation between flag leaves and seeds in rice 2012 Plant J National Key Laboratory of Plant Molecular Genetics and National Center for Plant Gene Research (Shanghai), Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. The plant vacuole is an important organelle for storing excess iron (Fe), though its contribution to increasing the Fe content in staple foods remains largely unexplored. In this study we report the isolation and functional characterization of two rice genes OsVIT1 and OsVIT2, orthologs of the Arabidopsis VIT1. Transient expression of OsVIT1:EGFP and OsVIT2:EGFP protein fusions revealed that OsVIT1 and OsVIT2 are localized to the vacuolar membrane. Ectopic expression of OsVIT1 and OsVIT2 partially rescued the Fe(2+) - and Zn(2+) -sensitive phenotypes in yeast mutant Deltaccc1 and Deltazrc1, and further increased vacuolar Fe(2+) , Zn(2+) and Mn(2+) accumulation. These data together suggest that OsVIT1 and OsVIT2 function to transport Fe(2+) , Zn(2+) and Mn(2+) across the tonoplast into vacuoles in yeast. In rice, OsVIT1 and OsVIT2 are highly expressed in flag leaf blade and sheath, respectively, and in contrast to OsVIT1, OsVIT2 is highly responsive to Fe treatments. Interestingly, functional disruption of OsVIT1 and OsVIT2 leads to increased Fe/Zn accumulation in rice seeds and a corresponding decrease in the source organ flag leaves, indicating an enhanced Fe/Zn translocation between source and sink organs, which might represent a novel strategy to biofortify Fe/Zn in staple foods. OsVIT1,OsVIT2 Xanthomonas oryzae pv. oryzae type III effector XopN targets OsVOZ2 and a putative thiamine synthase as a virulence factor in rice 2013 PLoS One Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea. Xanthomonasoryzae pv. oryzae (Xoo) is spread systemically through the xylem tissue and causes bacterial blight in rice. We evaluated the roles of Xanthomonas outer proteins (Xop) in the Xoo strain KXO85 in a Japonica-type rice cultivar, Dongjin. Five xop gene knockout mutants (xopQ KXO85 , xopX KXO85 , xopP1 KXO85 , xopP2 KXO85 , and xopN KXO85 ) were generated by EZ-Tn5 mutagenesis, and their virulence was assessed in 3-month-old rice leaves. Among these mutants, the xopN KXO85 mutant appeared to be less virulent than the wild-type KXO85; however, the difference was not statistically significant. In contrast, the xopN KXO85 mutant exhibited significantly less virulence in flag leaves after flowering than the wild-type KXO85. These observations indicate that the roles of Xop in Xoo virulence are dependent on leaf stage. We chose the xopN gene for further characterization because the xopN KXO85 mutant showed the greatest influence on virulence. We confirmed that XopNKXO85 is translocated into rice cells, and its gene expression is positively regulated by HrpX. Two rice proteins, OsVOZ2 and a putative thiamine synthase (OsXNP), were identified as targets of XopNKXO85 by yeast two-hybrid screening. Interactions between XopNKXO85 and OsVOZ2 and OsXNP were further confirmed in planta by bimolecular fluorescence complementation and in vivo pull-down assays. To investigate the roles of OsVOZ2 in interactions between rice and Xoo, we evaluated the virulence of the wild-type KXO85 and xopN KXO85 mutant in the OsVOZ2 mutant line PFG_3A-07565 of Dongjin. The wild-type KXO85 and xopN KXO85 mutant were significantly less virulent in the mutant rice line. These results indicate that XopNKXO85 and OsVOZ2 play important roles both individually and together for Xoo virulence in rice. OsVOZ2 Sequence and functional analyses of the rice gene homologous to the maize Vp1 1994 Plant Mol Biol Center for Molecular Biology and Genetics, Mie University, Tsu, Japan. The homologous gene of the maize Vp1 gene was isolated from rice (Oryza sativa). Sequence analysis revealed that the rice Vp1 gene (Osvp1) encodes a protein of 728 amino acids and is interupted by 5 introns at positions identical to those of the maize gene. Osvp1 transcript was detected in developing embryo as early as 10 days after flowering and decreased toward maturity. Osvp1 transcript was also detected in dry as well as imbided mature embryos. The ability of Osvp1 gene product to activate a target gene was shown by transient expression experiments in rice suspension-cultured cell protoplasts using a reporter gene construct carrying the bacterial beta-glucuronidase (GUS) gene fused to the promoter of OsEm gene, the rice homologue of the wheat Em gene. OSVP1|VP1 Short, direct repeats (SDRs)-mediated post-transcriptional processing of a transcription factor gene OsVP1 in rice (Oryza sativa) 2007 J Exp Bot Ministry of Education Key Laboratory for Bio-resource and Eco-environment, College of Life Science, Sichuan University, Chengdu 610064, China. The various degrees of preharvest sprouting occurring in hybrid rice is a limiting factor in the propagation and production of hybrid rice seeds. The phenotype of sprouted rice is very similar to that of the maize (Zea mays) seed-specific mutation viviparous 1. VP1 has been shown to be a transcription factor essential for seed maturation and dormancy induction. In this study, numerous truncated transcripts of OsVP1 resulting from an unusual post-transcriptional processing, were detected in four rice (Oryza sativa) cultivars. The observed events took place at a region spanning exons 1 to 5, and led to a variety of deletions that resulted in the loss of functional domain and frame-shifts with premature termination by introducing a stop codon. Various proportions of the transcripts expressed in both immature and mature embryos were observed to be incorrectly processed and associated with the genetic variation of preharvest sprouting rates among various rice varieties. In sprouting-susceptible rice cultivars, G46B and HeiB, many more incorrectly processed OsVP1 transcripts were expressed in immature than in mature embryos, indicating that the unusual post-transcriptional processing of the OsVP1 gene was developmentally regulated. In addition, comprehensive sequence analyses demonstrated the presence of paired short direct repeats (SDRs) at the junctions of the unusual excision sites in exons of OsVP1 gene. Site selection for the deletion of exon materials was altered along with the genotypes and developmental stages. OSVP1|VP1 The Seed-Specific Transcription Factor VP1 (OSVP1) Is Expressed in Rice Suspension-Cultured Cells 1996 Plant Cell Physiol Center for Molecular Biology and Genetics, Mie University 1515 Kamihama-cho, Tsu, 514 Japan A seed-specific transcriptional regulator, VP1, is required for the induction of ABA-regulated genes that include Lea (late embryogenesis abundant protein) genes. Although the induction of one rice Lea gene, Osem, by ABA is normally restricted to seed tissues, we found that the expression was strongly induced by ABA in the Oc line of rice suspension-cultured cells. Since this observation suggested that rice VP1 (OSVP1) protein or a functionally similar protein might be expressed in the cultured cells, we analyzed the expression of Osvpl in these cells at both the mRNA and the protein level, we detected Osvp1 mRNA and OSVP1 protein in the cultured cells at levels similar to or higher than those in developing embryos. In the cultured cells, neither the level of total cellular OSVP1 nor that of nuclear OSVP1 protein was affected by ABA. Based on the results, the mechanism for the transcriptional regulation of VP1-dependent ABA-inducible genes is discussed. OSVP1|VP1 A bZIP factor, TRAB1, interacts with VP1 and mediates abscisic acid-induced transcription 1999 Proc Natl Acad Sci U S A Center for Molecular Biology, Mie University, 1515 Kamihama-cho, Tsu 514-8507, Japan. The transcription factor VP1 regulates maturation and dormancy in plant seeds by activating genes responsive to the stress hormone abscisic acid (ABA). Although activation involves ABA-responsive elements (ABREs), VP1 itself does not specifically bind ABREs. Instead, we have identified and cloned a basic region leucine zipper (bZIP) factor, TRAB1, that interacts with both VP1 and ABREs. Transcription from a chimeric promoter with GAL4-binding sites was ABA-inducible if cells expressed a GAL4 DNA-binding domain::TRAB1 fusion protein. Results indicate that TRAB1 is a true trans-acting factor involved in ABA-regulated transcription and reveal a molecular mechanism for the VP1-dependent, ABA-inducible transcription that controls maturation and dormancy in plant embryos. OSVP1|VP1,TRAB1|OsbZIP66 Molecular cloning of Sdr4, a regulator involved in seed dormancy and domestication of rice 2010 Proc Natl Acad Sci U S A National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. Seed dormancy provides a strategy for flowering plants to survive adverse natural conditions. It is also an important agronomic trait affecting grain yield, quality, and processing performance. We cloned a rice quantitative trait locus, Sdr4, which contributes substantially to differences in seed dormancy between japonica (Nipponbare) and indica (Kasalath) cultivars. Sdr4 expression is positively regulated by OsVP1, a global regulator of seed maturation, and in turn positively regulates potential regulators of seed dormancy and represses the expression of postgerminative genes, suggesting that Sdr4 acts as an intermediate regulator of dormancy in the seed maturation program. Japonica cultivars have only the Nipponbare allele (Sdr4-n), which endows reduced dormancy, whereas both the Kasalath allele (Srd4-k) and Sdr4-n are widely distributed in the indica group, indicating prevalent introgression. Srd4-k also is found in the wild ancestor Oryza rufipogon, whereas Sdr4-n appears to have been produced through at least two mutation events from the closest O. rufipogon allele among the accessions examined. These results are discussed with respect to possible selection of the allele during the domestication process. OSVP1|VP1,Sdr4 Knockout of the VPS22 component of the ESCRT-II complex in rice (Oryza sativa L.) causes chalky endosperm and early seedling lethality 2013 Mol Biol Rep College of Agriculture, South China Agricultural University, Guangzhou 510642, China. In both yeast and mammals, the major constituent of the endosomal sorting complex required for transport-II (ESCRT-II) is the VPS22/EAP30 protein, which plays an important role in ubiquitin-mediated degradation of membrane proteins through the multivesicular body pathway. However, the functions of ESCRT-II subunits in plants are largely unknown. In this work, we report the genetic analysis and phenotypic characterization of mutants in OsVPS22 gene, which encodes a functional VPS22 homolog in rice. On the basis of a collection of T-DNA lines, we identified a T-DNA insertion mutant, which showed abnormal segregation ratios; we then found that the T-DNA insertion is located within the sixth intron of the OsVPS22 gene. Compared with the wild type, this vps22 mutant exhibited seedling lethality and severe reduction in shoot and root growth. In addition, the vps22 mutant had a chalky endosperm in the grain. In summary, our data suggest that OsVPS22 may be required for seedling viability and grain filling in rice, thus providing a valuable resource for further exploration of the functions of the ESCRTing machinery in plants. OsVPS22 The role of tocopherol cyclase in salt stress tolerance of rice (Oryza sativa) 2011 Sci China Life Sci State Key Laboratory of Plant Genomics and National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Tocopherols synthesized exclusively by photosynthetic organisms are major antioxidants in biomembranes. In plants, tocopherol cyclase (TC/VTE1) catalyzes the conversion of 2,3-dimethyl-5-phytyl-1,4-benzoquinone (DMPBQ) to gamma-tocopherol. In the present study, OsVTE1, which encodes a rice tocopherol cyclase ortholog, was cloned and characterized. OsVTE1 was induced significantly by abiotic stresses such as high salt, H(2)O(2), drought, cold and by the plant hormones ABA and salicylic acid. The tissue-specific expression pattern and OsVTE1-promoter GUS activity assay showed that OsVTE1 was mainly expressed in the leaf, and also could be detected in the root, stem and panicle. Compared with control plants, transgenic plants with Os-VTE1 RNA interference (OsVTE1-RNAi) were more sensitive to salt stress whereas, in contrast, transgenic plants overexpressing OsVTE1 (OsVTE1-OX) showed higher tolerance to salt stress. The DAB in vivo staining showed that OsVTE1-OX plants accumulated less H(2)O(2) than did control plants. OsVTE1 A novel wall-associated receptor-like protein kinase gene, OsWAK1, plays important roles in rice blast disease resistance 2009 Plant Mol Biol China Agricultural University, Beijing, 100193, China. Wall-associated protein kinases (WAKs) are a new group of receptor-like kinases (RLK) recently identified in Arabidopsis. A cDNA encoding a novel WAK was isolated from rice and was named OsWAK1 (Oryza sativa WAK). The deduced amino acid sequence of OsWAK1 showed 27.6% identity to WAK2 from Arabidopsis. OsWAK1 not only has the ability of autophosphorylation but also can phosphorylate OsRFP1, a putative transcription regulator recently identified in rice. OsRFP1 strongly interacts with the kinase domain of OsWAK1. This demonstrated that OsWAK1 is a functional protein kinase. A fusion protein of OsWAK1 with GFP was found to be localized on the cell surface. Plasmolysis experiments further revealed OsWAK1 is associated with the cell wall. Northern blotting analysis showed that infection of the rice blast fungus, Magnaporthe oryzae significantly induced the OsWAK1 transcripts, and the accumulation of OsWAK1 mRNA occurred earlier and was more abundant in rice leaves infected with an incompatible race than with a compatible race of the blast fungus. OsWAK1 was also induced after treatment by mechanical wounding, SA and MeJA, but not by ABA. These results imply that OsWAK1 is a novel gene involved in plant defense. Furthermore, six transgenic rice lines with constitutive expression of OsWAK1 became resistant to the compatible race. However, OsWAK1 expression was undetectable in leaves, stems and flowers but very weak in roots under normal growth conditions. This provides functional evidence that induction of OsWAK1 as a novel RLK plays important roles in plant disease resistance. OsWAK1 An ethylene response factor OsWR1 responsive to drought stress transcriptionally activates wax synthesis related genes and increases wax production in rice 2012 Plant Mol Biol Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China. Increasing evidence has revealed the major enzymes-involved in Arabidopsis and maize wax/cutin synthesis; however, there is limited information about the genes-associated with wax/cutin synthesis in rice. Here we report the characterization of an ethylene response factor gene in rice. This rice wax synthesis regulatory gene 1 (OsWR1) is a homolog of Arabidopsis wax/cutin synthesis regulatory gene WIN1/SHN1. Transcript analysis showed that OsWR1 is induced by drought, abscisic acid and salt, and is predominantly expressed in leaves. Functional analyses indicated that overexpressing OsWR1 (Ox-WR1) improved while RNA interference OsWR1 rice (RI-WR1) decreased drought tolerance, consistent with water loss and cuticular permeability, suggesting that OsWR1-triggered drought response might be associated with cuticular characteristics. In addition, OsWR1 activated the expression of the genes-related to oxidative stress response and membrane stability. Gas chromatograph-mass spectrometry analysis further showed that OsWR1 modulated the wax synthesis through alteration of long chain fatty acids and alkanes, evidencing the regulation of OsWR1 in wax synthesis. Detection with real-time PCR amplification indicated that Ox-WR1 enhanced while RI-WR1 decreased the expression of wax/cutin synthesis related genes. Furthermore, OsWR1 physically interacted with the DRE and GCC box in the promoters of wax related genes OsLACS2 and OsFAE1'-L, indicating that OsWR1 at least directly modulates the expression of these genes. Thus our results indicate that OsWR1 is a positive regulator of wax synthesis related genes in rice, and this regulation, distinct from its homology regulator of WIN1/SHN1 in cutin synthesis, subsequently contributes to reduced water loss and enhanced drought tolerance. OsWR1 Enhanced heat and drought tolerance in transgenic rice seedlings overexpressing OsWRKY11 under the control of HSP101 promoter 2009 Plant Cell Rep Laboratory of Environmental Biotechnology, Tohoku University, 1-1 Tsutumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555, Japan. An OsWRKY11 gene, which encodes a transcription factor with the WRKY domain, was identified as one of the genes that was induced by both heat shock and drought stresses in seedlings of rice (Oryza sativa L.). To determine if overexpression of OsWRKY11 confers heat and drought tolerance, OsWRKY11 cDNA was fused to the promoter of HSP101 of rice and introduced into a rice cultivar Sasanishiki. Overexpression of OsWRKY11 was induced by heat treatment. After heat pretreatment, the transgenic lines showed significant heat and drought tolerance, as indicated by the slower leaf-wilting and less-impaired survival rate of green parts of plants. They also showed significant desiccation tolerance, as indicated by the slower water loss in detached leaves. Our results indicate that the OsWRKY11 gene plays a role in heat and drought stress response and tolerance, and might be useful for improvement of stress tolerance. OsWRKY11 The WRKY Gene Family in Rice (Oryza sativa) 2007 J Integr Plant Biol Bioinformatics Core, School of Life Sciences, University of Nevada, Las Vegas, Nevada 89154, USA WRKY genes encode transcription factors that are involved in the regulation of various biological processes. These zinc-finger proteins, especially those members mediating stress responses, are uniquely expanded in plants. To facilitate the study of the evolutionary history and functions of this supergene family, we performed an exhaustive search for WRKY genes using HMMER and a Hidden Markov Model that was specifically trained for rice. This work resulted in a comprehensive list of WRKY gene models in Oryza sativa L. ssp. indica and L. ssp. japonica. Mapping of these genes to individual chromosomes facilitated elimination of the redundant, leading to the identification of 98 WRKY genes in japonica and 102 in indica rice. These genes were further categorized according to the number and structure of their zinc-finger domains. Based on a phylogenetic tree of the conserved WRKY domains and the graphic display of WRKY loci on corresponding indica and japonica chromosomes, we identified possible WRKY gene duplications within, and losses between the two closely related rice subspecies. Also reviewed are the roles of WRKY genes in disease resistance and responses to salicylic acid and jasmonic acid, seed development and germination mediated by gibberellins, other developmental processes including senescence, and responses to abiotic stresses and abscisic acid in rice and other plants. The signaling pathways mediating WRKY gene expression are also discussed. OsWRKY11,OsWRKY12,OsWRKY13,OsWRKY14|OsWRKY33,OsWRKY22,OsWRKY23,OsWRKY24,OsWRKY28,OsWRKY30,OsWRKY31,OsWRKY45,OsWRKY47,OsWRKY51,OsWRKY53,OsWRKY55|WRKY55a|WRKY55b,OsWRKY6,OsWRKY62,OsWRKY71,OsWRKY72,OsWRKY76,OsWRKY78,OsWRKY8,OsWRKY80,OsWRKY82,OsWRKY89 Annotations and functional analyses of the rice WRKY gene superfamily reveal positive and negative regulators of abscisic acid signaling in aleurone cells 2005 Plant Physiol Department of Biological Sciences, University of Nevada, Las Vegas, Nevada 89154, USA. The WRKY proteins are a superfamily of regulators that control diverse developmental and physiological processes. This family was believed to be plant specific until the recent identification of WRKY genes in nonphotosynthetic eukaryotes. We have undertaken a comprehensive computational analysis of the rice (Oryza sativa) genomic sequences and predicted the structures of 81 OsWRKY genes, 48 of which are supported by full-length cDNA sequences. Eleven OsWRKY proteins contain two conserved WRKY domains, while the rest have only one. Phylogenetic analyses of the WRKY domain sequences provide support for the hypothesis that gene duplication of single- and two-domain WRKY genes, and loss of the WRKY domain, occurred in the evolutionary history of this gene family in rice. The phylogeny deduced from the WRKY domain peptide sequences is further supported by the position and phase of the intron in the regions encoding the WRKY domains. Analyses for chromosomal distributions reveal that 26% of the predicted OsWRKY genes are located on chromosome 1. Among the dozen genes tested, OsWRKY24, -51, -71, and -72 are induced by abscisic acid (ABA) in aleurone cells. Using a transient expression system, we have demonstrated that OsWRKY24 and -45 repress ABA induction of the HVA22 promoter-beta-glucuronidase construct, while OsWRKY72 and -77 synergistically interact with ABA to activate this reporter construct. This study provides a solid base for functional genomics studies of this important superfamily of regulatory genes in monocotyledonous plants and reveals a novel function for WRKY genes, i.e. mediating plant responses to ABA. OsWRKY11,OsWRKY12,OsWRKY24,OsWRKY45,OsWRKY51,OsWRKY6,OsWRKY71,OsWRKY72,OsWRKY78,OsWRKY8,OsWRKY80 Exploring transcriptional signalling mediated by OsWRKY13, a potential regulator of multiple physiological processes in rice 2009 BMC Plant Biol National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, PR China. qiudeyun@hotmail.com BACKGROUND: Rice transcription regulator OsWRKY13 influences the functioning of more than 500 genes in multiple signalling pathways, with roles in disease resistance, redox homeostasis, abiotic stress responses, and development. RESULTS: To determine the putative transcriptional regulation mechanism of OsWRKY13, the putative cis-acting elements of OsWRKY13-influenced genes were analyzed using the whole genome expression profiling of OsWRKY13-activated plants generated with the Affymetrix GeneChip Rice Genome Array. At least 39 transcription factor genes were influenced by OsWRKY13, and 30 of them were downregulated. The promoters of OsWRKY13-upregulated genes were overrepresented with W-boxes for WRKY protein binding, whereas the promoters of OsWRKY13-downregulated genes were enriched with cis-elements putatively for binding of MYB and AP2/EREBP types of transcription factors. Consistent with the distinctive distribution of these cis-elements in up- and downregulated genes, nine WRKY genes were influenced by OsWRKY13 and the promoters of five of them were bound by OsWRKY13 in vitro; all seven differentially expressed AP2/EREBP genes and six of the seven differentially expressed MYB genes were suppressed by in OsWRKY13-activated plants. A subset of OsWRKY13-influenced WRKY genes were involved in host-pathogen interactions. CONCLUSION: These results suggest that OsWRKY13-mediated signalling pathways are partitioned by different transcription factors. WRKY proteins may play important roles in the monitoring of OsWRKY13-upregulated genes and genes involved in pathogen-induced defence responses, whereas MYB and AP2/EREBP proteins may contribute most to the control of OsWRKY13-downregulated genes. OsWRKY13 Identification of novel pathogen-responsive cis-elements and their binding proteins in the promoter of OsWRKY13, a gene regulating rice disease resistance 2008 Plant Cell Environ National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. The WRKY transcription factor superfamily controls diverse developmental and physiological processes in plants. However, little is known about the factors that directly regulate the function of WRKY genes. In this study, we identified cis-acting elements and their binding proteins of rice OsWRKY13, a gene that plays a pivotal role in disease resistance against bacterial and fungal pathogens. Two novel pathogen-responsive cis-elements, PRE2 and PRE4, were characterized from the promoter region of OsWRKY13. The two cis-elements negatively regulate gene expression without pathogen challenge, and positively regulate gene expression after pathogen-induced protein binding. OsWRKY13 binds to PRE4, which harbours a novel W-like box. Another five proteins (Rad51-like; tubby-like; SWIM zinc finger and nucleotide-binding adaptor shared by APAF-1, certain R proteins and CED-4 (NB-ARC) domain containing proteins; and an unknown protein) also bind to one of the two cis-elements. Different proteins interacting with the same cis-element appear to have different DNA-binding core sequences. These proteins localize in the nucleus and show differential expression upon pathogen challenge. These results suggest that OsWRKY13 expression is regulated by multiple factors to achieve disease resistance. OsWRKY13 Rice gene network inferred from expression profiling of plants overexpressing OsWRKY13, a positive regulator of disease resistance 2008 Mol Plant National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Accumulating information indicates that plant disease resistance signaling pathways frequently interact with other pathways regulating developmental processes or abiotic stress responses. However, the molecular mechanisms of these types of crosstalk remain poorly understood in most cases. Here we report that OsWRKY13, an activator of rice resistance to both bacterial and fungal pathogens, appears to function as a convergent point for crosstalk among the pathogen-induced salicylate-dependent defense pathway and five other physiologic pathways. Genome-wide analysis of the expression profiles of OsWRKY13-overexpressing lines suggests that OsWRKY13 directly or indirectly regulates the expression of more than 500 genes that are potentially involved in different physiologic processes according to the classification of the Gene Ontology database. By comparing the expression patterns of genes functioning in known pathways or cellular processes of pathogen infection and the phenotypes between OsWRKY13-overexpressing and wild-type plants, our data suggest that OsWRKY13 is also a regulator of other physiologic processes during pathogen infection. The OsWRKY13-associated disease resistance pathway synergistically interacts via OsWRKY13 with the glutathione/glutaredoxin system and flavonoid biosynthesis pathway to monitor redox homeostasis and to putatively enhance the biosynthesis of antimicrobial flavonoid phytoalexins, respectively, in OsWRKY13-overexpressing lines. Meanwhile, the OsWRKY13-associated disease resistance pathway appears to interact antagonistically with the SNAC1-mediated abiotic stress defense pathway, jasmonic acid signaling pathway, and terpenoid metabolism pathway via OsWRKY13 to suppress salt and cold defense responses as well as to putatively retard rice growth and development. OsWRKY13 Methanol is an endogenous elicitor molecule for the synthesis of tryptophan and tryptophan-derived secondary metabolites upon senescence of detached rice leaves 2011 Plant J Department of Biotechnology, Interdisciplinary Program of Graduate School for Bioenergy and Biomaterials, Bioenergy Research Center, Chonnam National University, Gwangju 500-757, Republic of Korea. During senescence of detached rice leaves, tryptophan (Trp) and Trp-derived secondary metabolites such as serotonin and 4-coumaroylserotonin accumulated in concert with methanol (MeOH) production. This senescence-induced MeOH induction was closely associated with levels of pectin methylesterase (PME)1 mRNA and PME enzyme activity. Exogenous challenge of detached rice leaves with 1% MeOH accelerated Trp and serotonin biosynthesis with induction of the corresponding genes. No other solvents, including ethanol, resulted in a Trp-inducing effect. This MeOH-induced Trp synthesis was positively regulated by abscisic acid but negatively regulated by cytokinin, suggesting hormonal involvement in the action of MeOH. Endogenous overproduction or suppression of MeOH either by PME1 overexpression or RNA interference (RNAi) gene silencing revealed that PME1 overexpressing lines produced twofold higher Trp levels with elevated Trp biosynthetic gene expression, whereas RNAi lines showed twofold reduction in Trp level in healthy control rice leaves, suggesting that MeOH acts as an endogenous elicitor to enhance Trp biosynthesis. Among many transcription factors induced following MeOH treatment, the WRKY family showed significant induction patterns, of which WRKY14 appeared to play a key regulatory role in MeOH-induced Trp and Trp-derived secondary metabolite biosynthesis. OsWRKY14|OsWRKY33 OsWRKY22, a monocot WRKY gene, plays a role in the resistance response to blast 2012 Mol Plant Pathol Rice Genomics Unit, Parco Tecnologico Padano, via Einstein, 26900 Lodi, Italy. pamela.abbruscato@tecnoparco.org With the aim of identifying novel regulators of host and nonhost resistance to fungi in rice, we carried out a systematic mutant screen of mutagenized lines. Two mutant wrky22 knockout lines revealed clear-cut enhanced susceptibility to both virulent and avirulent Magnaporthe oryzae strains and altered cellular responses to nonhost Magnaporthe grisea and Blumeria graminis fungi. In addition, the analysis of the pathogen responses of 24 overexpressor OsWRKY22 lines revealed enhanced resistance phenotypes on infection with virulent M. oryzae strain, confirming that OsWRKY22 is involved in rice resistance to blast. Bioinformatic analyses determined that the OsWRKY22 gene belongs to a well-defined cluster of monocot-specific WRKYs. The co-regulatory analysis revealed no significant co-regulation of OsWRKY22 with a representative panel of OsWRKYs, supporting its unique role in a series of transcriptional responses. In contrast, inquiring a subset of biotic stress-related Affymetrix data, a large number of resistance and defence-related genes were found to be putatively co-expressed with OsWRKY22. Taken together, all gathered experimental evidence places the monocot-specific OsWRKY22 gene at the convergence point of signal transduction circuits in response to both host and nonhost fungi encountering rice plants. OsWRKY22 Heterologous expression of OsWRKY23 gene enhances pathogen defense and dark-induced leaf senescence in Arabidopsis 2009 Plant Growth Regulation Xishuangbanna Tropical Botanical Garden, The Chinese Academy of Science, 88 Xuefu Road, 650223, Kunming, Yunnan, People’s Republic of China WRKY proteins are a superfamily of plant transcriptional factors with potential regulatory roles pertaining to a variety of biotic and abiotic stress responses. In this study, we investigated the expression profiles of OsWRKY23 under different developmental stages, pathogen infection, continuous-dark and hormone treatments. Under normal growth conditions, OsWRKY23 expressed exclusively in roots and senescing leaves. Under biotic and abiotic stresses treatments, OsWRKY23 was markedly induced by continuous-dark-induced leaf senescence and infection by rice pathogen Pyricularia oryzae Cav as well as salicylic acid (SA). Further analysis of 35S:OsWRK23 Arabidopsis plants showed that over-expression of OsWRKY23 resulted in enhanced expression of the pathogenesis-related (PR) genes and increased resistance to the bacterial pathogen Pseudomanas syringae. Furthermore, over-expression of OsWRKY23 accelerated leaf senescence in darkness. The senescence-associated marker genes SAG12 and SEN1 were altered in darkness in 35S:WRKY23 Arabidopsis plants. In conclusion, these results suggest that OsWRKY23 is a novel modulator of pathogen responses as well as dark-induced leaf senescence. OsWRKY23 A negative regulator encoded by a rice WRKY gene represses both abscisic acid and gibberellins signaling in aleurone cells 2009 Plant Mol Biol School of Life Sciences, University of Nevada, M/S 4004, 4505 Maryland Parkway, Las Vegas, NV 89154, USA. Abscisic acid (ABA) and gibberellins (GAs) control several developmental processes including seed maturation, dormancy, and germination. The antagonism of these two hormones is well-documented. However, recent data from transcription profiling studies indicate that they can function as agonists in regulating the expression of many genes although the underlying mechanism is unclear. Here we report a rice WRKY gene, OsWRKY24, which encodes a protein that functions as a negative regulator of both GA and ABA signaling. Overexpression of OsWRKY24 via particle bombardment-mediated transient expression in aleurone cells represses the expression of two reporter constructs: the beta-glucuronidase gene driven by the GA-inducible Amy32b alpha-amylase promoter (Amy32b-GUS) and the ABA-inducible HVA22 promoter (HVA22-GUS). OsWRKY24 is unlikely a general repressor because it has little effect on the expression of the luciferase reporter gene driven by a constitutive ubiquitin promoter (UBI-Luciferase). As to the GA signaling, OsWRKY24 differs from OsWRKY51 and -71, two negative regulators specifically function in the GA signaling pathway, in several ways. First, OsWRKY24 contains two WRKY domains while OsWRKY51 and -71 have only one; both WRKY domains are essential for the full repressing activity of OsWRKY24. Second, binding of OsWRKY24 to the Amy32b promoter appears to involve sequences in addition to the TGAC cores of the W-boxes. Third, unlike OsWRKY71, OsWRKY24 is stable upon GA treatment. Together, these data demonstrate that OsWRKY24 is a novel type of transcriptional repressor that inhibits both GA and ABA signaling. OsWRKY24,OsWRKY71 OsWRKY28, a PAMP-responsive transrepressor, negatively regulates innate immune responses in rice against rice blast fungus 2013 Plant Mol Biol Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan. WRKY transcription factors form a large family of plant-specific transcription factors and participate in plant defense responses either as positive or negative regulators. In this study, we comprehensively analyzed the role of one of the group IIa WRKY transcription factors in rice, OsWRKY28, in the regulation of basal defense responses to a compatible race of the rice blast fungus Magnaporthe oryzae, strain Ina86-137. The expression analyses of the group IIa WRKY transcription factors in rice revealed that OsWRKY28, together with OsWRKY71, exhibit an early-induced expression prior to the late-induced expressions of OsWRKY62 and OsWRKY76. The GFP-OsWRKY28 fusion protein localized mainly in the nuclei of onion epidermal cells, and the maltose-binding protein-fused OsWRKY28 recombinant protein specifically bound to W-box elements. A transient reporter gene assay clearly showed that OsWRKY28 functions as a transcriptional repressor. Overexpression of OsWRKY28 in rice plants resulted in enhanced susceptibility to Ina86-137. Finally, transcriptome analysis revealed that the induction of several defense-related genes in the wild type after Ina86-137 infection was counteracted in OsWRKY28-overexpressing rice plants. These results strongly suggest that OsWRKY28 is a negative regulator of basal defense responses against Ina86-137 and acts as a modulator to maintain the responses at an appropriate level by attenuating the activation of defense-related gene expression levels. OsWRKY28,OsWRKY62,OsWRKY71,OsWRKY76 OsWRKY IIa Transcription Factors Modulate Rice Innate Immunity 2010 Rice (N Y) Department of Plant Pathology, University of California, Davis, CA 95616 USA. WRKY transcription factors regulate diverse plant processes including responses to biotic stresses. Our previous studies indicate that OsWRKY62, an OsWRKY IIa subfamily member, functions as a negative regulator of the rice defense against Xanthomonas oryzae pv. oryzae. Here, we report that a large inverted repeat construct designed to knock down the expression of the four OsWRKY IIa subfamily members (OsWRKY62, OsWRKY28, OsWRKY71, and OsWRKY76) leads to overexpression of all four genes and disease resistance in some transgenic plants. These phenotypes are stably inherited as reflected by progeny analysis. A pathogenesis-related gene, PR10, is up-regulated in plants overexpressing the OsWRKY IIa genes. These results suggest that OsWRKY IIa proteins interact functionally to modulate plant innate immunity. OsWRKY28 A comprehensive expression analysis of the WRKY gene superfamily in rice plants during defense response 2006 Plant Cell Rep Graduate School of Biotechnology & Plant Metabolism Research Center, Kyung Hee University, Yongin, 449-701, Korea. To understand the transcriptional regulatory mechanism of host genes during the activation of defense responses in rice, we isolated WRKY transcription factors whose expressions were altered upon attack of the fungal pathogen Magnaporthe grisea, the causal agent of the devastating rice blast disease. A systematic expression analysis of OsWRKYs (Oryza sativa L. WRKYs) revealed that among 45 tested genes the expression of 15 genes was increased remarkably in an incompatible interaction between rice and M. grisea. Twelve of the M. grisea-inducible OsWRKY genes were also differentially regulated in rice plants infected with the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). In experiments with defense signaling molecules, the expression of two genes, OsWRKY45 and OsWRKY62, was increased in salicylic acid (SA)-treated leaves and the expression of three genes, OsWRKY10, OsWRKY82, and OsWRKY85 was increased by jasmonic acid (JA) treatment. OsWRKY30 and OsWRKY83 responded to both SA- and JA treatments. The expression profiles suggest that a large number of WRKY DNA-binding proteins are involved in the transcriptional activation of defense-related genes in response to rice pathogens. OsWRKY30 Over-expression of the stress-induced OsWRKY45 enhances disease resistance and drought tolerance in Arabidopsis 2009 Environmental and Experimental Botany Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming 650223, PR China The WRKY transcriptional factor superfamily regulates diverse functions, including processes such as plant development and stress response. In this study, we have shown that the rice WRKY45 (OsWRKY45) expression is markedly induced in response to stress-related hormone abscisic acid (ABA) and various stress factors, e.g., application of NaCl, PEG, mannitol or dehydration, treatment with 0 °C and 42 °C as well as infection by Pyricularia oryzae Cav. and Xanthomonas oryzae pv. oryzae. Together, these results indicate that the OsWRKY45 may be involved in the signal pathways of both biotic and abiotic stress response. Further analyses of 35S:OsWRK45 Arabidopsis plants have shown that ectopic, constitutive over-expression of the OsWRKY45 transgene confers a number of properties to transgenic plants. These properties include significantly increased expression of PR genes, enhanced resistance to the bacterial pathogen Pseudomonas syringae tomato DC3000, enhanced tolerance to salt and drought stresses, decreased sensitivity toward ABA signalling during seed germination and post-germination processes, and modulation of ABA/stress-regulated genes during drought induction. In addition, higher levels of OsWRKY45 expression in transgenic plants correlate positively with the strength of the abiotic and biotic responses mentioned above. More specifically, the decreased ABA sensitivities, the enhanced disease resistance and drought tolerances may be attributed, in part, to stomatal closure and induction of stress-related genes during drought induction. The relationship between OsWRKY45 expression and ABA signalling is discussed. OsWRKY45 OsWRKY45 alleles play different roles in abscisic acid signalling and salt stress tolerance but similar roles in drought and cold tolerance in rice 2011 J Exp Bot National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Although allelic diversity of genes has been shown to contribute to many phenotypic variations associated with different physiological processes in plants, information on allelic diversity of abiotic stress-responsive genes is limited. Here it is shown that the alleles OsWRKY45-1 and OsWRKY45-2 play different roles in abscisic acid (ABA) signalling and salt stress adaptation in rice. The two alleles had different transcriptional responses to ABA and salt stresses. OsWRKY45-1-overexpressing lines showed reduced ABA sensitivity, whereas OsWRKY45-1-knockout lines showed increased ABA sensitivity. OsWRKY45-1 transgenic plants showed no obvious difference from negative controls in response to salt stress. In contrast, OsWRKY45-2-overexpressing lines showed increased ABA sensitivity and reduced salt stress tolerance, and OsWRKY45-2-suppressing lines showed reduced ABA sensitivity and increased salt stress tolerance. OsWRKY45-1 and OsWRKY45-2 transgenic plants showed differential expression of a set of ABA- and abiotic stress-responsive genes, but they showed similar responses to cold and drought stresses. These results suggest that OsWRKY45-1 negatively and OsWRKY45-2 positively regulates ABA signalling and, in addition, OsWRKY45-2 but not OsWRKY45-1 negatively regulates rice response to salt stress. The different roles of the two alleles in ABA signalling and salt stress may be due to their transcriptional mediation of different signalling pathways. OsWRKY45,RD22|OsBURP03|OsBURP3 Rice WRKY45 plays important roles in fungal and bacterial disease resistance 2012 Mol Plant Pathol Disease Resistant Crops Research Unit, GMO Research Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. Plant 'activators', such as benzothiadiazole (BTH), protect plants from various diseases by priming the plant salicylic acid (SA) signalling pathway. We have reported previously that a transcription factor identified in rice, WRKY45 (OsWRKY45), plays a pivotal role in BTH-induced disease resistance by mediating SA signalling. Here, we report further functional characterization of WRKY45. Different plant activators vary in their action points, either downstream (BTH and tiadinil) or upstream (probenazole) of SA. Rice resistance to Magnaporthe grisea, induced by both types of plant activator, was markedly reduced in WRKY45-knockdown (WRKY45-kd) rice, indicating a universal role for WRKY45 in chemical-induced resistance. Fungal invasion into rice cells was blocked at most attempted invasion sites (pre-invasive defence) in WRKY45-overexpressing (WRKY45-ox) rice. Hydrogen peroxide accumulated within the cell wall underneath invading fungus appressoria or between the cell wall and the cytoplasm, implying a possible role for H(2)O(2) in pre-invasive defence. Moreover, a hypersensitive reaction-like reaction was observed in rice cells, in which fungal growth was inhibited after invasion (post-invasive defence). The two levels of defence mechanism appear to correspond to Type I and II nonhost resistances. The leaf blast resistance of WRKY45-ox rice plants was much higher than that of other known blast-resistant varieties. WRKY45-ox plants also showed strong panicle blast resistance. BTH-induced resistance to Xanthomonas oryzae pv. oryzae was compromised in WRKY45-kd rice, whereas WRKY45-ox plants were highly resistant to this pathogen. However, WRKY45-ox plants were susceptible to Rhizoctonia solani. These results indicate the versatility and limitations of the application of this gene. OsWRKY45 Rice WRKY45 plays a crucial role in benzothiadiazole-inducible blast resistance 2007 Plant Cell Plant Disease Resistance Research Unit, National Institute of Agrobiological Sciences, Ibaraki, Japan. Benzothiadiazole (BTH) is a so-called plant activator and protects plants from diseases by activating the salicylic acid (SA) signaling pathway. By microarray screening, we identified BTH- and SA-inducible WRKY transcription factor (TF) genes that were upregulated within 3 h after BTH treatment. Overexpression of one of them, WRKY45, in rice (Oryza sativa) markedly enhanced resistance to rice blast fungus. RNA interference-mediated knockdown of WRKY45 compromised BTH-inducible resistance to blast disease, indicating that it is essential for BTH-induced defense responses. In a transient expression system, WRKY45 activated reporter gene transcription through W-boxes. Epistasis analysis suggested that WRKY45 acts in the SA signaling pathway independently of NH1, a rice ortholog of Arabidopsis thaliana NPR1, which distinguishes WRKY45 from known Arabidopsis WRKY TFs. Two defense-related genes, encoding a glutathione S-transferase and a cytochrome P450, were found to be regulated downstream of WRKY45 but were not regulated by NH1, consistent with the apparent independence of the WRKY45- and NH1-dependent pathways. Defense gene expression in WRKY45-overexpressed rice plants varied with growth conditions, suggesting that some environmental factor(s) acts downstream of WRKY45 transcription. We propose a role for WRKY45 in BTH-induced and SA-mediated defense signaling in rice and its potential utility in improving disease resistance of rice, an importance food resource worldwide. OsWRKY45 A pair of allelic WRKY genes play opposite roles in rice-bacteria interactions 2009 Plant Physiol National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Although allelic diversity of genes has been reported to play important roles in different physiological processes, information on allelic diversity of defense-responsive genes in host-pathogen interactions is limited. Here, we report that a pair of allelic genes, OsWRKY45-1 and OsWRKY45-2, which encode proteins with a 10-amino acid difference, play opposite roles in rice (Oryza sativa) resistance against bacterial pathogens. Bacterial blight caused by Xanthomonas oryzae pv oryzae (Xoo), bacterial streak caused by Xanthomonas oryzae pv oryzicola (Xoc), and fungal blast caused by Magnaporthe grisea are devastating diseases of rice worldwide. OsWRKY45-1-overexpressing plants showed increased susceptibility and OsWRKY45-1-knockout plants showed enhanced resistance to Xoo and Xoc. In contrast, OsWRKY45-2-overexpressing plants showed enhanced resistance and OsWRKY45-2-suppressing plants showed increased susceptibility to Xoo and Xoc. Interestingly, both OsWRKY45-1- and OsWRKY45-2-overexpressing plants showed enhanced resistance to M. grisea. OsWRKY45-1-regulated Xoo resistance was accompanied by increased accumulation of salicylic acid and jasmonic acid and induced expression of a subset of defense-responsive genes, while OsWRKY45-2-regulated Xoo resistance was accompanied by increased accumulation of jasmonic acid but not salicylic acid and induced expression of another subset of defense-responsive genes. These results suggest that both OsWRKY45-1 and OsWRKY45-2 are positive regulators in rice resistance against M. grisea, but the former is a negative regulator and the latter is a positive regulator in rice resistance against Xoo and Xoc. The opposite roles of the two allelic genes in rice-Xoo interaction appear to be due to their mediation of different defense signaling pathways. OsWRKY45,ICS1|OsVDAC1,PAD4|OsPAD4 Blast resistance of CC-NB-LRR protein Pb1 is mediated by WRKY45 through protein-protein interaction 2013 Proc Natl Acad Sci U S A Disease Resistant Crops Research Unit, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan. Panicle blast 1 (Pb1) is a panicle blast resistance gene derived from the indica rice cultivar "Modan." Pb1 encodes a coiled-coil-nucleotide-binding site-leucine-rich repeat (CC-NB-LRR) protein and confers durable, broad-spectrum resistance to Magnaporthe oryzae races. Here, we investigated the molecular mechanisms underlying Pb1-mediated blast resistance. The Pb1 protein interacted with WRKY45, a transcription factor involved in induced resistance via the salicylic acid signaling pathway that is regulated by the ubiquitin proteasome system. Pb1-mediated panicle blast resistance was largely compromised when WRKY45 was knocked down in a Pb1-containing rice cultivar. Leaf-blast resistance by Pb1 overexpression (Pb1-ox) was also compromised in WRKY45 knockdown/Pb1-ox rice. Blast infection induced higher accumulation of WRKY45 in Pb1-ox than in control Nipponbare rice. Overexpression of Pb1-Quad, a coiled-coil domain mutant that had weak interaction with WRKY45, resulted in significantly weaker blast resistance than that of wild-type Pb1. Overexpression of Pb1 with a nuclear export sequence failed to confer blast resistance to rice. These results suggest that the blast resistance of Pb1 depends on its interaction with WRKY45 in the nucleus. In a transient system using rice protoplasts, coexpression of Pb1 enhanced WRKY45 accumulation and increased WRKY45-dependent transactivation activity, suggesting that protection of WRKY45 from ubiquitin proteasome system degradation is possibly involved in Pb1-dependent blast resistance. OsWRKY45,Pb1 Transcriptional Profiling of Rice Early Response to Magnaporthe oryzae Identified OsWRKYs as Important Regulators in Rice Blast Resistance 2013 PLoS One State Key Laboratory for Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China Rice blast disease is a major threat to rice production worldwide, but the mechanisms underlying rice resistance to the causal agent Magnaporthe oryzae remain elusive. Therefore, we carried out a transcriptome study on rice early defense response to M. oryzae. We found that the transcriptional profiles of rice compatible and incompatible interactions with M oryzae were mostly similar, with genes regulated more prominently in the incompatible interactions. The functional analysis showed that the genes involved in signaling and secondary metabolism were extensively up-regulated. In particular, WRKY transcription factor genes were significantly enriched among the up-regulated genes. Overexpressing one of these WRKY genes, OsWRKY47, in transgenic rice plants conferred enhanced resistance against rice blast fungus. Our results revealed the sophisticated transcriptional reprogramming of signaling and metabolic pathways during rice early response to M. oryzae and demonstrated the critical roles of WRKY transcription factors in rice blast resistance. OsWRKY47 Interactions of two abscisic-acid induced WRKY genes in repressing gibberellin signaling in aleurone cells 2006 Plant J Department of Biological Sciences, University of Nevada, Las Vegas, NV 89154, USA. Gibberellins (GA) promote while abscisic acid (ABA) inhibits seed germination and post-germination growth. To address the cross-talk of GA and ABA signaling, we studied two rice WRKY genes (OsWRKY51 and OsWRKY71) that are ABA-inducible and GA-repressible in embryos and aleurone cells. Over-expression of these two genes in aleurone cells specifically and synergistically represses induction of the ABA-repressible and GA-inducible Amy32b alpha-amylase promoter reporter construct (Amy32b-GUS) by GA or the GA-inducible transcriptional activator, GAMYB. The physical interactions of OsWRKY71 proteins themselves and that of OsWRKY71 and OsWRKY51 are revealed in the nuclei of aleurone cells using bimolecular fluorescence complementation (BiFC) assays. Although OsWRKY51 itself does not bind to the Amy32b promoter in vitro, it interacts with OsWRKY71 and enhances the binding affinity of OsWRKY71 to W boxes in the Amy32b promoter. The binding activity of OsWRKY71 is abolished by deleting the C-terminus containing the WRKY domain or substituting the key amino acids in the WRKY motif and the zinc finger region. However, two of these non-DNA-binding mutants are still able to repress GA induction by enhancing the binding affinity of the wild-type DNA-binding OsWRKY71 repressors. In contrast, the third non-DNA-binding mutant enhances GA induction of Amy32b-GUS, by interfering with the binding of the wild-type OsWRKY71 or the OsWRKY71/OsWRKY51 repressing complex. These data demonstrate the synergistic interaction of ABA-inducible WRKY genes in regulating GAMYB-mediated GA signaling in aleurone cells, thereby establishing a novel mechanism for ABA and GA signaling cross-talk. OsWRKY51,OsWRKY71 Involvement of the elicitor-induced gene OsWRKY53 in the expression of defense-related genes in rice 2007 Biochim Biophys Acta Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. We present a detailed characterization of the chitin oligosaccharide elicitor-induced gene OsWRKY53. OsWRKY53 was also induced in suspension-cultured rice cells by a fungal cerebroside elicitor and in rice plants by infection with the blast fungus Magnaporthe grisea. A fusion of OsWRKY53 with green fluorescent protein was detected exclusively in the nuclei of onion epidermal cells, and OsWRKY53 protein specifically bound to W-box elements. A transient assay using the particle bombardment method showed that OsWRKY53 is a transcriptional activator. A microarray analysis revealed that several defense-related genes, including pathogenesis-related protein genes such as PBZ1, were upregulated in rice cells overexpressing OsWRKY53. Finally, overexpression of OsWRKY53 in rice plants resulted in enhanced resistance to M. grisea. These results strongly suggest that OsWRKY53 is a transcription factor that plays important roles in elicitor-induced defense signaling pathways in rice. OsWRKY53 Promoter Analysis of the Elicitor-Induced WRKY GeneOsWRKY53, Which Is Involved in Defense Responses in Rice 2014 Bioscience, Biotechnology and Biochemistry Biotechnology Research Center, The University of Tokyo, Japan. OsWRKY53, a chitin oligosaccharide elicitor-responsive rice WRKY gene, has been found to be involved in defense responses in rice. We identified three tandem W-box elements, putative recognition sites for WRKY transcription factors, as cis elements that are essential to the elicitor-responsiveness of OsWRKY53 by deletion and mutation analysis of the promoter by dual luciferase assay. OsWRKY53 Heterologous expression of OsWRKY6 gene in Arabidopsis activates the expression of defense related genes and enhances resistance to pathogens 2011 Plant Sci National Academy of Agricultural Science, Rural Development Administration, Suwon 440-707, Republic of Korea. seonhh@gmail.com The WRKY proteins are a major family of plant transcription factors implicated in the regulation of plant defense mechanisms against pathogens. OsWRKY6 was isolated based on expression profiling data carried out with samples infected by Xanthomonas oryzae pv. oryzae (Xoo). OsWRKY6 encodes a DNA binding protein that contains one WRKY domain, a nuclear localization signal and C(2)H(2)-type zinc finger motif. OsWRKY6 is a member of the group II family of WRKY proteins. Based on the result of yeast one hybrid assay this OsWRKY6 protein binds to the typical W box ((T)TGACC/T). OsWRKY6 functions as a transcriptional activator in yeast. OsWRKY6 enhanced the expression of the reporter gene downstream of OsPR1 promoter, indicating that OsWRKY6 is a transcriptional activator in rice as well. Heterologous expression of OsWRKY6 enhanced disease resistance to pathogen. Defense-related genes were constitutively expressed in Arabidopsis transgenic lines overexpressing OsWRKY6. All together, OsWRKY6 functions as a positive transcriptional regulator of the plant defense response. OsWRKY6 OsWRKY62 is a negative regulator of basal and Xa21-mediated defense against Xanthomonas oryzae pv. oryzae in rice 2008 Mol Plant Department of Plant Pathology, University of California, Davis, CA 95616, USA. The rice Xa21 gene, which confers resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), encodes a receptor-like kinase. Few components involved in transducing the Xa21-mediated defense response have yet been identified. Here, we report that XA21 binds to a WRKY transcription factor, called OsWRKY62. The OsWRKY62 gene encodes two splice variants (OsWRKY62.1 and OsWRKY62.2). OsWRKY62.1:smGFP2 and OsWRKY62.2:smGFP2 fusion proteins partially localize to the nucleus. Transgenic plants overexpressing OsWRKY62.1 are compromised in basal defense and Xa21-mediated resistance to Xoo. Furthermore, overexpression of OsWRKY62.1 suppresses the activation of defense-related genes. These results imply that OsWRKY62 functions as a negative regulator of innate immunity in rice, and serves as a critical mediator of both basal and race-specific defense responses. OsWRKY62,xa21 Cleavage and nuclear localization of the rice XA21 immune receptor 2012 Nat Commun Department of Plant Pathology and the Genome Center, University of California Davis, Davis, California 95616, USA. Plants and animals carry specific receptors that recognize invading pathogens and respond by activating an immune response. The rice XA21 receptor confers broad-spectrum immunity to the Gram-negative bacterial pathogen, Xanthomonas oryzae pv. oryzae upon recognition of a small protein, Ax21, that is conserved in all Xanthomonas species and related genera. Here we demonstrate that XA21 is cleaved to release the intracellular kinase domain and that this intracellular domain carries a functional nuclear localization sequence. Bimolecular fluorescence complementation assays indicate that the XA21 intracellular domain interacts with the OsWRKY62 transcriptional regulator exclusively in the nucleus of rice protoplasts. In vivo cleavage of XA21 and translocalization of the intracellular kinase domain to the nucleus is required for the XA21-mediated immune response. These results suggest a new model for immune receptor function: on receptor recognition of conserved microbial signatures, the associated kinase translocates to the nucleus where it directly interacts with transcriptional regulators. OsWRKY62,xa21 Characterization of an Elicitor-Induced Rice WRKY Gene,OsWRKY71 2014 Bioscience, Biotechnology and Biochemistry Biotechnology Research Center, The University of Tokyo, Yayoi, Tokyo, Japan. Expression of OsWRKY71, a rice WRKY gene, was induced by biotic elicitors and pathogen infection. It was also found that OsWRKY71 has features characteristic of a transcriptional repressor. Microarray analysis revealed that several elicitor-induced defense-related genes were upregulated in rice cells overexpressing OsWRKY71. These results indicate that the activation of defense-related genes by OsWRKY71 was probably indirect. OsWRKY71 WRKY72-type transcription factors contribute to basal immunity in tomato and Arabidopsis as well as gene-for-gene resistance mediated by the tomato R gene Mi-1 2010 Plant J Graduate Program in Plant Pathology, University of California at Riverside, Riverside, CA 92521, USA. WRKY transcription factors play a central role in transcriptional reprogramming associated with plant immune responses. However, due to functional redundancy, typically the contribution of individual members of this family to immunity is only subtle. Using microarray analysis, we found that the paralogous tomato WRKY genes SlWRKY72a and b are transcriptionally up-regulated during disease resistance mediated by the R gene Mi-1. Virus-induced gene silencing of these two genes in tomato resulted in a clear reduction of Mi-1-mediated resistance as well as basal defense against root-knot nematodes (RKN) and potato aphids. Using Arabidopsis T-DNA insertion mutants, we found that their Arabidopsis ortholog, AtWRKY72, is also required for full basal defense against RKN as well as to the oomycete Hyaloperonospora arabidopsidis. Despite their similar roles in basal defense against RKN in both tested plant species, WRKY72-type transcription factors in tomato, but not in Arabidopsis, clearly contributed to basal defense against the bacterial pathogen Pseudomonas syringae. Of the five R genes that we tested in tomato and Arabidopsis, only Mi-1 appeared to be dependent on WRKY72-type transcription factors. Interestingly, AtWRKY72 target genes, identified by microarray analysis of H. arabidopsidis-triggered transcriptional changes, appear to be largely non-responsive to analogs of the defense hormone salicylic acid (SA). Thus, similarly to Mi-1, which in part acts independently of SA, AtWRKY72 appears to utilize SA-independent defense mechanisms. We propose that WRKY72-type transcription factors play a partially conserved role in basal defense in tomato and Arabidopsis, a function that has been recruited to serve Mi-1-dependent immunity. OsWRKY72 Overexpression of OsWRKY72 gene interferes in the abscisic acid signal and auxin transport pathway of Arabidopsis 2010 Journal of Biosciences Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming 650223, PR China. Through activating specific transcriptional programmes, plants can launch resistance mechanisms to stressful environments and acquire a new equilibrium between development and defence. To screen the rice WRKY transcription factor which functions in abiotic stress tolerance and modulates the abscisic acid (ABA) response, we generated a whole array of 35S-OsWRKY transgenic Arabidopsis. In this study, we report that 35S-OsWRKY72 transgenic Arabidopsis, whose seed germination was retarded under normal conditions, emerged more sensitive to mannitol, NaCl, ABA stresses and sugar starvation than vector plants. Meanwhile, 35S-OsWRKY72 transgenic Arabidopsis displayed early flowering, reduced apical dominance, lost high temperature-induced hypocotyl elongation response, and enhanced gravitropism response, which were similar to the auxin-related gene mutants aux1, axr1 and bud1. Further, semi-quantitative RT-PCR showed that the expression patterns of three auxin-related genes AUX1, AXR1 and BUD1 were significantly altered in rosette leaves and infl orescences of 35S-OsWRKY72 plants compared with control Arabidopsis, and two ABA-related genes ABA2 and ABI4 were induced in 35S-OsWRKY72 seedlings. In addition, northern blot analysis indicated that, in rice, OsWRKY72 was inducible by polyethylene glycol (PEG), NaCl, naphthalene acetic acid (NAA), ABA and 42 degrees C, similar to its orthologue AtWRKY75 in Arabidopsis, implying that these two WRKY genes might be required for multiple physiological processes in their plants. Together, these results suggest that OsWRKY72 interferes in the signal cross-talk between the ABA signal and auxin transport pathway in transgenic Arabidopsis. OsWRKY72 WRKY76 is a rice transcriptional repressor playing opposite roles in blast disease resistance and cold stress tolerance 2013 J Exp Bot Disease Resistant Crops Research Unit, GMO Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan. OsWRKY76 encodes a group IIa WRKY transcription factor of rice. The expression of OsWRKY76 was induced within 48h after inoculation with rice blast fungus (Magnaporthe oryzae), and by wounding, low temperature, benzothiadiazole, and abscisic acid. Green fluorescent protein-fused OsWRKY76 localized to the nuclei in rice epidermal cells. OsWRKY76 showed sequence-specific DNA binding to the W-box element in vitro and exhibited W-box-mediated transcriptional repressor activity in cultured rice cells. Overexpression of OsWRKY76 in rice plants resulted in drastically increased susceptibility to M. oryzae, but improved tolerance to cold stress. Microarray analysis revealed that overexpression of OsWRKY76 suppresses the induction of a specific set of PR genes and of genes involved in phytoalexin synthesis after inoculation with blast fungus, consistent with the observation that the levels of phytoalexins in the transgenic rice plants remained significantly lower than those in non-transformed control plants. Furthermore, overexpression of OsWRKY76 led to the increased expression of abiotic stress-associated genes such as peroxidase and lipid metabolism genes. These results strongly suggest that OsWRKY76 plays dual and opposing roles in blast disease resistance and cold tolerance. OsWRKY76 The WRKY transcription factor OsWRKY78 regulates stem elongation and seed development in rice 2011 Planta Key Laboratory of Plant Functional Genomics of the Ministry of Education, Jiangsu Key Laboratory of Crop Genetics and Physiology, College of Agriculture, Yangzhou University, Jiangsu 225009, China. WRKY proteins are a large super family of transcriptional regulators primarily involved in various plant physiological programs. In present study, the expression profile and putative function of the WRKY transcriptional factor, WRKY78, in rice were identified. Real-time RT-PCR analysis showed that OsWRKY78 transcript was most abundant in elongating stems though its expression was detected in all the tested organs. The expression profiles were further confirmed by using promoter-GUS analysis in transgenic rice. OsWRKY78::GFP fusion gene transient expression analysis demonstrated that OsWRKY78 targeted to the nuclei of onion epidermal cell. Furthermore, OsWRKY78 RNAi and overexpression transgenic rice lines were generated. Transgenic plants with OsWRKY78 overexpression exhibited a phenotype identical to the wild type, whereas inhibition of OsWRKY78 expression resulted in a semi-dwarf and small kernel phenotype due to reduced cell length in transgenic plants. In addition, a T-DNA insertion mutant line oswrky78 was identified and a phenotype similar to that of RNAi plants was also observed. Grain quality analysis data showed no significant differences, with the exception of minor changes in endosperm starch crystal structure in RNAi plants. Taken together, these results suggest that OsWRKY78 may acts as a stem elongation and seed development regulator in rice. OsWRKY78 Overexpression of the stress-induced OsWRKY08 improves osmotic stress tolerance in Arabidopsis 2010 Chinese Science Bulletin Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming, 650223, China Previous Northern blotting analyses of rice seedlings have screened several WRKY genes among the transcripts that are differentially regulated in the following treatments: high salinity, cold stress, polyethylene glycol (PEG) and heat shock. Here, we report characterization of a WRKY gene, OsWRKY08, in rice, which was found to be inducible by PEG, NaCl, Abscisic acid (ABA), and naphthalene acetic acid (NAA) as its ortholog AtWRKY28 in Arabidopsis. To determine whether overexpression of OsWRKY08 alters abiotic stress tolerance, 35S::OsWRKY08 recombinant was generated and transformed into Arabidopsis. Physiological tests indicated that 35S::OsWRKY08 transgenic Arabidopsis displayed increased tolerance to mannitol stress through increasing the lateral root number and primary root length during seeding root development. Further, semi-quantitative RT-PCR showed that AtCOR47 and AtRD21, two ABA-independent abiotic stress responded genes, were induced in 35S::OsWRKY08 transgenic plants. These results suggest OsWRKY08 improves the osmotic stress tolerance of transgenic Arabidopsis through an ABA-independent signaling pathway. OsWRKY8 Isolation and Expression Patterns of Rice WRKY82 Transcription Factor Gene Responsive to Both Biotic and Abiotic Stresses 2011 Agricultural Sciences in China School of Life Sciences, Hunan University of Science and Technology, Xiangtan 411201, P.R. China WRKY transcription factors are involved in the regulation of response to biotic and abiotic stresses in plants. A full-length cDNA clone of rice WRKY82 gene (OsWRKY82) was isolated from a cDNA library generated from leaves infected by Magnaporthe grisea. OsWRKY82 contained an entire open reading frame in length of 1701 bp, and was predicted to encode a polypeptide of 566 amino acid residues consisting of two WRKY domains, each with a zinc finger motif of C(2)H(2), belonging to the WRKY subgroup I. OsWRKY82 shared high identity at the amino acid level with those from Sorghum bicolor, Hordeum vulgare, and Zea mays. The transcript level of OsWRKY82 was relatively higher in stems, leaves, and flowers, and less abundant in grains. It was induced by inoculation with M grisea and Rhizoctonia solani. However, the inducible expression in incompatible rice-M grisea interactions was earlier and greater than that in compatible interactions. The expression of OsWRKY82 was up-regulated by methyl jasmonate and ethephon, whereas salicylic acid exerted no effects on its expression. Moreover, OsWRKY82 exhibited transcriptional activation ability in yeast. Additionally, OsWRKY82 transcripts could be induced by wounding and heat shocking, but not by abscisic acid, cold, high salinity and dehydration. By contrast, gibberellin suppressed the expression of OsWRKY82. These indicate that OsWRKY82 is a multiply stress-inducible gene responding to both biotic and abiotic stresses, and may be involved in the regulation of defense response to pathogens and tolerance against abiotic stresses by jasmonic acid/ethylene-dependent signaling pathway. OsWRKY82 Overexpression of rice WRKY89 enhances ultraviolet B tolerance and disease resistance in rice plants 2007 Plant Mol Biol Department of Plant Pathology, State Key Laboratory of Agrobiotechnology, China Agricultural University, Yuanmenyuan West Rd. 2, Beijing 100094, China. WRKY proteins are a large family of transcriptional regulators involved in a variety of biological processes in plants. Here we report functional characterization of a rice WRKY gene, OsWRKY89. RNA gel blot analysis indicated that OsWRKY89 was strongly induced by treatments of methyl jasmonate and UV-B radiation. The transient expression analysis of the OsWRKY89-eGFP reporter in onion epidermal cells revealed that OsWRKY89 was targeted to nuclei. Transcriptional activity assays of OsWRKY89 and its mutants fused with a GAL4 DNA binding domain indicated that the 67 C-terminal amino acids were required for the transcriptional activation and that the leucine zipper region at the N-terminus enhanced its transcriptional activity. Overexpression of OsWRKY89 led to growth retardation at the early stage and reduction of internode length. Scanning electron microscopy revealed an increase in wax deposition on leaf surfaces of the OsWRKY89 overexpression lines and a decrease in wax loading in the RNAi-mediated OsWRKY89 suppression lines. Moreover, extractable and cell-wall-bound phenolic compounds were decreased in the overexpressor lines, but its SA levels were increased. Lignin staining showed an increase in lignification in culms of the overexpressor lines. Interestingly, overexpression of the OsWRKY89 gene enhanced resistance to the rice blast fungus and white-backed planthopper as well as tolerance to UV-B irradiation. These results suggest that OsWRKY89 plays an important role in response to biotic and abiotic stresses. OsWRKY89 Induction of a novel XIP-type xylanase inhibitor by external ascorbic acid treatment and differential expression of XIP-family genes in rice 2007 Plant Cell Physiol Graduate School of Biosphere Sciences, Hiroshima University, Kagamiyama, Higashi-Hiroshima, 739-8528 Japan. Rice microarray analysis showed that a number of stress-related genes are induced by external addition of L-ascorbic acid (AsA). The gene designated as AK073843 which is homologous to class capital SHA, Cyrillic chitinase was found to exhibit the highest induction among these genes. However, its crucial residues within the chitinase active site are substituted with other residues, suggesting that the protein has no chitinase activity. The recombinant protein which is encoded by the AK073843 gene produced in Escherichia coli has xylanase inhibitor activity, indicating that the gene encodes a novel rice XIP-type xylanase inhibitor protein (OsXIP). The expression of OsXIP was enhanced not only by exogenous AsA treatment but also by various stresses such as citrate and sodium chloride treatments, and wounding; however, it was not influenced by increasing endogenous AsA content. External AsA treatment caused a significant increase in electrolyte leakage from rice root. These results suggested that OsXIP was induced by stress which is caused by external AsA treatment. Rice XIP-family genes, OsXIP, riceXIP and RIXI, showed differential organ-specific expression. Also, these genes were differentially induced by stress and stress-related phytohormones. The transcripts of OsXIP and riceXIP were undetectable under normal conditions, and were drastically induced by wounding and methyl jasmonate (MeJA) treatment in the root. RIXI was constitutively expressed in the shoot but not induced by wounding and stress-related phytohormones. Thus, XIP-type xylanase inhibitors were suggested to be specialized in their function and involved in defense mechanisms in rice. OsXIP,riceXIP,RIXI|C10701 RNAi-mediated knockdown of the XIP-type endoxylanase inhibitor gene, OsXIP, has no effect on grain development and germination in rice 2008 Plant Cell Physiol Graduate School of Biosphere Sciences, Hiroshima University, 1-4-4, Kagamiyama, Higashi-Hiroshima, 739-8528 Japan. OsXIP (Oryza sativa xylanase inhibitor protein) is a XIP-type xylanase inhibitor which was identified as a protein encoded by a wound stress-responsive gene in rice. Although the OsXIP gene was specifically expressed in mature grains under basal conditions, recombinant OsXIP had no effect on rice endogenous xylanases, and OsXIP-suppressed transgenic rice plants did not exhibit any change in grain development and germination, suggesting that rice development may be independent of OsXIP. Analysis using an OsXIP-specific antibody revealed that OsXIP is markedly accumulated in apoplast in rice root cells by wounding. These results reinforced the possibility that OsXIP is involved in plant defense mechanisms against phytopathogens. OsXIP Characterization of plant XRCC1 and its interaction with proliferating cell nuclear antigen 2008 Planta Department of Applied Biological Science, Tokyo University of Science, Chiba, 278-8510, Japan. In plants, there are no DNA polymerase beta (Pol beta) and DNA ligase III (Lig3) genes. Thus, the plant short-patch base excision repair (short-patch BER) pathway must differ considerably from that in mammals. We characterized the rice (Oryza Sativa L. cv. Nipponbare) homologue of the mammalian X-ray repair cross complementing 1 (XRCC1), a well-known BER protein. The plant XRCC1 lacks the N-terminal domain (NTD) which is required for Pol beta binding and is essential for mammalian cell survival. The recombinant rice XRCC1 (OsXRCC1) protein binds single-stranded DNA (ssDNA) as well as double-stranded DNA (dsDNA) and also interacts with rice proliferating cell nuclear antigen (OsPCNA) in a pull-down assay. Through immunoprecipitation, we demonstrated that OsXRCC1 forms a complex with PCNA in vivo. OsXRCC1 mRNA was expressed in all rice organs and was induced by application of bleomycin, but not of MMS, H(2)O(2) or UV-B. Bleomycin also increased the fraction of OsXRCC1 associated with chromatin. These results suggest that OsXRCC1 contributes to DNA repair pathways that differ from the mammalian BER system. OsXRCC1 Characterization of a xyloglucan endotransglucosylase gene that is up-regulated by gibberellin in rice 2004 Plant Physiol National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan. Xyloglucan endotransglucosylases/hydrolases (XTHs) that mediate cleavage and rejoining of the beta (1-4)-xyloglucans of the primary cell wall are considered to play an important role in the construction and restructuring of xyloglucan cross-links. A novel rice (Oryza sativa) XTH-related gene, OsXTH8, was cloned and characterized after being identified by cDNA microarray analysis of gibberellin-induced changes in gene expression in rice seedlings. OsXTH8 was a single copy gene; its full-length cDNA was 1,298 bp encoding a predicted protein of 290 amino acids. Phylogenetic analysis revealed that OsXTH8 falls outside of the three established subfamilies of XTH-related genes. OsXTH8 was preferentially expressed in rice leaf sheath in response to gibberellic acid. In situ hybridization and OsXTH8 promoter GUS fusion analysis revealed that OsXTH8 was highly expressed in vascular bundles of leaf sheath and young nodal roots where the cells are actively undergoing elongation and differentiation. OsXTH8 gene expression was up-regulated by gibberellic acid and there was very little effect of other hormones. In two genetic mutants of rice with abnormal height, the expression of OsXTH8 positively correlated with the height of the mutants. Transgenic rice expressing an RNAi construct of OsXTH8 exhibited repressed growth. These results indicate that OsXTH8 is differentially expressed in rice leaf sheath in relation to gibberellin and potentially involved in cell elongation processes. OsXTH8 Characterization of XET-related genes of rice 2000 Plant Physiol Graduate School of Bioagricultural Science and BioScience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan. uozu@nuagr1.agr.nagoya-u.ac.jp To elucidate the mechanism of internodal elongation in rice (Oryza sativa L.), we analyzed genes encoding xyloglucan endotransglycosylase (XET), a cell wall-loosening enzyme essential for cell elongation. Four rice XET-related (XTR) genes, OsXTR1, OsXTR2, OsXTR3, and OsXTR4, were isolated and their expression patterns in rice plants determined. The expression of the four XTR genes showed different patterns of organ specificity and responses to several plant hormones. OsXTR1 and OsXTR3 were up-regulated by gibberellin and brassinosteroids, whereas OsXTR2 and OsXTR4 showed no clear response to these hormones. Expression of the four XTR genes was also investigated in elongating internodes at different developmental stages. OsXTR1 and OsXTR3 were preferentially expressed in the elongating zone of internodes, while OsXTR2 and OsXTR4 were expressed in nodes and in the divisional and elongating zones of internodes. In three genetic mutants with abnormal heights, the expression of OsXTR1 and OsXTR3 correlated with the height of the mutants, whereas no such correlation was observed for OsXTR2 and OsXTR4. Based on these observations, we discuss the roles that OsXTR1 and OsXTR3 may play in internodal elongation in rice. OsXTR1|XTH2 A surprising diversity and abundance of xyloglucan endotransglucosylase/hydrolases in rice. Classification and expression analysis 2004 Plant Physiol Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai 980-8578, Japan. A search of the recently completed genomic database of rice (Oryza sativa) identified a 29-member xyloglucan endotransglucosylase/hydrolase (OsXTH) gene family. This first report of a complete XTH family from a monocotyledonous species reveals that the OsXTH family is comparable in size with that of the dicotyledon Arabidopsis thaliana, which consists of 33 AtXTH genes. This is surprising because xyloglucan, the specific substrate of XTHs, is considerably less abundant in cell walls of monocotyledons than dicotyledons and is not typically ascribed an important structural role in monocotyledons. As a first step toward determining the roles of rice XTHs, the expression patterns of all 29 OsXTH genes were examined using a quantitative DNA microarray procedure with gene-specific oligonucleotide probes. The analysis showed that most members of the rice XTH family exhibited organ- and growth stage-specific expression. This was confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction analysis of representative OsXTH members. This revealed in more detail the temporally and spatially controlled expression profiles of individual OsXTH genes at particular sites in rice. Previous reports indicated that grasses have relatively greater xyloglucan endotransglucosylase activities, one of the two enzyme activities catalyzed by XTHs, than in equivalent tissues in dicotyledons. This observation, together with the tissue-specific and growth stage-dependent expression of a large rice XTH gene family, suggests that xyloglucan metabolism plays a more central role in monocotyledon cell wall restructuring than has been reported previously. OsXTR1|XTH2 Molecular characterization the YABBY gene family in Oryza sativa and expression analysis of OsYABBY1 2007 Mol Genet Genomics Graduate School of Science, University of Tokyo, Tokyo, Japan. Members of the YABBY gene family have a general role that promotes abaxial cell fate in a model eudicot, Arabidopsis thaliana. To understand the function of YABBY genes in monocots, we have isolated all YABBY genes in Oryza sativa (rice), and revealed the spatial and temporal expression pattern of one of these genes, OsYABBY1. In rice, eight YABBY genes constitute a small gene family and are classified into four groups according to sequence similarity, exon-intron structure, and organ-specific expression patterns. OsYABBY1 shows unique spatial expression patterns that have not previously been reported for other YABBY genes, so far. OsYABBY1 is expressed in putative precursor cells of both the mestome sheath in the large vascular bundle and the abaxial sclerenchyma in the leaves. In the flower, OsYABBY1 is specifically expressed in the palea and lemma from their inception, and is confined to several cell layers of these organs in the later developmental stages. The OsYABBY1-expressing domains are closely associated with cells that subsequently differentiate into sclerenchymatous cells. These findings suggest that the function of OsYABBY1 is involved in regulating the differentiation of a few specific cell types and is unrelated to polar regulation of lateral organ development. OsYABBY1|OsYAB1,OsYABBY3|OsYAB4,OsYABBY6|OsYAB5,OsYABBY7 Ectopic expression of OsYAB1 causes extra stamens and carpels in rice 2004 Plant Mol Biol National Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Republic of Korea. Members in the YABBY gene family of proteins are plant-specific transcription factors that play critical roles in determining organ polarity. We have isolated a cDNA clone from rice that encodes a YABBY protein. This protein, OsYAB1, is similar to Arabidopsis YAB2 (50.3%) and YAB5 (47.6%). It carries a zinc-finger motif and a YABBY domain, as do those in Arabidopsis . A fusion protein between OsYAB1 and GFP is located in the nucleus. RNA gel-blot analysis showed that the OsYAB1 gene is preferentially expressed in flowers. In-situ hybridization experiments also indicated that the transcript accumulated in the stamen and carpel primordia. Unlike the Arabidopsis YABBY genes, however, the OsYAB1 gene does not show polar expression pattern in the tissues of floral organs. Our transgenic plants that ectopically expressed OsYAB1 were normal during the vegetative growth period, but then showed abnormalities in their floral structures. Spikelets contained supernumerary stamens and carpels compared with those of the wild types. These results suggest that OsYAB1 plays a major role in meristem development and maintenance of stamens and carpels, rather than in determining polarity. OsYABBY1|OsYAB1 A rice YABBY gene, OsYABBY4, preferentially expresses in developing vascular tissue 2007 Dev Genes Evol Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, the Chinese Academy of Sciences, Nanxincun 20, Xiangshan, Beijing, 100093, People's Republic of China. Developmental gene families have diversified during land plant evolution. The primary role of YABBY gene family is promoting abaxial fate in model eudicot, Arabidopsis thaliana. However recent results suggest that roles of YABBY genes are not conserved in the angiosperms. In this paper, a rice YABBY gene was isolated, and its expression patterns were analyzed in detail. Sequence characterization and phylogenetic analyses showed the gene is OsYABBY4, which is group-classified into FIL/YAB3 subfamily. Beta-glucuronidase reporter assay and in situ analysis consistently revealed that OsYABBY4 was expressed in the meristems and developing vascular tissue of rice, predominantly in the phloem tissue, suggesting that the function of the rice gene is different from those of its counterparts in eudicots. OsYABBY4 may have been recruited to regulate the development of vasculature in rice. However, transgenic Arabidopsis plants ectopically expressing OsYABBY4 behaved very like those over-expressing FIL or YAB3 with abaxialized lateral organs, suggesting the OsYABBY4 protein domain is conserved with its Arabidopsis counterparts in sequences. Our results also indicate that the functional diversification of OsYABBY4 may be associated with the divergent spatial-temporal expression patterns, and YABBY family members may have preserved different expression regulatory systems and functions during the evolution of different kinds of species. OsYABBY4 The YABBY gene TONGARI-BOUSHI1 is involved in lateral organ development and maintenance of meristem organization in the rice spikelet 2012 Plant Cell Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan. The meristem initiates lateral organs in a regular manner, and proper communication between the meristem and the lateral organs ensures the normal development of plants. Here, we show that mutation of the rice (Oryza sativa) gene TONGARI-BOUSHI1 (TOB1) results in pleiotropic phenotypes in spikelets, such as the formation of a cone-shaped organ instead of the lemma or palea, the development of two florets in a spikelet, or premature termination of the floret meristem, in addition to reduced growth of the lemma or palea and elongation of the awn. These phenotypes seem to result from not only failure in growth of the lateral organs, but also defects in maintenance and organization of the meristem. For example, the cone-shaped organ develops as a ring-like primordium from an initial stage, suggesting that regulation of organ initiation in the meristem may be compromised. TOB1 encodes a YABBY protein, which is closely related to FILAMENTOUS FLOWER in Arabidopsis thaliana, and is expressed in the lateral organ primordia without any patterns of polarization. No TOB1 expression is detected in the meristem, so TOB1 may act non-cell autonomously to maintain proper meristem organization and is therefore likely to play an important role in rice spikelet development. OsYABBY5|OsYAB3|TOB1 A WUSCHEL-LIKE HOMEOBOX gene represses a YABBY gene expression required for rice leaf development 2007 Plant Physiol National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China. YABBY and WUSCHEL-LIKE HOMEOBOX (WOX) genes have been shown to play important roles in lateral organ formation and meristem function. Here, we report the characterization of functional relationship between rice (Oryza sativa) YAB3 and WOX3 in rice leaf development. Rice YAB3 is closely related to maize (Zea mays) ZmYAB14 and Arabidopsis (Arabidopsis thaliana) FILAMENTOUS FLOWER (FIL), whereas rice WOX3 is highly conserved with maize narrow sheath1 (NS1) and NS2 and Arabidopsis PRESSED FLOWER (PRS). In situ hybridization experiments revealed that the expression of both genes was excluded from the shoot apical meristem, but the transcripts were detected in leaf primordia, young leaves, and reproductive organs without any polar distribution. The function of the two genes was studied by both overexpression and RNA interference (RNAi) in transgenic rice. YAB3 RNAi induced twisted and knotted leaves lacking specialized structures such as ligule and auricles, while no phenotypic change was observed in YAB3 overexpression plants, suggesting that rice YAB3 may be required for leaf cell growth and differentiation. Overexpression of WOX3 repressed YAB3 and showed a YAB3 RNAi phenotype. The expression of class I KNOTTED-LIKE HOMEOBOX (KNOX) genes was ectopically induced in leaves of YAB3 RNAi or WOX3 overexpression plants. Data from inducible WOX3 expression and DNA-protein interaction assays suggested that WOX3 acted as a transcriptional repressor of YAB3. These data reveal a regulatory network involving YAB3, WOX3, and KNOX genes required for rice leaf development. OsYABBY5|OsYAB3|TOB1,NAL2|OsWOX3A|WOX3 Evolutionary expansion and functional diversification of oligopeptide transporter gene family in rice 2012 Rice Key Laboratory of South China Agricultural Plant Genetics and Breeding, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, 510650, People's Republic of China Oligopeptide transporters (OPTs) play important roles in the mobilization of organic nitrogenous compounds and usually associate with tissues that show signs of rapid protein hydrolysis, such as germinating seeds and senescing leaves. This study is to investigate rice OPT genes. OsYSL12,OsYSL13,OsYSL15,OsYSL2 Rice OsYSL15 is an iron-regulated iron(III)-deoxymugineic acid transporter expressed in the roots and is essential for iron uptake in early growth of the seedlings 2009 J Biol Chem Graduate School of Agricultural and Life Sciences, the University of Tokyo, 1-1-1 Yayoi, Tokyo 113-8657, Japan. Graminaceous plants take up iron through YS1 (yellow stripe 1) and YS1-like (YSL) transporters using iron-chelating compounds known as mugineic acid family phytosiderophores. We examined the expression of 18 rice (Oryza sativa L.) YSL genes (OsYSL1-18) in the epidermis/exodermis, cortex, and stele of rice roots. Expression of OsYSL15 in root epidermis and stele was induced by iron deficiency and showed daily fluctuation. OsYSL15 restored a yeast mutant defective in iron uptake when supplied with iron(III)-deoxymugineic acid and transported iron(III)-deoxymugineic acid in Xenopus laevis oocytes. An OsYSL15-green fluorescent protein fusion was localized to the plasma membrane when transiently expressed in onion epidermal cells. OsYSL15 promoter-beta-glucuronidase analysis revealed that OsYSL15 expression in roots was dominant in the epidermis/exodermis and phloem cells under conditions of iron deficiency and was detected only in phloem under iron sufficiency. These results strongly suggest that OsYSL15 is the dominant iron(III)-deoxymugineic acid transporter responsible for iron uptake from the rhizosphere and is also responsible for phloem transport of iron. OsYSL15 was also expressed in flowers, developing seeds, and in the embryonic scutellar epithelial cells during seed germination. OsYSL15 knockdown seedlings showed severe arrest in germination and early growth and were rescued by high iron supply. These results demonstrate that rice OsYSL15 plays a crucial role in iron homeostasis during the early stages of growth. OsYSL15 Disruption of OsYSL15 leads to iron inefficiency in rice plants 2009 Plant Physiol Department of Integrative Bioscience and Biotechnology, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea. Uptake and translocation of metal nutrients are essential processes for plant growth. Graminaceous species release phytosiderophores that bind to Fe(3+); these complexes are then transported across the plasma membrane. We have characterized OsYSL15, one of the rice (Oryza sativa) YS1-like (YSL) genes that are strongly induced by iron (Fe) deficiency. The OsYSL15 promoter fusion to beta-glucuronidase showed that it was expressed in all root tissues when Fe was limited. In low-Fe leaves, the promoter became active in all tissues except epidermal cells. This activity was also detected in flowers and seeds. The OsYSL15:green fluorescent protein fusion was localized to the plasma membrane. OsYSL15 functionally complemented yeast strains defective in Fe uptake on media containing Fe(3+)-deoxymugineic acid and Fe(2+)-nicotianamine. Two insertional osysl15 mutants exhibited chlorotic phenotypes under Fe deficiency and had reduced Fe concentrations in their shoots, roots, and seeds. Nitric oxide treatment reversed this chlorosis under Fe-limiting conditions. Overexpression of OsYSL15 increased the Fe concentration in leaves and seeds from transgenic plants. Altogether, these results demonstrate roles for OsYSL15 in Fe uptake and distribution in rice plants. OsYSL15 Activation of rice Yellow Stripe1-Like 16 (OsYSL16) enhances iron efficiency 2012 Mol Cells Department of Biological Sciences, Dartmouth College, Hanover, NH, USA. Graminaceous plants release ferric-chelating phytosiderophores that bind to iron. These ferric-phytosiderophore complexes are transported across the plasma membrane by a protein produced from Yellow Stripe 1 (YS1). Here, we report the characterization of OsYSL16, one of the YS1-like genes in rice. Real-time analysis revealed that this gene was constitutively expressed irrespective of metal status. Promoter fusions of OsYSL16 to beta-glucuronidase (GUS) showed that OsYSL16 was highly expressed in the vascular tissues of the root, leaf, and spikelet, and in leaf mesophyll cells. The OsYSL16-green fluorescence protein (GFP) fusion protein was localized to the plasma membrane. From a pool of rice T-DNA insertional lines, we identified two independent activation-tagging mutants in OsYSL16. On an Fe-deficient medium, those mutants retained relatively high chlorophyll concentrations compared with the wild-type (WT) controls, indicating that they are more tolerant to a lack of iron. The Fe concentration in shoots was also higher in the OsYSL16 activation lines than in the WT. During germination, the rate of Fe-utilization from the seeds was higher in the OsYSL16 activation lines than in the WT seeds. Our results suggest that the function of OsYSL16 in Fe-homeostasis is to enable distribution of iron within a plant. OsYSL16 OsYSL16 plays a role in the allocation of iron 2012 Plant Mol Biol Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Graminaceous plants acquire iron by secreting mugineic acid family phytosiderophores into the rhizosphere and taking up complexes of iron and phytosiderophores through YSL (yellow stripe 1-like) transporters. Rice OsYSL15 is a transporter of the iron(III)-2'-deoxymugineic acid complex. OsYSL16 has 85 % similarity to both OsYSL15 and the iron(II)-nicotianamine transporter OsYSL2. In the present study, we show that OsYSL16 functionally complemented a yeast mutant defective in iron uptake when grown on medium containing iron(III)-deoxymugineic acid, but not when grown on medium containing iron(II)-nicotianamine. OsYSL16-knockdown seedlings were smaller than wild-type seedlings when only iron(III)chloride was supplied as an iron source. The iron concentration in shoots of OsYSL16-knockdown plants was similar to that of the wild type; however, they showed more severe chlorosis than wild-type plants under iron-deficient conditions. Furthermore, OsYSL16-knockdown plants accumulated more iron in the vascular bundles of the leaves. Expression of the OsYSL16 promoter fused to the beta-glucuronidase gene showed that OsYSL16 is expressed in the root epidermis and vascular bundles of whole plants. The expression was typically observed around the xylem. In the vascular bundles of unelongated nodes, it was detected in the xylem of old leaves and the phloem of new leaves. Graminaceous plants translocate iron from the roots to old leaves mainly via the xylem and to new leaves mainly via the phloem. Our results suggest that OsYSL16 plays a role in the allocation of iron(III)-deoxymugineic acid via the vascular bundles. OsYSL16 YSL16 is a phloem-localized transporter of the copper-nicotianamine complex that is responsible for copper distribution in rice 2012 Plant Cell Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan. Cu is an essential element for plant growth, but the molecular mechanisms of its distribution and redistribution within the plants are unknown. Here, we report that Yellow stripe-like16 (YSL16) is involved in Cu distribution and redistribution in rice (Oryza sativa). Rice YSL16 was expressed in the roots, leaves, and unelongated nodes at the vegetative growth stage and highly expressed in the upper nodes at the reproductive stage. YSL16 was expressed at the phloem of nodes and vascular tissues of leaves. Knockout of this gene resulted in a higher Cu concentration in the older leaves but a lower concentration in the younger leaves at the vegetative stage. At the reproductive stage, a higher Cu concentration was found in the flag leaf and husk, but less Cu was present in the brown rice, resulting in a significant reduction in fertility in the knockout line. Isotope labeling experiments with (65)Cu showed that the mutant lost the ability to transport Cu-nicotianamine from older to younger leaves and from the flag leaf to the panicle. Rice YSL16 transported the Cu-nicotianamine complex in yeast. Taken together, our results indicate that Os-YSL16 is a Cu-nicotianamine transporter that is required for delivering Cu to the developing young tissues and seeds through phloem transport. OsYSL16 OsYSL18 is a rice iron(III)-deoxymugineic acid transporter specifically expressed in reproductive organs and phloem of lamina joints 2009 Plant Mol Biol Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Iron uptake and translocation in plants are important processes for both plant and human nutrition, whereas relatively little is known about the molecular mechanisms of iron transport within the plant body. Several reports have shown that yellow stripe 1 (YS1) and YS1-like (YSL) transporters mediate metal-phytosiderophore uptake and/or metal-nicotianamine translocation. Among the 18 YSL genes in rice (OsYSLs), OsYSL18 is predicted to encode a polypeptide of 679 amino acids containing 13 putative transmembrane domains. An OsYSL18-green fluorescent protein (GFP) fusion was localized to the plasma membrane when transiently expressed in onion epidermal cells. Electrophysiological measurements using Xenopus laevis oocytes showed that OsYSL18 transports iron(III)-deoxymugineic acid, but not iron(II)-nicotianamine, zinc(II)-deoxymugineic acid, or zinc(II)-nicotianamine. Reverse transcriptase PCR analysis revealed more OsYSL18 transcripts in flowers than in shoots or roots. OsYSL18 promoter-beta-glucuronidase (GUS) analysis revealed that OsYSL18 was expressed in reproductive organs including the pollen tube. In vegetative organs, OsYSL18 was specifically expressed in lamina joints, the inner cortex of crown roots, and phloem parenchyma and companion cells at the basal part of every leaf sheath. These results suggest that OsYSL18 is an iron-phytosiderophore transporter involved in the translocation of iron in reproductive organs and phloem in joints. OsYSL18 OsYSL2 is a rice metal-nicotianamine transporter that is regulated by iron and expressed in the phloem 2004 Plant J Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. We identified 18 putative yellow stripe 1 (YS1)-like genes (OsYSLs) in the rice genome that exhibited 36-76% sequence similarity to maize iron(III)-phytosiderophore transporter YS1. Of particular interest was OsYSL2, the transcripts of which were not detected in the roots of either iron-sufficient or iron-deficient plants, but dramatic expression was induced in the leaves by iron deficiency. Based on the nucleotide sequence, OsYSL2 was predicted to encode a polypeptide of 674 amino acids containing 14 putative transmembrane domains. OsYSL2:green fluorescent protein (GFP) was localized in the plasma membrane of onion epidermal cells. Promoter:beta-glucuronidase (GUS) analysis revealed that OsYSL2 was expressed in companion cells in iron-sufficient roots. GUS activity was increased in companion cells, but no GUS staining was observed in epidermal or cortex cells, even in iron-deficient roots. In the leaves and leaf sheaths of iron-sufficient rice, GUS staining was observed in phloem cells of the vascular bundles. In iron-deficient leaves, the OsYSL2 promoter was active in all tissues with particularly strong GUS activity evident in companion cells. The phloem-specific expression of the OsYSL2 promoter suggests that OsYSL2 is involved in the phloem transport of iron. Strong OsYSL2 promoter activity was also detected in developing seeds. Electrophysiological measurements using Xenopus laevis oocytes showed that OsYSL2 transported iron(II)-nicotianamine (NA) and manganese(II)-NA, but did not transport iron(III)-phyosiderophore. These results suggest that OsYSL2 is a rice metal-NA transporter that is responsible for the phloem transport of iron and manganese, including the translocation of iron and manganese into the grain. OsYSL2 Rice metal-nicotianamine transporter, OsYSL2, is required for the long-distance transport of iron and manganese 2010 Plant J Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Rice (Oryza sativa) is indispensable in the diet of most of the world's population. Thus, it is an important target in which to alter iron (Fe) uptake and homeostasis, so as to increase Fe accumulation in the grain. We previously isolated OsYSL2, a functional iron [Fe(II)]- and manganese [Mn(II)]-nicotianamine complex transporter that is expressed in phloem cells and developing seeds. We produced RNAi (OsYSL2i) and overexpression lines (OXOsYSL2) of OsYSL2. At the vegetative stage in an OsYSL2i line, the Fe and Mn concentrations were decreased in the shoots, and the Fe concentration was increased in the roots. At the reproductive stage, positron-emitting tracer imaging system analysis revealed that Fe translocation to the shoots and seeds was suppressed in OsYSL2i. The Fe and Mn concentrations were decreased in the seeds of OsYSL2i, especially in the endosperm. Moreover, the Fe concentration in OXOsYSL2 was lower in the seeds and shoots, but higher in the roots, compared with the wild type. Furthermore, when OsYSL2 expression was driven by the sucrose transporter promoter, the Fe concentration in the polished rice was up to 4.4-fold higher compared with the wild type. These results indicate that the altered expression of OsYSL2 changes the localization of Fe, and that OsYSL2 is a critical Fe-nicotianamine transporter important for Fe translocation, especially in the shoots and endosperm. OsYSL2 Iron biofortification in rice by the introduction of multiple genes involved in iron nutrition 2012 Sci Rep Global Agricultural Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. To address the problem of iron-deficiency anemia, one of the most prevalent human micronutrient deficiencies globally, iron-biofortified rice was produced using three transgenic approaches: by enhancing iron storage in grains via expression of the iron storage protein ferritin using endosperm-specific promoters, enhancing iron translocation through overproduction of the natural metal chelator nicotianamine, and enhancing iron flux into the endosperm by means of iron(II)-nicotianamine transporter OsYSL2 expression under the control of an endosperm-specific promoter and sucrose transporter promoter. Our results indicate that the iron concentration in greenhouse-grown T(2) polished seeds was sixfold higher and that in paddy field-grown T(3) polished seeds was 4.4-fold higher than that in non-transgenic seeds, with no defect in yield. Moreover, the transgenic seeds accumulated zinc up to 1.6-times in the field. Our results demonstrate that introduction of multiple iron homeostasis genes is more effective for iron biofortification than the single introduction of individual genes. OsYSL2,GLB1 OsYSL6 is involved in the detoxification of excess manganese in rice 2011 Plant Physiol Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan. Yellow Stripe-Like (YSL) proteins belong to the oligopeptide transporter family and have been implicated in metal transport and homeostasis in different plant species. Here, we functionally characterized a rice (Oryza sativa) YSL member, OsYSL6. Knockout of OsYSL6 resulted in decreased growth of both roots and shoots only in the high-manganese (Mn) condition. There was no difference in the concentration of total Mn and other essential metals between the wild-type rice and the knockout line, but the knockout line showed a higher Mn concentration in the leaf apoplastic solution and a lower Mn concentration in the symplastic solution than wild-type rice. OsYSL6 was constitutively expressed in both the shoots and roots, and the expression level was not affected by either deficiency or toxicity of various metals. Furthermore, the expression level increased with leaf age. Analysis with OsYSL6 promoter-green fluorescent protein transgenic rice revealed that OsYSL6 was expressed in all cells of both the roots and shoots. Heterogolous expression of OsYSL6 in yeast showed transport activity for the Mn-nicotianamine complex but not for the Mn-mugineic acid complex. Taken together, our results suggest that OsYSL6 is a Mn-nicotianamine transporter that is required for the detoxification of excess Mn in rice. OsYSL6 Auxin biosynthesis by the YUCCA genes in rice 2007 Plant Physiol Bioscience and Biotechnology Center, Nagoya University Chikusa, Nagoya Aichi, 464-8601, Japan. Although indole-3-acetic acid (IAA), the predominant auxin in plants, plays a critical role in various plant growth and developmental processes, its biosynthesis and regulation have not been clearly elucidated. To investigate the molecular mechanisms of IAA synthesis in rice (Oryza sativa), we identified seven YUCCA-like genes (named OsYUCCA1-7) in the rice genome. Plants overexpressing OsYUCCA1 exhibited increased IAA levels and characteristic auxin overproduction phenotypes, whereas plants expressing antisense OsYUCCA1 cDNA displayed defects that are similar to those of rice auxin-insensitive mutants. OsYUCCA1 was expressed in almost all of the organs tested, but its expression was restricted to discrete areas, including the tips of leaves, roots, and vascular tissues, where it overlapped with expression of a beta-glucuronidase reporter gene controlled by the auxin-responsive DR5 promoter. These observations are consistent with an important role for the rice enzyme OsYUCCA1 in IAA biosynthesis via the tryptophan-dependent pathway. OsYUCCA1,OsYUCCA2,OsYUCCA3,OsYUCCA4,OsYUCCA5,OsYUCCA6,OsYUCCA7 Overexpression of OsZHD1, a zinc finger homeodomain class homeobox transcription factor, induces abaxially curled and drooping leaf in rice 2014 Planta National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing, 210095, China. Leaf rolling is receiving considerable attention as an important agronomic trait in rice (Oryza sativa L.). However, little has been known on the molecular mechanism of rice leaf rolling, especially the abaxial rolling. We identified a novel abaxially curled and drooping leaf-dominant mutant from a T(1) transgenic rice line. The abaxially curled leaf phenotypes, co-segregating with the inserted transferred DNA, were caused by overexpression of a zinc finger homeodomain class homeobox transcription factor (OsZHD1). OsZHD1 exhibited a constitutive expression pattern in wild-type plants and accumulated in the developing leaves and panicles. Artificial overexpression of OsZHD1 or its closest homolog OsZHD2 induced the abaxial leaf curling. Histological analysis indicated that both the increased number and the abnormal arrangement of bulliform cells in leaf were responsible for the abaxially curled leaves. We herein reported OsZHD1 with key roles in rice morphogenesis, especially in the modulating of leaf rolling, which provided a novel insight into the molecular mechanism of leaf development in rice. OsZHD1 Characterization of three rice basic/leucine zipper factors, including two inhibitors of EmBP-1 DNA binding activity 1996 J Biol Chem Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA. andre.nantel@br.nrc.ca The promoter of the wheat Em gene contains elements with a CACGTG core sequence (G-boxes), which are recognized by EmBP-1, a wheat basic/leucine zipper (bZIP) protein. G-boxes are required for Em expression in response to the phytohormone abscisic acid and for transactivation by the Viviparous-1 protein (VP1) using transient expression systems. In order to identify other factors that are part of the transcriptional complex that associates with G-boxes, we have screened a rice (Oryza sativa) cDNA library with biotinylated EmBP-1. We have isolated osZIP-1a, a homolog of EmBP-1 and other plant G-box-binding factors. We show that EmBP-1 and osZIP-1a will preferentially heterodimerize in vitro. Overexpression of osZIP-1a in rice protoplasts can enhance expression from the Em promoter only in the presence of abscisic acid. Two other clones have been identified by screening with EmBP-1: osZIP-2a and osZIP-2b. These osZIP-2 factors represent a novel class of bZIP proteins with an unusual DNA-binding domain that does not recognize G-boxes. The osZIP-2 factors can heterodimerize with EmBP-1 and prevent it from binding to the Em promoter. Interestingly, osZIP-1a does not heterodimerize with the osZIP-2 factors and its DNA binding activity is unaffected by their presence. Thus, osZIP-2 factors may be involved in sequestering a particular group of G-box-binding factors into inactive heterodimers. OsZIP-1a|OsbZIP86,OsZIP-2a|OsbZIP80,osZIP-2b Overexpression of the OsZIP4 zinc transporter confers disarrangement of zinc distribution in rice plants 2007 J Exp Bot Department of Global Agricultural Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657 Japan. Zinc (Zn), an essential nutrient in cells, plays a vital role in controlling cellular processes such as growth, development, and differentiation. Although the mechanisms of Zn translocation in rice plants (Oryza sativa) are not fully understood, it has recently received increased interest. OsZIP4 is a Zn transporter that localizes to apical cells. Transgenic rice plants overexpressing the OsZIP4 gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter were produced. The Zn concentration in roots of 35S-OsZIP4 transgenic plants was 10 times higher than in those of vector controls, but it was five times lower in shoots. The Zn concentration in seeds of 35S-OsZIP4 plants was four times lower compared with vector controls. Northern blot analysis and quantitative real-time reverse transcription-PCR revealed transcripts of OsZIP4 expression driven by the CaMV 35S promoter in roots and shoots of 35S-OsZIP4 plants, but levels of endogenous OsZIP4 transcripts were low in roots and high in shoots compared with vector controls. Microarray analysis revealed that the genes expressed in shoots of 35S-OsZIP4 plants coincided with those induced in shoots of Zn-deficient plants. These results indicate that constitutive expression of OsZIP4 changes the Zn distribution within rice plants, and that OsZIP4 is a critical Zn transporter that must be strictly regulated. OsZIP4 OsZIP5 is a plasma membrane zinc transporter in rice 2010 Plant Mol Biol Department of Plant Molecular Systems Biotechnology and Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Republic of Korea. Zinc is essential for normal plant growth and development. To understand its transport in rice, we characterized OsZIP5, which is inducible under Zn deficiency. OsZIP5 complemented the growth defect of a yeast Zn-uptake mutant, indicating that OsZIP5 is a Zn transporter. The OsZIP5-GFP fusion protein was localized to the plasma membrane. Transgenic plants overexpressing the gene grew less well. Overexpression of the gene decreased the Zn concentration in shoots, but caused it to rise in the roots. Knockout plants showed no visible phenotypic changes under either normal or deficient conditions. However, they were tolerant to excess Zn and contained less Zn. In contrast, overexpressing transgenics were sensitive to excess Zn. These results indicate that OsZIP5 plays a role in Zn distribution within rice. OsZIP5 Cloning and functional identification of two members of the ZIP (Zrt, Irt-like protein) gene family in rice (Oryza sativa L.) 2009 Mol Biol Rep State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China. Two ZIP (Zrt, Irt-like Protein) cDNAs were isolated from rice (Oryza sativa L.) by RT-PCR approach, and named as OsZIP7a and OsZIP8 respectively. The predicted proteins of OsZIP7a and OsZIP8 consist of 384 and 390 amino acid residues respectively, and display high similarity to other plant ZIP proteins. Each protein contains eight transmembrane (TM) domains and a highly conserved ZIP signature motif, with a histidine-rich region in the variable region between TM domains III and IV. By semi-quantitative RT-PCR approach, it was found that the expression of OsZIP7a was significantly induced in rice roots by iron-deficiency, while that of OsZIP8 induced in both rice roots and shoots by zinc-deficiency. When expressed in yeast cells, OsZIP7a and OsZIP8 could complement an iron-uptake-deficient yeast mutant and a zinc-uptake-deficient yeast mutant respectively. It suggested that the OsZIP7a and OsZIP8 might encode an iron and a zinc transporter protein in rice respectively. OsZIP7a,OsZIP8 Zinc deficiency-inducible OsZIP8 encodes a plasma membrane-localized zinc transporter in rice 2010 Mol Cells Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, 790-784, Korea. Zinc is an essential micronutrient for several physiological and biochemical processes. To investigate its transport in rice, we characterized OsZIP8, a rice ZIP (Zrt, Irt-like Protein) gene that is strongly up-regulated in shoots and roots under Zn deficiency. OsZIP8 could complement the growth defect of Zn-uptake yeast mutant. The OsZIP8-GFP fusion proteins were localized to the plasma membrane, suggesting that OsZIP8 is a plasma membrane zinc transporter in rice. Activation and overexpression of this gene disturbed the zinc distribution in rice plants, resulting in lower levels in shoots and mature seeds, but an increase in the roots. Field-grown transgenic plants were shorter than the WT. Under treatment with excess zinc, transgenics contained less zinc in their shoots but accumulated more in the roots. Altogether, these results demonstrate that OsZIP8 is a zinc transporter that functions in Zn uptake and distribution. Furthermore, zinc homeostasis is important to the proper growth and development of rice. OsZIP8 The novel gene HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS1 of rice encodes a putative coiled-coil protein required for homologous chromosome pairing in meiosis 2004 Plant Cell Experimental Farm/Plant Genetics Laboratory, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan. knonomur@lab.nig.ac.jp We have identified and characterized a novel gene, PAIR1 (HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS1), required for homologous chromosome pairing and cytokinesis in male and female meiocytes of rice (Oryza sativa). The pair1 mutation, tagged by the endogenous retrotransposon Tos17, exhibited meiosis-specific defects and resulted in complete sterility in male and female gametes. The PAIR1 gene encodes a 492-amino acid protein, which contains putative coiled-coil motifs in the middle, two basic regions at both termini, and a potential nuclear localization signal at the C terminus. Expression of the PAIR1 gene was detected in the early stages of flower development, in which the majority of the sporocytes had not entered meiosis. During prophase I of the pair1 meiocyte, all the chromosomes became entangled to form a compact sphere adhered to a nucleolus, and homologous pairing failed. At anaphase I and telophase I, chromosome nondisjunction and degenerated spindle formation resulted in multiple uneven spore production. However, chromosomal fragmentation frequent in plant meiotic mutants was never observed in all of the pair1 meiocytes. These observations clarify that the PAIR1 protein plays an essential role in establishment of homologous chromosome pairing in rice meiosis. PAIR1 An insertional mutation in the rice PAIR2 gene, the ortholog of Arabidopsis ASY1, results in a defect in homologous chromosome pairing during meiosis 2004 Mol Genet Genomics Experimental Farm and Plant Genetics Laboratory, National Institute of Genetics, Yata 1111, 411-8540 Mishima, Shizuoka, Japan. knonomur@lab.nig.ac.jp To elucidate the genetic system that establishes homologous chromosome pairing in monocot plants, we have isolated an asynaptic mutant of rice, designated pair2 (homologous pairing aberration in rice meiosis 2), in which 24 completely unpaired univalents are observed at pachytene and diakinesis. The mutation was caused by an insertion of the retrotransposon Tos17, as demonstrated by complementation of the mutation by transformation with the corresponding wild-type gene. The gene in which the element was inserted is orthologous to the ASY1 gene of Arabidopsis thaliana and the HOP1 gene of Saccharomyces cerevisiae. Mature PAIR2 mRNA and several splicing variants were found to be highly expressed in wild-type reproductive tissues, and lower expression was also detected in vegetative tissues. In situ hybridization and BrdU incorporation experiments revealed that PAIR2 expression is specifically enhanced in male and female meiocytes, but not in those at pre-meiotic S phase or in the pollen maturation stages. The results obtained in this study suggest that the PAIR2 gene is essential for homologous chromosome pairing in meiosis, as in the case of the genes ASY1 and HOP1. The study also suggested the possibility that a highly homologous copy of the PAIR2 gene located on a different chromosome is in fact a pseudogene. PAIR2 PAIR2 is essential for homologous chromosome synapsis in rice meiosis I 2006 J Cell Sci Experimental Farm/Plant Genetics Laboratory, National Institute of Genetics, Yata1111, Mishima, Shizuoka 411-8540, Japan. The PAIR2 gene is required for homologous chromosome synapsis at meiosis I in rice (Oryza sativa L.) and encodes a HORMA-domain protein that is homologous to Saccharomyces cerevisiae HOP1 and Arabidopsis ASY1. Immunocytological and electron microscopic analyses indicate that PAIR2 proteins associate with axial elements (AEs) at leptotene and zygotene, and is removed from the AEs of arm regions when homologous chromosomes have been synapsed. Immunocytology against a centromeric histone H3 variant revealed that PAIR2 remains at centromeres until diakinesis, by which time the homologous centromeres had already been synapsed. However, neither precocious segregation of sister chromatids nor kinetochore dysfunction is observed, and AEs are normally assembled in the mutant. In the pair2-null mutant, homologous chromosome synapsis is completely eliminated. This study provides the first description of AE-associated protein in monocot plants and indicates that PAIR2 plays an essential role in promoting homologous chromosome synapsis. However, PAIR2 does not play a role in AE formation, sister chromatid cohesion at centromeres or kinetochore assembly in meiosis I of rice. PAIR2 The central element protein ZEP1 of the synaptonemal complex regulates the number of crossovers during meiosis in rice 2010 Plant Cell State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. ZEP1, a transverse filament (TF) protein, is the rice (Oryza sativa) homolog of Arabidopsis thaliana ZYP1. In the Tos17-insertional zep1 mutants, homologous chromosomes align along the entire length of the chromosome, but the synaptonemal complex is not assembled in early prophase I. Crossovers are well formed, and 12 bivalents could be detected from diakinesis to metaphase I, which leads to equal chromosomal segregation in anaphase I. Moreover, the number of crossovers has a tendency to be increased compared with that in the wild type. These phenomena are different from the TF mutants identified so far in other organisms. Chiasma terminalization of the bivalent, which occurs frequently in the wild type, seldom occurred in zep1. Transmission electron micrographs and immunodetection using an antibody against ZEP1 showed that ZEP1 is the central element of the synaptonemal complex. Although PAIR2 and MER3 were loaded normally in zep1, their dissociation was delayed severely compared with the wild type. In addition, ZEP1 is reloaded onto chromosomes in early microspores as the chromosome decondense, suggesting that ZEP1 might have other biological functions during this process. PAIR2,MER3|RCK,ZEP1 Mutation of the rice gene PAIR3 results in lack of bivalent formation in meiosis 2009 Plant J National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Meiosis is essential for eukaryotic sexual reproduction and important for genetic diversity among individuals. Although a number of genes regulating homologous chromosome pairing and synapsis have been identified in the plant kingdom, their molecular basis remains poorly understood. In this study, we identified a novel gene, PAIR3 (HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS 3), required for homologous chromosome pairing and synapsis in rice. Two independent alleles, designated pair3-1 and pair3-2, were identified in our T-DNA insertional mutant library which could not form bivalents due to failure of homologous chromosome pairing and synapsis at diakinesis, resulting in sterility in both male and female gametes. Suppression of PAIR3 by RNAi produced similar results to the T-DNA insertion lines. PAIR3 encodes a protein that contains putative coiled-coil motifs, but does not have any close homologs in other organisms. PAIR3 is preferentially expressed in reproductive organs, especially in pollen mother cells and the ovule tissues during meiosis. Our results suggest that PAIR3 plays a crucial role in homologous chromosome pairing and synapsis in meiosis. PAIR3 Oryza sativa polyamine oxidase 1 back-converts tetraamines, spermine and thermospermine, to spermidine 2014 Plant Cell Rep Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba, Sendai, Miyagi, 980-8577, Japan. KEY MESSAGE: Oryza sativa polyamine oxidase 1 back-converts spermine (or thermospermine) to spermidine. Considering the previous work, major path of polyamine catabolism in rice plant is suggestive to be back-conversion but not terminal catabolism. Rice (Oryza sativa) contains seven genes encoding polyamine oxidases (PAOs), termed OsPAO1 to OsPAO7, based on their chromosomal number and gene ID number. We previously showed that three of these members, OsPAO3, OsPAO4 and OsPAO5, are abundantly expressed, that their products localize to peroxisomes and that they catalyze the polyamine back-conversion reaction. Here, we have focused on OsPAO1. The OsPAO1 gene product shares a high level of identity with those of Arabidopsis PAO5 and Brassica juncea PAO. Expression of OsPAO1 appears to be quite low under physiological conditions, but is markedly induced in rice roots by spermine (Spm) or T-Spm treatment. Consistent with the above finding, the recombinant OsPAO1 prefers T-Spm as a substrate at pH 6.0 and Spm at pH 8.5 and, in both cases, back-converts these tetraamines to spermidine, but not to putrescine. OsPAO1 localizes to the cytoplasm of onion epidermal cells. Differing in subcellular localization, four out of seven rice PAOs, OsPAO1, OsPAO3, OsPAO4 and OsPAO5, catalyze back-conversion reactions of PAs. Based on the results, we discuss the catabolic path(s) of PAs in rice plant. PAO1 Constitutively and highly expressed Oryza sativa polyamine oxidases localize in peroxisomes and catalyze polyamine back conversion 2012 Amino Acids Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba, Sendai, Miyagi, 980-8577, Japan. Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine (PA) catabolism. Recent studies have revealed that plant PAOs are not only active in the terminal catabolism of PAs as demonstrated for maize apoplastic PAO but also in a polyamine back-conversion pathway as shown for most Arabidopsis PAOs. We have characterized Oryza sativa PAOs at molecular and biochemical levels. The rice genome contains 7 PAO isoforms that are termed OsPAO1 to OsPAO7. Of the seven PAOs, OsPAO3, OsPAO4, and OsPAO5 transcripts were most abundant in 2-week-old seedlings and mature plants, while OsPAO1, OsPAO2, OsPAO6, and OsPAO7 were expressed at very low levels with different tissue specificities. The more abundantly expressed PAOs--OsPAO3, OsPAO4, and OsPAO5--were cloned, and their gene products were produced in Escherichia coli. The enzymatic activities of the purified OsPAO3 to OsPAO5 proteins were examined. OsPAO3 favored spermidine (Spd) as substrate followed by thermospermine (T-Spm) and spermine (Spm) and showed a full PA back-conversion activity. OsPAO4 substrate specificity was similar to that of OsPAO5 preferring Spm and T-Spm but not Spd. Those enzymes also converted Spm and T-Spm to Spd, again indicative of PA back-conversion activities. Lastly, we show that OsPAO3, OsPAO4, and OsPAO5 are localized in peroxisomes. Together, these data revealed that constitutively and highly expressed O. sativa PAOs are localized in peroxisomes and catalyze PA back-conversion processes. PAO1 Characterization of five polyamine oxidase isoforms in Arabidopsis thaliana 2010 Plant Cell Rep Laboratory of Plant Molecular and Cellular Biology, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba, Sendai, Miyagi, 980-8577, Japan. ytakahashi@ige.tohoku.ac.jp The genome of Arabidopsis thaliana contains five genes (AtPAO1 to AtPAO5) encoding polyamine oxidase (PAO) which is an enzyme responsible for polyamine catabolism. To understand the individual roles of the five AtPAOs, here we characterized their tissue-specific and space-temporal expression. AtPAO1 seems to have a specific function in flower organ. AtPAO2 was expressed in shoot meristem and root tip of seedlings, and to a higher extent in the later growth stage within restricted parts of the organs, such as shoot meristem, leaf petiole and also in anther. The expression of AtPAO3 was constitutive, but highest in flower organ. AtPAO3 promoter activity was detected in cotyledon, distal portion of root, boundary region of mature rosette leaf and in filaments of flower. AtPAO4 was expressed at higher level all over young seedlings including roots, and in the mature stage its expression was ubiquitous with rather lower level in stem. AtPAO5 expression was observed in the whole plant body throughout various growth stages. Its highest expression was in flowers, particularly in sepals, but not in petals. Furthermore, we determined the substrate specificity of AtPAO1 to AtPAO4. None of the AtPAO enzymes recognized putrescine (Put). AtPAO2 and AtPAO3 showed almost similar substrate recognition patterns in which the most preferable substrate is spermidine (Spd) followed by less specificity to other tetraamines tested. AtPAO4 seemed to be spermine (Spm)-specific. More interestingly, AtPAO1 preferred thermospermine (T-Spm) and norspermine (NorSpm) to Spm, but did not recognize Spd. Based on the results, the individual function of AtPAOs is discussed. PAO1 Identification of a dual-targeted protein belonging to the mitochondrial carrier family that is required for early leaf development in rice 2013 Plant Physiol State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, China. A dual-targeted protein belonging to the mitochondrial carrier family was characterized in rice (Oryza sativa) and designated 3'-Phosphoadenosine 5'-Phosphosulfate Transporter1 (PAPST1). The papst1 mutant plants showed a defect in thylakoid development, resulting in leaf chlorosis at an early leaf developmental stage, while normal leaf development was restored 4 to 6 d after leaf emergence. OsPAPST1 is highly expressed in young leaves and roots, while the expression is reduced in mature leaves, in line with the recovery of chloroplast development seen in the older leaves of papst1 mutant plants. OsPAPST1 is located on the outer mitochondrial membrane and chloroplast envelope. Whole-genome transcriptomic analysis reveals reduced expression of genes encoding photosynthetic components (light reactions) in papst1 mutant plants. In addition, sulfur metabolism is also perturbed in papst1 plants, and it was seen that PAPST1 can act as a nucleotide transporter when expressed in Escherichia coli that can be inhibited significantly by 3'-phosphoadenosine 5'-phosphosulfate. Given these findings, together with the altered phenotype seen only when leaves are first exposed to light, it is proposed that PAPST1 may act as a 3'-phosphoadenosine 5'-phosphosulfate carrier that has been shown to act as a retrograde signal between chloroplasts and the nucleus. OsPAPST1 Identification of a RFLP Marker Tightly Linked to the Panicle Blast Resistance Gene, Pb1, in Rice 2000 Breeding Science Aichi Prefectural Agriculture Research Center (AARC) We precisely mapped the Pb1 locus for the panicle blast resistence of a quantitative nature on rice chromosome 11 using RFLP markers. Based on the cosegregation of the resistant genotypes of Pb1 and Stvb-i, a rice stripe virus (RSV) resistance gene derived from an Indica cultivar Modan, we examined the linkage relationships between Pb1 and 13 Stvb-i-linked RFLP markers located on the long arm of chromosome 11. The Pb1 locus was mapped in the Modan-derived chromosomal region in the middle part of the long arm of chromosome 11. Pb1 was located on the telomere side in relation to the Stvb-i locus. The Pb1 gene was closely located at 1.2cM from three RFLP markers: S723, CDO226, C189. Then, we examined the graphical genotypes of 34 Modan-derived RSV-resistant cultivars with or without panicle blast resistance, and 12 susceptible Japonica cultivars, using 21 RFLP markers. Among them, cultivars with panicle blast resistance were classified into four types: A, B, C and D, and those without it into six types: E, F, G, H, I and J. In all of the panicle blast-resistant cultivars, the Modan-type bands were observed in S723, CDO226 and C189. On the other hand, in all the panicle blast-susceptible cultivars, the Japonica-type band was observed in S723, whereas the Modan-type bands were noticed in CDO226 and C189 in Type E cultivars. Consequently, only the genotypes of the S723 locus completely coincided with the genotypes of the Pb1 locus. Although, it was determined, based on linkage analysis, that S723, CDO226 and C189 were located at the same locus, graphical genotyping analysis, using many progeny cultivars derived from Modan, revealed that S723 was the closest marker to Pb1 among the three. Pb1 Durable panicle blast-resistance gene Pb1 encodes an atypical CC-NBS-LRR protein and was generated by acquiring a promoter through local genome duplication 2010 Plant J Plant Disease Resistance Research Unit, Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. Rice blast is one of the most widespread and destructive plant diseases worldwide. Breeders have used disease resistance (R) genes that mediate fungal race-specific 'gene-for-gene' resistance to manage rice blast, but the resistance is prone to breakdown due to high pathogenic variability of blast fungus. Panicle blast 1 (Pb1) is a blast-resistance gene derived from the indica cultivar 'Modan'. Pb1-mediated resistance, which is characterized by durability of resistance and adult/panicle blast resistance, has been introduced into elite varieties for commercial cultivation. We isolated the Pb1 gene by map-based cloning. It encoded a coiled-coil-nucleotide-binding-site-leucine-rich repeat (CC-NBS-LRR) protein. The Pb1 protein sequence differed from previously reported R-proteins, particularly in the NBS domain, in which the P-loop was apparently absent and some other motifs were degenerated. Pb1 was located within one of tandemly repeated 60-kb units, which presumably arose through local genome duplication. Pb1 transcript levels increased during the development of Pb1+ cultivars; this expression pattern accounts for their adult/panicle resistance. Promoter:GUS analysis indicated that genome duplication played a crucial role in the generation of Pb1 by placing a promoter sequence upstream of its coding sequence, thereby conferring a Pb1-characteristic expression pattern to a transcriptionally inactive 'sleeping' resistance gene. We discuss possible determinants for the durability of Pb1-mediated blast resistance. Pb1 PCF1 and PCF2 specifically bind to cis elements in the rice proliferating cell nuclear antigen gene 1997 Plant Cell National Institute of Agrobiological Resources, Ibaraki, Japan. We have previously defined the promoter elements, sites IIa and IIb, in the rice proliferating cell nuclear antigen (PCNA) gene that are essential for meristematic tissue-specific expression. In this study, we isolated and characterized cDNAs encoding proteins that specifically bind to sites IIa and IIb. The two DNA binding proteins, designated PCF1 and PCF2, share > 70% homology in common conserved sequences at the N-terminal regions. The conserved regions are responsible for DNA binding and homodimer and heterodimer formation, and they contain a putative basic helix-loop-helix (bHLH) motif. The structure and DNA binding specificity of the bHLH motif are distinguishable from those of other known bHLH proteins that bind to the E-box. The motif is > 70% homologous to several expressed sequence tags from Arabidopsis and rice, suggesting that PCF1 and PCF2 are members of a novel family of proteins that are conserved in monocotyledons and dicotyledons. A supershift assay using an anti-PCF2 antibody showed the involvement of PCF2 in site IIa (site IIb) binding activities in rice nuclear extracts, particularly in meristematic tissues. PCF1 and PCF2 were also more likely to form heterodimers than homodimers. Our results suggest that PCF1 and PCF2 are involved in meristematic tissue-specific expression of the rice PCNA gene through binding to sites IIa and IIb and formation of homodimers or heterodimers. PCF1,PCF2 Comparative overviews of clock-associated genes of Arabidopsis thaliana and Oryza sativa 2007 Plant Cell Physiol Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan. t2706775@mbox.nagoya-u.ac.jp In higher plants, circadian rhythms are highly relevant to a wide range of biological processes. To such circadian rhythms, the clock (oscillator) is central, and recent intensive studies on the model higher plant Arabidopsis thaliana have begun to shed light on the molecular mechanisms underlying the functions of the central clock. Such representative clock-associated genes of A. thaliana are the homologous CCA1 and LHY genes, and five PRR genes that belong to a small family of pseudo-response regulators including TOC1. Others are GI, ZTL, ELF3, ELF4, LUX/PCL1, etc. In this context, a simple question arose as to whether or not the molecular picture of the model Arabidopsis clock is conserved in other higher plants. Here we made an effort to answer the question with special reference to Oryza sativa, providing experimental evidence that this model monocot also has a set of highly conserved clock-associated genes, such as those designated as OsCCA1, OsPRR-series including OsTOC1/OsPRR1, OsZTLs, OsPCL1 as well as OsGI. These results will provide us with insight into the general roles of plant circadian clocks, such as those for the photoperiodic control of flowering time that has a strong impact on the reproduction and yield in many higher plants. OsPCL1,FKF1 Characterisation of a plant 3-phosphoinositide-dependent protein kinase-1 homologue which contains a pleckstrin homology domain 1999 FEBS Letters MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee DD1 5EH, UK A plant homologue of mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1) has been identified in Arabidopsis and rice which displays 40% overall identity with human 3-phosphoinositide-dependent protein kinase-1. Like the mammalian 3-phosphoinositide-dependent protein kinase-1, Arabidopsis 3-phosphoinositide-dependent protein kinase-1 and rice 3-phosphoinositide-dependent protein kinase-1 possess a kinase domain at N-termini and a pleckstrin homology domain at their C-termini. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 can rescue lethality in Saccharomyces cerevisiae caused by disruption of the genes encoding yeast 3-phosphoinositide-dependent protein kinase-1 homologues. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 interacts via its pleckstrin homology domain with phosphatidic acid, PtdIns3P, PtdIns(3,4,5)P3 and PtdIns(3,4)P2 and to a lesser extent with PtdIns(4,5)P2 and PtdIns4P. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 is able to activate human protein kinase Bα (PKB/AKT) in the presence of PtdIns(3,4,5)P3. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 is only the second plant protein reported to possess a pleckstrin homology domain and the first plant protein shown to bind 3-phosphoinositides. PDK1 A rice phenolic efflux transporter is essential for solubilizing precipitated apoplasmic iron in the plant stele 2011 J Biol Chem Department of Global Agricultural Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan. Iron deficiency is one of the major agricultural problems, as 30% of the arable land of the world is too alkaline for optimal crop production, rendering plants short of available iron despite its abundance. To take up apoplasmic precipitated iron, plants secrete phenolics such as protocatechuic acid (PCA) and caffeic acid. The molecular pathways and genes of iron uptake strategies are already characterized, whereas the molecular mechanisms of phenolics synthesis and secretion have not been clarified, and no phenolics efflux transporters have been identified in plants yet. Here we describe the identification of a phenolics efflux transporter in rice. We identified a cadmium-accumulating rice mutant in which the amount of PCA and caffeic acid in the xylem sap was dramatically reduced and hence named it phenolics efflux zero 1 (pez1). PEZ1 localized to the plasma membrane and transported PCA when expressed in Xenopus laevis oocytes. PEZ1 localized mainly in the stele of roots. In the roots of pez1, precipitated apoplasmic iron increased. The growth of PEZ1 overexpression lines was severely restricted, and these lines accumulated more iron as a result of the high solubilization of precipitated apoplasmic iron in the stele. We show that PEZ1 is responsible for an increase of PCA concentration in the xylem sap and is essential for the utilization of apoplasmic precipitated iron in the stele. PEZ1 The role of rice phenolics efflux transporter in solubilizing apoplasmic iron 2011 Plant Signal Behav Department of Global Agricultural Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan. Iron (Fe) is an essential micronutrient for plants whose deficiency presents a major worldwide agricultural problem. Moreover, Fe is not easily available in neutral to alkaline soils, rendering plants deficient in Fe despite its abundance. Plants secrete phenolics, such as protocatechuic acid (PCA) and caffeic acid (CA), to take up and utilize apoplasmic precipitated Fe, but despite the rapid progress in understanding cellular and subcellular Fe transport, the molecular mechanisms of phenolics synthesis and secretion are not clear. Recently, we isolated and characterized a phenolics efflux transporter in rice by characterizing a mutant in which the amount of PCA and CA in the xylem sap was dramatically reduced, which we hence named phenolics efflux zero 1 (pez1). PEZ1 is a plasma membrane protein that transports PCA when expressed in Xenopus laevis oocytes, and characterization of PEZ1 knockdown and overexpressing plants revealed that it plays an essential role in solubilizing precipitated apoplasmic Fe. The identification of PEZ1 will increase our understanding of apoplasmic Fe solubilization as well as promote research on phenolics efflux mechanisms in different organisms. PEZ1 PF1: an A-T hook-containing DNA binding protein from rice that interacts with a functionally defined d(AT)-rich element in the oat phytochrome A3 gene promoter 1994 Plant Cell University of California-Berkeley/United States Department of Agriculture, Plant Gene Expression Center, Albany 94710. Phytochrome-imposed down-regulation of the expression of its own phytochrome A gene (PHYA) is one of the fastest light-induced effects on transcription reported in plants to date. Functional analysis of the oat PHYA3 promoter in a transfection assay has revealed two positive elements, PE1 and PE3, that function synergistically to support high levels of transcription in the absence of light. We have isolated a rice cDNA clone (pR4) encoding a DNA binding protein that binds to the AT-rich PE1 element. We tested the selectivity of the pR4-encoded DNA binding activity using linker substitution mutations of PE1 that are known to disrupt positive expression supported by the PHYA3 promoter in vivo. Binding to these linker substitution mutants was one to two orders of magnitude less than to the native PE1 element. Because this is the behavior expected of positive factor 1 (PF1), the presumptive nuclear transcription factor that acts in trans at the PE1 element in vivo, the data support the conclusion that the protein encoded by pR4 is in fact rice PF1. The PF1 polypeptide encoded by pR4 is 213 amino acids long and contains four repeats of the A-T hook DNA binding motif found in high-mobility group I-Y (HMGI-Y) proteins. In addition, PF1 contains an 11-amino acid-long hydrophobic region characteristic of HMG I proteins, its N-terminal region shows strong similarities to a pea H1 histone sequence and a short peptide sequence from wheat HMGa, and it shows a high degree of similarity along its entire length to the HMG Y-like protein encoded by a soybean cDNA, SB16. In vitro footprinting and quantitative gel shift analyses showed that PF1 binds preferentially to the PE1 element but also at lower affinity to two other AT-rich regions upstream of PE1. This feature is consistent with the binding characteristics of HMG I-Y proteins that are known to bind to most runs of six or more AT base pairs. Taken together, the properties of PF1 suggest that it belongs to a newly described family of nuclear proteins containing both histone H1 domains and A-T hook DNA binding domains. PF1 PGR5-dependent cyclic electron transport around PSI contributes to the redox homeostasis in chloroplasts rather than CO(2) fixation and biomass production in rice 2012 Plant Cell Physiol Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502 Japan. The PGR5 (PROTON GRADIENT REGULATION 5) gene that is required for PSI cyclic electron transport in Arabidopsis was knocked down in rice (Oryza sativa). In three PGR5 knockdown (KD) lines, the PGR5 protein level was reduced to 5-8% of that in the wild type, resulting in a 50% reduction in PGRL1 (PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE 1) protein levels. In ruptured chloroplasts, ferredoxin-dependent plastoquinone reduction activity was partially impaired; the phenotype was mimicked by addition of antimycin A to wild-type chloroplasts. As occurred in the Arabidopsis pgr5 mutant, non-photochemical quenching of Chl fluorescence (NPQ) induction was impaired in the leaves, but the electron transport rate (ETR) was only mildly affected at high light intensity. The P700(+) level was reduced even at low light intensity, suggesting that the PGR5 function was severely disturbed as in the Arabidopsis pgr5 mutant and that the other alternative routes of electrons could not compensate the stromal redox balance. The amplitude of the light-dark electrochromic shift (ECS) signal (ECSt), which reflects the total size of the proton motive force in steady-state photosynthesis, was reduced by 13-25% at approximately the growth light intensity. The CO(2) fixation rate was only slightly reduced in the PGR5 KD lines. Despite the drastic reduction in NPQ and P700(+) levels, total biomass was only slightly reduced in PGR5 KD lines grown at 370 micromol photons m(-2) s(-1). These results suggest that CO(2) fixation and growth rate are very robust in the face of alterations in the fundamental reactions of photosynthesis under constant light conditions in rice. OsPGR5 A rice plastidial nucleotide sugar epimerase is involved in galactolipid biosynthesis and improves photosynthetic efficiency 2011 PLoS Genet State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. Photosynthesis is the final determinator for crop yield. To gain insight into genes controlling photosynthetic capacity, we selected from our large T-DNA mutant population a rice stunted growth mutant with decreased carbon assimilate and yield production named photoassimilate defective1 (phd1). Molecular and biochemical analyses revealed that PHD1 encodes a novel chloroplast-localized UDP-glucose epimerase (UGE), which is conserved in the plant kingdom. The chloroplast localization of PHD1 was confirmed by immunoblots, immunocytochemistry, and UGE activity in isolated chloroplasts, which was approximately 50% lower in the phd1-1 mutant than in the wild type. In addition, the amounts of UDP-glucose and UDP-galactose substrates in chloroplasts were significantly higher and lower, respectively, indicating that PHD1 was responsible for a major part of UGE activity in plastids. The relative amount of monogalactosyldiacylglycerol (MGDG), a major chloroplast membrane galactolipid, was decreased in the mutant, while the digalactosyldiacylglycerol (DGDG) amount was not significantly altered, suggesting that PHD1 participates mainly in UDP-galactose supply for MGDG biosynthesis in chloroplasts. The phd1 mutant showed decreased chlorophyll content, photosynthetic activity, and altered chloroplast ultrastructure, suggesting that a correct amount of galactoglycerolipids and the ratio of glycolipids versus phospholipids are necessary for proper chloroplast function. Downregulated expression of starch biosynthesis genes and upregulated expression of sucrose cleavage genes might be a result of reduced photosynthetic activity and account for the decreased starch and sucrose levels seen in phd1 leaves. PHD1 overexpression increased photosynthetic efficiency, biomass, and grain production, suggesting that PHD1 plays an important role in supplying sufficient galactolipids to thylakoid membranes for proper chloroplast biogenesis and photosynthetic activity. These findings will be useful for improving crop yields and for bioenergy crop engineering. PHD1 Mutation of the plastidial alpha-glucan phosphorylase gene in rice affects the synthesis and structure of starch in the endosperm 2008 Plant Cell Plant Genetic Resources, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan. hsatoh@agr.kyushu-u.ac.jp Plastidial phosphorylase (Pho1) accounts for approximately 96% of the total phosphorylase activity in developing rice (Oryza sativa) seeds. From mutant stocks induced by N-methyl-N-nitrosourea treatment, we identified plants with mutations in the Pho1 gene that are deficient in Pho1. Strikingly, the size of mature seeds and the starch content in these mutants showed considerable variation, ranging from shrunken to pseudonormal. The loss of Pho1 caused smaller starch granules to accumulate and modified the amylopectin structure. Variation in the morphological and biochemical phenotype of individual seeds was common to all 15 pho1-independent homozygous mutant lines studied, indicating that this phenotype was caused solely by the genetic defect. The phenotype of the pho1 mutation was temperature dependent. While the mutant plants grown at 30 degrees C produced mainly plump seeds at maturity, most of the seeds from plants grown at 20 degrees C were shrunken, with a significant proportion showing severe reduction in starch accumulation. These results strongly suggest that Pho1 plays a crucial role in starch biosynthesis in rice endosperm at low temperatures and that one or more other factors can complement the function of Pho1 at high temperatures. Pho1,Pho2 Rice phot1a mutation reduces plant growth by affecting photosynthetic responses to light during early seedling growth 2009 Plant Mol Biol Environmental Biotechnology and Research Center, Gyeongsang National University, Jinju, 660-701, South Korea. changhyogoh@hanmail.net The aim of this work was to characterize the phot1 mutant of rice during early seedling growth in various light conditions. We isolated the rice T-DNA insertion mutant phot1a-1 and compared it to the Tos17 insertion mutant phot1a-2. When phot1a mutants were grown under WL (100) and BL (40 miccromol m(-2) s(-1)), they demonstrated a considerable reduction in photosynthetic capacity, which included decreased leaf CO(2) uptake and plant growth. Pigment analysis showed no significant difference between wild-type and mutants in the Chl a:b ratios, whereas in the latter, total concentration was reduced (a 2-fold decrease). Carotenoid contents of the mutants were also decreased considerably, implying the involvement of phot1a in pigment degradation. Deletion of phot1a showed higher contents of H(2)O(2) in leaves. Chloroplastic APX and SOD activities were lower in the mutants whereas the activities of cytosolic enzymes were increased. Immunoblotting indicated reduced accumulation of photosystem proteins (D1, D2, CP43, Lhca2, and PsaC) relative to the other light-harvesting complexes in the mutant. We conclude that the defect of Os Phot1a affects degradation of chlorophylls and carotenoids, and under photosynthetically active photon fluxes, mutation of phot1a results in loss of photosynthetic capacity owing to the damage of photosystems caused by elevated H(2)O(2) accumulation, leading to a reduction in plant growth. phot1a Isolation and characterization of rice phytochrome A mutants 2001 Plant Cell Department of Plant Physiology, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan. mtakano@abr.affrc.go.jp To elucidate phytochrome A (phyA) function in rice, we screened a large population of retrotransposon (Tos17) insertional mutants by polymerase chain reaction and isolated three independent phyA mutant lines. Sequencing of the Tos17 insertion sites confirmed that the Tos17s interrupted exons of PHYA genes in these mutant lines. Moreover, the phyA polypeptides were not immunochemically detectable in these phyA mutants. The seedlings of phyA mutants grown in continuous far-red light showed essentially the same phenotype as dark-grown seedlings, indicating the insensitivity of phyA mutants to far-red light. The etiolated seedlings of phyA mutants also were insensitive to a pulse of far-red light or very low fluence red light. In contrast, phyA mutants were morphologically indistinguishable from wild type under continuous red light. Therefore, rice phyA controls photomorphogenesis in two distinct modes of photoperception--far-red light-dependent high irradiance response and very low fluence response--and such function seems to be unique and restricted to the deetiolation process. Interestingly, continuous far-red light induced the expression of CAB and RBCS genes in rice phyA seedlings, suggesting the existence of a photoreceptor(s) other than phyA that can perceive continuous far-red light in the etiolated seedlings. PHYA Serine-to-alanine substitutions at the amino-terminal region of phytochrome A result in an increase in biological activity 1992 Genes Dev Rockefeller University, Laboratory of Plant Molecular Biology, New York, New York 10021-6399. We have used a tobacco transgenic plant system to assay the structure/function relationship of phytochrome A (phyA), a plant photoreceptor. The amino terminus of phyA from different plant species is very rich in serine residues. To investigate whether these serine residues are required for phytochrome function, the first 10 serine codons encoding amino acid residues 2-4, 10-14, 19, and 20 in the amino-terminal domain of the rice phyA gene (phyA) were changed to alanine codons. The mutant (S/A phyA), as well as the wild-type phyA cDNA, was placed under the control of the 35S promoter, and the chimeric genes were transferred into the tobacco genome by Agrobacterium-mediated transformation. Transgenic tobacco plants expressing either wild-type or S/A phyA showed similar phenotypic alterations, including dwarfism and dark-green leaves. However, hypocotyl elongation experiments revealed that transgenic seedlings expressing S/A phyA showed a higher amplitude of the red light response with respect to the inhibition of hypocotyl elongation. The observed difference is not correlated with expression levels of the transgene. The chromophore is attached to the mutant phyA apoprotein (PHY A), and the mutant photoreceptor is photoreversible, giving a difference spectrum indistinguishable from that of the rice phyA. Our results indicate that the S/A mutant has a higher biological activity as compared with the wild-type rice phyA. PHYA Phytochromes are the sole photoreceptors for perceiving red/far-red light in rice 2009 Proc Natl Acad Sci U S A Department of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. mtakano@affrc.go.jp Phytochromes are believed to be solely responsible for red and far-red light perception, but this has never been definitively tested. To directly address this hypothesis, a phytochrome triple mutant (phyAphyBphyC) was generated in rice (Oryza sativa L. cv. Nipponbare) and its responses to red and far-red light were monitored. Since rice only has three phytochrome genes (PHYA, PHYB and PHYC), this mutant is completely lacking any phytochrome. Rice seedlings grown in the dark develop long coleoptiles while undergoing regular circumnutation. The phytochrome triple mutants also show this characteristic skotomorphogenesis, even under continuous red or far-red light. The morphology of the triple mutant seedlings grown under red or far-red light appears completely the same as etiolated seedlings, and they show no expression of the light-induced genes. This is direct evidence demonstrating that phytochromes are the sole photoreceptors for perceiving red and far-red light, at least during rice seedling establishment. Furthermore, the shape of the triple mutant plants was dramatically altered. Most remarkably, triple mutants extend their internodes even during the vegetative growth stage, which is a time during which wild-type rice plants never elongate their internodes. The triple mutants also flowered very early under long day conditions and set very few seeds due to incomplete male sterility. These data indicate that phytochromes play an important role in maximizing photosynthetic abilities during the vegetative growth stage in rice. PHYA,PHYB|OsphyB,PHYC,LHCB|LHCPII Distinct and cooperative functions of phytochromes A, B, and C in the control of deetiolation and flowering in rice 2005 Plant Cell Department of Plant Physiology, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan. mtakano@nias.affrc.go.jp We have isolated phytochrome B (phyB) and phyC mutants from rice (Oryza sativa) and have produced all combinations of double mutants. Seedlings of phyB and phyB phyC mutants exhibited a partial loss of sensitivity to continuous red light (Rc) but still showed significant deetiolation responses. The responses to Rc were completely canceled in phyA phyB double mutants. These results indicate that phyA and phyB act in a highly redundant manner to control deetiolation under Rc. Under continuous far-red light (FRc), phyA mutants showed partially impaired deetiolation, and phyA phyC double mutants showed no significant residual phytochrome responses, indicating that not only phyA but also phyC is involved in the photoperception of FRc in rice. Interestingly, the phyB phyC double mutant displayed clear R/FR reversibility in the pulse irradiation experiments, indicating that both phyA and phyB can mediate the low-fluence response for gene expression. Rice is a short-day plant, and we found that mutation in either phyB or phyC caused moderate early flowering under the long-day photoperiod, while monogenic phyA mutation had little effect on the flowering time. The phyA mutation, however, in combination with phyB or phyC mutation caused dramatic early flowering. PHYA,PHYB|OsphyB,PHYC Comprehensive metabolite profiling of phyA phyB phyC triple mutants to reveal their associated metabolic phenotype in rice leaves 2009 J Biosci Bioeng Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. The phytochrome photoreceptors regulate plant growth and development throughout their life cycle. Rice (Oryza sativa) possesses three phytochromes, phyA, phyB, and phyC. Physiological, genetic, and biochemical analyses of null mutants of each phytochrome have revealed the function of each in rice. However, few studies have investigated the relationship between phytochrome signaling and metabolism. In the present study, non-targeted metabolite analysis by gas chromatography time-of-flight mass spectrometry (GC/TOF-MS) and targeted metabolite analysis by capillary electrophoresis electrospray ionization mass spectrometry (CE/ESI-MS) were employed to investigate metabolic changes in rice phyA phyB phyC triple mutants. Distinct metabolic profiles between phyA phyB phyC triple mutants and the wild type (WT), as well as those between young and mature leaf blades, could be clearly observed by principal component analysis (PCA). The metabolite profiles indicated high accumulation of amino acids, organic acids, sugars, sugar phosphates, and nucleotides in the leaf blades of phyA phyB phyC triple mutants, especially in the young leaves, compared with those in the WT. Remarkable overaccumulation of monosaccharide, such as glucose (53.4-fold), fructose (42.5-fold), and galactose (24.5-fold), was observed in young leaves of phyA phyB phyC triple mutants. These metabolic phenotypes suggest that sugar metabolism, carbon partitioning, sugar transport, or some combination of these is impaired in the phyA phyB phyC triple mutants, and conversely, that phytochromes have crucial roles in sugar metabolism. PHYA,PHYB|OsphyB,PHYC Fine mapping and DNA marker-assisted pyramiding of the three major genes for blast resistance in rice 2000 TAG Theoretical and Applied Genetics International Rice Research Institute, P.O. Box 933, Manila, Philippines e-mail: maslab@satyam.net.in, PH Three major genes (Pi1, Piz-5 and Pita) for blast resistance on chromosomes 11, 6 and 12, respectively, were fine-mapped and closely linked RFLP markers identified. New markers for Pi1 and Pita were found that were flanking the genes. The three genes were pyramided using RFLP markers. A PCR-based SAP (sequence amplified polymorphism) marker was used to identify Piz-5 in the segregating population. The plants carrying the two- and three-gene combinations that were tested for resistance to leaf blast in the Philippines and India indicated that combinations including Piz-5 have enhanced resistance than when it is present alone. The genes from the pyramided lines are at present being deployed into agronomically superior ricevarieties by marker-aided selection (MAS). Pi1-5,Pi1-6,Pita|Pi-4a Identification of microsatellite markers linked to the blast resistance gene Pi-1(t) in rice 2007 Euphytica Escuela de Biología, Facultad de Ciencias, Universidad Industrial de Santander (UIS), Apartado Aéreo 678, Bucaramanga, Colombia The present work was conducted to identify microsatellite markers linked to the rice blast resistance gene Pi-1(t) for a marker-assisted selection program. Twenty-four primer pairs corresponding to 19 microsatellite loci were selected from the Gramene database (www. gramene.org) considering their relative proximity to Pi-1(t) gene in the current rice genetic map. Progenitors and DNA bulks of resistant and susceptible families from F3 segregating populations of a cross between the near-isogenic lines C101LAC (resistant) and C101A51 (susceptible) were used to identify polymorphic microsatellite markers associated to this gene through bulked segregant analysis. Putative molecular markers linked to the blast resistance gene Pi-1(t) were then used on the whole progeny for linkage analysis. Additionally, the diagnostic potential of the microsatellite markers associated to the resistance gene was also evaluated on 17 rice varieties planted in Latin America by amplification of the specific resistant alleles for the gene in each genotype. Comparing with greenhouse phenotypic evaluations for blast resistance, the usefulness of the highly linked microsatellite markers to identify resistant rice genotypes was evaluated. As expected, the phenotypic segregation in the F3 generation agreed to the expected segregation ratio for a single gene model. Of the 24 microsatellite sequences tested, six resulted polymorphic and linked to the gene. Two markers (RM1233*I and RM224) mapped in the same position (0.0 cM) with the Pi-1(t) gene. Other three markers corresponding to the same genetic locus were located at 18.5 cM above the resistance gene, while another marker was positioned at 23.8 cM below the gene. Microsatellite analysis on elite rice varieties with different genetic background showed that all known sources of blast resistance included in this study carry the specific Pi-1(t) allele. Results are discussed considering the potential utility of the microsatellite markers found, for MAS in rice breeding programs aiming at developing rice varieties with durable blast resistance based on a combination of resistance genes. Pi1-5,Pi1-6 Improving blast resistance of Jin 23B and its hybrid rice by marker-assisted gene pyramiding 2012 Molecular Breeding National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research and National Center of Crop Molecular Breeding, Huazhong Agricultural University, Wuhan, 430070, China Rice blast is one of the most serious diseases in rice (Oryza sativa L.) worldwide. Jin 23B is the maintainer line, a parent for a number of hybrid rice varieties used widely in China. However, Jin 23B is highly susceptible to rice blast. In this study, Pi1, Pi2, and D12 were introgressed to improve the blast resistance of Jin 23B and its derived hybrids, Jinyou 402 and Jinyou 207, by marker-assisted selection (MAS). The improved Jin 23B, which carried single, two, and three genes, were evaluated for their resistance to rice blast using natural inoculation methods in disease nursery of Xianfeng, Hubei, China. The results showed that, the greater the number of genes contained in the improved Jin 23B and hybrids, the higher the resistance to rice blast. Pi1, Pi2, and D12 showed a strong dosage effect on the resistance to blast in the hybrid background during the entire growth duration in the field condition, being very useful for breeding blast-resistant hybrids. The result of examining agronomic traits showed that the improved Jin 23B and its derived hybrid rice were taller than or similar to controls, when there was no disease stress. Pi1-5,Pi1-6 Molecular mapping of rice blast resistance gene Pi-1(t) in the elite indica variety Samba mahsuri 2009 World Journal of Microbiology and Biotechnology Department of Biotechnology, Directorate of Rice Research, Rajendranagar, Hyderabad, Andhra Pradesh, 500030, India Samba mahsuri (BPT 5204) is a cultivar of the medium slender grain indica variety of Oryza sativa grown across India for its high yield and quality. However, this cultivar is susceptible to several diseases and pests including rice blast. The analysis of near isogenic lines indicated the presence of a resistance gene, Pi-1(t) in the donor cultivar C101LAC which is highly resistant to the rice blast fungus Magnaporthe grisea (M. grisea). C101LAC was crossed with susceptible indica rice cultivar (BPT 5204) to generate the mapping population. A mendelian segregation ratio of 3:1 for resistant to susceptible F2 plants using bulk segregation analysis confirmed the presence of a major gene pi-1(t) by simple sequence repeats marker RM224 to the highly virulent blast isolate DRR 001. Pi1-5,Pi1-6 Rice Blast Resistance Gene Pi1 Identified by MRG4766 Marker in 173 Yunnan Rice Landraces 2012 Rice Genomics and Genetics Agricultural Environment and Resources Research Institute, Yunnan Academy of Agricultural Sciences, Kunming, 650205, P.R. China Rice Blast is one of the most serious rice diseases and caused great yield losses every year in the world. It had been proved that using resistant rice varieties would be the most effective way to control this disease; therefore, mining the resistant genes might be important foundational work in the breeding program. In the present study, we identified the existence of the Pi1 gene among the tested 173 landraces collected from Yunnan Province by using the SSR marker MRG4766. The results showed that the Pi1 gene was found in 64 landraces, accounting for 36.99% in total of 173 landraces. 33 of 102 indica landraces (32.35%) and 31 of 71 japonica landraces (43.66%) both contained Pi1 gene. The landraces harboring the Pi1 gene were distributed in 29 counties of 11 prefectures in Yunnan province. The distribution frequency of Pi1 gene was higher in paddy-upland rice region in South margin of Yunnan (41.03%) than that of in single and or double indica cropping region in south of Yunnan (32.69%). The distribution frequency of Pi1 gene was 35.29% in single indica and or japonica cropping region in center of Yunnan, 35.29% in japonica cropping region in northeast of Yunnan and 33.33% in japonica cropping region in northwest highland of Yunnan. Pi1-5,Pi1-6 Three types of defense-responsive genes are involved in resistance to bacterial blight and fungal blast diseases in rice 2003 Mol Genet Genomics National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding, Huazhong Agricultural University, 430070, Wuhan, China. Bacterial blight and fungal blast diseases of rice, caused by Xanthomonas oryzae pv. oryzae and Pyricularia grisea Sacc., respectively, are two of the most devastating diseases in rice worldwide. To study the defense responses to infection with each of these pathogens, expression profiling of 12 defense-responsive genes was performed using near-isogenic rice lines that are resistant or susceptible to bacterial blight and fungal blast, respectively, and rice cultivars that are resistant or susceptible to both pathogens. All 12 genes showed constitutive expression, but expression levels increased in response to infection. Based on their expression patterns in 12 host-pathogen combinations, these genes could be classified into three types, pathogen non-specific (6), pathogen specific but race non-specific (4) and race specific (2). Most of the 12 genes were only responsive during incompatible interactions. These results suggest that bacterial blight and fungal blast resistances share common pathway(s), but are also regulated by different defense pathways in rice. Activation of the corresponding R gene is the key step that initiates the action of these genes in defense responses. The chromosomal locations and pathogen specificities of seven of the 12 genes were consistent with those of previously identified quantitative trait loci for rice disease resistance, which indicates that some of the 12 genes studied may have a phenotypic impact on disease resistance in rice. Pi1-5,Pi1-6 Molecular mapping of genes for resistance to rice blast (Pyricularia grisea Sacc.) 1996 Theor Appl Genet Department of Plant Breeding and Biometry, Cornell University, 14853, Ithaca, NY, USA. Two dominant genes conferring complete resistance to specific isolates of the rice blast fungus, Pyricularia grisea Sacc., were located on the molecular map of rice in this study. Pi-l(t) is a blast resistance gene derived from the cultivar 'LAC23'. Its map location was determined using a pair of nearly isogenic lines (NILs) and a B6F3 segregating population from which the isoline was derived. RFLP analysis showed that Pi-l(t) is located near the end of chromosome 11, linked to RZ536 at a distance of 14.0+/-4.5 centiMorgans (cM). A second gene, derived from the cultivar 'Apura', was mapped using a rice doubled-haploid (DH) population. This gene was located on chromosome 12, flanked by RG457 and RG869, at a distance of 13.5+-4.3 cM and 17.7+-4.5 cM, respectively. The newly mapped gene on chromosome 12 may be allelic or closely linked toPi-ta. (=Pi-4(t)), a gene derived from Tetep that was previously reported to be linked to RG869 at a distance of 15.4+/-4.7 cM. The usefulness of markers linked to blast resistance genes will be discussed in the context of breeding for durable blast resistance. Pi1-5,Pi1-6 The isolation of Pi1, an allele at the Pik locus which confers broad spectrum resistance to rice blast 2012 Theor Appl Genet State Key Laboratory for Conservation and Utilization of Subtropic Agrobioresources, Laboratory of Plant Resistance and Genetics, College of Natural Resources and Environmental Sciences, South China Agricultural University, Guangzhou 510642, China. We report the isolation of Pi1, a gene conferring broad-spectrum resistance to rice blast (Magnaporthe oryzae). Using loss- and gain-of-function approaches, we demonstrate that Pi1 is an allele at the Pik locus. Like other alleles at this locus, Pi1 consists of two genes. A functional nucleotide polymorphism (FNP) was identified that allows differentiation of Pi1 from other Pik alleles and other non-Pik genes. A extensive germplasm survey using this FNP reveals that Pi1 is a rare allele in germplasm collections and one that has conferred durable resistance to a broad spectrum of pathogen isolates. Pi1-5,Pi1-6 QTL analysis and mapping of pi21 , a recessive gene for field resistance to rice blast in Japanese upland rice 2001 TAG Theoretical and Applied Genetics Department of Genetic Resources I, National Institute of Agrobiological Resources, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602 Japan Field resistance is defined as the resistance that allows effective control of a parasite under natural field conditions and is durable when exposed to new races of that parasite. To identify the genes for field resistance to rice blast, quantitative trait loci (QTLs) conferring field resistance to rice blast in Japanese upland rice were detected and mapped using RFLP and SSR markers. QTL analysis was carried out in F4 progeny lines from the cross between Nipponbare (moderately susceptible, lowland) and Owarihatamochi (resistant, upland). Two QTLs were detected on chromosome 4 and one QTL was detected on each of chromosomes 9 and 12. The phenotypic variation explained by each QTL ranged from 7.9 to 45.7% and the four QTLs explained 66.3% of the total phenotypic variation. Backcrossed progeny lines were developed to transfer the QTL with largest effect using the susceptible cultivar Aichiasahi as a recurrent parent. Among 82 F3 lines derived from the backcross, resistance segregated in the expected ratio of resistant 1 : heterozygous 2 : susceptible 1. The average score for blast resistance measured in the field was 4.2 ± 0.67, 7.5 ± 0.51and 8.2 ± 0.66, for resistant, heterozygous and susceptible groups, respectively. The resistance gene, designated pi21, was mapped on chromosome 4 as a single recessive gene between RFLP marker loci G271 and G317 at a distance of 5.0 cM and 8.5 cM, respectively. The relationship to previously reported major genes and QTLs conferring resistance to blasts, and the significance of marker-assisted selection to improve field resistance, are discussed. Pi21 Loss of function of a proline-containing protein confers durable disease resistance in rice 2009 Science QTL Genomics Research Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. fukusan@affrc.go.jp Blast disease is a devastating fungal disease of rice, one of the world's staple foods. Race-specific resistance to blast disease has usually not been durable. Here, we report the cloning of a previously unknown type of gene that confers non-race-specific resistance and its successful use in breeding. Pi21 encodes a proline-rich protein that includes a putative heavy metal-binding domain and putative protein-protein interaction motifs. Wild-type Pi21 appears to slow the plant's defense responses, which may support optimization of defense mechanisms. Deletions in its proline-rich motif inhibit this slowing. Pi21 is separable from a closely linked gene conferring poor flavor. The resistant pi21 allele, which is found in some strains of japonica rice, could improve blast resistance of rice worldwide. Pi21 A Pid3 allele from rice cultivar Gumei2 confers resistance to Magnaporthe oryzae 2011 J Genet Genomics State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China. Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases. Using map-based strategy and in silico approach we isolated a new rice (Oryza sativa L.) blast resistance allele of Pid3, designated Pi25, from a stable blast resistance cultivar Gumei2. Over-expression analysis and complementation test showed that Pi25 conferred blast resistance to M. oryzae isolate js001-20. Sequence analysis showed that Pi25 was an intronless gene of 2,772 nucleotides with single nucleotide substitution in comparison to Pid3 at the nucleotide position 459 and predicatively encoded a typical coiled coil--nucleotide binding site--leucine rich repeat (CC--NBS--LRR) protein of 924 amino acid residuals with 100% identity to Pid3 putative protein. The susceptible allele pi25 in Nipponbare contained a nonsense mutation at the nucleotide position 2,209 resulting in a truncated protein with 736 amino acid residuals. In addition, 14 nucleotide substitutions resulting in 10 amino acid substitutions were identified between Pi25 and pi25 upstream the premature stop codon in the susceptible allele. Although the mechanism of Pi25/Pid3-mediated resistance needs to be further investigated, the isolation of the allele would facilitate the utilization of Pi25/Pid3 in rice blast resistance breeding program via transgenic approach and marker assisted selection. Pi25|Pid3 Mapping of leaf and neck blast resistance genes with resistance gene analog, RAPD and RFLP in rice 2002 Euphytica China National Rice Research Institute, National Center for Rice Improvement, Hangzhou, 310006, China An F-8 recombinant inbred population was constructed using a commercial indica rice variety Zhong 156 as the female parent and a semidwarf indica variety Gumei 2 with durable resistance to rice blast as the male parent. Zhong 156 is resistant to the fungus race ZC(15) at the seedling stage but susceptible to the same race at the flowering stage. Gumei 2 is resistant to ZC15 at both stages. The blast resistance of 148 recombinant inbred lines was evaluated using the blast race ZC15. Genetic analysis indicated that the resistance to leaf blast was controlled by three genes and the presence of resistant alleles at any loci would result in resistance. One of the three genes did not have effects at the flowering stage. Two genes, tentatively assigned as Pi24(t) and Pi25(t), were mapped onto chromosome 12 and 6, respectively, based on RGA (resistance gene analog), RFLP and RAPD markers. Pi24( t) conferred resistance to leaf blast only, and its resistance allele was from Zhong 156. Pi25( t) conferred resistance to both leaf and neck blast, and its resistance allele was from Gumei 2. In a natural infection test in a blast hot-spot, Pi25(t) exhibited high resistance to neck blast, while Pi24(t) showed little effect. Pi25|Pid3 Identification of a new rice blast resistance gene, Pid3, by genomewide comparison of paired nucleotide-binding site--leucine-rich repeat genes and their pseudogene alleles between the two sequenced rice genomes 2009 Genetics State Key Laboratory of Plant Genomics and National Plant Gene Research Centre, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases. The two major subspecies of Asian cultivated rice (Oryza sativa L.), indica and japonica, have shown obvious differences in rice blast resistance, but the genomic basis that underlies the difference is not clear. We performed a genomewide comparison of the major class of resistant gene family, the nucleotide-binding site-leucine-rich repeat (NBS-LRR) gene family, between 93-11 (indica) and Nipponbare (japonica) with a focus on their pseudogene members. We found great differences in either constitution or distribution of pseudogenes between the two genomes. According to this comparison, we designed the PCR-based molecular markers specific to the Nipponbare NBS-LRR pseudogene alleles and used them as cosegregation markers for blast susceptibility in a segregation population from a cross between a rice blast-resistant indica variety and a susceptible japonica variety. Through this approach, we identified a new blast resistance gene, Pid3, in the indica variety, Digu. The allelic Pid3 loci in most of the tested japonica varieties were identified as pseudogenes due to a nonsense mutation at the nucleotide position 2208 starting from the translation initiation site. However, this mutation was not found in any of the tested indica varieties, African cultivated rice varieties, or AA genome-containing wild rice species. These results suggest that the pseudogenization of Pid3 in japonica occurred after the divergence of indica and japonica. Pi25|Pid3,Pi9|Piz-t|Pi2 Genetic control of rice blast resistance in the durably resistant cultivar Gumei 2 against multiple isolates 2005 Theor Appl Genet Chinese National Centre for Rice Improvement and State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China. To further our understanding of the genetic control of blast resistance in rice cultivar Gumei 2 and, consequently, to facilitate the utilization of this durably blast-resistant cultivar, we studied 304 recombinant inbred lines of indica rice cross Zhong 156/Gumei 2 and a linkage map comprising 181 markers. An analysis of segregation for resistance against five isolates of rice blast suggested that one gene cluster and three additional major genes that are independently inherited are responsible for the complete resistance of Gumei 2. The gene cluster was located to chromosome 6 and includes two genes mapped previously, Pi25(t), against Chinese rice blast isolate 92-183 (race ZC15) and Pi26(t) against Philippine rice blast isolate Ca89 (lineage 4), and a gene for resistance against Philippine rice blast isolate 92330-5 (lineage 17). Of the two genes conferring resistance against the Philippine isolates V86013 (lineage 15) and C923-39 (lineage 46), we identified one as Pi26(t) and mapped the other onto the distal end of chromosome 2 where Pib is located. We used three components of partial blast resistance, percentage diseased leaf area (DLA), lesion number and lesion size, all measured in the greenhouse, to measure the degree of susceptibility to isolates Ca89 and C923-39 and subsequently identified nine and eight quantitative trait loci (QTLs), respectively. Epistasis was determined to play an important role in partial resistance against Ca89. Using DLA measured on lines susceptible in a blast nursery, we detected six QTLs. While different QTLs were detected for partial resistance to Ca89 and C923-39, respectively, most were involved in the partial resistance in the field. Our results suggest that the blast resistance in Gumei 2 is controlled by multiple major genes and minor genes with epistatic effects. Pi25|Pid3 Study on the interaction between methyl jasmonate and the coiled-coil domain of rice blast resistance protein Pi36 by spectroscopic methods 2012 Spectrochim Acta A Mol Biomol Spectrosc Key Biotechnology Laboratory of State Ethnic Affairs Commission, College of Life Science, South-Central University for Nationalities, Wuhan, China. Interaction between the coiled-coil domain of rice blast resistance protein Pi36 and methyl-jasmonate (MeJA) was studied by fluorescence and UV-vis spectroscopic techniques. The quenching mechanism of fluorescence of MeJA by this domain was discussed to be a static quenching procedure. Fluorescence quenching was explored to measure the number of binding sites n and apparent binding constants K. The thermodynamics parameters DeltaH, DeltaG, DeltaS were also calculated. The results indicate the binding reaction was not entropy-driven but enthalpy-driven, and hydrophobic binding played major role in the interaction. The binding sites of MeJA with the coiled-coil structural domain of rice blast resistance protein Pi36 were found to approach the microenvironment of both Tyr and Trp by the synchronous fluorescence spectrometry. The distance r between donor (the coiled-coil domain of rice blast resistance protein Pi36) and acceptor (MeJA) was obtained according to Forster theory of non-radioactive energy transfer. Pi36 The in silico map-based cloning of Pi36, a rice coiled-coil nucleotide-binding site leucine-rich repeat gene that confers race-specific resistance to the blast fungus 2007 Genetics Laboratory of Plant Resistasnce and Genetics, College of Resources and Environmental Sciences, South China Agricultural University, Guangzhou 510642, China. The indica rice variety Kasalath carries Pi36, a gene that determines resistance to Chinese isolates of rice blast and that has been located to a 17-kb interval on chromosome 8. The genomic sequence of the reference japonica variety Nipponbare was used for an in silico prediction of the resistance (R) gene content of the interval and hence for the identification of candidate gene(s) for Pi36. Three such sequences, which all had both a nucleotide-binding site and a leucine-rich repeat motif, were present. The three candidate genes were amplified from the genomic DNA of a number of varieties by long-range PCR, and the resulting amplicons were inserted into pCAMBIA1300 and/or pYLTAC27 vectors to determine sequence polymorphisms correlated to the resistance phenotype and to perform transgenic complementation tests. Constructs containing each candidate gene were transformed into the blast-susceptible variety Q1063, which allowed the identification of Pi36-3 as the functional gene, with the other two candidates being probable pseudogenes. The Pi36-encoded protein is composed of 1056 amino acids, with a single substitution event (Asp to Ser) at residue 590 associated with the resistant phenotype. Pi36 is a single-copy gene in rice and is more closely related to the barley powdery mildew resistance genes Mla1 and Mla6 than to the rice blast R genes Pita, Pib, Pi9, and Piz-t. An RT-PCR analysis showed that Pi36 is constitutively expressed in Kasalath. Pi36 Genetic and physical mapping of Pi36(t), a novel rice blast resistance gene located on rice chromosome 8 2005 Mol Genet Genomics Laboratory of Plant Resistance and Genetics, College of Natural Resources and Environmental Sciences, South China Agricultural University, Guangzhou, 510642, China. Blast resistance in the indica cultivar (cv.) Q61 was inherited as a single dominant gene in two F2 populations, F2-1 and F2-2, derived from crosses between the donor cv. and two susceptible japonica cvs. Aichi Asahi and Lijiangxintuanheigu (LTH), respectively. To rapidly determine the chromosomal location of the resistance (R) gene detected in Q61, random amplified polymorphic DNA (RAPD) analysis was performed in the F2-1 population using bulked-segregant analysis (BSA) in combination with recessive-class analysis (RCA). One of the three linked markers identified, BA1126(550), was cloned and sequenced. The R gene locus was roughly mapped on rice chromosome 8 by comparison of the BA1126(550) sequence with rice sequences in the databases (chromosome landing). To confirm this finding, seven known markers, including four sequence-tagged-site (STS) markers and three simple-sequence repeat (SSR) markers flanking BA1126(550) on chromosome 8, were subjected to linkage analysis in the two F2 populations. The locus was mapped to a 5.8 cM interval bounded by RM5647 and RM8018 on the short arm of chromosome 8. This novel R gene is therefore tentatively designated as Pi36(t). For fine mapping of the Pi36(t) locus, five additional markers including one STS marker and four candidate resistance gene (CRG) markers were developed in the target region, based on the genomic sequence of the corresponding region of the reference japonica cv. Nipponbare. The Pi36(t) locus was finally localized to an interval of about 0.6 cM flanked by the markers RM5647 and CRG2, and co-segregated with the markers CRG3 and CRG4. To physically map this locus, the Pi36(t)-linked markers were mapped by electronic hybridization to bacterial artificial chromosome (BAC) or P1 artificial chromosome (PAC) clones of Nipponbare, and a contig map was constructed in silico through Pairwise BLAST analysis. The Pi36(t) locus was physically delimited to an interval of about 17.0 kb, based on the genomic sequence of Nipponbare. Pi36 The effect of the rice blast resistance gene Pi36 on the expression of disease resistance-related genes 2010 Chinese Science Bulletin Key Biotechnology Laboratory of State Ethnic Affairs Commission, College of Life Science, South-Central University for Nationalities, Wuhan, 430074, China The resistance gene Pi36 confers a stable and high level of resistance against Chinese isolates of the rice blast pathogen Magnaporthe oryzae. Quantitative real time RT-PCR was used to investigate the expression profiles of various resistance-related genes in both the Pi36/AvPi36 incompatible interactions. Among 17 members of the OsMAPK family, OsMAPK2, OsMAPK4, OsMAPK8 and OsMAPK15 were differentially expressed, as were OsWRKY30, OsWRKY32, OsWRKY64 and OsWRKY67 among the 13 OsWRKY members studied. The induction of the expression of genes from both of these families suggests that both the JA and SA pathways are involved in Pi36-mediated defence. Other genes differentially expressed included the OsbZIP transcription factors, OsNIF1 and OsNIF2, and the NPR1 homologue OsNH2, all of which were reported in the rice blast pathosystem. The involvement of these genes illustrates the complexity of the downstream signalling pathways involved in the Pi36/AvrPi36 interaction. Pi36 Genetic and physical mapping of Pi37(t), a new gene conferring resistance to rice blast in the famous cultivar St. No. 1 2005 Theor Appl Genet Laboratory of Plant Resistance and Genetics, College of Resources and Environmental Sciences, South China Agricultural University, Guangzhou 510642, China. The famous rice cultivar (cv.), St. No. 1, confers complete resistance to many isolates collected from the South China region. To effectively utilize the resistance, a linkage assay using microsatellite markers (SSR) was performed in the three F2 populations derived from crosses between the donor cv. St. No. 1 and each of the three susceptible cvs. C101PKT, CO39 and AS20-1, which segregated into 3R:1S (resistant/susceptible) ratio, respectively. A total of 180 SSR markers selected from each chromosome equally were screened. The result showed that the two markers RM128 and RM486 located on chromosome 1 were linked to the resistance gene in the respective populations above. This result is not consistent with those previously reported, in which a well-known resistance gene Pif in the St. No. 1 is located on chromosome 11. To confirm this result, additional four SSR markers, which located in the region lanked by RM128 and RM486, were tested. The results showed that markers RM543 and RM319 were closer to, and RM302 and RM212 completely co-segregated with the resistance locus detected in the present study. These results indicated that another resistance gene involved in the St. No. 1, which is located on chromosome 1, and therefore tentatively designated as Pi37(t). To narrow down genomic region of the Pi37(t) locus, eight markers were newly developed in the target region through bioinformatics analysis (BIA) using the publicly available sequences. The linkage analysis with these markers showed that the Pi37(t) locus was mapped to a approximately 0.8 centimorgans (cM) interval flanked by RM543 and FPSM1, where a total of seven markers co-segregated with it. To physically map the locus, the Pi37(t)-linked markers were landed on the reference sequence of cv. Nipponbare through BIA. A contig map corresponding to the locus was constructed based on the reference sequence aligned by the Pi37(t)-linked markers. Consequently, the Pi37(t) locus was defined to 374 kb interval flanking markers RM543 and FPSM1, where only four candidate genes with the resistance gene conserved structure (NBS-LRR) were further identified to a DNA fragment of 60 kb in length by BIA. Pi37 The blast resistance gene Pi37 encodes a nucleotide binding site leucine-rich repeat protein and is a member of a resistance gene cluster on rice chromosome 1 2007 Genetics Laboratory of Plant Resistance and Genetics, College of Resources and Environmental Sciences, South China Agricultural University, Guangzhou, 510642, China. The resistance (R) gene Pi37, present in the rice cultivar St. No. 1, was isolated by an in silico map-based cloning procedure. The equivalent genetic region in Nipponbare contains four nucleotide binding site-leucine-rich repeat (NBS-LRR) type loci. These four candidates for Pi37 (Pi37-1, -2, -3, and -4) were amplified separately from St. No. 1 via long-range PCR, and cloned into a binary vector. Each construct was individually transformed into the highly blast susceptible cultivar Q1063. The subsequent complementation analysis revealed Pi37-3 to be the functional gene, while -1, -2, and -4 are probably pseudogenes. Pi37 encodes a 1290 peptide NBS-LRR product, and the presence of substitutions at two sites in the NBS region (V239A and I247M) is associated with the resistance phenotype. Semiquantitative expression analysis showed that in St. No. 1, Pi37 was constitutively expressed and only slightly induced by blast infection. Transient expression experiments indicated that the Pi37 product is restricted to the cytoplasm. Pi37-3 is thought to have evolved recently from -2, which in turn was derived from an ancestral -1 sequence. Pi37-4 is likely the most recently evolved member of the cluster and probably represents a duplication of -3. The four Pi37 paralogs are more closely related to maize rp1 than to any of the currently isolated rice blast R genes Pita, Pib, Pi9, Pi2, Piz-t, and Pi36. Pi37 Use of Pi5(t) markers in marker-assisted selection to screen for cultivars with resistance to Magnaporthe grisea 2004 Theor Appl Genet National Yeongnam Agricultural Experiment Station, Rural Development Administration, 1085, Neidong, Milyang, 627-803, Korea. Identification of the PCR markers tightly linked to genes that encode important agronomic traits is useful for marker-assisted selection (MAS). The rice Pi5(t) locus confers broad-spectrum resistance to Magnaporthe grisea, the causal agent of rice blast disease. It has been hypothesized that the Pi5(t) locus carries the same gene as that encoded by the Pi3(t) and Pii(t) loci. We developed three PCR-based dominant markers (JJ80-T3, JJ81-T3, and JJ113-T3) from three previously identified BIBAC clones-JJ80, JJ81, and JJ113-that are linked to the Pi5(t) locus. PCR analysis of 24 monogenic lines revealed that these markers are present only in lines that carry Pi5(t), Pi3(t), and Pii(t). PCR and DNA gel-blot analysis of candidate resistance lines using JJ80-T3, JJ81-T3, and JJ113-T3 indicated that Tetep is the likely donor of Pi5(t). Of the 184 rice varieties tested, 34 carried the JJ80-T3-, JJ81-T3-, and JJ113-T3-specific bands. Disease evaluation of those 34 varieties revealed that all conferred resistance to PO6-6. The genomic structure of three of these resistant varieties (i.e., IR72, Taebaeg, Jahyangdo) is most similar to that of Pi5(t). Our results demonstrate the usefulness of the JJ80-T3, JJ81-T3, and JJ113-T3 markers for MAS for M. grisea resistance. Pi5-1,Pi5-2 Rice Pi5-mediated resistance to Magnaporthe oryzae requires the presence of two coiled-coil-nucleotide-binding-leucine-rich repeat genes 2009 Genetics Plant Metabolism Research Center and Graduate School of Biotechnology, Kyung Hee University, Yongin, Korea. Rice blast, caused by the fungus Magnaporthe oryzae, is one of the most devastating diseases of rice. To understand the molecular basis of Pi5-mediated resistance to M. oryzae, we cloned the resistance (R) gene at this locus using a map-based cloning strategy. Genetic and phenotypic analyses of 2014 F2 progeny from a mapping population derived from a cross between IR50, a susceptible rice cultivar, and the RIL260 line carrying Pi5 enabled us to narrow down the Pi5 locus to a 130-kb interval. Sequence analysis of this genomic region identified two candidate genes, Pi5-1 and Pi5-2, which encode proteins carrying three motifs characteristic of R genes: an N-terminal coiled-coil (CC) motif, a nucleotide-binding (NB) domain, and a leucine-rich repeat (LRR) motif. In genetic transformation experiments of a susceptible rice cultivar, neither the Pi5-1 nor the Pi5-2 gene was found to confer resistance to M. oryzae. In contrast, transgenic rice plants expressing both of these genes, generated by crossing transgenic lines carrying each gene individually, conferred Pi5-mediated resistance to M. oryzae. Gene expression analysis revealed that Pi5-1 transcripts accumulate after pathogen challenge, whereas the Pi5-2 gene is constitutively expressed. These results indicate that the presence of these two genes is required for rice Pi5-mediated resistance to M. oryzae. Pi5-1,Pi5-2 Genetic and physical mapping of Pi5(t), a locus associated with broad-spectrum resistance to rice blast 2003 Mol Genet Genomics Department of Plant Pathology, University of California, One Shields Avenue, Davis 95616, USA. To gain an understanding of the molecular basis for resistance to rice blast (Magnaporthe grisea), we have initiated a project to clone Pi5(t), a locus associated with broad-spectrum resistance to diverse blast isolates. AFLP-derived markers linked to Pi5(t)-mediated resistance were isolated using bulked segregant analysis of F(2) populations generated by crossing three recombinant inbred lines (RILs), RIL125, RIL249, and RIL260 with the susceptible line CO39. The most tightly linked AFLP marker, S04G03, was positioned on chromosome 9 of the fingerprint-based physical map of Nipponbare, a well-characterized rice genotype. Flanking BAC-based Nipponbare markers were generated for saturation mapping using four populations, the three initial RILs and an additional one derived from a cross between M202 and RIL260. A BIBAC (binary BAC) library was constructed from RIL260. Using these resources Pi5(t) was mapped to a 170-kb interval, and a contiguous set of BIBAC clones spanning this region was constructed. It had previously been suggested that Pi3(t) and Pi5(t) might be allelic, due to their identical resistance spectrum and tight linkage. We therefore compared genomic regions for lines containing Pi3(t) using the Pi5(t)-linked markers. DNA gel-blot analyses indicated that the region around Pi3(t) is identical to that of Pi5(t), suggesting that Pi3(t) and Pi5(t) are the same resistance gene. Pi5-1,Pi5-2 The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine-rich repeat class of plant disease resistance genes 1999 The Plant Journal Rice Genome Research Program, Institute of the Society for Techno-Innovation of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki, Japan. Rice blast, caused by the fungal pathogen Magnaporthe grisea, is one of the most serious diseases of rice. Here we describe the isolation and characterization of Pib, one of the rice blast resistance genes. The Pib gene was isolated by a map-based cloning strategy. The deduced amino acid sequence of the Pib gene product contains a nucleotide binding site (NBS) and leucine-rich repeats (LRRs); thus, Pib is a member of the NBS-LRR class of plant disease resistance genes. Interestingly, a duplication of the kinase 1a, 2 and 3a motifs of the NBS region was found in the N-terminal half of the Pib protein. In addition, eight cysteine residues are clustered in the middle of the LRRs, a feature which has not been reported for other R genes. Pib gene expression was induced upon altered environmental conditions, such as altered temperatures and darkness. Pib Development of pyramided lines with two resistance genes, Pish and Pib, for blast disease (Magnaporthe oryzae B. Couch) in rice (Oryza sativa L.) 2010 Plant Breeding International Rice Research Institute (IRRI), DAPO Box 7777, Metro Manila, Philippines Gene pyramiding is considered one of the most effective strategies for achieving durable resistance against blast disease (Magnaporthe oryzae B. Couch) in rice (Oryza sativa L.), although few studies have evaluated the combining effect of the resistance genes. We report the development of pyramided lines with two major blast resistance genes, Pish and Pib, and the evaluation of the combining effect of them. The two genes pyramided lines were selected from the progenies of a cross between one near isogenic line (NIL), which harbours Pish, and another NIL, which harbours Pib, in the genetic background of blast susceptible variety, CO 39. The presence of the resistance genes was confirmed by DNA markers linked to them. To obtain DNA markers for Pish, we genetically mapped the Pish locus. We confirmed the additive effect of Pish and Pib in the pyramided lines by their reaction patterns to blast isolates, suggesting the potential availabilities of the combinations of these genes. In addition, we provide DNA markers linked to Pish for marker aided selection in rice blast resistance breeding. Pib The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine-rich repeat class of plant disease resistance genes 1999 Plant J Rice Genome Research Program, Institute of the Society for Techno-Innovation of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki, Japan. Rice blast, caused by the fungal pathogen Magnaporthe grisea, is one of the most serious diseases of rice. Here we describe the isolation and characterization of Pib, one of the rice blast resistance genes. The Pib gene was isolated by a map-based cloning strategy. The deduced amino acid sequence of the Pib gene product contains a nucleotide binding site (NBS) and leucine-rich repeats (LRRs); thus, Pib is a member of the NBS-LRR class of plant disease resistance genes. Interestingly, a duplication of the kinase 1a, 2 and 3a motifs of the NBS region was found in the N-terminal half of the Pib protein. In addition, eight cysteine residues are clustered in the middle of the LRRs, a feature which has not been reported for other R genes. Pib gene expression was induced upon altered environmental conditions, such as altered temperatures and darkness. PIBH8 Expression of the Pib rice-blast-resistance gene family is up-regulated by environmental conditions favouring infection and by chemical signals that trigger secondary plant defences 2001 Plant Mol Biol Institute of the Society for Techno-Innovation of Agriculture, Forestry and Fisheries, Ippaizuka 446-1, Kamiyokoba, Tsukuba, Ibaraki, 305-0854, Japan The rice blast resistance gene Pib is a member of the nucleotide binding site (NBS) and leucine-rich repeat (LRR) class of plant disease resistance (R) genes and belongs to a small gene family. We describe here the isolation and characterization of a Pib homologue (PibH8), and extensive investigation of the expression of the Pib gene family (Pib, PibH8, HPibH8-1, HPibH8-2) under various environmental and chemical treatments. PibH8 shows 42% identity and 60% similarity to Pib and, like Pib, has a duplication of the kinase 1a, 2, and 3a motifs of the NBS region in the N-terminal half of the protein. Interestingly, genes of the Pib family exhibit a diurnal rhythm of expression. RNA gel blot analysis revealed that their expression was regulated dramatically by environmental signals, such as temperature, light and water availability. Their expression was also induced by chemical treatments, such as jasmonic acid, salicylic acid, ethylene and probenazole. Our findings suggest that expression of the Pib gene family is up-regulated by environmental conditions that would favour pathogen infection. This may reflect the evolution of anticipatory control of R gene expression. PIBH8 Genetic Transformation of Rice with Pi-d2 Gene Enhances Resistance to Rice Blast Fungus Magnaporthe oryzae 2010 Rice Science Rice Research Institute, Sichuan Agricultural University, Wenjiang 611130, China The gene Pi-d2, conferring gene-for-gene resistance to the Chinese blast strain ZB15, was isolated from a rice variety (Digu) by the map-based cloning strategy. Here, we constructed a control plasmid pZH01-pi-d2tp309 (pZH01-tp309) and three different expression constructs, pCB-Pi-d25.3kb (pCB5.3kb), pCB-Pi-d26.3kb (pCB6.3kb) and pZH01-Pi-d22.72kb (pZH01-2.72kb) of Pi-d2, driven by Pi-d2 gene's own promoter or CaMV35S promoter. These constructs were separately introduced into japonica rice varieties Lijiangxintuanhegu, Taipei 309, Nipponbare and Zhonghua 9 through Agrobacterium-mediated transformation. A total of 150 transgenic rice plants were obtained from the regenerated calli selected on hygromycin. PCR, RT-PCR and Southern-blotting assay showed that the gene of interest had been integrated into rice genome and stably inherited. Thirty-five transgenic lines independently derived from T1 progeny were inoculated with the rice blast strain ZB15. Transformants exhibited resistance to rice blast at various levels. The lesions on the transgenic plant leaves were less severe than those on the controls and the resistance level of transgenic plants harboring the gene of interest from three vectors had no difference. The own promoter of Pi-d2, about 2.2 kb or 3.2 kb, had the similar promoter function as CaMV35S. Field evaluation for three successive years supported the results of artificial trial, and some lines with high resistance to rice leaf blast and neck blast were obtained. OsPK10|Pid2 Rice Blast Resistance of Transgenic Rice Plants with Pi-d2 Gene 2010 Rice Science Institute of Plant Protection, Sichuan Academy of Agricultural Sciences, Chengdu 610066, China Resistance to rice blast of transgenic rice lines harboring rice blast resistance gene Pi-d2 transformed from three different expression vectors of pCB6.3kb, pCB5.3kb and pZH01-2.72kb were analyzed. Nine advanced-generation transgenic rice lines with Pi-d2 gene displayed various resistance to 39 rice blast strains, and the highest disease-resistant frequency reached 91.7%. Four early-generation homozygous transgenic lines with Pi-d2 gene exhibited resistance to more than 81.5% of 58 rice blast strains, showing the characteristic of wide-spectrum resistance. The transgenic embryonic calli selected by the crude toxin of rice blast fungus showed that the callus induction rate of immature embryo from transgenic rice plants decreased as the concentration of crude toxin in the culture medium increased. When the concentration of crude toxin reached 40%, the callus induction rate of immature embryo from transgenic lines was 49.3%, and that of the receptor control was 5%. The disease incidence of neck blast of the transgenic rice lines in fields under induction was 0% to 50%, indicating that the rice blast resistance of transgenic rice lines is much higher than that of the receptor control. OsPK10|Pid2 Expression of the chimeric receptor between the chitin elicitor receptor CEBiP and the receptor-like protein kinase Pi-d2 leads to enhanced responses to the chitin elicitor and disease resistance against Magnaporthe oryzae in rice 2013 Plant Mol Biol Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. We previously reported that rice plants expressing the chimeric receptor consisting of rice chitin oligosaccharides binding protein (CEBiP) and the intracellular protein kinase region of Xa21, which confers resistance to rice bacterial blight, showed enhanced cellular responses to a chitin elicitor N-acetylchitoheptaose and increased resistance to the rice blast fungus Magnaporthe oryzae. Here, we investigated whether CEBiP fused with another type of receptor-like protein kinase (RLK) also functions as a chimeric receptor. Fusion proteins CRPis consist of CEBiP and the intracellular protein kinase region of a true resistance gene Pi-d2. Transgenic rice expressing a CRPi showed enhanced cellular responses specifically to N-acetylchitoheptaose in cultured cells and increased levels of disease resistance against M. oryzae in plants. These responses depended on the amino acid sequences predicted to be essential for the protein kinase activity of CRPi. The structure of the transmembrane domain in CRPi affected the protein accumulation, cellular responses, and disease resistance in transgenic rice. These results suggest that the chimeric receptor consisting of CEBiP and Pi-d2 functions as a receptor for chitin oligosaccharides and CEBiP-based chimeric receptors fused with other RLKs may also act as functional receptors. OsPK10|Pid2 A B-lectin receptor kinase gene conferring rice blast resistance 2006 Plant J Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Rice blast, caused by the fungal pathogen Magnaporthe grisea, is one of the most devastating diseases in rice worldwide. The dominant resistance gene, Pi-d2 [previously named Pi-d(t)2], present in the rice variety Digu, confers gene-for-gene resistance to the Chinese blast strain, ZB15. Pi-d2 was previously mapped close to the centromere of chromosome 6. In this study, the Pi-d2 gene was isolated by a map-based cloning strategy. Pi-d2 encodes a receptor-like kinase protein with a predicted extracellular domain of a bulb-type mannose specific binding lectin (B-lectin) and an intracellular serine-threonine kinase domain. Pi-d2 is a single-copy gene that is constitutively expressed in the rice variety Digu. Transgenic plants carrying the Pi-d2 transgene confer race-specific resistance to the M. grisea strain, ZB15. The Pi-d2 protein is plasma membrane localized. A single amino acid difference at position 441 of Pi-d2 distinguishes resistant and susceptible alleles of rice blast resistance gene Pi-d2. Because of its novel extracellular domain, Pi-d2 represents a new class of plant resistance genes. OsPK10|Pid2 Molecular cloning and biochemical characterization of a receptor-like serine/threonine kinase from rice 1994 Plant Mol Biol Department of Botany, University of Maryland, College Park 20742-5815. A receptor-like protein kinase, OsPK10, has been cloned from rice (Oryza sativa). The 2.8 kb cDNA contains an open reading frame capable of encoding a peptide sequence of 824 amino acids. The topological features of the predicted OsPK10 protein include an N-terminal signal peptide, a cysteine-rich extracellular ligand-binding domain, a membrane-spanning segment, and a cytoplasmic domain possessing all the hallmarks of catalytic domains of eukaryotic protein kinases. The cytoplasmic domain was selectively expressed in Escherichia coli and assayed for kinase activity. The results show the protein is capable of autophosphorylation using either ATP or GTP as the phosphate donor. Phosphoamino acid analysis reveals phosphorylation of threonines, consistent with the substrate specificity indicated by sequence motifs in the catalytic core. A single amino acid substitution of Glu for Lys-528 completely abolishes autophosphorylation activity. DNA gel blot analyses suggest that the haploid rice genome contains a single copy of the OsPK10 gene. OsPK10 transcripts appear to be more abundant in shoots than in roots of rice seedlings. OsPK10|Pid2 Development of DNA markers suitable for marker assisted selection of three Pi genes conferring resistance to multiple Pyricularia grisea pathotypes 2004 Crop Science USDA-ARS, Rice Research, 1509 Aggie Drive, Beaumont, TX 77713, USA Rice blast, caused by the fungus Pyricularia grisea (Cooke) Sacc., is a serious rice (Oryza sativa L.)disease causing considerable economic damage worldwide. DNA markers for rice blast resistance have been developed, but most are not suitable for routine use in a marker-assisted selection breeding program involving large numbers of progeny. After identifying candidate microsatellite markers from public database sources, we have mapped these markers near the blast resistance genes Pi-b, Pi-k, and Pi-ta(2) on rice chromosomes 2, 11, and 12, respectively, using segregation information from hundreds of progeny in several crosses. Two microsatellite markers, RM208 and RM224, were found to cosegregate with the Pi-b and Pi-k genes, respectively, while additional microsatellites were found to closely flank these two genes and the Pi-tal gene. The new markers are polymorphic in the narrow crosses characteristic of applied breeding programs and appear to be ideally suited for marker assisted selection for blast resistance in rice because of their tight linkage with resistance genes and ease of use through analysis of amplification products. A dominant marker indicating the presence of the Pi-b gene, Pibdom, has also been developed on the basis of the sequence of the cloned Pi-b gene. These markers should facilitate the introgression and pyramiding of these three blast resistance genes into new rice cultivars and elite lines. Pik-1,Pik-2 The isolation and characterization of Pik, a rice blast resistance gene which emerged after rice domestication 2011 New Phytol Laboratory of Plant Resistance and Genetics, College of Resources and Environmental Sciences, South China Agricultural University, Guangzhou, China. * The rice-rice blast pathosystem is of great interest, not only because of the damaging potential of rice blast to the rice crop, but also because both the pathogen and its host are experimentally amenable. The rice blast resistance gene Pik, which is one of the five classical alleles located at the Pik locus on the long arm of chromosome 11, confers high and stable resistance to many Chinese rice blast isolates. * The isolation and functional characterization of Pik were performed in the present study through genetic and genomic approaches. * A combination of Pik-1 and Pik-2 is required for the expression of Pik resistance. Both Pik-1 and Pik-2 encode coiled-coil nucleotide binding site leucine-rich repeat (NBS-LRR) proteins, and each shares a very high level of protein identity with corresponding proteins encoded by the Pik-m and Pik-p alleles. Pik could be distinguished from other Pik alleles, including Pik-m and Pik-p, by the allele-specific, single-nucleotide polymorphism T1-2944G. * The coupled genes probably did not evolve as a result of a duplication event, and are far from any NBS-LRR R gene characterized. Pik is a younger allele at the locus that probably emerged after rice domestication. Pik-1,Pik-2 Characterization of the rice blast resistance gene Pik cloned from Kanto51 2011 Molecular Breeding NARO, Institute of Crop Science, Tsukuba, Ibaraki, 305-8518, Japan To study similar, but distinct, plant disease resistance (R) specificities exhibited by allelic genes at the rice blast resistance locus Pik/Pikm, we cloned the Pik gene from rice cultivar Kanto51 and compared its molecular features with those of Pikm and of another Pik gene cloned from cv. Kusabue. Like Pikm, Pik is composed of two adjacent NBS-LRR (nucleotide-binding site, leucine-rich repeat) genes: the first gene, Pik1-KA, and the second gene, Pik2-KA. Pik from Kanto51 and Pik from Kusabue were not identical; although the predicted protein sequences of the second genes were identical, the sequences differed by three amino acids within the NBS domain of the first genes. The Pik proteins from Kanto51 and Kusabue differed from Pikm in eight and seven amino acids, respectively. Most of these substituted amino acids were within the coiled-coil (CC) and NBS domains encoded by the first gene. Of these substitutions, all within the CC domain were conserved between the two Pik proteins, whereas all within the NBS domain differed between them. Comparison of the two Pik proteins and Pikm suggests the importance of the CC domain in determining the resistance specificities of Pik and Pikm. This feature contrasts with that of most allelic or homologous NBS-LRR genes characterized to date, in which the major specificity determinant is believed to lie in the highly diverged LRR domain. In addition, our study revealed high evolutionary flexibility in the genome at the Pik locus, which may be relevant to the generation of new R specificities at this locus. Pik-1,Pik-2 Genetic analysis of resistance/susceptibility in individual F3 families of rice against strains of Magnaporthe grisea containing different genes for avirulence 1999 Euphytica Plant Breeding and Plant Genetics Program and Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI, 53706, U.S.A A cross was made between rice cultivars Katy and Lemont. F3 families were produced from individual F2 plants. Approximately 25 plants of each F3 family were inoculated with each of 8 different strains of Magnaporthe grisea. Each strain of the pathogen was known to have different genes for avirulence on Katy or Lemont. Each F3 family was recorded as having all plants resistant, segregating for resistance, or all plants being susceptible. The results suggest that the ‘single gene for resistance in Katy is a tightly linked cluster of at least seven genes. Pik-1,Pik-2 Efficient authentic fine mapping of the rice blast resistance gene Pik-h in the Pik cluster, using new Pik-h-differentiating isolates 2008 Molecular Breeding Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannon-dai 2-1-2, Tsukuba, Ibaraki, 305-8602, Japan The Pik-h gene in rice confers resistance to several races of rice blast fungus (Magnaporthe oryzae), and has been classified as a member of the Pik cluster, one of the most resistance (R) gene-dense regions in the rice genome. However, the loss of a key mutant isolate has long made it difficult to differentiate Pik-h from other Pik group genes especially from Pik-m. We identified new natural isolates enabling the differentiation between Pik-h and Pik-m genes, and first confirmed the authenticity of the International Rice Research Institute (IRRI) “monogenic" line IRBLkh-K3, and then fine-mapped the Pik-h gene in the Pik cluster. Using 701 susceptible individuals among 3,060 siblings from a cross of IRBLkh-K3×CO39, the Pik-h region was delimited to 270 kb, the narrowest interval among the Pik group genes reported to date, in the cv. Nipponbare genome. Annotation of this genome region first revealed 6 NBS-LRR type R-gene analogs (RGAs), clustered within the central 120 kb, as possible counterparts of Pik-h and 6 other Pik group R genes. Interestingly, the Pik-h region and the cluster of RGAs were shown to be located 130 kb and 230 kb apart from Xa4 and Xa2 bacterial blight resistance genes, respectively, once classified as belonging to the Pik cluster. The closest recombination events were limited to the margins of the Pik-h region, and recombination was suppressed in the core interval with the RGA cluster. This fine-mapping, performed in a short time using an HEGS system, will facilitate utilization of the cluster’s genetic resources and help to elucidate the mechanism of evolution of R-genes. The presence of natural isolates also confirmed that evolution of Pik-h corresponds to pathogen evolution. pikh|pi54 The single functional blast resistance gene Pi54 activates a complex defence mechanism in rice 2012 J Exp Bot National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, Pusa Campus, New Delhi-110012, India. The Pi54 gene (Pi-k(h)) confers a high degree of resistance to diverse strains of the fungus Magnaporthe oryzae. In order to understand the genome-wide co-expression of genes in the transgenic rice plant Taipei 309 (TP) containing the Pi54 gene, microarray analysis was performed at 72 h post-inoculation of the M. oryzae strain PLP-1. A total of 1154 differentially expressing genes were identified in TP-Pi54 plants. Of these, 587 were up-regulated, whereas 567 genes were found to be down-regulated. 107 genes were found that were exclusively up-regulated and 58 genes that were down- regulated in the case of TP-Pi54. Various defence response genes, such as callose, laccase, PAL, and peroxidase, and genes related to transcription factors like NAC6, Dof zinc finger, MAD box, bZIP, and WRKY were found to be up-regulated in the transgenic line. The enzymatic activities of six plant defence response enzymes, such as peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, beta-glucosidase, beta-1,3-glucanase, and chitinase, were found to be significantly high in TP-Pi54 at different stages of inoculation by M. oryzae. The total phenol content also increased significantly in resistant transgenic plants after pathogen inoculation. This study suggests the activation of defence response and transcription factor-related genes and a higher expression of key enzymes involved in the defence response pathway in the rice line TP-Pi54, thus leading to incompatible host-pathogen interaction. pikh|pi54 Functional complementation of rice blast resistance gene Pi-k h (Pi54) conferring resistance to diverse strains of Magnaporthe oryzae 2011 Journal of Plant Biochemistry and Biotechnology National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi, 110012, India Blast disease of rice, caused by Magnaporthe oryzae is an explosive disease that can spread rapidly in conducive conditions. R-gene mediated resistance offers an environmentally sustainable solution for management of this important disease of rice. We have earlier identified a unique R-gene of rice, on chromosome 11 of Oryza sativa ssp. indica cultivar Tetep. In this study we report functional validation of the Pi-k h (Pi54) gene using complementation assay. The blast resistance candidate gene Pi-k h (Pi54) was cloned into a plant transformation vector and the construct was used to transform a japonica cultivar of rice Taipei 309, which is susceptible to M. oryzae. Transgenic lines containing Pi-k h (Pi54) gene were found to confer high degree of resistance to diverse isolates of M. oryzae. The callose deposition was analyzed and compared between the transgenic and non-transgenic rice plants and widespread deposition was observed at the infection sites in plants showing incompatible interaction. Successful complementation of Pi-k h (Pi54) gene confirmed that the gene is responsible for resistance to M. oryzae in transgenic lines developed during this study. Expression analysis of the gene in resistant plants revealed that the gene is pathogen inducible in nature and is not expressed constitutively. Detection of callose deposition in resistant plants containing Pi-k h (Pi54) gene implicates its involvement in the initiation of defense response cascade. pikh|pi54 High-resolution mapping, cloning and molecular characterization of the Pi-k ( h ) gene of rice, which confers resistance to Magnaporthe grisea 2005 Mol Genet Genomics National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 110012, India. trsharma@nrcpb.org In order to understand the molecular mechanisms involved in the gene-for-gene type of pathogen resistance, high-resolution genetic and physical mapping of resistance loci is required to facilitate map-based cloning of resistance genes. Here, we report the molecular mapping and cloning of a dominant gene (Pi-k ( h )) present in the rice line Tetep, which is associated with resistance to rice blast disease caused by Magnaporthe grisea. This gene is effective against M. grisea populations prevalent in the Northwestern Himalayan region of India. Using 178 sequence tagged microsatellite, sequence-tagged site, expressed sequence tag and simple sequence repeat (SSR) markers to genotype a population of 208 F(2) individuals, we mapped the Pi-k ( h ) gene between two SSR markers (TRS26 and TRS33) which are 0.7 and 0.5 cM away, respectively, and can be used in marker-assisted-selection for blast-resistant rice cultivars. We used the markers to identify the homologous region in the genomic sequence of Oryza sativa cv. Nipponbare, and a physical map consisting of two overlapping bacterial artificial chromosome and P1 artificial chromosome clones was assembled, spanning a region of 143,537 bp on the long arm of chromosome 11. Using bioinformatic analyses, we then identified a candidate blast-resistance gene in the region, and cloned the homologous sequence from Tetep. The putative Pi-k ( h ) gene cloned from Tetep is 1.5 kbp long with a single ORF, and belongs to the nucleotide binding site-leucine rich repeat class of disease resistance genes. Structural and expression analysis of the Pi-k ( h ) gene revealed that its expression is pathogen inducible. pikh|pi54 A novel blast resistance gene, Pi54rh cloned from wild species of rice, Oryza rhizomatis confers broad spectrum resistance to Magnaporthe oryzae 2012 Funct Integr Genomics National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 110 012, India. The dominant rice blast resistance gene, Pi54 confers resistance to Magnaporthe oryzae in different parts of India. In our effort to identify more effective forms of this gene, we isolated an orthologue of Pi54 named as Pi54rh from the blast-resistant wild species of rice, Oryza rhizomatis, using allele mining approach and validated by complementation. The Pi54rh belongs to CC-NBS-LRR family of disease resistance genes with a unique Zinc finger (C(3)H type) domain. The 1,447 bp Pi54rh transcript comprises of 101 bp 5'-UTR, 1,083 bp coding region and 263 bp 3'-UTR, driven by pathogen inducible promoter. We showed the extracellular localization of Pi54rh protein and the presence of glycosylation, myristoylation and phosphorylation sites which implicates its role in signal transduction process. This is in contrast to other blast resistance genes that are predicted to be intracellular NBS-LRR-type resistance proteins. The Pi54rh was found to express constitutively at basal level in the leaves, but upregulates 3.8-fold at 96 h post-inoculation with the pathogen. Functional validation of cloned Pi54rh gene using complementation test showed high degree of resistance to seven isolates of M. oryzae collected from different geographical locations of India. In this study, for the first time, we demonstrated that a rice blast resistance gene Pi54rh cloned from wild species of rice provides broad spectrum resistance to M. oryzae hence can be used in rice improvement breeding programme. pikh|pi54 Sequence variation at the rice blast resistance gene Pi-km locus: Implications for the development of allele specific markers 2010 Plant Science Department of Biochemistry, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, 305-8602, Tsukuba, Japan. We identified two new members of the GRAS gene family from rice, CIGR1 and CIGR2, which are rapidly induced upon N-acetylchitooligosaccharide elicitor perception. The predicated proteins encoded by CIGR1 and CIGR2 possess significant sequence similarity with previously identified members of the GRAS family, such as Arabidopsis SCARECROW, GAI, RGA, tomato Lateral suppressor, and rice SLR1, all of which have VHIID regions, likely to play a role in cellular signaling. Fusions of CIGR1 and CIGR2 with Green Fluorescent Protein were detected exclusively in the nuclei of onion epidermal cells. The expression of CIGR1 and CIGR2 was dependent on the structure of N-acetylchitooligosaccharides, which parallels the structural specificity for chitin binding to the plasma membrane-localized chitin-binding protein, and independent of de novo protein synthesis. Co-cultivation of rice cells with rice blast fungus strongly induced the expression of CIGR1 and CIGR2, whereas inoculation of suspension cells with phytopathogenic bacteria did not. We hypothesize that CIGR1 and CIGR2 act as transcriptional regulators in the early events of the elicitor-induced defense response in rice. Pikm1-TS,Pikm2-TS Two adjacent nucleotide-binding site-leucine-rich repeat class genes are required to confer Pikm-specific rice blast resistance 2008 Genetics QTL Genomics Research Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. ashikawa@affrc.go.jp The rice blast resistance gene Pikm was cloned by a map-based cloning strategy. High-resolution genetic mapping and sequencing of the gene region in the Pikm-containing cultivar Tsuyuake narrowed down the candidate region to a 131-kb genomic interval. Sequence analysis predicted two adjacently arranged resistance-like genes, Pikm1-TS and Pikm2-TS, within this candidate region. These genes encoded proteins with a nucleotide-binding site (NBS) and leucine-rich repeats (LRRs) and were considered the most probable candidates for Pikm. However, genetic complementation analysis of transgenic lines individually carrying these two genes negated the possibility that either Pikm1-TS or Pikm2-TS alone was Pikm. Instead, it was revealed that transgenic lines carrying both of these genes expressed blast resistance. The results of the complementation analysis and an evaluation of the resistance specificity of the transgenic lines to blast isolates demonstrated that Pikm-specific resistance is conferred by cooperation of Pikm1-TS and Pikm2-TS. Although these two genes are not homologous with each other, they both contain all the conserved motifs necessary for an NBS-LRR class gene to function independently as a resistance gene. Pikm1-TS,Pikm2-TS The Pik m gene, conferring stable resistance to isolates of Magnaporthe oryzae, was finely mapped in a crossover-cold region on rice chromosome 11 2007 Molecular Breeding Laboratory of Plant Resistance and Genetics, College of Natural Resources and Environmental Sciences, South China Agricultural University, Guangzhou, 510642, China The Pik m gene in rice confers a high and stable resistance to many isolates of Magnaporthe oryzae collected from southern China. This gene locus was roughly mapped to the long arm of rice chromosome 11 with restriction fragment length polymorphic (RFLP) markers in the previous study. To effectively utilize the resistance, a linkage analysis was performed in a mapping population consisting of 659 highly susceptible plants collected from four F2 populations using the publicly available simple sequence repeat (SSR) markers. The result showed that the locus was linked to the six SSR markers and defined by RM254 and RM144 with ≈13.4 and ≈1.2 cM, respectively. To fine map this locus, additional 10 PCR-based markers were developed in a region flanked by RM254 and RM144 through bioinformatics analysis (BIA) using the reference sequence of cv. Nipponbare. The linkage analysis with these 10 markers showed that the locus was further delimited to a 0.3-cM region flanked by K34 and K10, in which three markers, K27, K28, and K33, completely co-segregated with the locus. To physically map the locus, the Pik m -linked markers were anchored to bacterial artificial chromosome clones of the reference cv. Nipponbare by BIA. A physical map spanning ≈278 kb in length was constructed by alignment of sequences of the clones anchored by BIA, in which only six candidate genes having the R gene conserved structure, protein kinase, were further identified in an 84-kb segment. Pikm1-TS,Pikm2-TS The Pik-p resistance to Magnaporthe oryzae in rice is mediated by a pair of closely linked CC-NBS-LRR genes 2011 Theor Appl Genet Laboratory of Plant Resistance and Genetics, College of Resources and Environmental Sciences, South China Agricultural University, Guangzhou, China. The blast resistance gene Pik-p, mapping to the Pik locus on the long arm of rice chromosome 11, was isolated by map-based in silico cloning. Four NBS-LRR genes are present in the target region of cv. Nipponbare, and a presence/absence analysis in the Pik-p carrier cv. K60 excluded two of these as candidates for Pik-p. The other two candidates (KP3 and KP4) were expressed in cv. K60. A loss-of-function experiment by RNAi showed that both KP3 and KP4 are required for Pik-p function, while a gain-of-function experiment by complementation test revealed that neither KP3 nor KP4 on their own can impart resistance, but that resistance was expressed when both were introduced simultaneously. Both Pikp-1 (KP3) and Pikp-2 (KP4) encode coiled-coil NBS-LRR proteins and share, respectively, 95 and 99% peptide identity with the two alleles, Pikm1-TS and Pikm2-TS. The Pikp-1 and Pikp-2 sequences share only limited homology. Their sequence allowed Pik-p to be distinguished from Pik, Pik-s, Pik-m and Pik-h. Both Pikp-1 and Pikp-2 were constitutively expressed in cv. K60 and only marginally induced by blast infection. Pikp-1,Pikp-2 Characterization of rice blast resistance genes in the Pik cluster and fine mapping of the Pik-p locus 2009 Phytopathology Laboratory of Plant Resistance and Genetics, College of Natural Resources and Environmental Sciences, South China Agricultural University, Guangzhou, China. Pik-p is carried by cv. K60, which is one of the Japanese differentials widely used in both Japan and China since the 1980s. Its utility and specificity was evaluated with a total of 612 isolates of Magnaporthe oryzae collected from various regions in China in combination with 16 main resistance genes being used in the breeding programs. Pik-p is an independently and dominantly acting gene in the Pik cluster, which conditions differential reactions against many isolates and contains higher resistance in Guangdong, Jiangsu, and Sichuan provinces, China, indicating that this gene could be still used in these regions. A high-resolution genetic map of Pik-p was constructed using genomic position-ready markers. A set of 47 recombinants out of 681 F(2) plants derived from the crosses cv. K60 (resistant) x cv. AS20-1 (susceptible) and x cv. Kasalath (susceptible) was identified in the genetic interval defined by the markers RM5926 and K37 which flank the Pik gene cluster. This set was then genotyped with seven markers known to reside within the interval. The closest markers to Pik-p were K28 (approximately 0.60 centimorgans [cM]) and K39 (approximately 0.07 cM). A further four markers in the K28-K39 interval were developed from an in silico analysis based on the cv. Nipponbare genome sequence, and these all co-segregated with Pik-p. This 0.67-cM region is equivalent to a physical separation in cv. Nipponbare of approximately 126 kb, plus an as-yet-unfilled genomic gap of unknown length. Four nucleotide-binding site leucine-rich repeat-type resistance genes are present in this interval, and these represent good candidates for Pik-p. Pikp-1,Pikp-2 Characterization of a rice gene induced by Pseudomonas syringae pv. syringae: requirement for the bacterial lemA gene function 1995 Physiological and Molecular Plant Pathology Institute of Plant Biology, University of Zürich, Zollikerstrasse 107, CH-8008 Zürich, Switzerland and Ciba-Geigy Limited, Agricultural Division, CH-4002 Basel, Switzerland. To characterize the acquired resistance response in rice (Oryza sativa) we have cloned cDNAs corresponding to transcripts that accumulate concomitantly with the establishment of induced resistance following leaf infiltration with Pseudomonas syringae pv. syringae. Here we describe the structure and regulation of one of these cDNAs and two homologous genes. The cDNA represents a transcript from one of these genes, Pir7b, and encodes a putative protein that is 70% identical to the one encoded by Pir7a . Both of these proteins contain a lipase sequence motif. While Pir7a was not expressed under any of the circumstances that were checked, transcripts from Pir7b accumulated transiently after infiltration of leaves with P. syringae pv. syringae. In contrast to other defence-related transcripts, the accumulation of Pir7b-specific mRNA depended on the lemA gene function of the bacterial strain infiltrated. It did not occur upon treatment of the plants with other resistance-inducing agents. Thus, the Pir7b gene product does not appear to play a role in acquired resistance. Its function is more likely related to a specific property of the inducing P. syringae pv. syringae strains that is under the control of the two-component regulatory system for which lemA encodes the sensor. A hypothesis about this function is discussed. Pir7b,Pir7a Syringolin-mediated activation of the Pir7b esterase gene in rice cells is suppressed by phosphatase inhibitors 2000 Mol Plant Microbe Interact Institute of Plant Biology, University of Zurich, Switzerland. Inoculation of rice plants (Oryza sativa) with the nonhost pathogen Pseudomonas syringae pv. syringae leads to the activation of defense-related genes and ultimately to induced resistance against the rice blast fungus Pyricularia oryzae. One of the molecular determinants of P. syringae pv. syringae that is recognized by the plant cells and evokes these defense responses is syringolin A, an elicitor that is secreted by the bacteria under appropriate conditions. In order to investigate signal transduction events elicited by syringolin A, the response of cultured rice cells to syringolin A application was analyzed. Cultured rice cells were able to sense syringolin A at concentrations in the nanomolar range as observed by the transient accumulation of Pir7b esterase transcripts. Syringolin A-mediated Pir7b transcript accumulation was inhibited by cycloheximide, indicating that de novo protein synthesis was required. Calyculin and okadaic acid, two protein phosphatase inhibitors, blocked Pir7b gene induction, whereas the serine/threonine protein kinase inhibitors staurosporine and K-252a had no effect on Pir7b transcript levels. Actin transcript levels were essentially not affected by inhibitor treatments over the experimental time span. These results imply that dephosphorylation of a phosphoprotein is an important step in the syringolin A-triggered signal transduction pathway. Pir7b Blast resistance in rice: a review of conventional breeding to molecular approaches 2013 Mol Biol Rep Laboratory of Food Crops, Institute of Tropical Agriculture, Universiti Putra Malaysia (UPM), 43400, UPM Serdang, Selangor, Malaysia. Blast disease caused by the fungal pathogen Magnaporthe oryzae is the most severe diseases of rice. Using classical plant breeding techniques, breeders have developed a number of blast resistant cultivars adapted to different rice growing regions worldwide. However, the rice industry remains threatened by blast disease due to the instability of blast fungus. Recent advances in rice genomics provide additional tools for plant breeders to improve rice production systems that would be environmentally friendly. This article outlines the application of conventional breeding, tissue culture and DNA-based markers that are used for accelerating the development of blast resistant rice cultivars. The best way for controlling the disease is to incorporate both qualitative and quantitative genes in resistant variety. Through conventional and molecular breeding many blast-resistant varieties have been developed. Conventional breeding for disease resistance is tedious, time consuming and mostly dependent on environment as compare to molecular breeding particularly marker assisted selection, which is easier, highly efficient and precise. For effective management of blast disease, breeding work should be focused on utilizing the broad spectrum of resistance genes and pyramiding genes and quantitative trait loci. Marker assisted selection provides potential solution to some of the problems that conventional breeding cannot resolve. In recent years, blast resistant genes have introgressed into Luhui 17, G46B, Zhenshan 97B, Jin 23B, CO39, IR50, Pusa1602 and Pusa1603 lines through marker assisted selection. Introduction of exotic genes for resistance induced the occurrence of new races of blast fungus, therefore breeding work should be concentrated in local resistance genes. This review focuses on the conventional breeding to the latest molecular progress in blast disease resistance in rice. This update information will be helpful guidance for rice breeders to develop durable blast resistant rice variety through marker assisted selection. PISH Pi35(t), a new gene conferring partial resistance to leaf blast in the rice cultivar Hokkai 188 2006 Theor Appl Genet Fungal Disease Laboratory, Department of Plant Pathology, National Agricultural Research Organization, Kannondai 3-1-1, Tsukuba, Ibaraki 305-8666, Japan. The japonica rice cultivar Hokkai 188 shows a high level of partial resistance to leaf blast. For mapping genes conferring the resistance, a set of 190 F2 progeny/F3 families was developed from the cross between the indica rice cultivar Danghang-Shali, with a low level of partial resistance, and Hokkai 188. Partial resistance to leaf blast in the F3 families was assessed in upland nurseries. From a primary microsatellite (SSR) linkage map and QTL analysis using a subset of 126 F2 progeny/F3 families randomly selected from the above set, one major QTL located on chromosome 1 was detected in the vicinity of SSR marker RM1216. This QTL was responsible for 69.4% of the phenotypic variation, and Hokkai 188 contributed the resistance allele. Segregation analysis in the F3 families for partial resistance to leaf blast was in agreement with the existence of a major gene, and the gene was designated as Pi35(t). Another QTL detected on chromosome 8 was minor, explained 13.4% of the phenotypic variation, and an allele of Danghang-Shali increased the level of resistance in this QTL. Additional SSR markers of the targeted Pi35(t) region were further surveyed in the 190 F2 plants, and Pi35(t) was placed in a 3.5-cM interval flanked by markers RM1216 and RM1003. PISH Unique features of the rice blast resistance Pish locus revealed by large scale retrotransposon-tagging 2010 BMC Plant Biol Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences, Ibaraki, 305-8602, Japan. BACKGROUND: R gene-mediated resistance is one of the most effective mechanisms of immunity against pathogens in plants. To date some components that regulate the primary steps of plant immunity have been isolated, however, the molecular dissection of defense signaling downstream of the R proteins remains to be completed. In addition, R genes are known to be highly variable, however, the molecular mechanisms responsible for this variability remain obscure. RESULTS: To identify novel factors required for R gene-mediated resistance in rice, we used rice insertional mutant lines, induced by the endogenous retrotransposon Tos17, in a genetic screening involving the rice blast fungus Magnaporthe oryzae. We inoculated 41,119 mutant lines with the fungus using a high throughput procedure, and identified 86 mutant lines with diminished resistance. A genome analysis revealed that 72 of the 86 lines contained mutations in a gene encoding a nucleotide binding site (NBS) and leucine rich repeat (LRR) domain-containing (NBS-LRR) protein. A genetic complementation analysis and a pathogenesis assay demonstrated that this NBS-LRR gene encodes Pish, which confers resistance against races of M. oryzae containing avrPish. The other 14 lines have intact copies of the Pish gene, suggesting that they may contain mutations in the signaling components downstream of Pish. The genome analysis indicated that Pish and its neighboring three NBS-LRR genes are high similar to one another and are tandemly located. An in silico analysis of a Tos17 flanking sequence database revealed that this region is a "hot spot" for insertion. Intriguingly, the insertion sites are not distributed evenly among these four NBS-LRR genes, despite their similarity at the sequence and expression levels. CONCLUSIONS: In this work we isolated the R gene Pish, and identified several other mutants involved in the signal transduction required for Pish-mediated resistance. These results indicate that our genetic approach is efficient and useful for unveiling novel aspects of defense signaling in rice. Furthermore, our data provide experimental evidence that R gene clusters have the potential to be highly preferred targets for transposable element insertions in plant genomes. Based on this finding, a possible mechanism underlying the high variability of R genes is discussed. PISH Identification and characterization of a new blast resistance gene located on rice chromosome 1 through linkage and differential analyses 2004 Phytopathology None The Chinese native cv. Q14 expresses a high level of resistance to many isolates of Pyricularia grisea collected from Japan, Thailand, and China. Q14 was crossed to an indica-susceptible cultivar, Q61 . To rapidly determine the chromosomal location of the major resistance gene present in the cultivar, a linkage analysis using microsatellite markers was performed in the F-2 population segregating 3R:1S (resistant/susceptible) through bulked-segregant analysis (BSA) in combination with recessive-class analysis (RCA). A total of 189 microsatellite markers selected from each chromosome equally (with approximate to10 centimorgans) were tested with the BSA approach. Only two markers. RM151 and RM259, located on chromosome 1 showed positive and negative polymorphisms, respectively, for a resistance gene segregating in the population. To confirm the polymorphic markers, a total of 155 viable susceptible individuals were tested with the RCA approach. The markers RM151 and RM259 were found to link to the resistance gene with recombination frequencies of 11.9 +/- 2.8% and 9.7 +/- 8.0%, respectively. For further characterization of the resistance gene, 3 resistance genes mapped on chromosome 1, as well as 15 major resistance genes that might be employed in the breeding program, were selected for differential tests with 85 Chinese isolates. The resistance gene identified in this research conveys reactions distinct front those conditioned by the 18 resistance genes. This new resistance gene tentatively was designated Pi27(t). PISH Identification of the blast resistance gene Pit in rice cultivars using functional markers 2010 Theor Appl Genet National Agricultural Research Center, National Agriculture and Food Research Organization (NARO), Tsukuba, Japan. keko1@affrc.go.jp DNA markers that allow for identification of resistance genes in rice germplasm have a great advantage in resistance breeding because they can assess the existence of the genes without laborious inoculation tests. Functional markers (FMs), which are designed from functional polymorphisms within the sequence of genes, are unaffected by nonfunctional allelic variation and make it possible to identify an individual gene. We previously showed that the resistance function of the rice blast resistance gene Pit in a resistant cultivar, K59, was mainly acquired by up-regulated promoter activity through the insertion of a long terminal repeat (LTR) retrotransposon upstream of Pit. Here, we developed PCR-based DNA markers derived from the LTR-retrotransposon sequence and used these markers to screen worldwide accessions of rice germplasm. We identified 5 cultivars with the LTR-retrotransposon insertion out of 68 rice accessions. The sequence and expression pattern of Pit in the five cultivars were the same as those in K59 and all showed Pit-mediated blast resistance. The results suggest that the functional Pit identified using the markers was derived from a common progenitor. Additionally, comparison of the Pit coding sequences between K59 and susceptible cultivars revealed that one nucleotide polymorphism, which caused an amino acid substitution, offered another target for a FM. These results indicate that our DNA markers should enhance prediction of Pit function and be applicable to a range of rice varieties/landraces cultivated in various regions worldwide and belonging to the temperate japonica, tropical japonica, and indica groups. Pit Refunctionalization of the ancient rice blast disease resistance gene Pit by the recruitment of a retrotransposon as a promoter 2009 Plant J National Agricultural Research Center, National Agriculture and Food Research Organization, 1-2-1 Inada, Jo-etsu, Niigata 943 0193, Japan. The plant genome contains a large number of disease resistance (R) genes that have evolved through diverse mechanisms. Here, we report that a long terminal repeat (LTR) retrotransposon contributed to the evolution of the rice blast resistance gene Pit. Pit confers race-specific resistance against the fungal pathogen Magnaporthe grisea, and is a member of the nucleotide-binding site leucine-rich repeat (NBS-LRR) family of R genes. Compared with the non-functional allele Pit(Npb), the functional allele Pit(K59) contains four amino acid substitutions, and has the LTR retrotransposon Renovator inserted upstream. Pathogenesis assays using chimeric constructs carrying the various regions of Pit(K59) and Pit(Npb) suggest that amino acid substitutions might have a potential effect in Pit resistance; more importantly, the upregulated promoter activity conferred by the Renovator sequence is essential for Pit function. Our data suggest that transposon-mediated transcriptional activation may play an important role in the refunctionalization of additional 'sleeping' R genes in the plant genome. Pit Molecular diversity in rice blast resistance gene Pi-ta makes it highly effective against dynamic population of Magnaporthe oryzae 2013 Funct Integr Genomics National Research Centre on Plant Biotechnology, IARI, Pusa Campus, New Delhi 110012, India. Rice blast is one of the important diseases of rice which can be effectively managed by the deployment of resistance genes. Pi-ta is one of the major blast resistant genes effective against pathogen populations in different parts of India. We analysed allelic variants of Pi-ta from 48 rice lines selected after phenotyping of 529 rice landraces across three eco-geographical blast hot spot regions. Besides, Pi-ta orthologue sequences of 220 rice accessions belonging to wild and cultivated species of rice were also included in the study for a better evo-devo perspective of the diversity present in the gene and the selection pressures acting on this locus. We obtained high nucleotide variations (SNPs and insertion-deletions) in the intronic region. We also identified 64 haplotypes based on nucleotide polymorphism in these alleles. Pi-ta orthologues of Indian landraces were scattered in eight major haplotypes indicating its heterogenous nature. We identified a total of 47 different Pi-ta protein variants on the basis of deduced amino acid residues amongst the orthologues. Five unique and novel Pi-ta variants were identified for the first time in rice landraces exhibiting different reaction types against the Magnaporthe oryzae population. A high value of Pi(non/syn) was observed only in the leucine-rich domain of the alleles cloned from Indian landraces, indicating strong selective forces acting on this region. The detailed molecular analysis of the Pi-ta orthologues provides insights to a high degree of inter- and intraspecific relationships amongst the Oryza species. We identified rice landraces possessing the effective alleles of this resistance gene which can be used in future blast resistance breeding programmes. Pita|Pi-4a Direct interaction of resistance gene and avirulence gene products confers rice blast resistance 2000 EMBO J DuPont Agricultural Products, PO Box 80402, Wilmington, DE 19880-0402, USA. Rice expressing the Pi-ta gene is resistant to strains of the rice blast fungus, Magnaporthe grisea, expressing AVR-Pita in a gene-for-gene relationship. Pi-ta encodes a putative cytoplasmic receptor with a centrally localized nucleotide-binding site and leucine-rich domain (LRD) at the C-terminus. AVR-Pita is predicted to encode a metalloprotease with an N-terminal secretory signal and pro-protein sequences. AVR-Pita(176) lacks the secretory and pro-protein sequences. We report here that transient expression of AVR-Pita(176) inside plant cells results in a Pi-ta-dependent resistance response. AVR-Pita(176) protein is shown to bind specifically to the LRD of the Pi-ta protein, both in the yeast two-hybrid system and in an in vitro binding assay. Single amino acid substitutions in the Pi-ta LRD or in the AVR-Pita(176) protease motif that result in loss of resistance in the plant also disrupt the physical interaction, both in yeast and in vitro. These data suggest that the AVR-Pita(176) protein binds directly to the Pi-ta LRD region inside the plant cell to initiate a Pi-ta-mediated defense response. Pita|Pi-4a Molecular evolution of the Pi-ta gene resistant to rice blast in wild rice (Oryza rufipogon) 2008 Genetics Institute of Plant Biology, National Taiwan University, Taipei 106, Taiwan. Rice blast disease resistance to the fungal pathogen Magnaporthe grisea is triggered by a physical interaction between the protein products of the host R (resistance) gene, Pi-ta, and the pathogen Avr (avirulence) gene, AVR-pita. The genotype variation and resistant/susceptible phenotype at the Pi-ta locus of wild rice (Oryza rufipogon), the ancestor of cultivated rice (O. sativa), was surveyed in 36 locations worldwide to study the molecular evolution and functional adaptation of the Pi-ta gene. The low nucleotide polymorphism of the Pi-ta gene of O. rufipogon was similar to that of O. sativa, but greatly differed from what has been reported for other O. rufipogon genes. The haplotypes can be subdivided into two divergent haplogroups named H1 and H2. H1 is derived from H2, with nearly no variation and at a low frequency. H2 is common and is the ancestral form. The leucine-rich repeat (LRR) domain has a high pi(non)/pi(syn) ratio, and the low polymorphism of the Pi-ta gene might have primarily been caused by recurrent selective sweep and constraint by other putative physiological functions. Meanwhile, we provide data to show that the amino acid Ala-918 of H1 in the LRR domain has a close relationship with the resistant phenotype. H1 might have recently arisen during rice domestication and may be associated with the scenario of a blast pathogen-host shift from Italian millet to rice. Pita|Pi-4a Development of Dominant Rice Blast Resistance Gene Markers 2002 Crop Science USDA-ARS, Dale Bumpers National Rice Research Center, P. O. Box 1090, Stuttgart, AR 72160-0287 Incorporation of resistance genes into existing rice (Oryza sativa L.) cultivars is a powerful strategy and is commonly applied in breeding rice resistance to blast disease [caused by Pyricularia grisea Sacc. = P. oryzae Cavara (teleomorph: Magnaporthe grisea (Hebert) Barr)]. The rice blast resistance gene, Pi-ta, originally introgressed into japonica from indica rice is important in breeding for rice blast resistance worldwide. In the southern USA, the rice cultivar Katy contains Pi-ta and is resistant to the predominant blast M. grisea races IB-49 and IC-17 and has been used as the blast resistant breeding parent. Three pairs of DNA primers specific to the dominant indica Pi-ta gene were designed to amplify the Pi-ta DNA fragments by polymerase chain reaction (PCR). PCR products amplified by these Pi-ta specific primers were cloned and sequenced. Sequence analysis confirmed the presence of the dominant indica Pi-ta allele. These Pi-ta primers were used to examine the presence of Pi-ta alleles in advanced Arkansas rice breeding lines. The Pi-ta containing rice lines, as determined by PCR analysis, were resistant to both IB-49 and IC-17 in standard pathogenicity assays. In contrast, lines lacking the Pi-ta genes failed to protect rice plants against both races IB-49 and IC-17. The presence of Pi-ta markers correlated with the Pi-ta resistance spectrum. Thus, the Pi-ta gene markers provide a basis for stacking other blast resistance genes into high yielding and good quality advanced breeding rice lines. Pita|Pi-4a Characterization ofPi-tablast resistance gene in an international rice core collection 2009 Plant Breeding State Key Lab of Rice Biology, IAEA Collaborating Center, Zhejiang University, Hangzhou, China The Pi-ta gene in rice prevents the infections by races of Magnaporthe oryzae containing AVR-Pita. In the present study, 1790 accessions were characterized for Pi-ta, and Pi-ta independent resistance genes using marker analysis, disease evaluation with the race IB-49 carrying AVR-Pita, and IE-1k not carrying AVR-Pita and sequence analysis. A total of 183 accessions were identified using a Pi-ta-indel marker from the intron region. Sequence analysis revealed that resistance functional nucleotide polymorphism (FNP) was present in 163 accessions including reference cultivars. Among them, 89 were resistant to IB-49 and susceptible to IE-1k indicating that these accessions contain Pi-ta R alleles. Four accessions were susceptible to IB-49 suggesting that components were not intact in Pi-ta-mediated resistance. In contrast, 14 accessions with the susceptible FNP were resistant to IB-49 suggesting these 14 accessions may contain Pi-ta independent new R genes. Together, 83 accessions were identified to contain new R gene to IE-1k. These accessions were genetically distinct determined by simple sequence repeat markers. The results could impact breeding for blast resistance worldwide. Pita|Pi-4a Molecular evolution of the rice blast resistance gene Pi-ta in invasive weedy rice in the USA 2011 PLoS One Rice Research Extension Center, University of Arkansas, Stuttgart, Arkansas, United States of America. The Pi-ta gene in rice has been effectively used to control rice blast disease caused by Magnaporthe oryzae worldwide. Despite a number of studies that reported the Pi-ta gene in domesticated rice and wild species, little is known about how the Pi-ta gene has evolved in US weedy rice, a major weed of rice. To investigate the genome organization of the Pi-ta gene in weedy rice and its relationship to gene flow between cultivated and weedy rice in the US, we analyzed nucleotide sequence variation at the Pi-ta gene and its surrounding 2 Mb region in 156 weedy, domesticated and wild rice relatives. We found that the region at and around the Pi-ta gene shows very low genetic diversity in US weedy rice. The patterns of molecular diversity in weeds are more similar to cultivated rice (indica and aus), which have never been cultivated in the US, rather than the wild rice species, Oryza rufipogon. In addition, the resistant Pi-ta allele (Pi-ta) found in the majority of US weedy rice belongs to the weedy group strawhull awnless (SH), suggesting a single source of origin for Pi-ta. Weeds with Pi-ta were resistant to two M. oryzae races, IC17 and IB49, except for three accessions, suggesting that component(s) required for the Pi-ta mediated resistance may be missing in these accessions. Signatures of flanking sequences of the Pi-ta gene and SSR markers on chromosome 12 suggest that the susceptible pi-ta allele (pi-ta), not Pi-ta, has been introgressed from cultivated to weedy rice by out-crossing. Pita|Pi-4a Identification of a new locus, Ptr(t), required for rice blast resistance gene Pi-ta-mediated resistance 2008 Mol Plant Microbe Interact United States Department of Agriculture-Agriculture Research Service, Dale Bumpers National Rice Research Center, Stuttgart, AR 72160, USA. Yulin.Jia@ars.usda.gov Resistance to the blast pathogen Magnaporthe oryzae is proposed to be initiated by physical binding of a putative cytoplasmic receptor encoded by a nucleotide binding site-type resistance gene, Pi-ta, to the processed elicitor encoded by the corresponding avirulence gene AVR-Pita. Here, we report the identification of a new locus, Ptr(t), that is required for Pi-ta-mediated signal recognition. A Pi-ta-expressing susceptible mutant was identified using a genetic screen. Putative mutations at Ptr(t) do not alter recognition specificity to another resistance gene, Pi-k(s), in the Pi-ta homozygote, indicating that Ptr(t) is more likely specific to Pi-ta-mediated signal recognition. Genetic crosses of Pi-ta Ptr(t) and Pi-ta ptr(t) homozygotes suggest that Ptr(t) segregates as a single dominant nuclear gene. A ratio of 1:1 (resistant/susceptible) of a population of BC1 of Pi-ta Ptr(t) with pi-ta ptr(t) homozygotes indicates that Pi-ta and Ptr(t) are linked and cosegregate. Genotyping of mutants of pi-ta ptr(t) and Pi-ta Ptr(t) homozygotes using ten simple sequence repeat markers at the Pi-ta region determined that Pi-ta and Ptr(t) are located within a 9-megabase region and are of indica origin. Identification of Ptr(t) is a significant advancement in studying Pi-ta-mediated signal recognition and transduction. Pita|Pi-4a tA single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta 2000 Plant Cell DuPont Agricultural Products, P.O. Box 80402, Wilmington, Delaware 19880-0402, USA. The rice blast resistance (R) gene Pi-ta mediates gene-for-gene resistance against strains of the fungus Magnaporthe grisea that express avirulent alleles of AVR-Pita. Using a map-based cloning strategy, we cloned Pi-ta, which is linked to the centromere of chromosome 12. Pi-ta encodes a predicted 928-amino acid cytoplasmic receptor with a centrally localized nucleotide binding site. A single-copy gene, Pi-ta shows low constitutive expression in both resistant and susceptible rice. Susceptible rice varieties contain pi-ta(-) alleles encoding predicted proteins that share a single amino acid difference relative to the Pi-ta resistance protein: serine instead of alanine at position 918. Transient expression in rice cells of a Pi-ta(+) R gene together with AVR-Pita(+) induces a resistance response. No resistance response is induced in transient assays that use a naturally occurring pi-ta(-) allele differing only by the serine at position 918. Rice varieties reported to have the linked Pi-ta(2) gene contain Pi-ta plus at least one other R gene, potentially explaining the broadened resistance spectrum of Pi-ta(2) relative to Pi-ta. Molecular cloning of the AVR-Pita and Pi-ta genes will aid in deployment of R genes for effective genetic control of rice blast disease. Pita|Pi-4a The broad-spectrum blast resistance gene Pi9 encodes a nucleotide-binding site-leucine-rich repeat protein and is a member of a multigene family in rice 2006 Genetics Department of Plant Pathology, Ohio State University, Columbus 43210, USA. The broad-spectrum rice blast resistance gene Pi9 was cloned using a map-based cloning strategy. Sequencing of a 76-kb bacterial artificial chromosome (BAC) contig spanning the Pi9 locus led to identification of six tandemly arranged resistance-like genes with a nucleotide-binding site (NBS) and leucine-rich repeats (LRRs) (Nbs1-Pi9-Nbs6-Pi9). Analysis of selected Pi9 deletion mutants and transformation of a 45-kb fragment from the BAC contig into the susceptible rice cultivar TP309 narrowed down Pi9 to the candidate genes Nbs2-Pi9 and Nbs3-Pi9. Disease evaluation of the transgenic lines carrying the individual candidate genes confirmed that Nbs2-Pi9 is the Pi9 gene. Sequence comparison analysis revealed that the six paralogs at the Pi9 locus belong to four classes and gene duplication might be one of the major evolutionary forces contributing to the formation of the NBS-LRR gene cluster. Semiquantitative reverse transcriptase (RT)-PCR analysis showed that Pi9 was constitutively expressed in the Pi9-resistant plants and was not induced by blast infection. The cloned Pi9 gene provides a starting point to elucidate the molecular basis of the broad-spectrum disease resistance and the evolutionary mechanisms of blast resistance gene clusters in rice. Pi9|Piz-t|Pi2 Diverse response of rice and maize genes encoding homologs of WPK4, an SNF1-related protein kinase from wheat, to light, nutrients, low temperature and cytokinins 2000 Mol Gen Genet Laboratory of Plant Molecular Breeding, Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Ikoma, Japan. The wheat gene WPK4 encodes a 56-kDa protein kinase that belongs to group 3 of the SNF1-related protein kinase family (SnRK3), and is up-regulated by light and cytokinins and down-regulated by sucrose. In order to determine whether or not this particular regulation pattern is general among plant species, we isolated and characterized homologous genes from rice and maize. Two rice genes, OsPK4 and OsPK7, encode proteins comprising 508 and 520 amino acids, and show, respectively, 75% and 76% sequence similarity to WPK4. OsPK4 and OsPK7 proteins produced in Escherichia coli were able to phosphorylate themselves and myelin basic proteins, the reaction requiring magnesium and/or manganese ions. Transcripts of OsPK4 were detected in all tissues tested, and amounts were increased upon illumination, nutrient deprivation and treatment with cytokinins. In contrast, transcripts of OsPK7 were not found in any tissues except in mature leaves at low levels, and did not accumulate under any of the stress conditions examined. A maize gene, ZmPK4, encodes a protein with 518 amino acids that shows 74% similarity to WPK4. Its transcripts were constitutively expressed in all tissues, regardless of light, nutrient and cytokinin status, but were increased upon exposure to low temperature. These results indicate that, despite the sequence similarity between their products, genes for SnRK3 proteins are differentially regulated in response to environmental stimuli. PK4|OsPK4|OsCIPK19 Rice PLASTOCHRON genes regulate leaf maturation downstream of the gibberellin signal transduction pathway 2012 Planta Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, 113-8657, Japan. Rice PLASTOCHRON 1 (PLA1) and PLA2 genes regulate leaf maturation and plastochron, and their loss-of-function mutants exhibit small organs and rapid leaf emergence. They encode a cytochrome P450 protein CYP78A11 and an RNA-binding protein, respectively. Their homologs in Arabidopsis and maize are also associated with plant development/organ size. Despite the importance of PLA genes in plant development, their molecular functions remain unknown. Here, we investigated how PLA1 and PLA2 genes are related to phytohormones. We found that gibberellin (GA) is the major phytohormone that promotes PLA1 and PLA2 expression. GA induced PLA1 and PLA2 expression, and conversely the GA-inhibitor uniconazole suppressed PLA1 and PLA2 expression. In pla1-4 and pla2-1 seedlings, expression levels of GA biosynthesis genes and the signal transduction gene were similar to those in wild-type seedlings. GA treatment slightly down-regulated the GA biosynthesis gene GA20ox2 and up-regulated the GA-catabolizing gene GA2ox4, whereas the GA biosynthesis inhibitor uniconazole up-regulated GA20ox2 and down-regulated GA2ox4 both in wild-type and pla mutants, suggesting that the GA feedback mechanism is not impaired in pla1 and pla2. To reveal how GA signal transduction affects the expression of PLA1 and PLA2, PLA expression in GA-signaling mutants was examined. In GA-insensitive mutant, gid1 and less-sensitive mutant, Slr1-d1, PLA1 and PLA2 expression was down-regulated. On the other hand, the expression levels of PLA1 and PLA2 were highly enhanced in a GA-constitutive-active mutant, slr1-1, causing ectopic overexpression. These results indicate that both PLA1 and PLA2 act downstream of the GA signal transduction pathway to regulate leaf development. PLA1,PLA2|LHD2 PLASTOCHRON3/GOLIATH encodes a glutamate carboxypeptidase required for proper development in rice 2009 Plant J Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. Most aerial parts of the plant body are products of the continuous activity of the shoot apical meristem (SAM). Leaves are the major component of the aerial plant body, and their temporal and spatial distribution mainly determines shoot architecture. Here we report the identification of the rice gene PLASTOCHRON3 (PLA3)/GOLIATH (GO) that regulates various developmental processes including the rate of leaf initiation (the plastochron). PLA3/GO encodes a glutamate carboxypeptidase, which is thought to catabolize small acidic peptides and produce small signaling molecules. pla3 exhibits similar phenotypes to pla1 and pla2- a shortened plastochron, precocious leaf maturation and rachis branch-to-shoot conversion in the reproductive phase. However, in contrast to pla1 and pla2, pla3 showed pleiotropic phenotypes including enlarged embryo, seed vivipary, defects in SAM maintenance and aberrant leaf morphology. Consistent with these pleiotropic phenotypes, PLA3 is expressed in the whole plant body, and is involved in plant hormone homeostasis. Double mutant analysis revealed that PLA1, PLA2 and PLA3 are regulated independently but function redundantly. Our results suggest that PLA3 modulates various signaling pathways associated with a number of developmental processes. PLA1,PLA2|LHD2,PLA3 A recessive heterochronic mutation, plastochron1, shortens the plastochron and elongates the vegetative phase in rice 1998 Plant Cell Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. We describe two recessive alleles of a rice heterochronic gene, plastochron1-1 (pla1-1) and pla1-2, that reduce the length of the plastochron to approximately half that of the wild type. Because the onset of the reproductive phase in pla1 was not temporally affected, the number of leaves produced in the vegetative phase was nearly twice that produced in the wild type. Panicle development was severely disturbed in pla1 mutants. In pla1-1, many primordia of primary rachis branches were converted into vegetative shoots. These ectopic shoots repeated the initiation of panicle development and the conversion of primary rachis branches into shoots. In the weak allele pla1-2, however, only the basal one or two primordia developed as vegetative shoots, and the remaining primordia developed to produce a truncated panicle. These results indicate that both vegetative and reproductive programs are expressed simultaneously during the reproductive phase of pla1; however, the degree varied depending on the strength of the allele. Accordingly, pla1 is a heterochronic mutation that extends the vegetative period. The shoot apical meristem of pla1 was larger than that of the wild type, although the shape was not modified. An in situ hybridization experiment using the histone H4 gene as a probe revealed that cell divisions are accelerated in the pla1 meristem. The PLA1 gene is considered to regulate the duration of the vegetative phase by controlling the rate of leaf production in the meristem. PLA1 PLASTOCHRON1, a timekeeper of leaf initiation in rice, encodes cytochrome P450 2004 Proc Natl Acad Sci U S A Plant Genetics Laboratory, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan. During postembryonic development of higher plants, the shoot apical meristem produces lateral organs in a regular spacing (phyllotaxy) and a regular timing (plastochron). Molecular analysis of mutants associated with phyllotaxy and plastochron would greatly increase understanding of the developmental mechanism of plant architecture because phyllotaxy and plastochron are fundamental regulators of plant architecture. pla1 of rice is not only a plastochron mutant showing rapid leaf initiation without affecting phyllotaxy, but also a heterochronic mutant showing ectopic shoot formation in the reproductive phase. Thus, pla1 provides a tool for analyzing the molecular basis of temporal regulation in leaf development. In this work, we isolated the PLA1 gene by map-based cloning. The identified PLA1 gene encodes a cytochrome P450, CYP78A11, which potentially catalyzes substances controlling plant development. PLA1 is expressed in developing leaf primordia, bracts of the panicle, and elongating internodes, but not in the shoot apical meristem. The expression pattern and mutant phenotype suggest that the PLA1 gene acting in developing leaf primordia affects the timing of successive leaf initiation and the termination of vegetative growth. PLA1 PLASTOCHRON2 Regulates Leaf Initiation and Maturation in Rice 2006 The Plant Cell Online Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan In higher plants, leaves initiate in constant spatial and temporal patterns. Although the pattern of leaf initiation is a key element of plant shoot architecture, little is known about how the time interval between initiation events, termed plastochron, is regulated. Here, we present a detailed analysis of plastochron2 (pla2), a rice (Oryza sativa) mutant that exhibits shortened plastochron and precocious maturation of leaves during the vegetative phase and ectopic shoot formation during the reproductive phase. The corresponding PLA2 gene is revealed to be an orthologue of terminal ear1, a maize (Zea mays) gene that encodes a MEI2-like RNA binding protein. PLA2 is expressed predominantly in young leaf primordia. We show that PLA2 normally acts to retard the rate of leaf maturation but does so independently of PLA1, which encodes a member of the P450 family. Based on these analyses, we propose a model in which plastochron is determined by signals from immature leaves that act non-cell-autonomously in the shoot apical meristem to inhibit the initiation of new leaves. PLA1,PLA2|LHD2 Plant low-molecular-weight phospholipase A2s (PLA2s) are structurally related to the animal secretory PLA2s and are present as a family of isoforms in rice (Oryza sativa) 1999 Plant Mol Biol Department of Plant Biology, Swedish University of Agricultural Sciences, P.O. Box 7080, 750 07 , Uppsala, Sweden Recently, we purified to homogeneity and characterized a low-molecular-weight calcium-dependent phospholipase A2 (PLA2) from developing elm seed endosperm. This represented the first purified and characterized PLA2 from a plant tissue. The full sequences of two distinct but homologous rice (Oryza sativa) cDNAs are given here. These encode mature proteins of 119 amino acids (PLA2-I, preceded by a 19 amino acid signal peptide) and 128 amino acids (PLA2-II, preceded by a 25 amino acid signal peptide), and were derived from four expressed sequence tag (EST) clones. Both proteins were homologous to the N-terminal amino acid sequence of the elm PLA2. They contained twelve conserved cysteine residues and sequences that are likely to represent the Ca2+-binding loop and active-site motif, which are characteristic of animal secretory PLA2s. A soluble PLA2 activity was purified 145 000-fold from green rice shoots. This had the same biochemical characteristics as the elm and animal secretory PLA2s. The purified rice PLA2 consisted of two proteins, with a molecular weight of 12 440 and 12 920, that had identical N-terminal amino acid sequences. This sequence was different from but homologous to the PLA2-I and PLA2-II sequences. Taken together, the results suggest that at least three different low-molecular-weight PLA2s are expressed in green rice shoots. Southern blot analysis suggested that multiple copies of such genes are likely to occur in the rice and in other plant genomes. PLA2-I,PLA2-II Leafy head2, which encodes a putative RNA-binding protein, regulates shoot development of rice 2006 Cell Res State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. During vegetative development, higher plants continuously form new leaves in regular spatial and temporal patterns. Mutants with abnormal leaf developmental patterns not only provide a great insight into understanding the regulatory mechanism of plant architecture, but also enrich the ways to its modification by which crop yield could be improved. Here, we reported the characterization of the rice leafy-head2 (lhd2) mutant that exhibits shortened plastochron, dwarfism, reduced tiller number, and failure of phase transition from vegetative to reproductive growth. Anatomical and histological study revealed that the rapid emergence of leaves in lhd2 was resulted from the rapid initiation of leaf primordia whereas the reduced tiller number was a consequence of the suppression of the tiller bud outgrowth. The molecular and genetic analysis showed that LHD2 encodes a putative RNA binding protein with 67% similarity to maize TE1. Comparison of genome-scale expression profiles between wild-type and lhd2 plants suggested that LHD2 may regulate rice shoot development through KNOX and hormone-related genes. The similar phenotypes caused by LHD2 mutation and the conserved expression pattern of LHD2 indicated a conserved mechanism in controlling the temporal leaf initiation in grass. PLA2|LHD2 Photoperiod- and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA 2012 Cell Res State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, China. Photoperiod- and thermo-sensitive genic male sterility (PGMS and TGMS) are the core components for hybrid breeding in crops. Hybrid rice based on the two-line system using PGMS and TGMS lines has been successfully developed and applied widely in agriculture. However, the molecular mechanism underlying the control of PGMS and TGMS remains obscure. In this study, we mapped and cloned a major locus, p/tms12-1 (photo- or thermo-sensitive genic male sterility locus on chromosome 12), which confers PGMS in the japonica rice line Nongken 58S (NK58S) and TGMS in the indica rice line Peiai 64S (PA64S, derived from NK58S). A 2.4-kb DNA fragment containing the wild-type allele P/TMS12-1 was able to restore the pollen fertility of NK58S and PA64S plants in genetic complementation. P/TMS12-1 encodes a unique noncoding RNA, which produces a 21-nucleotide small RNA that we named osa-smR5864w. A substitution of C-to-G in p/tms12-1, the only polymorphism relative to P/TMS12-1, is present in the mutant small RNA, namely osa-smR5864m. Furthermore, overexpression of a 375-bp sequence of P/TMS12-1 in transgenic NK58S and PA64S plants also produced osa-smR5864w and restored pollen fertility. The small RNA was expressed preferentially in young panicles, but its expression was not markedly affected by different day lengths or temperatures. Our results reveal that the point mutation in p/tms12-1, which probably leads to a loss-of-function for osa-smR5864m, constitutes a common cause for PGMS and TGMS in the japonica and indica lines, respectively. Our findings thus suggest that this noncoding small RNA gene is an important regulator of male development controlled by cross-talk between the genetic networks and environmental conditions. pms3 Localization of pms3, a gene for photoperiod-sensitive genic male sterility, to a 28.4-kb DNA fragment 2005 Mol Genet Genomics National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research Wuhan, Huazhong Agricultural University, Wuhan 430070, China. Photoperiod-sensitive genic male-sterile (PSGMS) rice, in which pollen fertility is regulated by day-length, originally arose as a natural mutant in the rice cultivar Nongken 58 (Oryza sativa ssp. japonica). Previous studies identified pms3 on chromosome 12 as the locus of the original PSGMS mutation. In this study we have assigned the pms3 locus to a 28.4-kb DNA fragment by genetic and physical mapping. A cross between Nongken 58S (PSGMS line) and DH80 was used to produce an F2 population of about 7000 plants, from which 892 highly sterile individuals were obtained for recombination analysis. By analyzing recombination events in the sterile individuals using a total of 157 RFLP probes from a BAC contig covering the pms3 region, the pms3 locus was localized to a sub-region of less than 1.7 cM. Further analysis of recombination events using 49 additional probes isolated from this sub-region identified markers flanking the pms3 region on each side; these markers are only 28.4-kb apart. Sequence analysis of this fragment predicted the presence of five ORFs, found high homology with two ESTs in public databases, and detected three SNPs between the mutant and the wild-type parents, which may be helpful for identifying a candidate gene for pms3. pms3 A long noncoding RNA regulates photoperiod-sensitive male sterility, an essential component of hybrid rice 2012 Proc Natl Acad Sci U S A National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agriculture University, Wuhan 430070, China. Hybrid rice has greatly contributed to the global increase of rice productivity. A major component that facilitated the development of hybrids was a mutant showing photoperiod-sensitive male sterility (PSMS) with its fertility regulated by day length. Transcriptome studies have shown that large portions of the eukaryotic genomic sequences are transcribed to long noncoding RNAs (lncRNAs). However, the potential roles for only a few lncRNAs have been brought to light at present. Thus, great efforts have to be invested to understand the biological functions of lncRNAs. Here we show that a lncRNA of 1,236 bases in length, referred to as long-day-specific male-fertility-associated RNA (LDMAR), regulates PSMS in rice. We found that sufficient amount of the LDMAR transcript is required for normal pollen development of plants grown under long-day conditions. A spontaneous mutation causing a single nucleotide polymorphism (SNP) between the wild-type and mutant altered the secondary structure of LDMAR. This change brought about increased methylation in the putative promoter region of LDMAR, which reduced the transcription of LDMAR specifically under long-day conditions, resulting in premature programmed cell death (PCD) in developing anthers, thus causing PSMS. Thus, a lncRNA could directly exert a major effect on a trait like a structure gene, and a SNP could alter the function of a lncRNA similar to amino acid substitution in structural genes. Molecular elucidating of PSMS has important implications for understanding molecular mechanisms of photoperiod regulation of many biological processes and also for developing male sterile germplasms for hybrid crop breeding. pms3 Dissection of a QTL reveals an adaptive, interacting gene complex associated with transgressive variation for flowering time in rice 2010 Theor Appl Genet Department of Plant Breeding and Genetics, Cornell University, 162 Emerson Hall, Ithaca, NY 14853, USA. A days to heading QTL (dth1.1) located on the short arm of rice chromosome 1 was sub-divided into eight sub-introgression lines (SILs) to analyze the genetic basis of transgressive variation for flowering time. Each SIL contained one or more introgression(s) from O. rufipogon in the genetic background of the elite Oryza sativa cultivar, Jefferson. Each introgression was defined at high resolution using molecular markers and those in the dth1.1 region were associated with the presence of one or more flowering time genes (GI, SOC1, FT-L8, EMF1, and PNZIP). SILs and controls were evaluated for flowering time under both short- and long-day growing conditions. Under short-day lengths, lines with introgressions carrying combinations of linked flowering time genes (GI/SOC1, SOC1/FT-L8, GI/SOC1/FT-L8 and EMF1/PNZIP) from the late parent, O. rufipogon, flowered earlier than the recurrent parent, Jefferson, while recombinant lines carrying smaller introgressions marked by the presence of GI, SOC1, EMF1 or PNZIP alone no longer flowered early. Under long-day length, lines carrying SOC1/FT-L8, SOC1 or PNZIP flowered early, while those carrying GI or EMF1 delayed flowering. Across all experiments and in the field, only SIL_SOC1/FT-L8 was consistently early. A preliminary yield evaluation indicated that the transgressive early flowering observed in several of the SILs was also associated with a measurable and positive effect on yield. These SILs represent a new source of variation that can be used in breeding programs to manipulate flowering time in rice cultivars without the reduction in yield that is often associated with early maturing phenotypes. PNZIP Substitution mapping of dth1.1, a flowering-time quantitative trait locus (QTL) associated with transgressive variation in rice, reveals multiple sub-QTL 2006 Genetics Department of Plant Breeding and Genetics, Cornell University, Ithaca, New York 14853, USA. A quantitative trait locus (QTL), dth1.1, was associated with transgressive variation for days to heading in an advanced backcross population derived from the Oryza sativa variety Jefferson and an accession of the wild rice relative Oryza rufipogon. A series of near-isogenic lines (NILs) containing different O. rufipogon introgressions across the target region were constructed to dissect dth1.1 using substitution mapping. In contrast to the late-flowering O. rufipogon parent, O. rufipogon alleles in the substitution lines caused early flowering under both short- and long-day lengths and provided evidence for at least two distinct sub-QTL: dth1.1a and dth1.1b. Potential candidate genes underlying these sub-QTL include genes with sequence similarity to Arabidopsis GI, FT, SOC1, and EMF1, and Pharbitis nil PNZIP. Evidence from families with nontarget O. rufipogon introgressions in combination with dth1.1 alleles also detected an early flowering QTL on chromosome 4 and a late-flowering QTL on chromosome 6 and provided evidence for additional sub-QTL in the dth1.1 region. The availability of a series of near-isogenic lines with alleles introgressed from a wild relative of rice provides an opportunity to better understand the molecular basis of transgressive variation in a quantitative trait. PNZIP Differential induction of a peroxidase gene family during infection of rice by Xanthomonas oryzae pv. oryzae 1997 Mol Plant Microbe Interact Department of Plant Pathology, Kansas State University, Manhattan 66506, USA. Induction of peroxidase has been correlated with resistant interactions between rice and Xanthomonas oryzae pv. oryzae. To assist in analysis of the role of rice peroxidases in plant defense against the bacterial pathogen, three peroxidase genes, POX22.3, POX8.1, and POX5.1, were identified from a rice cDNA library that was constructed from leaves of plants undergoing a resistant reaction. These genes were highly similar in nucleic acid and amino acid sequences and belonged to a gene family. The three genes showed differential expression in infiltrated rice leaves during pathogen interactions and mechanical stress. Only two peroxidase genes, POX8.1 and POX22.3, were predominantly expressed during resistant interactions. These two genes also were expressed during susceptible interactions, but induction was delayed compared with resistant interactions. POXgX9, a fourth peroxidase gene that was isolated from a genomic library, is adjacent to POX22.3 in the rice genome and has greater than 90% similarity in nucleotide and amino acid sequence identity to POX22.3. Interestingly, POXgX9 was expressed only in the roots of rice plants. While POX22.3 was expressed in both leaves and roots, POX8.1 and POX5.1 were not detected in roots but were induced in leaves by mechanical wounding at different times after treatment. POX22.3, POX8.1, and POX5.1 were estimated to be present in single copies in rice haploid genome. These results indicate that different members of the rice peroxidase gene family are distinctly regulated in response to various environmental cues. POX5.1|prx113,POX8.1|prx110,POXGX9|prx112,POX3006|prx114 The class III peroxidase multigenic family in rice and its evolution in land plants 2004 Phytochemistry Laboratory of Plant Physiology, University of Geneva, Quai Ernest-Ansermet 30, CH-1211 Geneva 4, Switzerland. Plant peroxidases (class III peroxidases, E.C. 1.11.1.7) are secreted glycoproteins known to be involved in the mechanism of cell elongation, in cell wall construction and differentiation, and in the defense against pathogens. They usually form large multigenic families in angiosperms. The recent completion of rice (Oryza sativa japonica c.v. Nipponbare) genome sequencing allowed drawing up the full inventory of the genes encoding class III peroxidases in this plant. We found 138 peroxidase genes distributed among the 12 rice chromosomes. In contrast to several other gene families studied so far, peroxidase genes are twice as numerous in rice as in Arabidopsis. This large number of genes results from various duplication events that were tentatively traced back using a phylogenetic tree based on the alignment of conserved amino acid sequences. We also searched for peroxidase encoding genes in the major phyla of plant kingdom. In addition to gymnosperms and angiosperms, sequences were found in liverworts, mosses and ferns, but not in unicellular green algae. Two rice and one Arabidopsis peroxidase genes appeared to be rather close to the only known sequence from the liverwort Marchantia polymorpha. The possible relationship of these peroxidases with the putative ancestor of peroxidase genes is discussed, as well as the connection between the development of the class III peroxidase multigenic family and the emergence of the first land plants. POX5.1|prx113,POX8.1|prx110,POXGX9|prx112,OsPOD|prx11,POX3006|prx114,POXA|prx126,POXN|prx38 Protein phosphatase 2A: identification in Oryza sativa of the gene encoding the regulatory A subunit 2001 Plant Mol Biol National Center For Gene Research, Chinese Academy of Sciences, Shanghai. A 2225 bp cDNA, designated RPA1, was isolated from an Oryza sativa cDNA library. Analysis revealed a 1761 bp coding sequence with 15 non-identical repeat units. The ORF encoded the A regulatory subunit of protein phosphatase 2A (PP2A-A) as ascertained by complementation of the yeast tpd3 mutant defective in this gene. The corresponding genomic DNA from a rice genome BAC library revealed that the gene contains eleven introns. The rice genome contains only a single copy of this gene as judged by Southern blot analysis. The PP2A protein is highly conserved in nature; the rice protein shows 88% amino acid identity with its counterparts in Arabidopsis or Nicotiana tabacum. PP2A-A Posttranslational regulation of pyruvate, orthophosphate dikinase in developing rice (Oryza sativa) seeds 2006 Planta Department of Biosciences, Minnesota State University-Moorhead, Moorhead, MN 56563, USA. chastain@mnstate.edu Pyruvate, orthophosphate dikinase (PPDK; E.C.2.7.9.1) is most well known as a photosynthetic enzyme in C4 plants. The enzyme is also ubiquitous in C3 plant tissues, although a precise non-photosynthetic C3 function(s) is yet to be validated, owing largely to its low abundance in most C3 organs. The single C3 organ type where PPDK is in high abundance, and, therefore, where its function is most amenable to elucidation, are the developing seeds of graminaceous cereals. In this report, we suggest a non-photosynthetic function for C3 PPDK by characterizing its abundance and posttranslational regulation in developing Oryza sativa (rice) seeds. Using primarily an immunoblot-based approach, we show that PPDK is a massively expressed protein during the early syncitial-endosperm/-cellularization stage of seed development. As seed development progresses from this early stage, the enzyme undergoes a rapid, posttranslational down-regulation in activity and amount via regulatory threonyl-phosphorylation (PPDK inactivation) and protein degradation. Immunoblot analysis of separated seed tissue fractions (pericarp, embryo + aleurone, seed embryo) revealed that regulatory phosphorylation of PPDK occurs in the non-green seed embryo and green outer pericarp layer, but not in the endosperm + aleurone layer. The modestly abundant pool of inactive PPDK (phosphorylated + dephosphorylated) that was found to persist in mature rice seeds was shown to remain largely unchanged (inactive) upon seed germination, suggesting that PPDK in rice seeds function in developmental rather than in post-developmental processes. These and related observations lead us to postulate a putative function for the enzyme that aligns its PEP to pyruvate-forming reaction with biosynthetic processes that are specific to early cereal seed development. FLO4|OsPPDKB|ppdk White-core endosperm floury endosperm-4 in rice is generated by knockout mutations in the C-type pyruvate orthophosphate dikinase gene (OsPPDKB) 2005 Plant J Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea. We have isolated a floury endosperm-4 (flo4) rice mutant with a floury-white endosperm but a normal outer portion. Scanning electron microscopic analysis revealed that this abnormal endosperm consisted of loosely packed starch granules. The mutant phenotype was generated by T-DNA insertion into the fifth intron of the OsPPDKB gene encoding pyruvate orthophosphate dikinase (PPDK). Plants containing flo4-1 produced no OsPPDKB transcript or the OsPPDKB protein in their developing kernels and leaves. We obtained two additional alleles, flo4-2 and flo4-3, that also showed the same white-core endosperm phenotype. The flo4 kernels weighed about 6% less than wild-type ones. Starch contents in both kernel types were similar, but the total protein content was slightly higher in the mutant kernels. Moreover, lipid contents were significantly increased in the flo4 kernels. Expression analyses demonstrated that the cytosolic mRNA of OsPPDKB was induced in the reproductive organs after pollination, and greatly increased until about 10 days after fertilization. This mRNA was localized mainly in the endosperm, aleurone, and scutellum of the developing kernel. Our results suggest that cytosolic PPDK functions in rice to modulate carbon metabolism during grain filling. FLO4|OsPPDKB|ppdk Characterization of the gene for pyruvate, orthophosphate dikinase from rice, a C3 plant, and a comparison of structure and expression between C3 and C4 genes for this protein 1997 Plant Mol Biol Japan Turfgrass Inc., 3-6-2 Akanehama, Narashino, Chiba, 275, Japan To investigate the molecular changes that might have occurred in genes for pyruvate,orthophosphate dikinase (PPDK) during the evolution of C4 plants from C3 plants, we isolated a full-length cDNA and the corresponding gene for a C4-like PPDK from rice, a C3 gramineous plant and compared their structures and promoter activities to those of the corresponding gene from maize, a C4 gramineous plant. As in maize, there are at least two ppdk genes in rice and one of them was very similar to the maize C4-type ppdk. The deduced amino acid sequence of the rice PPDK was 88% homologous to the maize C4-type PPDK in the mature peptide region and 56% homologous in the transit peptide region. The C4-like ppdk in rice contained 21 exons, which were interrupted by twenty introns, and the positions of the introns were essentially the same as those in the gene from maize, with the except in that the gene from rice had two extra introns. Such extra introns were also found in the C4-type ppdk from a dicot, Flaveria, at the same positions. These results strongly suggest that the two introns were present in an ancestral gene before the divergence of monocot and dicot plants. The C4-like ppdk in rice contained two functionally independent promoters had generated a larger transcript with the transit peptide region and a smaller transcript without this region. The unusual dual-promoter system for transcription has been conserved in the C4-type ppdk gene from maize, indicating that the dual-promoter system is a common feature of ppdk genes in both C3 and C4 plants. The patterns of expression of the two transcripts in rice were different: the larger transcript was expressed exclusively in green leaves at a low level whereas the smaller transcript was expressed in some reproductive organs at a high level. Essentially the same patterns of expression were observed in maize, but the level of expression of the larger transcript in maize green leaves was much higher than that in green leaves of rice. The promoter activities of the rice and maize genes for PPDK were examined directly in a transient expression assay in maize mesophyll protoplasts after electroporation with promoter::beta-glucuronidase chimeric genes. The rice promoter for the smaller transcript was very active in the protoplasts but the rice promoter for the larger transcript had relatively low activity. By contrast, both promoters of the maize gene had high activity. Taken together, these results demonstrate that the rice C4-like ppdk is very similar to the maize C4-type ppdk, not only in terms of primary structure but also in terms of the regulation of expression, with the exception that the strength of the maize promoter for the larger transcript is higher. The results strongly suggest that the genetic alterations required to give rise to the C4-type ppdk gene were relatively limited. FLO4|OsPPDKB|ppdk Isolation and Molecular Characterization of the COP1 Gene Homolog from Rice,OryzasativaL. subsp.Indicavar. Pusa Basmati 2001 DNA Res Centre for Plant Molecular Biology and Department of Plant Molecular Biology, University of Delhi, New Delhi, India. The COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1) gene has been identified earlier from dicot species namely Arabidopsis, tomato and pea. The protein encoded by this gene acts as a molecular switch that negatively regulates the transition from the skotomorphogenic to the photomorphogenic mode of plant development. We have isolated and characterized the COP1 homolog from a monocot species, i.e. rice (var. Pusa Basmati 1). All the functional domains (Zn-binding RING finger motif, coiled-coil region, WD-40 repeats, cytoplasmic/nuclear localization sequences and protein-protein interaction domains) that are known in the COP1 proteins from dicots are conserved in COP1 from rice as well. The transcript levels of COP1 vary in various tissues of the rice plant. These variations were found to be development-dependent and do not solely depend on the light conditions. COP1|PPS The COP1 ortholog PPS regulates the juvenile-adult and vegetative-reproductive phase changes in rice 2011 Plant Cell Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. Because plant reproductive development occurs only in adult plants, the juvenile-to-adult phase change is an indispensable part of the plant life cycle. We identified two allelic mutants, peter pan syndrome-1 (pps-1) and pps-2, that prolong the juvenile phase in rice (Oryza sativa) and showed that rice PPS is an ortholog of Arabidopsis thaliana CONSTITUTIVE PHOTOMORPHOGENIC1. The pps-1 mutant exhibits delayed expression of miR156 and miR172 and the suppression of GA biosynthetic genes, reducing the GA(3) content in this mutant. In spite of its prolonged juvenile phase, the pps-1 mutant flowers early, and this is associated with derepression of RAP1B expression in pps-1 plants independently of the Hd1-Hd3a/RFT1 photoperiodic pathway. PPS is strongly expressed in the fourth and fifth leaves, suggesting that it regulates the onset of the adult phase downstream of MORI1 and upstream of miR156 and miR172. Its ability to regulate the vegetative phase change and the time of flowering suggests that rice PPS acquired novel functions during the evolution of rice/monocots. COP1|PPS Role of rice PPS in late vegetative and reproductive growth 2012 Plant Signal Behav Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan. The rice peter pan syndrome-1 (pps-1) mutant shows a prolonged juvenile phase and early flowering. Although the early vegetative phase and flowering time of pps-1 have been closely examined, the phenotypes in the late vegetative and reproductive phases are not yet well understood. In the ninth leaf blade of pps-1, the relative length of the midrib was comparable to the sixth leaf blade of wild-type. Moreover, pps-1 had a small inflorescence meristem and small panicles. These phenotypes indicate that in pps-1 the juvenile phase coexists with the late vegetative phase, resulting in small panicles. Gibberellin is known to promote the juvenile-adult phase transition. d18-k is dwarf and has a prolonged juvenile phase. Double mutant (d18-k pps-1) showed the same phenotype as the pps-1, indicating that PPS is upstream of GA biosynthetic genes. COP1|PPS Transgenic rice with inducible ethylene production exhibits broad-spectrum disease resistance to the fungal pathogens Magnaporthe oryzae and Rhizoctonia solani 2013 Plant Biotechnol J Department of Plant Pathology and Huck Institutes of Life Sciences, Pennsylvania State University, University Park, PA, USA. Rice blast (Magnaporthe oryzae) and sheath blight (Rhizoctonia solani) are the two most devastating diseases of rice (Oryza sativa), and have severe impacts on crop yield and grain quality. Recent evidence suggests that ethylene (ET) may play a more prominent role than salicylic acid and jasmonic acid in mediating rice disease resistance. In this study, we attempt to genetically manipulate endogenous ET levels in rice for enhancing resistance to rice blast and sheath blight diseases. Transgenic lines with inducible production of ET were generated by expressing the rice ACS2 (1-aminocyclopropane-1-carboxylic acid synthase, a key enzyme of ET biosynthesis) transgene under control of a strong pathogen-inducible promoter. In comparison with the wild-type plant, the OsACS2-overexpression lines showed significantly increased levels of the OsACS2 transcripts, endogenous ET and defence gene expression, especially in response to pathogen infection. More importantly, the transgenic lines exhibited increased resistance to a field isolate of R. solani, as well as different races of M. oryzae. Assessment of the growth rate, generational time and seed production revealed little or no differences between wild type and transgenic lines. These results suggest that pathogen-inducible production of ET in transgenic rice can enhance resistance to necrotrophic and hemibiotrophic fungal pathogens without negatively impacting crop productivity. OsPR5|Pir2|PR-5|PR5-1 Ultra low-dose radiation: stress responses and impacts using rice as a grass model 2009 Int J Mol Sci Health Technology Research Center, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan. rakwal-68@aist.go.jp We report molecular changes in leaves of rice plants (Oryza sativa L. - reference crop plant and grass model) exposed to ultra low-dose ionizing radiation, first using contaminated soil from the exclusion zone around Chernobyl reactor site. Results revealed induction of stress-related marker genes (Northern blot) and secondary metabolites (LC-MS/MS) in irradiated leaf segments over appropriate control. Second, employing the same in vitro model system, we replicated results of the first experiment using in-house fabricated sources of ultra low-dose gamma (gamma) rays and selected marker genes by RT-PCR. Results suggest the usefulness of the rice model in studying ultra low-dose radiation response/s. OsPR5|Pir2|PR-5|PR5-1,CHS Control of a key transition from prostrate to erect growth in rice domestication 2008 Nat Genet State Key Laboratory of Plant Physiology and Biochemistry, National Center for Evaluation of Agricultural Wild Plants (Rice), Department of Plant Genetics and Breeding, China Agricultural University, Beijing, 100094, China. The transition from the prostrate growth of ancestral wild rice (O. rufipogon Griff.) to the erect growth of Oryza sativa cultivars was one of the most critical events in rice domestication. This evolutionary step importantly improved plant architecture and increased grain yield. Here we find that prostrate growth of wild rice from Yuanjiang County in China is controlled by a semi-dominant gene, PROG1 (PROSTRATE GROWTH 1), on chromosome 7 that encodes a single Cys(2)-His(2) zinc-finger protein. prog1 variants identified in O. sativa disrupt the prog1 function and inactivate prog1 expression, leading to erect growth, greater grain number and higher grain yield in cultivated rice. Sequence comparison shows that 182 varieties of cultivated rice, including 87 indica and 95 japonica cultivars from 17 countries, carry identical mutations in the prog1 coding region that may have become fixed during rice domestication. prog1 Genetic control of rice plant architecture under domestication 2008 Nat Genet National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China. The closely related wild rice species Oryza rufipogon is considered the progenitor of cultivated rice (Oryza sativa). The transition from the characteristic plant architecture of wild rice to that of cultivated rice was one of the most important events in rice domestication; however, the molecular basis of this key domestication transition has not been elucidated. Here we show that the PROG1 gene controls aspects of wild-rice plant architecture, including tiller angle and number of tillers. The gene encodes a newly identified zinc-finger nuclear transcription factor with transcriptional activity and is mapped on chromosome 7. PROG1 is predominantly expressed in the axillary meristems, the site of tiller bud formation. Rice transformation experiments demonstrate that artificial selection of an amino acid substitution in the PROG1 protein during domestication led to the transition from the plant architecture of wild rice to that of domesticated rice. prog1 Dependence of reaction center-type energy-dependent quenching on photosystem II antenna size 2007 Biochim Biophys Acta Department of Molecular Biology, Pusan National University, Jangjeon-dong, Keumjung-ku, Busan 609-735, South Korea. izulfugarov@pusan.ac.kr The effects of photosystem II antenna size on reaction center-type energy-dependent quenching (qE) were examined in rice plants grown under two different light intensities using both wild type and qE-less (OsPsbS knockout) mutant plants. Reaction center-type qE was detected by measuring non-photochemical quenching at 50 micromol photons m(-2) s(-1) white light intensity. We observed that in low light-grown rice plants, reaction center-type qE was higher than in high light-grown plants, and the amount of reaction center-type qE did not depend on zeaxanthin accumulation. This was confirmed in Arabidopsis npq1-2 mutant plants that lack zeaxanthin due to a mutation in the violaxanthin de-epoxidase enzyme. Although the electron transport rate measured at a light intensity of 50 micromol photons m(-2) s(-1) was the same in high light- and low light-grown wild type and mutant plants lacking PsbS protein, the generation of energy-dependent quenching was completely impaired only in mutant plants. Analyses of the pigment content, Lhcb proteins and D1 protein of PSII showed that the antenna size was larger in low light-grown plants, and this correlated with the amount of reaction center-type qE. Our results mark the first time that the reaction center-type qE has been shown to depend on photosystem II antenna size and, although it depends on the existence of PsbS protein, the extent of reaction center-type qE does not correlate with the transcript levels of PsbS protein. The presence of reaction center-type energy-dependent quenching, in addition to antenna-type quenching, in higher plants for dissipation of excess light energy demonstrates the complexity and flexibility of the photosynthetic apparatus of higher plants to respond to different environmental conditions. OsPsbS|psbS1 The photoprotective protein PsbS exerts control over CO(2) assimilation rate in fluctuating light in rice 2012 Plant J Division of Plant and Crop Science, School of Biosciences, Sutton Bonington Campus, University of Nottingham, LE12 5RD, Nottingham, UK. A direct impact of chloroplastic protective energy dissipation (qE) on photosynthetic CO(2) assimilation has not been shown directly in plants in the absence of photoinhibition. To test this empirically we transformed rice to possess higher (overexpressors, OE) and lower (RNA interference, RNAi) levels of expression of the regulatory psbS gene and analysed CO(2) assimilation in transformants in a fluctuating measurement light regime. Western blots showed a several-fold difference in levels of PsbS protein between RNAi and OE plants with the wild type (WT) being intermediate. At a growth light intensity of 600 mumol m(-2) sec(-1) , the carboxylation capacity, electron transport capacity and dark adapted F(v)/F(m) (ratio of variable to maximum fluorescence) were inhibited in RNAi plants compared with WT and OE. The PsbS content had a significant impact on qE (measured here as non-photochemical quenching, NPQ) but the strongest effect was observed transiently, immediately following the application of light. This capacity for qE was several-fold lower in RNAi plants and significantly higher in OE plants during the first 10 min of illumination. At steady state the differences were reduced: notably at 500 mumol m(-2) sec(-1) all plants had the same NPQ values regardless of PsbS content. During a series of light-dark transitions the induction of CO(2) assimilation was inhibited in OE plants, reducing integrated photosynthesis during the light period. We conclude that the accumulation of PsbS and the resultant qE exerts control over photosynthesis in fluctuating light, showing that optimization of photoprotective processes is necessary for maximum photosynthetic productivity even in the absence of photoinhibitory stress. OsPsbS|psbS1 Allocation of absorbed light energy in PSII to thermal dissipations in the presence or absence of PsbS subunits of rice 2011 Plant Cell Physiol Division of Integrated Life Sciences, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan. The thermal dissipation (TD) of absorbed light energy in PSII is considered to be an important photoprotection process in photosynthesis. A major portion of TD has been visualized through the analysis of Chl fluorescence as energy quenching (qE) which depends on the presence of the PsbS subunit. Although the physiological importance of qE-associated TD (qE-TD) has been widely accepted, it is not yet clear how much of the absorbed light energy is dissipated through a qE-associated mechanism. In this study, the fates of absorbed light energy in PSII with regard to different TD processes, including qE-TD, were quantitatively estimated by the typical energy allocation models using transgenic rice in which psbS genes were silenced by RNA interference (RNAi). The silencing of psbS genes resulted in a decrease in the light-inducible portion of TD, whereas the allocation of energy to electron transport did not change over a wide range of light intensities. The allocation models indicate that the energy allocated to qE-TD under saturating light is 30-50%. We also showed that a large portion of absorbed light energy is thermally dissipated in manners that are independent of qE. The nature of such dissipations is discussed. OsPsbS|psbS1,psbS2 Expression and characterization of rice putativePAUSEDgene 2008 Acta Biochimica et Biophysica Sinica State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai 200031, China In Arabidopsis, PAUSED (PSD) encodes the ortholog of los1p/exportin-t, which mediates the nuclear export of transfer RNA (tRNA) in yeast and mammals. However, in monocot plants such as rice, knowledge of the corresponding ortholog is limited, and its effects on growth development and productivity remain unknown. In this study, we verified a rice transfer-DNAinsertional mutant psd line and analyzed its phenotypes; the mutant displayed severe morphological defects including retarded development and low fertility compared with wild-type rice. Examining intronless tRNA-Tyr and intron-containing pre-tRNA-Ala expression levels in cytoplasmic and nuclear fraction with Northern blot analysis between wild-type and mutant leaf tissue suggested that rice PSD might be involved in tRNA export from the nucleus to the cytoplasm. Additionally, reverse transcription-polymerase chain reaction analysis revealed that PSD transcript was expressed throughout normal rice plant development, and subcellular localization assays showed that rice PSD protein was present in both the nucleus and cytoplasm. In summary, our data implied that the putative PSD gene might be indispensable for normal rice development and its function might be the same as that of Arabidopsis PSD. PSD Isolation of a rice regeneration quantitative trait loci gene and its application to transformation systems 2005 Proc Natl Acad Sci U S A Honda Research Institute Japan, 2-1-4 Kazusa-Kamatari, Kisarazu-shi, Chiba 292-0818, Japan. asuka.nishimura@jp.honda-ri.com Regeneration of plant organs is often the essential step in genetic transformation; however, the regeneration ability of a plant varies depending on the genetic background. By conventional crosses of low-regeneration rice strain Koshihikari with high-regeneration rice strain Kasalath, we identified some quantitative trait loci, which control the regeneration ability in rice. Using a map-based cloning strategy, we isolated a main quantitative trait loci gene encoding ferredoxin-nitrite reductase (NiR) that determines regeneration ability in rice. Molecular analyses revealed that the poor regeneration ability of Koshihikari is caused by lower expression than in Kasalath and the specific activity of NiR. Using the NiR gene as a selection marker, we succeeded in selectively transforming a foreign gene into rice without exogenous marker genes. Our results demonstrate that nitrate assimilation is an important process in rice regeneration and also provide an additional selectable marker for rice transformation. PSR1 Fine mapping of pss1, a pollen semi-sterile gene in rice (Oryza sativa L.) 2007 Theor Appl Genet State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, 210095, China. During routine seed increase procedures in rice, semi-sterile plants are common; however, such semi-sterility mutants in rice varieties have been only rarely analyzed genetically. W207-2 is a semi-sterile selection from the japonica rice variety Nipponbare. In this report, we found the female gamete of W207-2 was normal, and its semi-sterility was unaffected by growth duration but was conditioned by a recessive nuclear gene whose action leads to pollen semi-sterility and anther indehiscence, and the gene was named as pss1 (pollen semi-sterile). Using an F(2) population derived from the two parents W207-2 and Dular and a pooled DNA strategy, pss1 was mapped to an interval on chromosome 8 defined by the two SSR loci RM6356 and RS41. The position of pss1 was confirmed in another F(2) population derived from the cross W207-2 x Nipponbare. Over 2,000 homozygous pss1 segregants from the large W207-2 x Dular F(2) population were used to fine map pss1 to a 0.04 cM segment flanked by a CAPs marker L2 and a dCAPs L3 marker. Sequences for both markers are present on a single PAC clone, and the physical distance between them is about 28 kb. Analysis of the PAC sequence predicts the presence of five open reading frames, they are as follows: putative ribonuclease PH, putative avr9 elicitor response protein, kinesin1-like protein, putative protein RNP-D precursor and putative 40S ribosomal protein S13. This result would be helpful in cloning the pss1 gene. PSS1 Pollen semi-sterility1 encodes a kinesin-1-like protein important for male meiosis, anther dehiscence, and fertility in rice 2011 Plant Cell National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, China. In flowering plants, male meiosis produces four microspores, which develop into pollen grains and are released by anther dehiscence to pollinate female gametophytes. The molecular and cellular mechanisms regulating male meiosis in rice (Oryza sativa) remain poorly understood. Here, we describe a rice pollen semi-sterility1 (pss1) mutant, which displays reduced spikelet fertility (~40%) primarily caused by reduced pollen viability (~50% viable), and defective anther dehiscence. Map-based molecular cloning revealed that PSS1 encodes a kinesin-1-like protein. PSS1 is broadly expressed in various organs, with highest expression in panicles. Furthermore, PSS1 expression is significantly upregulated during anther development and peaks during male meiosis. The PSS1-green fluorescent protein fusion is predominantly localized in the cytoplasm of rice protoplasts. Substitution of a conserved Arg (Arg-289) to His in the PSS1 motor domain nearly abolishes its microtubule-stimulated ATPase activity. Consistent with this, lagging chromosomes and chromosomal bridges were found at anaphase I and anaphase II of male meiosis in the pss1 mutant. Together, our results suggest that PSS1 defines a novel member of the kinesin-1 family essential for male meiotic chromosomal dynamics, male gametogenesis, and anther dehiscence in rice. PSS1 Development and application of gene-based markers for the major rice QTL Phosphorus uptake 1 2010 Theor Appl Genet International Rice Research Institute (IRRI), Plant Breeding, Genetics, and Biotechnology Division (PBGB), DAPO Box 7777, Metro Manila, Philippines. Marker-assisted breeding is a very useful tool for breeders but still lags behind its potential because information on the effect of quantitative trait loci (QTLs) in different genetic backgrounds and ideal molecular markers are unavailable. Here, we report on some first steps toward the validation and application of the major rice QTL Phosphate uptake 1 (Pup1) that confers tolerance of phosphorus (P) deficiency in rice (Oryza sativa L.). Based on the Pup1 genomic sequence of the tolerant donor variety Kasalath that recently became available, markers were designed that target (1) putative genes that are partially conserved in the Nipponbare reference genome and (2) Kasalath-specific genes that are located in a large insertion-deletion (INDEL) region that is absent in Nipponbare. Testing these markers in 159 diverse rice accessions confirmed their diagnostic value across genotypes and showed that Pup1 is present in more than 50% of rice accessions adapted to stress-prone environments, whereas it was detected in only about 10% of the analyzed irrigated/lowland varieties. Furthermore, the Pup1 locus was detected in more than 80% of the analyzed drought-tolerant rice breeding lines, suggesting that breeders are unknowingly selecting for Pup1. A hydroponics experiment revealed genotypic differences in the response to P deficiency between upland and irrigated varieties but confirmed that root elongation is independent of Pup1. Contrasting Pup1 near-isogenic lines (NILs) were subsequently grown in two different P-deficient soils and environments. Under the applied aerobic growth conditions, NILs with the Pup1 locus maintained significantly higher grain weight plant(-1) under P deprivation in comparison with intolerant sister lines without Pup1. Overall, the data provide evidence that Pup1 has the potential to improve yield in P-deficient and/or drought-prone environments and in diverse genetic backgrounds. PSTOL1 The protein kinase Pstol1 from traditional rice confers tolerance of phosphorus deficiency 2012 Nature International Rice Research Institute, DAPO Box 7777 Metro, Manila 1301, Philippines. As an essential macroelement for all living cells, phosphorus is indispensable in agricultural production systems. Natural phosphorus reserves are limited, and it is therefore important to develop phosphorus-efficient crops. A major quantitative trait locus for phosphorus-deficiency tolerance, Pup1, was identified in the traditional aus-type rice variety Kasalath about a decade ago. However, its functional mechanism remained elusive until the locus was sequenced, showing the presence of a Pup1-specific protein kinase gene, which we have named phosphorus-starvation tolerance 1 (PSTOL1). This gene is absent from the rice reference genome and other phosphorus-starvation-intolerant modern varieties. Here we show that overexpression of PSTOL1 in such varieties significantly enhances grain yield in phosphorus-deficient soil. Further analyses show that PSTOL1 acts as an enhancer of early root growth, thereby enabling plants to acquire more phosphorus and other nutrients. The absence of PSTOL1 and other genes-for example, the submergence-tolerance gene SUB1A-from modern rice varieties underlines the importance of conserving and exploring traditional germplasm. Introgression of this quantitative trait locus into locally adapted rice varieties in Asia and Africa is expected to considerably enhance productivity under low phosphorus conditions. PSTOL1 Developing rice with high yield under phosphorus deficiency: Pup1 sequence to application 2011 Plant Physiol International Rice Research Institute, Plant Breeding, Genetics, and Biotechnology Division, 7777 Metro Manila, Philippines. The major quantitative trait locus (QTL) Phosphorus uptake1 (Pup1) confers tolerance of phosphorus deficiency in soil and is currently one of the most promising QTLs for the development of tolerant rice (Oryza sativa) varieties. To facilitate targeted introgression of Pup1 into intolerant varieties, the gene models predicted in the Pup1 region in the donor variety Kasalath were used to develop gene-based molecular markers that are evenly distributed over the fine-mapped 278-kb QTL region. To validate the gene models and optimize the markers, gene expression analyses and partial allelic sequencing were conducted. The markers were tested in more than 80 diverse rice accessions revealing three main groups with different Pup1 allele constitution. Accessions with tolerant (group I) and intolerant (group III) Pup1 alleles were distinguished from genotypes with Kasalath alleles at some of the analyzed loci (partial Pup1; group II). A germplasm survey additionally confirmed earlier data showing that Pup1 is largely absent from irrigated rice varieties but conserved in varieties and breeding lines adapted to drought-prone environments. A core set of Pup1 markers has been defined, and sequence polymorphisms suitable for single-nucleotide polymorphism marker development for high-throughput genotyping were identified. Following a marker-assisted backcrossing approach, Pup1 was introgressed into two irrigated rice varieties and three Indonesian upland varieties. First phenotypic evaluations of the introgression lines suggest that Pup1 is effective in different genetic backgrounds and environments and that it has the potential to significantly enhance grain yield under field conditions. PSTOL1 Nucleotide diversity and molecular evolution of the PSY1 gene in Zea mays compared to some other grass species 2010 Theor Appl Genet National Maize Improvement Center of China, Key Laboratory of Crop Genomics and Genetic Improvement, China Agricultural University, Yuanmingyuan West Road, Haidian, Beijing, China. Phytoene synthase (PSY), which is encoded by the phytoene synthase 1 (PSY1) gene, is the first rate-limiting enzyme in the plant carotenoid biosynthetic pathway. In order to examine the genetic diversity and evolution pattern of PSY1 within the Andropogoneae, sequences of 76 accessions from 5 species (maize, teosinte, tripsacum, coix, and sorghum) of the Andropogoneae were tested, along with 4 accessions of rice (Oryza sativa L.) included as outliers. Both the number and the order of exons and introns were relatively conserved across the species tested. Three domains were identified in the coding sequence, including signal peptide (SP), PSY, and highly conserved squalene synthase (SQS) domain. Although no positive selection signal was detected at an overall coding level among all species tested, the SP domain and the region upstream of the SQS-PSY domain appear to have undergone rapid evolution, as evidenced by a high d (N)/d (S) ratio (>1.0). At the nucleotide level, positive selection and balancing selection were detected only among the yellow maize germplasm and the white maize germplasm, respectively. The phylogenetic tree based on full-length sequences of PSY1-like regions supported the monophyletic theory of the Andropogoneae and the closest relationship between Zea and Tripsacum among the Andropogoneae. Coix, which was theorized to have a closer relationship with maize due to similarities in morphology and chromosome number, has been shown in this study to have diverged relatively early from the other Andropogoneae, including maize. PSY1 Gene duplication in the carotenoid biosynthetic pathway preceded evolution of the grasses 2004 Plant Physiol Department of Biological Sciences, Lehman College, The City University of New York, Bronx, New York 10468, USA. Despite ongoing research on carotenoid biosynthesis in model organisms, there is a paucity of information on pathway regulation operating in the grasses (Poaceae), which include plants of world-wide agronomic importance. As a result, efforts to either breed for or metabolically engineer improvements in carotenoid content or composition in cereal crops have led to unexpected results. In comparison to maize (Zea mays), rice (Oryza sativa) accumulates no endosperm carotenoids, despite having a functional pathway in chloroplasts. To better understand why these two related grasses differ in endosperm carotenoid content, we began to characterize genes encoding phytoene synthase (PSY), since this nuclear-encoded enzyme appeared to catalyze a rate-controlling step in the plastid-localized biosynthetic pathway. The enzyme had been previously associated with the maize Y1 locus thought to be the only functional gene controlling PSY accumulation, though function of the Y1 gene product had never been demonstrated. We show that both maize and rice possess and express products from duplicate PSY genes, PSY1 (Y1) and PSY2; PSY1 transcript accumulation correlates with carotenoid-containing endosperm. Using a heterologous bacterial system, we demonstrate enzyme function of PSY1 and PSY2 that are largely conserved in sequence except for N- and C-terminal domains. By database mining and use of ortholog-specific universal PCR primers, we found that the PSY duplication is prevalent in at least eight subfamilies of the Poaceae, suggesting that this duplication event preceded evolution of the Poaceae. These findings will impact study of grass phylogeny and breeding of enhanced carotenoid content in an entire taxonomic group of plant crops critical for global food security. PSY1,PSY2 Natural variation in PTB1 regulates rice seed setting rate by controlling pollen tube growth 2013 Nat Commun Rice Research Institute, Sichuan Agricultural University, 211 Huimin Road, Wenjiang 611130, Sichuan, China Grain number, panicle seed setting rate, panicle number and grain weight are the most important components of rice grain yield. To date, several genes related to grain weight, grain number and panicle number have been described in rice. However, no genes regulating the panicle seed setting rate have been functionally characterized. Here we show that the domestication-related POLLEN TUBE BLOCKED 1 (PTB1), a RING-type E3 ubiquitin ligase, positively regulates the rice panicle seed setting rate by promoting pollen tube growth. The natural variation in expression of PTB1 which is affected by the promoter haplotype and the environmental temperature, correlates with the rice panicle seed setting rate. Our results support the hypothesis that PTB1 is an important maternal sporophytic factor of pollen tube growth and a key modulator of the rice panicle seed setting rate. This finding has implications for the improvement of rice yield. PTB1 PERSISTENT TAPETAL CELL1 encodes a PHD-finger protein that is required for tapetal cell death and pollen development in rice 2011 Plant Physiol School of Life Sciences and Biotechnology , Shanghai Jiao Tong University, Shanghai 200240, China. In higher plants, timely degradation of tapetal cells, the innermost sporophytic cells of the anther wall layer, is a prerequisite for the development of viable pollen grains. However, relatively little is known about the mechanism underlying programmed tapetal cell development and degradation. Here, we report a key regulator in monocot rice (Oryza sativa), PERSISTANT TAPETAL CELL1 (PTC1), which controls programmed tapetal development and functional pollen formation. The evolutionary significance of PTC1 was revealed by partial genetic complementation of the homologous mutation MALE STERILITY1 (MS1) in the dicot Arabidopsis (Arabidopsis thaliana). PTC1 encodes a PHD-finger (for plant homeodomain) protein, which is expressed specifically in tapetal cells and microspores during anther development in stages 8 and 9, when the wild-type tapetal cells initiate a typical apoptosis-like cell death. Even though ptc1 mutants show phenotypic similarity to ms1 in a lack of tapetal DNA fragmentation, delayed tapetal degeneration, as well as abnormal pollen wall formation and aborted microspore development, the ptc1 mutant displays a previously unreported phenotype of uncontrolled tapetal proliferation and subsequent commencement of necrosis-like tapetal death. Microarray analysis indicated that 2,417 tapetum- and microspore-expressed genes, which are principally associated with tapetal development, degeneration, and pollen wall formation, had changed expression in ptc1 anthers. Moreover, the regulatory role of PTC1 in anther development was revealed by comparison with MS1 and other rice anther developmental regulators. These findings suggest a diversified and conserved switch of PTC1/MS1 in regulating programmed male reproductive development in both dicots and monocots, which provides new insights in plant anther development. PTC1 The tillering phenotype of the rice plastid terminal oxidase (PTOX) loss-of-function mutant is associated with strigolactone deficiency 2014 New Phytol Iwate Biotechnology Research Center, Narita 22-174-4, Kitakami, Iwate, 024-0003, Japan. The significance of plastid terminal oxidase (PTOX) in phytoene desaturation and chloroplast function has been demonstrated using PTOX-deficient mutants, particularly in Arabidopsis. However, studies on its role in monocots are lacking. Here, we report cloning and characterization of the rice (Oryza sativa) PTOX1 gene. Using Ecotype Targeting Induced Local Lesions IN Genomes (EcoTILLING) and TILLING as forward genetic tools, we identified the causative mutation of an EMS mutant characterized by excessive tillering, semi-dwarfism and leaf variegation that corresponded to the PTOX1 gene. The tillering and semi-dwarf phenotypes of the ptox1 mutant are similar to phenotypes of known strigolactone (SL)-related rice mutants, and both phenotypic traits could be rescued by application of the synthetic SL GR24. The ptox1 mutant accumulated phytoene in white leaf sectors with a corresponding deficiency in beta-carotene, consistent with the expected function of PTOX1 in promoting phytoene desaturase activity. There was also no accumulation of the carotenoid-derived SL ent-2'-epi-5-deoxystrigol in root exudates. Elevated concentrations of auxin were detected in the mutant, supporting previous observations that SL interaction with auxin is important in shoot branching control. Our results demonstrate that PTOX1 is required for both carotenoid and SL synthesis resulting in SL-deficient phenotypes in rice. PTOX1 Isolation and Characterization of a Water Stress-Specific Genomic Gene, pwsi18, from Rice 1998 Plant Cell Physiol Laboratory of Plant Genetic Engineering, Akita Prefectural College of Agriculture, Japan. One of the water stress-specific cDNA clones of rice characterised previously, wsi18, was selected for further study. The wsi18 gene can be induced by water stress conditions such as mannitol, NaCl, and dryness, but not by ABA, cold, or heat. A genomic clone for wsi18, pwsi18, contained about 1.7 kbp of the 5' upstream sequence, two introns, and the full coding sequence. The 5'-upstream sequence of pwsi18 contained putative cis-acting elements, namely an ABA-responsive element (ABRE), three G-boxes, three E-boxes, a MEF-2 sequence, four direct and two inverted repeats, and four sequences similar to DRE, which is involved in the dehydration response of Arabidopsis genes. The gusA reporter gene under the control of the pwsi18 promoter showed transient expression in response to water stress. Deletion of the downstream DRE-like sequence between the distal G-boxes-2 and -3 resulted in rather low GUS expression. pwsi18|WSI18 Functional analysis of the C-terminal region of the vacuolar cadmium-transporting rice OsHMA3 2014 FEBS Lett Graduate School of Bioresource Sciences, Akita Prefectural University, Kaidoubata-Nishi 241-438, Shimoshinjyo-Nakano, Akita 010-0195, Japan. Rice OsHMA3 is a vacuolar cadmium (Cd) transporter belonging to the P1B-ATPase family and has a long (273aa) C-terminal region. We analyzed the function of the region related to Cd using the transgenic Arabidopsis Col-0 ecotype, which is sensitive to Cd. The OsHMA3 variant containing a truncated (58aa) C-terminal region did not confer Cd tolerance, whereas an OsHMA3 variant containing a longer truncated (105aa) C-terminal region conferred Cd tolerance to transgenic Arabidopsis. We conclude that the C-terminal region, particularly the region containing the first 105aa, has an important role in OsHMA3 activity. qCdT7|OsHMA3 OsHMA3, a P1B-type of ATPase affects root-to-shoot cadmium translocation in rice by mediating efflux into vacuoles 2011 New Phytol Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, Shimoshinjyo-Nakano, Akita, Japan. * The cadmium (Cd) over-accumulating rice (Oryza sativa) cv Cho-Ko-Koku was previously shown to have an enhanced rate of root-to-shoot Cd translocation. This trait is controlled by a single recessive allele located at qCdT7. * In this study, using positional cloning and transgenic strategies, heavy metal ATPase 3 (OsHMA3) was identified as the gene that controls root-to-shoot Cd translocation rates. The subcellular localization and Cd-transporting activity of the gene products were also investigated. * The allele of OsHMA3 that confers high root-to-shoot Cd translocation rates (OsHMA3mc) encodes a defective P(1B) -ATPase transporter. OsHMA3 fused to green fluorescent protein was localized to vacuolar membranes in plants and yeast. An OsHMA3 transgene complemented Cd sensitivity in a yeast mutant that lacks the ability to transport Cd into vacuoles. By contrast, OsHMA3mc did not complement the Cd sensitivity of this yeast mutant, indicating that the OsHMA3mc transport function was lost. * We propose that the root cell cytoplasm of Cd-overaccumulating rice plants has more Cd available for loading into the xylem as a result of the lack of OsHMA3-mediated transportation of Cd to the vacuoles. This defect results in Cd translocation to the shoots in higher concentrations. These data demonstrate the importance of vacuolar sequestration for Cd accumulation in rice. qCdT7|OsHMA3 Physiological, genetic, and molecular characterization of a high-Cd-accumulating rice cultivar, Jarjan 2011 J Exp Bot Institute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki 710-0046, Japan. Cadmium (Cd) in rice is a major source of Cd intake for people on a staple rice diet. The mechanisms underlying Cd accumulation in rice plant are still poorly understood. Here, we characterized the physiology and genetics of Cd transport in a high-Cd-accumulating cultivar (Jarjan) of rice (Oryza sativa). Jarjan showed 5- to 34-fold higher Cd accumulation in the shoots and grains than the cultivar Nipponbare, when it was grown in either a non-Cd-contaminated or a Cd-contaminated soil. A short-term uptake experiment showed no significant difference in Cd uptake by the roots between the two cultivars. However, Jarjan translocated 49% of the total Cd taken up to the shoots, whereas Nipponbare retained most of the Cd in the roots. In both concentration- and time-dependent experiments, Jarjan showed a superior capacity for root-to-shoot translocation of Cd. These results indicate that the high-Cd-accumulation phenotype in Jarjan results from efficient translocation of Cd from roots to shoots. Genetic analysis using an F(2) population derived from Jarjan and Nipponbare revealed that plants showing high- and low-Cd-accumulation phenotypes segregated in a 1:3 ratio, indicating that high accumulation in Jarjan is controlled by a single recessive gene. Furthermore, we isolated OsHMA3, a gene encoding a tonoplast-localized Cd transporter from Jarjan. The OsHMA3 protein was localized in all roots cells, but the sequence has a mutation leading to loss of function. Therefore, failure to sequester Cd into the root vacuoles by OsHMA3 is probably responsible for high Cd accumulation in Jarjan. qCdT7|OsHMA3 Gene limiting cadmium accumulation in rice 2010 Proc Natl Acad Sci U S A Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan. Intake of toxic cadmium (Cd) from rice caused Itai-itai disease in the past and it is still a threat for human health. Therefore, control of the accumulation of Cd from soil is an important food-safety issue, but the molecular mechanism for the control is unknown. Herein, we report a gene (OsHMA3) responsible for low Cd accumulation in rice that was isolated from a mapping population derived from a cross between a high and low Cd-accumulating cultivar. The gene encodes a transporter belonging to the P(1B)-type ATPase family, but shares low similarity with other members. Heterologous expression in yeast showed that the transporter from the low-Cd cultivar is functional, but the transporter from the high-Cd cultivar had lost its function, probably because of the single amino acid mutation. The transporter is mainly expressed in the tonoplast of root cells at a similar level in both the low and high Cd-accumulating cultivars. Overexpression of the functional gene from the low Cd-accumulating cultivar selectively decreased accumulation of Cd, but not other micronutrients in the grain. Our results indicated that OsHMA3 from the low Cd-accumulating cultivar limits translocation of Cd from the roots to the above-ground tissues by selectively sequestrating Cd into the root vacuoles. qCdT7|OsHMA3 A single recessive gene controls cadmium translocation in the cadmium hyperaccumulating rice cultivar Cho-Ko-Koku 2010 Theor Appl Genet Laboratory of Plant Genetics and Breeding, Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, Kaidoubata-Nishi 241-438, Shimoshinjyo-Nakano, Akita, 010-0195, Japan. The heavy metal cadmium (Cd) is highly toxic to humans and can enter food chains from contaminated crop fields. Understanding the molecular mechanisms of Cd accumulation in crop species will aid production of safe Cd-free food. Here, we identified a single recessive gene that allowed higher Cd translocation in rice, and also determined the chromosomal location of the gene. The Cd hyperaccumulator rice variety Cho-Ko-Koku showed 3.5-fold greater Cd translocation than the no-accumulating variety Akita 63 under hydroponics. Analysis of an F(2) population derived from these cultivars gave a 1:3 segregation ratio for high:low Cd translocation. This indicates that a single recessive gene controls the high Cd translocation phenotype. A QTL analysis identified a single QTL, qCdT7, located on chromosome 7. On a Cd-contaminated field, Cd accumulation in the F(2) population showed continuous variation with considerable transgression. Three QTLs for Cd accumulation were identified and the peak of the most effective QTL mapped to the same region as qCdT7. Our data indicate that Cd translocation mediated by the gene on qCdT7 plays an important role in Cd accumulation on contaminated soil. qCdT7|OsHMA3 Control of grain size, shape and quality by OsSPL16 in rice 2012 Nat Genet The State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, National Centre for Plant Gene Research, Beijing, China. Grain size and shape are important components of grain yield and quality and have been under selection since cereals were first domesticated. Here, we show that a quantitative trait locus GW8 is synonymous with OsSPL16, which encodes a protein that is a positive regulator of cell proliferation. Higher expression of this gene promotes cell division and grain filling, with positive consequences for grain width and yield in rice. Conversely, a loss-of-function mutation in Basmati rice is associated with the formation of a more slender grain and better quality of appearance. The correlation between grain size and allelic variation at the GW8 locus suggests that mutations within the promoter region were likely selected in rice breeding programs. We also show that a marker-assisted strategy targeted at elite alleles of GS3 and OsSPL16 underlying grain size and shape can be effectively used to simultaneously improve grain quality and yield. qGW8|OsSPL16 Isolation and characterization of a rice WUSCHEL-type homeobox gene that is specifically expressed in the central cells of a quiescent center in the root apical meristem 2003 The Plant Journal Nagoya University, BioScience Center, Chikusa, Nagoya 464-8601, Japan. The Arabidopsis WUSCHEL (WUS) protein, which plays an important role in the specification of the stem cells in the shoot apical meristem (SAM), contains an 'atypical' homeodomain (HD) with extra residues in its loop and turn regions. We speculated that a WUS-type atypical HD protein might also be involved in the specification and maintenance of the root apical meristem (RAM) stem cells of rice. To investigate this possibility, we isolated and characterized a rice WUS-type homeobox gene designated quiescent-center-specific homeobox (QHB) gene. Using transformants carrying the QHB promoter-GUS and in situ hybridization, we found that QHB was specifically expressed in the central cells of a quiescent center (QC) of the root. During embryogenesis and crown root formation, QHB expression was observed prior to the morphological differentiation of the root. However, we detected different QHB expression patterns in the process of the RAM development, specifically between radicle and crown root formation, suggesting that the cell-fate determination of the QC may be controlled by different mechanisms. We also produced transformants that overexpress QHB or Arabidopsis WUS. These transformants did not form crown roots, but developed multiple shoots from ectopic SAMs with malformed leaves. On the basis of these observations, we propose that the WUS-type homeobox gene is involved in the specification and maintenance of the stem cells (QC cells) in the RAM, by a mechanism similar to that for WUS in the SAM. QHB Genome-wide analysis of genes targeted by qLTG3-1 controlling low-temperature germinability in rice 2010 Plant Mol Biol Plant Breeding & Production Division, Agricultural Research Institute, Hokuren Federation of Agricultural Cooperatives, Naganuma, Hokkaido, 0691317, Japan. fujino-kenji@hokuren.jp The control of seed germination under environmental conditions, where plants will be grown, is important for the adaptability of plants. Low-temperature is one of the most common environmental stress factors that affect plant growth and development and places a major limit on crop productivity in cultivated areas. Previously, qLTG3-1, a major quantitative trait locus controlling low-temperature tolerance at the germination stage in rice (called low-temperature germinability) was identified, which encodes a protein of unknown function. To identify genes targeted by qLTG3-1, a genome-wide expression profiling analysis using the 44 K Rice Oligo microarray was performed. Because the expression of qLTG3-1 was dramatically increased at 1 day after incubation, the expression profiles at this time were compared between Hayamasari, which has a loss-of-function qLTG3-1 allele, and a near isogenic line with a functional allele. A total of 4,587 genes showed significant differences between their expression levels in the two lines. Most of these genes might be involved in the process of seed germination itself, and then a focus was made on qLTG3-1 dependently induced or suppressed genes, defined as 'qLTG3-1 dependent' genes. Twenty-nine 'qLTG3-1 dependent' genes with diverse functions were categorized, implying that disruption of cellular homeostasis leads to a wide range of metabolic alterations and diverse cross-talk between various signaling pathways. In particular, genes involved in defense responses were up-regulated by qLTG3-1, indicating that qLTG3-1 expression is required for the expression of defense response genes in low-temperature germinability in rice. qLTG3-1 Molecular identification of a major quantitative trait locus, qLTG3-1, controlling low-temperature germinability in rice 2008 Proc Natl Acad Sci U S A Plant Breeding and Production Division, Agricultural Research Institute, HOKUREN Federation of Agricultural Cooperatives, Naganuma, Hokkaido 0691317, Japan. fujino-kenji@hokuren.jp Tolerance to abiotic stress is an important agronomic trait in crops and is controlled by many genes, which are called quantitative trait loci (QTLs). Identification of these QTLs will contribute not only to the understanding of plant biology but also for plant breeding, to achieve stable crop production around the world. Previously, we mapped three QTLs controlling low-temperature tolerance at the germination stage (called low-temperature germinability). To understand the molecular basis of one of these QTLs, qLTG3-1 (quantitative trait locus for low-temperature germinability on chromosome 3), map-based cloning was performed, and this QTL was shown to be encoded by a protein of unknown function. The QTL qLTG3-1 is strongly expressed in the embryo during seed germination. Before and during seed germination, specific localization of beta-glucuronidase staining in the tissues covering the embryo, which involved the epiblast covering the coleoptile and the aleurone layer of the seed coat, was observed. Expression of qLTG3-1 was tightly associated with vacuolation of the tissues covering the embryo. This may cause tissue weakening, resulting in reduction of the mechanical resistance to the growth potential of the coleoptile. These phenomena are quite similar to the model system of seed germination presented by dicot plants, suggesting that this model may be conserved in both dicot and monocot plants. qLTG3-1 An SNP caused loss of seed shattering during rice domestication 2006 Science Institute of the Society for Techno-Innovation of Agriculture, Forestry, and Fisheries, 446-1 Ippaizuka, Kamiyokoba Tsukuba, Ibaraki 305-0854, Japan. Loss of seed shattering was a key event in the domestication of major cereals. We revealed that the qSH1 gene, a major quantitative trait locus of seed shattering in rice, encodes a BEL1-type homeobox gene and demonstrated that a single-nucleotide polymorphism (SNP) in the 5' regulatory region of the qSH1 gene caused loss of seed shattering owing to the absence of abscission layer formation. Haplotype analysis and association analysis in various rice collections revealed that the SNP was highly associated with shattering among japonica subspecies of rice, implying that it was a target of artificial selection during rice domestication. qSH1 Selection on grain shattering genes and rates of rice domestication 2009 New Phytol State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. Molecular cloning of major quantitative trait loci (QTLs) responsible for the reduction of rice grain shattering, a hallmark of cereal domestication, provided opportunities for in-depth investigation of domestication processes. Here, we studied nucleotide variation at the shattering loci, sh4 and qSH1, for cultivated rice, Oryza sativa ssp. indica and Oryza sativa ssp. japonica, and the wild progenitors, Oryza nivara andOryza rufipogon. The nonshattering sh4 allele was fixed in all rice cultivars, with levels of sequence polymorphism significantly reduced in both indica and japonica cultivars relative to the wild progenitors. The sh4 phylogeny together with the neutrality tests and coalescent simulations suggested that sh4 had a single origin and was fixed by artificial selection during the domestication of rice. Selection on qSH1 was not detected in indica and remained unclear in japonica. Selection on sh4 could be strong enough to have driven its fixation in a population of cultivated rice within a period of c. 100 yr. The slow fixation of the nonshattering phenotype observed at the archeological sites might be a result of relatively weak selection on mutations other than sh4 in early rice cultivation. The fixation of sh4 could have been achieved later through strong selection for the optimal phenotype. qSH1,sh4|SHA1 The Rice Cyclin-Dependent Kinase -Activating Kinase R2 Regulates S-Phase Progression 2002 The Plant Cell Online Institut für Allgemeine Botanik, Universität Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany. Cyclin-dependent kinases (CDKs) are the central components of eukaryotic cell cycle regulation. Phosphorylation of CDKs at a conserved threonine residue is required for their full activity and is mediated by a CDK-activating kinase (CAK). The CAK R2 from rice belongs to those CAKs that phosphorylate not only CDKs but also the C-terminal domain (CTD) of RNA polymerase II. We showed that R2 is a nuclear protein with increased expression and increased CTD kinase activity in S-phase. Increasing R2 abundance through a transgenic approach accelerated S-phase progression and overall growth rate in suspension cells. In planta, the CTD kinase activity of R2 was induced by a growth-promoting signal. R2 regulation, therefore, may constitute a plant-specific adaptive mechanism that is used to adjust the rate of cell proliferation in response to a changing environment. R2 Genome-wide identification of BURP domain-containing genes in rice reveals a gene family with diverse structures and responses to abiotic stresses 2009 Planta National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, 430070 Wuhan, China. Increasing evidence suggests that a gene family encoding proteins containing BURP domains have diverse functions in plants, but systematic characterization of this gene family have not been reported. In this study, 17 BURP family genes (OsBURP01-17) were identified and analyzed in rice (Oryza sativa L.). These genes have diverse exon-intron structures and distinct organization of putative motifs. Based on the phylogenetic analysis of BURP protein sequences from rice and other plant species, the BURP family was classified into seven subfamilies, including two subfamilies (BURP V and BURP VI) with members from rice only and one subfamily (BURP VII) with members from monocotyledons only. Two BURP gene clusters, belonging to BURP V and BURP VI, were located in the duplicated region on chromosome 5 and 6 of rice, respectively. Transcript level analysis of BURP genes of rice in various tissues and organs revealed different tempo-spatial expression patterns, suggesting that these genes may function at different stages of plant growth and development. Interestingly, all the genes of the BURP VII subfamily were predominantly expressed in flower organs. We also investigated the expression patterns of BURP genes of rice under different stress conditions. The results suggested that, except for two genes (OsBURP01 and OsBURP13), all other members were induced by at least one of the stresses including drought, salt, cold, and abscisic acid treatment. Two genes (OsBURP05 and OsBURP16) were responsive to all the stress treatments and most of the OsBURP genes were responsive to salt stress. Promoter sequence analysis revealed an over-abundance of stress-related cis-elements in the stress-responsive genes. The data presented here provide important clues for elucidating the functions of genes of this family. RA8,RAFTIN1,RD22|OsBURP03|OsBURP3 Isolation and characterization of an anther-specific gene, RA8, from rice (Oryza sativa L.) 1999 Plant Mol Biol Department of Life Science, Pohang University of Science and Technology, Korea. An anther-specific cDNA clone of rice, RA8, was isolated from an anther cDNA library by differential screening. RNA blot analysis indicated that the RA8 transcript is present specifically in anthers and the transcript level increased as flowers matured, reaching the highest level in mature flowers. The RA8 clone contains an open reading frame of 264 amino acid residues with a hydrophobic N-terminal region. The deduced amino acid sequences did not show significant homology to any known sequences. Genomic DNA blot analysis showed that RA8 is a single-copy gene. A genomic clone corresponding to the RA8 cDNA was isolated and its promoter region was fused to the beta-glucuronidase (GUS) gene. Transgenic rice plants exhibited anther-specific expression of the GUS reporter gene. Histochemical GUS analysis showed that the RA8 promoter was active in the tapetum, endothecium, and connective tissues of anthers. Experiments showed that expression of the gene starts when microspores are released from tetrads, and it reaches to the maximum level at the late vacuolated-pollen stage. The RA8 promoter may be useful for controlling gene expression in anthers of cereal plants and for generating male-sterile plants. RA8 Abscisic acid and water-stress induce the expression of a novel rice gene 1988 EMBO J Laboratory of Plant Molecular Biology, Rockefeller University, New York, NY 10021. We have identified a novel rice gene, called RAB 21, which is induced when plants are subject to water-stress. This gene encodes a basic, glycine-rich protein (mol. wt 16,529) which has a duplicated domain structure. Immunoblots probed with antibodies raised against beta-galactosidase/RAB 21 fusion protein detect RAB 21 protein only in cytosolic cell fractions. RAB 21 mRNA and protein accumulate in rice embryos, leaves, roots and callus-derived suspension cells upon treatment with NaCl (200 mM) and/or the plant hormone abscisic acid (10 microM ABA). The effects of NaCl and ABA are not cumulative, suggesting that these two inducers share a common response pathway. Induction of RAB 21 mRNA accumulation by ABA is rapid (less than 15 min in suspension cells) and does not require protein synthesis, indicating that preformed nuclear and/or cytosolic factors mediate the response to this hormone. We have characterized the RAB 21 gene by determining the complete nucleotide sequence of a nearly full-length cDNA and corresponding genomic copy, and by mapping the start site of its major transcript. The proximal promoter region contains various GC-rich repeats. RAB21|Rab16A Overexpression of Rab16A gene in indica rice variety for generating enhanced salt tolerance 2012 Plant Signal Behav Plant Molecular Biology and Biotechnology Laboratory, Department of Botany, University of Calcutta, Kolkata, India. We report here the overexpression of Rab16A full length gene (promoter + ORF), from the salt-tolerant indica rice Pokkali, in the salt-susceptible indica rice variety Khitish, via particle bombardment. Molecular analysis of the transgenics revealed stable integration of the transgene upto T2 generation. High level of expression of the transgene (driven by its own stress-inducible promoter), as well as the protein, was detectable in the leaves under simulated salinity stress (250 mM NaCl, 24 h); the expression level being higher than wild type (WT) plants. The Rab16A transcript also increased gradually with seed maturity, with its maximal accumulation at 30 d after pollination (DAP) i.e., fully matured seeds, explaining the protective role of Rab16A gene during seed maturation. Enhanced tolerance to salinity was observed in the plants transformed with Rab16A. The superior physiological performances of the transgenics under salt treatment were also reflected in lesser shoot or root length inhibition, reduced chlorophyll damages, lesser accumulation of Na(+) and reduced loss of K(+), increased proline content as compared with the WT plants. All these results indicated that the overproduction of RAB16A protein in the transgenics enable them to display enhanced tolerance to salinity stress with improved physiological traits. Our work demonstrates the profound potential of Group 2 LEA proteins (to which RAB16A belongs to) in conferring stress tolerance in crop plants through their genetic manipulation. RAB21|Rab16A The rice RAD51C gene is required for the meiosis of both female and male gametocytes and the DNA repair of somatic cells 2012 J Exp Bot National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. The RecA/RAD51 family of rice (Oryza sativa) consists of at least 13 members. However, the functions of most of these members are unknown. Here the functional characterization of one member of this family, RAD51C, is reported. Knockout (KO) of RAD51C resulted in both female and male sterility in rice. Transferring RAD51C to the RAD51C-KO line restored fertility. Cytological analyses showed that the sterility of RAD51C-KO plants was associated with abnormal early meiotic processes in both megasporocytes and pollen mother cells (PMCs). PMCs had an absence of normal pachytene chromosomes and had abnormal chromosome fragments. The RAD51C-KO line showed no obvious difference from wild-type plants in mitosis in the anther wall cells, which was consistent with the observation that the RAD51C-KO line did not have obviously abnormal morphology during vegetative development. However, the RAD51C-KO line was sensitive to different DNA-damaging agents. These results suggest that RAD51C is essential for reproductive development by regulating meiosis as well as for DNA damage repair in somatic cells. RAD51C Gene structure and expression of rice seed allergenic proteins belonging to the alpha-amylase/trypsin inhibitor family 1993 Plant Mol Biol Department of Food Science and Technology, School of Agriculture, Nagoya University, Japan. Genomic and two novel cDNA clones for rice seed allergenic protein (RA) belonging to the alpha-amylase/trypsin inhibitor family were isolated and their nucleotide sequences determined. Ten cysteine residues deduced from nucleotide sequences were completely conserved among three cDNA clones including a clone, RA17, reported previously. One genomic clone, lambda 4, contained two RA genes, RAG1 and RAG2. Although RAG1 was cloned at the 5' portion only, two RA genes were arranged divergently. Nucleotide sequencing and DNA blotting analyses showed that RA are encoded by a multigene family consisting of at least four members. The transcriptional initiation site of RAG1 was localized at A, 26 bp upstream of the putative translational initiation codon, ATG, by the primer extension assay. The putative TATA box and CAAT box existed about 45 bp and 147 bp upstream of the transcription initiation site, respectively. A conserved sequence (ATGCAAAA) which was similar to the sequence (TGCAAAA) identified in rice glutelin promoters was observed in the 5' region of the two genes. In addition, RNA blotting analyses provided that RA genes specifically expressed in ripening seed and their transcripts accumulated maximally between 15 and 20 days after flowering. RAG1 A new rice zinc-finger protein binds to the O2S box of the alpha-amylase gene promoter 2004 Eur J Biochem Shanghai Key Laboratory of Agricultural Genetic and Breeding, Agro-Biotechnology Research Center, Shanghai Academy of Agricultural Sciences, China. pengrihe69@yahoo.com A putative transcription factor, named RAMY, that binds to the 20-bp O2S sequences of the regulatory region of the Amy2 gene promoter has been identified using the yeast one-hybrid system from a rice library. The full length RAMY cDNA clone encodes a 218-amino acid protein and is homologous to the late embryogenesis-abundant protein (LEA5). In vitro mutagenesis and electrophoretic mobility shift assays confirmed that RAMY can bind with O2S specifically through an unusual zinc finger with a CXCX(4)CX(2)H consensus sequence. Low levels of RAMY mRNAs were detected in rice leaves and roots by Northern blot hybridization. The plant hormone gibberellin (GA) induces expression of both RAMY and Amy2 genes, as performed by Northern blot hybridization, but the increase in RAMY mRNA level occurs prior to that of the Amy2 mRNA level in the GA-treated aleurone tissues. These data suggest that RAMY may act as a trans-acting protein and is probably involved in the GA-induced expression of the rice alpha-amylase gene. RAMY Functional dissection of a sugar-repressed α-amylase gene (RAmy1A) promoter in rice embryos 1998 FEBS Letters Bioscience Center, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan The gibberellin-inducible rice α-amylase gene, RAmy1A, was demonstrated to be sugar repressed in rice embryos and functional dissection of the promoter of RAmy1A in relation of its sugar-modulated expression was performed. Gibberellin-response cis-elements of GARE (TAACAAA) and pyrimidine box (CCTTTT) were partially involved in the sugar repression. AmyI-1|RAmy1A Involvement of alpha-amylase I-1 in starch degradation in rice chloroplasts 2005 Plant Cell Physiol Laboratories of Plant and Microbial Genome Control, Graduate School of Science and Technology, Niigata University, Niigata, 950-2181 Japan. To determine the role of alpha-amylase isoform I-1 in the degradation of starch in rice leaf chloroplasts, we generated a series of transgenic rice plants with suppressed expression or overexpression of alpha-amylase I-1. In the lines with suppressed expression of alpha-amylase I-1 at both the mRNA and protein levels, seed germination and seedling growth were markedly delayed in comparison with those in the wild-type plants. However, the growth retardation was overcome by supplementation of sugars. Interestingly, a significant increase of starch accumulation in the young leaf tissues was observed under a sugar-supplemented condition. In contrast, the starch content of leaves was reduced in the plants overexpressing alpha-amylase I-1. In immunocytochemical analysis with specific anti-alpha-amylase I-1 antiserum, immuno-gold particles deposited in the chloroplasts and extracellular space in young leaf cells. We further examined the expression and targeting of alpha-amylase I-1 fused with the green fluorescent protein in re-differentiated green cells, and showed that the fluorescence of the expressed fusion protein co-localized with the chlorophyll autofluorescence in the transgenic cells. In addition, mature protein species of alpha-amylase I-1 bearing an oligosaccharide side chain were detected in the isolated chloroplasts. Based on these results, we concluded that alpha-amylase I-1 targets the chloroplasts through the endoplasmic reticulum-Golgi system and plays a significant role in the starch degradation in rice leaves. AmyI-1|RAmy1A The rice alpha-amylase glycoprotein is targeted from the Golgi apparatus through the secretory pathway to the plastids 2009 Plant Cell Graduate School of Science and Technology, Niigata University, Niigata, Japan. The well-characterized secretory glycoprotein, rice (Oryza sativa) alpha-amylase isoform I-1 (AmyI-1), was localized within the plastids and proved to be involved in the degradation of starch granules in the organelles of rice cells. In addition, a large portion of transiently expressed AmyI-1 fused to green fluorescent protein (AmyI-1-GFP) colocalized with a simultaneously expressed fluorescent plastid marker in onion (Allium cepa) epidermal cells. The plastid targeting of AmyI-1 was inhibited by both dominant-negative and constitutively active mutants of Arabidopsis thaliana ARF1 and Arabidopsis SAR1, which arrest endoplasmic reticulum-to-Golgi traffic. In cells expressing fluorescent trans-Golgi and plastid markers, these fluorescent markers frequently colocalized when coexpressed with AmyI-1. Three-dimensional time-lapse imaging and electron microscopy of high-pressure frozen/freeze-substituted cells demonstrated that contact of the Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids occur within the cells. The transient expression of a series of C-terminal-truncated AmyI-1-GFP fusion proteins in the onion cell system showed that the region from Trp-301 to Gln-369 is necessary for plastid targeting of AmyI-1. Furthermore, the results obtained by site-directed mutations of Trp-302 and Gly-354, located on the surface and on opposite sides of the AmyI-1 protein, suggest that multiple surface regions are necessary for plastid targeting. Thus, Golgi-to-plastid traffic appears to be involved in the transport of glycoproteins to plastids and plastid targeting seems to be accomplished in a sorting signal-dependent manner. AmyI-1|RAmy1A Rice bifunctional alpha-amylase/subtilisin inhibitoe: cloning and characterization of the recombinant inhibitor expressed in Escherichia coli 2006 Bioscience, biotechnology, and biochemistry Laboratory of Biochemistry, Faculty of Agriculture, Kobe University, Japan. The complete nucleotide sequences of the cDNA and its gene that encode a bifunctional alpha-amylase/subtilisin inhibitor of rice (Oryza sativa L.) (RASI) were analyzed. RASI cDNA (939 bp) encoded a 200-residue polypeptide with a molecular mass of 21,417Da, including a signal peptide of 22 amino acids. Sequence comparison and phylogenetic analysis showed that RASI is closely related to alpha-amylase/subtilisin inhibitors from barley and wheat. RASI was found to be expressed only in seeds, suggesting that it has a seed-specific function. A coding region of RASI cDNA without the signal peptide was introduced into Escherichia coli and was expressed as a His-tagged protein. Recombinant RASI was purified to homogeneity in a single step by Ni-chelating affinity column chromatography and characterized to elucidate the target enzyme. The recombinant inhibitor had strong inhibitory activity toward subtilisin, with an equimolar relationship, comparable with that of native RASI, and weak inhibitory activity toward some microbial alpha-amylases, but not toward animal or insect alpha-amylases. These results suggest that RASI might function in the defense of the seed against microorganisms. RASI Association between seed dormancy and pericarp color is controlled by a pleiotropic gene that regulates abscisic acid and flavonoid synthesis in weedy red rice 2011 Genetics Plant Science Department, South Dakota State University, Brookings, South Dakota 57007, USA. xingyou.gu@sdstate.edu Seed dormancy has been associated with red grain color in cereal crops for a century. The association was linked to qSD7-1/qPC7, a cluster of quantitative trait loci for seed dormancy/pericarp color in weedy red rice. This research delimited qSD7-1/qPC7 to the Os07g11020 or Rc locus encoding a basic helix-loop-helix family transcription factor by intragenic recombinants and provided unambiguous evidence that the association arises from pleiotropy. The pleiotropic gene expressed in early developing seeds promoted expression of key genes for biosynthesis of abscisic acid (ABA), resulting in an increase in accumulation of the dormancy-inducing hormone; activated a conserved network of eight genes for flavonoid biosynthesis to produce the pigments in the lower epidermal cells of the pericarp tissue; and enhanced seed weight. Thus, the pleiotropic locus most likely controls the dormancy and pigment traits by regulating ABA and flavonoid biosynthetic pathways, respectively. The dormancy effect could be eliminated by a heat treatment, but could not be completely overcome by gibberellic acid or physical removal of the seed maternal tissues. The dormancy-enhancing alleles differentiated into two groups basically associated with tropical and temperate ecotypes of weedy rice. Of the pleiotropic effects, seed dormancy could contribute most to the weed adaptation. Pleiotropy prevents the use of the dormancy gene to improve resistance of white pericarp cultivars against pre-harvest sprouting through conventional breeding approaches. Rc|qSD7-1|qPC7 Inference of the japonica rice domestication process from the distribution of six functional nucleotide polymorphisms of domestication-related genes in various landraces and modern cultivars 2008 Plant Cell Physiol Plant Genome Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan. Crop domestication can serve as a model of plant evolutionary processes. It involves a series of selection events from standing natural variation and newly occurring mutations and combinations of mutations as a result of natural crossings in populations during local adaptation and propagation of plant lines to other cultivation areas. Our earlier identification of three functional nucleotide polymorphisms (FNPs) of distinct genes involved in the rice domestication process led us to propose a model of the japonica rice domestication process. Here, we examined three more FNPs in two domestication-related genes involved in pigment synthesis during the development of seed pericarp color (Rc and Rd) in 91 landraces (and some modern cultivars) of japonica rice collected from throughout the area of distribution of rice. These polymorphisms were assigned by using genome-wide patterns of restriction fragment length polymorphisms (RFLPs) and the local origins of the landraces. The results led us to infer the process of japonica rice domestication in more detail and propose a more refined model of the japonica domestication process. In this model, the critical role of the Rc FNP at an early step of the domestication process was highlighted. Independent artificial selections of two defective Rd alleles were found, suggesting a role for Rd other than in pigment synthesis during rice domestication. Rc|qSD7-1|qPC7 The Rc and Rd genes are involved in proanthocyanidin synthesis in rice pericarp 2007 Plant J Genetic Diversity Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan. Different colors, such as purple, brown, red and white, occur in the pericarp of rice. Here, two genes affecting proanthocyanidin synthesis in red- and brown-colored rice were elucidated. Genetic segregation analysis suggested that the Rd and A loci are identical, and both encode dihydroflavonol-4-reductase (DFR). The introduction of the DFR gene into an Rcrd mutant resulted in red-colored rice, which was brown in the original mutant, demonstrating that the Rd locus encodes the DFR protein. Accumulation of proanthocyanidins was observed in the transformants by the introduction of the Rd gene into the rice Rcrd line. Protein blot analysis showed that the DFR gene was translated in seeds with alternative translation initiation. A search for the Rc gene, which encodes a transacting regulatory factor, was conducted using available DNA markers and the Rice Genome Automated Annotation System program. Three candidate genes were identified and cloned from a rice RcRd line and subsequently introduced into a rice rcrd line. Brown-colored seeds were obtained from transgenic plants by the introduction of a gene containing the basic helix-loop-helix (bHLH) motif, demonstrating that the Rc gene encodes a bHLH protein. Comparison of the Rc locus among rice accessions showed that a 14-bp deletion occurred only in the rc locus. Rc|qSD7-1|qPC7 The molecular basis of white pericarps in African domesticated rice: novel mutations at the Rc gene 2010 J Evol Biol Department of Biology, Washington University in St. Louis, St. Louis, MO, USA Repeated phenotypic evolution can occur at both the inter- and intraspecific level and is especially prominent in domesticated plants, where artificial selection has favoured the same traits in many different species and varieties. The question of whether repeated evolution reflects changes at the same or different genes in each lineage can now be addressed using the domestication and improvement genes that have been identified in a variety of crops. Here, we document the genetic basis of nonpigmented (‘white') pericarps in domesticated African rice (Oryza glaberrima) and compare it with the known genetic basis of the same trait in domesticated Asian rice (Oryza sativa). In some cases, white pericarps in African rice are apparently caused by unique mutations at the Rc gene, which also controls pericarp colour variation in Asian rice. In one case, white pericarps appear to reflect changes at a different gene or potentially a cis-regulatory region. Rc|qSD7-1|qPC7 Global dissemination of a single mutation conferring white pericarp in rice 2007 PLoS Genet Department of Plant Breeding and Genetics, Cornell University, Ithaca, New York, United States of America. Here we report that the change from the red seeds of wild rice to the white seeds of cultivated rice (Oryza sativa) resulted from the strong selective sweep of a single mutation, a frame-shift deletion within the Rc gene that is found in 97.9% of white rice varieties today. A second mutation, also within Rc, is present in less than 3% of white accessions surveyed. Haplotype analysis revealed that the predominant mutation originated in the japonica subspecies and crossed both geographic and sterility barriers to move into the indica subspecies. A little less than one Mb of japonica DNA hitchhiked with the rc allele into most indica varieties, suggesting that other linked domestication alleles may have been transferred from japonica to indica along with white pericarp color. Our finding provides evidence of active cultural exchange among ancient farmers over the course of rice domestication coupled with very strong, positive selection for a single white allele in both subspecies of O. sativa. Rc|qSD7-1|qPC7 Caught red-handed: Rc encodes a basic helix-loop-helix protein conditioning red pericarp in rice 2006 Plant Cell Department of Plant Breeding and Genetics, Cornell University, Ithaca, New York 14953-1901, USA. Rc is a domestication-related gene required for red pericarp in rice (Oryza sativa). The red grain color is ubiquitous among the wild ancestors of O. sativa, in which it is closely associated with seed shattering and dormancy. Rc encodes a basic helix-loop-helix (bHLH) protein that was fine-mapped to an 18.5-kb region on rice chromosome 7 using a cross between Oryza rufipogon (red pericarp) and O. sativa cv Jefferson (white pericarp). Sequencing of the alleles from both mapping parents as well as from two independent genetic stocks of Rc revealed that the dominant red allele differed from the recessive white allele by a 14-bp deletion within exon 6 that knocked out the bHLH domain of the protein. A premature stop codon was identified in the second mutant stock that had a light red pericarp. RT-PCR experiments confirmed that the Rc gene was expressed in both red- and white-grained rice but that a shortened transcript was present in white varieties. Phylogenetic analysis, supported by comparative mapping in rice and maize (Zea mays), showed that Rc, a positive regulator of proanthocyanidin, is orthologous with INTENSIFIER1, a negative regulator of anthocyanin production in maize, and is not in the same clade as rice bHLH anthocyanin regulators. Rc|qSD7-1|qPC7 A natural mutation in rc reverts white-rice-pericarp to red and results in a new, dominant, wild-type allele: Rc-g 2008 Theor Appl Genet USDA ARS, Dale Bumpers National Rice Research Center, 2890 Hwy. 130 E., Stuttgart, Arkansas 72160, USA. ricegenes@mac.com The Rc locus regulates pigmentation of the rice bran layer, and selection for the rc allele (white pericarp) occurred during domestication of the crop. White bran is now ubiquitous among cultivated varieties throughout rice growing regions of the world. We identified a new allele that arose by natural mutation within the rc pseudogene of the cultivar 'Wells'. The mutation restored the reading frame of the gene, and reverted the bran layer pigmentation to red (wild-type). By sequencing the Rc locus in plants derived from red seeds, and linkage analysis in a segregating population, we were able to demonstrate that mutation within rc resulted in the new, dominant, wild-type allele Rc-g. Rc|qSD7-1|qPC7 Regulation, expression and function of a new basic chitinase gene in rice (Oryza sativa L.) 1996 Plant Mol Biol Plant Biology Laboratory, The Salk Institute for Biological Studies, San Diego, CA, 92186, USA. A new basic chitinase gene, designated RC24, was isolated from a rice genomic library. The predicted RC24 protein contains 322 amino acid residues and exhibits 68% to 95% amino acid identity with known class I rice chitinases. RC24 protein expressed in Escherichia coli exhibited chitinase activity and strongly inhibited bacterial growth. Two transcription start sites of the RC24 gene were mapped by primer extension analysis of both rice native RNA and in vitro transcribed RNA using a RC24 promoter/GUS (beta-glucuronidase) gene fusion as a template. The 5'-flanking region of RC24 contained several putative stress-responsive cis-acting elements. A basal level of RC24 transcripts was detected in rice root and stem tissues, but not in leaf tissues. RC24 transcripts rapidly accumulated within 1 h after fungal elicitor treatment of suspension-cultured cells, and the levels continued to increase for at least 9 h. RC24 transcript accumulation was also observed in intact leaf tissues upon wounding, Transgenic rice plants containing the RC24/GUS gene fusion further confirmed that the RC24 gene showed a tissue-specific expression pattern and that transcription of the RC24 propmoter was sensitively and rapidly activated by wounding. RC24 Functional conservation of the meiotic genes SDS and RCK in male meiosis in the monocot rice 2009 Cell Res National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. The Arabidopsis SDS (SOLO DANCERS) and RCK (ROCK-N-ROLLERS) genes are important for male meiosis, but it is still unknown whether they represent conserved functions in plants. We have performed phylogenetic analyses of SDS and RCK and their respective homologs, and identified their putative orthologs in poplar and rice. Quantitative real-time RT-PCR analysis indicated that rice SDS and RCK are expressed preferentially in young flowers, and transgenic RNAi rice lines with reduced expression of these genes exhibited normal vegetative development, but showed significantly reduced fertility with partially sterile flowers and defective pollens. SDS deficiency also caused a decrease in pollen amounts. Further cytological examination of male meiocytes revealed that the SDS deficiency led to defects in homolog interaction and bivalent formation in meiotic prophase I, and RCK deficiency resulted in defective meiotic crossover formation. These results indicate that rice SDS and RCK genes have similar functions to their Arabidopsis orthologs. Because rice and Arabidopsis, respectively, are members of monocots and eudicots, two largest groups of flowering plants, our results suggest that the functions of SDS and RCK are likely conserved in flowering plants. MER3|RCK,SDS ZIP4 in homologous chromosome synapsis and crossover formation in rice meiosis 2012 J Cell Sci State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. In budding yeast, the ZMM complex is closely associated with class I crossovers and synaptonemal complex (SC) formation. However, the relationship between the ZMM genes remains unclear in most higher eukaryotes. Here, we identify the rice ZIP4 homolog, a member of the ZMM gene group, and explore its relationship with two other characterized ZMM genes, MER3 and ZEP1. Our results show that in the rice zip4 mutant, the chiasma frequency is greatly reduced, although synapsis proceeds with only mild defects. Immunocytological analyses of wild-type rice reveal that ZIP4 presents as punctuate foci and colocalizes with MER3 in prophase I meiocytes. Additionally, ZIP4 is essential for the loading of MER3 onto chromosomes, but not vice versa. Double-mutant analyses show that zip4 mer3 displays a greater decrease in the mean number of chiasmata than either of the zip4 or mer3 single mutants, suggesting that ZIP4 and MER3 work cooperatively to promote CO formation but their individual contributions are not completely identical in rice. Although zep1 alone gives an increased chiasma number, both zip4 zep1 and mer3 zep1 show a much lower chiasma number than the zip4 or mer3 single mutants. These results imply that the normal functions of ZIP4 and MER3 are required for the regulation of COs by ZEP1. MER3|RCK,ZEP1,ZIP4|SPO22 The rice REDUCED CULM NUMBER11 gene controls vegetative growth under low-temperature conditions in paddy fields independent of RCN1/OsABCG5 2013 Plant Sci Department of Crop Science, Obihiro University of Agricultural and Veterinary Medicine, 2-11 Nishi, Inada, Obihiro, Hokkaido, Japan. Low temperature tolerance during vegetative growth is an important objective in rice (Oryza sativa L.) breeding programs. We isolated a novel reduced culm number mutant, designated reduced culm number11 (rcn11), by screening under low-temperature condition in a paddy fields. Since the shoot architecture of the rcn11 was very similar to that of the rcn1, we examined whether RCN11 is involved in RCN1/OsABCG5-associated vegetative growth control. The rcn11 mutant has no mutation in the RCN1/OsABCG5 gene and rcn11 has no effect on RCN1/OsABCG5 gene expression. In the rcn1 mutant, RCN1/OsABCG5 was upregulated showing that RCN1/OsABCG5 is controlled by negative feedback regulation. Absence of an effect of rcn11 on RCN1/OsABCG5 feedback regulation supported that RCN11 is not involved in the RCN1/OsABCG5-associated transport system. A genetic allelism test and molecular mapping study showed that rcn11 is independent of rcn1 on rice chromosome 3 and located on chromosome 8. The rcn1 rcn11 phenotype suggests that RCN11 acts on vegetative growth independent of RCN1/OsABCG5. A root development comparison between rcn1 and rcn11 in young seedlings represented that rcn11 reduced crown root number and elongation, whereas rcn1 reduced lateral root density and elongation. Thus, rcn11 will shed new light on vegetative growth control under low temperature. Rcn1 Overexpression ofRCN1andRCN2, riceTERMINAL FLOWER 1/CENTRORADIALIShomologs, confers delay of phase transition and altered panicle morphology in rice 2002 The Plant Journal Nara Institute of Science and Technology, Ikoma, Japan. TERMINAL FLOWER 1 (TFL1)/CENTRORADIALIS (CEN)-like genes play important roles in determining plant architecture, mainly by controlling the timing of phase transition. To investigate the possibility of similar mechanisms operating in the control of inflorescence architecture in rice, we analysed the functions of RCN1 and RCN2, rice TFL1/CEN homologs. Constitutive overexpression of RCN1 or RCN2 in Arabidopsis caused a late-flowering and highly branching phenotype, indicating that they possess conserved biochemical functions as TFL1. In 35S::RCN1 and 35S::RCN2 transgenic rice plants, the delay of transition to the reproductive phase was observed. The transgenic rice plants exhibited a more branched, denser panicle morphology. Detailed observation of the panicle structure revealed that the phase change from the branch shoot to the floral meristem state was also delayed, leading to the generation of higher-order panicle branches. These results suggest rice has a pathway that can respond to the overexpressed TFL1/CEN-like functions, and the molecular mechanisms controlling the phase transition of meristems are conserved between grass and dicot species, at least to some extent. Rcn1,RCN2 Rice shoot branching requires an ATP-binding cassette subfamily G protein 2009 New Phytol Department of Crop Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan. * Shoot branching is important for the establishment of plant architecture and productivity. * Here, characterization of rice (Oryza sativa) reduced culm number 1 (rcn1) mutants revealed that Rcn1 positively controls shoot branching by promoting the outgrowth of lateral shoots. Molecular studies revealed that Rcn1 encodes a novel member of ATP-binding cassette protein subfamily G (ABCG subfamily), also known as the white-brown complex (WBC) subfamily, and is designated OsABCG5. * Rcn1 is expressed in leaf primordia of main and axillary shoots, and in the vascular cells and leaf epidermis of older leaves. In addition, Rcn1 is expressed in the crown root primordia, endodermis, pericycle and stele in the root. No effect on Rcn1 expression in shoots or roots was seen when the roots were treated with auxins. Phenotypic analyses of rcn1 and tillering dwarf 3 (d3) double mutants at the seedling stage clarified that Rcn1 works independently of D3 in the branching inhibitor pathway. * Rcn1 is the first functionally defined plant ABCG protein gene that controls shoot branching and could thus be significant in future breeding for high-yielding rice. Rcn1 Circadian clock- and phytochrome-regulated Dof-like gene, Rdd1, is associated with grain size in rice 2009 Plant Cell Environ Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan. iwamas@nias.affrc.go.jp We report here on the characterization of a putative Dof transcription factor gene in rice (Oryza sativa)--rice Dof daily fluctuations 1 (Rdd1). Daily oscillations in Rdd1 expression were retained after transferring to continuous dark (DD) or light (LL) conditions, indicating circadian regulation. However, Rdd1 showed arrhythmic expression in etiolated coleoptiles. Experiments revealed that the Rdd1 transcript accumulated up to 1 h after transferring from DD to LL conditions and decreased thereafter. We examined Rdd1 expression using phytochrome (phy)-deficient mutants, and the results showed that phyA and most likely phyB contributed to the regulation of Rdd1 expression. To further examine the role of Rdd1, transgenic rice plants were produced that carried Rdd1 in either a sense (RDD1-S) or antisense (RDD1-AS) orientation, driven by a constitutive promoter. The expression of endogenous Rdd1 in response to far-red light was found to be modified in RDD1-AS plants compared with wild-type (WT) or RDD1-S plants. In addition, RDD1-AS plants were smaller and flowered later than WT or RDD1-S plants; decreases in grain length, width and 1000-grain weight were also recorded. This study demonstrates that Rdd1 is a circadian clock and phy-regulated gene, which is associated with grain size in rice. RDD1 A rice functional transcriptional activator, RISBZ1, responsible for endosperm-specific expression of storage protein genes through GCN4 motif 2001 J Biol Chem Department of Biotechnology, National Institute of Agrobiological Resources, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. The GCN4 motif, a cis-element that is highly conserved in the promoters of cereal seed storage protein genes, plays a central role in controlling endosperm-specific expression. This motif is the recognition site for a basic leucine zipper transcriptional factor that belongs to the group of maize Opaque-2 (O2)-like proteins. Five different basic leucine zipper cDNA clones, designated RISBZ1-5, have been isolated from a rice seed cDNA library. The predicted gene products can be divided into two groups based on their amino acid sequences. Although all the RISBZ proteins are able to interact with the GCN4 motif, only RISBZ1 is capable of activating (more than 100-fold expression) the expression of a reporter gene under a minimal promoter fused to a pentamer of the GCN4 motif. Loss-of-function and gain-of-function experiments using the yeast GAL4 DNA binding domain revealed that the proline-rich N-terminal domain (27 amino acids in length) is responsible for transactivation. The RISBZ1 protein is capable of forming homodimers as well as heterodimers with other RISBZ subunit proteins. RISBZ1 gene expression is restricted to the seed, where it precedes the expression of storage protein genes. When the RISBZ1 promoter was transcriptionally fused to the beta-glucuronidase reporter gene and the chimeric gene was introduced into rice, the beta-glucuronidase gene is specifically expressed in aleurone and subaleurone layer of the developing endosperm. These findings suggest that the specific expression of transcriptional activator RISBZ1 gene may determine the endosperm specificity of the storage protein genes. REB,RISBZ1|OsbZIP58,RITA1 Characterization of a novel rice bZIP protein which binds to the alpha-globulin promoter 1997 Plant Mol Biol Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Japan. Many plant basic leucine-zipper (bZIP) proteins have been isolated several of which have been shown to play a role in seed-specific gene expression. We isolated a novel bZIP protein (REB) gene encoding 425 amino acid residues from rice endosperm, which is similar to Opaque-2 heterodimerizing protein (OHP) of maize. The gene product, termed REB, contains Pro- and Gly-rich regions at its N terminus, followed by the typical basic and leucine-repeat regions. Recombinant REB binds to the region from -754 to -562 in the alpha-globulin gene promoter, but not to promoters of other major storage genes such as glutelin, prolamin and albumin. The 5' region of the alpha-globulin gene possesses three binding sites for REB, which were determined as GCCACGT(A/C)AG, by using synthetic oligonucleotides. A Super-shift assay using anti-REB antibody suggested that REB is a major DNA-binding protein for the alpha-globulin gene promoter in rice endosperm. REB Expression of the REB transcriptional activator in rice grains improves the yield of recombinant proteins whose genes are controlled by a Reb-responsive promoter 2001 Proc Natl Acad Sci U S A Applied Phytologics, Incorporated, Sacramento, CA 95834, USA. The gene encoding the rice transcription factor, REB (rice endosperm bZIP) was cloned from a bacterial artificial chromosome library of rice. The cloned 6,227-bp-long Reb gene is composed of six exons and five introns and is flanked by a 1.2-kb 5' promoter and a 1.2-kb 3' terminator region. The function of the Reb gene was explored by a transient assay by using a rice immature endosperm system. The effector constructs containing the native gene or fusion genes linking Reb to the rice actin (Act) or globulin (Glb) gene promoters and the reporter gene construct Glb-beta-glucuronidase (GUS) were used in this study. When these effector constructs were cotransferred with the reporter uidA gene encoding GUS under the control of the Glb promoter into immature rice endosperm cells, the Glb promoter was activated. The transient GUS expression was 2.0 to 2.5-fold higher with the effector construct than without. When the upstream activation sequence containing the GCCACGT(A/C)AG motifs of the Glb promoter was deleted, the activation by REB was abolished. On the other hand, a gain-of-function experiment showed that inserting the upstream activation sequence into the glutelin-1 (Gt1) promoter made it responsive to activation by REB. When cotransformed with Reb gene, mature transgenic rice grains containing the human lysozyme gene driven by the Glb promoter produced 3.7-fold more lysozyme. Accumulation of recombinant lysozyme in mature seed ranged from 30.57 to 279.61 microg.mg(-1) total soluble protein in individual transformants from 30 independent transformation events. Thus, our results show that REB is not only a transcriptional activator, it can also be used to increase the expression of recombinant protein in transgenic rice grains. REB Identification, cDNA cloning and possible roles of seed-specific rice asparaginyl endopeptidase, REP-2 2003 Planta Department of Biological Sciences, Tokyo Metropolitan University, Minami-ohsawa 1-1, Hachioji-shi, 192-0397 Tokyo, Japan. We previously showed that two major cysteine endopeptidases, REP-1 and REP-2, were present in germinated rice ( Oryza sativa L.) seeds, and that REP-1 was the enzyme that digests seed storage proteins. The present study shows that REP-2 is an asparaginyl endopeptidase that acts as an activator of REP-1, and we separated it into two forms, REP-2alpha (39 kDa) and REP-2beta (40 kDa), using ion-exchange chromatography and gel filtration chromatography. Although analysis of the amino terminals revealed that 10 amino acids of both forms were identical, their isoelectric points were different. SDS-PAGE/immunoblot analysis using an antiserum raised against legumain, an asparaginyl endopeptidase from jack bean, indicated that both forms were present in maturing and germinating rice seeds, and that their amounts transiently decreased in dry seeds. Northern blot analysis indicated that REP-2 mRNA was expressed in both maturing and germinating seeds. In germinating seeds, the mRNA was detected in aleurone layers but not in shoot and root tissues. Incubation of the de-embryonated seeds in 10(-6) M gibberellic acid induced the production of large amounts of REP-1, whereas REP-2beta levels declined rapidly. Southern blot analysis showed that there is one gene for REP-2 in the genome, indicating that both REP-2 enzymes are generated from a single gene. The structure of the gene was similar to that of beta-VPE and gamma-VPE isolated from Arabidopsis thaliana. OsVPE3|REP-2 RETARDED PALEA1 controls palea development and floral zygomorphy in rice 2009 Plant Physiol School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. Poaceae, one of the largest flowering plant families in angiosperms, evolved distinct inflorescence and flower morphology diverging from eudicots and other monocots. However, the mechanism underlying the specification of flower morphology in grasses remains unclear. Here we show that floral zygomorphy along the lemma-palea axis in rice (Oryza sativa) is partially or indirectly determined by the CYCLOIDEA (CYC)-like homolog RETARDED PALEA1 (REP1), which regulates palea identity and development. The REP1 gene is only expressed in palea primordium during early flower development, but during later floral stages is radially dispersed in stamens and the vascular bundles of the lemma and palea. The development of palea is significantly retarded in the rep1 mutant and its palea has five vascular bundles, which is similar to the vascular pattern of the wild-type lemma. Furthermore, ectopic expression of REP1 caused the asymmetrical overdifferentiation of the palea cells, altering their floral asymmetry. This work therefore extends the function of the TCP gene family members in defining the diversification of floral morphology in grasses and suggests that a common conserved mechanism controlling floral zygomorphy by CYC-like genes exists in both eudicots and the grasses. REP1 Abscisic acid- and stress-induced highly proline-rich glycoproteins regulate root growth in rice 2013 Plant Physiol Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan, Republic of China. In the root of rice (Oryza sativa), abscisic acid (ABA) treatment, salinity, or water deficit stress induces the expression of a family of four genes, REPETITIVE PROLINE-RICH PROTEIN (RePRP). These genes encode two subclasses of novel proline-rich glycoproteins with highly repetitive PX(1)PX(2) motifs, RePRP1 and RePRP2. RePRP orthologs exist only in monocotyledonous plants, and their functions are virtually unknown. Rice RePRPs are heavily glycosylated with arabinose and glucose on multiple hydroxyproline residues. They are significantly different from arabinogalactan proteins that have glycan chains composed of arabinose and galactose. Transient and stable expressions of RePRP-green fluorescent protein reveal that a fraction of this protein is localized to the plasma membrane. In rice roots, ABA treatment increases RePRP expression preferentially in the elongation zone. Overexpression of RePRP in transgenic rice reduces root cell elongation in the absence of ABA, similar to the effect of ABA on wild-type roots. Conversely, simultaneous knockdown of the expression of RePRP1 and RePRP2 reduces the root sensitivity to ABA, indicating that RePRP proteins play an essential role in ABA/stress regulation of root growth and development. Moreover, rice RePRPs specifically interact with a polysaccharide, arabinogalactan, in a dosage-dependent manner. It is suggested that RePRP1 and RePRP2 are functionally redundant suppressors of root cell expansion and probably act through interactions with cell wall components near the plasma membrane. OsPRP|RePRP1.1,RePRP1.2,RePRP2.1,RePRP2.2 Isolation and characterization of a gene for a repetitive proline rich protein (OsPRP) down-regulated during submergence in rice (Oryza sativa) 2003 Physiologia Plantarum Department of Low Temperature Science, National Agricultural Research Center for Hokkaido Region, 1 Hitsujigaoka, Toyohira, Sapporo, Hokkaido 062-8555, Japan Differential screening was performed on a cDNA library made from RNAs isolated from rice seedlings (variety Yukihikari) grown in submergence condition. A cDNA clone (OsPRP) encoding a repetitive proline-rich protein of 358 amino acids and molecular weight of 39 kDa was obtained. The C-terminal region of OsPRP contains proline (P) residues (40%), and high levels of glutamic acid (E) and lysine (K). The N-terminus was free from proline residues. Sequence analysis indicated that 40 distinct PEPK repetitive sequence motifs were present in the sequence along with other repetitive motifs. Furthermore, four phosphorylation sites were predicted from the sequence. The domain structure of OsPRP was compared with proline-rich proteins from other monocot species. The N-terminus of OsPRP is highly hydrophobic indicating the presence of a signal peptide. Southern blot data indicate that in the Yukihikari genome OsPRP is most likely encoded by a single gene. The expression of OsPRP was relatively uniform in the young seedlings. However, tissue-specific expression of OsPRP indicated that shoot tissue accumulates more mRNA, and the root has relatively low expression. In addition, OsPRP expression is regulated by various environmental factors and is down-regulated under submergence stress. Exogenous application of growth hormones, namely abscisic acid (ABA), methyl jasmonate (MeJ) and ethephon caused significant repression of the OsPRP transcript. OsPRP|RePRP1.1 RERJ1, a jasmonic acid-responsive gene from rice, encodes a basic helix-loop-helix protein 2004 Biochem Biophys Res Commun Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Differential screening of a cDNA library constructed using poly(A)(+) RNA from suspension-cultured rice cells treated with jasmonic acid (JA) for 1/2h yielded a cDNA of a gene tentatively named RERJ1 that is upregulated in response to exogenous JA. Northern blot analysis indicated that the RERJ1 mRNA levels peaked at 1/2-1h after the addition of jasmonic acid and then decreased gradually. RERJ1 encodes a transcriptional regulator with a basic helix-loop-helix motif. The phenotypes of transgenic rice plants overexpressing sense or antisense RERJ1 mRNA demonstrated that RERJ1 is involved in the growth inhibition of rice shoots caused by JA. Other biological functions of RERJ1 are discussed from an evolutionary standpoint. RERJ1 Stress-induced expression of the transcription factor RERJ1 is tightly regulated in response to jasmonic acid accumulation in rice 2013 Protoplasma Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. aa097045@mail.ecc.u-tokyo.ac.jp The plant hormone jasmonic acid (JA) regulates various developmental processes and plant defence responses to environmental stresses. We previously reported that RERJ1, a JA-inducible transcription factor in rice, is up-regulated by exposure to wounding and drought stress. Here, we demonstrated that the expression of RERJ1 after wounding is regulated in a JA-dependent manner in rice, based on histochemical analysis of RERJ1 promoter-GUS transgenic plants. RERJ1 expression was induced only at the region of injury after wounding, whereas expression was induced in the entire leaf after drought. According to JA measurements of stressed leaves, high accumulation of endogenous JA was only detected around the wound site in a rice leaves, whereas the drought treatment led to uniform accumulation of JA in the entire leaf, suggesting that RERJ1 will be a useful marker gene for studies on localization of JA in rice. Nuclear localization and transactivation ability of RERJ1 were also demonstrated. These results suggest that RERJ1 plays a role as a transcriptional activator for regulating stress-inducible gene expression, with a strong correlation to JA accumulation in the stressed region. RERJ1 Involvement of the Basic Helix-Loop-Helix Transcription Factor RERJ1 in Wounding and Drought Stress Responses in Rice Plants 2014 Bioscience, Biotechnology and Biochemistry Biotechnology Research Center, The University of Tokyo The jasmonic acid (JA)-responsive gene RERJ1 isolated from suspension-cultured rice cells encodes a transcription factor with a basic helix-loop-helix motif. In this study, we found that RERJ1 is also expressed in rice plants in response to JA, and that its expression in rice leaves is up-regulated by exposure to wounding and drought stress. It is also suggested that JA but not abscisic acid is involved in the up-regulation of RERJ1 expression caused by wounding and drought stress. RERJ1 Suppressed expression of Retrograde-Regulated Male Sterility restores pollen fertility in cytoplasmic male sterile rice plants 2009 Proc Natl Acad Sci U S A Laboratory of Environmental Biotechnology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan. Conflict/reconciliation between mitochondria and nuclei in plants is manifested by the fate of pollen (viable or nonviable) in the cytoplasmic male sterility (CMS)/fertility restoration (Rf) system. Through positional cloning, we identified a nuclear candidate gene, RETROGRADE-REGULATED MALE STERILITY (RMS) for Rf17, a fertility restorer gene for Chinese wild rice (CW)-type CMS in rice (Oryza sativa L.). RNA interference-mediated gene silencing of RMS restored fertility to a CMS plant, whereas its overexpression in the fertility restorer line induced pollen abortion. The mRNA expression level of RMS in mature anthers depended on cytoplasmic genotype, suggesting that RMS is a candidate gene to be regulated via retrograde signaling. We found that a reduced-expression allele of the RMS gene restored fertility in haploid pollen, whereas a normal-expression allele caused pollen to die in the CW-type CMS. RMS encodes a mitochondrial protein, 178 aa in length, of unknown function, unlike the majority of other Rf genes cloned thus far, which encode pentatricopeptide repeat proteins. The unique features of RMS provide novel insights into retrograde signaling and CMS. Rf17|RMS Mapping of the nuclear fertility restorer gene for HL cytoplasmic male sterility in rice using microsatellite markers 2000 Chinese Science Bulletin College of Life Science, Wuhan University, 430072, Wuhan, China Bulked segregant analysis (BSA) of a BC, population derived from Congguang 41A//Miyang 23/Congguang 41B was used to map the nuclear fertility restorer gene for Honglian (HL) cytoplasmic male sterility. One hundred and fifty-nine microsatellite primer pairs were screened for polymorphisms between the parents and between two bulks representing fertile and sterile plants. One microsatellite marker RM258 produced polymorphic products. The nuclear fertility restorer gene for HL cytoplasmic male sterility was mapped on chromosome 10, 7.8cM from RM258. The restorer gene may be clustered on chromosome. Rf1a|Rf5 Positional cloning of the rice Rf-1 gene, a restorer of BT-type cytoplasmic male sterility that encodes a mitochondria-targeting PPR protein 2004 Theor Appl Genet Laboratory of Plant Breeding and Genetics, Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, Kaidoubata-Nishi 241-7, Shimoshinjyo-Nakano, 010-0195 Akita, Japan. akagi@akita-pu.ac.jp The combination of cytoplasmic male sterility (CMS) in one parent and a restorer gene ( Rf) to restore fertility in another are indispensable for the development of hybrid varieties. We have found a rice Rf-1 gene that restores BT-type CMS by applying a positional cloning strategy. Using linkage analysis in combination with 6,104 BC(1)F(3) progeny derived from a cross between two near-isogenic lines (NILs) differing only at the Rf-1 locus, we delimited the Rf-1 gene to a 22.4-kb region in the rice genome. Duplicate open reading frames ( Rf-1A and Rf-1B) with a pentatricopeptide (PPR) motif were found in this region. Since several insertions and/or deletions were found in the regions corresponding to both the Rf-1A and Rf-1B genes in the maintainer's allele, they may have lost their function. Rf-1A protein had a mitochondria-targeting signal, whereas Rf-1B did not. The Rf-1B gene encoded a shorter polypeptide that was determined by a premature stop codon. Based on the function of the Rf-1 gene, its product is expected to target mitochondria and may process the transcript from an atp6/orf79 region in the mitochondrial genome. Since the Rf-1A gene encodes a 791-amino acid protein with a signal targeting mitochondria and has 16 repeats of the PPR motif, we concluded that Rf-1A is the Rf-1 gene. Nine duplications of Rf-1A homologs were found around the Rf-1 locus in the Nipponbare genome. However, while some of them encoded proteins with the PPR motif, they do not restore BT-type CMS based on the lack of co-segregation with the restoration phenotype. These duplicates may have played diversified roles in RNA processing and/or recombination in mitochondria during the co-evolution of these genes and the mitochondrial genome. Rf1a|Rf5 Two non-allelic nuclear genes restore fertility in a gametophytic pattern and enhance abiotic stress tolerance in the hybrid rice plant 2012 Theor Appl Genet State Key Laboratory of Hybrid Rice, Wuhan University, Wuhan, China. In indica rice, the HongLian (HL)-type combination of cytoplasmic male sterility (CMS) and fertility restoration (Rf) is widely used for the production of commercial hybrid seeds in China, Laos, Vietnam and other Southeast Asian countries. Generally, any member of the gametophytic fertility restoration system, 50% of the pollen in hybrid F(1) plants displays recovered sterility. In this study, however, a HL-type hybrid variety named HongLian You6 had approximately 75% normal (viable) pollen rather than the expected 50%. To resolve this discrepancy, several fertility segregation populations, including F(2) and BC(1)F(1) derived from the HL-CMS line Yuetai A crossed with the restorer line 9311, were constructed and subjected to genetic analysis. A gametophytic restoration model was discovered to involve two non-allelic nuclear restorer genes, Rf5 and Rf6. The Rf5 had been previously identified using a positional clone strategy. The Rf6 gene represents a new restorer gene locus, which was mapped to the short arm of chromosome 8. The hybrid F(1) plants containing one restorer gene, either Rf5 or Rf6, displayed 50% normal pollen grains with I(2)-KI solution; however, those with both Rf5 and Rf6 displayed 75% normal pollens. We also established that the hybrid F(1) plants including both non-allelic restorer genes exhibited an increased stable seed setting when subjected to stress versus the F(1) plants with only one restorer gene. Finally, we discuss the breeding scheme for the plant gametophytic CMS/Rf system. Rf1a|Rf5 A rapid PCR-aided selection of a rice line containing the Rf-1 gene which is involved in restoration of the cytoplasmic male sterility 1997 Molecular Breeding Life Science Institute, Mitsui Toatsu Chemicals, Inc., Togo 1144, Mobara, 297, Japan Cytoplasmic male sterility (CMS) is widely known in higher plants, the mechanism of which is believed to involve incompatibility between nuclei and cytoplasms. In rice lines with the CMS trait, fertility is restored by the aid of a nuclear-encoded gene, Rf-1, whose locus has been determined in chromosome 10. We found a particular PCR-amplified fragment, designated fL601, that specifically amplified using the DNAs from Rf-1 lines tested as templates. RFLP mapping of the fL601 locus revealed that there are two loci for the fL601, and that both are tightly linked to the Rf-1 locus. Progeny analysis also showed high frequency of their co-segregation. Southern analysis of the genomic DNA demonstrated that the Rf-1 lines shared a unique sequence in the fL601 region. These results enabled us to construct a system for specific detection of the corresponding regions. Utilizing this detection system, we established a simple PCR-mediated selection method for the Rf-1 lines, which may facilitate the breeding for hybrid rice. Rf1a|Rf5 A codominant DNA marker closely linked to the rice nuclear restorer gene, RF-1, identified with inter-SSR fingerprinting 1996 Genome Plant Biotechnology Laboratory, Mitsui Toatsu Chemicals Incorporated, Mobara, Japan. A new molecular marker (OSRRf) closely linked to the nuclear restorer gene (Rf-1) for fertility in rice has been found. The Rf-1 gene is essential for hybrid rice seed production. A PCR-fingerprinting technique using simple sequence repeats (SSRs) was applied to compare two near-isogenic lines with (MTC-10R) or without (MTC-10A) the Rf-1 gene. Of 76 inter-SSR primers tested, only one primer, (AG)8YC, generated polymorphisms. The tetranucleotide repeats generating polymorphisms were found within each amplicon. the genetic distance between OSRRf and Rf-1 was 3.7 +/- 1.1 cM. As in the case of a codominant marker, this marker will be applied not only to breeding both restorer lines and maintainer lines, but also to the purity management of hybrid rice seeds. Rf1a|Rf5,Rf1b Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing 2006 Plant Cell Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Province, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. Cytoplasmic male sterility (CMS) and nucleus-controlled fertility restoration are widespread plant reproductive features that provide useful tools to exploit heterosis in crops. However, the molecular mechanism underlying this kind of cytoplasmic-nuclear interaction remains unclear. Here, we show in rice (Oryza sativa) with Boro II cytoplasm that an abnormal mitochondrial open reading frame, orf79, is cotranscribed with a duplicated atp6 (B-atp6) gene and encodes a cytotoxic peptide. Expression of orf79 in CMS lines and transgenic rice plants caused gametophytic male sterility. Immunoblot analysis showed that the ORF79 protein accumulates specifically in microspores. Two fertility restorer genes, Rf1a and Rf1b, were identified at the classical locus Rf-1 as members of a multigene cluster that encode pentatricopeptide repeat proteins. RF1A and RF1B are both targeted to mitochondria and can restore male fertility by blocking ORF79 production via endonucleolytic cleavage (RF1A) or degradation (RF1B) of dicistronic B-atp6/orf79 mRNA. In the presence of both restorers, RF1A was epistatic over RF1B in the mRNA processing. We have also shown that RF1A plays an additional role in promoting the editing of atp6 mRNAs, independent of its cleavage function. Rf1a|Rf5,Rf1b The fertility restorer gene, Rf2, for Lead Rice-type cytoplasmic male sterility of rice encodes a mitochondrial glycine-rich protein 2011 Plant J Laboratory of Environmental Plant Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. Cytoplasmic male sterility (CMS) is associated with a mitochondrial mutation that causes an inability to produce fertile pollen. The fertility of CMS plants is restored in the presence of a nuclear-encoded fertility restorer (Rf) gene. In Lead Rice-type CMS, discovered in the indica variety 'Lead Rice', fertility of the CMS plant is restored by the single nuclear-encoded gene Rf2 in a gametophytic manner. We performed map-based cloning of Rf2, and proved that it encodes a protein consisting of 152 amino acids with a glycine-rich domain. Expression of Rf2 mRNA was detected in developing and mature anthers. An RF2-GFP fusion was shown to be targeted to mitochondria. Replacement of isoleucine by threonine at amino acid 78 of the RF2 protein was considered to be the cause of functional loss in the rf2 allele. As Rf2 does not encode a pentatricopeptide repeat protein, unlike a majority of previously identified Rf genes, the data from this study provide new insights into the mechanism for restoring fertility in CMS. Rf2 RF2a, a bZIP transcriptional activator of the phloem-specific rice tungro bacilliform virus promoter, functions in vascular development 1997 EMBO J Division of Plant Biology, The Scripps Research Institute, BCC206, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Rice tungro bacilliform virus (RTBV) replicates only in phloem cells in infected rice plants and its promoter drives strong phloem-specific reporter gene expression in transgenic rice plants. We isolated a cDNA encoding a basic leucine zipper (bZIP) protein, RF2a, which binds to the Box II cis element that is important for expression from the promoter. RF2a, which stimulates Box II-dependent transcription in a homologous in vitro transcription system, accumulates in nuclei of phloem and certain other cell types in shoots, but is found at only very low levels in roots. Transgenic antisense plants in which RF2a accumulation was suppressed had normal roots but stunted, twisted leaves with small, disorganized vascular bundles, an enlarged sclerenchyma and large air spaces. We propose that the RTBV promoter exploits a host transcription factor that is critical for leaf tissue differentiation and vascular development for its expression. RF2a|OsbZIP75 Functional analysis of RF2a, a rice transcription factor 2003 J Biol Chem Donald Danforth Plant Science Center, St. Louis, Missouri 63132, USA. RF2a is a bZIP transcription factor that regulates expression of the promoter of rice tungro bacilliform badnavirus. RF2a is predicted to include three domains that contribute to its function. The results of transient assays with mutants of RF2a from which one or more domains were removed demonstrated that the acidic domain was essential for the activation of gene expression, although the proline-rich and glutamine-rich domains each played a role in this function. Studies using fusion proteins of different functional domains of RF2a with the 2C7 synthetic zinc finger DNA-binding domain showed that the acidic region is a relatively strong activation domain, the function of which is dependent on the context in which the domain is placed. Data from transgenic plants further supported the conclusion that the acidic domain was important for maintaining the biological function of RF2a. RF2a and TBP (TATA-binding protein) synergistically activate transcription in vitro (Zhu, Q., Ordiz, M. I., Dabi, T., Beachy, R. N., and Lamb, C. (2002) Plant Cell 14, 795-803). In vitro and in vivo assays showed that RF2a interacts with TBP through the glutamine-rich domain but not the acidic domain. Functional analysis of such interactions indicates that the acidic domain activates transcription through mechanisms other than via the direct recruitment of TBP. RF2a|OsbZIP75 Functional analysis of the activation domain of RF2a, a rice transcription factor 2010 Plant Biotechnol J Donald Danforth Plant Science Center, St Louis, MO 63132, USA. Rice transcription factor RF2a binds to the BoxII cis element of the promoter of rice tungro bacilliform virus and activates promoter expression. The acidic acid-rich domain of RF2a is a transcription activator and has been partially characterized (Dai et al., 2003). The RF2a acidic domain (A; amino acids 49-116) was fused with the synthetic zinc finger ZF-TF 2C7 and was co-introduced with a reporter gene into transgenic Arabidopsis plants. Expression of the reporter gene was increased up to seven times by the effector. In transient assays in tobacco BY-2 protoplasts, we identified a subdomain comprising amino acids 56-84 (A5) that was equally as effective as an activator as the entire acidic domain. A chemically inducible system was used to show determined that A and A5 domains are equally as effective in transcription activation as the well-characterized VP16 activation domain. Bioinformatics analyses revealed that the A5 domain is present only in b-ZIP transcription factors. In dicots, the A domain contains an insertion of four amino acids that is not present in monocot proteins. The A5 domain, and similar domains in other b-ZIP transcription factors, is predicted to form an anti-parallel beta sheet structure. RF2a|OsbZIP75 Transcription factor RF2a alters expression of the rice tungro bacilliform virus promoter in transgenic tobacco plants 2001 Proc Natl Acad Sci U S A The Scripps Research Institute, La Jolla, CA 92037, USA. The promoter from rice tungro bacilliform badnavirus (RTBV) is expressed only in phloem tissues in transgenic rice plants. RF2a, a b-Zip protein from rice, is known to bind to the Box II cis element near the TATA box of the promoter. Here, we report that the full-length RTBV promoter and a truncated fragment E of the promoter, comprising nucleotides -164 to +45, result in phloem-specific expression of beta-glucuronidase (GUS) reporter genes in transgenic tobacco plants. When a fusion gene comprising the cauliflower mosaic virus 35S promoter and RF2a cDNA was coexpressed with the GUS reporter genes, GUS activity was increased by 2-20-fold. The increase in GUS activity was positively correlated with the amount of RF2a, and the expression pattern of the RTBV promoter was altered from phloem-specific to constitutive. Constitutive expression of RF2a did not induce morphological changes in the transgenic plants. In contrast, constitutive overexpression of the b-ZIP domain of RF2a had a strong effect on the development of transgenic plants. These studies suggest that expression of the b-Zip domain can interfere with the function of homologues of RF2a that regulate development of tobacco plants. RF2a|OsbZIP75 RF2b, a rice bZIP transcription activator, interacts with RF2a and is involved in symptom development of rice tungro disease 2004 Proc Natl Acad Sci U S A The Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO 63132, USA. The phloem-specific promoter of rice tungro bacilliform virus (RTBV) is regulated in part by sequence-specific DNA-binding proteins that bind to Box II, an essential cis element. Previous studies demonstrated that the bZIP protein RF2a is involved in transcriptional regulation of the RTBV promoter. Here we report the identification and functional characterization of a second bZIP protein, RF2b. RF2b, identified by its interaction with RF2a, binds to Box II in in vitro assays as a homodimer and as RF2a/RF2b heterodimers. Like RF2a, RF2b activates the RTBV promoter in transient assays and in transgenic tobacco plants. Both RF2a and RF2b are predominantly expressed in vascular tissues. However, RF2a and RF2b have different DNA-binding affinities to Box II, show distinctive expression patterns in different rice organs, and exhibit different patterns of subcellular localization. Furthermore, transgenic rice plants with reduced levels of RF2b exhibit a disease-like phenotype. We propose that the regulation of phloem-specific expression of the RTBV promoter and potentially the control of RTBV replication are mainly achieved via interactions of the Box II cis element with multiple host factors, including RF2a and RF2b. We also propose that quenching/titration of these and perhaps other transcription factors by RTBV is involved in the development of the symptoms of rice tungro disease. RF2a|OsbZIP75,RF2b|OsbZIP30 Transgenic rice plants that overexpress transcription factors RF2a and RF2b are tolerant to rice tungro virus replication and disease 2008 Proc Natl Acad Sci U S A Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO 63132, USA. sdai@danforthcenter.org Rice tungro disease (RTD) is a significant yield constraint in rice-growing areas of South and Southeast Asia. Disease symptoms are caused largely by infection by the rice tungro bacilliform virus (RTBV). Two host transcription factors, RF2a and RF2b, regulate expression of the RTBV promoter and are important for plant development. Expression of a dominant negative mutant of these factors in transgenic rice resulted in phenotypes that mimic the symptoms of RTD, whereas overexpression of RF2a and RF2b had essentially no impact on plant development. Conversely, lines with elevated expression of RF2a or RF2b showed weak or no symptoms of infection after Agrobacterium inoculation of RTBV, whereas control plants showed severe stunting and leaf discoloration. Furthermore, transgenic plants exhibited reduced accumulation of RTBV RNA and viral DNA compared with nontransgenic plants. Similar results were obtained in studies after virus inoculation by green leafhoppers. Gaining disease resistance by elevating the expression of host regulators provides another strategy against RTD and may have implications for other pararetrovirus infections. RF2a|OsbZIP75,RF2b|OsbZIP30 The expression of the large rice FK506 binding proteins (FKBPs) demonstrate tissue specificity and heat stress responsiveness 2006 Plant Science Department of Plant Sciences, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel The FK506 binding proteins (FKBPs) are abundant and ubiquitous proteins belonging to the large peptidyl prolyl cis–trans isomerase superfamily. In this study we have identified and characterized the expression of three large FKBPs in rice: the rice rFKBP64, rFKBP65 and rFKBP75. These FKBPs contain three FKBP12-like domains and a tetratricopeptide repeat (TPR) domain. The expression of the rice FKBPs was found to be regulated by heat stress in various organs. The expression of rFKBP64 at RNA level was elevated by heat stress in roots and shoots and low in mature leaves. The expression of rFKBP65 was detected at the RNA level only after heat stress in all cultivars whereas at the protein level there were differences in the expression between the rice cultivars. The rFKBP75 was expressed at the RNA in all tissues before and after heat stress and the rFKBP75 protein appears to be more abundant after heat stress. The only FKBP to be expressed in seeds was the rFKBP75 which was higher in the embryos and endosperm of dry seeds then in the same organs separated from imbibed seeds, indicating that the protein is important in the steps of seed maturation. In this study we have characterized three large rice FKBPs and have demonstrated that rice FKBPs are heat stress induced and differentially expressed in various tissues indicating specific physiological functions. rFKBP64,rFKBP65,rFKBP75 Down-regulation of RFL, the FLO/LFY homolog of rice, accompanied with panicle branch initiation 1998 Proc Natl Acad Sci U S A Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, 630-01 Japan. FLORICAULA (FLO) of Antirrhinum and LEAFY (FLY) of Arabidopsis regulate the formation of floral meristems. To examine whether same mechanisms control floral development in distantly related species such as grasses, we isolated RFL, FLO-LFY homolog of rice, and examined its expression and function. Northern analysis showed that RFL is expressed predominantly in very young panicle but not in mature florets, mature leaves, or roots. In situ hybridization revealed that RFL RNA was expressed in epidermal cells in young leaves at vegetative growth stage. After the transition to reproductive stage, RFL RNA was detected in all layers of very young panicle including the apical meristem, but absent in the incipient primary branches. As development of branches proceeds, RFL RNA accumulation localized in the developing branches except for the apical meristems of the branches and secondary branch primordia. Expression pattern of RFL raised a possibility that, unlike FLO and LFY, RFL might be involved in panicle branching. Transgenic Arabidopsis plants constitutively expressing RFL from the cauliflower mosaic virus 35S promoter were produced to test whether 35S-RFL would cause similar phenotype as observed in 35S-LFY plants. In 35S-RFL plants, transformation of inflorescence meristem to floral meristem was rarely observed. Instead, development of cotyledons, rosette leaves, petals, and stamens was severely affected, demonstrating that RFL function is distinct from that of LFY. Our results suggest that mechanisms controlling floral development in rice might be diverged from that of Arabidopsis and Antirrhinum. RFL|APO2 Natural variation of the RICE FLOWERING LOCUS T 1 contributes to flowering time divergence in rice 2013 PLoS One Agrogenomics Research Center, National Institute of Agrobiological Sciences, Kannondai Tsukuba, Ibaraki, Japan. In rice (Oryza sativa L.), there is a diversity in flowering time that is strictly genetically regulated. Some indica cultivars show extremely late flowering under long-day conditions, but little is known about the gene(s) involved. Here, we demonstrate that functional defects in the florigen gene RFT1 are the main cause of late flowering in an indica cultivar, Nona Bokra. Mapping and complementation studies revealed that sequence polymorphisms in the RFT1 regulatory and coding regions are likely to cause late flowering under long-day conditions. We detected polymorphisms in the promoter region that lead to reduced expression levels of RFT1. We also identified an amino acid substitution (E105K) that leads to a functional defect in Nona Bokra RFT1. Sequencing of the RFT1 region in rice accessions from a global collection showed that the E105K mutation is found only in indica, and indicated a strong association between the RFT1 haplotype and extremely late flowering in a functional Hd1 background. Furthermore, SNPs in the regulatory region of RFT1 and the E105K substitution in 1,397 accessions show strong linkage disequilibrium with a flowering time-associated SNP. Although the defective E105K allele of RFT1 (but not of another florigen gene, Hd3a) is found in many cultivars, relative rate tests revealed no evidence for differential rate of evolution of these genes. The ratios of nonsynonymous to synonymous substitutions suggest that the E105K mutation resulting in the defect in RFT1 occurred relatively recently. These findings indicate that natural mutations in RFT1 provide flowering time divergence under long-day conditions. RFT1 A multifaceted genomics approach allows the isolation of the rice Pia-blast resistance gene consisting of two adjacent NBS-LRR protein genes 2011 Plant J Iwate Biotechnology Research Center, Kitakami, Iwate 024-0003, Japan. The Oryza sativa (rice) resistance gene Pia confers resistance to the blast fungus Magnaporthe oryzae carrying the AVR-Pia avirulence gene. To clone Pia, we employed a multifaceted genomics approach. First, we selected 12 R-gene analog (RGA) genes encoding nucleotide binding site-leucine rich repeats (NBS-LRRs) proteins from a region on chromosome 11 that shows linkage to Pia. By using seven rice accessions, we examined the association between Pia phenotypes and DNA polymorphisms in the 10 genes, which revealed three genes (Os11gRGA3-Os11gRGA5) exhibiting a perfect association with the Pia phenotypes. We also screened ethyl methane sulfonate (EMS)-treated mutant lines of the rice cultivar 'Sasanishiki' harboring Pia, and isolated two mutants that lost the Pia phenotype. DNA sequencing of Os11gRGA3-Os11gRGA5 from the two mutant lines identified independent mutations of major effects in Os11gRGA4. The wild-type 'Sasanishiki' allele of Os11gRGA4 (SasRGA4) complemented Pia function in both mutants, suggesting that SasRGA4 is necessary for Pia function. However, when the rice cultivar 'Himenomochi' lacking Pia was transfected with SasRGA4, the Pia phenotype was not recovered. An additional complementation study revealed that the two NBS-LRR-type R genes, SasRGA4 and SasRGA5, that are located next to each other and oriented in the opposite direction are necessary for Pia function. A population genetics analysis of SasRGA4 and SasRGA5 suggests that the two genes are under long-term balancing selection. RGA4,RGA5 The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding 2013 Plant Cell INRA, UMR 385 Biologie et Genetique des Interactions Plante-Parasite, F-34398 Montpellier, France. Resistance (R) proteins recognize pathogen avirulence (Avr) proteins by direct or indirect binding and are multidomain proteins generally carrying a nucleotide binding (NB) and a leucine-rich repeat (LRR) domain. Two NB-LRR protein-coding genes from rice (Oryza sativa), RGA4 and RGA5, were found to be required for the recognition of the Magnaporthe oryzae effector AVR1-CO39. RGA4 and RGA5 also mediate recognition of the unrelated M. oryzae effector AVR-Pia, indicating that the corresponding R proteins possess dual recognition specificity. For RGA5, two alternative transcripts, RGA5-A and RGA5-B, were identified. Genetic analysis showed that only RGA5-A confers resistance, while RGA5-B is inactive. Yeast two-hybrid, coimmunoprecipitation, and fluorescence resonance energy transfer-fluorescence lifetime imaging experiments revealed direct binding of AVR-Pia and AVR1-CO39 to RGA5-A, providing evidence for the recognition of multiple Avr proteins by direct binding to a single R protein. Direct binding seems to be required for resistance as an inactive AVR-Pia allele did not bind RGA5-A. A small Avr interaction domain with homology to the Avr recognition domain in the rice R protein Pik-1 was identified in the C terminus of RGA5-A. This reveals a mode of Avr protein recognition through direct binding to a novel, non-LRR interaction domain. RGA4,RGA5 Characterization and fine mapping of the rice blast resistance gene Pia 2011 Sci China Life Sci Laboratory of Plant Resistance and Genetics, College of Natural Resources and Environment Sciences, South China Agricultural University, Guangzhou 510642, China. Blast, caused by Magnaporthe oryzae, is one of the most widespread and destructive diseases of rice. Breeding durable resistant cultivars (cvs) can be achieved by pyramiding of various resistance (R) genes. Pia, carried by cv. Aichi Asahi, was evaluated against 612 isolates of M. oryzae collected from 10 Chinese provinces. The Pia gene expresses weak resistance in all the provinces except for Jiangsu. Genomic position-ready marker-based linkage analysis was carried out in a mapping population consisting of 800 F(2) plants derived from a cross of Aichi AsahixKasalath. The locus was defined in an interval of approximately 90 kb, flanked by markers A16 and A21. Four candidate genes (Pia-1, Pia-2, Pia-3, and Pia-4), all having the R gene conserved structure, were predicted in the interval using the cv. Nipponbare genomic sequence. Four candidate resistance gene (CRG) markers (A17, A25, A26, and A27), derived from the four candidates, were subjected to genotyping with the recombinants detected at the flanking markers. The first three markers completely co-segregated with the Pia locus, and the fourth was absent in the Aichi Asahi genome and disordered with the Pia locus and its flanking markers, indicating that the fourth candidate gene, Pia-4, could be excluded. Co-segregation marker-based genotyping of the three sets of differentials with known R gene genotypes revealed that the genotype of A26 (Pia-3) perfectly matched the R gene genotype of Pia, indicating that Pia-3 is the strongest candidate gene for Pia. RGA4 A metallothionein-like protein of rice (rgMT) functions in E. coli and its gene expression is induced by abiotic stresses 2006 Biotechnol Lett Alkali Soil Natural Environmental Science Center (ASNESC), Stress Molecular Biology Laboratory, Northeast Forestry University, Harbin 150040, P. R. China. A metallothionein-like (rgMT) gene was isolated from a rice (Oryza sativa L.) root cDNA library that was prepared from plants grown under NaHCO3 stress. The rgMT gene expression was induced in rice leaves and roots under several abiotic stresses from salts (NaCl and NaHCO3), drought (PEG) and metals (CuCl2, ZnCl2, CdCl2). The results suggested that the rgMT gene was expressed in response to environmental stresses. The rgMT gene was expressed in Escherichia coli, and the final yield of the purified rgMT protein was 4.8 mg g(-1) dry cells. Tolerance of E. coli expressing GST-rgMT fusion protein to Cu2+, Zn2+ and Cd2+ was enhanced, and cells dry weight increased 0.04 mg, 0.17 mg and 0.07 mg in 1 ml culture treated with either CuCl2, ZnCl2 or CdCl2, respectively, compared with control after 6 h culture. OsMT1a|rgMT OsMT1a, a type 1 metallothionein, plays the pivotal role in zinc homeostasis and drought tolerance in rice 2009 Plant Mol Biol State Key Laboratory of Plant Genomics and National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, The Chinese Academy of Sciences, 100101, Beijing, China. Metallothioneins (MTs) are small, cysteine-rich, metal-binding proteins that may be involved in metal homeostasis and detoxification in both plants and animals. OsMT1a, encoding a type 1 metallothionein, was isolated via suppression subtractive hybridization from Brazilian upland rice (Oryza sativa L. cv. Iapar 9). Expression analysis revealed that OsMT1a predominantly expressed in the roots, and was induced by dehydration. Interestingly, the OsMT1a expression was also induced specifically by Zn(2+) treatment. Both transgenic plants and yeasts harboring OsMT1a accumulated more Zn(2+) than wild type controls, suggesting OsMT1a is most likely to be involved in zinc homeostasis. Transgenic rice plants overexpressing OsMT1a demonstrated enhanced tolerance to drought. The examination of antioxidant enzyme activities demonstrated that catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) were significantly elevated in transgenic plants. Furthermore, the transcripts of several Zn(2+)-induced CCCH zinc finger transcription factors accumulated in OsMT1a transgenic plants, suggesting that OsMT1a not only participates directly in ROS scavenging pathway but also regulates expression of the zinc finger transcription factors via the alteration of Zn(2+) homeostasis, which leads to improved plant stress tolerance. OsMT1a|rgMT A reversibly glycosylated polypeptide (RGP1) possibly involved in plant cell wall synthesis: purification, gene cloning, and trans-Golgi localization 1997 Proc Natl Acad Sci U S A Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA. We purified from pea (Pisum sativum) tissue an approximately 40 kDa reversibly glycosylated polypeptide (RGP1) that can be glycosylated by UDP-Glc, UDP-Xyl, or UDP-Gal, and isolated a cDNA encoding it, apparently derived from a single-copy gene (Rgp1). Its predicted translation product has 364 aminoacyl residues and molecular mass of 41.5 kDa. RGP1 appears to be a membrane-peripheral protein. Immunogold labeling localizes it specifically to trans-Golgi dictyosomal cisternae. Along with other evidence, this suggests that RGP1 is involved in synthesis of xyloglucan and possibly other hemicelluloses. Corn (Zea mays) contains a biochemically similar and structurally homologous RGP1, which has been thought (it now seems mistakenly) to function in starch synthesis. The expressed sequence database also reveals close homologs of pea Rgp1 in Arabidopsis and rice (Oryza sativa). Rice possesses, in addition, a distinct but homologous sequence (Rgp2). RGP1 provides a polypeptide marker for Golgi membranes that should be useful in plant membrane studies. RGP1 A novel ras-related rgp1 gene encoding a GTP-binding protein has reduced expression in 5-azacytidine-induced dwarf rice 1991 MGG Molecular & General Genetics Biotechnology Institute, Akita Prefectural College of Agriculture, 010-04, Akita, Japan Exposure of normal, tall rice (Oryza sativa) seedlings to 5-azacytidine, a powerful inhibitor of DNA methylation in vivo, induced both demethylation of genomic DNA and dwarf plants. Genes that had been affected by treatment were identified by differential screening of a cDNA library, and a ras-related gene, rgp1, was subsequently isolated. The cDNA of rgp1 was found to encode a deduced protein sequence of 226 amino acids with a relative molecular mass of 24850, which was most closely related to the ras-related ypt3 protein of fission yeast, Shizosaccharomyces pombe. The rgp1 protein, expressed in transformed Escherichia coli, clearly showed GTP-binding activity. During seedling growth, rgp1 expression was first observed 14 days after germination, reaching a maximum level between 28 and 42 days, and gradually decreased thereafter until 63 days when it attained the same level of expression as in 14-day-old seedlings. Expression of rgp1 was found to be markedly reduced throughout the growth period of both 5-azacytidine-induced dwarf plants and their progenies, relative to levels in untreated tall control plants. These results suggest that expression of rgp1 may be influenced, either directly or indirectly, by DNA methylation, and that the rgp1 protein may play an important role in plant growth and development. RGP1 Molecular characterization of rgp2, a gene encoding a small GTP-binding protein from rice 1993 Mol Gen Genet Laboratory of Molecular Genetics, Akita Prefectural College of Agriculture, Japan. We previously reported the isolation of rgp1, a gene from rice, which encodes a ras-related GTP-binding protein, and subsequently showed that the gene induces specific morphological changes in transgenic tobacco plants. Here, we report the isolation and characterization of an rgp1 homologue, rgp2, from rice. The deduced rgp2 protein sequence shows 53% identity with the rice rgp1 protein, but 63% identity with both the marine ray ora3 protein, which is closely associated with synaptic vesicles of neuronal tissue, and the mammalian rab11 protein. Conservation of particular amino acid sequence motifs places rgp2 in the rab/ypt subfamily, which has been implicated in vesicular transport. Northern blot analysis of rgp1 and rgp2 suggests that both genes show relatively high, but differential, levels of expression in leaves, stems and panicles, but low levels in roots. In addition, whereas rgp1 shows maximal expression at a particular stage of plantlet growth, rgp2 is constitutively expressed during the same period. Southern blot analysis suggests that, in addition to rgp1 and rgp2, several other homologues exist in rice and these may constitute a small multigene family. rgp2 A Rice HAL2-like Gene Encodes a Ca[IMAGE]-sensitive 3`(2`),5`-Diphosphonucleoside 3`(2`)-Phosphohydrolase and Complements Yeast met22 and Escherichia coli cysQ Mutations 1995 Journal of Biological Chemistry Department of Plant Biology, Plant Biotechnology Center, Ohio State University, Columbus 43210-1002, USA. A plant homolog of yeast HAL2 gene (RHL) was cloned from rice (Orizya sativa L.). The RHL cDNA complemented an Escherichia coli cysteine auxotrophic mutant, cysQ, and the yeast HAL2 mutant, met22. The latter is a methionine auxotroph and cannot use sulfate, sulfite, or sulfide as sulfur sources but exhibits wild-type activities of the enzymes necessary to assimilate sulfate and has normal sulfur uptake system. These results demonstrated that HAL2, cysQ, and RHL genes encode proteins with similar function in sulfur assimilatory pathway. The RHL cDNA expressed a 40-kDa protein that was shown to catalyze the conversion of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) to adenosine 5'-phosphosulfate (APS) and 3'(2')-phosphoadenosine 5'-phosphate (PAP) to AMP. The enzyme activity is Mg(2+)-dependent, sensitive to Ca2+, Li+, and Na+ and activated by K+. The inhibition by Ca2+ depends on the Mg2+/Ca2+ ratio and is reversible by high Mg2+ concentration. The substrate specificity and kinetics of RHL enzyme are very similar to the Chlorella 3'(2'),5'-diphosphonucleoside 3'(2')-phosphohydrolase (DPNPase). Our evidence suggests that this enzyme regulates the flux of sulfur in the sulfur-activation pathway by converting PAPS to APS. Several residues that are essential for the activity of this enzyme were identified by site-directed mutagenesis, and the possible role of DPNPase in salt tolerance is discussed. OsSAL1|RHL sal1 determines the number of aleurone cell layers in maize endosperm and encodes a class E vacuolar sorting protein 2003 Proc Natl Acad Sci U S A Pioneer Hi-Bred International, Johnston, IA 50131, USA. A microscopy-based screen of a large collection of maize Mutator (Mu) transposon lines identified the supernumerary aleurone layers 1-1 (sal1-1) mutant line carrying up to seven layers of aleurone cells in defective kernel endosperm compared with only a single layer in wild-type grains. Normal, well filled endosperm that is homozygous for the sal1-1 mutant allele contains two to three layers of aleurone cells. Cloning of the sal1 gene was accomplished by using Mu tagging, and the identity of the cloned gene was confirmed by isolating an independent sal1-2 allele by reverse genetics. Homozygous sal1-2 endosperm has two to three layers of aleurone cells in normal, well filled grains. In situ hybridization experiments reveal that the sal1 gene is ubiquitously expressed in vegetative as well as zygotic grain tissues, with no difference being detected between aleurone cells and starchy endosperm cells. Northern blot analysis failed to detect the sal1-2 transcript in leaves of homozygous plants, suggesting that the allele is a true sal1 knockout allele. The sal1 gene encodes a homologue of the human Chmp1 gene, a member of the conserved family of the class E vacuolar protein sorting genes implicated in membrane vesicle trafficking. In mammals, CHMP1 functions in the pathway targeting plasma membrane receptors and ligands to lysosomes for proteolytic degradation. Possible roles for the function of the sal1 gene in aleurone signaling, including a defect in endosome trafficking, are discussed. OsSAL1|RHL Distribution patterns of HSP 90 protein in rice 1997 Plant Science Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, Dhaula Kuan. New Delhi-110021, India Rice seedlings exposed to high and low temperatures, salinity and water stress accumulate 87 and 85 kDa stress-associated-proteins (SAP), collectively referred to as SAP 90. In the present study, we provide evidence that SAP 87 of rice is a member of the eukaryotic stress 90 family. Further, high uninduced levels of rice 90 exist in lemma, palea and culm tissues. Dry seeds of rice, wheat, maize and sorghum also contain high uninduced amounts of this protein. Seed-derived callus tissue show high constitutive levels of this protein as well. When seed and callus tissues were subjected to heat shock, the levels of this protein declined. Tissue print-immunoblot analysis using anti SAP 90 antibodies revealed higher accumulation of 90 in the vascular bundles/procambial cells and in the outermost cell layers of various leaves as well as in the meristematic cells of the stem apex. In seeds, this protein was most abundant in the seed coat and whole embryo. Hsp90|rHsp90 Yeast HSP104 homologue rice HSP110 is developmentally- and stress-regulated 1997 Plant Science Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, Dhaula Kuan, New Delhi-110021, India HSP104 plays a critical role in the development of thermotolerance in yeast cells. Rice (Oryza sativa L.) HSP110 (OsHSP110) is previously shown to be an immunological homologue of yeast HSP104. This protein accumulates in rice seedlings in response to heat shock. However, no such high temperature-induced accumulation of OsHSP110 was found in the topmost leaf of 90-day-old (just prior to flowering) plants of the cultivated species of rice (Oryza sativa L.) in our earlier study. In this paper, we show that, at the comparable growth stage, leaves of O. australiensis accumulated this protein to a marginal extent in response to heat shock, while levels of HSP110 were either markedly declined or remained unaltered in 14 other wild rice species. Further, different organs of the mature cultivated rice plant accumulated differential levels of OsHSP110, constitutively as well as in response to heat shock. In particular, upper portions of culm, grains and developing embryos showed significantly high constitutive levels of OsHSP110. Tissue print immunolocalization studies showed that OsHSP110 is distributed in the vascular bundles in the shoot tissues and in seeds it is specifically localized in the seec coat (outermost layer) only. Importantly, OsHSP110 accumulated in shoots of rice seedlings in response to salinity, desiccation and low temperature stress also; furthermore, salinity stress (NaCl) caused nearly a three-fold higher accumulation of this protein than high temperature stress. Hsp90|rHsp90 rHsp90 gene expression in response to several environmental stresses in rice (Oryza sativa L.) 2006 Plant Physiol Biochem Alkali Soil Natural Environmental Science Center (ASNESC), Stress Molecular Biology Laboratory, Northeast Forestry University, Harbin 150040, PR China. In this study, the gene for a rice (Oryza sativa L.) 90 kDa heat shock protein (rHsp90, GenBank accession no. AB037681) was identified by screening rice root cDNAs that were up-regulated under carbonate (NaHCO(3)) stress using the method of differential display, and cloned. The open-reading-frame of rHsp90-cDNA was predicted to encode a protein containing 810 amino acids, which showed high similarity to proteins in Hordeum vulgare (accession no. X67960) and Catharathus roseus (accession no. L14594). Further studies showed that rHsp90 mRNA accumulated following exposure to several abiotic stresses, including salts (NaCl, NaHCO(3) and Na(2)CO(3)), desiccation (using polyethylene glycol), high pH (8.0 and 11.0) and high temperature (42 and 50 degrees C). Yeast (Saccharomyces cerevisiae) over-expressing rHsp90 exhibited greater tolerance to NaCl, Na(2)CO(3) and NaHCO(3) and tobacco seedlings over-expressing rHsp90 could tolerate salt concentrations as high as 200 mM NaCl, whereas untransformed control seedlings couldn't. These results suggest that rHsp90 plays an important role in multiple environmental stresses. Hsp90|rHsp90 Plant Hsp90 family with special reference to rice 1998 Journal of Biosciences Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, Dhaula Kuan, 110 021, New Delhi, India Hsp90 family represents a group of highly conserved and strongly expressed proteins present in almost all biological species. Heat shock proteins in the range of 90 kDa have been detected in a range of plant species andhsp90 genes have been cloned and characterized in selected instances. However, the expression characteristics of plant Hsp90 are poorly understood. Work on expression characteristics of rice Hsp90 is reviewed in this paper. Experimental evidence is provided for indicating that while the rice 87 kDa protein is transiently synthesized within initial 2 h of heat shock, high steady-state levels of this protein are retained even under prolonged high temperature stress conditions or recovery following 4 h heat shock. It is further shown that fifteen different wild rices accumulate differential levels of these proteins in response to heat shock treatment. Hsp90|rHsp90 RID1, encoding a Cys2/His2-type zinc finger transcription factor, acts as a master switch from vegetative to floral development in rice 2008 Proc Natl Acad Sci U S A National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China. cywu@mail.hzau.edu.cn Transition from the vegetative phase to reproductive phase is a crucial process in the life cycle of higher plants. Although the molecular mechanisms of flowering regulation have been extensively characterized in a number of plant species, little is known regarding how the transition process initiates. Here, we show that the Rice Indeterminate 1 (RID1) gene acts as the master switch for the transition from the vegetative to reproductive phase. RID1 encodes a Cys-2/His-2-type zinc finger transcription factor that does not have an ortholog in Arabidopsis spp. A RID1 knockout (rid1), mutated by T-DNA insertion, never headed after growing for >500 days under a range of growth conditions and is thus referred to as a never-flowering phenotype. This mutation-suppressed expression of the genes is known to be involved in flowering regulation, especially in the Ehd1/Hd3a pathway and a series of RFT homologs. RID1 seems to be independent of the circadian clock. A model was proposed to place RID1 in the molecular pathways of flowering regulation in rice, for which there are two indispensable elements. In the first, RID1 is controlling the phase transition and initiation of floral induction. In the other, the Hd3a/RFL1/FTL complex acts as the immediate inducer of flowering. Loss of function in either element would cause never-flowering. Once the phase transition is induced with the activation of RID1, flowering signal is transduced and regulated through the various pathways and eventually integrated with FT-like proteins to induce flowering. Ehd2|RID1 A point mutation in the zinc finger motif of RID1/EHD2/OsID1 protein leads to outstanding yield-related traits in japonica rice variety Wuyunjing 7 2013 Rice (N Y) State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310006, China. dalizeng@126.com. BACKGROUND: Flowering time, which is often associated with the length of the growth period in rice, determines the adaptability of a plant to various environments. However, little is known about how flowering-time genes affect panicle development and yield formation potential in rice after inducing the transition from vegetative growth to reproductive growth. RESULTS: To explore the relationship between floral induction and yield formation and the molecular mechanism of panicle development in rice, a novel mutant, ghd10, was identified from japonica variety Wuyunjing 7 plants subjected to ethyl methane sulfonate (EMS) treatment. The ghd10 mutant exhibited delayed flowering time, tall stalks and increased panicle length and primary branch number. Map-based cloning revealed that Ghd10 encodes a transcription factor with Cys-2/His-2-type zinc finger motifs. Ghd10 is orthologous to INDETERMINATE1 (ID1), which promotes flowering in maize (Zea mays) and is identical to the previously cloned genes Rice Indeterminate1 (RID1), Early heading date2 (Ehd2) and OsId1. Transient expression analysis of the Ghd10-GFP fusion protein in tobacco mesophyll cells showed that this protein is expressed in the nucleus. Ghd10 mRNA accumulated most abundantly in developing leaves and panicle structures, but rarely in roots. Expression analysis revealed that the expression levels of Ehd1, Hd1, RFT1, Hd3a and OsMADS15 decreased dramatically under both short-day and long-day conditions in ghd10. CONCLUSION: These results indicate that Ghd10, which encodes a promoter of flowering, influences plant height and panicle development by regulating the expression levels of some flowering-related genes, such as Ehd1, Hd1, OsMADS15 and others. The ghd10 allele is a useful resource for improvement of panicle traits in rice grown in tropical and low-latitude areas. Ehd2|RID1 Disruption of a novel gene for a NAC-domain protein in rice confers resistance to Rice dwarf virus 2009 Plant J National Agricultural Research Center, Tsukuba, Ibaraki 305-8666, Japan. Rice dwarf virus (RDV) is a serious viral pest that is transmitted to rice plants (Oryza sativa L.) by leafhoppers and causes a dwarfism in infected plants. To identify host factors involved in the multiplication of RDV, we screened Tos17 insertion mutant lines of rice for mutants with reduced susceptibility to RDV. One mutant, designated rim1-1, did not show typical disease symptoms upon infection with RDV. The accumulation of RDV capsid proteins was also drastically reduced in inoculated rim1-1 mutant plants. Co-segregation and complementation analyses revealed that the rim1-1 mutation had been caused by insertion of Tos17 in an intron of a novel NAC gene. The rim1-1 mutant remained susceptible to the two other viruses tested, one of which is also transmitted by leafhoppers, suggesting that the multiplication rather than transmission of RDV is specifically impaired in this mutant. We propose that RIM1 functions as a host factor that is required for multiplication of RDV in rice. RIM1 The NAC transcription factor RIM1 of rice is a new regulator of jasmonate signaling 2010 Plant J National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. Jasmonates (JAs) are lipid-derived regulators that play crucial roles in both host immunity and development. We recently identified the NAC transcription factor RIM1 as a host factor involved in multiplication of rice dwarf virus (RDV). Here, we report that RIM1 functions as a transcriptional regulator of JA signaling and is degraded in response to JA treatment via a 26S proteasome-dependent pathway. Plants carrying rim1 mutations show a phenotype of root growth inhibition. The expression profiles of the mutants were significantly correlated with those of JA-treated wild-type plants without accumulation of endogenous JA, indicating that RIM1 functions as a component of JA signaling. The expression of genes encoding JA biosynthetic enzymes (lipoxygenase (LOX), allene oxide synthase 2 (AOS2) and OPDA reductase 7 (OPR7)) was up-regulated in the rim1 mutants under normal conditions, and a rapid and massive accumulation of endogenous JA was detected in the mutants after wounding. These results suggest that RIM1 may represent a new molecular link in jasmonate signaling, and may thereby provide new insights into the well-established coronatine-insensitive 1 (COI1)-Jasmonate ZIM-domain (JAZ) JA signaling pathway. RIM1 The rice Rim2 transcript accumulates in response to Magnaporthe grisea and its predicted protein product shares similarity with TNP2-like proteins encoded by CACTA transposons 2000 Molecular and General Genetics MGG Department of Plant Pathology, University of California, Davis, CA 95616, USA A rice transcript, Rim2, was identified that accumulated in both incompatible and compatible interactions between rice and Magnaporthe grisea. The Rim2 transcript also accumulated in response to treatment with a cell wall elicitor derived from M. grisea. A 3.3-kb RIM2 cDNA clone was isolated and is predicted to encode a protein of 653 amino acids, which shares 32 55% identity with TNP2-like proteins encoded by CACTA transposons of other plants. A 1.05-kb segment of the Rim2 sequence shows 82% nucleotide sequence identity with sequences flanking the A1 and C members of the rice Xa21 disease resistance gene family. The 5'-upstream region of Rim2 was cloned and the transcriptional start sites were identified. The 5' and 3' noncoding termini of Rim2 are AT-rich. A cis-element showing similarity to a sequence that mediates defense-associated transcriptional activation of the tobacco retrotransposon Tnt1, and four motifs that fit the consensus sequence of the elicitor-responsive elements in the promoters of the parsley PR-1 genes were found in the 5'-upstream region. Four imperfect tandem repeats were identified in the 3' noncoding terminus. Southern analysis with genomic DNA from different rice species indicated that Rim2 is present in 3-4 copies per genome. These results suggest that Rim2 may be one component of a large CACTA-like element, whose transcript accumulates in response to attack by pathogens. Rim2 Rice Immature Pollen 1 (RIP1) is a regulator of late pollen development 2006 Plant Cell Physiol National Research Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Republic of Korea. We isolated a pollen-preferential gene, RICE IMMATURE POLLEN 1 (RIP1), from a T-DNA insertional population of japonica rice that was trapped by a promoterless beta-glucuronidase (GUS) gene. Semi-quantitative reverse transcription-PCR (RT-PCR) analyses confirmed that the RIP1 transcript was abundant at the late stages of pollen development. Transgenic plants carrying a T-DNA insertion in the RIP1 gene displayed the phenotype of segregation distortion of the mutated rip1 gene. Moreover, rip1/rip1 homozygous progeny were not present. Reciprocal crosses between Rip1/rip1 heterozygous plants and the wild type showed that the rip1 allele could not be transmitted through the male. Microscopic analysis demonstrated that development in the rip1 pollen was delayed, starting at the early vacuolated stage. Close examination of that pollen by transmission electron microscopy also showed delayed formation of starch granules and the intine layer. In addition, development of the mitochondria, Golgi apparatus, lipid bodies, plastids and endoplasmic reticulum was deferred in the mutant pollen. Under in vitro conditions, germination of this mutant pollen did not occur, whereas the rate for wild-type pollen was >90%. These results indicate that RIP1 is necessary for pollen maturation and germination. This gene encodes a protein that shares significant homology with a group of proteins containing five WD40 repeat sequences. The green fluorescent protein (GFP)-RIP1 fusion protein is localized to the nucleus. Therefore, RIP1 is probably a nuclear protein that may form a functional complex with other proteins and carry out essential cellular and developmental roles during the late stage of pollen formation. RIP1 Constitutive expression of the defense-related Rir1b gene in transgenic rice plants confers enhanced resistance to the rice blast fungus Magnaporthe grisea 2000 Plant Mol Biol Institute of Plant Biology, University of Zurich, Switzerland. The Rirlb gene of rice (Oryza sativa) is one of a set of putative defense genes whose transcripts accumulate upon inoculation of rice with the non-host pathogen Pseudomonas syringae pv. syringae. It belongs to a family of genes encoding small extracellular proteins so far only identified in cereals. To assess the function of the Rirlb gene in rice blast resistance, it was placed under the control of the CaMV 35S promoter and transferred into rice plants of the japonica variety Taipei 309 by biolistic transformation of immature embryos. Two out of 12 hygromycin-resistant regenerated plants (OE1 and OE3) were fertile. DNA gel blot analysis suggested that these two T0 plants were independent transformants, each of which had stably incorporated one complete copy of the transgene into the genome. In addition, the OE1 plant appeared also a contain a rearranged copy or incomplete copy. T1 plants homozygous for the transgene were identified by DNA gel blot analysis of individual T2 progeny and further propagated. Expression analysis of the transgene showed that the transgene was active both in T1 plants and homozygous decendants. Challenge inoculation of homozygous transgenic plants with Magnaporthe grisea, the causal agent of rice blast disease, revealed that both independent transgenic lines were more resistant than the untransformed wild type, suggesting that over-expression of the Rirlb gene confers partial resistance against this important pathogen. Rir1b Synergism between RPBF Dof and RISBZ1 bZIP activators in the regulation of rice seed expression genes 2006 Plant Physiol Transgenic Crop Research and Development Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. The Dof (DNA binding with one finger) transcriptional activator rice (Oryza sativa) prolamin box binding factor (RPBF), which is involved in gene regulation of rice seed storage proteins, has been isolated from rice cDNA expressed sequence tag clones containing the conserved Dof. RPBF is found as a single gene per haploid genome. Comparison of RPBF genomic and cDNA sequences revealed that the genomic copy is interrupted by one long intron of 1,892 bp in the 5' noncoding region. We demonstrated by transient expression in rice callus protoplasts that the isolated RPBF trans-activated several storage protein genes via an AAAG target sequence located within their promoters, and with methylation interference experiments the additional AAAG-like sequences in promoters of genes expressed in maturing seeds were recognized by the RPBF protein. Binding was sequence specific, since mutation of the AAAG motif or its derivatives decreased both binding and trans-activation by RPBF. Synergism between RPBF and RISBZ1 recognizing the GCN4 motif [TGA(G/C)TCA] was observed in the expression of many storage protein genes. Overexpression of both transcription factors gave rise to much higher levels of expression than the sum of individual activities elicited by either RPBF or RISBZ1 alone. Furthermore, mutation of recognition sites suppressed reciprocal trans-activation ability, indicating that there are mutual interactions between RISBZ1 and RPBF. The RPBF gene is predominantly expressed in maturing endosperm and coordinately expressed with seed storage protein genes, and is involved in the quantitative regulation of genes expressed in the endosperm in cooperation with RISBZ1. RISBZ1|OsbZIP58,RPBF|OsDof3 Emergence of a subfamily of xylanase inhibitors within glycoside hydrolase family 18 2005 FEBS J Institute of Food Research (IFR), Norwich, UK. The xylanase inhibitor protein I (XIP-I), recently identified in wheat, inhibits xylanases belonging to glycoside hydrolase families 10 (GH10) and 11 (GH11). Sequence and structural similarities indicate that XIP-I is related to chitinases of family GH18, despite its lack of enzymatic activity. Here we report the identification and biochemical characterization of a XIP-type inhibitor from rice. Despite its initial classification as a chitinase, the rice inhibitor does not exhibit chitinolytic activity but shows specificities towards fungal GH11 xylanases similar to that of its wheat counterpart. This, together, with an analysis of approximately 150 plant members of glycosidase family GH18 provides compelling evidence that xylanase inhibitors are largely represented in this family, and that this novel function has recently emerged based on a common scaffold. The plurifunctionality of GH18 members has major implications for genomic annotations and predicted gene function. This study provides new information which will lead to a better understanding of the biological significance of a number of GH18 'inactivated' chitinases. RIXI|C10701 Rice class III chitinase homologues isolated by random cloning of rice cDNAs 1997 DNA Res Rice Genome Research Program, Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, Ibaraki, Japan. We obtained seven kinds of cDNA clones putatively identified as encoding class III chitinases from cDNA libraries constructed from dichlorophenoxyacetic acid (2,4-D)- and benzyl adenine (BA)- treated rice callus. Putative amino acid sequences encoded in these cDNA clones were compared with those of known chitinases of other plants. Two clones coded for homologues that show high similarity to class III chitinases. These clones contained the common glutamic acid at the active site and were classified as true homologues. The other five clones, however, showed relatively low similarity to class III chitinases and their active sites contained aspartic acid instead of glutamic acid. These clones may correspond to relatives of a super family of class III chitinases. The location of the genes coding for these homologues on the rice genome has been determined by genetic linkage analysis with restriction fragment length polymorphism. RIXI|C10701 Rolling-leaf14 is a 2OG-Fe (II) oxygenase family protein that modulates rice leaf rolling by affecting secondary cell wall formation in leaves 2012 Plant Biotechnol J Rice Research Institute, Chongqing Key Laboratory of Application and Safety Control of Genetically Modified Crops, Southwest University, Chongqing, China. hegh1968@yahoo.com.cn As an important agronomic trait, leaf rolling in rice (Oryza sativa L.) has attracted much attention from plant biologists and breeders. Moderate leaf rolling increases the amount of photosynthesis in cultivars and hence raises grain yield. Here, we describe the map-based cloning of the gene RL14, which was found to encode a 2OG-Fe (II) oxygenase of unknown function. rl14 mutant plants had incurved leaves because of the shrinkage of bulliform cells on the adaxial side. In addition, rl14 mutant plants displayed smaller stomatal complexes and decreased transpiration rates, as compared with the wild type. Defective development could be rescued functionally by the expression of wild-type RL14. RL14 was transcribed in sclerenchymatous cells in leaves that remained wrapped inside the sheath. In mature leaves, RL14 accumulated mainly in the mesophyll cells that surround the vasculature. Expression of genes related to secondary cell wall formation was affected in rl14-1 mutants, and cellulose and lignin content were altered in rl14-1 leaves. These results reveal that the RL14 gene affects water transport in leaves by affecting the composition of the secondary cell wall. This change in water transport results in water deficiency, which is the major reason for the abnormal shape of the bulliform cells. RL14 cDNA Cloning and Expression Analysis of the Rice ( Oryza sativa L.) RNase L Inhibitor 2003 DNA Sequence - The Journal of Sequencing and Mapping Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, People's Republic of China. A subtractive cDNA library was constructed using rice (Oryza sativa L.) callus cDNA as driver and differentiating callus cDNA as tester. A novel cDNA fragment encoding RNase L inhibitor (RLI) was isolated by screening the subtractive library, which had a higher expression level in differentiating callus than in callus. The full-length cDNA of rice-RLI was obtained by the method of rapid amplification of cDNA ends, which contained a 1812-bp open reading frame encoding a 604 amino acid polypeptide. Homologous analysis showed that rice-RLI contained the conserved motifs (two repeated P-loops, two ATP-binding boxes and an iron-sulfur binding motif). The fluorescence quantitative PCR analysis showed that it was a constitutive expressed gene but up-regulated in abiotic stress (low temperature and NaCl treatment) and down-regulated under the treatments of NAA and IAA. RLI A novel protein RLS1 with NB-ARM domains is involved in chloroplast degradation during leaf senescence in rice 2012 Mol Plant National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Leaf senescence, a type of programmed cell death (PCD) characterized by chlorophyll degradation, is important to plant growth and crop productivity. It emerges that autophagy is involved in chloroplast degradation during leaf senescence. However, the molecular mechanism(s) involved in the process is not well understood. In this study, the genetic and physiological characteristics of the rice rls1 (rapid leaf senescence 1) mutant were identified. The rls1 mutant developed small, yellow-brown lesions resembling disease scattered over the whole surfaces of leaves that displayed earlier senescence than those of wild-type plants. The rapid loss of chlorophyll content during senescence was the main cause of accelerated leaf senescence in rls1. Microscopic observation indicated that PCD was misregulated, probably resulting in the accelerated degradation of chloroplasts in rls1 leaves. Map-based cloning of the RLS1 gene revealed that it encodes a previously uncharacterized NB (nucleotide-binding site)-containing protein with an ARM (armadillo) domain at the carboxyl terminus. Consistent with its involvement in leaf senescence, RLS1 was up-regulated during dark-induced leaf senescence and down-regulated by cytokinin. Intriguingly, constitutive expression of RLS1 also slightly accelerated leaf senescence with decreased chlorophyll content in transgenic rice plants. Our study identified a previously uncharacterized NB-ARM protein involved in PCD during plant growth and development, providing a unique tool for dissecting possible autophagy-mediated PCD during senescence in plants. RLS1 Identification of a mitochondrial ATP synthase small subunit gene (RMtATP6) expressed in response to salts and osmotic stresses in rice (Oryza sativa L.) 2006 J Exp Bot Alkali Soil Natural Environmental Science Center (ASNESC), Stress Molecular Biology Laboratory, Northeast Forestry University, Harbin 150040, PR China. Large areas of northern China have alkaline soil due to the accumulation of sodium carbonates (NaHCO3, Na2CO3). To understand better how plants can tolerate alkaline soil, a cDNA library was prepared from rice (Oryza sativa L.) roots grown in the presence of NaHCO3 stress. A cDNA clone isolated from this library was identified by a homology search as a mitochondrial ATP synthase 6 kDa subunit gene (RMtATP6; GenBank accession nos AB055076, BAB21526). In transformed yeast and tobacco protoplasts, the RMtATP6 protein was localized in mitochondria using the green fluorescent protein (GFP) marker. Analysis of RMtATP6 mRNA levels suggested that the expression of this gene was induced by stress from sodium carbonates and other sodium salts. Transgenic tobacco overexpressing the RMtATP6 gene had greater tolerance to salt stress at the seedling stage than untransformed tobacco. Among the other genes for F1F0-ATPase of rice, some were found to be up-regulated by some environmental stresses and some were not. These data suggest that the RMtATP6 protein acts as a subunit of ATP synthase, and is expressed in response to stress from several salts, with the other genes coding for the subunits of the same ATP-synthase. RMtATP6 Rice virescent3 and stripe1 encoding the large and small subunits of ribonucleotide reductase are required for chloroplast biogenesis during early leaf development 2009 Plant Physiol Department of Plant Science, Plant Genomics and Breeding Institute, and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea. The virescent3 (v3) and stripe1 (st1) mutants in rice (Oryza sativa) produce chlorotic leaves in a growth stage-dependent manner under field conditions. They are temperature-conditional mutants that produce bleached leaves at a constant 20 degrees C or 30 degrees C but almost green leaves under diurnal 30 degrees C/20 degrees C conditions. Here, we show V3 and St1, which encode the large and small subunits of ribonucleotide reductase (RNR), RNRL1, and RNRS1, respectively. RNR regulates the rate of deoxyribonucleotide production for DNA synthesis and repair. RNRL1 and RNRS1 are highly expressed in the shoot base and in young leaves, and the expression of the genes that function in plastid transcription/translation and in photosynthesis is altered in v3 and st1 mutants, indicating that a threshold activity of RNR is required for chloroplast biogenesis in developing leaves. There are additional RNR homologs in rice, RNRL2 and RNRS2, and eukaryotic RNRs comprise alpha(2)beta(2) heterodimers. In yeast, RNRL1 interacts with RNRS1 (RNRL1:RNRS1) and RNRL2:RNRS2, but no interaction occurs between other combinations of the large and small subunits. The interacting activities are RNRL1:RNRS1 > RNRL1:rnrs1(st1) > rnrl1(v3):RNRS1 > rnrl1(v3):rnrs1(st1), which correlate with the degree of chlorosis for each genotype. This suggests that missense mutations in rnrl1(v3) and rnrs1(st1) attenuate the first alphabeta dimerization. Moreover, wild-type plants exposed to a low concentration of an RNR inhibitor, hydroxyurea, produce chlorotic leaves without growth retardation, reminiscent of v3 and st1 mutants. We thus propose that upon insufficient activity of RNR, plastid DNA synthesis is preferentially arrested to allow nuclear genome replication in developing leaves, leading to continuous plant growth. RNRL1|V3,RNRL2,RNRS1|St1,RNRS2 Position dependent expression of GL2-type homeobox gene, Roc1: significance for protoderm differentiation and radial pattern formation in early rice embryogenesis 2002 The Plant Journal BioScience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan. In early plant embryogenesis, the determination of cell fate in the protodermal cell layer is considered to be the earliest event in radial pattern formation. To elucidate the mechanisms of epidermal cell fate determination and radial pattern formation in early rice embryogenesis, we have isolated a GL2-type homeobox gene Roc1 (Rice outermost cell-specific gene1), which is specifically expressed in the protoderm (epidermis). In early rice embryogenesis, cell division occurs randomly and the morphologically distinct layer structure of the protoderm cannot be observed until the embryo reaches more than 100 microm in length. Nonetheless, in situ hybridization analyses revealed that specific expression of Roc1 in the outermost cells is established shortly after fertilization, much earlier than protoderm differentiation. In the regeneration process from callus, the Roc1 gene is also expressed in the outermost cells of callus in advance of tissue and organ differentiation, and occurs independently of whether the cells will differentiate into epidermis in the future or not. Furthermore, this cell-specific Roc1 expression could be induced flexibly in the newly produced outermost cells when we cut the callus. These findings suggest that the expression of Roc1 in the outermost cells may be dependent on the positional information of cells in the embryo or callus prior to the cell fate determination of the protoderm (epidermis). Furthermore, the Roc1 expression is downregulated in the inner cells of ligule, which have previously been determined as protodermal cells, also suggesting that the Roc1 expression is position dependent and that this position dependent Roc1 expression is important also in post-embryonic protoderm (epidermis) differentiation. Roc1 Leaf rolling controlled by the homeodomain leucine zipper class IV gene Roc5 in rice 2011 Plant Physiol Biotechnology Research Institute/National Key Facility for Genetic Resources and Gene Improvement, Chinese Academy of Agricultural Sciences, Beijing 100081, People's Republic of China. Leaf rolling is considered an important agronomic trait in rice (Oryza sativa) breeding. To understand the molecular mechanism controlling leaf rolling, we screened a rice T-DNA insertion population and isolated the outcurved leaf1 (oul1) mutant showing abaxial leaf rolling. The phenotypes were caused by knockout of Rice outermost cell-specific gene5 (Roc5), an ortholog of the Arabidopsis (Arabidopsis thaliana) homeodomain leucine zipper class IV gene GLABRA2. Interestingly, overexpression of Roc5 led to adaxially rolled leaves, whereas cosuppression of Roc5 resulted in abaxial leaf rolling. Bulliform cell number and size increased in oul1 and Roc5 cosuppression plants but were reduced in Roc5-overexpressing lines. The data indicate that Roc5 negatively regulates bulliform cell fate and development. Gene expression profiling, quantitative polymerase chain reaction, and RNA interference (RNAi) analyses revealed that Protodermal Factor Like (PFL) was probably down-regulated in oul1. The mRNA level of PFL was increased in Roc5-overexpressing lines, and PFL-RNAi transgenic plants exhibit reversely rolling leaves by reason of increases of bulliform cell number and size, indicating that Roc5 may have a conserved function. These are, to our knowledge, the first functional data for a gene encoding a homeodomain leucine zipper class IV transcriptional factor in rice that modulates leaf rolling. Roc5|oul1,PFL A null mutation of ROS1a for DNA demethylation in rice is not transmittable to progeny 2012 Plant J National Institute for Basic Biology, Okazaki 444-8585, Japan. Genes that promote DNA methylation and demethylation in plants have been characterized mainly in Arabidopsis. Arabidopsis DNA demethylation is mediated by bi-functional DNA enzymes with glycosylase activity that removes 5-methylcytosine and lyase activity that nicks double-stranded DNA at an abasic site. Homologous recombination-promoted knock-in targeting of the ROS1a gene, the longest of six putative DNA demethylase genes in the rice genome, by fusing its endogenous promoter to the GUS reporter gene, led to reproducibly disrupted ROS1a in primary (T(0)) transgenic plants in the heterozygous condition. These T(0) plants exhibited no overt morphological phenotypes during the vegetative phase, and GUS staining showed ROS1a expression in pollen, unfertilized ovules and meristematic cells. Interestingly, neither the maternal nor paternal knock-in null allele, ros1a-GUS1, was virtually detected in the progeny; such an intransmittable null mutation is difficult to isolate by conventional mutagenesis techniques that are usually used to identify and isolate mutants in the progeny population. Even in the presence of the wild-type paternal ROS1a allele, the maternal ros1a-GUS1 allele caused failure of early-stage endosperm development, resulting in incomplete embryo development, with embryogenesis producing irregular but viable embryos that failed to complete seed dormancy, implying non-equivalent maternal and paternal contribution of ROS1a in endosperm development. The paternal ros1a-GUS1 allele was not transmitted to progeny, presumably because of a male gametophytic defect(s) prior to fertilization. Thus, ROS1a is indispensable in both male and female gametophytes, and DNA demethylation must plays important roles in both gametophytes. ROS1a Genetic dissection of the biotic stress response using a genome-scale gene network for rice 2011 Proc Natl Acad Sci U S A Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, 262 Seongsanno, Seodaemun-gu, Seoul 120-749, Korea Rice is a staple food for one-half the world's population and a model for other monocotyledonous species. Thus, efficient approaches for identifying key genes controlling simple or complex traits in rice have important biological, agricultural, and economic consequences. Here, we report on the construction of RiceNet, an experimentally tested genome-scale gene network for a monocotyledonous species. Many different datasets, derived from five different organisms including plants, animals, yeast, and humans, were evaluated, and 24 of the most useful were integrated into a statistical framework that allowed for the prediction of functional linkages between pairs of genes. Genes could be linked to traits by using guilt-by-association, predicting gene attributes on the basis of network neighbors. We applied RiceNet to an important agronomic trait, the biotic stress response. Using network guilt-by-association followed by focused protein-protein interaction assays, we identified and validated, in planta, two positive regulators, LOC_Os01g70580 (now Regulator of XA21; ROX1) and LOC_Os02g21510 (ROX2), and one negative regulator, LOC_Os06g12530 (ROX3). These proteins control resistance mediated by rice XA21, a pattern recognition receptor. We also showed that RiceNet can accurately predict gene function in another major monocotyledonous crop species, maize. RiceNet thus enables the identification of genes regulating important crop traits, facilitating engineering of pathways critical to crop productivity. ROX1,ROX2,ROX3 Nucleotide sequence of a rice (Oryza sativa) prolamin storage protein gene, RP6 1993 Plant Physiol Institute of Botany, Academia Sinica, National Taiwan University, Taipei, Republic of China. None RP6 Replication protein A2c coupled with replication protein A1c regulates crossover formation during meiosis in rice 2013 Plant Cell National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, Hubei Province, China. Replication protein A (RPA) is a conserved heterotrimeric protein complex comprising RPA1, RPA2, and RPA3 subunits involved in multiple DNA metabolism pathways attributable to its single-stranded DNA binding property. Unlike other species possessing a single RPA2 gene, rice (Oryza sativa) possesses three RPA2 paralogs, but their functions remain unclear. In this study, we identified RPA2c, a rice gene preferentially expressed during meiosis. A T-DNA insertional mutant (rpa2c) exhibited reduced bivalent formation, leading to chromosome nondisjunction. In rpa2c, chiasma frequency is reduced by ~78% compared with the wild type and is accompanied by loss of the obligate chiasma. The residual ~22% chiasmata fit a Poisson distribution, suggesting loss of crossover control. RPA2c colocalized with the meiotic cohesion subunit REC8 and the axis-associated protein PAIR2. Localization of REC8 was necessary for loading of RPA2c to the chromosomes. In addition, RPA2c partially colocalized with MER3 during late leptotene, thus indicating that RPA2c is required for class I crossover formation at a late stage of homologous recombination. Furthermore, we identified RPA1c, an RPA1 subunit with nearly overlapping distribution to RPA2c, required for ~79% of chiasmata formation. Our results demonstrate that an RPA complex comprising RPA2c and RPA1c is required to promote meiotic crossovers in rice. RPA1c,RPA2c Identification of Dof proteins with implication in the gibberellin-regulated expression of a peptidase gene following the germination of rice grains 2001 Biochim Biophys Acta Laboratory of Environmental Molecular Biology, Division of Bioscience, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan. washi@ees.hokudai.ac.jp Type III carboxypeptidase (CPD3) is one of the hydrolytic enzymes whose expression is up-regulated by gibberellins (GA) in the aleurones of germinated cereal grains. A number of pyrimidine boxes and a sequence resembling the gibberellic acid response element (GARE) are observed in the region upstream of the transcription initiation site of the CPD3 gene, showing a characteristic of cereal GA-responsive genes. Transient gene expression assays in germinated rice aleurone demonstrated that the CPD3 promoter was able to confer hormonally responses on the expression of the reporter gene. By southwestern screening, several cDNAs encoding the Dof class proteins were isolated from a rice aleurone library. Each mRNA accumulation for five novel members of Dof proteins (OsDof1--5) occurs with a different time course and in a tissue-specific manner following the germination of grains. Of these, the expression of the OsDof3 gene is abundant in aleurones where it precedes that of the CPD3 gene, implying that this is an early response gene of GA. The OsDof3 protein, expressed in Escherichia coli, selectively bound AAAG motifs of the pyrimidine boxes through the DNA-binding activity of its Dof domain. Co-expression experiments in aleurones suggested that the OsDof3 protein should play a regulatory role in the expression of the CPD3 gene under the control of GA. RPBF|OsDof3 RPE, a plant gene involved in early developmental steps of nematode feeding cells 1998 EMBO J INRA, Laboratoire de Biologie des Invertebres, 123 bd F. Meilland, 06600 Antibes, France. Sedentary plant-parasitic nematodes are able to induce the redifferentiation of root cells into multinucleate nematode feeding sites (NFSs). We have isolated by promoter trapping an Arabidopsis thaliana gene that is essential for the early steps of NFS formation induced by the root-knot nematode Meloidogyne incognita. Its pattern of expression is similar to that of key regulators of the cell cycle, but it is not observed with the cyst nematode. Later in NFS development, this gene is induced by both root-knot and cyst nematodes. It encodes a protein similar to the D-ribulose-5-phosphate 3-epimerase (RPE) (EC 5.1.3.1), a key enzyme in the reductive Calvin cycle and the oxidative pentose phosphate pathway (OPPP). Quantitative RT-PCR showed the accumulation of RPE transcripts in potato, as in Arabidopsis NFS. Homozygous rpe plants have a germination mutant phenotype that can be rescued in dwarf plants on sucrose-supplemented medium. During root development, this gene is expressed in the meristems and initiation sites of lateral roots. These results suggest that the genetic control of NFSs and the first stages of meristem formation share common steps and confirms the previous cytological observations which indicate that root cells undergo metabolic reprogramming when they turn into NFSs. RPE Transcriptional downregulation of rice rpL32 gene under abiotic stress is associated with removal of transcription factors within the promoter region 2011 PLoS One Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, Delhi, India. BACKGROUND: The regulation of ribosomal proteins in plants under stress conditions has not been well studied. Although a few reports have shown stress-specific post-transcriptional and translational mechanisms involved in downregulation of ribosomal proteins yet stress-responsive transcriptional regulation of ribosomal proteins is largely unknown in plants. METHODOLOGY/PRINCIPAL FINDINGS: In the present work, transcriptional regulation of genes encoding rice 60S ribosomal protein L32 (rpL32) in response to salt stress has been studied. Northern and RT-PCR analyses showed a significant downregulation of rpL32 transcripts under abiotic stress conditions in rice. Of the four rpL32 genes in rice genome, the gene on chromosome 8 (rpL32_8.1) showed a higher degree of stress-responsive downregulation in salt sensitive rice variety than in tolerant one and its expression reverted to its original level upon withdrawal of stress. The nuclear run-on and promoter:reporter assays revealed that the downregulation of this gene is transcriptional and originates within the promoter region. Using in vivo footprinting and electrophoretic mobility shift assay (EMSA), cis-elements in the promoter of rpL32_8.1 showing reduced binding to proteins in shoots of salt stressed rice seedlings were identified. CONCLUSIONS: The present work is one of the few reports on study of stress downregulated genes. The data revealed that rpL32 gene is transcriptionally downregulated under abiotic stress in rice and that this transcriptional downregulation is associated with the removal of transcription factors from specific promoter elements. rpL32_8.1 A putative acyl-CoA-binding protein is a major phloem sap protein in rice (Oryza sativa L.) 2006 J Exp Bot Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Tokyo 113-8657, Japan. suzui.nobuo@jaea.go.jp The N-terminal amino-acid sequence of a major rice phloem-sap protein, named RPP10, was determined. RPP10 is encoded by a single gene in the rice genome. Its complete amino-acid sequence, predicted from the corresponding rice full-length cDNA, showed high similarity to plant acyl-CoA-binding proteins (ACBPs). Western blot analysis using anti-ACBP antiserum revealed that putative ACBP is abundant in the phloem sap of rice plants, and is also present in sieve-tube exudates of winter squash (Cucurbita maxima), oilseed rape (Brassica napus), and coconut palm (Cocos nucifera). These findings give rise to the idea that ACBP may involve lipid metabolism and regulation in the phloem. RPP10 Rice C2-domain proteins are induced and translocated to the plasma membrane in response to a fungal elicitor 2003 Biochemistry Division of Applied Life Science, Gyeongsang National University, Jinju, Korea Hundreds of proteins involved in signaling pathways contain a Ca(2+)-dependent membrane-binding motif called the C2-domain. However, no small C2-domain proteins consisting of a single C2-domain have been reported in animal cells. We have isolated two cDNA clones, OsERG1a and OsERG1b, that encode two small C2-domain proteins of 156 and 159 amino acids, respectively, from a fungal elicitor-treated rice cDNA library. The clones are believed to have originated from a single gene by alternative splicing. Transcript levels of the OsERG1 gene are dramatically elevated by a fungal elicitor prepared from Magnaporthe grisea or by Ca(2+) ions. The OsERG1 protein produced in Escherichia coli binds to phospholipid vesicles in a Ca(2+)-dependent manner and is translocated to the plasma membrane of plant cells by treatment with either a fungal elicitor or a Ca(2+) ionophore. These results suggest that OsERG1 proteins containing a single C2-domain are involved in plant defense signaling systems. Rpp17|OsERG1 Identification of rice blast fungal elicitor-responsive genes by differential display analysis 2000 Mol Plant Microbe Interact Department of Biochemistry, Gyeongsang National University, Chinju, Korea. In order to study molecular interactions that occur between rice and rice blast fungus upon infection, we isolated fungal elicitor-responsive genes from rice (Oryza sativa cv. Milyang 117) suspension-cultured cells treated with fungal elicitor prepared from the rice blast fungus (Magnaporthe grisea) employing a method that combined mRNA differential display and cDNA library screening. Data base searches with the isolated cDNA clones revealed that the OsERG1 and OsERG2 cDNAs share significant similarities with the mammalian Ca2+-dependent lipid binding (C2) domains. The OsCPX1 cDNA is highly homologous to peroxidases. The OsHin1 cDNA exhibits homology to the tobacco hin1 gene, whose expression is induced by avirulent pathogens. The OsLPL1 and OsMEK1 cDNAs share homologies with lysophospholipases and serine/threonine mitogen-activated protein (MAP) kinase kinases, respectively. The OsWRKY1 and OsEREBP1 cDNAs are homologous to transcription factors, such as the WRKY protein family and the AP2/EREBP family, respectively. Transcripts of the OsERG1, OsHin1, and OsMEK1 genes were specifically elevated only in response to the avirulent race KJ301 of the rice blast fungus. Our study yielded a number of elicitor-responsive genes that will not only provide molecular probes, but also contribute to our understanding of host defense mechanisms against the rice blast fungus. Rpp17|OsERG1 Cloning and characterization of the gene for a phloem-specific glutathione S-transferase from rice leaves 2004 Physiol Plant Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan. The N-terminal amino-acid sequence was determined for a major rice phloem protein with a molecular mass of 31 kDa, named RPP31. The corresponding full-length rice EST-clone was cloned based on the amino acid sequence. The predicted total amino-acid sequence of RPP31 shared high similarity with plant glutathione S-transferases (GSTs). Recombinant RPP31 produced in Escherichia coli and rice phloem sap showed GST activity. Immunocytological analysis indicated that RPP31 is localized in the phloem region of leaves. In mature leaves, the signal was restricted to sieve element-companion cell complexes, and was stronger in sieve elements than in companion cells. Although some plant GSTs are known to be induced by xenobiotics, the amount of RPP31 was not affected by treatments with an herbicide, pretilachlor, and/or its safener, fenclorim. These results suggest that RPP31 is an active GST restricted to the phloem region of normal rice leaves. RPP31 Isolation and characterization of a rice glutathione S-transferase gene promoter regulated by herbicides and hormones 2011 Plant Cell Rep Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, 400044 Chongqing, People's Republic of China. tzhu2002@yahoo.com.cn OsGSTL2, encoding glutathione S-transferase, is a lambda class gene on chromosome 3 of rice (Oryza sativa L.). RNA blot analysis and semi-quantitative RT-PCR assays demonstrated that the transcription of OsGSTL2 in rice roots treated with chlorsulfuron increased significantly. To further understand OsGSTL2 promoter activity, a DNA fragment (GST2171) of 2,171 bp upstream of the OsGSTL2 coding region was isolated. In silico sequence analysis revealed that this fragment contains stress-regulated regulatory elements, hormone-responsive elements and three transposable elements. To define the core promoter sequence, a series of 5' truncation derivatives of GST2171 were fused to uidA gene. The chimeric genes were introduced into rice plants via Agrobacterium-mediated transformation. The expression of the GST2171::GUS transgene varied considerably. GUS staining indicated that the uidA gene is expressed in young seedlings, older leaves, flowering glumes and seeds, but not in older roots. Quantitative fluorescence assays revealed that the expression of the uidA gene is strong in young seedlings and decreases gradually over a period of 25 days. To our surprise, among the 5' truncation derivatives, the shortest promoter GST525 showed the highest GUS expression, and the second shortest promoter GST962 showed the lowest GUS expression. The uidA gene expression in the roots of transgenic rice seedlings is upregulated by chlorsulfuron, glyphosate, salicylic acid (SA) and naphthalene acetic acid (NAA). The possible roles of the repetitive elements on the OsGSTL2 promoter were discussed in terms of transcription repression and promoter induction by herbicides and hormones. RPP31 Chemical induction of disease resistance in rice is correlated with the expression of a gene encoding a nucleotide binding site and leucine-rich repeats 1999 Plant Mol Biol Mitsui Plant Biotechnology Research Institute, TCI D-21, Sengen 2-1-6, Tsukuba, 305-0047, Japan Probenazole (3-allyloxy-1,2-benzisothiazole-1,1-dioxide) is an agricultural chemical primarily used to prevent rice blast disease. Probenazole-treated rice acquires resistance to blast fungus irrespective of the rice variety. The chemical is applied prophylactically, and is thought to induce or bolster endogenous plant defenses. However, the mechanisms underlying this effect have not been established. To understand the mode of the chemical's action, we screened for novel probenazole-responsive genes in rice by means of differential display and identified a candidate gene, RPR1. RPR1 contains a nucleotide binding site and leucine-rich repeats, thus sharing structural similarity with known disease resistance genes. The expression of RPR1 in rice can be up-regulated by treatment with chemical inducers of systemic acquired resistance (SAR) and by inoculation with pathogens. RPR1-related sequences in rice varieties seem to be varied in sequence and/or expression, indicating that RPR1 itself is not a crucial factor for induced resistance in rice. However, Southern blot analysis revealed the existence of homologous sequences in all varieties examined. While the role of RPR1 has yet to be clarified, this is the first report of the identification of a member of this gene class and its induction during the systemic expression of induced disease resistance. RPR1 A single nuclear transcript encoding mitochondrial RPS14 and SDHB of rice is processed by alternative splicing: Common use of the same mitochondrial targeting signal for different proteins 1999 Proc Natl Acad Sci U S A Faculty of Horticulture, Chiba University, Matsudo 648, Matsudo, Chiba 271-0092, Japan. The rice mitochondrial genome has a sequence homologous to the gene for ribosomal protein S14 (rps14), but the coding sequence is interrupted by internal stop codons. A functional rps14 gene was isolated from the rice nuclear genome, suggesting a gene-transfer event from the mitochondrion to the nucleus. The nuclear rps14 gene encodes a long N-terminal extension showing significant similarity to a part of mitochondrial succinate dehydrogenase subunit B (SDHB) protein from human and a malarial parasite (Plasmodium falciparum). Isolation of a functional rice sdhB cDNA and subsequent sequence comparison to the nuclear rps14 indicate that the 5' portions of the two cDNAs are identical. The sdhB genomic sequence shows that the SDHB-coding region is divided into two exons. Surprisingly, the RPS14-coding region is located between the two exons. DNA gel blot analysis indicates that both sdhB and rps14 are present at a single locus in the rice nucleus. These findings strongly suggest that the two gene transcripts result from a single mRNA precursor by alternative splicing. Protein blot analysis shows that the size of the mature RPS14 is 16.5 kDa, suggesting removal of the N-terminal 22.6-kDa peptide region. Considering that the rice mitochondrial genome lacks the sdhB gene but contains the rps14-related sequence, transfer of the sdhB gene seems to have occurred before the transfer of the rps14 gene. The migration of the mitochondrial rps14 sequence into the already existing sdhB gene could bestow the capacity for nuclear expression and mitochondrial targeting. RPS14 Cloning and characterization of a jasmonic acid-responsive gene encoding 12-oxophytodienoic acid reductase in suspension-cultured rice cells 2003 Planta Biotechnology Research Center, The University of Tokyo, Tokyo 113-8657, Japan. In suspension-cultured rice ( Oryza sativaL.) cells, jasmonic acid (JA) functions as a signal transducer in elicitor N-acetylchitoheptaose-induced phytoalexin production. Differential screening of a cDNA library constructed using poly(A)(+) RNA from suspension-cultured rice cells treated with JA (10(-4) M) for 2 h yielded a cDNA for a gene that responded to exogenous JA by an increase in mRNA level. Nucleotide sequence analysis indicated that the cDNA encodes an homologue of the yeast Old Yellow Enzyme. The deduced amino acid sequence was very similar to the sequences of 12-oxophytodienoic acid reductases (OPR) 1 and 2 from Arabidopsis thaliana(AtOPR1 and AtOPR2) and OPR1 from tomato ( Lycopersicon esculentum) (LeOPR1). The cDNA-encoded protein purified from recombinant Escherichia coli cells as a hexahistidine-tagged fusion protein exhibited OPR activity similar to that of AtOPR1, AtOPR2, and LeOPR1, which catalyze reduction of (-)- cis-12-oxophytodienoic acid (OPDA) preferentially over (+)- cis-OPDA, a natural precursor of JA. Thus the rice enzyme was termed OsOPR1. The physiological roles of OsOPR1 are discussed. This is the first report of the cloning of an OPR gene from a monocot plant. RRJ1 Molecular Cloning and Characterization of a cDNA and a Gene for Subtilisin-like Serine Proteases from Rice (Oryza sativaL.) andArabidopsis thaliana 2014 Bioscience, Biotechnology and Biochemistry Laboratory of Biological Chemistry, Faculty of Agriculture, Kobe University The complete nucleotide sequences of a cDNA (RSP1) that encodes a subtilisin-like serine protease (subtilase) of rice (Oryza sativa L.) and a gene (ASP48) for Arabidopsis subtilase were analyzed. The RSP1 cDNA and ASP48 DNA encoded 736- and 757-residue pre-pro-polypeptides including a signal peptide with molecular masses of 78,668 Da and 79,414 Da, respectively. RSP1 is the first known serine protease in rice, and ASP48 is a gene for ara12 cDNA. Sequence comparison and phylogenetic analysis showed that RSP1 is distantly related to all other plant subtilases and ASP48 is closely related to a tomato subtilase, SBT1. The ASP48 gene was found to lack introns. The Arabidopsis subtilase gene appears to consist of a small gene family. The RSP1 was found to be expressed in seed and shoots of seedlings while ASP48 transcripts was found to be accumulated in immature silique and flowers, indicating that both RSP1 and ASP48 are organ-specific and may be involved in the specific proteolytic events that occur during organ development. RSP1 Coexpression analysis identifies Rice Starch Regulator1, a rice AP2/EREBP family transcription factor, as a novel rice starch biosynthesis regulator 2010 Plant Physiol National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. Starch biosynthesis is important for plant development and is a critical factor in crop quality and nutrition. As a complex metabolic pathway, the regulation of starch biosynthesis is still poorly understood. We here present the identification of candidate regulators for starch biosynthesis by gene coexpression analysis in rice (Oryza sativa). Starch synthesis genes can be grouped into type I (in seeds; sink tissues) and type II (in vegetative tissues; source tissues), and 307 and 621 coexpressed genes are putatively involved in the regulation of starch biosynthesis in rice seeds and vegetative tissues, respectively. Among these genes, Rice Starch Regulator1 (RSR1), an APETALA2/ethylene-responsive element binding protein family transcription factor, was found to negatively regulate the expression of type I starch synthesis genes, and RSR1 deficiency results in the enhanced expression of starch synthesis genes in seeds. Seeds of the knockout mutant rsr1 consistently show the increased amylose content and altered fine structure of amylopectin and consequently form the round and loosely packed starch granules, resulting in decreased gelatinization temperature. In addition, rsr1 mutants have a larger seed size and increased seed mass and yield. In contrast, RSR1 overexpression suppresses the expression of starch synthesis genes, resulting in altered amylopectin structure and increased gelatinization temperature. Interestingly, a decreased proportion of A chains in rsr1 results in abnormal starch granules but reduced gelatinization temperature, whereas an increased proportion of A chains in RSR1-overexpressing plants leads to higher gelatinization temperatures, which is novel and different from previous reports, further indicating the complicated regulation of starch synthesis and determination of the physicochemical properties of starch. These results demonstrate the potential of coexpression analysis for studying rice starch biosynthesis and the regulation of a complex metabolic pathway and provide informative clues, including the characterization of RSR1, to facilitate the improvement of rice quality and nutrition. RSR1 Cloning and expression of rice (Oryza sativa) sucrose synthase 1 (RSs1) in developing seed endosperm 1996 Plant Science Department of Nutritional Sciences, 231 Morgan Hall, University of California, Berkeley, CA 94720, USA A previously cloned cDNA to rice (Oryza sativa L.) sucrose synthase 1 (RSs1) was used to determine spatial expression of the gene in rice tissues and temporal expression in developing rice endosperm. RSs1 was expressed predominantly in the endosperm of milky stage rice seeds with maximum expression at 3–5 days after pollination which were 8–10-fold over leaf levels. The peak in RSs1 transcript levels preceded the peak of enzyme activity which occured 9–11 days after pollination. RSs1 transcript and activity levels were analyzed in two starch deficient mutants of rice to determine if the lesion in these mutants resides at the locus for RSs1. No correlation between sucrose synthase activity and starch biosynthesis was seen in these mutants, although slight elevations of RSs1 transcript levels were observed. RSS1 Molecular characterization of the rice protein RSS1 required for meristematic activity under stressful conditions 2012 Plant Physiol Biochem Bioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan. Post embryonic growth of plants depends on cell division activity in the shoot and root meristems, in conjunction with subsequent cell differentiation. Under environmental stress conditions, where plant growth is moderately impaired, the meristematic activity is maintained by mechanisms as yet unknown. We previously showed that the rice protein RSS1, whose stability is regulated depending on the cell cycle phases, is a key factor for the maintenance of meristematic activity under stressful conditions. RSS1 interacts with a catalytic subunit of protein phosphatase 1 (PP1), but other molecular characteristics are largely unknown. Here we show that RSS1 interacts with all the PP1 expressed in the shoot apex of rice. This interaction requires one of the conserved regions of RSS1, which is important for RSS1 function. Interestingly, the recombinant RSS1 protein is highly resistant to heat with respect to its anti-coagulability and binding activity to PP1. The features of RSS1 are reminiscent of those of inhibitor-2 of animals, although it is likely that the mode of function of RSS1 is different from that of inhibitor-2. Noticeably, RSS1 binds to PP1 under extremely high ionic strength conditions in vitro. Therefore, RSS1 possibly functions by forming a stable complex with PP1. RSS1 RSS1 regulates the cell cycle and maintains meristematic activity under stress conditions in rice 2011 Nat Commun Bioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan. Plant growth and development are sustained by continuous cell division in the meristems, which is perturbed by various environmental stresses. For the maintenance of meristematic functions, it is essential that cell division be coordinated with cell differentiation. However, it is unknown how the proliferative activities of the meristems and the coordination between cell division and differentiation are maintained under stressful conditions. Here we show that a rice protein, RSS1, whose stability is controlled by cell cycle phases, contributes to the vigour of meristematic cells and viability under salinity conditions. These effects of RSS1 are exerted by regulating the G1-S transition, possibly through an interaction of RSS1 with protein phosphatase 1, and are mediated by the phytohormone, cytokinin. RSS1 is conserved widely in plant lineages, except eudicots, suggesting that RSS1-dependent mechanisms might have been adopted in specific lineages during the evolutionary radiation of angiosperms. RSS1 RICE SALT SENSITIVE3 forms a ternary complex with JAZ and class-C bHLH factors and regulates jasmonate-induced gene expression and root cell elongation 2013 Plant Cell Bioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan. Plasticity of root growth in response to environmental cues and stresses is a fundamental characteristic of land plants. However, the molecular basis underlying the regulation of root growth under stressful conditions is poorly understood. Here, we report that a rice nuclear factor, RICE SALT SENSITIVE3 (RSS3), regulates root cell elongation during adaptation to salinity. Loss of function of RSS3 only moderately inhibits cell elongation under normal conditions, but it provokes spontaneous root cell swelling, accompanied by severe root growth inhibition, under saline conditions. RSS3 is preferentially expressed in the root tip and forms a ternary complex with class-C basic helix-loop-helix (bHLH) transcription factors and JASMONATE ZIM-DOMAIN proteins, the latter of which are the key regulators of jasmonate (JA) signaling. The mutated protein arising from the rss3 allele fails to interact with bHLH factors, and the expression of a significant portion of JA-responsive genes is upregulated in rss3. These results, together with the known roles of JAs in root growth regulation, suggest that RSS3 modulates the expression of JA-responsive genes and plays a crucial role in a mechanism that sustains root cell elongation at appropriate rates under stressful conditions. RSS3 An expression analysis profile for the entire sucrose synthase gene family in rice 2008 Plant Science National Agricultural Research Center, 1-2-1 Inada, Joetsu, Niigata 943-0193, Japan In rice three genes encoding for sucrose synthase (Sus) (EC 2.4.1.13) have previously been characterized, and a further three sequences for putative Sus genes reported. The current work confirms that these six genes comprise the entire rice Sus gene family. An expression analysis was conducted in various rice tissues using real-time quantitative RT-PCR. Sus transcripts were detected in a wide range of tissues and at different developmental stages, indicating that Sus is involved in numerous and diverse growth processes in the rice plant. SUS1 was predominantly expressed in elongating tissues including roots, developing leaves and internodes; a potential role for SUS1 in cellulose synthesis is discussed. SUS2 was shown to be expressed in a wide range of tissues, suggesting that it may perform a number of housekeeping role(s). In addition, overall Sus activity and expression of SUS2 increased significantly in seedlings germinated and grown in hypoxia (submerged conditions), suggesting a role in increasing sucrose metabolism as a response to environmental stress. SUS3 and SUS4 were predominantly expressed in the caryopsis, indicating potential roles in carbon allocation within the filling grain. Transcript levels of SUS5 and SUS6 were low in all tissues examined, and were suppressed in germinating shoots under submergence. RSUS3|SUS3 Complete Structures of Three Rice Sucrose Synthase Isogenes and Differential Regulation of Their Expressions 1996 Biosci Biotechnol Biochem Department of Agricultural Chemistry, National Taiwan University 2) Department of Agricultural Chemistry, National Taiwan University By cloning and sequencing cDNA and genomic DNA and transcription initiation site mapping, the total structures including at least 1 kb of putative regulatory sequences upstream of the transcription initiation sites of three genes encoding rice sucrose synthase isoprotomers were either newly established or amended. The third type of SS gene, RSus3, has not been found in other plants. The structures of the three genes and the gene products were compared and their evolutionary sequence was proposed. Specific probes for the three SS mRNA's were developed and used for analyzing their steady state levels at different organs and under some physiological stress conditions. It appears that RSus2 is a house-keeping gene, RSus3 is highly specific to the grain, and the expression of RSus1 shows a tendency to complement that of RSus3. A possible cause of the presence of the third rice SS gene was discussed. We also reported a novel method to synthesize single-stranded DNA for S1 mapping of a transcription initiation site associated with extended secondary structures. RSUS3|SUS3 Single nucleotide polymorphisms and haplotype diversity in rice sucrose synthase 3 2011 J Hered Department of Plant Science, Plant Genomics and Breeding Institute, Seoul National University, Seoul 151-921, Korea. Rice sucrose synthase 3 (RSUS3) is expressed predominantly in rice seed endosperm and is thought to play an important role in starch filling during the milky stage of rice seed ripening. Because the genetic diversity of this locus is not known yet, the full sequence of RSUS3 from 43 rice varieties was amplified to examine the distribution of DNA polymorphisms. A total of 254 sequence variants, including SNPs and insertion/deletions, were successfully identified in the 7733 bp sequence that comprises the promoter, exons and introns, and 3' downstream nontranscribed region (NTR). Eleven haplotypes were distinguished among the 43 rice varieties based on nucleotide variation in the 3 defined regions (5' NTR, transcript, and 3' NTR). The promoter region showed evidence of a base change on a cis-element that might influence the functional role of the motif in seed-specific expression. The genetic diversity of the RSUS3 gene sequences in the rice germplasm used in this study appears to be the result of nonrandom processes. Analysis of polymorphism sites indicated that at least 11 recombinations have occurred, primarily in the transcribed region. This finding provides insight into the development of a cladistic approach for establishing future genetic association studies of the RSUS3 locus. RSUS3|SUS3 Solution structure of the DNA binding domain of rice telomere binding protein RTBP1 2009 Biochemistry Department of Biochemistry and Biology, Protein Network Research Center, Yonsei University, Seoul 120-749, Korea. RTBP1 is a rice telomeric protein that binds to the duplex array of TTTAGGG repeats at chromosome ends. The DNA binding domain of RTBP1 contains a Myb-type DNA binding motif and a highly conserved C-terminal Myb extension that is unique to plant telomeric proteins. Using an electrophoretic mobility shift assay, we identified the C-terminal 110-amino acid region (RTBP1(506-615)) as the minimal telomeric DNA binding domain, suggesting that the Myb extension is required for binding plant telomeric DNA. Like other telomeric proteins such as human TRF1 and yeast Rap1, RTBP1 induced a DNA bending in the telomeric repeat sequence, suggesting that RTBP1 may play a role in establishing and/or maintaining an active telomere configuration in vivo. To elucidate the DNA binding mode of RTBP1, we determined the three-dimensional structure of RTBP1(506-615) in solution by NMR spectroscopy. The overall structure of RTBP1(506-615) is composed of four alpha-helices and stabilized by three hydrophobic patches. The second and third helices in RTBP1 form a helix-turn-helix motif that interacts directly with DNA. The fourth helix located in the Myb extension is essential for binding to telomeric DNA via stabilization of the overall structure of the RTBP1 DNA binding domain. When DNA bound to RTBP1(506-615), large chemical shift perturbations were induced in the N-terminal arm, helix 3, and the loop between helices 3 and 4. These results suggest that helix 3 functions as a sequence-specific recognition helix while the N-terminal arm stabilizes the DNA binding. RTBP1 Suppression of RICE TELOMERE BINDING PROTEIN 1 results in severe and gradual developmental defects accompanied by genome instability in rice 2007 Plant Cell Department of Biology, College of Science, Yonsei University, Seoul, Korea. Although several potential telomere binding proteins have been identified in higher plants, their in vivo functions are still unknown at the plant level. Both knockout and antisense mutants of RICE TELOMERE BINDING PROTEIN1 (RTBP1) exhibited markedly longer telomeres relative to those of the wild type, indicating that the amount of functional RTBP1 is inversely correlated with telomere length. rtbp1 plants displayed progressive and severe developmental abnormalities in both germination and postgermination growth of vegetative organs over four generations (G1 to G4). Reproductive organ formation, including panicles, stamens, and spikelets, was also gradually and severely impaired in G1 to G4 mutants. Up to 11.4, 17.2, and 26.7% of anaphases in G2, G3, and G4 mutant pollen mother cells, respectively, exhibited one or more chromosomal fusions, and this progressively increasing aberrant morphology was correlated with an increased frequency of anaphase bridges containing telomeric repeat DNA. Furthermore, 35S:anti-RTBP1 plants expressing lower levels of RTBP1 mRNA exhibited developmental phenotypes intermediate between the wild type and mutants in all aspects examined, including telomere length, vegetative and reproductive growth, and degree of genomic anomaly. These results suggest that RTBP1 plays dual roles in rice (Oryza sativa), as both a negative regulator of telomere length and one of positive and functional components for proper architecture of telomeres. RTBP1 Alteration of TGA factor activity in rice results in enhanced tolerance to Xanthomonas oryzae pv. oryzae 2005 Plant J Department of Plant Pathology, University of California, Davis, CA 95616, USA. In dicotyledonous plants broad-spectrum resistance to pathogens is established after the induction of the systemic acquired resistance (SAR) response. In Arabidopsis the NPR1 protein can regulate SAR by interacting with members of the TGA class of basic, leucine-zipper transcription factors to alter pathogenesis-related (PR) gene expression. Overexpression of (At)NPR1 in Arabidopsis enhances resistance to multiple pathogens. Similarly, overexpression of (At)NPR1 in rice enhances resistance to the bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo). These results suggest that components of the (At)NPR1-mediated SAR defense response may be conserved between monocots and dicots. To determine whether or not rice TGA factors are involved in disease resistance responses, the effect of altering the function of rice TGA2.1 was analyzed in transgenic plants. Transgenic rice overexpressing an rTGA2.1 mutant, that can no longer bind DNA, and transgenic rice that have the endogenous rTGA2.1 silenced by dsRNA-mediated silencing were generated. Both types of transgenic rice displayed increased tolerance to Xoo, were dwarfed, and had altered accumulation of PR genes. The results presented in this study suggest that wild-type rTGA2.1 has primarily a negative role in rice basal defense responses to bacterial pathogens. rTGA2.1|OsbZIP63|OsNIF1 Molecular cloning of a root hairless gene rth1 in rice 2009 Breeding Science Faculty of Agriculture, Kagawa University Root hairs, projections from the epidermal cells of the roots, are contributing to water and nutrient uptake and anchorage to the soil. To better understand genetic control of root hair formation in rice, we analyzed root hairless 1 (rth1) mutant that was induced by NaN3 treatment. SEM observation showed that in rth1 plants, root hair elongation was abolished after the formation of bulge. High-resolution mapping using 2,088 segregants revealed three predicted genes in a 38-kb candidate interval on chromosome 7. Sequences comparison of the three genes between wild-type Oochikara and rth1 detected a nucleotide substitution only in apyrase (OsAPY). This nucleotide substitution (G -> A) lies in the junction between the third intron and the forth exon, and results in the splicing anomaly to the rth1 cDNA sequence. Transgenic plants with introduced OsAPY allele restored normal root hairs and plant growth, showing a complementation of rth1 phenotype. We concluded that the root hairless phenotype of rth1 is caused by a mutation of OsAPY. OsAPY appears to be an important gene for root hair elongation and plant growth in rice. rth1|OsAPY RTS, a rice anther-specific gene is required for male fertility and its promoter sequence directs tissue-specific gene expression in different plant species 2006 Plant Mol Biol Department of Genetics, Biochemistry and Life Science Studies, Clemson University, 100 Jordan Hall, Clemson, SC 29634, USA. hluo@clemson.edu A tapetum-specific gene, RTS, has been isolated by differential screening of a cDNA library from rice panicles. RTS is a unique gene in the rice genome. RNA blot analysis and in situ hybridization indicates that this gene is predominantly expressed in the anther's tapetum during meiosis and disappears before anthesis. RTS has no introns and encodes a putative polypeptide of 94 amino acids with a hydrophobic N-terminal region. The nucleotide and deduced amino acid sequence of the gene do not show significant homology to any known sequences. However, a sequence in the promoter region, GAATTTGTTA, differs only by one or two nucleotides from one of the conserved motifs in the promoter region of two pollen-specific genes of tomato. Several other sequence motifs found in other anther-specific promoters were also identified in the promoter of the RTS gene. Transgenic and antisense RNA approaches revealed that RTS gene is required for male fertility in rice. The promoter region of RTS, when fused to the Bacillus amyloliquefaciens ribonuclease gene, barnase, or the antisense of the RTS gene, is able to drive tissue-specific expression of both genes in rice, creeping bentgrass (Agrostis stolonifera L.) and Arabidopsis, conferring male sterility to the transgenic plants. Light and near-infrared confocal microscopy of cross-sections through developing flowers of male-sterile transgenics shows that tissue-specific expression of barnase or the antisense RTS genes interrupts tapetal development, resulting in deformed non-viable pollen. These results demonstrate a critical role of the RTS gene in pollen development in rice and the versatile application of the RTS gene promoter in directing anther-specific gene expression in both monocotyledonous and dicotyledonous plants, pointing to a potential for exploiting this gene and its promoter for engineering male sterility for hybrid production of various plant species. RTS Expression enhancement of a rice polyubiquitin gene promoter 2006 Plant Mol Biol Department of Crop Science, North Carolina State University, Raleigh, NC 27695-7620, USA. An 808 bp promoter from a rice polyubiquitin gene, rubi3, has been isolated. The rubi3 gene contained an open reading frame of 1,140 bp encoding a pentameric polyubiquitin arranged as five tandem, head-to-tail repeats of 76 aa. The 1,140 bp 5' UTR intron of the gene enhanced its promoter activity in transient expression assays by 20-fold. Translational fusion of the GUS reporter gene to the coding sequence of the ubiquitin monomer enhanced GUS enzyme activity in transient expression assays by 4.3-fold over the construct containing the original rubi3 promoter (including the 5' UTR intron) construct. The enhancing effect residing in the ubiquitin monomer coding sequence has been narrowed down to the first 9 nt coding for the first three amino acid residues of the ubiquitin protein. Mutagenesis at the third nucleotide of this 9 nt sequence still maintains the enhancing effect, but leads to translation of the native GUS protein rather than a fusion protein. The resultant 5' regulatory sequence, consisting of the rubi3 promoter, 5' UTR exon and intron, and the mutated first 9 nt coding sequence, has an activity nearly 90-fold greater than the rubi3 promoter only (without the 5' UTR intron), and 2.2-fold greater than the maize Ubi1 gene promoter (including its 5' UTR intron). The newly created expression vector is expected to enhance transgene expression in monocot plants. Considering the high conservation of the polyubiquitin gene structure in higher plants, the observed enhancement in gene expression may apply to 5' regulatory sequences of other plant polyubiquitin genes. RUBI3 Kiddo, a new transposable element family closely associated with rice genes 2001 Mol Genet Genomics Institute of Developmental and Molecular Biology, Texas A & M University, College Station, TX 77843-3155, USA. The promoter region of the rice ubiquitin2 (rubq2) gene was found to be polymorphic between japonica (T309) and indica (IR24) lines as the result of a 270-bp deletion in T309. A TTATA footprint in the T309 rubq2 promoter suggested that an excision event had occurred, and inspection of the 270-bp region present in IR24 revealed that it had all the characteristics of a miniature inverted repeat transposable element (MITE). Database searches showed that this element is a member of a new MITE family, which we have named Kiddo. Thirty-five complete Kiddo sequences were identified in existing rice genomic sequence databases. They could be arranged into four groups, within-group sequence identity was over 90%, with 65-75% identity between groups. The high sequence similarity within a group indicates that some Kiddo members were recently mobile and may still be active. An additional 24 decayed Kiddo sequences were detected. Interestingly, approximately 80% of 18 Kiddo members from annotated accessions lie within 530 bp of a coding sequence. That approximately 40% of Kiddo members present in genic regions reside in introns suggests that Kiddo transposition entails the use of both DNA and RNA intermediates, and may provide some insight into the origins of individual groups. DNA blot analysis showed that Kiddo is a rice-specific element, although one sequence with limited (72%) similarity to Kiddo group A was detected as a wheat EST. Kiddo family members may represent new molecular and phylogenetic markers, as well as representing valuable materials for studying the molecular mechanisms of MITE transposition. rubq2 A two-edged role for the transposable element Kiddo in the rice ubiquitin2 promoter 2005 Plant Cell Institute of Developmental and Molecular Biology, Texas A&M University, College Station, Texas 77843-3155, USA. Miniature inverted repeat transposable elements (MITEs) are thought to be a driving force for genome evolution. Although numerous MITEs are found associated with genes, little is known about their function in gene regulation. Whereas the rice ubiquitin2 (rubq2) promoter in rice (Oryza sativa) line IR24 contains two nested MITEs (Kiddo and MDM1), that in line T309 has lost Kiddo, providing an opportunity to understand the role of MITEs in promoter function. No difference in endogenous rubq2 transcript levels between T309 and IR24 was evident using RT-PCR. However, promoter analysis using both transient and stably transformed calli revealed that Kiddo contributed some 20% of the total expression. Bisulfite genomic sequencing of the rubq2 promoters revealed specific DNA methylation at both symmetric and asymmetric cytosine residues on the MITE sequences, possibly induced by low levels of homologous transcripts. When methylation of the MITEs was blocked by 5-azacytidine treatment, a threefold increase in the endogenous rubq2 transcript level was detected in IR24 compared with that in T309. Together with the observed MITE methylation pattern, the detection of low levels of transcripts, but not small RNAs, corresponding to Kiddo and MDM1 suggested that RNA-dependent DNA methylation is induced by MITE transcripts. We conclude that, although Kiddo enhances transcription from the rubq2 promoter, this effect is mitigated by sequence-specific epigenetic modification. rubq2 Molecular cloning, expression and characterization of a glycosyltransferase from rice 2006 Plant Cell Rep Bio/Molecular Informatics Center, Department of Molecular Biotechnology, Konkuk University, Seoul, 143-701, South Korea. hydekjh@msn.com Secondary plant metabolites undergo several modification reactions, including glycosylation. Glycosylation, which is mediated by UDP-glycosyltransferase (UGT), plays a role in the storage of secondary metabolites and in defending plants against stress. In this study, we cloned one of the glycosyltransferases from rice, RUGT-5 resulting in 40-42% sequence homology with UGTs from other plants. RUGT-5 was functionally expressed as a glutathione S-transferase fusion protein in Escherichia coli and was then purified. Eight different flavonoids were used as tentative substrates. HPLC profiling of reaction products displayed at least two peaks. Glycosylation positions were located at the hydroxyl groups at C-3, C-7 or C-4' flavonoid positions. The most efficient substrate was kaempferol, followed by apigenin, genistein and luteolin, in that order. According to in vitro results and the composition of rice flavonoids the in vivo substrate of RUGT-5 was predicted to be kaempferol or apigenin. To our knowledge, this is the first time that the function of a rice UGT has been characterized. RUGT-5 Gene Cloning, Bacterial Expression, and Purification of a Novel Rice (Oryza sativaL.) Ubiquitin-Related Protein, RURM1 2014 Bioscience, Biotechnology and Biochemistry Laboratory of Plant Breeding, Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Japan. A rice ubiquitin-related modifier-1 (Rurm1) gene was cloned and transformed in Escherichia coli. We successfully expressed the RURM1 protein as a glutathione S-transferase (GST)-fusion protein by cultivating the E. coli cells at 16 degrees C for 16 h. After cleavage of GST, we obtained a single protein of 12 kDa. This protein was identified as the RURM1 protein by western blot analysis. RURM1 Molecular cloning of a novel water channel from rice: its products expression in Xenopus oocytes and involvement in chilling tolerance 2000 Plant Science Laboratory of Plant Genetic Engineering, Biotechnology Institute, Akita Prefectural University, Ogata, Akita, Japan Water channel proteins, aquaporins, play a fundamental role in transmembrane water movements in plants. We isolated rice cDNA, rwc1, by screening a rice (Oryza sativa cv. Josaeng Tongil) cDNA library using a conserved motif of aquaporins. Like other aquaporin genes, rwc1 encodes a 290-residue protein with six putative transmembrane domains. The derived amino acid sequence of RWC1 shows high homology with PIP1 (plasma membrane intrinsic protein 1) subfamily members, which suggest it is localized in the plasma membrane. Injection of its cRNA into Xenopus oocytes increased the osmotic water permeability of the oocytes 2-3 times. Northern analysis showed that rice aquaporin genes are expressed in rice seedling leaves and roots, but that it disappeared from the root 6 h after osmotic stress began and that the transcript level remained low for about 24 h, then recovered. The time course of rice aquaporin gene-expression under osmotic stress was correlated with time course of turgor transition in plant. On the other hand, the levels of rice aquaporin gene-transcripts in leaves under chilling and recovery temperature depend on the pretreatment of mannitol for short time. This variation of the transcripts shown that rice aquaporin genes may play an important role in response to water stress-induced chilling tolerance. OsPIP1;1|RWC1 Aquaporin OsPIP1;1 promotes rice salt resistance and seed germination 2013 Plant Physiol Biochem Graduate School of Bioresource Sciences, Akita Prefectural University, Shimoshinjo, Akita 010-0195, Japan. OsPIP1;1 is one of the most abundant aquaporins in rice leaves and roots and is highly responsible to environmental stresses. However, its biochemical and physiological functions are still largely unknown. The oocyte assay data showed OsPIP1;1 had lower water channel activity in contrast to OsPIP2;1. EGFP and immunoelectron microscopy studies revealed OsPIP1;1 was predominantly localized in not only plasma membrane but also in some ER-like intracellular compartments in the cells. OsPIP1;1 exhibited low water channel activity in Xenopus oocytes but coexpression of OsPIP2;1 significantly enhanced its water permeability. Stop-flow assay indicated that 10His-OsPIP1;1-reconstituted proteoliposomes had significantly higher water permeability than the control liposomes. Overexpression of OsPIP1;1 greatly altered many physiological features of transgenic plants in a dosage-dependent manner. Moderate expression of OsPIP1;1 increased rice seed yield, salt resistance, root hydraulic conductivity, and seed germination rate. This work suggests OsPIP1;1 functions as an active water channel and plays important physiological roles. OsPIP1;1|RWC1 The role of aquaporin RWC3 in drought avoidance in rice 2004 Plant Cell Physiol Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032, PR China. Although the discovery of aquaporins in plants has resulted in a paradigm shift in the understanding of plant water relations, the relationship between aquaporins and drought resistance still remains elusive. From an agronomic viewpoint, upland rice is traditionally considered as showing drought avoidance. In the investigation of different morphological and physiological responses of upland rice (Oryza sativa L. spp indica cv. Zhonghan 3) and lowland rice (O. sativa L. spp japonica cv. Xiushui 63) to water deficit, we observed young leaf rolling and the remarkable decline of cumulative transpiration in the upland rice. The expression of water channel protein RWC3 mRNA was increased in upland rice at the early response (up to 4 h) to the 20% polyethylene glycol (PEG) 6000 treatment, whereas there was no significant expression changes in lowland rice. Protein levels were increased in upland rice and decreased in lowland rice at 10 h after the water deficit. The up-regulation of RWC3 in upland rice fits well with the knowledge that upland rice adopts the mechanism of drought avoidance. The physiological significance of this RWC3 up-regulation was then explored with the over-expression of RWC3 in transgenic lowland rice (O. sativa L. spp japonica cv. Zhonghua 11) controlled by a stress-inducible SWPA2 promoter. Compared to the wild-type plant, the transgenic lowland rice exhibited higher root osmotic hydraulic conductivity (Lp), leaf water potential and relative cumulative transpiration at the end of 10 h PEG treatment. These results indicated that RWC3 probably played a role in drought avoidance in rice. RWC3|OsPIP1;3 Mutations in the eIF(iso)4G translation initiation factor confer high resistance of rice to Rice yellow mottle virus 2006 Plant J UMR 5096, IRD/CNRS/Universite de Perpignan, BP 64501, 34394 Montpellier CEDEX 5, France. laurence.albar@mpl.ird.fr We report here evidence of the role that the isoform of the eukaryotic translation initiation factor 4G (eIF(iso)4G) plays in naturally occurring resistance in plant/virus interactions. A genetic and physical mapping approach was developed to isolate the Rymv1 locus controlling the high recessive resistance to Rice yellow mottle virus (RYMV) in the rice (Oryza sativa) variety Gigante. The locus was mapped to a 160-kb interval containing a gene from the eIF(iso)4G family. The stable transformation of a resistant line with the cDNA of this gene, derived from a susceptible variety, resulted in the loss of resistance in transgenic plants. The allelic variability of this gene was analysed in three resistant and 17 susceptible varieties from different cultivated rice species or subspecies. Compared with susceptible varieties, resistant varieties present specific alleles, characterized by either amino acid substitutions or short amino-acid deletions in the middle domain of the protein. The structure of this domain was modelled and showed that the substitutions were clustered on a small surface patch. This suggests that this domain may be involved in an interaction with the virus. Rymv1 Independent evolution of a new allele of F1 pollen sterility gene S27 encoding mitochondrial ribosomal protein L27 in Oryza nivara 2011 Theor Appl Genet Plant Breeding Laboratory, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581, Japan. Loss of function of duplicated genes plays an important role in the evolution of postzygotic reproductive isolation. The widespread occurrence of gene duplication followed by rapid loss of function of some of the duplicate gene copies suggests the independent evolution of loss-of-function alleles of duplicate genes in divergent lineages of speciation. Here, we found a novel loss-of-function allele of S27 in the Asian annual wild species Oryza nivara, designated S27-niv (s), that leads to F(1) pollen sterility in a cross between O. sativa and O. nivara. Genetic linkage analysis and complementation analysis demonstrated that S27-niv (s) lies at the same locus as the previously identified S27 locus and S27-niv (s) is a loss-of-function allele of S27. S27-niv (s) is composed of two tandem mitochondrial ribosomal protein L27 genes (mtRPL27a and mtRPL27b), both of which are inactive. The coding and promoter regions of S27-niv (s) showed a number of nucleotide differences from the functional S27-T65 (+) allele. The structure of S27-niv (s) is different from that of a previously identified null S27 allele, S27-glum (s), in the South American wild rice species O. glumaepatula, in which mtRPL27a and mtRPL27b are absent. These results show that the mechanisms for loss-of-function of S27-niv (s) and S27-glum (s) are different. Our results provide experimental evidence that different types of loss-of-function alleles are distributed in geographically and phylogenetically isolated species and represent a potential mechanism for postzygotic isolation in divergent species. S27 Mitochondrial gene in the nuclear genome induces reproductive barrier in rice 2010 Proc Natl Acad Sci U S A Faculty of Agriculture, Kyushu University, Hakozaki, Higashi, Fukuoka 812-8581, Japan. Hybrid incompatibility in F(1) hybrids or later generations is often observed as sterility or inviability. This incompatibility acts as postzygotic reproductive isolation, which results in the irreversible divergence of species. Here, we show that the reciprocal loss of duplicated genes encoding mitochondrial ribosomal protein L27 causes hybrid pollen sterility in F(1) hybrids of the cultivated rice Oryza sativa and its wild relative O. glumaepatula. Functional analysis revealed that this gene is essential for the later stage of pollen development, and distribution analysis suggests that the gene duplication occurred before the divergence of the AA genome species. On the basis of these results, we discuss the possible contribution of the "founder effect" in establishing this reproductive barrier. S27,S28 Molecular analysis of the inheritance of the S-5 locus, conferring wide compatibility in Indica/Japonica hybrids of rice (O. sativa L.) 1995 Theor Appl Genet Department of Plant Breeding and Biometry, Cornell University, 14853-1902, Ithaca, NY, USA. RFLP analysis was conducted on a population derived from a three-way cross to determine the location of the hybrid sterility locus, S-5, in relation to mapped molecular markers and to identify markers that would be useful for selection in breeding. S-5 is of interest to rice breeders because it is associated with spikelet sterility of F1 hybrids in Indica/Japonica crosses. Identification of an S-5 allele which confers fertility in Indica/Japonica hybrids when introgressed into either the Indica or the Japonica parent has been reported. Varieties carrying this S-5 (n) allele are known as "wide compatibility varieties (WCV)". Our data suggests that RFLP marker RG213 on chromosome 6 is closely linked to the S-5 locus and can be efficiently used to identify wide compatibility (WC) lines. RG213 is a single-copy genomic clone that detects three bands of different molecular weights in DNA from Japonica ('Akihikari') and Indica ('IR36') varieties and WC line ('Nekken 2'). We demonstrate that the three alleles detected by this marker could be used to trace the inheritance of the "wide compatible" phenotype in breeders' material. S5(ORF3),S5(ORF4),S5(ORF5) A triallelic system of S5 is a major regulator of the reproductive barrier and compatibility of indica-japonica hybrids in rice 2008 Proc Natl Acad Sci U S A National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China. Hybrid sterility is a major form of postzygotic reproductive isolation. Although reproductive isolation has been a key issue in evolutionary biology for many decades in a wide range of organisms, only very recently a few genes for reproductive isolation were identified. The Asian cultivated rice (Oryza sativa L.) is divided into two subspecies, indica and japonica. Hybrids between indica and japonica varieties are usually highly sterile. A special group of rice germplasm, referred to as wide-compatibility varieties, is able to produce highly fertile hybrids when crossed to both indica and japonica. In this study, we cloned S5, a major locus for indica-japonica hybrid sterility and wide compatibility, using a map-based cloning approach. We show that S5 encodes an aspartic protease conditioning embryo-sac fertility. The indica (S5-i) and japonica (S5-j) alleles differ by two nucleotides. The wide compatibility gene (S5-n) has a large deletion in the N terminus of the predicted S5 protein, causing subcellular mislocalization of the protein, and thus is presumably nonfunctional. This triallelic system has a profound implication in the evolution and artificial breeding of cultivated rice. Genetic differentiation between indica and japonica would have been enforced because of the reproductive barrier caused by S5-i and S5-j, and species coherence would have been maintained by gene flow enabled by the wide compatibility gene. S5(ORF3),S5(ORF4),S5(ORF5) Molecular basis underlying the S5-dependent reproductive isolation and compatibility of indica/japonica rice hybrids 2012 Plant Physiol National Maize Improvement Center of China, China Agricultural University, Beijing 100193, People's Republic of China. The S5 locus regulates spikelet fertility of indica/japonica hybrid rice (Oryza sativa). There are three alleles at the S5 locus, including an indica allele (S5i), a japonica allele (S5j), and a wide-compatibility allele (S5n). This study analyzed the molecular basis for S5-dependent reproductive isolation and compatibility of indica/japonica rice hybrids. Three S5 alleles were expressed at extremely low levels, and only in the ovary. S5n was more similar to S5i in both RNA and protein expression profiles. The S5 locus was not essential for embryo sac development, although deleterious interactions between S5i and S5j resulted in reduced rates of spikelet fertility. The yeast two-hybrid system was used to test direct interactions between S5-encoded proteins. The results indicated that the S5i- and S5j-encoded eukaryotic aspartyl proteases formed both homodimers and heterodimers, whereas the S5n-encoded aspartyl protease was incapable of dimerization. Site-directed mutagenesis revealed that a single amino acid difference between S5i- and S5j-encoded aspartyl proteases (phenylalanine/leucine at residue 273) was primarily responsible for embryo sac abortion. The S5 locus may have promoted the subspeciation of indica and japonica, but it also enables gene flow between them. S5(ORF3),S5(ORF4),S5(ORF5) A genome-wide analysis of wide compatibility in rice and the precise location of the S 5 locus in the molecular map 1997 TAG Theoretical and Applied Genetics National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China, CN The discovery of wide-compatibility varieties (WCVs) that are able to produce normal fertility hybrids when crossed both to indica and japonica rice has enabled the fertility barrier between indica and japonica subspecies to be broken and provided the possibility of developing inter-subspecific hybrids in rice breeding programs. However, a considerable variation in the fertility level of hybrids from the same WCV crossed to different varieties has often been observed. One hypothesis for this variable fertility is that additional genes are involved in hybrid fertility besides the wide-compatibility gene (WCG). To assess such a possibility, we performed a genome-wide analysis by assaying a large population from a three-way cross ‘02428’/‘Nanjing 11’//‘Balilla’ using a total of 171 RFLP probes detecting 191 polymorphic loci distributed throughout the entire rice linkage map. Our analysis recovered 3 loci conferring significant effects on hybrid fertility. The major locus on chromosome 6 coincided in chromosomal location with the previously identified S 5 locus, and the 2 minor loci that mapped to chromosomes 2 and 12, respectively, were apparently distinct from all previously reported hybrid sterility genes. Interaction between the indica and japonica alleles at each of the loci caused a reduction in hybrid fertility. The joint effect of the 2 minor loci could lead to partial sterility even in the presence of the WCG. The location of the S 5 locus on the molecular marker linkage map was determined to be approximately 1.0 cM from the RFLP locus R2349. This tight linkage will be useful for marker-aided transfer of the WCG in hybrid rice breeding and for map-based cloning. S5(ORF3),S5(ORF4),S5(ORF5) Delimitation of the rice wide compatibility gene S5 ( n ) to a 40-kb DNA fragment 2005 Theor Appl Genet National Key Laboratory of Crop Genetic Improvement, and National Center of Plant Gene Research-Wuhan, Huazhong Agricultural University, Wuhan 430070, China. Wide compatibility varieties (WCVs) are a special class of rice (Oryza sativa L.) germplasm that produces hybrids with normal pollen and spikelet fertility when crossed with both indica and japonica subspecies. The wide compatibility gene S5 ( n ) has been used extensively in inter-subspecific hybrid breeding programs. We previously mapped the S5 locus to a 2.2-cM genomic region between RM253 and R2349 on chromosome 6, using a population of 356 F(1) plants derived from the three-way cross 02428/Nanjing11//Balilla. In this study, a chromosome walking strategy was employed to construct a physical map covering this genomic region using these two closest markers as the starting points. A physical map consisting of six overlapping BAC clones was formed, spanning a genomic region of 540-kb in length. By analyzing recombination events from a population of 8,000 F(1) plants derived from a three-way cross based on near isogenic lines of the S5 locus, the S5 locus was localized to a DNA fragment of 40-kb in length, flanked by two shotgun subclones, 7B1 and 15D2. Sequence analysis of this fragment predicted five open reading frames, encoding xyloglucan fucosyltransferases, dnak-type molecular chaperone BiP, a putative eukaryotic aspartyl protease, and a hypothetical protein. This result will be very useful in molecular cloning of the S5 ( n ) allele and marker-assisted transferring of the wide compatibility gene in rice breeding programs. S5(ORF3),S5(ORF4),S5(ORF5) Complex evolution of S5, a major reproductive barrier regulator, in the cultivated rice Oryza sativa and its wild relatives 2011 New Phytol National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China. * The hybrid sterility gene S5 comprises three types of alleles in cultivated rice. Such tri-allelic system provided a unique opportunity to study the molecular bases of evolutionary changes underlying reproductive isolation in plants. * We analysed the sequence diversity and evolutionary history of S5 in 138 Oryza accessions. We also examined the effect of the two functional variations (C819A and C1412T) in determining hybrid sterility by transformation. * Nineteen haplotypes were identified, which were classified into the indica-like, the japonica-like and the wide-compatibility gene (WCG)-like group, according to the sequence features of the tri-allelic system. The origin and evolutionary course of the three allelic groups were investigated, thus confirming the independent origins of indica and japonica subspecies. There were perfect associations between C819A and C1412T in the rice germplasm assayed, and the combination of C819 and C1412 was required for hybrid sterility. Evidence of positive selection in the WCG-like alleles suggested that they might have been favored by selection for higher compatibility in hybrids. * The complex evolution of S5 revealed the counteractive function of the three allelic groups at the species level. S5 might perform an important primary function in an evolutionary scale, and hybrid sterility acts as a 'byproduct' of this speciation gene. S5(ORF3),S5(ORF4),S5(ORF5) A killer-protector system regulates both hybrid sterility and segregation distortion in rice 2012 Science National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China. Hybrid sterility is a major form of postzygotic reproductive isolation that restricts gene flow between populations. Cultivated rice (Oryza sativa L.) consists of two subspecies, indica and japonica; inter-subspecific hybrids are usually sterile. We show that a killer-protector system at the S5 locus encoded by three tightly linked genes [Open Reading Frame 3 (ORF3) to ORF5] regulates fertility in indica-japonica hybrids. During female sporogenesis, the action of ORF5+ (killer) and ORF4+ (partner) causes endoplasmic reticulum (ER) stress. ORF3+ (protector) prevents ER stress and produces normal gametes, but ORF3- cannot prevent ER stress, resulting in premature programmed cell death and leads to embryo-sac abortion. Preferential transmission of ORF3+ gametes results in segregation distortion in the progeny. These results add to our understanding of differences between indica and japonica rice and may aid in rice genetic improvement. S5(ORF3),S5(ORF4),S5(ORF5) Hybrid male sterility in rice controlled by interaction between divergent alleles of two adjacent genes 2008 Proc Natl Acad Sci U S A Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Province, College of Life Sciences, South China Agricultural University, Wushan, Guangzhou 510642, China. Sterility is common in hybrids between divergent populations, such as the indica and japonica subspecies of Asian cultivated rice (Oryza sativa). Although multiple loci for plant hybrid sterility have been identified, it remains unknown how alleles of the loci interact at the molecular level. Here we show that a locus for indica-japonica hybrid male sterility, Sa, comprises two adjacent genes, SaM and SaF, encoding a small ubiquitin-like modifier E3 ligase-like protein and an F-box protein, respectively. Most indica cultivars contain a haplotype SaM(+)SaF(+), whereas all japonica cultivars have SaM(-)SaF(-) that diverged by nucleotide variations in wild rice. Male semi-sterility in this heterozygous complex locus is caused by abortion of pollen carrying SaM(-). This allele-specific gamete elimination results from a selective interaction of SaF(+) with SaM(-), a truncated protein, but not with SaM(+) because of the presence of an inhibitory domain, although SaM(+) is required for this male sterility. Lack of any one of the three alleles in recombinant plants does not produce male sterility. We propose a two-gene/three-component interaction model for this hybrid male sterility system. The findings have implications for overcoming male sterility in inter-subspecific hybrid rice breeding. SaF,SaM Characterization of a rice gene showing organ-specific expression in response to salt stress and drought 1990 Plant Cell Laboratorium voor Genetica, Rijksuniversiteit Gent, Belgium. Protein changes induced by salinity stress were investigated in the roots of the salt-sensitive rice cultivar Taichung native 1. We found eight proteins to be induced and obtained partial sequences of one with a molecular mass of 15 kilodaltons and an isoelectric point of 5.5. Using an oligonucleotide probe based on this information, a cDNA clone, salT, was selected and found to contain an open reading frame coding for a protein of 145 amino acid residues. salT mRNA accumulates very rapidly in sheaths and roots from mature plants and seedlings upon treatment with Murashige and Skoog salts (1%), air drying, abscisic acid (20 microM), polyethylene glycol (5%), sodium chloride (1%), and potassium chloride (1%). Generally, no induction was seen in the leaf lamina even when the stress should affect all parts of the plant uniformly. The organ-specific response of salT is correlatable with the pattern of Na+ accumulation during salt stress. SALT The submergence tolerance regulator SUB1A mediates crosstalk between submergence and drought tolerance in rice 2011 Plant Cell Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California, Riverside, California 92521, USA. Submergence and drought are major constraints to rice (Oryza sativa) production in rain-fed farmlands, both of which can occur sequentially during a single crop cycle. SUB1A, an ERF transcription factor found in limited rice accessions, dampens ethylene production and gibberellic acid responsiveness during submergence, economizing carbohydrate reserves and significantly prolonging endurance. Here, we evaluated the functional role of SUB1A in acclimation to dehydration. Comparative analysis of genotypes with and without SUB1A revealed that SUB1A enhanced recovery from drought at the vegetative stage through reduction of leaf water loss and lipid peroxidation and increased expression of genes associated with acclimation to dehydration. Overexpression of SUB1A augmented ABA responsiveness, thereby activating stress-inducible gene expression. Paradoxically, vegetative tissue undergoes dehydration upon desubmergence even though the soil contains sufficient water, indicating that leaf desiccation occurs in the natural progression of a flooding event. Desubmergence caused the upregulation of gene transcripts associated with acclimation to dehydration, with higher induction in SUB1A genotypes. SUB1A also restrained accumulation of reactive oxygen species (ROS) in aerial tissue during drought and desubmergence. Consistently, SUB1A increased the abundance of transcripts encoding ROS scavenging enzymes, resulting in enhanced tolerance to oxidative stress. Therefore, in addition to providing robust submergence tolerance, SUB1A improves survival of rapid dehydration following desubmergence and water deficit during drought. SALT,Sub1A,Sub1B Exogenous application of brassinosteroid offers tolerance to salinity by altering stress responses in rice variety Pusa Basmati-1 2013 Plant Physiol Biochem Department of Biotechnology, Guru Nanak Dev University, Amritsar 143005, Punjab, India. ishashrikhand@yahoo.com Plant steroidal hormones, brassinosteroids, play a pivotal role in variety of plant developmental processes and adaptation to various environmental stresses. The present work investigates the response of various stress markers upon exogenous application of 24-epibrassinolide (EBL) on Pusa Basmati-1, a commercially important rice variety, under salt stress conditions. Rice seeds after treatment with different concentrations of NaCl alone or in combination with different concentrations of 24-epibrassinolide (EBL) were analysed for various growth parameters, protein, proline and malondialdehyde content (MDA) and antioxidant enzymes activities. The seedlings exposed to NaCl exhibited a significant decline in growth parameters and changes in the levels of antioxidant enzymes, however, treatment with EBL showed an improvement in growth, levels of protein and proline content and antioxidant enzymes activity. The enhanced levels of MDA content during salt stress in rice seedlings was decreased with EBL treatment. Further, the treatment with EBL increased the expression of various oxidative stress marker genes, although to different levels. Expression of various brassinosteroids (OsBRI1, OsDWF4) and salt (SalT) responsive genes, revealed the down regulation of OsDWF4 with application of EBL and upregulation of SalT in presence of salt stress thereby confirming the efficacy of the treatments. Interestingly, a significant down regulation of SalT gene was observed on application of EBL along with salt compared to salt treatment alone. On the other hand, the application of EBL alone and in combination with salt has resulted in upregulation of OsBRI1. SALT Identification of sucrose-regulated genes in cultured rice cells using mRNA differential display 1995 Gene Institute of Life Science, National Tsing-Hua University, Hsinchu, Taiwan 300, ROC In order to get more information about carbon metabolite regulation pathways, cloning and sequence analysis of sucrose-regulated genes from rice-suspension-cultured cells were performed. We used a new method, mRNA differential display, to screen differentially expressed genes under conditions of 3% and no sucrose in the cultured medium. Six candidate clones were identified and sequenced. Clones SI1 and SI2 were repressed by sucrose starvation, while clones SR1, SR2, SR3 and SR4 were induced by sucrose starvation. Nucleotide sequence analysis showed that clone SR2 has 94.8% homology to the salT gene, and clones SI1 and SR3 show 88.3 and 96.9% identity, respectively, to partial cDNA sequences in the GenBank database. The results suggest that mRNA differential display provides an easy and quick way to clone genes involved in the carbon metabolite regulation pathway. SALT Differential Activation of the Rice Sucrose Nonfermenting1-Related Protein Kinase2 Family by Hyperosmotic Stress and Abscisic Acid 2004 The Plant Cell Online Bioscience and Biotechnology Center, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan To date, a large number of sequences of protein kinases that belong to the sucrose nonfermenting1–related protein kinase2 (SnRK2) family are found in databases. However, only limited numbers of the family members have been characterized and implicated in abscisic acid (ABA) and hyperosmotic stress signaling. We identified 10 SnRK2 protein kinases encoded by the rice (Oryza sativa) genome. Each of the 10 members was expressed in cultured cell protoplasts, and its regulation was analyzed. Here, we demonstrate that all family members are activated by hyperosmotic stress and that three of them are also activated by ABA. Surprisingly, there were no members that were activated only by ABA. The activation was found to be regulated via phosphorylation. In addition to the functional distinction with respect to ABA regulation, dependence of activation on the hyperosmotic strength was different among the members. We show that the relatively diverged C-terminal domain is mainly responsible for this functional distinction, although the kinase domain also contributes to these differences. The results indicated that the SnRK2 protein kinase family has evolved specifically for hyperosmotic stress signaling and that individual members have acquired distinct regulatory properties, including ABA responsiveness by modifying the C-terminal domain. SAPK1,SAPK10,SAPK2,SAPK3|REK,SAPK4|OSPDK,SAPK5,SAPK6|OSRK1,SAPK7,SAPK8,SAPK9 Abscisic acid-activated SNRK2 protein kinases function in the gene-regulation pathway of ABA signal transduction by phosphorylating ABA response element-binding factors 2005 Plant J Bioscience and Biotechnology Center, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan. The plant hormone abscisic acid (ABA) induces gene expression via the ABA-response element (ABRE) present in the promoters of ABA-regulated genes. A group of bZIP proteins have been identified as ABRE-binding factors (ABFs) that activate transcription through this cis element. A rice ABF, TRAB1, has been shown to be activated via ABA-dependent phosphorylation. While a large number of signalling factors have been identified that are involved in stomatal regulation by ABA, relatively less is known about the ABA-signalling pathway that leads to gene expression. We have shown recently that three members of the rice SnRK2 protein kinase family, SAPK8, SAPK9 and SAPK10, are activated by ABA signal as well as by hyperosmotic stress. Here we show that transient overexpression in cultured cell protoplasts of these ABA-activated SnRK2 protein kinases leads to the activation of an ABRE-regulated promoter, suggesting that these kinases are involved in the gene-regulation pathway of ABA signalling. We further show several lines of evidence that these ABA-activated SnRK2 protein kinases directly phosphorylate TRAB1 in response to ABA. Kinetic analysis of SAPK10 activation and TRAB1 phosphorylation indicated that the latter immediately followed the former. TRAB1 was found to be phosphorylated not only in response to ABA, but also in response to hyperosmotic stress, which was interpreted as the consequence of phosphorylation of TRAB1 by hyperosmotically activated SAPKs. Physical interaction between TRAB1 and SAPK10 in vivo was demonstrated by a co-immunoprecipitation experiment. Finally, TRAB1 was phosphorylated in vitro by the ABA-activated SnRK2 protein kinases at Ser102, which is phosphorylated in vivo in response to ABA and is critical for the activation function. SAPK10,SAPK8,SAPK9 SAUR39, a small auxin-up RNA gene, acts as a negative regulator of auxin synthesis and transport in rice 2009 Plant Physiol Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1. The phytohormone auxin plays a critical role for plant growth by regulating the expression of a set of genes. One large auxin-responsive gene family of this type is the small auxin-up RNA (SAUR) genes, although their function is largely unknown. The expression of the rice (Oryza sativa) SAUR39 gene showed rapid induction by transient change in different environmental factors, including auxin, nitrogen, salinity, cytokinin, and anoxia. Transgenic rice plants overexpressing the SAUR39 gene resulted in lower shoot and root growth, altered shoot morphology, smaller vascular tissue, and lower yield compared with wild-type plants. The SAUR39 gene was expressed at higher levels in older leaves, unlike auxin biosynthesis, which occurs largely in the meristematic region. The transgenic plants had a lower auxin level and a reduced polar auxin transport as well as the down-regulation of some putative auxin biosynthesis and transporter genes. Biochemical analysis also revealed that transgenic plants had lower chlorophyll content, higher levels of anthocyanin, abscisic acid, sugar, and starch, and faster leaf senescence compared with wild-type plants at the vegetative stage. Most of these phenomena have been shown to be negatively correlated with auxin level and transport. Transcript profiling revealed that metabolic perturbations in overexpresser plants were largely due to transcriptional changes of genes involved in photosynthesis, senescence, chlorophyll production, anthocyanin accumulation, sugar synthesis, and transport. The lower growth and yield of overexpresser plants was largely recovered by exogenous auxin application. Taken together, the results suggest that SAUR39 acts as a negative regulator for auxin synthesis and transport. SAUR39 Crystal structure of the rice branching enzyme I (BEI) in complex with maltopentaose 2012 Biochem Biophys Res Commun Laboratory of Structural Biology, Graduate School of Systems Life Sciences, Kyushu University, Hakozaki 6-10-1, Fukuoka 812-8581, Japan. Starch branching enzyme (SBE) catalyzes the cleavage of alpha-1,4-linkages and the subsequent transfer of alpha-1,4 glucan to form an alpha-1,6 branch point in amylopectin. We determined the crystal structure of the rice branching enzyme I (BEI) in complex with maltopentaose at a resolution of 2.2A. Maltopentaose bound to a hydrophobic pocket formed by the N-terminal helix, carbohydrate-binding module 48 (CBM48), and alpha-amylase domain. In addition, glucose moieties could be observed at molecular surfaces on the N-terminal helix (alpha2) and CBM48. Amino acid residues involved in the carbohydrate bindings are highly conserved in other SBEs, suggesting their generally conserved role in substrate binding for SBEs. BEI|SBE1 Interaction of rice bZIP protein REB with the 5'- upstream re-gion of both rice sbe1 gene and waxy gene 2002 Chinese Science Bulletin State Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 200032, Shanghai, China Rice starch branching enzyme 1 (SBE1) and granule-bound starch synthase (GBSS) catalyzed the biosynthesis of amylopectin and amylose in rice developing endosperm respectively, and the genes encoding these two enzymes, sbe1 and waxy, are mainly expressed in the endosperm. Within the 5'-upstream region of rice sbe1 gene, we identified a 53 bp fragment C53 which could interact with the nuclear proteins extracted from rice endosperm. We also found that fragment Ha-2 in the 5'-upstream region of rice waxy gene could compete with C53 for its interaction. Further experiments demonstrated that rice bZIP protein REB could interact with two ACGT elements (G-box and Hex) in the fragment C53 as well as three ACGT elements (WG1, WG2, and WG3) in the fragment Ha-2. The WG1 element could compete well with Hex element for its interaction with REB. These results suggested that the expression of sbe1 and waxy gene in rice developing endosperm may be coordinately regulated by REB-like bZIP transcriptional factors. BEI|SBE1 Biochemical and Crystallographic Characterization of the Starch Branching Enzyme I (BEI) fromOryza sativaL 2014 Bioscience, Biotechnology and Biochemistry Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan. Starch branching enzyme (SBE) catalyzes the cleavage of alpha-1.4-linkages and the subsequent transfer of alpha-1.4 glucan to form an alpha-1.6 branch point in amylopectin. We overproduced rice branching enzyme I (BEI) in Escherichia coli cells, and the resulting enzyme (rBEI) was characterized with respect to biochemical and crystallographic properties. Specific activities were calculated to be 20.8 units/mg and 2.5 units/mg respectively when amylose and amylopectin were used as substrates. Site-directed mutations of Tyr235, Asp270, His275, Arg342, Asp344, Glu399, and His467 conserved in the alpha-amylase family enzymes drastically reduced catalytic activity of rBEI. This result suggests that the structures of BEI and the other alpha-amylase family enzymes are similar and that they share common catalytic mechanisms. Crystals of rBEI were grown under appropriate conditions and the crystals diffracted to a resolution of 3.0 A on a synchrotron X-ray source. BEI|SBE1 The Action of Rice Branching Enzyme I (BEI) on Starches 2014 Bioscience, Biotechnology and Biochemistry Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan. The rice branching enzyme I (BEI) overproduced in Escherichia coli cells was investigated with respect to action on starches. BEI treatment decreased the turbidity of starch suspensions with distinct pasting behaviors from a native starch. This result suggests the great potential of BEI as a molecular tool for the production of a novel glucan polymer. BEI|SBE1 cDNA Cloning and Sequence Analysis of Rice Sbe1 and Sbe3 Genes 2004 RICE SCIENCE Agricultural College, Yangzhou University, Yangzhou 225009, China Two starch-branching enzyme (SBE) in rice, is known to be a key enzyme in amylopectin biosynthesis. The cDNA of two SBE(starch-branching enzyme) genes Sbe1 and Sbe3 encoding SBE I and SBE III (two major isoforms in rice) were cloned by an improved RT-PCR technique, from a template cDNA library derived from the total mRNAs extracted from the immature seeds of a japonica rice Wuyunjing 7. DNA sequence analysis showed that the size of the cloned Sbe1 and Sbe3 cDNAs were 2490 and 2481 bp long, respectively, including their entire coding sequences. Comparison analysis indicated that the nucleotide sequence of Sbe3 was the same as that of sbe3 (Genbank Accession No. D16201) as reported previously. There were only four base-pairs difference, which resulted in changes of two deduced amino acids between the cloned Sbe1 cDNA and the reported sbe1 (Genbank Accession No. D11082). The cloned Sbe1 and Sbe3 cDNAs make it possible to improve rice starch quality through genetic engineering. BEI|SBE1 Green revolution: a mutant gibberellin-synthesis gene in rice 2002 Nature Bioscience Center, Nagoya University, Nagoya 464-8601, Japan. makoto@nuagr1.agr.nagoya-u.ac.jp The chronic food shortage that was feared after the rapid expansion of the world population in the 1960s was averted largely by the development of a high-yielding semi-dwarf variety of rice known as IR8, the so-called rice 'green revolution'. The short stature of IR8 is due to a mutation in the plant's sd1 gene, and here we identify this gene as encoding an oxidase enzyme involved in the biosynthesis of gibberellin, a plant growth hormone. Gibberellin is also implicated in green-revolution varieties of wheat, but the reduced height of those crops is conferred by defects in the hormone's signalling pathway. sd1|GA20ox2 Positional Cloning of Rice Semidwarfing Gene, sd-1: Rice "Green Revolution Gene" Encodes a Mutant Enzyme Involved in Gibberellin Synthesis 2002 DNA Research Plant Genome Center 1-25-2 Kannondai, Tsukuba, Ibaraki 305-0856, Japan A rice semidwarfing gene, sd-1, known as the “green revolution gene" was isolated by positional cloning and revealed to encode gibberellin 20-oxidase, the key enzyme in the gibberellin biosynthesis pathway. Analysis of 3477 segregants using several PCR-based marker technologies, including cleaved amplified polymorphic sequence, derived-CAPS, and single nucleotide polymorphisms revealed 1 ORF in a 6-kb candidate interval. Normal-type rice cultivars have an identical sequence in this region, consisting of 3 exons (558, 318, and 291 bp) and 2 introns (105 and 1471 bp). Dee-Geo-Woo-Gen-type sd-1 mutants have a 383-bp deletion from the genome (278-bp deletion from the expressed sequence), from the middle of exon 1 to upstream of exon 2, including a 105-bp intron, resulting in a frame-shift that produces a termination codon after the deletion site. The radiation-induced sd-1 mutant Calrose 76 has a 1-bp substitution in exon 2, causing an amino acid substitution (Leu [CTC] to Phe [TTC]). Expression analysis suggests the existence of at least one more locus of gibberellin 20-oxidase which may prevent severe dwarfism from developing in sd-1 mutants. sd1|GA20ox2 Semidwarf (sd-1), "green revolution" rice, contains a defective gibberellin 20-oxidase gene 2002 Proc Natl Acad Sci U S A Division of Plant Industry, Commonwealth Scientific and Industrial Research Organization, GPO Box 1600, Canberra ACT 2601, Australia. wolfgang.spielmeyer@csiro.au The introduction of semidwarf rice (Oryza sativa L.) led to record yield increases throughout Asia in the 1960s. The major semidwarfing allele, sd-1, is still extensively used in modern rice cultivars. The phenotype of sd-1 is consistent with dwarfism that results from a deficiency in gibberellin (GA) plant growth hormones. We propose that the semidwarf (sd-1) phenotype is the result of a deficiency of active GAs in the elongating stem arising from a defective 20-oxidase GA biosynthetic enzyme. Sequence data from the rice genome was combined with previous mapping studies to locate a putative GA 20-oxidase gene (Os20ox2) at the predicted map location of sd-1 on chromosome 1. Two independent sd-1 alleles contained alterations within Os20ox2: a deletion of 280 bp within the coding region of Os20ox2 was predicted to encode a nonfunctional protein in an indica type semidwarf (Doongara), whereas a substitution in an amino acid residue (Leu-266) that is highly conserved among dioxygenases could explain loss of function of Os20ox2 in a japonica semidwarf (Calrose76). The quantification of GAs in elongating stems by GC-MS showed that the initial substrate of GA 20-oxidase activity (GA53) accumulated, whereas the content of the major product (GA20) and of bioactive GA1 was lower in semidwarf compared with tall lines. We propose that the Os20ox2 gene corresponds to the sd-1 locus. sd1|GA20ox2 The endoplasmic reticulum-quality control component SDF2 is essential for XA21-mediated immunity in rice 2013 Plant Sci Department of Plant Pathology and The Genome Center, University of California Davis, Davis, CA, United States. Plant genomes contain large number of plasma membrane (PM)-localized immune receptors, also called pattern recognition receptors (PRRs). PRRs are synthesized in the endoplasmic reticulum (ER) and then translocated to the PM, where they recognize conserved pathogen-associated molecular patterns (PAMPs) and activate innate immune response. The rice XA21 immune receptor confers resistance to the Gram-negative bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo). To identify components that mediate XA21-mediated signaling, we performed co-purification experiments using C-terminal GFP tagged XA21 protein. Several endoplasmic reticulum-quality control (ER-QC) proteins including stromal-derived factor 2 (SDF2) co-purified with XA21. Silencing of the SDF2 genes in the XA21 rice genetic background compromises resistance to Xoo but does not affect plant growth and development. OsSDF2-1,OsSDF2-2,xa21 SDG714 regulates specific gene expression and consequently affects plant growth via H3K9 dimethylation 2010 J Integr Plant Biol State Key Laboratory of Genetic Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China. Histone lysine methylation is known to be involved in the epigenetic regulation of gene expression in all eukaryotes including plants. Here we show that the rice SDG714 is primarily responsible for dimethylation but not trimethylation on histone H3K9 in vivo. Overexpression of YFP-SDG714 in Arabidopsis significantly inhibits plant growth and this inhibition is associated with an enhanced level of H3K9 dimethylation. Our microarray results show that many genes essential for the plant growth and development were downregulated in transgenic Arabidopsis plants overexpressing YFP-SDG714. By chromatin immunoprecipitation analysis, we show that YFP-SDG714 is targeted to specific chromatin regions and dimethylate the H3K9, which is linked with heterochromatinization and the downregulation of genes. Most interestingly, when YFP-SDG714 production is stopped, the inhibited plants can partially restore their growth, suggesting that the perturbation of gene expression caused by YFP-SDG714 is revertible. Taken together, our results point to an important role of SDG714 in H3K9 dimethylation, suppression of gene expression and plant growth, and provide a potential method to regulate gene expression and plant development by an on-off switch of SDG714 expression. SDG714 Rice SUVH histone methyltransferase genes display specific functions in chromatin modification and retrotransposon repression 2010 Mol Plant National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, 430070 Wuhan, China. Histone lysine methylation plays an important role in heterochromatin formation and reprogramming of gene expression. SET-domain-containing proteins are shown to have histone lysine methyltransferase activities. A large number of SET-domain genes are identified in plant genomes. The function of most SET-domain genes is not known. In this work, we studied the 12 rice (Oryza sativa) homologs of Su(var)3-9, the histone H3 lysine 9 (H3K9) methyltransferase identified in Drosophila. Several rice SUVHs (i.e. SDG714, SDG727, and SDG710) were found to have an antagonistic function to the histone H3K9 demethylase JMJ706, as down-regulation of these genes could partially complement the jmj706 phenotype and reduced histone H3K9 methylation. Down-regulation of a rice Su(var)3-9 homolog (SUVH), namely SDG728, decreased H3K9 methylation and altered seed morphology. Overexpression of the gene increased H3K9 methylation. SDG728 and other SUVH genes were found to be involved in the repression of retrotransposons such as Tos17 and a Ty1-copia element. Analysis of histone methylation suggested that SDG728-mediated H3K9 methylation may play an important role in retrotransposon repression. SDG714,OsSET22|SDG728|OsSUVH8 SDG714, a histone H3K9 methyltransferase, is involved in Tos17 DNA methylation and transposition in rice 2007 Plant Cell State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Although the role of H3K9 methylation in rice (Oryza sativa) is unclear, in Arabidopsis thaliana the loss of histone H3K9 methylation by mutation of Kryptonite [also known as SU(VAR)3-9 homolog] reduces genome-wide DNA methylation and increases the transcription of transposable elements. Here, we report that rice SDG714 (for SET Domain Group Protein714) encodes a histone H3K9-specific methyltransferase. The C terminus of SDG714 confers enzymatic activity and substrate specificity, whereas the N terminus localizes it in the nucleus. Loss-of-function mutants of SDG714 (SDG714IR transformants) generated by RNA interference display a mostly glabrous phenotype as a result of the lack of macro trichomes in glumes, leaves, and culms compared with control plants. These mutants also show decreased levels of CpG and CNG cytosine methylation as well as H3K9 methylation at the Tos17 locus, a copia-like retrotransposon widely used for the generation of rice mutants. Most interestingly, loss of function of SDG714 can enhance transcription and cause the transposition of Tos17. Together, these results suggest that histone H3K9 methylation mediated by SDG714 is involved in DNA methylation, the transposition of transposable elements, and genome stability in rice. SDG714 H3K36 Methylation Is Involved in Promoting Rice Flowering 2013 Mol Plant State Key Laboratory of Genetic Engineering, Institute of Plant Biology, Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai 200433, PR China None SDG725 Genome-wide identification, phylogenetic and co-expression analysis of OsSET gene family in rice 2013 PLoS One College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan, PR China. BACKGROUND: SET domain is responsible for the catalytic activity of histone lysine methyltransferases (HKMTs) during developmental process. Histone lysine methylation plays a crucial and diverse regulatory function in chromatin organization and genome function. Although several SET genes have been identified and characterized in plants, the understanding of OsSET gene family in rice is still very limited. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a systematic analysis was performed and revealed the presence of at least 43 SET genes in rice genome. Phylogenetic and structural analysis grouped SET proteins into five classes, and supposed that the domains out of SET domain were significant for the specific of histone lysine methylation, as well as the recognition of methylated histone lysine. Based on the global microarray, gene expression profile revealed that the transcripts of OsSET genes were accumulated differentially during vegetative and reproductive developmental stages and preferentially up or down-regulated in different tissues. Cis-elements identification, co-expression analysis and GO analysis of expression correlation of 12 OsSET genes suggested that OsSET genes might be involved in cell cycle regulation and feedback. CONCLUSIONS/SIGNIFICANCE: This study will facilitate further studies on OsSET family and provide useful clues for functional validation of OsSETs. OsSET22|SDG728|OsSUVH8,SDG736|OsSET9 Genome-wide analysis of cyclin family in rice (Oryza Sativa L.) 2006 Mol Genet Genomics Rice Functional Genomics, Joint Laboratory of Temasek Life Sciences Laboratory of Singapore and Institute of Genetics and Developmental Biology, The Chinese Academy of Sciences, 100101, Beijing, China. The cyclins together with highly conserved cyclin-dependent kinases regulate cell cycle progression in plants. Although extensive and systematic study on cell cycle mechanisms and cyclin functions in yeasts and animals has been carried out, only a small number of plant cyclins have been characterized and classified functionally and phylogenetically. We identified several types of cyclin genes in the rice genome and characterized them by phylogenetic, tandem and segmental duplications analyses. Our results indicated that there were at least 49 predicted rice cyclin genes in the rice genome, and they were distributed on 12 chromosomes. Of these cyclins, one possessed only cyclin_C domain and no cyclin_N domain, and the remaining 48 cyclins with cyclin_N domains were classified as nine types based on evolutionary relationships. Eight of these nine types were common between rice and Arabidopsis, whereas only one, known as F-type cyclins, was unique to rice. No homologues of the F-type cyclins in plants could be retrieved from the public databases, and reverse transcription-PCR analysis supported an existence of the F-type cyclin genes. Sequence alignment suggested that the cyclin genes in the rice genome experienced a mass of gene tandem and segmental duplications occurred on seven chromosomes related to the origins of new cyclin genes. Our study provided an opportunity to facilitate assessment and classification of new members, serving as a guide for further functional elucidation of rice cyclins. SDS Complete loss of photoperiodic response in the rice mutant line X61 is caused by deficiency of phytochrome chromophore biosynthesis gene 2011 Theor Appl Genet Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyou, Kyoto, 606-8502, Japan. In rice (Oryza sativa), a short-day plant, photoperiod is the most favorable external signal for floral induction because of the constant seasonal change throughout the years. Compared with Arabidopsis, however, a large part of the regulation mechanism of the photoperiodic response in rice still remains unclear due mainly to the lack of induced mutant genes. An induced mutant line X61 flowers 35 days earlier than its original variety Gimbozu under a natural photoperiod in Kyoto (35 degrees 01'N). We attempted to identify the mutant gene conferring early heading to X61. Experimental results showed that the early heading of X61 was conferred by a complete loss of photoperiodic response due to a novel single recessive mutant gene se13. This locus interacts with two crucial photoperiod sensitivity loci, Se1 and E1. Wild type alleles at these two loci do not function in coexistence with se13 in a homozygous state, suggesting that Se13 is an upstream locus of the Se1 and E1 loci. Linkage analysis showed that Se13 is located in a 110 kb region between the two markers, INDEL3735_1 and INDEL3735_3 on chromosome 1. A database search suggested that the Se13 gene is identical to AK101395 (=OsHY2), which encodes phytochromobilin synthase, a key enzyme in phytochrome chromophore biosynthesis. Subsequent sequence analysis revealed that X61 harbors a 1 bp insertion in exon 1 of OsHY2, which induces a frame-shift mutation producing a premature stop codon. It is therefore considered that the complete loss of photoperiodic response of X61 is caused by a loss of function of the Se13 (OsHY2) gene involved in phytochrome chromophore biosynthesis. Se13|OsHY2 Occurrence of D-serine in rice and characterization of rice serine racemase 2009 Phytochemistry Department of Life Science and Biotechnology, Faculty of Chemistry, Materials, and Bioengineering, Kansai University, 3-3-35 Yamate-Cho, Suita, Osaka-Fu 564-8680, Japan. Germinated, unpolished rice was found to contain a substantial amount of D-serine, with the ratio of the D-enantiomer to the L-enantiomer being higher for serine than for other amino acids. The relative amount of D-serine (D/(D+L)%) reached approximately 10% six days after germination. A putative serine racemase gene (serr, clone No. 001-110-B03) was found in chromosome 4 of the genomic DNA of Oryza sativa L. ssp. Japonica cv. Nipponbare. This was expressed as serr in Escherichia coli and its gene product (SerR) was purified to apparent homogeneity. SerR is a homodimer with a subunit molecular mass of 34.5kDa, and is highly specific for serine. In addition to a serine racemase reaction, SerR catalyzes D- and L-serine dehydratase reactions, for which the specific activities were determined to be 2.73 and 1.42nkatal/mg, respectively. The optimum temperature and pH were respectively determined for the racemase reaction (35 degrees C and pH9.0) and for the dehydratase reaction (35 degrees C and pH9.5). SerR was inhibited by PLP-enzyme inhibitors. ATP decreased the serine racemase activity of SerR but increased the serine dehydratase activity. Kinetic analysis showed that Mg(2+) increases the catalytic efficiency of the serine racemase activity of SerR and decreases that of the serine dehydratase activity. Fluorescence-quenching analysis of the tryptophan residues in SerR indicated that the structure of SerR is distorted by the addition of Mg(2+), and this structural change probably regulates the two enzymatic activities. SerR Short grain1 decreases organ elongation and brassinosteroid response in rice 2012 Plant Physiol National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. We identified a short-grain mutant (Short grain1 (Sg1) Dominant) via phenotypic screening of 13,000 rice (Oryza sativa) activation-tagged lines. The causative gene, SG1, encodes a protein with unknown function that is preferentially expressed in roots and developing panicles. Overexpression of SG1 in rice produced a phenotype with short grains and dwarfing reminiscent of brassinosteroid (BR)-deficient mutants, with wide, dark-green, and erect leaves. However, the endogenous BR level in the SG1 overexpressor (SG1:OX) plants was comparable to the wild type. SG1:OX plants were insensitive to brassinolide in the lamina inclination assay. Therefore, SG1 appears to decrease responses to BRs. Despite shorter organs in the SG1:OX plants, their cell size was not decreased in the SG1:OX plants. Therefore, SG1 decreases organ elongation by decreasing cell proliferation. In contrast to the SG1:OX plants, RNA interference knockdown plants that down-regulated SG1 and a related gene, SG1-LIKE PROTEIN1, had longer grains and internodes in rachis branches than in the wild type. Taken together, these results suggest that SG1 decreases responses to BRs and elongation of organs such as seeds and the internodes of rachis branches through decreased cellular proliferation. SG1,SGL1 The Stay-Green Rice like (SGRL) gene regulates chlorophyll degradation in rice 2013 J Plant Physiol Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, PR China. The Stay-Green Rice (SGR) protein is encoded by the SGR gene and has been shown to affect chlorophyll (Chl) degradation during natural and dark-induced leaf senescence. An SGR homologue, SGR-like (SGRL), has been detected in many plant species. We show that SGRL is primarily expressed in green tissues, and is significantly downregulated in rice leaves undergoing natural and dark-induced senescence. As the light intensity increases during the natural photoperiod, the intensity of SGRL expression declines while that of SGR expression increases. Overexpression of SGRL reduces the levels of Chl and Chl-binding proteins in leaves, and accelerates their degradation in dark-induced senescence leaves in rice. Our results suggest that the SGRL protein is also involved in Chl degradation. The relationship between SGRL and SGR and their effects on the degradation of the light-harvesting Chl a/b-binding protein are also discussed. SGR,OsSGRL Overexpression of SGR results in oxidative stress and lesion-mimic cell death in rice seedlings 2011 J Integr Plant Biol Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China. It is thought that the Stay Green Rice (SGR) gene is involved in the disaggregation of the light harvesting complex and in the subsequent breakdown of chlorophyll and apo-protein during senescence. In this study, we found that overexpression of SGR (Ov-SGR) resulted in the generation of singlet oxygen and other reactive oxygen species and produced a chlorophyll-dependent regional cell death phenotype on leaves of rice seedlings. Transcriptome analyses using Affymetrix Rice GeneChips revealed that Ov-SGR rice seedlings exhibited a number of signs of singlet oxygen response. The genes and their associated biochemical pathways identified provide an insight into how rice plants respond to singlet oxygen at the molecular and physiologic level. SGR Origin of seed shattering in rice (Oryza sativa L.) 2007 Planta Department of Plant Genetics and Breeding and State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, China. A critical evolutionary step during rice domestication was the elimination of seed shattering. Wild rice disperses seeds freely at maturity to guarantee the propagation, while cultivated rice retains seeds on the straws to make easy harvest and decrease the loss of production. The molecular basis for this key event during rice domestication remains to be elucidated. Here we show that the seed shattering is controlled by a single dominant gene, Shattering1 (SHA1), encoding a member of the trihelix family of plant-specific transcription factors. SHA1 was mapped to a 5.5 kb genomic fragment, which contains a single open reading frame, using a backcrossed population between cultivated rice Teqing and an introgression line IL105 with the seed shattering habit derived from perennial common wild rice, YJCWR. The predicted amino acid sequence of SHA1 in YJCWR and IL105 is distinguished from that in eight domesticated rice cultivars, including Teqing, by only a single amino acid substitution (K79N) caused by a single nucleotide change (g237t). Further sequence verification on the g237t mutation site revealed that the g237t mutation is present in all the domesticated rice cultivars, including 92 indica and 108 japonica cultivars, but not in any of the 24 wild rice accessions examined. Our results demonstrate that the g237t mutation in SHA1 accounts for the elimination of seed shattering, and that all the domesticated rice cultivars harbor the mutant sha1 gene and therefore have lost the ability to shed their seeds at maturity. In addition, our data support the theory that the non-shattering trait selection during rice domestication occurred prior to the indica-japonica differentiation in rice evolutionary history. sh4|SHA1 Molecular evolution of shattering loci in U.S. weedy rice 2010 Mol Ecol Biology Department, University of Massachusetts, Amherst, MA 01003, USA. Cultivated rice fields worldwide are plagued with weedy rice, a conspecific weed of cultivated rice (Oryza sativa L.). The persistence of weedy rice has been attributed, in part, to its ability to shatter (disperse) seed prior to crop harvesting. In the United States, separately evolved weedy rice groups have been shown to share genomic identity with exotic domesticated cultivars. Here, we investigate the shattering phenotype in a collection of U.S. weedy rice accessions, as well as wild and cultivated relatives. We find that all U.S. weedy rice groups shatter seeds easily, despite multiple origins, and in contrast to a decrease in shattering ability seen in cultivated groups. We assessed allelic identity and diversity at the major shattering locus, sh4, in weedy rice; we find that all cultivated and weedy rice, regardless of population, share similar haplotypes at sh4, and all contain a single derived mutation associated with decreased seed shattering. Our data constitute the strongest evidence to date of an evolution of weeds from domesticated backgrounds. The combination of a shared cultivar sh4 allele and a highly shattering phenotype, suggests that U.S. weedy rice have re-acquired the shattering trait after divergence from their progenitors through alternative genetic mechanisms. sh4|SHA1 Genetic analysis of rice domestication syndrome with the wild annual species, Oryza nivara 2006 New Phytol Department of Plant Biology, Michigan State University, East Lansing, MI 48824, USA. With a small and sequenced genome, rice provides an excellent system for studying the genetics of cereal domestication. We conducted a quantitative trait locus (QTL) analysis of key domestication traits using an F2 population derived from a cross between the cultivated rice, Oryza sativa, and the annual wild species, O. nivara. We found that the QTL of large phenotypic effects were targeted by domestication selection for effective harvest and planting, including a reduction in seed shattering and seed dormancy and the synchronization of seed maturation. Selection for higher yield was probably responsible for the fixation of mutations at a cluster of QTL on chromosome 7 and a few other chromosomal locations that could have substantially improved plant architecture and panicle structure, resulting in fewer erect tillers and longer and more highly branched panicles in cultivated rice. In comparison with the wild perennial species, O. rufipogon, rice domestication from O. nivara would have involved QTL with a greater degree of chromosomal co-localization and required little genetic change associated with life history or mating system transitions. The genetic analyses of domestication traits with both wild relatives will open opportunities for the improvement of rice cultivars utilizing natural germplasm. sh4|SHA1 Genetic control of seed shattering in rice by the APETALA2 transcription factor shattering abortion1 2012 Plant Cell National Center for Gene Research, National Center for Plant Gene Research Shanghai and Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200233, China. Seed shattering is an important agricultural trait in crop domestication. SH4 (for grain shattering quantitative trait locus on chromosome 4) and qSH1 (for quantitative trait locus of seed shattering on chromosome 1) genes have been identified as required for reduced seed shattering during rice (Oryza sativa) domestication. However, the regulatory pathways of seed shattering in rice remain unknown. Here, we identified a seed shattering abortion1 (shat1) mutant in a wild rice introgression line. The SHAT1 gene, which encodes an APETALA2 transcription factor, is required for seed shattering through specifying abscission zone (AZ) development in rice. Genetic analyses revealed that the expression of SHAT1 in AZ was positively regulated by the trihelix transcription factor SH4. We also identified a frameshift mutant of SH4 that completely eliminated AZs and showed nonshattering. Our results suggest a genetic model in which the persistent and concentrated expression of active SHAT1 and SH4 in the AZ during early spikelet developmental stages is required for conferring AZ identification. qSH1 functioned downstream of SHAT1 and SH4, through maintaining SHAT1 and SH4 expression in AZ, thus promoting AZ differentiation. sh4|SHA1,SHAT1 Rice domestication by reducing shattering 2006 Science Department of Plant Biology, Michigan State University, East Lansing, MI 48824, USA. Crop domestication frequently began with the selection of plants that did not naturally shed ripe fruits or seeds. The reduction in grain shattering that led to cereal domestication involved genetic loci of large effect. The molecular basis of this key domestication transition, however, remains unknown. Here we show that human selection of an amino acid substitution in the predicted DNA binding domain encoded by a gene of previously unknown function was primarily responsible for the reduction of grain shattering in rice domestication. The substitution undermined the gene function necessary for the normal development of an abscission layer that controls the separation of a grain from the pedicel. sh4|SHA1 Identification of genetic factors controlling domestication-related traits of rice using an F 2 population of a cross between Oryza sativa and O. rufipogon 1999 TAG Theoretical and Applied Genetics National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural Domesticated rice differs from the wild progenitor in large arrays of morphological and physiological traits. The present study was conducted to identify the genetic factors controlling the differences between cultivated rice and its wild progenitor, with the intention to assess the genetic basis of the changes associated with the processes of rice domestication. A total of 19 traits, including seven qualitative and 12 quantitative traits, that are related to domestication were scored in an F2 population from a cross between a variety of the Asian cultivated rice (Oryza sativa) and an accession of the common wild rice (O. rufipogon). Loci controlling the inheritance of these traits were determined by making use of a molecular linkage map consisting of 348 molecular-marker loci (313 RFLPs, 12 SSRs and 23 AFLPs) based on this F2 population. All seven qualitative traits were each controlled by a single Mendelian locus. Analysis of the 12 quantitative traits resolved a total of 44 putative QTLs with an average of 3.7 QTLs per trait. The amount of variation explained by individual QTLs ranged from a low of 6.9% to a high of 59.8%, and many of the QTLs accounted for more than 20% of the variation. Thus, genes of both major and minor effect were involved in the differences between wild and cultivated rice. The results also showed that most of the genetic factors (qualitative or QTLs) controlling the domestication-related traits were concentrated in a few chromosomal blocks. Such a clustered distribution of the genes may provide explanations for the genetic basis of the “domestication syndrome” observed in evolutionary studies and also for the “linkage drag” that occurs in many breeding programs. The information on the genetic basis of some desirable traits possessed by the wild parent may also be useful for facilitating the utilization of these traits in rice-breeding programs. sh4|SHA1 The BEL1-type homeobox gene SH5 induces seed shattering by enhancing abscission-zone development and inhibiting lignin biosynthesis 2014 Plant J Crop Biotech Institute, Kyung Hee University, Yongin, 446-701, Korea; Department of Life Science, Pohang University of Science and Technology, Pohang, 790-784, Korea. Seed shattering is an important trait that influences grain yield. A major controlling quantitative trait locus in rice is qSH1. Although the degree of shattering is correlated with the level of expression of qSH1, some qSH1-defective cultivars display moderate shattering while others show a non-shattering phenotype. Os05 g38120 (SH5) on chromosome 5 is highly homologous to qSH1. Although we detected SH5 transcripts in various organs, this gene was highly expressed at the abscission zone (AZ) in the pedicels. When expression of this gene was suppressed in easy-shattering 'Kasalath', development of the AZ was reduced and thereby so was seed loss. By contrast, the extent of shattering, as well as AZ development, was greatly enhanced in moderate-shattering 'Dongjin' rice when SH5 was overexpressed. Likewise, overexpression of SH5 in the non-shattering 'Ilpum' led to an increase in seed shattering because lignin levels were decreased in the basal region of spikelets in the absence of development of an AZ. We also determined that two shattering-related genes, SHAT1 and Sh4, which are necessary for proper formation of an AZ, were induced by SH5. Based on these observations, we propose that SH5 modulates seed shattering by enhancing AZ development and inhibiting lignin biosynthesis. SH5 Over-expression of rice OsAGO7 gene induces upward curling of the leaf blade that enhanced erect-leaf habit 2007 Planta National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai , 200032, China. High-yield cultivars are characterized by erect leaf canopies that optimize photosynthesis and thus favor increased biomass. Upward curling of the leaf blade (called rolled leaf) can result in enhanced erect-leaf habit, increase erect duration and promote an overall erect leaf canopy. The rice mutant R05, induced through transferred DNA (T-DNA) insertion, had the rolled-leaf trait. The leaves in the wild type demonstrated natural drooping tendencies, resulting in decreasing leaf erection indices (LEIs) during senescence at the 20th day after flowering. Conversely, LEIs of the leaves in R05 remained high, even 20-day post-flowering. We applied T-DNA tagging and isolated a rolled-leaf gene from rice which, when over-expressed, could induce upward curling of the leaf blade. This gene encodes for a protein of 1,048 amino acids including the PAZ and PIWI conserved domains, belonging to the Argonaute (AGO) family. There are at least 18 members of the AGO family in rice. According to high-sequence conservation, the rolled-leaf gene in rice could be orthologous to the Arabidopsis ZIP/Ago7 gene, so we called it OsAGO7. These results provide a possible opportunity for implementing OsAGO7 gene in crop improvement. OsAGO7|shl4|SHO2 The SHOOT ORGANIZATION2 gene coordinates leaf domain development along the central-marginal axis in rice 2008 Plant Cell Physiol Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, 113-8657 Japan. We describe how rice leaves are regionalized and regulated along the central-marginal axis. The shoot organization2 (sho2) mutant, a weak allele of SHOOTLESS4 that is a ZIPPY/ARGONAUTE7 homolog in rice, shows a variety of leaf abnormalities; filamentous leaves, bi- or trifurcated leaves, separation of the filamentous structure from the leaf blade or deletion of the margin. All of these phenotypes can be interpreted as combinatorial defects in the growth of the central, lateral and marginal domains along the central-marginal axis, on the condition that the growth of the central domain is predominant. The leaf founder cells for the lateral and marginal domains are recruited normally in sho2, indicating that sho2 is defective in the growth of leaf domains after the founder cells are recruited. The expression pattern of SHO2 in the outer layer of the shoot apical meristem and the adaxial surface of the leaf, as well as the altered expression of HD-ZIP III and ETTIN homologs in the central domain of sho2 leaves, suggest that normal development of the central domain is a prerequisite for the synchronous growth of the three domains. This synchrony is thought to be mediated by a small interfering RNA-dependent process. OsAGO7|shl4|SHO2 Oryza sativa dicer-like4 reveals a key role for small interfering RNA silencing in plant development 2007 Plant Cell State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. MicroRNAs and small interfering RNAs (siRNAs) are two classes of small regulatory RNAs derived from different types of precursors and processed by distinct Dicer or Dicer-like (DCL) proteins. During evolution, four Arabidopsis thaliana DCLs and six rice (Oryza sativa) DCLs (Os DCLs) appear to have acquired specialized functions. The Arabidopsis DCLs are well characterized, but those in rice remain largely unstudied. Here, we show that both knockdown and loss of function of rice DCL4, the homolog of Arabidopsis DCL4, lead to vegetative growth abnormalities and severe developmental defects in spikelet identity. These phenotypic alterations appear to be distinct from those observed in Arabidopsis dcl4 mutants, which exhibit accelerated vegetative phase change. The difference in phenotype between rice and Arabidopsis dcl4 mutants suggests that siRNA processing by DCL4 has a broader role in rice development than in Arabidopsis. Biochemical and genetic analyses indicate that Os DCL4 is the major Dicer responsible for the 21-nucleotide siRNAs associated with inverted repeat transgenes and for trans-acting siRNA (ta-siRNA) from the endogenous TRANS-ACTING siRNA3 (TAS3) gene. We show that the biogenesis mechanism of TAS3 ta-siRNA is conserved but that putative direct targets of Os DCL4 appear to be differentially regulated between monocots and dicots. Our results reveal a critical role of Os DCL4-mediated ta-siRNA biogenesis in rice development. SHO1|OsDCL4 Genome-wide gene expression profiling of introgressed indica rice alleles associated with seedling cold tolerance improvement in a japonica rice background 2012 BMC Genomics Institute of Crop Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing 100081, China ABSTRACT: BACKGROUND: Rice in tropical and sub-tropical areas is often subjected to cold stress at the seedling stage, resulting in poor growth and yield loss. Although japonica rice is generally more cold tolerant (CT) than indica rice, there are several favorable alleles for CT exist in indica that can be used to enhance CT in rice with a japonica background. Genome-wide gene expression profiling is an efficient way to decipher the molecular genetic mechanisms of CT enhancement and to provide valuable information for CT improvement in rice molecular breeding. In this study, the transcriptome of the CT introgression line (IL) K354 and its recurrent parent C418 under cold stress were comparatively analyzed to explore the possible CT enhancement mechanisms of K354. RESULTS: A total of 3184 differentially expressed genes (DEGs), including 195 transcription factors, were identified in both lines under cold stress. About half of these DEGs were commonly regulated and involved in major cold responsive pathways associated with OsDREB1 and OsMyb4 regulons. K354-specific cold-induced genes were functionally related to stimulus response, cellular cell wall organization, and microtubule-based movement processes that may contribute to increase CT. A set of genes encoding membrane fluidity and defensive proteins were highly enriched only in K354, suggesting that they contribute to the inherent CT of K354. Candidate gene prediction based on introgressed regions in K354 revealed genotype-dependent CT enhancement mechanisms, associated with Sir2, OsFAD7, OsWAK112d, and programmed cell death (PCD) related genes, present in CT IL K354 but absent in its recurrent parent C418. In K354, a number of DEGs were co-localized onto introgressed segments associated with CT QTLs, providing a basis for gene cloning and elucidation of molecular mechanisms responsible for CT in rice. CONCLUSIONS: Genome-wide gene expression analysis revealed that genotype-specific cold induced genes and genes with higher basal expression in the CT genotype contribute jointly to CT improvement. The molecular genetic pathways of cold stress tolerance uncovered in this study, as well as the DEGs co-localized with CT-related QTLs, will serve as useful resources for further functional dissection of the molecular mechanisms of cold stress response in rice. Sir2 The Receptor-Like Kinase SIT1 Mediates Salt Sensitivity by Activating MAPK3/6 and Regulating Ethylene Homeostasis in Rice 2014 Plant Cell Hebei Key Laboratory of Molecular and Cellular Biology, Hebei 050024, P.R. China Key Laboratory of Molecular and Cellular Biology of Ministry of Education, College of Life Science, Hebei Normal University, Hebei 050024, P.R. China Hebei Collaboration Innovation Center for Cell Signaling, Shijiazhuang, Hebei 050024, P.R. China. High salinity causes growth inhibition and shoot bleaching in plants that do not tolerate high salt (glycophytes), including most crops. The molecules affected directly by salt and linking the extracellular stimulus to intracellular responses remain largely unknown. Here, we demonstrate that rice (Oryza sativa) Salt Intolerance 1 (SIT1), a lectin receptor-like kinase expressed mainly in root epidermal cells, mediates salt sensitivity. NaCl rapidly activates SIT1, and in the presence of salt, as SIT1 kinase activity increased, plant survival decreased. Rice MPK3 and MPK6 function as the downstream effectors of SIT1. SIT1 phosphorylates MPK3 and 6, and their activation by salt requires SIT1. SIT1 mediates ethylene production and salt-induced ethylene signaling. SIT1 promotes accumulation of reactive oxygen species (ROS), leading to growth inhibition and plant death under salt stress, which occurred in an MPK3/6- and ethylene signaling-dependent manner in Arabidopsis thaliana. Our findings demonstrate the existence of a SIT1-MPK3/6 cascade that mediates salt sensitivity by affecting ROS and ethylene homeostasis and signaling. These results provide important information for engineering salt-tolerant crops. SIT1 Increased leaf photosynthesis caused by elevated stomatal conductance in a rice mutant deficient in SLAC1, a guard cell anion channel protein 2012 J Exp Bot Department of Biology, Faculty of Science, Kyushu University Fukuoka 812-8581 Japan. kkususcb@kyushu-u.org In rice (Oryza sativa L.), leaf photosynthesis is known to be highly correlated with stomatal conductance; however, it remains unclear whether stomatal conductance dominantly limits the photosynthetic rate. SLAC1 is a stomatal anion channel protein controlling stomatal closure in response to environmental [CO(2)]. In order to examine stomatal limitations to photosynthesis, a SLAC1-deficient mutant of rice was isolated and characterized. A TILLING screen of N-methyl-N-nitrosourea-derived mutant lines was conducted for the rice SLAC1 orthologue gene Os04g0674700, and four mutant lines containing mutations within the open reading frame were obtained. A second screen using an infrared thermography camera revealed that one of the mutants, named slac1, had a constitutive low leaf temperature phenotype. Measurement of leaf gas exchange showed that slac1 plants grown in the greenhouse had significantly higher stomatal conductance (g (s)), rates of photosynthesis (A), and ratios of internal [CO(2)] to ambient [CO(2)] (C (i)/C (a)) compared with wild-type plants, whereas there was no significant difference in the response of photosynthesis to internal [CO(2)] (A/C (i) curves). These observations demonstrate that in well-watered conditions, stomatal conductance is a major determinant of photosynthetic rate in rice. OsBIABP1|SLAC1 Molecular characterization of a defense-related AMP-binding protein gene, OsBIABP1, from rice 2009 J Zhejiang Univ Sci B State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310029, China. We cloned and characterized a rice gene OsBIABP1 encoding an AMP-binding protein. The full-length cDNA of OsBIABP1 is 1912-bp long and is predicted to encode a 558-aa protein. OsBIABP1 contains a typical AMP-binding signature motif and shows high similarity to members of AMP-binding protein family. OsBIABP1 is expressed in stems, leaves and flowers of rice plants, but is not expressed, or expressed at a very low level, in rice roots. The expression of OsBIABP1 was induced by some defense-related signal molecules, e.g., salicylic acid (SA), benzothiadiazole, jasmonic acid (JA), and 1-amino cyclopropane-1-carboxylic acid, which mediate SA- and JA/ethylene (ET)-dependent defense signaling pathways, respectively. Furthermore, the expression of OsBIABP1 is activated by the infection of Magnaporthe oryzae, and the induced expression is quicker and stronger during early stages of pathogenesis in incompatible interaction than that in compatible interaction between rice and M. oryzae. Our results suggest that OsBIABP1 may be a defense-related AMP-binding protein that is involved in the regulation of defense response through SA and/or JA/ET signaling pathways. OsBIABP1|SLAC1 Natural variation of rice strigolactone biosynthesis is associated with the deletion of two MAX1 orthologs 2014 Proc Natl Acad Sci U S A Laboratory of Plant Physiology, Wageningen University, 6708 PB, Wageningen, The Netherlands. Rice (Oryza sativa) cultivar Azucena--belonging to the Japonica subspecies--exudes high strigolactone (SL) levels and induces high germination of the root parasitic plant Striga hermonthica. Consistent with the fact that SLs also inhibit shoot branching, Azucena is a low-tillering variety. In contrast, Bala, an Indica cultivar, is a low-SL producer, stimulates less Striga germination, and is highly tillered. Using a Bala x Azucena F6 population, a major quantitative trait loci--qSLB1.1--for the exudation of SL, tillering, and induction of Striga germination was detected on chromosome 1. Sequence analysis of the corresponding locus revealed a rearrangement of a 51- to 59-kbp stretch between 28.9 and 29 Mbp in the Bala genome, resulting in the deletion of two cytochrome P450 genes--SLB1 and SLB2--with high homology to the Arabidopsis SL biosynthesis gene, MAX1. Both rice genes rescue the Arabidopsis max1-1 highly branched mutant phenotype and increase the production of the SL, ent-2'-epi-5-deoxystrigol, when overexpressed in Bala. Furthermore, analysis of this region in 367 cultivars of the publicly available Rice Diversity Panel population shows that the rearrangement at this locus is a recurrent natural trait associated with the Indica/Japonica divide in rice. SLB1,SLB2 SLL1, which encodes a member of the stearoyl-acyl carrier protein fatty acid desaturase family, is involved in cell elongation in lateral roots via regulation of fatty acid content in rice 2013 Plant Sci Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi 464-8601, Japan. shelleyisrat@yahoo.com We have identified a gene, SHORT LATERAL ROOT LENGTH1 (SLL1), which is important for the elongation of lateral roots in rice. An sll1 mutant has decreased lateral root growth due to a defect in the cell elongation. The SLL1 gene encodes a member of the stearoyl-acyl carrier protein fatty acid desaturase family that is the key regulator of overall fatty acid desaturation in plants. We measured the fatty acid content and found that the 18:0 content in the sll1 mutant root was approximately 4 times that in the wild-type root. When the sll1 mutant was grown at 33 degrees C, it complemented the mutant phenotype to a moderate level, which reflects the importance of the low 18:0 content in maintaining the cell membrane structure. The SLL1 gene was expressed at the lateral root tip, whereas SLL1 expression was not detected in the elongation zone of the crown roots. These results indicate that the lateral root specific defect in sll1 mutant is caused by the different expression patterns of SLL1 in lateral and crown roots. In addition, SLL1 over-expressers produced significantly longer lateral roots compared to the wild-type, and thus SLL1 gene would be very useful for improving rice root architecture. RL9|SLL1 SHALLOT-LIKE1 is a KANADI transcription factor that modulates rice leaf rolling by regulating leaf abaxial cell development 2009 Plant Cell State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, Zhejiang, China. As an important agronomic trait, rice (Oryza sativa L.) leaf rolling has attracted much attention from plant biologists and breeders. Moderate leaf rolling increases the photosynthesis of cultivars and hence raises grain yield. However, the relevant molecular mechanism remains unclear. Here, we show the isolation and functional characterization of SHALLOT-LIKE1 (SLL1), a key gene controlling rice leaf rolling. sll1 mutant plants have extremely incurved leaves due to the defective development of sclerenchymatous cells on the abaxial side. Defective development can be functionally rescued by expression of SLL1. SLL1 is transcribed in various tissues and accumulates in the abaxial epidermis throughout leaf development. SLL1 encodes a SHAQKYF class MYB family transcription factor belonging to the KANADI family. SLL1 deficiency leads to defective programmed cell death of abaxial mesophyll cells and suppresses the development of abaxial features. By contrast, enhanced SLL1 expression stimulates phloem development on the abaxial side and suppresses bulliform cell and sclerenchyma development on the adaxial side. Additionally, SLL1 deficiency results in increased chlorophyll and photosynthesis. Our findings identify the role of SLL1 in the modulation of leaf abaxial cell development and in sustaining abaxial characteristics during leaf development. These results should facilitate attempts to use molecular breeding to increase the photosynthetic capacity of rice, as well as other crops, by modulating leaf development and rolling. RL9|SLL1 ROLLED LEAF 9, encoding a GARP protein, regulates the leaf abaxial cell fate in rice 2008 Plant Mol Biol The Key Laboratory of Plant Functional Genomics, Ministry of Education of China, Jiangsu Key Laboratory of Crop Genetics and Physiology, Agricultural College of Yangzhou University, 12 East Wenhui Road, Jiangsu 225009, People's Republic of China. Leaves, the collective organ produced by the shoot apical meristem (SAM), are polarized along their adaxial-abaxial axis. In this study, we characterized two rice (Oryza sativa) allelic rolled-leaf mutants, rolled leaf 9-1 (rl9-1) and rl9-2, which display very similar phenotypes with completely adaxialized leaves and malformed spikelets. We cloned the RL9 gene by way of a map-based cloning strategy. Molecular studies have revealed that RL9 encodes a GARP protein, an orthologue of Arabidopsis KANADIs. RL9 is mainly expressed in roots, leaves, and flowers. The transient expression of a RL9-GFP (green fluorescent protein) fusion protein has indicated that RL9 protein is localized in the nucleus, suggesting that RL9 acts as a putative transcription factor. RL9|SLL1 A rice mutant displaying a heterochronically elongated internode carries a 100 kb deletion 2011 J Genet Genomics National Institute for Basic Biology, Okazaki 444-8585, Japan. We have isolated a recessive rice mutant, designated as indeterminate growth (ing), which displays creeping and apparent heterochronic phenotypes in the vegetative period with lanky and winding culms. Rough mapping and subsequent molecular characterization revealed that the ing mutant carries a large deletion, which corresponds to a 103 kb region in the Nipponbare genome, containing nine annotated genes on chromosome 3. Of these annotated genes, the SLR1 gene encoding a DELLA protein is the only one that is well characterized in its function, and its null mutation, which is caused by a single base deletion in the middle of the intronless SLR1 gene, confers a slender phenotype that bears close resemblance to the ing mutant phenotype. The primary cause of the ing mutant phenotype is the deletion of the SLR1 gene, and the ing mutant appears to be the first characterized mutant having the entire SLR1 sequence deleted. Our results also suggest that the deleted region of 103 kb does not contain an indispensable gene, whose dysfunction must result in a lethal phenotype. SLR1|OsGAI Isolation and characterization of dominant dwarf mutants, Slr1-d, in rice 2009 Mol Genet Genomics Bioscience and Biotechnology Center, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan. sd1 is known as the 'green revolution' gene in rice because its application in rice breeding has dramatically increased rice yield. Since the 'green revolution,' sd1 has been extensively used to produce modern semi-dwarf varieties. The extensive use of limited dwarfing sources may, however, cause a bottleneck effect in the genetic background of rice varieties. To circumvent this problem, novel and useful sources of dwarf genes must be identified. In this study, we identified three semi-dominant dwarf mutants. These mutants were categorized as dn-type dwarf mutants according to the elongation pattern of internodes. Gibberellin (GA) response tests showed that the mutants were still responsive to GA, although at a reduced rate. Map-based cloning revealed that the dwarf phenotype in these mutants was caused by gain-of-function mutations in the N-terminal region of SLR1. Degradation of the SLR1 protein in these mutants occurred later than in the wild type. Reduced interaction abilities of the SLR1 protein in these mutants with GID1 were also observed using the yeast two-hybrid system. Crossing experiments indicated that with the use of an appropriate genetic background, the semi-dominant dwarf alleles identified in this study could be used to alleviate the deficiency of dwarfing genes for breeding applications. SLR1|OsGAI Submergence tolerance conferred by Sub1A is mediated by SLR1 and SLRL1 restriction of gibberellin responses in rice 2008 Proc Natl Acad Sci U S A Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California, Riverside, CA 92521, USA. Submergence-tolerant rice maintains viability during complete submergence by limiting underwater elongation until floodwaters recede. Acclimation responses to submergence are coordinated by the submergence-inducible Sub1A, which encodes an ethylene-responsive factor-type transcription factor (ERF). Sub1A is limited to tolerant genotypes and sufficient to confer submergence tolerance to intolerant accessions. Here we evaluated the role of Sub1A in the integration of ethylene, abscisic acid (ABA), and gibberellin (GA) signaling during submergence. The submergence-stimulated decrease in ABA content was Sub1A-independent, whereas GA-mediated underwater elongation was significantly restricted by Sub1A. Transgenics that ectopically express Sub1A displayed classical GA-insensitive phenotypes, leading to the hypothesis that Sub1A limits the response to GA. Notably Sub1A increased the accumulation of the GA signaling repressors Slender Rice-1 (SLR1) and SLR1 Like-1 (SLRL1) and concomitantly diminished GA-inducible gene expression under submerged conditions. In the Sub1A overexpression line, SLR1 protein levels declined under prolonged submergence but were accompanied by an increase in accumulation of SLRL1, which lacks the DELLA domain. In the presence of Sub1A, the increase in these GA signaling repressors and decrease in GA responsiveness were stimulated by ethylene, which promotes Sub1A expression. Conversely, ethylene promoted GA responsiveness and shoot elongation in submergence-intolerant lines. Together, these results demonstrate that Sub1A limits ethylene-promoted GA responsiveness during submergence by augmenting accumulation of the GA signaling repressors SLR1 and SLRL1. SLR1|OsGAI,OsSLRL1,Sub1A The Gibberellin Signaling Pathway Is Regulated by the Appearance and Disappearance of SLENDER RICE1 in Nuclei 2002 The Plant Cell Online BioScience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan. The slender rice1 mutant (slr1) shows a constitutive gibberellin (GA) response phenotype. To investigate the mode of action of SLR1, we generated transgenic rice expressing a fusion protein consisting of SLR1 and green fluorescent protein (SLR1-GFP) and analyzed the phenotype of the transformants and the subcellular localization of GFP in vivo. SLR1-GFP worked in nuclei to repress the GA signaling pathway; its overproduction caused a dwarf phenotype. Application of GA(3) to SLR1-GFP overproducers induced GA actions such as shoot elongation, downregulation of GA 20-oxidase expression, and upregulation of SLR1 expression linked with the disappearance of the nuclear SLR1-GFP protein. We also performed domain analyses of SLR1 using transgenic plants overproducing different kinds of truncated SLR1 proteins. The analyses revealed that the SLR1 protein can be divided into four parts: a GA signal perception domain located at the N terminus, a regulatory domain for its repression activity, a dimer formation domain essential for signal perception and repression activity, and a repression domain at the C terminus. We conclude that GA signal transduction is regulated by the appearance or disappearance of the nuclear SLR1 protein, which is controlled by the upstream GA signal. SLR1|OsGAI slender Rice, a Constitutive Gibberellin Response Mutant, Is Caused by a Null Mutation of the SLR1 Gene, an Ortholog of the Height-Regulating Gene GAI/RGA/RHT/D8 2001 The Plant Cell Online BioScience Center and Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan The rice slender mutant (slr1-1) is caused by a single recessive mutation and results in a constitutive gibberellin (GA) response phenotype. The mutant elongates as if saturated with GAs. In this mutant, (1) elongation was unaffected by an inhibitor of GA biosynthesis, (2) GA-inducible α-amylase was produced by the aleurone layers without gibberellic acid application, and (3) endogenous GA content was lower than in the wild-type plant. These results indicate that the product of the SLR1 gene is an intermediate of the GA signal transduction pathway. SLR1 maps to OsGAI in rice and has significant homology with height-regulating genes, such as RHT-1Da in wheat, D8 in maize, and GAI and RGA in Arabidopsis. The GAI gene family is likely to encode transcriptional factors belonging to the GRAS gene superfamily. DNA sequence analysis revealed that the slr1-1 mutation is a single basepair deletion of the nuclear localization signal domain, resulting in a frameshift mutation that abolishes protein production. Furthermore, introduction of a 6-kb genomic DNA fragment containing the wild-type SLR1 gene into the slr1-1 mutant restored GA sensitivity to normal. These results indicate that the slr1-1 mutant is caused by a loss-of-function mutation of the SLR1 gene, which is an ortholog of GAI, RGA, RHT, and D8. We also succeeded in producing GA-insensitive dwarf rice by transforming wild-type rice with a modified SLR1 gene construct that has a 17–amino acid deletion affecting the DELLA region. Thus, we demonstrate opposite GA response phenotypes depending on the type of mutations in SLR1. SLR1|OsGAI GA Perception and Signal Transduction: Molecular Interactions of the GA Receptor GID1 with GA and the DELLA Protein SLR1 in Rice 2007 The Plant Cell Online None None SLR1|OsGAI The slender rice mutant, with constitutively activated gibberellin signal transduction, has enhanced capacity for abscisic acid level 2002 Plant and Cell Physiology Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku N10-W8, Sapporo, 060-0810 Japan The slender rice (slr1-1) mutant, carrying a lethal and recessive single mutation, has a constitutive gibberellin (GA)-response phenotype and behaves as if it were saturated with GAs [Ikeda et al. (2001) Plant Cell 13, 999]. The SLR1 gene, with sequence homology to members of the plant-specific GRAS gene family, is a mediator of the GA signal transduction process. In the slender rice, GA-inducible a-amylase was produced from the aleurone layer without applying GA. GA-independent alpha-amylase production in the mutant was inhibited by applying abscisic acid (ABA). Shoot elongation in the mutant was also suppressed by ABA, indicating that the slender rice responds normally to ABA. Interestingly, shoot ABA content was 10-fold higher in the mutant than in the wild type, while there was no difference in root ABA content. Expression of the Rab16A gene, which is known to be ABA inducible, was about 10-fold higher in shoots of the mutant than in those of the wild type. These results indicate that constitutive activation of the GA signal transduction pathway by the slr1-1 mutation promotes the endogenous ABA level. SLR1|OsGAI Comprehensive transcriptome analysis of phytohormone biosynthesis and signaling genes in microspore/pollen and tapetum of rice 2008 Plant Cell Physiol Bioscience and Biotechnology Center, Nagoya University, Nagoya, 464-8601 Japan. To investigate the involvement of phytohormones during rice microspore/pollen (MS/POL) development, endogenous levels of IAA, gibberellins (GAs), cytokinins (CKs) and abscisic acid (ABA) in the mature anther were analyzed. We also analyzed the global expression profiles of genes related to seven phytohormones, namely auxin, GAs, CKs, brassinosteroids, ethylene, ABA and jasmonic acids, in MS/POL and tapetum (TAP) using a 44K microarray combined with a laser microdissection technique (LM-array analysis). IAA and GA(4) accumulated in a much higher amount in the mature anther compared with the other tissues, while CKs and ABA did not. LM-array analysis revealed that sets of genes required for IAA and GA synthesis were coordinately expressed during the later stages of MS/POL development, suggesting that these genes are responsible for the massive accumulation of IAA and GA(4) in the mature anther. In contrast, genes for GA signaling were preferentially expressed during the early developmental stages of MS/POL and throughout TAP development, while their expression was down-regulated at the later stages of MS/POL development. In the case of auxin signaling genes, such mirror-imaged expression observed in GA synthesis and signaling genes was not observed. IAA receptor genes were mostly expressed during the late stages of MS/POL development, and various sets of AUX/IAA and ARF genes were expressed during the different stages of MS/POL or TAP development. Such cell type-specific expression profiles of phytohormone biosynthesis and signaling genes demonstrate the validity and importance of analyzing the expression of phytohormone-related genes in individual cell types independently of other cells/tissues. OsSLRL1 The overexpression of OsNAC9 alters the root architecture of rice plants enhancing drought resistance and grain yield under field conditions 2012 Plant Biotechnol J School of Biotechnology and Environmental Engineering, Myongji University, Yongin, Korea. Drought conditions limit agricultural production by preventing crops from reaching their genetically predetermined maximum yields. Here, we present the results of field evaluations of rice overexpressing OsNAC9, a member of the rice NAC domain family. Root-specific (RCc3) and constitutive (GOS2) promoters were used to overexpress OsNAC9 and produced the transgenic RCc3:OsNAC9 and GOS2:OsNAC9 plants. Field evaluations over two cultivating seasons showed that grain yields of the RCc3:OsNAC9 and the GOS2:OsNAC9 plants were increased by 13%-18% and 13%-32% under normal conditions, respectively. Under drought conditions, RCc3:OsNAC9 plants showed an increased grain yield of 28%-72%, whilst the GOS2:OsNAC9 plants remained unchanged. Both transgenic lines exhibited altered root architecture involving an enlarged stele and aerenchyma. The aerenchyma of RCc3:OsNAC9 roots was enlarged to a greater extent than those of GOS2:OsNAC9 and non-transgenic (NT) roots, suggesting the importance of this phenotype for enhanced drought resistance. Microarray experiments identified 40 up-regulated genes by more than threefold (P < 0.01) in the roots of both transgenic lines. These included 9-cis-epoxycarotenoid dioxygenase, an ABA biosynthesis gene, calcium-transporting ATPase, a component of the Ca(2+) signalling pathway involved in cortical cell death and aerenchyma formation, cinnamoyl CoA reductase 1, a gene involved in lignin biosynthesis, and wall-associated kinases genes involved in cell elongation and morphogenesis. Interestingly, O-methyltransferase, a gene necessary for barrier formation, was specifically up-regulated only in the RCc3:OsNAC9 roots. Such up-regulated genes that are commonly and specifically up-regulated in OsNAC9 transgenic roots may account for the altered root architecture conferring increased drought resistance phenotype. OsNAC19|SNAC1|OsNAC9 Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice 2006 Proc Natl Acad Sci U S A National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research, Wuhan 430070, China. Drought and salinity are major abiotic stresses to crop production. Here, we show that overexpression of stress responsive gene SNAC1 (STRESS-RESPONSIVE NAC 1) significantly enhances drought resistance in transgenic rice (22-34% higher seed setting than control) in the field under severe drought stress conditions at the reproductive stage while showing no phenotypic changes or yield penalty. The transgenic rice also shows significantly improved drought resistance and salt tolerance at the vegetative stage. Compared with WT, the transgenic rice are more sensitive to abscisic acid and lose water more slowly by closing more stomatal pores, yet display no significant difference in the rate of photosynthesis. SNAC1 is induced predominantly in guard cells by drought and encodes a NAM, ATAF, and CUC (NAC) transcription factor with transactivation activity. DNA chip analysis revealed that a large number of stress-related genes were up-regulated in the SNAC1-overexpressing rice plants. Our data suggest that SNAC1 holds promising utility in improving drought and salinity tolerance in rice. OsNAC19|SNAC1|OsNAC9 OsNAC6, a member of the NAC gene family, is induced by various stresses in rice 2005 Genes Genet Syst Graduate School of Agricultural and Life Sciences, The University of Tokyo Members of the NAC gene family encode plant-specific transcription factors and are widely distributed in plant species. The OsNAC6 gene is one of many NAC genes in rice and has high similarity to genes in the ATAF subfamily. Here we show that OsNAC6 is induced by cold, salt, drought and abscisic acid (ABA). We found that OsNAC6 is also induced by wounding. The response of OsNAC6 to wounding is very rapid and strong. OsNAC6 was also induced by jasmonic acid (JA), a plant hormone that activates defense responses against herbivores and pathogens. Our results imply that OsNAC6, besides having a role in plant adaptation to abiotic stresses, also integrates signals derived from both abiotic and biotic stresses. OsNAC6|SNAC2 Characterization of transcription factor gene SNAC2 conferring cold and salt tolerance in rice 2008 Plant Mol Biol National Center of Plant Gene Research Wuhan, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China. Plants respond to adverse environment by initiating a series of signaling processes including activation of transcription factors that can regulate expression of arrays of genes for stress response and adaptation. NAC (NAM, ATAF, and CUC) is a plant specific transcription factor family with diverse roles in development and stress regulation. In this report, a stress-responsive NAC gene (SNAC2) isolated from upland rice IRA109 (Oryza sativa L. ssp japonica) was characterized for its role in stress tolerance. SNAC2 was proven to have transactivation and DNA-binding activities in yeast and the SNAC2-GFP fusion protein was localized in the rice nuclei. Northern blot and SNAC2 promoter activity analyses suggest that SNAC2 gene was induced by drought, salinity, cold, wounding, and abscisic acid (ABA) treatment. The SNAC2 gene was over-expressed in japonica rice Zhonghua 11 to test the effect on improving stress tolerance. More than 50% of the transgenic plants remained vigorous when all WT plants died after severe cold stress (4-8 degrees C for 5 days). The transgenic plants had higher cell membrane stability than wild type during the cold stress. The transgenic rice had significantly higher germination and growth rate than WT under high salinity conditions. Over-expression of SNAC2 can also improve the tolerance to PEG treatment. In addition, the SNAC2-overexpressing plants showed significantly increased sensitivity to ABA. DNA chip profiling analysis of transgenic plants revealed many up-regulated genes related to stress response and adaptation such as peroxidase, ornithine aminotransferase, heavy metal-associated protein, sodium/hydrogen exchanger, heat shock protein, GDSL-like lipase, and phenylalanine ammonia lyase. Interestingly, none of the up-regulated genes in the SNAC2-overexpressing plants matched the genes up-regulated in the transgenic plants over-expressing other stress responsive NAC genes reported previously. These data suggest SNAC2 is a novel stress responsive NAC transcription factor that possesses potential utility in improving stress tolerance of rice. OsNAC6|SNAC2 Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice 2007 Plant J Biological Resources Division, Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki 305-8686, Japan. The OsNAC6 gene is a member of the NAC transcription factor gene family in rice. Expression of OsNAC6 is induced by abiotic stresses, including cold, drought and high salinity. OsNAC6 gene expression is also induced by wounding and blast disease. A transactivation assay using a yeast system demonstrated that OsNAC6 functions as a transcriptional activator, and transient localization studies with OsNAC6-sGFP fusion protein revealed its nuclear localization. Transgenic rice plants over-expressing OsNAC6 constitutively exhibited growth retardation and low reproductive yields. These transgenic rice plants showed an improved tolerance to dehydration and high-salt stresses, and also exhibited increased tolerance to blast disease. By utilizing stress-inducible promoters, such as the OsNAC6 promoter, it is hoped that stress-inducible over-expression of OsNAC6 in rice can improve stress tolerance by suppressing the negative effects of OsNAC6 on growth under normal growth conditions. The results of microarray analysis revealed that many genes that are inducible by abiotic and biotic stresses were upregulated in rice plants over-expressing OsNAC6. A transient transactivation assay showed that OsNAC6 activates the expression of at least two genes, including a gene encoding peroxidase. Collectively, these results indicate that OsNAC6 functions as a transcriptional activator in response to abiotic and biotic stresses in plants. We conclude that OsNAC6 may serve as a useful biotechnological tool for the improvement of stress tolerance in various kinds of plants. OsNAC6|SNAC2 The rice heterochronic gene SUPERNUMERARY BRACT regulates the transition from spikelet meristem to floral meristem 2007 Plant J National Research Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Korea. Regulating the transition of meristem identity is a critical step in reproductive development. After the shoot apical meristem (SAM) acquires inflorescence meristem identity, it goes through a sequential transition to second- and higher-order meristems that can eventually give rise to floral organs. Despite ample information on the molecular mechanisms that control the transition from SAM to inflorescence meristems, little is known about the mechanism for inflorescence development, especially in monocots. Here, we report the identification of the SUPERNUMERARY BRACT (SNB) gene controlling the transition from spikelet meristem to floral meristem and the floral organ development. This gene encodes a putative transcription factor carrying two AP2 domains. The SNB:GFP fusion protein is localized to the nucleus. SNB is expressed in all the examined tissues, but most strongly in the newly emerging spikelet meristems. In SNB knockout plants, the transition from spikelet meristems to floral meristems is delayed, resulting in the production of multiple rudimentary glumes in an alternative phyllotaxy. The development of additional bracts interferes with subsequent floral architecture. In some spikelets, the empty glumes and lodicules are transformed into lemma/palea-like organs. Occasionally, the number of stamens and carpels is altered and an ectopic floret occurs in the axil of the rachilla. These phenotypes suggest that snb is a heterochronic mutant, affecting the phase transition of spikelet meristems, the pattern formation of floral organs and spikelet meristem determinancy. SNB Rice Snl6, a cinnamoyl-CoA reductase-like gene family member, is required for NH1-mediated immunity to Xanthomonas oryzae pv. oryzae 2010 PLoS Genet Department of Plant Pathology, University of California Davis, Davis, California, USA. Rice NH1 (NPR1 homolog 1) is a key mediator of innate immunity. In both plants and animals, the innate immune response is often accompanied by rapid cell death at the site of pathogen infection. Over-expression of NH1 in rice results in resistance to the bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo), constitutive expression of defense related genes and enhanced benzothiadiazole (BTH)- mediated cell death. Here we describe a forward genetic screen that identified a suppressor of NH1-mediated lesion formation and resistance, snl6. Comparative genome hybridization and fine mapping rapidly identified the genomic location of the Snl6 gene. Snl6 is a member of the cinnamoyl-CoA reductase (CCR)-like gene family. We show that Snl6 is required for NH1-mediated resistance to Xoo. Further, we show that Snl6 is required for pathogenesis-related gene expression. In contrast to previously described CCR family members, disruption of Snl6 does not result in an obvious morphologic phenotype. Snl6 mutants have reduced lignin content and increased sugar extractability, an important trait for the production of cellulosic biofuels. These results suggest the existence of a conserved group of CCR-like genes involved in the defense response, and with the potential to alter lignin content without affecting development. Snl6 The ethylene response factors SNORKEL1 and SNORKEL2 allow rice to adapt to deep water 2009 Nature Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan. Living organisms must acquire new biological functions to adapt to changing and hostile environments. Deepwater rice has evolved and adapted to flooding by acquiring the ability to significantly elongate its internodes, which have hollow structures and function as snorkels to allow gas exchange with the atmosphere, and thus prevent drowning. Many physiological studies have shown that the phytohormones ethylene, gibberellin and abscisic acid are involved in this response, but the gene(s) responsible for this trait has not been identified. Here we show the molecular mechanism of deepwater response through the identification of the genes SNORKEL1 and SNORKEL2, which trigger deepwater response by encoding ethylene response factors involved in ethylene signalling. Under deepwater conditions, ethylene accumulates in the plant and induces expression of these two genes. The products of SNORKEL1 and SNORKEL2 then trigger remarkable internode elongation via gibberellin. We also demonstrate that the introduction of three quantitative trait loci from deepwater rice into non-deepwater rice enabled the latter to become deepwater rice. This discovery will contribute to rice breeding in lowland areas that are frequently flooded during the rainy season. SNORKEL1,SNORKEL2 Molecular Cloning of the Gene (SodCc7) that Encodes a Cytosolic Copper/Zinc-Superoxide Dismutase from Rice 1995 Plant Physiol Biochem Department of Biochemistry, College of Agriculture, Kyoto Prefectural University, Japan. None SodCc7 Isolation of a novel mutant gene for soil-surface rooting in rice (Oryza sativa L.) 2013 Rice (N Y) Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, Miyagi 980-8577, Japan. tadashi@ige.tohoku.ac.jp. BACKGROUND: Root system architecture is an important trait affecting the uptake of nutrients and water by crops. Shallower root systems preferentially take up nutrients from the topsoil and help avoid unfavorable environments in deeper soil layers. We have found a soil-surface rooting mutant from an M2 population that was regenerated from seed calli of a japonica rice cultivar, Nipponbare. In this study, we examined the genetic and physiological characteristics of this mutant. RESULTS: The primary roots of the mutant showed no gravitropic response from the seedling stage on, whereas the gravitropic response of the shoots was normal. Segregation analyses by using an F2 population derived from a cross between the soil-surface rooting mutant and wild-type Nipponbare indicated that the trait was controlled by a single recessive gene, designated as sor1. Fine mapping by using an F2 population derived from a cross between the mutant and an indica rice cultivar, Kasalath, revealed that sor1 was located within a 136-kb region between the simple sequence repeat markers RM16254 and 2935-6 on the terminal region of the short arm of chromosome 4, where 13 putative open reading frames (ORFs) were found. We sequenced these ORFs and detected a 33-bp deletion in one of them, Os04g0101800. Transgenic plants of the mutant transformed with the genomic fragment carrying the Os04g0101800 sequence from Nipponbare showed normal gravitropic responses and no soil-surface rooting. CONCLUSION: These results suggest that sor1, a rice mutant causing soil-surface rooting and altered root gravitropic response, is allelic to Os04g0101800, and that a 33-bp deletion in the coding region of this gene causes the mutant phenotypes. SOR1 Short panicle1 encodes a putative PTR family transporter and determines rice panicle size 2009 Plant J National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. The architecture of the rice inflorescence, which is determined mainly by the number and length of primary and secondary inflorescence branches, is of importance in both agronomy and developmental biology. The position and number of primary branches are established during the phase transition from vegetative to reproductive growth, and several of the genes identified as participating in this process do so by regulating the meristemic activities of inflorescence. However, little is known about the molecular mechanism that controls inflorescence branch elongation. Here, we report on a novel rice mutant, short panicle1 (sp1), which is defective in rice panicle elongation, and thus leads to the short-panicle phenotype. Gene cloning and characterization indicate that SP1 encodes a putative transporter that belongs to the peptide transporter (PTR) family. This conclusion is based on the findings that SP1 contains a conserved PTR2 domain consisting of 12 transmembrane domains, and that the SP1-GFP fusion protein is localized in the plasma membrane. The SP1 gene is highly expressed in the phloem of the branches of young panicles, which is consistent with the predicted function of SP1 and the sp1 phenotype. Phylogenetic analysis implies that SP1 might be a nitrate transporter. However, neither nitrate transporter activity nor any other compounds transported by known PTR proteins could be detected in either a Xenopus oocyte or yeast system, in our study, suggesting that SP1 may need other component(s) to be able to function as a transporter, or that it transports unknown substrates in the monocotyledonous rice plant. SP1 RNA maturation of the rice SPK gene may involve trans-splicing 1999 The Plant Journal Mitsui Plant Biotehcnology Research Institute, TCI D-21, Sengen, Tsukuba, Japan. A gene encoding a calcium-dependent seed-specific protein kinase (SPK) is abundantly expressed in developing rice seeds (Kawasaki, T et al. Gene (1993) 129, 183-189). Rice genomic clones encoding SPK were isolated using the entire cDNA fragment as a probe. Physical mapping of these genomic clones indicated that the genomic region corresponding to the entire cDNA was divided into two different regions, SPK-A and SPK-B, located on different rice chromosomes. The results of RACE-PCR analyses showed that the respective transcripts from SPK-A and SPK-B contained additional sequences which were not found in the SPK cDNA, and that these sequences were removed like introns during maturation of the SPK mRNA. These results suggest that two different RNAs were independently transcribed from SPK-A and SPK-B and joined, possibly by trans-splicing. SPK Rice SPK, a Calmodulin-Like Domain Protein Kinase, Is Required for Storage Product Accumulation during Seed Development: Phosphorylation of Sucrose Synthase Is a Possible Factor 2002 The Plant Cell Online Department of Biological Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda 278-8510, Japan Suc, an end product of photosynthesis, is metabolized by Suc synthase in sink organs as an initial step in the biosynthesis of storage products. Suc synthase activity is known to be regulated by reversible phosphorylation, but the details of this process are unclear at present. Rice SPK, a calcium-dependent protein kinase, is expressed uniquely in the endosperm of immature seed, and its involvement in the biosynthetic pathways of storage products was suggested. Antisense SPK transformants lacked the ability to accumulate storage products such as starch, but produced watery seed with a large amount of Suc instead, as the result of an inhibition of Suc degradation. Analysis of in vitro phosphorylation indicated that SPK phosphorylated specifically a Ser residue in Suc synthase that has been shown to be important for its activity in the degradation of Suc. This finding suggests that SPK is involved in the activation of Suc synthase. It appears that SPK is a Suc synthase kinase that may be important for supplying substrates for the biosynthesis of storage products. SPK Spotted leaf11, a negative regulator of plant cell death and defense, encodes a U-box/armadillo repeat protein endowed with E3 ubiquitin ligase activity 2004 Plant Cell Department of Plant Pathology, Ohio State University, Columbus, Ohio 43210, USA. The rice (Oryza sativa) spotted leaf11 (spl11) mutant was identified from an ethyl methanesulfonate-mutagenized indica cultivar IR68 population and was previously shown to display a spontaneous cell death phenotype and enhanced resistance to rice fungal and bacterial pathogens. Here, we have isolated Spl11 via a map-based cloning strategy. The isolation of the Spl11 gene was facilitated by the identification of three additional spl11 alleles from an IR64 mutant collection. The predicted SPL11 protein contains both a U-box domain and an armadillo (ARM) repeat domain, which were demonstrated in yeast and mammalian systems to be involved in ubiquitination and protein-protein interactions, respectively. Amino acid sequence comparison indicated that the similarity between SPL11 and other plant U-box-ARM proteins is mostly restricted to the U-box and ARM repeat regions. A single base substitution was detected in spl11, which results in a premature stop codon in the SPL11 protein. Expression analysis indicated that Spl11 is induced in both incompatible and compatible rice-blast interactions. In vitro ubiquitination assay indicated that the SPL11 protein possesses E3 ubiquitin ligase activity that is dependent on an intact U-box domain, suggesting a role of the ubiquitination system in the control of plant cell death and defense. SPL11 Regulatory mechanisms of ROI generation are affected by rice spl mutations 2006 Plant Cell Physiol Department of Biology, Faculty of Sciences, Kyushu University, Hakozaki, Fukuoka, 812-8581 Japan. Reactive oxygen intermediates (ROIs) play a pivotal role in the hypersensitive response (HR) in disease resistance. NADPH oxidase is a major source of ROI; however, the mechanisms of its regulation are unclear. Rice spl mutants spontaneously form lesions which resemble those occurring during the HR, suggesting that the mutations affect regulation of the HR. We found that spl2, spl7 and spl11 mutant cells accumulated increased amounts of H(2)O(2) in response to rice blast fungal elicitor. Increased accumulation of ROIs was suppressed by inhibition of NADPH oxidase in the spl cells, and was also observed in the ozone-exposed spl plants. These mutants have sufficient activities of ROI-scavenging enzymes compared with the wild type. In addition, spl7 mutant cells accumulated higher amounts of H(2)O(2) when treated with calyculin A (CA), an inhibitor of protein phosphatase. Furthermore, spl2 mutant plants exhibited accelerated accumulation of H(2)O(2) and increased rates of cell death in response to wounding. These results suggest that the spl2, spl7 and spl11 mutants are defective in the regulation of NADPH oxidase, and the spl7 mutation may give rise to enhancement of the signaling pathway which protein dephosphorylation controls, while the spl2 mutation affects both the pathogen-induced and wound-induced signaling pathways. SPL11,Spl7 SPL28 encodes a clathrin-associated adaptor protein complex 1, medium subunit micro 1 (AP1M1) and is responsible for spotted leaf and early senescence in rice (Oryza sativa) 2010 New Phytol Department of Plant Science, Research Institute for Agriculture and Life Sciences, and Plant Genomics and Breeding Institute, Seoul National University, Seoul, 151-921, Republic of Korea. To expand our understanding of cell death in plant defense responses, we isolated a novel rice (Oryza sativa) spotted leaf mutant (spl28) that displays a lesion mimic phenotype in the absence of pathogen attack through treatment of Hwacheongbyeo (an elite Korean japonica cultivar) with N-methyl-N-nitrosourea (MNU). Early stage development of the spl28 mutant was normal. However, after flowering, spl28 mutants exhibited a significant decrease in chlorophyll content, soluble protein content, and photosystem II efficiency, and high concentrations of reactive oxygen species (ROS), phytoalexin, callose, and autofluorescent phenolic compounds that localized in or around the lesions. The spl28 mutant also exhibited significantly enhanced resistance to rice blast and bacterial blight. Using a map-based cloning approach, we determined that SPL28 encodes a clathrin-associated adaptor protein complex 1, medium subunit micro 1 (AP1M1), which is involved in the post-Golgi trafficking pathway. A green fluorescent protein (GFP) fusion protein of SPL28 (SPL28::GFP) localized to the Golgi apparatus, and expression of SPL28 complemented the membrane trafficking defect of apm1-1 Delta yeast mutants. SPL28 was ubiquitously expressed and contained a highly conserved adaptor complex medium subunit (ACMS) family domain. SPL28 appears to be involved in the regulation of vesicular trafficking, and SPL28 dysfunction causes the formation of hypersensitive response (HR)-like lesions, leading to the initiation of leaf senescence. SPL28 SPL5, a cell death and defense-related gene, encodes a putative splicing factor 3b subunit 3 (SF3b3) in rice 2011 Molecular Breeding College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua, 321004, China A lesion-mimic phenotype in rice (Oryza sativa L.) spotted leaf 5 (spl5) indicates that wild-type SPL5 negatively regulates cell death and resistance responses. Previously, the spl5 gene was already mapped to the 80-kb region between two markers SSR7 and RM7121 through a map-based cloning approach. Here, we further showed that the spl5 gene was delimitated into a 15.1-kb genomic region by the high-resolution sequence target site (STS) markers. Subsequent sequencing in this region of spl5 mutant revealed that one candidate gene harbored a single-base deletion, resulting in a frame-shift mutation and a premature stop codon. Bioinformatic analysis showed that SPL5 gene encodes a putative splicing factor 3b subunit 3 (SF3b3) and might be involved in splicing reactions of pre-mature RNAs participating in the regulation of cell death and resistance responses. Further analysis showed that wild-type SPL5 did functionally complement the spl5 phenotype. The data presented here clearly indicate that the SPL5 negatively regulates cell death and resistance responses via modulating RNA splicing in plants. spl5|SF3b3 A rice spotted leaf gene, Spl7, encodes a heat stress transcription factor protein 2002 Proc Natl Acad Sci U S A Institute of the Society for Techno-Innovation of Agriculture, Forestry, and Fisheries, Tsukuba, Ibaraki 305-0854, Japan. A rice spotted leaf (lesion-mimic) gene, Spl7, was identified by map-based cloning. High-resolution mapping with cleaved amplified polymorphic sequence markers enabled us to define a genomic region of 3 kb as a candidate for Spl7. We found one ORF that showed high similarity to a heat stress transcription factor (HSF). Transgenic analysis verified the function of the candidate gene for Spl7: leaf spot development was suppressed in spl7 mutants with a wild-type Spl7 transgene. Thus, we conclude that Spl7 encodes the HSF protein. The transcript of spl7 was observed in mutant plants. The levels of mRNAs (Spl7 in wild type and spl7 in mutant) increased under heat stress. Sequence analysis revealed only one base substitution in the HSF DNA-binding domain of the mutant allele, causing a change from tryptophan to cysteine. Spl7 A rice stromal processing peptidase regulates chloroplast and root development 2010 Plant Cell Physiol State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, PR China. The stromal processing peptidase (SPP) is a metalloendopeptidase that cleaves a broad range of precursor substrates. In this study, we isolated a rice mutant showing leaf chlorosis at the early seedling stage but inhibition of root growth during the whole growth period. Genetic analysis demonstrates that the phenotypes of the mutant were caused by a recessive single gene mutation. The mutated gene was cloned by map-based cloning, and was shown to encode an SPP. Sequence analysis showed a glutamate deletion in the highly conserved C-terminus of SPP in the mutant. The mutation of SPP in the mutant was verified by transgenic complementation. SPP is constitutively expressed in all tissues. Subcellular localization analysis indicates that SPP is targeted to the chloroplast. The expression of some genes associated with chloroplast development was decreased in young seedlings of the spp mutant, but not in 14-day-old seedlings. Western blot analysis revealed that the Rubisco small subunit is not precisely processed in the spp mutant in 7-day-old seedlings, but the processing activity in the spp mutant is restored in 14-day-old seedlings. Moreover, the expression levels of Cab1R and Cab2R for the light-harvesting chlorophyll a/b-binding protein (LHCP) were highly up-regulated in the transgenic plants with overexpression of SPP. The present results reveal that SPP is essential for chloroplast biogenesis at the early growth stage and for rice root development; this is the first report on the function of SPP in monocot plants. SPP Characterization of a rice sucrose-phosphate synthase-encoding gene 1996 Gene Departamento de Ingenieria Genetica de Plantas, Centro de Investigacion y Estudios Avanzados del I.P.N., Unidad Irapuato, Guanajuato, Argentina. A rice genomic clone (sps1) coding for sucrose phosphate synthase (SPS) was isolated and sequenced. Rice sps1 contains 13 exons and 12 introns, an unusually long 366-bp leader region with a highly organized primary structure and a promoter region with no obvious homology with eukaryotic promoter consensus sequences. Southern blot analysis showed that SPS is encoded by a single-copy gene in the rice genome. Comparison of the rice, maize, potato and spinach SPS deduced amino acid (aa) sequences showed that these enzymes have a well conserved region comprising their first 700 aa, and a variable C-terminal region. Analysis of rice sps1 expression showed that mRNA levels change during leaf development. SPS activity and mRNA were undetectable in roots. sps1|SPS Tissue-specific and developmental pattern of expression of the rice sps1 gene 2000 Plant Physiol Departamento de Ingenieria Genetica de Plantas, Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional, Unidad Irapuato, Irapuato, Guanajuato, Mexico. Sucrose-phosphate synthase (SPS) is one of the key regulatory enzymes in carbon assimilation and partitioning in plants. SPS plays a central role in the production of sucrose in photosynthetic cells and in the conversion of starch or fatty acids into sucrose in germinating seeds. To explore the mechanisms that regulate the tissue-specific and developmental distribution of SPS, the expression pattern of rice (Oryza sativa) sps1 (GenBank accession no. U33175) was examined by in situ reverse transcriptase-polymerase chain reaction and the expression directed by the sps1 promoter using the beta-glucuronidase reporter gene. It was found that the expression of the rice sps1 gene is limited to mesophyll cells in leaves, the scutellum of germinating seedlings, and pollen of immature inflorescences. During leaf development, the sps1 promoter directs a basipetal pattern of expression that coincides with the distribution of SPS activity during the leaf sink-to-source transition. It was also found that during the vegetative part of the growth cycle, SPS expression and enzymatic activity are highest in the youngest fully expanded leaf. Additionally, it was observed that the expression of the sps1 promoter is regulated by light and dependent on plastid development in photosynthetic tissues, whereas expression in scutellum is independent of both light and plastid development. sps1|SPS Identification of a new gene controlling plant height in rice using the candidate-gene strategy 2004 Planta National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, 305-8602 Ibaraki, Japan. kenshi@nias.affrc.go.jp A gene underlying a quantitative trait locus (QTL) controlling plant height on chromosome 1 (QTLph1) in rice ( Oryza sativa L.) was identified using the candidate-gene strategy. First, the function of a targeted gene was analyzed using near isogenic lines (NILs) in which the chromosomal region of a targeted QTL was substituted with that of another line. Second, for physiological information, the candidate gene was selected in the annotation data by the genome sequencing. Physiological analyses of an NIL-expressing QTLph1 (NIL6) suggested that the targeted gene controls plant height by enabling higher amounts of sucrose to be translocated in leaves. The results indicated that the gene for sucrose phosphate synthase (SPS; EC 2.4.1.14), the major limiting enzyme for sucrose synthesis, is a candidate gene for QTLph1 among the annotation results of the region of QTLph1. The higher level of SPS transcripts and the activity of SPS in NIL6 compared to control plants, and the fact that the relative SPS activity per SPS protein content was almost the same between NIL6 and Nipponbare suggested that the higher plant height in NIL6 compared to Nipponbare was due to the high SPS activity in NIL6. In agreement with this hypothesis, transgenic rice plants with a maize SPS gene that had about 3 times the SPS activity of that in Nipponbare (control plants) were significantly taller than Nipponbare from the early growth stage. From these results and the physiological data from NIL6, we concluded that SPS is the targeted gene underlying QTLph1. sps1|SPS Promoter shuffling at a nuclear gene for mitochondrial RPL27. Involvement of interchromosome and subsequent intrachromosome recombinations 2006 Plant Physiol Genetic Diversity Department , National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. The Reclinomonas americana mitochondrial genome contains a mitochondrial ribosomal protein L27 (rpl27) gene, whereas the rpl27 gene is absent from all plant mitochondrial genomes examined to date. This suggests that plant mitochondrial rpl27 genes have been transferred previously from the mitochondrial genome to the nuclear genome. A nuclear cDNA encoding mitochondrial RPL27 was identified in rice (Oryza sativa). Three similar sequences were identified: rpl27-1 and rpl27-2 on chromosome 8 and rpl27-3 on chromosome 4. Harr plot analysis suggests that they were generated by inter- and intrachromosomal duplications. Interestingly, the transcribed rpl27 gene (rpl27-1) acquired a promoter sequence that was derived from the rice spt16 (Osspt16) gene, the homolog of a global transcription factor in yeast (Saccharomyces cerevisiae) located downstream from the rpl27-3 sequence on chromosome 4, after inter- and intrachromosomal recombination. Reverse transcription-PCR and promoter assay revealed that the rpl27 mRNAs were mainly transcribed from rpl27-1. A repeat of seven nucleotides (AATAGTT) was identified at the junction of rpl27-1 and rpl27-2 on chromosome 8, and the same repeat was also identified at the 5' end of rpl27-2 and the 3' end of rpl27-1. This repeat (AATAGTT) contains the hot-spot sequence AGTT, which is preferentially recognized by topoisomerase I in wheat (Triticum aestivum) germ, suggesting the involvement of topoisomerase I in this recombination. We here report the example of promoter shuffling and show that this promoter shuffling resulted from a recent segmental duplication through inter- and intrachromosomal recombination events. Osspt16 RNAi-mediated disruption of squalene synthase improves drought tolerance and yield in rice 2012 J Exp Bot Division of Plant Sciences, University of Missouri, Columbia, MO 65211, USA. About one-third of the world's rice area is in rain-fed lowlands and most are prone to water shortage. The identification of genes imparting tolerance to drought in the model cereal plant, rice, is an attractive strategy to engineer improved drought tolerance not only rice but other cereals as well. It is demonstrated that RNAi-mediated disruption of a rice farnesyltransferase/squalene synthase (SQS) by maize squalene synthase improves drought tolerance at both the vegetative and reproductive stages. Twenty-day-old seedlings of wild type (Nipponbare) and seven independent events of transgenic RNAi lines showed no difference in morphology. When subjected to water stress for a period of 32 d under growth chamber conditions, transgenic positives showed delayed wilting, conserved more soil water, and improved recovery. When five independent events along with wild-type plants were subjected to drought at the reproductive stage under greenhouse conditions, the transgenic plants lost water more slowly compared with the wild type, through reduced stomatal conductance and the retention of high leaf relative water content (RWC). After 28 d of slow progressive soil drying, transgenic plants recovered better and flowered earlier than wild-type plants. The yield of water-stressed transgenic positive plants ranged from 14-39% higher than wild-type plants. When grown in plates with Yoshida's nutrient solution with 1.2% agar, transgenic positives from three independent events showed increased root length and an enhanced number of lateral roots. The RNAi-mediated inactivation produced reduced stomatal conductance and subsequent drought tolerance. SQS Semi-rolled leaf1 encodes a putative glycosylphosphatidylinositol-anchored protein and modulates rice leaf rolling by regulating the formation of bulliform cells 2012 Plant Physiol National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032 Shanghai, China. Leaf rolling is an important agronomic trait in rice (Oryza sativa) breeding and moderate leaf rolling maintains the erectness of leaves and minimizes shadowing between leaves, leading to improved photosynthetic efficiency and grain yields. Although a few rolled-leaf mutants have been identified and some genes controlling leaf rolling have been isolated, the molecular mechanisms of leaf rolling still need to be elucidated. Here we report the isolation and characterization of SEMI-ROLLED LEAF1 (SRL1), a gene involved in the regulation of leaf rolling. Mutants srl1-1 (point mutation) and srl1-2 (transferred DNA insertion) exhibit adaxially rolled leaves due to the increased numbers of bulliform cells at the adaxial cell layers, which could be rescued by complementary expression of SRL1. SRL1 is expressed in various tissues and is expressed at low levels in bulliform cells. SRL1 protein is located at the plasma membrane and predicted to be a putative glycosylphosphatidylinositol-anchored protein. Moreover, analysis of the gene expression profile of cells that will become epidermal cells in wild type but probably bulliform cells in srl1-1 by laser-captured microdissection revealed that the expression of genes encoding vacuolar H(+)-ATPase (subunits A, B, C, and D) and H(+)-pyrophosphatase, which are increased during the formation of bulliform cells, were up-regulated in srl1-1. These results provide the transcript profile of rice leaf cells that will become bulliform cells and demonstrate that SRL1 regulates leaf rolling through inhibiting the formation of bulliform cells by negatively regulating the expression of genes encoding vacuolar H(+)-ATPase subunits and H(+)-pyrophosphatase, which will help to understand the mechanism regulating leaf rolling. SRL1 A novel kinesin 13 protein regulating rice seed length 2010 Plant Cell Physiol Department of Bioscience, Fukui Prefectural University, Yoshida-gun, Fukui, Japan. The causal gene of a novel small and round seed mutant phenotype (srs3) in rice was identified by map-based cloning and named the SRS3 gene. The SRS3 gene was grouped as a member of the kinesin 13 subfamily. The SRS3 gene codes for a protein of 819 amino acids that contains a kinesin motor domain and a coiled-coil structure. Using scanning electron microscopy, we determined that the cell length of seeds in the longitudinal direction in srs3 is shorter than that in the wild type. The number of cells of seeds in the longitudinal direction in srs3 was not very different from that in the wild type. The result suggests that the small and round seed phenotype of srs3 is due to a reduction in cell length of seeds in the longitudinal direction. The SRS3 protein, which is found in the crude microsomal fraction, is highly expressed in developing organs. SRS3 Gene SGL, encoding a kinesin-like protein with transactivation activity, is involved in grain length and plant height in rice 2014 Plant Cell Rep State Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing, 210095, China. Grain shape, a complex agronomic trait, plays an important role in determining yield and quality in rice. In the present study, a mutant named short grain length (sgl) was identified among explants of tissue cultured japonica variety Kita-ake. It exhibited reduced plant height (about 72 % of WT) and short grain length (about 80 % of WT). The reduced length was due to decreased cell elongation. The Short Grain Length (SGL) gene was isolated via map-based cloning and identified to encode a kinesin-like protein. SGL was expressed in the whole plant, especially in the stem and panicles. SGL was shown to have transcriptional activity. In onion epidermal cells, SGL protein was found mainly in the nucleus. Real-time PCR analyses showed that expression levels of genes involved in gibberellin metabolic pathways were affected in the sgl mutant. These data suggested that SGL protein may be involved in regulating GA synthesis and response genes, that in turn, regulates grain length and plant height. SRS3 A dominant mutation of TWISTED DWARF 1 encoding an α-tubulin protein causes severe dwarfism and right helical growth in rice 2009 Genes Genet Syst Bioscience and Biotechnology Center, Nagoya University Dwarfism is a common type of mutation in many plant species. The pathways and factors regulating biosynthesis and signaling of several plant growth regulators have been clarified through analyses of dwarf mutants in rice, Arabidopsis, pea, and maize. However, the genetic mechanisms controlling dwarfism are not well characterized, and the causal genes underlying most dwarf mutants are still uncovered. Here, we report a dominant mutant, Twisted dwarf 1-1 (Tid1-1), showing dwarfism and twisted growth in rice. Tid1-1 exhibit right helical growth of the leaves and stem and shortening of the roots. They also show an increased number of cells in the shoot apical meristem. Cells in the leaves of Tid1-1 are often ill-shapen, possibly owing to irregular cell division. Cell elongation in roots is suppressed in the elongation zone, and cells in the root apical meristem are enlarged. Map-based cloning of TID1 revealed that it encodes an α-tubulin protein comprising microtubules and is an ortholog of Arabidopsis LEFTY genes. Our analysis of the Tid1-1 mutant revealed that the dynamics of microtubules affects not only anisotropic growth in both dicots and monocots, but also meristematic activity and gross plant morphology. TID1|SRS5 Analysis of the rice SHORT-ROOT5 gene revealed functional diversification of plant neutral/alkaline invertase family 2009 Plant Science Hokuriku Research Center, National Agricultural Research Center, 1-2-1 Inada, Niigata 943-0193, Japan Neutral/alkaline invertase (Inv-N) catalyzes the hydrolysis of sucrose to produce glucose and fructose. Although the rice genome contains eight Inv-N-like genes, their functions in plant development are unknown. We previously described a rice mutant, srt5, which exhibits extremely stunted post-embryonic root growth that is rescuable by metabolizable sugars. In the present study, we performed a functional analysis of the SRT5 gene. We showed that SRT5 encodes a putative cytosolic Inv-N that cleaves sucrose at pH 7.0 and 8.0. An SRT5-GFP fusion protein was localized to the cytosol. Sucrose levels in srt5 root cells were elevated, consistent with a crucial role for SRT5 in cytosolic sucrose cleavage at early root developmental stages. SRT5 was found to be ubiquitously expressed in various plant organs, whereas the other seven rice Inv-Ns were differentially expressed, suggesting that the functions of these latter proteins are distinct from that of SRT5. In support of this view, molecular evolutionary analysis revealed that all of the most closely related paralogs of SRT5 (OsNIN5, OsNIN6, and OsNIN7) have multiple non-conservative amino acid substitutions not found in SRT5. These results suggest that SRT5 is the key isoform of Inv-Ns required for carbon and energy supply during early root development. OsCYT-INV1|SRT5 OsCYT-INV1 for alkaline/neutral invertase is involved in root cell development and reproductivity in rice (Oryza sativa L.) 2008 Planta The State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Zi Jin Gang Campus, 310058 , Hangzhou, China. A short root mutant was isolated from an EMS-generated rice mutant library. Under normal growth conditions, the mutant exhibited short root, delayed flowering, and partial sterility. Some sections of the roots revealed that the cell length along the longitudinal axis was reduced and the cell shape in the root elongation zone shrank. Genetic analysis indicated that the short root phenotype was controlled by a recessive gene. Map-based cloning revealed that a nucleotide substitution causing an amino acid change from Gly to Arg occurred in the predicted rice gene (Os02g0550600). It coded an alkaline/neutral invertase and was homologous to Arabidopsis gene AtCyt-inv1. This gene was designated as OsCyt-inv1. The results of carbohydrate analysis showed an accumulation of sucrose and reduction of hexose in the Oscyt-inv1 mutant. Exogenously supplying glucose could rescue the root growth defects of the Oscyt-inv1 mutant. These results indicated that OsCyt-inv1 played important roles in root cell development and reproductivity in rice. OsCYT-INV1|SRT5 SRWD: a novel WD40 protein subfamily regulated by salt stress in rice (OryzasativaL.) 2008 Gene State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China. By analysis with microarray data, we found that a gene encoding a novel protein containing five WD40 repeats, was regulated by salt stress in rice and named as SRWD1 (Salt responsive WD40 protein 1). By database searching, additional four SRWD1-like genes (SRWD2-SRWD5) were found in rice genome, and these five SRWD genes formed a novel WD40 subfamily. Phylogenetic analysis showed that plant SRWD proteins divided into four groups. The significant functional divergences during SRWD evolution were found. The tissue-specific and salt responsive expression profiling for SRWD genes was investigated based on microarray data. It was found that all five SRWD genes in rice were regulated by salt stress. Further, we found that SRWD1 was regulated with different patterns by salt stress in two rice cultivars responding differently to salt stress. Our study correlates WD40 proteins with salt stress in plants and provides fundamental information for the further investigation of plant SRWD proteins. SRWD1,SRWD2,SRWD3,SRWD4,SRWD5 Stress repressive expression of rice SRZ1 and characterization of plant SRZ gene family 2008 Plant Science State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China A zinc finger protein gene was cloned from rice and designated as SRZ1 (stress repressive zinc finger protein 1). SRZ1 encodes a protein with three C2C2-type zinc finger motifs that are structurally similar to human ZNF265 zinc finger motifs. SRZ1 was expressed with high level in leaves and markedly repressed by salt, cold, drought and abcisic acid stresses but not by salicylic acid and blast inoculation in rice seedlings. Ectopic expression of SRZ1 in tobacco plants repressed expression of abiotic stress-related genes including osmotin, NtERB10B, NtERB10C, and increased plant sensitivity to cold and salt stresses. Database searches showed SRZ genes were widely distributed among plant species and their expressions in rice and Arabidopsis were significantly down-regulated by at least one type of abiotic stresses. This study indicates that SRZ1 and its relatives in plants may play negative roles in abiotic stress signaling and the down-regulation of them might be crucial for plant tolerance against stresses. SRZ1 SSD1, which encodes a plant-specific novel protein, controls plant elongation by regulating cell division in rice 2010 Proceedings of the Japan Academy, Series B Bioscience and Biotechnology Center, Nagoya University, Aichi, Japan. Plant height is one of the most important traits in crop improvement. Therefore revealing the mechanism of plant elongation and controlling plant height in accordance with breeding object is important. In this study we analyzed a novel dwarf mutant, ssd1, of which phenotype is different from typical GA- or BR-related dwarf phenotype. ssd1 exhibits pleiotropic defects in elongation of various organs such as stems, roots, leaves, and flowers. ssd1 also shows abnormal cell files and shapes, which suggests defects of normal cell division in the mutant. Map-based cloning and complementation test demonstrated that the dwarf phenotype in ssd1 mutant was caused by insertion of retrotransposon in a gene, which encodes plant-specific protein with unknown biochemical function. A BLAST search revealed that SSD1-like genes exist in diverse plant species, including monocots and dicots, but not fern and moss. Our results demonstrate that SSD1 controls plant elongation by controlling cell division in higher plants. SSD1 Amyloplast-localized SUBSTANDARD STARCH GRAIN4 protein influences the size of starch grains in rice endosperm 2014 Plant Physiol Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan. Starch is a biologically and commercially important polymer of glucose and is synthesized to form starch grains (SGs) inside amyloplasts. Cereal endosperm accumulates starch to levels that are more than 90% of the total weight, and most of the intracellular space is occupied by SGs. The size of SGs differs depending on the plant species and is one of the most important factors for industrial applications of starch. However, the molecular machinery that regulates the size of SGs is unknown. In this study, we report a novel rice (Oryza sativa) mutant called substandard starch grain4 (ssg4) that develops enlarged SGs in the endosperm. Enlargement of SGs in ssg4 was also observed in other starch-accumulating tissues such as pollen grains, root caps, and young pericarps. The SSG4 gene was identified by map-based cloning. SSG4 encodes a protein that contains 2,135 amino acid residues and an amino-terminal amyloplast-targeted sequence. SSG4 contains a domain of unknown function490 that is conserved from bacteria to higher plants. Domain of unknown function490-containing proteins with lengths greater than 2,000 amino acid residues are predominant in photosynthetic organisms such as cyanobacteria and higher plants but are minor in proteobacteria. The results of this study suggest that SSG4 is a novel protein that influences the size of SGs. SSG4 will be a useful molecular tool for future starch breeding and biotechnology. SSG4 A bacterial-type ABC transporter is involved in aluminum tolerance in rice 2009 Plant Cell Research Institute for Bioresources, Okayama University, Kurashiki 710-0046, Japan. Aluminum (Al) toxicity is a major factor limiting crop production in acidic soil, but the molecular mechanisms of Al tolerance are poorly understood. Here, we report that two genes, STAR1 (for sensitive to Al rhizotoxicity1) and STAR2, are responsible for Al tolerance in rice. STAR1 encodes a nucleotide binding domain, while STAR2 encodes a transmembrane domain, of a bacterial-type ATP binding cassette (ABC) transporter. Disruption of either gene resulted in hypersensitivity to aluminum toxicity. Both STAR1 and STAR2 are expressed mainly in the roots and are specifically induced by Al exposure. Expression in onion epidermal cells, rice protoplasts, and yeast showed that STAR1 interacts with STAR2 to form a complex that localizes to the vesicle membranes of all root cells, except for those in the epidermal layer of the mature zone. When expressed together in Xenopus laevis oocytes, STAR1/2 shows efflux transport activity specific for UDP-glucose. Furthermore, addition of exogenous UDP-glucose rescued root growth in the star1 mutant exposed to Al. These results indicate that STAR1 and STAR2 form a complex that functions as an ABC transporter, which is required for detoxification of Al in rice. The ABC transporter transports UDP-glucose, which may be used to modify the cell wall. STAR1,STAR2 The half-size ABC transporters STR1 and STR2 are indispensable for mycorrhizal arbuscule formation in rice 2012 Plant J Department of Plant Molecular Biology, University of Lausanne, Switzerland. The central structure of the symbiotic association between plants and arbuscular mycorrhizal (AM) fungi is the fungal arbuscule that delivers minerals to the plant. Our earlier transcriptome analyses identified two half-size ABCG transporters that displayed enhanced mRNA levels in mycorrhizal roots. We now show specific transcript accumulation in arbusculated cells of both genes during symbiosis. Presently, arbuscule-relevant factors from monocotyledons have not been reported. Mutation of either of the Oryza sativa (rice) ABCG transporters blocked arbuscule growth of different AM fungi at a small and stunted stage, recapitulating the phenotype of Medicago truncatula stunted arbuscule 1 and 2 (str1 and str2) mutants that are deficient in homologous ABCG genes. This phenotypic resemblance and phylogenetic analysis suggest functional conservation of STR1 and STR2 across the angiosperms. Malnutrition of the fungus underlying limited arbuscular growth was excluded by the absence of complementation of the str1 phenotype by wild-type nurse plants. Furthermore, plant AM signaling was found to be intact, as arbuscule-induced marker transcript accumulation was not affected in str1 mutants. Strigolactones have previously been hypothesized to operate as intracellular hyphal branching signals and possible substrates of STR1 and STR2. However, full arbuscule development in the strigolactone biosynthesis mutants d10 and d17 suggested strigolactones to be unlikely substrates of STR1/STR2. Interestingly, rice STR1 is associated with a cis-natural antisense transcript (antiSTR1). Analogous to STR1 and STR2, at the root cortex level, the antiSTR1 transcript is specifically detected in arbusculated cells, suggesting unexpected modes of STR1 regulation in rice. STR1,STR2 The submergence tolerance regulator Sub1A mediates stress-responsive expression of AP2/ERF transcription factors 2010 Plant Physiol Department of Plant Pathology, University of California, Davis, California 95616, USA. We previously characterized the rice (Oryza sativa) Submergence1 (Sub1) locus encoding three ethylene-responsive factor (ERF) transcriptional regulators. Genotypes carrying the Sub1A-1 allele are tolerant of prolonged submergence. To elucidate the mechanism of Sub1A-1-mediated tolerance, we performed transcriptome analyses comparing the temporal submergence response of Sub1A-1-containing tolerant M202(Sub1) with the intolerant isoline M202 lacking this gene. We identified 898 genes displaying Sub1A-1-dependent regulation. Integration of the expression data with publicly available metabolic pathway data identified submergence tolerance-associated pathways governing anaerobic respiration, hormone responses, and antioxidant systems. Of particular interest were a set of APETALA2 (AP2)/ERF family transcriptional regulators that are associated with the Sub1A-1-mediated response upon submergence. Visualization of expression patterns of the AP2/ERF superfamily members in a phylogenetic context resolved 12 submergence-regulated AP2/ERFs into three putative functional groups: (1) anaerobic respiration and cytokinin-mediated delay in senescence via ethylene accumulation during submergence (three ERFs); (2) negative regulation of ethylene-dependent gene expression (five ERFs); and (3) negative regulation of gibberellin-mediated shoot elongation (four ERFs). These results confirm that the presence of Sub1A-1 impacts multiple pathways of response to submergence. Sub1A,Sub1C The submergence tolerance gene SUB1A delays leaf senescence under prolonged darkness through hormonal regulation in rice 2012 Plant Physiol Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California, Riverside, California 92521, USA. Leaf senescence is a natural age-dependent process that is induced prematurely by various environmental stresses. Typical alterations during leaf senescence include breakdown of chlorophyll, a shift to catabolism of energy reserves, and induction of senescence-associated genes, all of which can occur during submergence, drought, and constant darkness. Here, we evaluated the influence of the submergence tolerance regulator, SUBMERGENCE1A (SUB1A), in the acclimation responses during leaf senescence caused by prolonged darkness in rice (Oryza sativa). SUB1A messenger RNA was highly induced by prolonged darkness in a near-isogenic line containing SUB1A. Genotypes with conditional and ectopic overexpression of SUB1A significantly delayed loss of leaf color and enhanced recovery from dark stress. Physiological analysis revealed that SUB1A postpones dark-induced senescence through the maintenance of chlorophyll and carbohydrate reserves in photosynthetic tissue. This delay allowed leaves of SUB1A genotypes to recover photosynthetic activity more quickly upon reexposure to light. SUB1A also restricted the transcript accumulation of representative senescence-associated genes. Jasmonate and salicylic acid are positive regulators of leaf senescence, but ectopic overexpression of SUB1A dampened responsiveness to both hormones in the context of senescence. We found that ethylene accelerated senescence stimulated by darkness and jasmonate, although SUB1A significantly restrained dark-induced ethylene accumulation. Overall, SUB1A genotypes displayed altered responses to prolonged darkness by limiting ethylene production and responsiveness to jasmonate and salicylic acid, thereby dampening the breakdown of chlorophyll, carbohydrates, and the accumulation of senescence-associated messenger RNAs. A delay of leaf senescence conferred by SUB1A can contribute to the enhancement of tolerance to submergence, drought, and oxidative stress. Sub1A Development of submergence-tolerant rice cultivars: the Sub1 locus and beyond 2009 Ann Bot International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines. BACKGROUND AND AIMS: Submergence is a recurring problem in the rice-producing rainfed lowlands of south and south-east Asia. Developing rice cultivars with tolerance of submergence and with agronomic and quality traits acceptable to farmers is a feasible approach to address this problem. The objectives of this study were to (a) develop mega varieties with Sub1 introgression that are submergence tolerant, (b) assess the performance of Sub1 in different genetic backgrounds, (c) determine the roles of the Sub1A and Sub1C genes in conferring tolerance, and (d) assess the level of tolerance in F(1) hybrids heterozygous for the Sub1A-1-tolerant allele. METHODS: Tolerant varieties were developed by marker-assisted backcrossing through two or three backcrosses, and their performance was evaluated to determine the effect of Sub1 in different genetic backgrounds. The roles of Sub1A and Sub1C in conferring the tolerant phenotype were further investigated using recombinants identified within the Sub1 gene cluster based on survival and gene expression data. KEY RESULTS: All mega varieties with Sub1 introgression had a significantly higher survival rate than the original parents. An intolerant Sub1C allele combined with the tolerant Sub1A-1 allele did not significantly reduce the level of tolerance, and the Sub1C-1 expression appeared to be independent of the Sub1A allele; however, even when Sub1C-1 expression is completely turned off in the presence of Sub1A-2, plants remained intolerant. Survival rates and Sub1A expression were significantly lower in heterozygotes compared with the homozygous tolerant parent. CONCLUSIONS: Sub1 provided a substantial enhancement in the level of tolerance of all the sensitive mega varieties. Sub1A is confirmed as the primary contributor to tolerance, while Sub1C alleles do not seem important. Lack of dominance of Sub1 suggests that the Sub1A-1 allele should be carried by both parents for developing tolerant rice hybrids. Sub1A,Sub1B,Sub1C Expression of rice SUB1A and SUB1C transcription factors in Arabidopsis uncovers flowering inhibition as a submergence tolerance mechanism 2011 Plant J Center for Plant Cell Biology and Department of Botany and Plant Sciences, University of California, Riverside, CA 92521, USA. Submergence of plant organs perturbs homeostasis by limiting diffusion of oxygen, carbon dioxide and ethylene. In rice (Oryza sativa L.), the haplotype at the multigenic SUBMERGENCE1 (SUB1) locus determines whether plants survive prolonged submergence. SUB1 encodes two or three transcription factors of the group VII ethylene response factor family: SUB1A, SUB1B and SUB1C. The presence of SUB1A-1 and its strong submergence-triggered ethylene-mediated induction confers submergence tolerance through a quiescence survival strategy that inhibits gibberellin (GA)-induced carbohydrate consumption and elongation growth. SUB1C is invariably present and acts downstream of the enhancement of GA responsiveness during submergence. In this study, heterologous ectopic expression of rice SUB1A and SUB1C in Arabidopsis thaliana was used to explore conserved mechanisms of action associated with these genes using developmental, physiological and molecular metrics. As in rice transgenic plants that ectopically express SUB1A-1, Arabidopsis transgenic plants that constitutively express SUB1A displayed GA insensitivity and abscisic acid hypersensitivity. Ectopic SUB1C expression had more limited effects on development, stress responses and the transcriptome. Observation of a delayed flowering phenotype in lines over-expressing SUB1A led to the finding that inhibition of floral initiation is a component of the quiescence survival strategy in rice. Together, these analyses demonstrate conserved as well as specific roles for group VII ethylene response factors in integration of abiotic responses with development. Sub1A,Sub1C Sub1A is an ethylene-response-factor-like gene that confers submergence tolerance to rice 2006 Nature Department of Plant Pathology, University of California, Davis, California 95616, USA. Most Oryza sativa cultivars die within a week of complete submergence--a major constraint to rice production in south and southeast Asia that causes annual losses of over US 1 billion dollars and affects disproportionately the poorest farmers in the world. A few cultivars, such as the O. sativa ssp. indica cultivar FR13A, are highly tolerant and survive up to two weeks of complete submergence owing to a major quantitative trait locus designated Submergence 1 (Sub1) near the centromere of chromosome 9 (refs 3, 4, 5-6). Here we describe the identification of a cluster of three genes at the Sub1 locus, encoding putative ethylene response factors. Two of these genes, Sub1B and Sub1C, are invariably present in the Sub1 region of all rice accessions analysed. In contrast, the presence of Sub1A is variable. A survey identified two alleles within those indica varieties that possess this gene: a tolerance-specific allele named Sub1A-1 and an intolerance-specific allele named Sub1A-2. Overexpression of Sub1A-1 in a submergence-intolerant O. sativa ssp. japonica conferred enhanced tolerance to the plants, downregulation of Sub1C and upregulation of Alcohol dehydrogenase 1 (Adh1), indicating that Sub1A-1 is a primary determinant of submergence tolerance. The FR13A Sub1 locus was introgressed into a widely grown Asian rice cultivar using marker-assisted selection. The new variety maintains the high yield and other agronomic properties of the recurrent parent and is tolerant to submergence. Cultivation of this variety is expected to provide protection against damaging floods and increase crop security for farmers. Sub1A Molecular marker survey and expression analyses of the rice submergence-tolerance gene SUB1A 2010 Theor Appl Genet Plant Breeding, Genetics, and Biotechnology Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines. The major rice quantitative-trait locus Submergence 1 (Sub1) confers tolerance of submergence for about 2 weeks. To identify novel sources of tolerance, we have conducted a germplasm survey with allele-specific markers targeting SUB1A and SUB1C, two of the three transcription-factor genes within the Sub1 locus. The objective was to identify tolerant genotypes without the SUB1A gene or with the intolerant SUB1A-2 allele. The survey revealed that all tolerant genotypes possessed the tolerant Sub1 haplotype (SUB1A-1/SUB1C-1), whereas all accessions without the SUB1A gene were intolerant. Only the variety James Wee with the SUB1A-2 allele was moderately tolerant. However, some intolerant genotypes with the SUB1A-1 allele were identified and RT-PCR analyses were conducted to compare gene expression in tolerant and intolerant accessions. Initial analyses of leaf samples failed to reveal a clear association of SUB1A transcript abundance and tolerance. Temporal and spatial gene expression analyses subsequently showed that SUB1A expression in nodes and internodes associated best with tolerance across representative genotypes. In James Wee, transcript abundance was high in all tissues, suggesting that some level of tolerance might be conferred by high expression of the SUB1A-2 allele. To further assess tissue-specific expression, we have expressed the GUS reporter gene under the control of the SUB1A-1 promoter. The data revealed highly specific GUS expression at the base of the leaf sheath and in the leaf collar region. Specific expression in the growing part of rice leaves is well in agreement with the role of SUB1A in suppressing leaf elongation under submergence. Sub1A Identification and characterization of SHORTENED UPPERMOST INTERNODE 1, a gene negatively regulating uppermost internode elongation in rice 2011 Plant Mol Biol State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, 359 Tiyuchang Road, Hangzhou, China. In rice, the elongated internodes are derived from the vegetative shoot apical meristem (SAM), and the transition of the SAM from the vegetative to the reproductive stage induces internode elongation. In this study, we characterize two shortened uppermost internode mutants (sui1-1 and sui1-2). During the seedling and tillering stages, sui1 plants are morphologically similar to wild-type plants. However, at the heading stage, the sui1-1 mutant exhibits a shortened uppermost internode and a partly sheathed panicle, and the sui1-2 mutant shows an extremely shortened uppermost internode and a fully sheathed panicle. Gibberellin treatment results in elongation of every internode, but the shortened uppermost internode phenotype remains unaltered. Microscopic analysis indicates that cell length of sui1-1 uppermost internode exhibits decreased. Map-based cloning revealed that SUI1 is located on Chromosome 1, and encodes a putative phosphatidyl serine synthase (PSS) family protein. Searches for matches in protein databases showed that OsSUI1 contains the InterPro domain IPR004277, which is conserved in both animal and plant kingdoms. Introduction of a wild-type SUI1 gene fully rescued the mutant phenotype of sui1-1 and sui1-2, confirming the identity of the cloned gene. Consistent with these results, the SUI1-RNAi transgenic plants displayed decreased elongation of the uppermost internode. Our results suggest that SUI1 plays an important role in regulating uppermost internode length by decreasing longitudinal cell length in rice. OsSUI1 Molecular evolution of the TAC1 gene from rice (Oryza sativa L.) 2012 J Genet Genomics State Key Laboratory of Plant Physiology and Biochemistry, National Centre for Evaluation of Agricultural Wild Plants (Rice), Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China. Tiller angle is a key feature of the architecture of cultivated rice (Oryza sativa), since it determines planting density and influences rice yield. Our previous work identified Tiller Angle Control 1 (TAC1) as a major quantitative trait locus that controls rice tiller angle. To further clarify the evolutionary characterization of the TAC1 gene, we compared a TAC1-containing 3164-bp genomic region among 113 cultivated varieties and 48 accessions of wild rice, including 43 accessions of O. rufipogon and five accessions of O. nivara. Only one single nucleotide polymorphism (SNP), a synonymous substitution, was detected in TAC1 coding regions of the cultivated rice varieties, whereas one synonymous and one nonsynonymous SNP were detected among the TAC1 coding regions of wild rice accessions. These data indicate that little natural mutation and modification in the TAC1 coding region occurred within the cultivated rice and its progenitor during evolution. Nucleotide diversities in the TAC1 gene regions of O. sativa and O. rufipogon of 0.00116 and 0.00112, respectively, further indicate that TAC1 has been highly conserved during the course of rice domestication. A functional nucleotide polymorphism (FNP) of TAC1 was only found in the japonica rice group. A neutrality test revealed strong selection, especially in the 3'-flanking region of the TAC1 coding region containing the FNP in the japonica rice group. However, no selection occurred in the indica and wild-rice groups. A phylogenetic tree derived from TAC1 sequence analysis suggests that the indica and japonica subspecies arose independently during the domestication of wild rice. TAC1 TAC1, a major quantitative trait locus controlling tiller angle in rice 2007 Plant J Department of Plant Genetics and Breeding and State Key Laboratory of Plant Physiology and Biochemistry, China Agricultural University, Beijing 100094, China. A critical step during rice (Oryza sativa) cultivation is dense planting: a wider tiller angle will increase leaf shade and decrease photosynthesis efficiency, whereas a narrower tiller angle makes for more efficient plant architecture. The molecular basis of tiller angle remains unknown. This research demonstrates that tiller angle is controlled by a major quantitative trait locus, TAC1 (Tiller Angle Control 1). TAC1 was mapped to a 35-kb region on chromosome 9 using a large F(2) population from crosses between an indica rice, IR24, which displays a relatively spread-out plant architecture, and an introgressed line, IL55, derived from japonica rice Asominori, which displays a compact plant architecture with extremely erect tillers. Genetic complementation further identified the TAC1 gene, which harbors three introns in its coding region and a fourth 1.5-kb intron in the 3'-untranslated region. A mutation in the 3'-splicing site of this 1.5-kb intron from 'AGGA' to 'GGGA' decreases the level of tac1, resulting in a compact plant architecture with a tiller angle close to zero. Further sequence verification of the mutation in the 3'-splicing site of the 1.5-kb intron revealed that the tac1 mutation 'GGGA' was present in 88 compact japonica rice accessions and TAC1 with 'AGGA' was present in 21 wild rice accessions and 43 indica rice accessions, all with the spread-out form, indicating that tac1 had been extensively utilized in densely planted rice grown in high-latitude temperate areas and at high altitudes where japonica rice varieties are widely cultivated. TAC1 TAWAWA1, a regulator of rice inflorescence architecture, functions through the suppression of meristem phase transition 2013 Proc Natl Acad Sci U S A Graduate School of Agriculture and Life Sciences, The University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan. Inflorescence structures result from the activities of meristems, which coordinate both the renewal of stem cells in the center and organ formation at the periphery. The fate of a meristem is specified at its initiation and changes as the plant develops. During rice inflorescence development, newly formed meristems acquire a branch meristem (BM) identity, and can generate further meristems or terminate as spikelets. Thus, the form of rice inflorescence is determined by a reiterative pattern of decisions made at the meristems. In the dominant gain-of-function mutant tawawa1-D, the activity of the inflorescence meristem (IM) is extended and spikelet specification is delayed, resulting in prolonged branch formation and increased numbers of spikelets. In contrast, reductions in TAWAWA1 (TAW1) activity cause precocious IM abortion and spikelet formation, resulting in the generation of small inflorescences. TAW1 encodes a nuclear protein of unknown function and shows high levels of expression in the shoot apical meristem, the IM, and the BMs. TAW1 expression disappears from incipient spikelet meristems (SMs). We also demonstrate that members of the SHORT VEGETATIVE PHASE subfamily of MADS-box genes function downstream of TAW1. We thus propose that TAW1 is a unique regulator of meristem activity in rice and regulates inflorescence development through the promotion of IM activity and suppression of the phase change to SM identity. TAW1 Importance of the rice TCD9 encoding alpha subunit of chaperonin protein 60 (Cpn60alpha) for the chloroplast development during the early leaf stage 2014 Plant Sci Development Center of Plant Germplasm Resources, College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, China. The chloroplast development governs plant growth and metabolism. This study characterized a novel rice thermo-sensitive chloroplast development 9 (tcd9) mutant, which exhibited the albino phenotype before the 3-leaf stage grown below 24 degrees C whereas displayed normal green at over 28 degrees C or even at 20 degrees C after 5-leaf stage. The obvious decrease in Chl levels, abnormal chloroplasts with few thylakoid lamella and abnormal thylakoids were observed for the albino mutant seedlings at low temperature, but the mutant was apparently normal green at high temperature, suggesting the thermo-sensitivity of albino phenotype. Genetic analysis showed that the albino phenotype was controlled by a single recessive nuclear gene (tcd9). The map-based cloning and molecular complementation tests revealed that the mutation of TCD9 encoding alpha subunit of Cpn60 protein (Cpn60alpha), localized in chloroplasts, was responsible for albino phenotype. In addition, TCD9 exhibited the high expression in all tested tissues, especially in young leaves. The transcriptional analysis indicated that all expression levels of the studied genes related to chloroplast development in tcd9 mutant were seriously affected in the albino seedlings at 20 degrees C, whereas some of them recovered into normal levels in green-seedlings at 32 degrees C. Our observations suggest that the nuclear-encoded Cpn60alpha protein TCD9 plays a crucial role in chloroplast development at early leaf stage of rice. TCD9 Senescence-induced serotonin biosynthesis and its role in delaying senescence in rice leaves 2009 Plant Physiol Department of Biotechnology, Interdisciplinary Program for Bioenergy and Biomaterials of Graduate School, Agricultural Plant Stress Research Center, Chonnam National University, Gwangju 500-757, South Korea. Serotonin, which is well known as a pineal hormone in mammals, plays a key role in conditions such as mood, eating disorders, and alcoholism. In plants, although serotonin has been suggested to be involved in several physiological roles, including flowering, morphogenesis, and adaptation to environmental changes, its regulation and functional roles are as yet not characterized at the molecular level. In this study, we found that serotonin is greatly accumulated in rice (Oryza sativa) leaves undergoing senescence induced by either nutrient deprivation or detachment, and its synthesis is closely coupled with transcriptional and enzymatic induction of the tryptophan biosynthetic genes as well as tryptophan decarboxylase (TDC). Transgenic rice plants that overexpressed TDC accumulated higher levels of serotonin than the wild type and showed delayed senescence of rice leaves. However, transgenic rice plants, in which expression of TDC was suppressed through an RNA interference (RNAi) system, produced less serotonin and senesced faster than the wild type, suggesting that serotonin is involved in attenuating leaf senescence. The senescence-retarding activity of serotonin is associated with its high antioxidant activity compared to either tryptophan or chlorogenic acid. Results of TDC overexpression and TDC RNAi plants suggest that TDC plays a rate-limiting role for serotonin accumulation, but the synthesis of serotonin depends on an absolute amount of tryptophan accumulation by the coordinate induction of the tryptophan biosynthetic genes. In addition, immunolocalization analysis revealed that serotonin was abundant in the vascular parenchyma cells, including companion cells and xylem-parenchyma cells, suggestive of its involvement in maintaining the cellular integrity of these cells for facilitating efficient nutrient recycling from senescing leaves to sink tissues during senescence. TDC1,TDC2 Serotonin accumulation in transgenic rice by over-expressing tryptophan decarboxylase results in a dark brown phenotype and stunted growth 2012 Plant Mol Biol Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan. A mutant M47286 with a stunted growth, low fertility and dark-brown phenotype was identified from a T-DNA-tagged rice mutant library. This mutant contained a copy of the T-DNA tag inserted at the location where the expression of two putative tryptophan decarboxylase genes, TDC-1 and TDC-3, were activated. Enzymatic assays of both recombinant proteins showed tryptophan decarboxylase activities that converted tryptophan to tryptamine, which could be converted to serotonin by a constitutively expressed tryptamine 5' hydroxylase (T5H) in rice plants. Over-expression of TDC-1 and TDC-3 in transgenic rice recapitulated the stunted growth, darkbrown phenotype and resulted in a low fertility similar to M47286. The degree of stunted growth and dark-brown color was proportional to the expression levels of TDC-1 and TDC-3. The levels of tryptamine and serotonin accumulation in these transgenic rice lines were also directly correlated with the expression levels of TDC-1 and TDC-3. A mass spectrometry assay demonstrated that the darkbrown leaves and hulls in the TDC-overexpressing transgenic rice were caused by the accumulation of serotonin dimer and that the stunted growth and low fertility were also caused by the accumulation of serotonin and serotonin dimer, but not tryptamine. These results represent the first evidence that over-expression of TDC results in stunted growth, low fertility and the accumulation of serotonin, which when converted to serotonin dimer, leads to a dark brown plant color. TDC1,TDC3 Loss of function of the IAA-glucose hydrolase gene TGW6 enhances rice grain weight and increases yield 2013 Nat Genet National Institute of Agrobiological Sciences, Tsukuba, Japan. kenshi@nias.affrc.go.jp Increases in the yield of rice, a staple crop for more than half of the global population, are imperative to support rapid population growth. Grain weight is a major determining factor of yield. Here, we report the cloning and functional analysis of THOUSAND-GRAIN WEIGHT 6 (TGW6), a gene from the Indian landrace rice Kasalath. TGW6 encodes a novel protein with indole-3-acetic acid (IAA)-glucose hydrolase activity. In sink organs, the Nipponbare tgw6 allele affects the timing of the transition from the syncytial to the cellular phase by controlling IAA supply and limiting cell number and grain length. Most notably, loss of function of the Kasalath allele enhances grain weight through pleiotropic effects on source organs and leads to significant yield increases. Our findings suggest that TGW6 may be useful for further improvements in yield characteristics in most cultivars. TGW6 THIS1 is a putative lipase that regulates tillering, plant height, and spikelet fertility in rice 2013 J Exp Bot Key Laboratory of Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China. Proper branching and successful reproductive growth is of great importance for rice productivity. Substantial progress has been made in uncovering the molecular mechanisms underlying tillering control and spikelet sterility. However, rice tillering is developmentally controlled, and how it is regulated coordinately with reproductive growth remains unclear. This study characterized a rice mutant, the most obvious phenotypes of which are high tillering, reduced height, and infertile spikelets (named this1). Similarly to the high tiller number and dwarf mutants in rice, the increased tiller number of this1 plants is ascribed to the release of tiller bud outgrowth rather than to increased tiller bud formation. In the this1 mutant, however, the accelerated rate of branching was delayed until the stem elongation stage, while other mutants lost the ability to control branching at all developmental stages. The seed-setting rate of this1 was less than half that of the wild type, owing to defects in pollen maturation, anther dehiscence, and flower opening. Histological analyses showed that the mutation in this1 resulted in anisotropic cell expansion and cell division. Using a map-based cloning approach, This1 was found to encode a class III lipase. Homology searches revealed that THIS1 is conserved in both monocots and eudicots, suggesting that it plays fundamental role in regulating branch and spikelet fertility, as well as other aspects of developmental control. The relative change in expression of marker genes highlighted the possibility that This1 is involved in phytohormone signalling pathways, such as those for strigolactone and auxin. Thus, This1 provides joint control between shoot branching and reproductive development. THIS1 The bHLH142 Transcription Factor Coordinates with TDR1 to Modulate the Expression of EAT1 and Regulate Pollen Development in Rice 2014 Plant Cell Academia Sinica Biotechnology Center in Southern Taiwan, Tainan 741, Taiwan Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan Male sterility plays an important role in F1 hybrid seed production. We identified a male-sterile rice (Oryza sativa) mutant with impaired pollen development and a single T-DNA insertion in the transcription factor gene bHLH142. Knockout mutants of bHLH142 exhibited retarded meiosis and defects in tapetal programmed cell death. RT-PCR and in situ hybridization analyses showed that bHLH142 is specifically expressed in the anther, in the tapetum, and in meiocytes during early meiosis. Three basic helix-loop-helix transcription factors, UDT1 (bHLH164), TDR1 (bHLH5), and EAT1/DTD1 (bHLH141) are known to function in rice pollen development. bHLH142 acts downstream of UDT1 and GAMYB but upstream of TDR1 and EAT1 in pollen development. In vivo and in vitro assays demonstrated that bHLH142 and TDR1 proteins interact. Transient promoter assays demonstrated that regulation of the EAT1 promoter requires bHLH142 and TDR1. Consistent with these results, 3D protein structure modeling predicted that bHLH142 and TDR1 form a heterodimer to bind to the EAT1 promoter. EAT1 positively regulates the expression of AP37 and AP25, which induce tapetal programmed cell death. Thus, in this study, we identified bHLH142 as having a pivotal role in tapetal programmed cell death and pollen development. TIP2|bHLH142 The Rice Basic Helix-Loop-Helix Transcription Factor TDR INTERACTING PROTEIN2 Is a Central Switch in Early Anther Development 2014 Plant Cell State Key Laboratory of Hybrid Rice, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China In male reproductive development in plants, meristemoid precursor cells possessing transient, stem cell-like features undergo cell divisions and differentiation to produce the anther, the male reproductive organ. The anther contains centrally positioned microsporocytes surrounded by four distinct layers of wall: the epidermis, endothecium, middle layer, and tapetum. Here, we report that the rice (Oryza sativa) basic helix-loop-helix (bHLH) protein TDR INTERACTING PROTEIN2 (TIP2) functions as a crucial switch in the meristemoid transition and differentiation during early anther development. The tip2 mutants display undifferentiated inner three anther wall layers and abort tapetal programmed cell death, causing complete male sterility. TIP2 has two paralogs in rice, TDR and EAT1, which are key regulators of tapetal programmed cell death. We revealed that TIP2 acts upstream of TDR and EAT1 and directly regulates the expression of TDR and EAT1. In addition, TIP2 can interact with TDR, indicating a role of TIP2 in later anther development. Our findings suggest that the bHLH proteins TIP2, TDR, and EAT1 play a central role in regulating differentiation, morphogenesis, and degradation of anther somatic cell layers, highlighting the role of paralogous bHLH proteins in regulating distinct steps of plant cell-type determination. TIP2|bHLH142 ZINC-INDUCED FACILITATOR-LIKE family in plants: lineage-specific expansion in monocotyledons and conserved genomic and expression features among rice (Oryza sativa) paralogs 2011 BMC Plant Biol Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenida Bento Goncalves 9500, Porto Alegre, Brazil. BACKGROUND: Duplications are very common in the evolution of plant genomes, explaining the high number of members in plant gene families. New genes born after duplication can undergo pseudogenization, neofunctionalization or subfunctionalization. Rice is a model for functional genomics research, an important crop for human nutrition and a target for biofortification. Increased zinc and iron content in the rice grain could be achieved by manipulation of metal transporters. Here, we describe the ZINC-INDUCED FACILITATOR-LIKE (ZIFL) gene family in plants, and characterize the genomic structure and expression of rice paralogs, which are highly affected by segmental duplication. RESULTS: Sequences of sixty-eight ZIFL genes, from nine plant species, were comparatively analyzed. Although related to MSF_1 proteins, ZIFL protein sequences consistently grouped separately. Specific ZIFL sequence signatures were identified. Monocots harbor a larger number of ZIFL genes in their genomes than dicots, probably a result of a lineage-specific expansion. The rice ZIFL paralogs were named OsZIFL1 to OsZIFL13 and characterized. The genomic organization of the rice ZIFL genes seems to be highly influenced by segmental and tandem duplications and concerted evolution, as rice genome contains five highly similar ZIFL gene pairs. Most rice ZIFL promoters are enriched for the core sequence of the Fe-deficiency-related box IDE1. Gene expression analyses of different plant organs, growth stages and treatments, both from our qPCR data and from microarray databases, revealed that the duplicated ZIFL gene pairs are mostly co-expressed. Transcripts of OsZIFL4, OsZIFL5, OsZIFL7, and OsZIFL12 accumulate in response to Zn-excess and Fe-deficiency in roots, two stresses with partially overlapping responses. CONCLUSIONS: We suggest that ZIFL genes have different evolutionary histories in monocot and dicot lineages. In rice, concerted evolution affected ZIFL duplicated genes, possibly maintaining similar expression patterns between pairs. The enrichment for IDE1 boxes in rice ZIFL gene promoters suggests a role in Zn-excess and Fe-deficiency up-regulation of ZIFL transcripts. Moreover, this is the first description of the ZIFL gene family in plants and the basis for functional studies on this family, which may play important roles in Zn and Fe homeostasis in plants. TOM1|OsZIFL4 Expressed sequence tags from cultured cells of rice (Oryza sativa L.) under stressed conditions: analysis of transcripts of genes engaged in ATP-generating pathways 1994 Plant Mol Biol Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan. Large-scale sequencing of randomly selected cDNA clones was used to isolate numerous genes in rice (Oryza sativa L.). Total RNA used for cDNA synthesis was prepared from suspension-cultured cells of rice grown under stressed conditions, such as in saline or nitrogen-starvation conditions. A total of 780 cDNA clones were partially sequenced and about 15% could be identified as putative genes. In the library constructed under saline conditions, we identified several genes associated with signal transduction, such as protein kinase and small GTP-binding protein genes. Many stress-related genes were isolated from both the saline and nitrogen-starvation libraries. These results indicate that stress treatment of suspension-cultured cells makes it possible to efficiently isolate various types of plant genes. To examine the usefulness of such tagged cDNAs for the study of gene expression in a specific metabolic pathway, we analyzed mRNA levels of genes engaged in the ATP-generating pathways in cultured cells of rice under different stresses, such as 20% sucrose, salt stress, cold stress and nitrogen-starvation stress. The results suggest that the coordinated induction of several genes in key steps under stressed conditions may be essential for activation of the entire energy-producing pathway to maintain homeostasis in rice cells. Expressed sequence tags identified by random cDNA sequencing provide the opportunity to generate a transcript map of rice genes. TPI,PGI1 Cytosolic triosephosphate isomerase is a single gene in rice 1993 Plant Physiol Institute of Developmental and Molecular Biology, Texas A&M University, College Station 77843-3155. A cDNA clone encoding rice (Oryza sativa L.) cytosolic triosephosphate isomerase (TPI), an important glycolytic enzyme, was isolated and characterized. The clone (pRTPI-6) contains an open reading frame of 759 base pairs, encoding a polypeptide chain of 253 amino acid residues (M(r) 27,060). The identity of this clone was defined by its high homology (85% nucleotide sequence and 89% amino acid sequence identical match) with a maize mRNA sequence encoding the cytosolic TPI and with TPIs from other species. Genomic DNA blot analysis using the cDNA as a probe showed that the cytosolic TPI gene is present as a single copy per haploid rice genome, as opposed to that found in maize, in which multiple TPI gene copies exist. A single TPI mRNA species of about 1100 nucleotides was detected by gel blot hybridization analysis of RNA isolated from root, culm, and leaf tissues, indicating that its expression is ubiquitous. Based on sequence comparison and molecular analysis, we propose that the chloroplast-located TPI may be encoded by divergent structural nuclear genes in rice. TPI Abscisic Acid-Induced Transcription Is Mediated by Phosphorylation of an Abscisic Acid Response Element Binding Factor, TRAB1 2002 The Plant Cell Online Center for Molecular Biology and Genetics, Mie University, 1515 Kamihama-cho, Tsu 514-8507, Japan. The rice basic domain/Leu zipper factor TRAB1 binds to abscisic acid (ABA) response elements and mediates ABA signals to activate transcription. We show that TRAB1 is phosphorylated rapidly in an in vivo labeling experiment and by phosphatase-sensitive mobility shifts on SDS-polyacrylamide gels. We had shown previously that a chimeric promoter containing GAL4 binding sites became ABA inducible when a GAL4 binding domain-TRAB1 fusion protein was present. This expression system allowed us to assay the ABA response function of TRAB1. Using this system, we show that Ser-102 of TRAB1 is critical for this function. Because no ABA-induced mobility shift was observed when Ser-102 was replaced by Ala, we suggest that this Ser residue is phosphorylated in response to ABA. Cell fractionation experiments, as well as fluorescence microscopy observations of transiently expressed green fluorescent protein-TRAB1 fusion protein, indicated that TRAB1 was localized in the nucleus independently of ABA. Our results suggest that the terminal or nearly terminal event of the primary ABA signal transduction pathway is the phosphorylation in the nucleus of preexisting TRAB1. TRAB1|OsbZIP66 Characterization of rice tryptophan decarboxylases and their direct involvement in serotonin biosynthesis in transgenic rice 2007 Planta Department of Molecular Biotechnology, Agricultural Plant Stress Research Center, Biotechnology Research Institute, Chonnam National University, Gwangju, South Korea. L-Tryptophan decarboxylase (TDC) and L-tyrosine decarboxylase (TYDC) belong to a family of aromatic L-amino acid decarboxylases and catalyze the conversion of tryptophan and tyrosine into tryptamine and tyramine, respectively. The rice genome has been shown to contain seven TDC or TYDC-like genes. Three of these genes for which cDNA clones were available were characterized to assign their functions using heterologous expression in Escherichia coli and rice (Oryza sativa cv. Dongjin). The purified products of two of the genes were expressed in E. coli and exhibited TDC activity, whereas the remaining gene could not be expressed in E. coli. The recombinant TDC protein with the greatest TDC activity showed a K (m) of 0.69 mM for tryptophan, and its activity was not inhibited by phenylalanine or tyrosine, indicating a high level of substrate specificity toward tryptophan. The ectopic expression of the three cDNA clones in rice led to the abundant production of the products of the encoded enzymes, tyramine and tryptamine. The overproduction of TYDC resulted in stunted growth and a lack of seed production due to tyramine accumulation, which increased as the plant aged. In contrast, transgenic plants that produced TDC showed a normal phenotype and contained 25-fold and 11-fold higher serotonin in the leaves and seeds, respectively, than the wild-type plants. The overproduction of either tyramine or serotonin was not strongly related to the enhanced synthesis of tyramine or serotonin derivatives, such as feruloyltyramine and feruloylserotonin, which are secondary metabolites that act as phytoalexins in plants. TYDC Rice Undeveloped Tapetum1 is a major regulator of early tapetum development 2005 Plant Cell National Research Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea. jkhim@postech.ac.kr The tapetum, the innermost of four sporophytic layers in the anther wall, comes in direct contact with the developing male gametophyte and is thought to play a crucial role in the development and maturation of microspores. Here, we report the identification of rice (Oryza sativa) Undeveloped Tapetum1 (Udt1), which is required for the differentiation of secondary parietal cells to mature tapetal cells. T-DNA or retrotransposon Tos17 insertions in the Udt1 gene caused male sterility. The anther walls and meiocytes of the mutants were normal during the early premeiosis stage, but their tapeta failed to differentiate and became vacuolated during the meiotic stage. In addition, meiocytes did not develop to microspores, and middle layer degeneration was inhibited. Consequently, the anther locules contained no pollen. The UDT1:green fluorescent protein fusion protein was localized to the nucleus. This, together with its homology with other basic helix-loop-helix proteins, suggests that UDT1 is a transcription factor. DNA microarray analysis identified 958 downregulated and 267 upregulated genes in the udt1-1 anthers, suggesting that Udt1 plays a major role in maintaining tapetum development, starting in early meiosis. Udt1 Inactivation of the UGPase1 gene causes genic male sterility and endosperm chalkiness in rice (Oryza sativa L.) 2008 Plant J Department of Plant Science and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea. A rice genic male-sterility gene ms-h is recessive and has a pleiotropic effect on the chalky endosperm. After fine mapping, nucleotide sequencing analysis of the ms-h gene revealed a single nucleotide substitution at the 3'-splice junction of the 14th intron of the UDP-glucose pyrophosphorylase 1 (UGPase1; EC2.7.7.9) gene, which causes the expression of two mature transcripts with abnormal sizes caused by the aberrant splicing. An in vitro functional assay showed that both proteins encoded by the two abnormal transcripts have no UGPase activity. The suppression of UGPase by the introduction of a UGPase1-RNAi construct in wild-type plants nearly eliminated seed set because of the male defect, with developmental retardation similar to the ms-h mutant phenotype, whereas overexpression of UGPase1 in ms-h mutant plants restored male fertility and the transformants produced T(1) seeds that segregated into normal and chalky endosperms. In addition, both phenotypes were co-segregated with the UGPase1 transgene in segregating T(1) plants, which demonstrates that UGPase1 has functional roles in both male sterility and the development of a chalky endosperm. Our results suggest that UGPase1 plays a key role in pollen development as well as seed carbohydrate metabolism. OsUgp1|Ugp1|UGPase1 Cloning of cDNA for UDP-glucose pyrophosphorylase and the expression of mRNA in rice endosperm 2002 Theor Appl Genet Laboratory of Plant Molecular Genetics and Breeding, Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, 1-23 Wakabamachi, Tsuruoka, 997-8555 Japan, tabe@tds1.tr.yamagata-u.ac.jp Rice endosperm UDP-glucose pyrophosphorylase (UGPase) cDNA clones were isolated by screening a lambda ZAP II library prepared from poly (A(+)) RNA of japonica rice (cv Sasanishiki) endosperm with a probe of potato UGPase cDNA. One cDNA clone, possessing about 1,700 nucleotides, contained the complete open reading frame of rice UGPase. At the nucleotide-sequence level, the UGPase cDNA of rice endosperm had high homology with the UGPase cDNA of barley endosperm (84%) and potato tuber (71%). The calculated molecular weight (50 kDa) agrees with the value determined by SDS-PAGE (51 kDa). At the amino-acid sequence level, rice UGPase has high homology with the UGPase of barley (92%) and potato (85%). The enzyme contained conserved sequence elements which are thought to be involved in substrate binding and catalytic activity. A Southern-blot analysis indicated that the gene existed as a single copy. Expression of the enzyme in rice endosperm examined by Northern-blot analysis was high at 10-15 days after heading. OsUgp1|Ugp1|UGPase1 Identification and characterization of proteins involved in rice urea and arginine catabolism 2010 Plant Physiol Key Laboratory of Plant and Soil Interactions, College of Resources and Environmental Sciences, China Agricultural University, Beijing, China. Rice (Oryza sativa) production relies strongly on nitrogen (N) fertilization with urea, but the proteins involved in rice urea metabolism have not yet been characterized. Coding sequences for rice arginase, urease, and the urease accessory proteins D (UreD), F (UreF), and G (UreG) involved in urease activation were identified and cloned. The functionality of urease and the urease accessory proteins was demonstrated by complementing corresponding Arabidopsis (Arabidopsis thaliana) mutants and by multiple transient coexpression of the rice proteins in Nicotiana benthamiana. Secondary structure models of rice (plant) UreD and UreF proteins revealed a possible functional conservation to bacterial orthologs, especially for UreF. Using amino-terminally StrepII-tagged urease accessory proteins, an interaction between rice UreD and urease could be shown. Prokaryotic and eukaryotic urease activation complexes seem conserved despite limited protein sequence conservation for UreF and UreD. In plant metabolism, urea is generated by the arginase reaction. Rice arginase was transiently expressed as a carboxyl-terminally StrepII-tagged fusion protein in N. benthamiana, purified, and biochemically characterized (K(m) = 67 mm, k(cat) = 490 s(-1)). The activity depended on the presence of manganese (K(d) = 1.3 microm). In physiological experiments, urease and arginase activities were not influenced by the external N source, but sole urea nutrition imbalanced the plant amino acid profile, leading to the accumulation of asparagine and glutamine in the roots. Our data indicate that reduced plant performance with urea as N source is not a direct result of insufficient urea metabolism but may in part be caused by an imbalance of N distribution. UreG|OsUreG Isolation of a novel UVB-tolerant rice mutant obtained by exposure to carbon-ion beams 2013 J Radiat Res Graduate School of Life Sciences, Tohoku University, Sendai, 980-8577, Japan. UVB radiation suppresses photosynthesis and protein biosynthesis in plants, which in turn decreases growth and productivity. Here, an ultraviolet-B (UVB)-tolerant rice mutant, utr319 (UV Tolerant Rice 319), was isolated from a mutagenized population derived from 2500 M1 seeds (of the UVB-resistant cultivar 'Sasanishiki') that were exposed to carbon ions. The utr319 mutant was more tolerant to UVB than the wild type. Neither the levels of UVB-induced cyclobutane pyrimidine dimers (CPDs) or (6-4) pyrimidine-pyrimidone photodimers [(6-4) photoproducts], nor the repair of CPDs or (6-4) photoproducts, was altered in the utr319 mutant. Thus, the utr319 mutant may be impaired in the production of a previously unidentified factor that confers UVB tolerance. To identify the mutated region in the utr319 mutant, microarray-based comparative genomic hybridization analysis was performed. Two adjacent genes on chromosome 7, Os07g0264900 and Os07g0265100, were predicted to represent the mutant allele. Sequence analysis of the chromosome region in utr319 revealed a deletion of 45 419 bp. RNAi analysis indicated that Os07g0265100 is most likely the mutated gene. Database analysis indicated that the Os07g0265100 gene, UTR319, encodes a putative protein with unknown characteristics or function. In addition, the homologs of UTR319 are conserved only among land plants. Therefore, utr319 is a novel UVB-tolerant rice mutant and UTR319 may be crucial for the determination of UVB sensitivity in rice, although the function of UTR319 has not yet been determined. UTR319 Cloning and expression of a UDP-glucuronic acid decarboxylase gene in rice 2003 J Exp Bot Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Gakuen-cho 1-1, Sakai, Osaka 599-8531, Japan. A cDNA fragment was cloned from rice immature seeds by the RT-PCR method. The deduced amino acid sequence of the cDNA showed a high degree of identity with UDP-d-glucuronic acid decarboxylase (UXS) from other plants and was most similar to the soluble UXS from Arabidopsis. The recombinant protein, expressed in an Escherichia coli system, catalysed the conversion of UDP-d-glucuronic acid to UDP-d-xylose, confirming that the gene encoded UXS. The uxs gene was expressed in mature, harvested rice seeds as well as in immature seeds 14 d post-anthesis, suggesting that the uxs gene is necessary at the beginning of the germination period. This is the first report of the cloning of the uxs gene from monocots. UXS,UXS-4 The rice nuclear gene, VIRESCENT 2, is essential for chloroplast development and encodes a novel type of guanylate kinase targeted to plastids and mitochondria 2007 Plant J Department of Biological Sciences, Faculty of Sciences, Kyushu University, Fukuoka 812-8581, Japan. Guanylate kinase (GK) is a critical enzyme in guanine nucleotide metabolism pathways, catalyzing the phosphorylation of (d)GMP to (d)GDP. Here we show that a novel gene, VIRESCENT 2 (V2), encodes a new type of GK (designated pt/mtGK) that is localized in plastids and mitochondria. We initially identified the V2 gene by positional cloning of the rice v2 mutant. The v2 mutant is temperature-sensitive and develops chlorotic leaves at restrictive temperatures. The v2 mutation causes inhibition of chloroplast differentiation; in particular, it disrupts the chloroplast translation machinery during early leaf development [Sugimoto et al. (2004)Plant Cell Physiol. 45, 985]. In the bacterial and animal species studied to date, GK is localized in the cytoplasm and participates in maintenance of the guanine nucleotide pools required for many fundamental cellular processes. Phenotypic analysis of rice seedlings with RNAi knockdown of cytosolic GK (designated cGK) showed that cGK is indispensable for the growth and development of plants, but not for chloroplast development. Thus, rice has two types of GK, as does Arabidopsis, suggesting that higher plants have two types of GK. Our results suggest that, of the two types of GK, only pt/mtGK is essential for chloroplast differentiation. v2 WAVY LEAF1, an ortholog of Arabidopsis HEN1, regulates shoot development by maintaining MicroRNA and trans-acting small interfering RNA accumulation in rice 2010 Plant Physiol Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan. In rice (Oryza sativa), trans-acting small interfering RNA (ta-siRNA) is essential for shoot development, including shoot apical meristem (SAM) formation and leaf morphogenesis. The rice wavy leaf1 (waf1) mutant has been identified as an embryonic mutant resembling shoot organization1 (sho1) and sho2, homologs of a loss-of-function mutant of DICER-LIKE4 and a hypomorphic mutant of ARGONAUTE7, respectively, which both act in the ta-siRNA production pathway. About half of the waf1 mutants showed seedling lethality due to defects in SAM maintenance, but the rest survived to the reproductive phase and exhibited pleiotropic phenotypes in leaf morphology and floral development. Map-based cloning of WAF1 revealed that it encodes an RNA methyltransferase, a homolog of Arabidopsis (Arabidopsis thaliana) HUA ENHANCER1. The reduced accumulation of small RNAs in waf1 indicated that the stability of the small RNA was decreased. Despite the greatly reduced level of microRNAs and ta-siRNA, microarray and reverse transcription-polymerase chain reaction experiments revealed that the expression levels of their target genes were not always enhanced. A double mutant between sho and waf1 showed an enhanced SAM defect, suggesting that the amount and/or quality of ta-siRNA is crucial for SAM maintenance. Our results indicate that stabilization of small RNAs by WAF1 is indispensable for rice development, especially for SAM maintenance and leaf morphogenesis governed by the ta-siRNA pathway. In addition, the inconsistent relationship between the amount of small RNAs and the level of the target mRNA in waf1 suggest that there is a complex regulatory mechanism that modifies the effects of microRNA/ta-siRNA on the expression of the target gene. WAF1|OsHEN1 Wax-deficient anther1 is involved in cuticle and wax production in rice anther walls and is required for pollen development 2006 Plant Cell National Research Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea. In vegetative leaf tissues, cuticles including cuticular waxes are important for protection against nonstomatal water loss and pathogen infection as well as for adaptations to environmental stress. However, their roles in the anther wall are rarely studied. The innermost layer of the anther wall (the tapetum) is essential for generating male gametes. Here, we report the characterization of a T-DNA insertional mutant in the Wax-deficient anther1 (Wda1) gene of rice (Oryza sativa), which shows significant defects in the biosynthesis of very-long-chain fatty acids in both layers. This gene is strongly expressed in the epidermal cells of anthers. Scanning electron microscopy analyses showed that epicuticular wax crystals were absent in the outer layer of the anther and that microspore development was severely retarded and finally disrupted as a result of defective pollen exine formation in the mutant anthers. These biochemical and developmental defects in tapetum found in wda1 mutants are earlier events than those in other male-sterile mutants, which showed defects of lipidic molecules in exine. Our findings provide new insights into the biochemical and developmental aspects of the role of waxes in microspore exine development in the tapetum as well as the role of epicuticular waxes in anther expansion. WDA1 Mutation in Wilted Dwarf and Lethal 1 (WDL1) causes abnormal cuticle formation and rapid water loss in rice 2010 Plant Mol Biol Department of Life Science, Pohang University of Science and Technology, Republic of Korea. Epidermal cell layers play important roles in plant defenses against various environmental stresses. Here we report the identification of a cuticle membrane mutant, wilted dwarf and lethal 1 (wdl1), from a rice T-DNA insertional population. The mutant is dwarf and die at seedling stage due to increased rates of water loss. Stomatal cells and pavement cells are smaller in the mutant, suggesting that WDL1 affects epidermal cell differentiation. T-DNA was inserted into a gene that encodes a protein belonging to the SGNH subfamily, within the GDSL lipase superfamily. The WDL1-sGFP signal coincided with the RFP signal driven by AtBIP-mRFP, indicating that WDL1 is an ER protein. SEM analyses showed that their leaves have a disorganized crystal wax layer. Cross-sectioning reveals loose packing of the cuticle and irregular thickness of cell wall. Detailed analyses of the epicuticular wax showed no significant changes either in the total amount and amounts of each monomer or in the levels of lipid polymers, including cutin and other covalently bound lipids, attached to the cell wall. We propose that WDL1 is involved in cutin organization, affecting depolymerizable components. WDL1 The rice nuclear gene WLP1 encoding a chloroplast ribosome L13 protein is needed for chloroplast development in rice grown under low temperature conditions 2014 Plant Mol Biol State Key Laboratory of Rice Biology, Key Laboratory of Rice Biology and Breeding of Ministry of Agriculture, China National Rice Research Institute, Hangzhou, 310006, China. Plastidial ribosome proteins (PRPs) form the major component of the plastidial ribosome. Here we describe a rice mutant named wlp1 (white leaf and panicles 1) selected from a population of tissue culture regenerants. The early seedling leaves of the mutant were albino, as was the immature panicle at heading, and the phenotype was more strongly expressed in plants exposed to low temperature conditions. Changes in the leaf pigmentation of the mutant were due to altered chlorophyll content and chloroplast development. Positional cloning of WLP1, followed by complementation and knock-down experiments, showed that it encodes a 50S ribosome L13 protein. The WLP1 protein localized to the chloroplast. WLP1 was mainly transcribed in green tissues and particularly abundantly in the early seedling leaves. In addition, the expression level of WLP1 was induced by the low temperature. The transcription pattern of a number of genes involved in plastidial transcription/translation and in photosynthesis was altered in the wlp1 mutants. These results reveal that WLP1 is required for normal chloroplast development, especially under low temperature conditions. This is the first report on the function of PRPs in rice. WLP1 The WUSCHEL-related homeobox gene WOX11 is required to activate shoot-borne crown root development in rice 2009 Plant Cell National Key Laboratory for Crop Genetic Improvement, Huazhong Agricultural University, 430070 Wuhan, China. In rice (Oryza sativa), the shoot-borne crown roots are the major root type and are initiated at lower stem nodes as part of normal plant development. However, the regulatory mechanism of crown root development is poorly understood. In this work, we show that a WUSCHEL-related Homeobox (WOX) gene, WOX11, is involved in the activation of crown root emergence and growth. WOX11 was found to be expressed in emerging crown roots and later in cell division regions of the root meristem. The expression could be induced by exogenous auxin or cytokinin. Loss-of-function mutation or downregulation of the gene reduced the number and the growth rate of crown roots, whereas overexpression of the gene induced precocious crown root growth and dramatically increased the root biomass by producing crown roots at the upper stem nodes and the base of florets. The expressions of auxin- and cytokinin-responsive genes were affected in WOX11 overexpression and RNA interference transgenic plants. Further analysis showed that WOX11 directly repressed RR2, a type-A cytokinin-responsive regulator gene that was found to be expressed in crown root primordia. The results suggest that WOX11 may be an integrator of auxin and cytokinin signaling that feeds into RR2 to regulate cell proliferation during crown root development. WOX11 A novel chloroplast-localized pentatricopeptide repeat protein involved in splicing affects chloroplast development and abiotic stress response in rice 2014 Mol Plant State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha 410082, P.R. China b National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China. Pentatricopeptide repeat (PPR) proteins comprise a large family in higher plants and modulate organellar gene expression by participating in various aspects of organellar RNA metabolism. In rice, the family contains 477 members, and the majority of their functions remain unclear. In this study, we isolated and characterized a rice mutant, white stripe leaf (wsl), which displays chlorotic striations early in development. Map-based cloning revealed that WSL encodes a newly identified rice PPR protein which targets the chloroplasts. In wsl mutants, PEP-dependent plastid gene expression was significantly down-regulated, and plastid rRNAs and translation products accumulate to very low levels. Consistently with the observations, wsl shows a strong defect in the splicing of chloroplast transcript rpl2, resulting in aberrant transcript accumulation and its product reduction in the mutant. The wsl shows enhanced sensitivity to ABA, salinity, and sugar, and it accumulates more H2O2 than wild-type. These results suggest the reduced translation efficiency may affect the response of the mutant to abiotic stress. WSL Wax Crystal-Sparse Leaf1 encodes a beta-ketoacyl CoA synthase involved in biosynthesis of cuticular waxes on rice leaf 2008 Planta State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, 100101 Beijing, People's Republic of China. Cuticular waxes, forming the plant/atmosphere interface of plants colonizing the terrestrial environment, are complex mixtures of very-long chain fatty acids (VLCFAs) and their derivatives. In VLCFAs biosynthesis, beta-ketoacyl CoA synthase (E.C.2.3.1.119, KCS) is the key enzyme. Using T-DNA insertional mutagenesis, we identified a cuticle-deficient rice mutant, which displayed a pleiotropic phenotype including reduced growth, leaf fusion, sparse wax crystals, enhanced sensitivity to drought and low fertility. Further analysis indicated that T-DNA was inserted in the 5'-UTR intron of the affected gene, Wax Crystal-Sparse Leaf1 (WSL1), and abnormal transcript caused the loss-of-function of WSL1 gene. Genetic complementation experiment confirmed the function of the candidate gene. WSL1 was predicted to encode a polypeptide containing a conserved FAE1_CUT1_RppA domain typical of the KCS family proteins. Qualitative and quantitative wax composition analyses by gas chromatography-mass spectrometry (GC-MS) demonstrated a marked reduction of total cuticular wax load on wsl1 leaf blades and sheaths, and VLCFA precursors of C20-C24 decreased in both. Moreover, ubiquitous expression of the WSL1 gene gave a hint that WSL1-catalyzed elongation of VLCFAs might participate in a wide range of rice growth and development processes beyond biosynthesis of cuticular waxes. WSL1 Wax crystal-sparse leaf2, a rice homologue of WAX2/GL1, is involved in synthesis of leaf cuticular wax 2012 Planta National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agriculture Sciences, Beijing, 100081, China. Epicuticular wax in plants limits non-stomatal water loss, inhibits postgenital organ fusion, protects plants against damage from UV radiation and imposes a physical barrier against pathogen infection. Here, we give a detailed description of the genetic, physiological and morphological consequences of a mutation in the rice gene WSL2, based on a comparison between the wild-type and an EMS mutant. The mutant's leaf cuticle membrane is thicker and less organized than that of the wild type, and its total wax content is diminished by ~80%. The mutant is also more sensitive to drought stress. WSL2 was isolated by positional cloning, and was shown to encode a homologue of the Arabidopsis thaliana genes CER3/WAX2/YRE/FLP1 and the maize gene GL1. It is expressed throughout the plant, except in the root. A transient assay carried out in both A. thaliana and rice protoplasts showed that the gene product is deposited in the endoplasmic reticulum. An analysis of the overall composition of the wax revealed that the mutant produces a substantially reduced quantity of C22-C32 fatty acids, which suggests that the function of WSL2 is associated with the elongation of very long-chain fatty acids. OsGL1-1|WSL2 Rice OsGL1-1 is involved in leaf cuticular wax and cuticle membrane 2011 Mol Plant State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Cuticular wax forms a hydrophobic barrier on aerial plant organs; it plays an important role in protecting a plant from damage caused by many forms of environmental stress. In the present study, we characterized a rice leaf wax-deficient mutant osgl1-1 derived from a spontaneous mutation, which exhibited a wax-deficient and highly hydrophilic leaf phenotype. We cloned the OsGL1-1 gene by the map-based cloning method and performed a complementation test to confirm the function of the candidate gene. Molecular studies revealed that OsGL1-1 was a member of the OsGL1 family, and contained regions that were homologous to some regions in sterol desaturases and short-chain dehydrogenases/reductases. Compared to the wild-type, the osgl1-1 mutant showed decreased cuticular wax deposition, thinner cuticular membrane, decreased chlorophyll leaching, increased rate of water loss, and enhanced sensitivity to drought. OsGL1-1 is expressed ubiquitously in rice. The transient expression of OsGL1-1-green fluorescent protein fusion protein indicated that OsGL1-1 is localized in the cytoplasm, plasma membrane, and nucleus. OsGL1-1|WSL2 Potential of Waxy gene microsatellite and single-nucleotide polymorphisms to develop japonica varieties with desired amylose levels in rice (Oryza sativa L.) 2007 Journal of Cereal Science Laboratory of Plant Genetic Engineering, Instituto de Tecnologia Química e Biológica (ITQB)/Instituto de Biologia Experimental e Tecnológica (IBET), Quinta do Marquês, 2784-505 Oeiras, Portugal The Waxy (Wx) gene is responsible for the synthesis of amylose, a key determinant of the cooking and processing qualities of rice. Polymorphisms of CT-microsatellite and G–T single-nucleotide polymorphism (SNP) in the Wx gene and their relationship to amylose content (Ac) were explored using 178 non-waxy rice genotypes. Nine Wx microsatellite alleles, namely (CT)10 and 11, and (CT)14–20 were identified and 11 haplotypes were recognised by different combinations of CT-microsatellite and G–T SNP. Amylose content analysed in a random set of 39 genotypes was correlated with different microsatellite alleles/haplotypes. The highest Ac levels (>30%) correlated with (CT)10 and 16, high (26–30%) with (CT)11, 15 and 20, and intermediate (21–25%) with (CT)14, in all cases with G at the G–T SNP. The CT-classes (CT)17 and 18 (mean Ac value of 21%), could be subdivided into low amylose haplotypes (16–20%) for 17T and 18T and intermediate amylose haplotypes (21–25%) for 17G and 18G. The use of haplotypes proved to discriminate between intermediate and low amylose accessions within the same microsatellite class. Analyses of a segregating population of a cross between low and high Ac parents showed that CT-microsatellite may help to classify breeding lines and identify pollen contamination. We suggest that CT-microsatellite together with G–T SNP may be used as molecular marker by breeders to develop varieties with desired amylose levels. Wx Genetic studies of speciation in cultivated rice. 5. Inter- and intraspecific differentiation in the waxy gene expression of rice 1986 Euphytica National Institute of Genetics, 411, Misima, Japan To get an insight in the gene regulation at the waxy locus of rice, the Wx gene product (Wx protein) controlling the synthesis of amylose was examined by electrophoretic techniques. Among nonwaxy rice strains, two different alleles, Wx a and Wx b, were found at the waxy locus. Wx a drastically enhanced the quantitative level of Wx protein as well as the amylose content in endosperm starch as compared with Wx b. The alleles acted additively in triploid endosperms. This implies that regulatory elements responsible for the Wx gene expression are on the same chromosome. The distribution patterns of Wx a and Wx b in five species of Oryza revealed that the regulatory changes are closely related to racial differentiation within a common rice species (O. sativa), suggesting that Wx b might have been selected for through the difference in grain quality during domestication. Wx Differential regulation of waxy gene expression in rice endosperm 1984 Theor Appl Genet National Institute of Genetics, 411, Misima, Japan. In order to examine the effects of different alleles on the gene expression at the waxy locus, the Wx gene product which controls the synthesis of amylose was isolated from endosperm starch of rice plants and analysed by electrophoretic techniques. The major protein bound to starch granules was absent in most of waxy strains and increased with the number of Wx alleles in triploid endosperms, suggesting that the major protein is the Wx gene product. In addition to wx alleles which result in the absence or drastic reduction of the Wx gene product and amylose, differentiation of Wx alleles seemed to have occurred among nonwaxy rice strains. At least two Wx alleles with different efficiencies in the production of the major protein as well as amylose were detected. These alleles are discussed in relation to regulation of the gene expression. Wx Map-based cloning proves qGC-6, a major QTL for gel consistency of japonica/indica cross, responds by Waxy in rice (Oryza sativa L.) 2011 Theor Appl Genet State Key Laboratory of Rice Biology, China National Rice Research Institute, 31006, Hangzhou, China. In this study, one major QTL affecting gel consistency (GC) of japonica/indica cross was identified on chromosome 6 using a DH population. To understand the molecular mechanism that regulates GC in rice grains, the major QTL (qGC-6) was isolated through a map-based cloning approach utilizing chromosome segment substitution lines (CSSLs). Using 64 plants with extremely soft GC that were selected on recombinant break points between two SSR markers, RM540 and RM8200 in a BC4F2 population, qGC-6 was mapped to a 60-kb DNA region between two STS markers, S26 and S27. These two markers were then used to further identify recombination break points. Finally, qGC-6 was delimited in an interval of a 11-kb region. Gene prediction analysis of the 11-kb DNA sequence containing qGC-6 identified only one putative ORF, which encodes granule-bound starch synthesis protein (Wx protein). Results of sequencing analysis and complementation experiment confirmed that this candidate ORF is responsible for rice GC. Genetic evidences revealed that Wx might contribute equally to the grain amylose content-controlling gene as well as gel consistency. This new information is important to breed rice varieties with improved grain quality. Wx Altered tissue-specific expression at the Wx gene of the opaque mutants in rice 1999 Euphytica Laboratory of Plant Breeding, Faculty of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan Amylose content is a major determinant of the eating quality in rice. To elucidate the allelic diversity at the Waxy (Wx) gene which controls the amylose synthesis, two cultivated strains having opaque endosperms were studied. The gene responsible for opaque endosperms was introduced into the genetic background of the Japonica type of rice by successive backcrosses, and the two near-isogenic lines (NILs) were selected from the B5 generation. The genetic experiments revealed that an allele, Wxop, controls opaque endosperms which show chalky as wx endosperms in spite of the production of amylose. Immunoblotting analysis was carried out to compare the gene expression by using the NILs with 4 different alleles (Wxa, Wxb, Wxop and wx). The level of the gene product bound to starch granules was slightly lower in the NILs with Wxop than that with Wxb, showing a positive correlation with amylose content in the endosperm. Extracts from mature anthers indicated that the gene product was markedly reduced in the NILs with Wxop as well as that with wx, showing an altered expression in the tissue specificity in the Wxop lines. Sequence analysis suggested that the Wxop had been derived from Wxa, independently of the origin of Wxb. The importance of the gene regulation was discussed in relation to diversified phenotypes established during the domestication process. Wx Molecular insights into how a deficiency of amylose affects carbon allocation--carbohydrate and oil analyses and gene expression profiling in the seeds of a rice waxy mutant 2012 BMC Plant Biol College of Life Science, China JiLiang University, Hangzhou 310018, China. BACKGROUND: Understanding carbon partitioning in cereal seeds is of critical importance to develop cereal crops with enhanced starch yields for food security and for producing specified end-products high in amylose, beta-glucan, or fructan, such as functional foods or oils for biofuel applications. Waxy mutants of cereals have a high content of amylopectin and have been well characterized. However, the allocation of carbon to other components, such as beta-glucan and oils, and the regulation of the altered carbon distribution to amylopectin in a waxy mutant are poorly understood. In this study, we used a rice mutant, GM077, with a low content of amylose to gain molecular insight into how a deficiency of amylose affects carbon allocation to other end products and to amylopectin. We used carbohydrate analysis, subtractive cDNA libraries, and qPCR to identify candidate genes potentially responsible for the changes in carbon allocation in GM077 seeds. RESULTS: Carbohydrate analysis indicated that the content of amylose in GM077 seeds was significantly reduced, while that of amylopectin significantly rose as compared to the wild type BP034. The content of glucose, sucrose, total starch, cell-wall polysaccharides and oil were only slightly affected in the mutant as compared to the wild type. Suppression subtractive hybridization (SSH) experiments generated 116 unigenes in the mutant on the wild-type background. Among the 116 unigenes, three, AGP, ISA1 and SUSIBA2-like, were found to be directly involved in amylopectin synthesis, indicating their possible roles in redirecting carbon flux from amylose to amylopectin. A bioinformatics analysis of the putative SUSIBA2-like binding elements in the promoter regions of the upregulated genes indicated that the SUSIBA2-like transcription factor may be instrumental in promoting the carbon reallocation from amylose to amylopectin. CONCLUSION: Analyses of carbohydrate and oil fractions and gene expression profiling on a global scale in the rice waxy mutant GM077 revealed several candidate genes implicated in the carbon reallocation response to an amylose deficiency, including genes encoding AGPase and SUSIBA2-like. We believe that AGP and SUSIBA2 are two promising targets for classical breeding and/or transgenic plant improvement to control the carbon flux between starch and other components in cereal seeds. Wx Molecular Characterization of Wx-mq, a Novel Mutant Gene for Low-amylose Content in Endosperm of Rice (Oryza sativa L.) 2002 Breeding Science National Institute of Crop Science In this paper, we characterized the Wx-mq gene for low amylose content in a rice variety, Milky Queen, at the molecular level. The Wx-mq gene was cloned by RT-PCR, and a nearly full-length cDNA sequence of the gene was determined. Sequence comparison between the Wx-mq gene and the wild type allele (Wx-b), cloned from cv. Koshihikari, revealed that two base changes existed within the coding region; a G to A base change at nucleotide position 497 and a T to C base change at nucleotide position 595. Each nucleotide substitution should generate a missense base change (an Arg-158 to His-158 change in exon4, and a Tyr-191 to His-191 change in exon5). However, it is not known which missense mutation is essential for the activity of the WX protein. To identify rice varieties and lines, which harbored the Wx-mq gene, PCR primers were designed at the gene level. These primers were able to amplify the Wx-mq specific 741 bp band in Milky Queen, and in other rice variety and lines, Milky Princess, Joiku 436 and Etsunan 190, all of which have the same pedigree as that of Milky Queen. On the other hand, no 741 bp band was amplified with the primers in Koshihikari which harbored the wild type allele (Wx-b), and the other low-amylose content variety and line, Snow Pearl and NM391, which do not have the pedigree. Thus, it is possible to detect the Wx-mq gene by PCR. Wx Genetic Polymorphism of Wx Gene and Its Correlation with Main Grain Quality Characteristics in Rice 2007 Rice Science Rice Research Institute, Sichuan Agricultural University, Wenjiang 611130, China The allelic variation of the Wx gene in 50 non-glutinous rice varieties (lines) was analyzed by using the microsatellite marker RM190 [for (CT)n simple sequence repeat (SSR)] and cleaved amplified polymorphic sequence(CAPS) marker 484/W2R-ACC?[for G/T single nucleotide polymorphism (SNP)]. Six homozygous (CT)n types, namely (CT)20, (CT)19, (CT)18, (CT)17, (CT)16, (CT)14, (CT)11 and (CT)10, and a heterozygous genotype (CT)11/(CT)18 were detected for RM190, of which (CT)11 and (CT)18 were predominant. Two homozygous Wx genotypes (G/G and T/T) and one heterozygous (G/T) were detected using 484/W2R-ACC?. Most of the materials with a RM190 of (CT)11 were G/G for SNP of 484/W2R-ACC I, while T/T for SNP was predominantly appeared in materials with (CT)18. The materials tested could be grouped into 10 categories using the two markers together. Results indicated that 59.3% variance of amylose content was attributed to the polymorphism of Wx gene revealed by RM190, while 56.1% and 24.6% of the variances in amylose content and gel consistency were respectively to the polymorphism of Wx gene revealed by 484/W2R-ACC I. Furthermore, with both SSR and CAPS markers, 72.4% of the variance in amylose content could be explained. In addition, the application prospects of the two markers in breeding were also discussed. Wx A naturally occurring functional allele of the rice waxy locus has a GT to TT mutation at the 5' splice site of the first intron 1998 The Plant Journal Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Takayama, Japan. In cultivated rice two wild-type alleles, Wxa and Wxb, predominate at the waxy locus, which encodes granule-bound starch synthase. The activity of Wxa is 10-fold higher than that of Wxb at the level of both protein and mRNA. Wxb has a +1G to T mutation at the 5' splice site of the first intron. Sequence analysis of Wxb transcripts revealed that splicing occurs at the mutant AG/UU site and at two cryptic sites: the first is A/GUU, one base upstream of the original site and the second is AG/GU found approximately 100 bases upstream of the mutant splice site. We introduced single base mutations to the 5' splice sites of both Wxa and Wxb, fused with the gus reporter gene and introduced them into rice protoplasts. Analysis of GUS activities and transcripts indicated that a G to T mutation in Wxa reduced GUS activity and the level of spliced RNA. Conversely, a T to G mutation of Wxb restored GUS activity and the level of spliced RNA to that of wild-type Wxa. These results demonstrated that the low level expression of Wxb results from a single base mutation at the 5' splice site of the first intron. It is of interest that the Wxb allele of rice carrying the G to T mutation of intron 1 has been conserved in the history of rice cultivation because there is a low amylose content of the seed caused by this mutation. Wx A single base change altered the regulation of the Waxy gene at the posttranscriptional level during the domestication of rice 1998 Mol Biol Evol National Institute of Genetics, Mishima, Japan. ahirano@hongo.ecc.u-tokyo.ac.jp The rice waxy (wx) locus has two functional alleles, Wxa and Wxb, which are defined by a large difference in the amount of the gene product, called Wx protein, that accumulates in mature seeds. To elucidate the molecular mechanism underlying this difference and to identify the base change causing the alteration of the regulation of the Wx gene during rice evolution, we determined the nucleotide sequences of the regulatory region of Wx alleles and analyzed their function in a transient assay system using rice protoplasts. All Wxa alleles from Oryza sativa Indica, O. rufipogon, and O. glaberrima have a normal sequence of GT at the 5' splice junction of the first intron, representing a high expression level of the Wx transcripts in the endosperm and a high beta-glucuronidase (GUS) activity in protoplasts. On the other hand, Wxb alleles from two strains of O. sativa Japonica have TT at the 5' splice junction, representing a low expression level of the mature transcripts and a low GUS activity. Northern blot analysis also indicated that a larger transcript, consisting of the unspliced first intron, is closely correlated with the function of the Wxb allele. These results suggest that a single base change at the 5' splice junction causes inefficient splicing and, as a result, reduces the level of mature transcript and the GUS activity in the Wxb allele. The Wxb allele in O. saativa Japonica may have been differentiated from the Wxa allele of O. rufipogon, its wild progenitor, by this mutation, and, therefore, a single base change that has altered the regulation of the Wx gene at the posttranscriptional level probably occurred during the domestication of rice. Wx Granule-bound starch synthase I is responsible for biosynthesis of extra-long unit chains of amylopectin in rice 2008 Plant Cell Physiol Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065 Japan. A rice Wx gene encoding a granule-bound starch synthase I (GBSSI) was introduced into the null-mutant waxy (wx) rice, and its effect on endosperm starches was examined. The apparent amylose content was increased from undetectable amounts for the non-transgenic wx cultivars to 21.6-22.2% of starch weight for the transgenic lines. The increase was in part due to a significant amount of extra-long unit chains (ELCs) of amylopectin (7.5-8.4% of amylopectin weight), that were absent in the non-transgenic wx cultivars. Thus, actual amylose content was calculated to be 14.9-16.0% for the transgenic lines. Only slight differences were found in chain-length distribution for the chains other than ELCs, indicating that the major effect of the Wx transgene on amylopectin structure was ELC formation. ELCs isolated from debranched amylopectin exhibited structures distinct from amylose. Structures of amylose from the transgenic lines were slightly different from those of cv. Labelle (Wx(a)) in terms of a higher degree of branching and size distribution. The amylose and ELC content of starches of the transgenic lines resulted in the elevation of pasting temperature, a 50% decrease in peak viscosity, a large decrease in breakdown and an increase in setback. As yet undetermined factors other than the GBSSI activity are thought to be involved in the control of formation and/or the amount of ELCs. Structural analysis of the Wx gene suggested that the presence of a tyrosine residue at position 224 of GBSSI correlates with the formation of large amounts of ELCs in cultivars carrying Wx(a). Wx Allelic diversities in rice starch biosynthesis lead to a diverse array of rice eating and cooking qualities 2009 Proc Natl Acad Sci U S A State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. More than half of the world's population uses rice as a source of carbon intake every day. Improving grain quality is thus essential to rice consumers. The three main properties that determine rice eating and cooking quality--amylose content, gel consistency, and gelatinization temperature--correlate with one another, but the underlying mechanism of these properties remains unclear. Through an association analysis approach, we found that genes related to starch synthesis cooperate with each other to form a fine regulating network that controls the eating and cooking quality and defines the correlation among these three properties. Genetic transformation results verified the association findings and also suggested the possibility of developing elite cultivars through modification with selected major and/or minor starch synthesis-related genes. Wx,SSIIB,SSIIC Identification of SNPs in the waxy gene among glutinous rice cultivars and their evolutionary significance during the domestication process of rice 2004 Theor Appl Genet Gene Research Center, Institute of Biological Sciences, University of Tsukuba, Tennodai 1-1-1, 305-8572 Tsukuba, Japan. Common non-waxy ( Wx) rice cultivars contain two different alleles at the waxy locus, designated Wx(a) and Wx(b), which encode different levels of granule-bound starch synthases and are hence involved in the control of endosperm amylose content. The Wx(a) allele was predominant in non-waxy indica cultivars, whereas the Wx(b) allele was common to the non-waxy japonica variety. Recently, some of the molecular mechanisms underlying the differentiation of Wx(a) from Wx(b) have been characterized. One structural difference between these two alleles was shown to be due to alternative splicing caused by a single-base substitution (AG GT to AG TT) at a donor site of the first intron within the Wx gene. In the case of waxy ( wx) rice, it was not possible to distinguish whether the each wx allele was derived from Wx(a) or Wx(b) alleles by phenotypic analysis. However, we succeeded in developing a derived cleaved amplified polymorphic sequence (dCAPS) marker for the detection of the one-base splicing mutation without the need for sequencing. A mismatch primer was used to generate a restriction site in the Wx(a) allele (AGGT) but not in the Wx(b) allele (AGTT). Three hundred fifty-three waxy rice strains that are widely found in Asia were then employed for analysis using this dCAPS marker. Our findings suggested that waxy rice strains have both Wx(a)- and Wx(b)-derived alleles, but that the Wx(b)-derived allele was predominant, and its distribution was independent of indica- japonica differentiation. The wild relatives of cultivated rice all possessed the AGGT allele. It was concluded that the waxy mutations, and the corresponding rice cultivation, originated from japonica during the evolution and domestication process of rice and was preferentially selected by most Asian peoples. Wx Nucleotide sequence of a long cDNA from the rice waxy gene 1992 Plant Mol Biol Laboratory of Genetics, University of Wisconsin, Madison 53706. None Wx A novel wx mutation caused by insertion of a retrotransposon-like sequence in a glutinous cultivar of rice (Oryza sativa) 2007 Theor Appl Genet Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-Amamiyamachi 1-1, Aoba-ku, Sendai, 981-8555, Japan. DNA polymorphism of the Wx gene in glutinous rice cultivars was investigated by PCR-RF-SSCP and heteroduplex cleavage analysis using Brassica petiole extract, and the nucleotide sequence variations were identified. Most japonica-type glutinous rice was found to have a 23-bp duplication in the second exon, which causes loss of the function of granule-bound starch synthase (GBSS) encoded by the Wx gene. Without the 23-bp duplication, there was an insertion of 7,764 bp in the ninth exon of the wx allele of 'Oragamochi'. Expression analysis of the wx allele using RT-PCR and Northern blot analysis revealed that transcripts of the 'Oragamochi' wx allele are about 1-kb shorter and that the deduced amino acid sequence of the transcript lacks a motif important for GBSS. Therefore, this insertion was considered to be the cause of the glutinous trait of 'Oragamochi'. This 7,764-bp insertion had long terminal repeats, a primer binding site, and a polypurine tract, but no sequence homologous with gag and pol, suggesting that it is a non-autonomous element. Furthermore, it had a structure similar to Dasheng and may be a member of Dasheng. Wx A single nucleotide polymorphism in the Waxy gene explains a significant component of gel consistency 2011 Theor Appl Genet Grain Quality and Nutrition Centre, International Rice Research Institute, DAPO 7777, Manila, Metro Manila, Philippines. Gel consistency (GC) is a standard assay used in rice improvement programmes to determine whether rice cultivars/breeding lines of high amylose content are soft or firm textured when cooked. In this study, we show that sequence variation in exon 10 of the Waxy (Wx) gene associates with GC using RILs derived from parents with high amylose content that differ in GC. The association was validated using a diverse set of traditional varieties, selected on the basis of amylose content, from the generation challenge programme. Structural investigations to explain how the mutation leads to differences in GC showed a strong association between GC and the proportion of amylose that leaches. It was shown that cooked rices of hard GC do not change in hardness over 24 h, whereas rices of soft GC retrograde significantly over 24 h. This leads to the conclusion that the mutation on exon 10 of the Wx gene affects the proportion of amylose bound to amylopectin and the proportion able to leach, and these structural differences alter the composition of the gel, which affects the amount of time the gel takes to reach a final hardness. The SNP described here completes the set of markers required to genotype for the current traits of cooking quality, but selecting the allele for soft texture has the negative result of also selecting for retrogradation potential. Wx Identification and characterization of a novel Waxy allele from a Yunnan rice landrace 2009 Plant Mol Biol State Key Laboratory for Crop Genetics & Germplasm Enhancement, Jiangsu Provincial Center of Plant Gene Engineering, Nanjing Agricultural University, Weigang 1, 210095 Nanjing, China. Low amylose content (AC) is a desirable trait for rice (Oryza sativa L.) cooking quality and is selected in soft rice breeding. To gain a better understanding of the molecular mechanism controlling AC formation, we screened 83 Yunnan rice landraces in China and identified a rice variety, Haopi, with low AC. Genetic analyses and transgenic experiments revealed that low AC in Haopi was controlled by a novel allele of the Wx locus, Wx(hp), encoding a granule-bound starch synthase (GBSSI). Sequence comparisons of Wx(hp) and Wx(b) alleles (from Nipponbare) showed several nucleotide changes in the upstream regulatory regions (including the promoter, 5'-untranslated region, and first intron 5' splicing junction site). Interestingly, these changes had no obvious effect on the expression level and splicing efficiency of Wx transcripts. In addition, an examination of the coding region revealed that the Wx(hp) allele carries an A-to-G change at nucleotide position +497 from the start codon, resulting in an Asp(165)/Gly(165) substitution. The amino acid substitution had no detectable effects on GBSSI activity in vitro; however, it notably reduced the binding of GBSSI to starch granules, resulting in a reduction of AC in rice seeds. Moreover, three other Yunnan landraces with low AC also carry a nucleotide substitution identical to Haopi at the +497 position of the Wx gene, suggesting common ancestry. Based on the single-nucleotide polymorphism, we have developed a new derived cleaved amplified polymorphic sequence marker for use in breeding practice to manipulate AC in rice endosperm. Wx Identification of a YAC clone carrying the Xa-1 allele, a bacterial blight resistance gene in rice 1996 Theor Appl Genet Department of Agriculture, Kyushu University, 6-10-1, Hakozaki, Higashiku, 812, Fukuoka, Japan. Map-based cloning methods have been applied for isolation of Xa-1, one of the bacterial blight resistance genes in rice.Xa-1 was previously mapped on chromosome 4 using molecular markers. For positional cloning of Xa-1, a high-resolution genetic map was made for theXa-1 region using an F2 population of 402 plants and additional molecular markers. Three restriction fragment length polymorphism (RFLP) markers, XNpb235, XNpb264 and C600 were found to be linked tightly to Xa-1, with no recombinants, and U08 750 was mapped 1.5 cM from Xa-1. The screening of a yeast artificial chromosome (YAC) library using theseXa-1-linked RFLP markers resulted in the identification of ten contiguous YAC clones. Among these, one YAC clone, designated Y5212, with an insert of 340 kb, hybridized with all three tightly linked markers. This YAC was confirmed to possess the Xa-1 allele by mapping the Xa-1 gene between both end clones of this YAC (Y5212R and Y5212L). Xa1 Expression of Xa1, a bacterial blight-resistance gene in rice, is induced by bacterial inoculation 1998 Proc Natl Acad Sci U S A Rice Genome Research Program, National Institute of Agrobiological Resources/Society for Techno-innovation of Agriculture, Forestry and Fisheries, Kannondai, Tsukuba, Ibaraki 305, Japan. The Xa1 gene in rice confers resistance to Japanese race 1 of Xanthomonas oryzae pv. oryzae, the causal pathogen of bacterial blight (BB). We isolated the Xa1 gene by a map-based cloning strategy. The deduced amino acid sequence of the Xa1 gene product contains nucleotide binding sites (NBS) and a new type of leucine-rich repeats (LRR); thus, Xa1 is a member of the NBS-LRR class of plant disease-resistance genes, but quite different from Xa21, another BB-resistance gene isolated from rice. Interestingly, Xa1 gene expression was induced on inoculation with a bacterial pathogen and wound, unlike other isolated resistance genes in plants, which show constitutive expression. The induced expression may be involved in enhancement of resistance against the pathogen. Xa1 The rice TAL effector-dependent resistance protein XA10 triggers cell death and calcium depletion in the endoplasmic reticulum 2014 Plant Cell Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604, Republic of Singapore. The recognition between disease resistance (R) genes in plants and their cognate avirulence (Avr) genes in pathogens can produce a hypersensitive response of localized programmed cell death. However, our knowledge of the early signaling events of the R gene-mediated hypersensitive response in plants remains limited. Here, we report the cloning and characterization of Xa10, a transcription activator-like (TAL) effector-dependent R gene for resistance to bacterial blight in rice (Oryza sativa). Xa10 contains a binding element for the TAL effector AvrXa10 (EBEAvrXa10) in its promoter, and AvrXa10 specifically induces Xa10 expression. Expression of Xa10 induces programmed cell death in rice, Nicotiana benthamiana, and mammalian HeLa cells. The Xa10 gene product XA10 localizes as hexamers in the endoplasmic reticulum (ER) and is associated with ER Ca(2+) depletion in plant and HeLa cells. XA10 variants that abolish programmed cell death and ER Ca(2+) depletion in N. benthamiana and HeLa cells also abolish disease resistance in rice. We propose that XA10 is an inducible, intrinsic terminator protein that triggers programmed cell death by a conserved mechanism involving disruption of the ER and cellular Ca(2+) homeostasis. XA10 Targeting xa13, a recessive gene for bacterial blight resistance in rice 2006 Theor Appl Genet National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, 430070 Wuhan, China. Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious diseases of rice worldwide. Thirty bacterial blight resistance (R) genes (21 dominant genes and 9 recessive genes) in rice have been identified. They are the main sources for the genetic improvement of rice for resistance to Xoo. However, little is known about the recessive R genes. To clone and characterize the recessive R genes, we fine-mapped xa13, a fully recessive gene for Xoo resistance, to a DNA fragment of 14.8 kb using the map-based cloning strategy and a series of sequence-based molecular markers. Sequence analysis of this fragment indicated that this region contains only two apparently intact candidate genes (an extensin-like gene and a homologue of nodulin MtN3) and the 5' end of a predicted hypothetical gene. These results will greatly facilitate the isolation and characterization of xa13. Four PCR-based markers, E6a, SR6, ST9 and SR11 that were tightly linked to the xa13 locus, were also developed. These markers will be useful tools for the marker-assisted selection of xa13 in breeding programs. Os8N3|xa13 Genetic and physical mapping of xa13 , a recessive bacterial blight resistance gene in rice 1999 TAG Theoretical and Applied Genetics Plant Breeding, Genetics and Biochemistry Division, International Rice Research Institute (IRRI), P.O. Box 933, 1099 Manila, The Philippines The recessive gene, xa13, confers resistance to Philippine race 6 (PXO99) of the bacterial blight pathogen Xanthomonas oryzae pv oryzae. Fine genetic mapping and physical mapping were conducted as initial steps in an effort to isolate the gene. Using nine selected DNA markers and two F2 populations of 132 and 230 plants, xa13 was fine-mapped to a genomic region <4 cM on the long arm of rice chromosome 8, flanked by two RFLP markers, RG136 and R2027. Four DNA markers, RG136, R2027, S14003, and G1149, in the target region were used to identify bacterial artificial chromosome (BAC) clones potentially harboring the xa13 locus from a rice BAC library. A total of 11 BACs were identified, forming four separate contigs including a single-clone contig, 29I3, associated with the RG136 STS marker, the S14003 contig consisting of four clones (44F8, 41O2, 12A16, and 12F20), the G1149 contig with two clones, 23D11 and 21H18, and the R2027 contig consisting of four overlapping clones, 42C23, 30B5, 6B7 and 21H14. Genetic mapping indicated that the xa13 locus was contained in the R2027 contig. Chromosomal walking on the R2027 contig resulted in two more clones, 33C7 and 14L3. DNA fingerprinting showed that the six clones of the R2027 contig were overlapping. Clone 44F8 hybridized with a single fragment from the clone 14L3, integrating the R2027 and S14003 contigs into a single contig consisting of ten BAC clones with a total size of approximately 330 kb. The physical presence of the xa13 locus in the contig was determined by mapping the ends of the BAC inserts generated by TAIL-PCR. In an F2 population of 230 plants, the BAC-end markers 42C23R and 6B7F flanked the xa13 locus. The probes 21H14F and 21H14R derived from BAC clone 21H14 were found to flank xa13 at a distance of 0.5 cM on either side, using a second F2 population of 132 plants. Thus, genetic mapping indicated that the contig and the 96-kb clone, 21H14, contained the xa13 locus. Os8N3|xa13 Gene silencing using the recessive rice bacterial blight resistance gene xa13 as a new paradigm in plant breeding 2012 Plant Cell Rep National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Resistant germplasm resources are valuable for developing resistant varieties in agricultural production. However, recessive resistance genes are usually overlooked in hybrid breeding. Compared with dominant traits, however, they may confer resistance to different pathogenic races or pest biotypes with different mechanisms of action. The recessive rice bacterial blight resistance gene xa13, also involved in pollen development, has been cloned and its resistance mechanism has been recently characterized. This report describes the conversion of bacterial blight resistance mediated by the recessive xa13 gene into a dominant trait to facilitate its use in a breeding program. This was achieved by knockdown of the corresponding dominant allele Xa13 in transgenic rice using recently developed artificial microRNA technology. Tissue-specific promoters were used to exclude most of the expression of artificial microRNA in the anther to ensure that Xa13 functioned normally during pollen development. A battery of highly bacterial blight resistant transgenic plants with normal seed setting rates were acquired, indicating that highly specific gene silencing had been achieved. Our success with xa13 provides a paradigm that can be adapted to other recessive resistance genes. Os8N3|xa13 Promoter mutations of an essential gene for pollen development result in disease resistance in rice 2006 Genes Dev National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China. Disease resistance and sexual reproductive development are generally considered as separate biological processes, regulated by different sets of genes. Here we show that xa13, a recessive allele conferring disease resistance against bacterial blight, one of the most devastating rice diseases worldwide, plays a key role in both disease resistance and pollen development. The dominant allele, Xa13, is required for both bacterial growth and pollen development. Promoter mutations in Xa13 cause down-regulation of expression during host-pathogen interaction, resulting in the fully recessive xa13 that confers race-specific resistance. The recessive xa13 allele represents a new type of plant disease resistance. Os8N3|xa13 Os8N3 is a host disease-susceptibility gene for bacterial blight of rice 2006 Proc Natl Acad Sci U S A Department of Plant Pathology, Kansas State University, Manhattan, KS 66506, USA. Many bacterial diseases of plants depend on the interaction of type III effector genes of the pathogen and disease-susceptibility genes of the host. The host susceptibility genes are largely unknown. Here, we show that expression of the rice gene Os8N3, a member of the MtN3 gene family from plants and animals, is elevated upon infection by Xanthomonas oryzae pv. oryzae strain PXO99(A) and depends on the type III effector gene pthXo1. Os8N3 resides near xa13, and PXO99(A) failed to induce Os8N3 in rice lines with xa13. Silencing of Os8N3 by inhibitory RNA produced plants that were resistant to infection by strain PXO99(A) yet remained susceptible to other strains of the pathogen. The effector gene avrXa7 from strain PXO86 enabled PXO99(A) compatibility on either xa13- or Os8N3-silenced plants. The findings indicate that Os8N3 is a host susceptibility gene for bacterial blight targeted by the type III effector PthXo1. The results support the hypothesis that X. oryzae pv. oryzae commandeers the regulation of otherwise developmentally regulated host genes to induce a state of disease susceptibility. Furthermore, the results support a model in which the pathogen induces disease susceptibility in a gene-for-gene manner. Os8N3|xa13 Characterization of Xanthomonas oryzae-responsive cis-acting element in the promoter of rice race-specific susceptibility gene Xa13 2011 Mol Plant National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. The rice Xa13 gene, whose promoter harbors a UPT (up-regulated by transcription activator-like [TAL] effector) box, UPT(PthXo1), plays a pivotal role in the race-specific pathogenicity caused by Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99. PXO99 causes rice disease by inducing Xa13. It is unknown, however, whether the UPT(PthXo1) box is the only PXO99-responsive cis-regulating elements in the activation of Xa13 expression. We analyzed the expression of a series of end- and site-truncated and site-mutated Xa13 promoters in rice and the binding of PXO99 protein to the intact, partial, or site-mutated UPT(PthXo1) boxes. In the Xa13 promoter, UPT(PthXo1) box is the only Xoo-responsive cis-acting element that results in PXO99-induced Xa13 expression. The 5'-terminal second, third, and fourth nucleotides of the box are important for bacterial protein binding and gene activation; mutation of any one of these sites abolished PXO99-induced gene expression. Furthermore, the 3'-half of the UPT(PthXo1) box is also required for protein binding and gene activation. These findings will enhance our understanding of the molecular mechanism of the interaction of rice and Xoo via UPT boxes and TAL effectors. Os8N3|xa13 Identification of Resistance Genes Effective Against Rice Bacterial Blight Pathogen in Eastern India 2001 Plant Disease Central Rice Research Institute (CRRI), Cuttack 753 006, India Breeding for bacterial blight resistance in rice requires an understanding of the contemporary pathogen populations in the locations where resistance genes are to be deployed. We characterized 450 strains of Xanthomonas oryzae pv. oryzae collected from three states of India using polymerase chain reaction fingerprinting and virulence analysis. This pathogen collection was differentiated into 17 haplotypes (12 lineages at 80% similarity level). Significant differences in the distribution of haplotypes were observed among regions. Virulence analysis of the pathogen collection revealed nine pathotypes. Among the populations from three regions, the Orissa population was the most diverse, consisting of 11 out of 17 haplotypes and five out of nine pathotypes detected in the total collection. Representative pathotypes were used to evaluate seven near-isogenic lines carrying individual bacterial blight resistance genes (Xa3, Xa4, xa5, Xa7, Xa10, xa13, and Xa21) and gene pyramids. Pathogen strains compatible to individual genes were present in detectable frequencies, although no single strain could overcome all resistance genes. Gene combinations Xa4 + xa5, xa5 + Xa21, and Xa4 + xa5 + Xa21 conferred a broad spectrum of resistance to all the strains evaluated, supporting the strategy of pyramiding appropriate resistance genes. Os8N3|xa13,xa21 Plasma membrane localization and potential endocytosis of constitutively expressed XA21 proteins in transgenic rice 2010 Mol Plant National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China. The rice pattern recognition receptor (PRR) XA21 confers race-specific resistance in leaf infection by bacterial blight Xathomonas oryzae pv. oryzae (Xoo), and was shown to be primarily localized to the endoplasmic reticulum (ER) when expressed with its native promoter or overexpressed in the protoplast. However, whether the protein is still ER-localization in the intact cell when overexpressed remains to be identified. Here, we showed that XA21, its kinase-dead mutant XA21P(K736EP), and the triple autophosphorylation mutant XA21P(S686A/T688A/S699A) GFP fusions were primarily localized to the plasma membrane (PM) when overexpressed in the intact transgenic rice cell, and also localized to the ER in the transgenic protoplast. The transgenic plants constitutively expressing the wild-type XA21 or its GFP fusion displayed race-specific resistance to Xoo at the adult and seedling stages. XA21 and XA21P(K736EP) could be internalized probably via the SCAMP-positive early endosomal compartment in the protoplast, suggesting that XA21 might be endocytosed to initiate resistance responses during pathogen infection. We also established a root infection system and demonstrated that XA21 also mediated race-specific resistance responses to Xoo in the root. Our current study provides an insight into the nature of the XA21-mediated resistance and a practical approach using the root cell system to further dissect the cellular signaling of the PRR during the rice-Xoo interaction. xa21 Biochemical characterization of the kinase domain of the rice disease resistance receptor-like kinase XA21 2002 J Biol Chem Department of Plant Pathology, University of Florida, Gainesville, Florida 32611, USA. The rice disease resistance gene, Xa21, encodes a receptor kinase-like protein consisting of leucine-rich repeats in the putative extracellular domain and a serine/threonine kinase in the putative intracellular domain. The putative XA21 kinase domain was expressed as maltose-binding and glutathione S-transferase fusion proteins in Escherichia coli. The fusion proteins are capable of autophosphorylation. Phosphoamino acid analysis of the glutathione S-transferase fusion protein indicates that only serine and threonine residues are phosphorylated. The relative phosphorylation rate of the XA21 kinase against increasing enzyme concentrations follows a first-order rather than second-order kinetics, indicating an intramolecular phosphorylation mechanism. Moreover, the active XA21 kinase cannot phosphorylate a kinase-deficient mutant of XA21 kinase. The enzymatic activity of the XA21 kinase in a buffer containing Mn(2+) is at least 15 times higher than that with Mg(2+). The K(m) and V(max) of XA21 kinase for ATP are 0.3 microm and 8.4 nmol/mg/min, respectively. Tryptic phosphopeptide mapping reveals that multiple sites on the XA21 kinase are phosphorylated. Finally, our data suggest that the region of XA21 kinase corresponding to the RD kinase activation domain is not phosphorylated, revealing a distinct mode of action compared with the tomato Pto serine/threonine kinase conferring disease resistance. xa21 Transcriptional characteristics of Xa21-mediated defense responses in rice 2011 J Integr Plant Biol Institute of Genetics and Developmental Biology, the Chinese Academy of Sciences, Beijing 100101, China. Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most destructive bacterial disease of rice. The cloned rice gene Xa21 confers resistance to a broad spectrum of Xoo races. To identify genes involved in Xa21-mediated immunity, a whole-genome oligonucleotide microarray of rice was used to profile the expression of rice genes between incompatible interactions and mock treatments at 0, 4, 8, 24, 72 and 120 h post inoculation (hpi) or between incompatible and compatible interactions at 4 hpi, respectively. A total of 441 differentially expressed genes, designated as XDGs (Xa21 mediated differentially expressed genes), were identified. Based on their functional annotations, the XDGs were assigned to 14 categories, including defense-related, signaling, transcriptional regulators. Most of the defense-related genes belonged to the pathogenesis-related gene family, which was induced dramatically at 72 and 120 hpi. Interestingly, most signaling and transcriptional regulator genes were downregulated at 4 and 8 hpi, suggesting that negative regulation of cellular signaling may play a role in the Xa21-mediated defense response. Comparison of expression profiles between Xa21- and other R gene-mediated defense systems revealed interesting common responses. Representative XDGs with supporting evidences were also discussed. xa21 The cloned gene, Xa21, confers resistance to multiple Xanthomonas oryzae pv. oryzae isolates in transgenic plants 1996 Mol Plant Microbe Interact Department of Plant Pathology, University of California, Davis 95616, USA. The cloned rice gene, Xa21, confers resistance to multiple pathogen isolates of Xanthomonas oryzae pv. oryzae in transgenic plants. The resistance phenotype was stably transmitted to T1 progeny and inherited as a single locus. The T1 progeny were tested for resistance to 32 X. oryzae pv. oryzae isolates from eight countries. Both the engineered line and the donor line showed resistance to 29 isolates and susceptibility to three isolates. The identical resistance spectrum of both lines indicates that the presence of a single member of a multigene family, Xa21, is sufficient to confer multi-isolate resistance. The results presented here have important implications for engineering disease resistance in crop plants. xa21 Ectopic expression of rice Xa21 overcomes developmentally controlled resistance to Xanthomonas oryzae pv. oryzae 2010 Plant Sci Department of Plant Pathology, University of California Davis, Davis, California, United States of America. Recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) activates the innate immune response. The rice PRR, XA21, confers robust resistance at adult stages to most strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). Seedlings are still easily infected by Xoo, causing severe yield losses. Here we report that Xa21 is induced by Xoo infection and that ectopic expression of Xa21 confers resistance at three leaf stage (three-week-old), overcoming the developmental limitation of XA21-mediated resistance. Ectopic expression of Xa21 also up-regulates a larger set of defense-related genes as compared to Xa21 driven by the native promoter. These results indicate that altered regulation of Xa21 expression is useful for developing enhanced resistance to Xoo at multiple developmental stages. xa21 Rice XB15, a protein phosphatase 2C, negatively regulates cell death and XA21-mediated innate immunity 2008 PLoS Biol Department of Plant Pathology, University of California Davis, Davis, California, USA. Perception of extracellular signals by cell surface receptors is of central importance to eukaryotic development and immunity. Kinases that are associated with the receptors or are part of the receptors themselves modulate signaling through phosphorylation events. The rice (Oryza sativa L.) XA21 receptor kinase is a key recognition and signaling determinant in the innate immune response. A yeast two-hybrid screen using the intracellular portion of XA21, including the juxtamembrane (JM) and kinase domain as bait, identified a protein phosphatase 2C (PP2C), called XA21 binding protein 15 (XB15). The interaction of XA21 and XB15 was confirmed in vitro and in vivo by glutathione-S-transferase (GST) pull-down and co-immunoprecipitation assays, respectively. XB15 fusion proteins purified from Escherichia coli and from transgenic rice carry PP2C activity. Autophosphorylated XA21 can be dephosphorylated by XB15 in a temporal- and dosage-dependent manner. A serine residue in the XA21 JM domain is required for XB15 binding. Xb15 mutants display a severe cell death phenotype, induction of pathogenesis-related genes, and enhanced XA21-mediated resistance. Overexpression of Xb15 in an XA21 rice line compromises resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae. These results demonstrate that Xb15 encodes a PP2C that negatively regulates the XA21-mediated innate immune response. xa21,XB15|PP2C An ATPase promotes autophosphorylation of the pattern recognition receptor XA21 and inhibits XA21-mediated immunity 2010 Proc Natl Acad Sci U S A Department of Plant Pathology, University of California, Davis, CA 95616, USA. Cell-surface pattern recognition receptors (PRRs) are key components of the innate immune response in animals and plants. These receptors typically carry or associate with non-RD kinases to control early events of innate immunity signaling. Despite their importance, the mode of regulation of PRRs is largely unknown. Here we show that the rice PRR, XA21, interacts with XA21 binding protein 24 (XB24), a previously undescribed ATPase. XB24 promotes autophosphorylation of XA21 through its ATPase activity. Rice lines silenced for Xb24 display enhanced XA21-mediated immunity, whereas rice lines overexpressing XB24 are compromised for immunity. XB24 ATPase enzyme activity is required for XB24 function. XA21 is degraded in the presence of the pathogen-associated molecular pattern Ax21 when XB24 is overexpressed. These results demonstrate a function for this large class of broadly conserved ATPases in PRR-mediated immunity. xa21,XB24 Short communication: developmental control of Xa21-mediated disease resistance in rice 1999 Plant J Department of Biology, San Francisco State University, CA 94132, USA. The rice resistance gene Xa21 confers resistance against the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). The molecular genetic mechanism controlling the integration of the Xa21-mediated disease resistance response with the developmental program in rice is under study in this model system. Reproducible means of infecting plants at certain developmental stages were designed based on the timing of full expansion of the leaf. Xa21-resistance progressively increases from the susceptible juvenile leaf 2 stage through later stages, with 100% resistance at the adult leaf 9/10 stage. We found that Xa21 expression is independent of plant developmental stage, infection with Xoo, or wounding. Expression of the Xa21 gene transcript is not correlated with expression of Xa21 disease resistance indicating that the developmental regulation of Xa21-resistance is either controlled post-transcriptionally or by other factors. xa21 A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21 1995 Science Department of Plant Pathology, University of California, Davis 95616, USA. The rice Xa21 gene, which confers resistance to Xanthomonas oryzae pv. oryzae race 6, was isolated by positional cloning. Fifty transgenic rice plants carrying the cloned Xa21 gene display high levels of resistance to the pathogen. The sequence of the predicted protein, which carries both a leucine-rich repeat motif and a serine-threonine kinase-like domain, suggests a role in cell surface recognition of a pathogen ligand and subsequent activation of an intracellular defense response. Characterization of Xa21 should facilitate understanding of plant disease resistance and lead to engineered resistance in rice. xa21 Xa21D Encodes a Receptor-like Molecule with a Leucine-Rich Repeat Domain That Determines Race-Specific Recognition and Is Subject to Adaptive Evolution 1998 The Plant Cell Online Department of Plant Pathology, University of California at Davis, 1 Shields Avenue, Davis, California 95616, USA. The rice Xa21 gene confers resistance to Xanthomonas oryzae pv oryzae in a race-specific manner. Analysis of the inheritance patterns and resistance spectra of transgenic plants carrying six Xa21 gene family members indicated that one member, designated Xa21D, displayed a resistance spectrum identical to that observed for Xa21 but conferred only partial resistance. Xa21D encodes a receptor-like protein carrying leucine-rich repeat (LRR) motifs in the presumed extracellular domain. The Xa21D transcript terminates shortly after the stop codon introduced by the retrotransposon Retrofit. Comparison of nucleotide substitutions in the LRR coding regions of Xa21 and Xa21D provided evidence of adaptive selection. Both functional and evolutionary evidence indicates that the Xa21D LRR domain controls race-specific pathogen recognition. xa21 Genetic and physical analysis of the rice bacterial blight disease resistance locus, Xa21 1992 Mol Gen Genet Department of Plant Breeding and Biometry, Cornell University, Ithaca, NY 14853. Nearly isogenic lines (NILs) of rice (Oryza sativa) differing at a locus conferring resistance to the pathogen Xanthomonas oryzae pv. oryzae were surveyed with 123 DNA markers and 985 random primers using restriction fragment length plymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analysis. One chromosome 11 marker (RG103) detected polymorphism between the NILs that cosegregated with Xa21. All other chromosome 11 DNA markers tested were monomorphic between the NILs, localizing the Xa21 introgressed region to an 8.3 cM interval on chromosome 11. Furthermore, we identified two polymerase chain reaction (PCR) products (RAPD2148 and RAPD818) that detected polymorphisms between the NILs. Genomic sequences hybridizing with RAPD818, RAPD248 and RG103 were duplicated specifically in the Xa21 NIL. All three markers cosegregated with the resistance locus, Xa21, in a F2 population of 386 progeny. Based on the frequency with which we recovered polymorphic Xa21-linked markers, we estimated the physical size of the introgressed region to be approximately 800 kb. This estimation was supported by physical mapping (using pulsed field gel electrophoresis) of the sequences hybridizing with the three Xa21-linked DNA markers. The results showed that the three Xa21-linked markers are physically close to each other, with one copy of the RAPD818 sequences located within 60 kb of RAPD248 and the other copy within 270 kb of RG103. None of the enzymes tested generated a DNA fragment that hybridized with all three of the markers indicating that the introgressed region containing the resistance locus Xa21 is probably larger than 270 kb. xa21 Rice XA21 binding protein 3 is a ubiquitin ligase required for full Xa21-mediated disease resistance 2006 Plant Cell Department of Plant Pathology, University of Florida, Gainesville, Florida 32611, USA. XA21 is a receptor-like kinase protein in rice (Oryza sativa) that confers gene-for-gene resistance to specific races of the causal agent of bacterial blight disease, Xanthomonas oryzae pv oryzae. We identified XA21 binding protein 3 (XB3), an E3 ubiquitin ligase, as a substrate for the XA21 Ser and Thr kinase. The interaction between XB3 and the kinase domain of XA21 has been shown in yeast and in vitro, and the physical association between XB3 and XA21 in vivo has also been confirmed by coimmunoprecipitation assays. XB3 contains an ankyrin repeat domain and a RING finger motif that is sufficient for its interaction with the kinase domain of XA21 and for its E3 ubiquitin ligase activity, respectively. Transgenic plants with reduced expression of the Xb3 gene are compromised in resistance to the avirulent race of X. oryzae pv oryzae. Furthermore, reduced levels of Xb3 lead to decreased levels of the XA21 protein. These results indicate that Xb3 is necessary for full accumulation of the XA21 protein and for Xa21-mediated resistance. xa21,Xb3 The XA21 binding protein XB25 is required for maintaining XA21-mediated disease resistance 2013 Plant J Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA. Plant genomes encode a large number of proteins that potentially function as immune receptors in the defense against pathogen invasion. As a well-characterized receptor kinase consisting of 23 tandem leucine-rich repeats, a transmembrane domain and a serine/threonine kinase, the rice (Oryza sativa) protein XA21 confers resistance to a broad spectrum of Xanthomonas oryzae pv. oryzae (Xoo) races that cause bacterial blight disease. We report here that XA21 binding protein 25 (XB25) belongs to the plant-specific ankyrin-repeat (PANK) family. XB25 physically interacts, in vitro, with the transmembrane domain of XA21 through its N-terminal binding to transmembrane and positively charged domain (BTMP) repeats. In addition, XB25 associates with XA21 in planta. The downregulation of Xb25 results in reduced levels of XA21 and compromised XA21-mediated disease resistance at the adult stage. Moreover, the accumulation of XB25 is induced by Xoo infection. Taken together, these results indicate that XB25 is required for maintaining XA21-mediated disease resistance. xa21,OsBIANK1|XB25 New gene for bacterial blight resistance in rice located on chromosome 12 identified from minghui 63, an elite restorer line 2002 Phytopathology None Bacterial blight, caused by Xanthomonas oryzae pv. oryzae, is a serious disease of rice worldwide. A new dominant gene for bacterial blight resistance in rice, Xa25(t), was identified from Minghui 63, a restorer line for a number of rice hybrids that are widely cultivated in China. This gene conferred resistance to Philippine race 9 (PXO339) of X. oryzae pv. oryzae in both seedling and adult stages. It was mapped to the centromeric region of chromosome 12, 2.5 cM from a disease resistance gene-homologous sequence, NBS109, and 7.3 cM from a restriction fragment length polymorphism marker, G1314. The genomic location of this gene is similar to the previously identified blast resistance genes, Pi-ta and Pi-ta2. Xa25 A paralog of the MtN3/saliva family recessively confers race-specific resistance to Xanthomonas oryzae in rice 2011 Plant Cell Environ National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China. Approximately one third of the identified 34 rice major disease resistance (R) genes conferring race-specific resistance to different strains of Xanthomonas oryzae pv. oryzae (Xoo), which causes rice bacterial blight disease, are recessive genes. However, only two of the recessive resistance genes have been characterized thus far. Here we report the characterization of another recessive resistance gene, xa25, for Xoo resistance. The xa25, localized in the centromeric region of chromosome 12, mediates race-specific resistance to Xoo strain PXO339 at both seedling and adult stages by inhibiting Xoo growth. It encodes a protein of the MtN3/saliva family, which is prevalent in eukaryotes, including mammals. Transformation of the dominant Xa25 into a resistant rice line carrying the recessive xa25 abolished its resistance to PXO339. The encoding proteins of recessive xa25 and its dominant allele Xa25 have eight amino acid differences. The expression of dominant Xa25 but not recessive xa25 was rapidly induced by PXO339 but not other Xoo strain infections. The nature of xa25-encoding protein and its expression pattern in comparison with its susceptible allele in rice-Xoo interaction indicate that the mechanism of xa25-mediated resistance appears to be different from that conferred by most of the characterized R proteins. Xa25 Xa3, conferring resistance for rice bacterial blight and encoding a receptor kinase-like protein, is the same as Xa26 2006 Theor Appl Genet National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, 430070, China. Xa3-mediated resistance for rice bacterial blight, one of the most devastating rice diseases worldwide, is influenced by genetic background. Xa3 is genetically tightly linked to Xa26, another gene for bacterial blight resistance. Xa26 belongs to a clustered multigene family encoding leucine-rich repeat (LRR) receptor kinase-like proteins. To characterize Xa3, we fine mapped it using a population segregating for only one resistance gene and markers developed from Xa26 family. Genetic analysis showed that Xa3 co-segregated with the marker of Xa26 gene and segregated from the markers of other members of Xa26 family. DNA fingerprinting revealed that rice line IRBB3 carrying Xa3 had the same copy numbers of Xa26 family members as rice line Minghui 63 carrying Xa26. Phenotypic comparison showed that all the rice lines carrying either Xa3 or Xa26 developed dark brown deposition at the border between the lesion caused by incompatible-pathogen infection and health leaf tissue, while other rice lines did not show this dark brown deposition in either incompatible or compatible interactions. These results suggest that Xa3 and Xa26 is the same gene. We name it Xa3/Xa26 to indicate the relationship between the two gene symbols. The putative encoding products of Xa3/Xa26 and its susceptible allele xa3/xa26 shared 92% sequence identity. The sequence difference occurred in the LRR domains, specifically at the solvent-exposed amino acid residues, might be the major cause that differentiates the resistant and susceptible proteins. Xa26|Xa3 The expression pattern of a rice disease resistance gene xa3/xa26 is differentially regulated by the genetic backgrounds and developmental stages that influence its function 2007 Genetics National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Genetic background and developmental stage influence the function of some disease resistance (R) genes. The molecular mechanisms of these modifications remain elusive. Our results show that the two factors are associated with the expression of the R gene in rice Xa3 (also known as Xa26)-mediated resistance to Xanthomonas oryzae pv. oryzae (Xoo), which in turn influences the expression of defense-responsive genes. The background of japonica rice, one of the two major subspecies of Asian cultivated rice, facilitates the function of Xa3 more than the background of indica rice, another rice subspecies. Xa3 expression gradually increases from early seedling stage to adult stage. Japonica plants carrying Xa3 regulated by the native promoter showed an enlarged resistance spectrum (i.e., resistance to more Xoo races), an increased resistance level (i.e., further reduced lesion length), and whole-growth-stage resistance compared to the indica rice; this enhanced resistance was associated with an increased expression of Xa3 throughout the growth stages in the japonica plants, which resulted in enhanced expression of defense-responsive genes. Overexpressing Xa3 with a constitutive strong promoter further enhanced rice resistance due to further increased Xa3 transcripts in both indica and japonica backgrounds, whereas regulating Xa3 with a pathogen-induced weak promoter impaired rice resistance. Xa26|Xa3 Functional analysis of Xa3/Xa26 family members in rice resistance to Xanthomonas oryzae pv. oryzae 2007 Theor Appl Genet National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Plant disease resistant (R) genes are frequently clustered in the genome. The diversity of members in a complex R-gene family may provide variation in resistance specificity. Rice Xa3/Xa26, conferring resistance to Xanthomonas oryzae pv. oryzae (Xoo) encodes a leucine-rich repeat (LRR) receptor kinase-type protein and belongs to a multigene family, consisting of Xa3/Xa26, MRKa, MRKc and MRKd in rice cultivar Minghui 63. MRKa and MRKc are intact genes, while MRKd is a pseudogene. Complementary analyses showed that MRKa and MRKc could not mediate resistance to Xoo when regulated by their native promoters, but MRKa not MRKc conferred partial resistance to Xoo when regulated by a strong constitutive promoter. Plants carrying truncated XA3/XA26, which lacked the kinase domain, were compromised in their resistance to Xoo. However, the kinase domain of MRKa could partially restore the function of the truncated XA3/XA26 in resistance. MRKa and MRKc showed similar expression pattern as Xa3/Xa26, which expressed only in the vascular systems of different tissues. The expressional characteristic of MRKa and MRKc perfectly fits the function of genes conferring resistance to Xoo, a vascular pathogen. These results suggest that although MRKa and MRKc cannot mediate bacterial blight resistance nowadays, they may be once effective genes for Xoo resistance. Their expressional characteristic and sequence similarity to Xa3/Xa26 will provide templates for generating novel recognition specificity to face the evolution of Xoo. In addition, both LRR and kinase domains encoded by Xa3/Xa26 and MRKa are the functional determinants and MRKa-mediated resistance is dosage-dependent. Xa26|Xa3 Ortholog alleles at Xa3/Xa26 locus confer conserved race-specific resistance against Xanthomonas oryzae in rice 2012 Mol Plant National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. The rice disease resistance (R) gene Xa3/Xa26 (having also been named Xa3 and Xa26) against Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight disease, belongs to a multiple gene family clustered in chromosome 11 and is from an AA genome rice cultivar (Oryza sativa L.). This family encodes leucine-rich repeat (LRR) receptor kinase-type proteins. Here, we show that the orthologs (alleles) of Xa3/Xa26, Xa3/Xa26-2, and Xa3/Xa26-3, from wild Oryza species O. officinalis (CC genome) and O. minuta (BBCC genome), respectively, were also R genes against Xoo. Xa3/Xa26-2 and Xa3/Xa26-3 conferred resistance to 16 of the 18 Xoo strains examined. Comparative sequence analysis of the Xa3/Xa26 families in the two wild Oryza species showed that Xa3/Xa26-3 appeared to have originated from the CC genome of O. minuta. The predicted proteins encoded by Xa3/Xa26, Xa3/Xa26-2, and Xa3/Xa26-3 share 91-99% sequence identity and 94-99% sequence similarity. Transgenic plants carrying a single copy of Xa3/Xa26, Xa3/Xa26-2, or Xa3/Xa26-3, in the same genetic background, showed a similar resistance spectrum to a set of Xoo strains, although plants carrying Xa3/Xa26-2 or Xa3/Xa26-3 showed lower resistance levels than the plants carrying Xa3/Xa26. These results suggest that the Xa3/Xa26 locus predates the speciation of A and C genome, which is approximately 7.5 million years ago. Thus, the resistance specificity of this locus has been conserved for a long time. Xa26|Xa3 Xa26, a gene conferring resistance toXanthomonas oryzaepv.oryzaein rice, encodes an LRR receptor kinase-like protein 2004 The Plant Journal National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding, Huazhong Agricultural University, Wuhan 430070, China. Rice bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious rice diseases worldwide. A rice gene, Xa26, conferring resistance against Xoo at both seedling and adult stages was isolated by map-based cloning strategies from the rice cultivar Minghui 63. Xa26 belongs to a multigene family consisting of four members. It encodes a leucine-rich repeat (LRR) receptor kinase-like protein and is constitutively expressed. Sequence analysis revealed that IRBB3 and Zhachanglong lines that are resistant to a broad range of Xoo strains, also carry Xa26. However, significant difference in lesion length was observed among these lines after inoculation with a set of Xoo strains. Moreover, transgenic plants carrying Xa26 showed enhanced resistance compared with the donor line of the gene in both seedling and adult stages. These results suggest that the resistance conferred by Xa26 is influenced by the genetic background. Xa26|Xa3 Multiple gene loci affecting genetic background-controlled disease resistance conferred by R gene Xa3/Xa26 in rice 2009 Theor Appl Genet National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, 430070, Wuhan, China. The function of bacterial-blight resistance gene Xa3/Xa26 in rice is influenced by genetic background; the Oryza sativa L. ssp. japonica background can increase Xa3/Xa26 expression, resulting in an enhanced resistance. To identify whether Xa3/Xa26 transcript level is the only factor contributing to genetic background-controlled resistance, we screened an F(2) population that was developed from a cross between Oryza sativa L. ssp. indica and japonica rice lines and was segregating for Xa3/Xa26, and compared the expression profiles of a pair of indica and japonica rice lines that both carried Xa3/Xa26. Eight quantitative trait loci (QTLs), in addition to Xa3/Xa26, were identified as contributing to the bacterial resistance of this population. Four of the eight QTLs were contributed to the japonica line. The resistance of this population was also affected by epistatic effects. Some F(2) individuals showed significantly increased Xa3/Xa26 transcripts, but the increased transcripts did not completely correlate with the reduced disease in this population. The analysis of the expression profile of Xa3/Xa26-mediated resistance using a microarray containing approximate 7,990 rice genes identified 44 differentially expressed genes. Thirty-five genes were rapidly activated in the japonica background, but not in the indica background, during disease resistance. These results suggest that multiple factors, including the one resulting in increased Xa3/Xa26 expression, may contribute to the enhanced resistance in the japonica background. These factors can cause a variation in gene expression profile that differs from that in the indica background during disease resistance. Xa26|Xa3 Genetic and physical mapping of a new gene for bacterial blight resistance in rice 2003 Theor Appl Genet National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding, Huazhong Agricultural University, Wuhan 430070, China. The inheritance of resistance for bacterial blight, caused by Xanthomonas oryzae pv. oryzae ( Xoo), was studied in Minghui 63, an elite restorer line for a number of widely used rice hybrids in China. A new dominant gene against a Chinese Xoo strain JL691 in both the seedling and adult stages was identified in Minghui 63 and designated as Xa26( t). Using a total of 477 highly susceptible individuals from an F(2) population, the Xa26( t) locus was mapped to a region of about 1.68 cM. This locus co-segregated with marker R1506 and was 0.21 cM from marker RM224 on one side and 1.47 cM from marker Y6855RA on the other side, in rice chromosome 11. A contig map, composed of five non-redundant bacterial artificial chromosome (BAC) clones and spanning approximately 500 kb in length, was constructed. Analysis of recombination events in the Xa26( t) region with the highly susceptible F(2) individuals anchored the gene locus to a region covered by three overlapped BAC clones. Assay of the lines showing a double crossover in marker loci flanking Xa26( t), in a population of recombinant inbred lines carrying Xa26( t), further delineated the gene to a 20-kb fragment. The Xa26( t) locus is tightly linked to another bacterial blight resistance gene locus, Xa4. Xa26|Xa3 High-resolution genetic mapping of Xa27(t), a new bacterial blight resistance gene in rice, Oryza sativa L 2004 Theor Appl Genet Temasek Life Sciences Laboratory, 1 Research Link, The National University of Singapore, 117604, Singapore, Republic of Singapore. Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae ( Xoo) (Ishyama) Dye, is one of the serious diseases prevalent throughout Asia. In a previous study, a resistance ( R) locus was transferred from the tetraploid wild rice Oryza minuta to the cultivated rice species, Oryza sativa L. Here, we report the fine genetic mapping of the R locus, tentatively designated as Xa27(t). We performed disease evaluation with an Xa27(t) near-isogenic line, IRBB27, testing 35 Xoo strains collected from 11 countries. The Xa27(t) locus conferred a high level of resistance to 27 strains and moderate resistance to three strains. Resistance of the Xa27(t) gene was developmentally regulated in IRBB27 and showed semi-dominant or a dosage effect in the cv. CO39 genetic background. As a prelude to cloning Xa27(t), a molecular mapping strategy was employed with a large mapping population consisting of 3,875 gametes. Three molecular markers, M336, M1081, and M1059, closely linked to Xa27(t), were identified to facilitate the mapping of Xa27(t) to the long arm of chromosome 6. The Xa27(t) locus was confirmed by chromosome landing of M1081 and M1095 markers on the rice genome. Markers derived from the genomic sequence of O. sativa cv. Nipponbare were used to further saturate the Xa27(t) genomic region. Xa27(t) was finally located within a genetic interval of 0.052 cM, flanked by markers M964 and M1197, and co-segregated with markers M631, M1230, and M449. Xa27 R gene expression induced by a type-III effector triggers disease resistance in rice 2005 Nature Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore 117604, Singapore. Disease resistance (R) genes in plants encode products that specifically recognise incompatible pathogens and trigger a cascade of events leading to disease resistance in the host plant. R-gene specificity is dictated by both host R genes and cognate avirulence (avr) genes in pathogens. However, the basis of gene-for-gene specificity is not well understood. Here, we report the cloning of the R gene Xa27 from rice and the cognate avr gene avrXa27 from Xanthomonas oryzae pv. oryzae. Resistant and susceptible alleles of Xa27 encode identical proteins. However, expression of only the resistant allele occurs when a rice plant is challenged by bacteria harbouring avrXa27, whose product is a nuclear localized type-III effector. Induction of Xa27 occurs only in the immediate vicinity of infected tissue, whereas ectopic expression of Xa27 resulted in resistance to otherwise compatible strains of the pathogen. Thus Xa27 specificity towards incompatible pathogens involves the differential expression of the R gene in the presence of the AvrXa27 effector. Xa27 XA27 depends on an amino-terminal signal-anchor-like sequence to localize to the apoplast for resistance to Xanthomonas oryzae pv oryzae 2008 Plant Physiol Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604, Republic of Singapore. The rice (Oryza sativa) gene Xa27 confers resistance to Xanthomonas oryzae pv oryzae, the causal agent of bacterial blight disease in rice. Sequence analysis of the deduced XA27 protein provides little or no clue to its mode of action, except that a signal-anchor-like sequence is predicted at the amino (N)-terminal region of XA27. As part of an effort to characterize the biochemical function of XA27, we decided to determine its subcellular localization. Initial studies showed that a functional XA27-green fluorescent protein fusion protein accumulated in vascular elements, the host sites where the bacterial blight pathogens multiply. The localization of XA27-green fluorescent protein to the apoplast was verified by detection of the protein on cell walls of leaf sheath and root cells after plasmolysis. Similarly, XA27-FLAG localizes to xylem vessels and cell walls of xylem parenchyma cells, revealed by immunogold electron microscopy. XA27-FLAG could be secreted from electron-dense vesicles in cytoplasm to the apoplast via exocytosis. The signal-anchor-like sequence has an N-terminal positively charged region including a triple arginine motif followed by a hydrophobic region. Deletion of the hydrophobic region or substitution of the triple arginine motif with glycine or lysine residues abolished the localization of the mutated proteins to the cell wall and impaired the plant's resistance to X. oryzae pv oryzae. These results indicate that XA27 depends on the N-terminal signal-anchor-like sequence to localize to the apoplast and that this localization is important for resistance to X. oryzae pv oryzae. Xa27 Analysis of nucleotide diversity among alleles of the major bacterial blight resistance gene Xa27 in cultivars of rice (Oryza sativa) and its wild relatives 2013 Planta Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Prof. C. R. Rao Road, Gachibowli, Hyderabad, India. bimowai@gmail.com Xa27 is one of the important R-genes, effective against bacterial blight disease of rice caused by Xanthomonas oryzae pv. oryzae (Xoo). Using natural population of Oryza, we analyzed the sequence variation in the functionally important domains of Xa27 across the Oryza species. DNA sequences of Xa27 alleles from 27 rice accessions revealed higher nucleotide diversity among the reported R-genes of rice. Sequence polymorphism analysis revealed synonymous and non-synonymous mutations in addition to a number of InDels in non-coding regions of the gene. High sequence variation was observed in the promoter region including the 5'UTR with 'pi' value 0.00916 and 'theta w ' = 0.01785. Comparative analysis of the identified Xa27 alleles with that of IRBB27 and IR24 indicated the operation of both positive selection (Ka/Ks > 1) and neutral selection (Ka/Ks approximately 0). The genetic distances of alleles of the gene from Oryza nivara were nearer to IRBB27 as compared to IR24. We also found the presence of conserved and null UPT (upregulated by transcriptional activator) box in the isolated alleles. Considerable amino acid polymorphism was localized in the trans-membrane domain for which the functional significance is yet to be elucidated. However, the absence of functional UPT box in all the alleles except IRBB27 suggests the maintenance of single resistant allele throughout the natural population. Xa27 High resolution genetic mapping and candidate gene identification at the xa5 locus for bacterial blight resistance in rice ( Oryza sativa L.) 2003 Theor Appl Genet Department of Plant Breeding, 240 Emerson Hall, Cornell University, Ithaca, NY 14853-1901, USA. The xa5 resistance gene from rice provides recessive, race-specific resistance to bacterial blight of rice caused by the pathogen Xanthomonas oryzae pv oryzae. A high-resolution genetic map of the chromosomal region surrounding xa5 was developed by placing 44 DNA markers on the distal end of rice chromosome 5. The basis for mapping was a PCR-based screening of 1,016 F(2) individuals derived from a cross between a near-isogenic line (NIL) and its corresponding recurrent parent to identify recombinants in the region. Recombinant F(2) individuals were progeny tested using F(3) families inoculated with the Philippine strain PXO 61 of bacterial blight pathogen. The xa5 gene was mapped to a 0.5-cM interval between the markers RS7 and RM611, which spanned an interval of approximately 70 kb and contained a total of 11 open reading frames. Sequence data for the locus was generated from an Indica (the IR24 isoline, IRBB21) BAC covering part of the region and compared to other overlapping Indica (cv 93-11) and Japonica (cv Nipponbare) sequences. Candidate-gene analysis revealed that a basal transcription factor (TFIIa), an ABC transporter, a tRNA synthase, a MAP kinase and a cysteine protease, as well as four unknown, hypothetical or putative proteins, are encoded at the locus and could be potential candidates for the resistance gene product. The mechanism by which these genes could provide recessive, race-specific resistance will be elucidated by map-based cloning of the xa5 gene. Xa5 Microsatellite and sequence-tagged site markers diagnostic for the rice bacterial leaf blight resistance gene xa-5 1997 TAG Theoretical and Applied Genetics Department of Plant Breeding, 252 Emerson Hall, Cornell University, Ithaca, NY 14853-1901, USA, US Microsatellite and sequence-tagged site (STS) markers tightly linked to the bacterial leaf blight (BLB) resistance gene xa-5 were identified in this study. A survey was conducted to find molecular markers that detected polymorphisms between the resistant (IRBB5) and susceptible (‘IR24’) nearly isogenic lines for xa-5, and between Chinsurah Boro II (CBII), an alternative source of xa-5, and a widely planted variety (‘IR64’) that lacks xa-5. Two F2 populations, from the crosses ‘IR24’×IRBB5 and CBIIבIR64’, were used to estimate linkage based on marker genotype and reaction to disease inoculation with Xanthomonas oryzae pv. oryzae. Two RFLP clones, RZ390 and RG556, were found to co-segregate with xa-5 and were converted into STS markers. A microsatellite marker, RM390, was developed based on a simple sequence repeat in the 5′ untranslated region of the cDNA probe, RZ390, and found to co-segregate with resistance. Two other microsatellites, RM122 and RM13, were located 0.4 cM and 14.1 cM away from xa-5. A germplasm survey of diverse lines containing BLB resistance genes using automated fluorescent detection indicated the range of allelic diversity for each of the microsatellite loci linked to xa-5 and confirmed their usefulness in following genes through the narrow crosses typical of a breeding program. The limited number of alleles observed at the microsatellite loci linked to the resistance gene in 35 xa-5-containing accessions suggested either a single ancestral origin or a few independent origins of the xa-5 gene. PCR-based markers, like the ones developed in this study, are economical and easy to use, and have applicability in efforts to pyramid the recessive xa-5 gene with other BLB resistance genes. Xa5 Construction of a BAC contig containing the xa5 locus in rice 1998 TAG Theoretical and Applied Genetics International Rice Research Institute, P.O. Box 933, 1099 Manila, The Philippines, XX The recessive gene xa5 confers resistance to bacterial blight in rice. To generate a physical map of the xa5 locus, three RFLP markers RG556, RG207 and RZ390, closely linked to xa5, were used to screen a rice bacterial artificial chromosome (BAC) library. The identified overlapping BAC clones formed two small contigs which were extended to both sides by chromosome walking. The final physical map consisted of 14 BAC clones and covered 550 kb. Genetic analysis with an F2 population showed that two RFLP markers 28N22R and 40F20R, derived from the BAC clones in the contig, flanked the xa5 locus. To further delimit the location of the xa5 locus, RFLP markers RG556 and RG207 were converted to sequence tagged sites and used to perform genetic analysis. The results indicated that the xa5 locus was most likely located between RG207 and RG556. Among the BAC clones in the contig, one clone, 44B4, hybridized to both RG207 and RG556. This suggests that BAC clone 44B4 carried the xa5 locus. Xa5 Testifying the rice bacterial blight resistance gene xa5 by genetic complementation and further analyzing xa5 (Xa5) in comparison with its homolog TFIIAgamma1 2006 Mol Genet Genomics Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 100101 Beijing, China. The recessive gene xa5 for resistance to bacterial blight resistance of rice is located on chromosome 5, and evidence based on genetic recombination has been shown to encode a small subunit of the basal transcription factor IIA (Iyer and McCouch in MPMI 17(12):1348-1354, 2004). However, xa5 has not been demonstrated by a complementation test. In this study, we introduced the dominant allele Xa5 into a homozygous xa5-line, which was developed from a cross between IRBB5 (an indica variety with xa5) and Nipponbare (a japonica variety with Xa5). Transformation of Xa5 and subsequent segregation analysis confirmed that xa5 is a V39E substitution variant of the gene for TFIIAgamma on chromosome 5 (TFIIAgamma5 or Xa5). The rice has an addition gene for TFIIAgamma exists on chromosome 1 (TFIIAgamma1). Analysis of the expression patterns of Xa5 (TFIIAgamma5)/xa5 and TFIIAgamma1 revealed that both the genes are constitutively expressed in different rice organs. However, no expression of TFIIAgamma1 could be detected in the panicle by reverse transcriptase-polymerase chain reaction. To compare the structural difference between the Xa5/xa5 and TFIIAgamma1 proteins, 3-D structures were predicted using computer-aided modeling techniques. The modeled structures of Xa5 (xa5) and TFIIAgamma1 fit well with the structure of TFIIA small subunit from human, suggesting that they may all act as a small subunit of TFIIA. The E39V substitution in the xa5 protein occurs in the alpha-helix domain, a supposed conservative substitutable site, which should not affect the basal transcription function of TFIIAgamma. The structural analysis indicates that xa5 and Xa5 potentially retain their basic transcription factor function, which, in turn, may mediate the novel pathway for bacterial blight resistance and susceptibility, respectively. Xa5 Genetic and functional characterization of the rice bacterial blight disease resistance gene xa5 2008 Phytopathology Department of Plant Breeding and Genetics, Cornell University, Ithaca, NY 14853, USA. Xanthomonas oryzae pv. oryzae is the causal agent of rice bacterial blight, a destructive rice disease worldwide. The gene xa5 provides race-specific resistance to X. oryzae pv. oryzae, and encodes the small subunit of transcription factor IIA. How xa5 functions in bacterial blight resistance is not well understood, and its recessive gene action is disputed. Here we show that xa5 is inherited in a completely recessive manner and the susceptible allele Xa5 is fully dominant. In accordance with this, bacterial growth in heterozygous and homozygous susceptible lines is not significantly different. Further, one allele of Xa5 is sufficient to promote disease in previously resistant plants; additional copies are not predictive of increased lesion length. Surprisingly, a resistant nearly isogenic line (NIL) of an indica variety sustains high levels of bacterial populations compared to the susceptible NIL, yet the resistant plants restrict symptom expression. In contrast, in japonica NILs, bacterial population dynamics differ in resistant and susceptible genotypes. However, both resistant indica and japonica plants delay bacterial movement down the leaf. These results support a model in which xa5-mediated recessive resistance is the result of restricted bacterial movement, but not restricted multiplication. Xa5 Molecular characterization of rice OsBIANK1, encoding a plasma membrane-anchored ankyrin repeat protein, and its inducible expression in defense responses 2010 Mol Biol Rep State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, 310029, Hangzhou, Zhejiang, People's Republic of China. A rice gene, OsBIANK1, encoding a protein containing a typical ankyrin repeat domain, was cloned and identified. The OsBIANK1 protein, consisting of 329 amino acids, contains a conserved ankyrin repeat domain with two ankyrin repeats organized in tandem and was showed to be localized on cytoplasmic membrane during transient expression in onion epidermal cells. Expression of OsBIANK1 was induced by treatment with benzothiadiazole (BTH), a chemical inducer capable of inducing disease resistance response in rice. In BTH-treated rice seedlings, expression of OsBIANK1 was further induced by infection with Magnaporthe grisea, the rice blast fungus, as compared with those in water-treated seedlings. Our preliminary results confirm previous evidences that OsBIANK1 may be involved in regulation of disease resistance response in rice. OsBIANK1|XB25 XIAO is involved in the control of organ size by contributing to the regulation of signaling and homeostasis of brassinosteroids and cell cycling in rice 2012 Plant J National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China. Organ size is determined by cell number and size, and involves two fundamental processes: cell proliferation and cell expansion. Although several plant hormones are known to play critical roles in shaping organ size by regulating the cell cycle, it is not known whether brassinosteroids (BRs) are also involved in regulating cell division. Here we identified a rice T-DNA insertion mutant for organ size, referred to as xiao, that displays dwarfism and erect leaves, typical BR-related phenotypes, together with reduced seed setting. XIAO is predicted to encode an LRR kinase. The small stature of the xiao mutant resulted from reduced organ sizes due to decreased cell numbers resulting from reduced cell division rate, as supported by the observed co-expression of XIAO with a number of genes involved in cell cycling. The xiao mutant displayed a tissue-specific enhanced BR response and greatly reduced BR contents at the whole-plant level. These results indicated that XIAO is a regulator of BR signaling and cell division. Thus, XIAO may provide a possible connection between BRs and cell-cycle regulation in controlling organ growth. XIAO A chlorophyll-deficient rice mutant with impaired chlorophyllide esterification in chlorophyll biosynthesis 2007 Plant Physiol National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, China. Chlorophyll (Chl) synthase catalyzes esterification of chlorophyllide to complete the last step of Chl biosynthesis. Although the Chl synthases and the corresponding genes from various organisms have been well characterized, Chl synthase mutants have not yet been reported in higher plants. In this study, a rice (Oryza Sativa) Chl-deficient mutant, yellow-green leaf1 (ygl1), was isolated, which showed yellow-green leaves in young plants with decreased Chl synthesis, increased level of tetrapyrrole intermediates, and delayed chloroplast development. Genetic analysis demonstrated that the phenotype of ygl1 was caused by a recessive mutation in a nuclear gene. The ygl1 locus was mapped to chromosome 5 and isolated by map-based cloning. Sequence analysis revealed that it encodes the Chl synthase and its identity was verified by transgenic complementation. A missense mutation was found in a highly conserved residue of YGL1 in the ygl1 mutant, resulting in reduction of the enzymatic activity. YGL1 is constitutively expressed in all tissues, and its expression is not significantly affected in the ygl1 mutant. Interestingly, the mRNA expression of the cab1R gene encoding the Chl a/b-binding protein was severely suppressed in the ygl1 mutant. Moreover, the expression of some nuclear genes associated with Chl biosynthesis or chloroplast development was also affected in ygl1 seedlings. These results indicate that the expression of nuclear genes encoding various chloroplast proteins might be feedback regulated by the level of Chl or Chl precursors. YGL1,CAB2R YGL138(t), encoding a putative signal recognition particle 54 kDa protein, is involved in chloroplast development of rice 2013 Rice College of Life Sciences, Jiangxi Normal University, Nanchang, 330022, China Background: Normal development of chloroplast is vitally important to plants, but its biological mechanism is still far from fully being understood, especially in rice. YGL138(t) A knockdown mutation of YELLOW-GREEN LEAF2 blocks chlorophyll biosynthesis in rice 2013 Plant Cell Rep National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing, 210095, China. KEY MESSAGE: An insert mutation of YELLOW-GREEN LEAF2 , encoding Heme Oxygenase 1 , results in significant reduction of its transcript levels, and therefore impairs chlorophyll biosynthesis in rice. Heme oxygenase (HO) in higher plants catalyzes the degradation of heme to synthesize phytochrome precursor and its roles conferring the photoperiodic control of flowering in rice have been revealed. However, its involvement in regulating rice chlorophyll (Chl) synthesis is not fully explored. In this study, we isolated a rice mutant named yellow-green leaf 2 (ygl2) from a (60)Co-irradiated population. Normal grown ygl2 seedlings showed yellow-green leaves with reduced contents of Chl and tetrapyrrole intermediates whereas an increase of Chl a/b ratio. Ultrastructural analyses demonstrated grana were poorly stacked in ygl2 mutant, resulting in underdevelopment of chloroplasts. The ygl2 locus was mapped to chromosome 6 and isolated via map-based cloning. Sequence analysis indicated that it encodes the rice HO1 and its identity was verified by transgenic complementation test and RNA interference. A 7-Kb insertion was found in the first exon of YGL2/HO1, resulting in significant reduction of YGL2 expressions in the ygl2 mutant. YGL2 was constitutively expressed in a variety of rice tissues with the highest levels in leaves and regulated by temperature. In addition, we found expression levels of some genes associated with Chl biosynthesis and photosynthesis were concurrently altered in ygl2 mutant. These results provide direct evidence that YGL2 has a vital function in rice Chl biosynthesis. Se5|OsHY1|OsHO1|YGL2 Phytochromes confer the photoperiodic control of flowering in rice (a short-day plant) 2000 The Plant Journal Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Takayama, Ikoma, Nara 630-0101. The photoperiodic sensitivity 5 (se5) mutant of rice, a short-day plant, has a very early flowering phenotype and is completely deficient in photoperiodic response. We have cloned the SE5 gene by candidate cloning and demonstrated that it encodes a putative heme oxygenase. Lack of responses of coleoptile elongation by light pulses and photoreversible phytochromes in crude extracts of se5 indicate that SE5 may function in phytochrome chromophore biosynthesis. Ectopic expression of SE5 cDNA by the CaMV 35S promoter restored the photoperiodic response in the se5 mutant. Our results indicate that phytochromes confer the photoperiodic control of flowering in rice. Comparison of se5 with hy1, a counterpart mutant of Arabidopsis, suggests distinct roles of phytochromes in the photoperiodic control of flowering in these two species. Se5|OsHY1|OsHO1|YGL2 Heme oxygenase is involved in nitric oxide- and auxin-induced lateral root formation in rice 2012 Plant Cell Rep Department of Agronomy, National Taiwan University, Taipei, Taiwan, ROC. Lateral root (LR) development performs the essential tasks of providing water, nutrients, and physical support to plants. Therefore, understanding the regulation of LR development is of agronomic importance. In this study, we examined the effect of nitric oxide (NO), auxin, and hemin (Hm) on LR formation in rice. Treatment with Hm [a highly effective heme oxygenase (HO) inducer], sodium nitroprusside (SNP, an NO donor), or indole-3-butyric acid (IBA, a naturally occurring auxin) induced LR formation and HO activity. LR formation and HO activity induced by SNP and IBA but not Hm was reduced by the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. As well, Hm, SNP, and IBA could induce OsHO1 mRNA expression. Zn protoporphyrin IX (the specific inhibitor of HO) and hemoglobin (the carbon monoxide/NO scavenger) reduced LR number and HO activity induced by Hm, SNP, and IBA. Our data suggest that HO is required for Hm-, auxin-, and NO-induced LR formation in rice. Se5|OsHY1|OsHO1|YGL2 RNAi knockdown of rice SE5 gene is sensitive to the herbicide methyl viologen by the down-regulation of antioxidant defense 2012 Plant Mol Biol College of Life Sciences, Cooperative Demonstration Laboratory of Centrifuge Technique, Nanjing Agricultural University, Nanjing 210095, People's Republic of China. Plant heme oxygenase (HO) catalyzes the oxygenation of heme to biliverdin, carbon monoxide (CO), and free iron (Fe(2+))-and Arabidopsis and rice (Oryza sativa) HOs are involved in light signaling. Here, we identified that the rice PHOTOPERIOD SENSITIVITY 5 (SE5) gene, which encoded a putative HO with high similarity to HO-1 from Arabidopsis (HY1), exhibited HO activity, and localized in the chloroplasts. Rice RNAi mutants silenced for SE5 were generated and displayed early flowering under long-day conditions, consistent with phenotypes of the null mutation in SE5 gene reported previously (se5 and s73). The herbicide methyl viologen (MV), which produces reactive oxygen species (ROS), was applied to determine whether SE5 regulates oxidative stress response. Compared with wild-type, SE5 RNAi transgenic plants aggravated seedling growth inhibition, chlorophyll loss and ROS overproduction, and decreased the transcripts of some representative antioxidative genes. By contrast, administration of exogenous CO partially rescued corresponding MV hypersensitivity in the SE5 RNAi plants. Alleviation of seed germination inhibition, chlorophyll loss and ROS overproduction, as well as the induction of antioxidant defense were further observed when SE5 or HY1 was overexpressed in transgenic Arabidopsis plants, indicating that SE5 may be useful for molecular breeding designed to improve plant tolerance to oxidative stress. Se5|OsHY1|OsHO1|YGL2 Young Leaf Chlorosis 1, a chloroplast-localized gene required for chlorophyll and lutein accumulation during early leaf development in rice 2013 Planta National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, China. Chlorophyll (Chl) and lutein are the two most abundant and essential components in photosynthetic apparatus, and play critical roles in plant development. In this study, we characterized a rice mutant named young leaf chlorosis 1 (ylc1) from a (6)(0)Co-irradiated population. Young leaves of the ylc1 mutant showed decreased levels of Chl and lutein compared to those of wild type, and transmission electron microscopy analysis revealed that the thylakoid lamellar structures were obviously loosely arranged. Whereas, the mutant turns green gradually and approaches normal green at the maximum tillering stage. The Young Leaf Chlorosis 1 (YLC1) gene was isolated via map-based cloning and identified to encode a protein of unknown function belonging to the DUF3353 superfamily. Complementation and RNA-interference tests confirmed the role of the YLC1 gene, which expressed in all tested rice tissues, especially in the leaves. Real-time PCR analyses showed that the expression levels of the genes associated with Chl biosynthesis and photosynthesis were affected in ylc1 mutant at different temperatures. In rice protoplasts, the YLC1 protein displayed a typical chloroplast location pattern. The N-terminal 50 amino acid residues were confirmed to be necessary and sufficient for chloroplast targeting. These data suggested that the YLC1 protein may be involved in Chl and lutein accumulation and chloroplast development at early leaf development in rice. YLC1 Disruption of a rice pentatricopeptide repeat protein causes a seedling-specific albino phenotype and its utilization to enhance seed purity in hybrid rice production 2012 Plant Physiol National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, People's Republic of China. The pentatricopeptide repeat (PPR) gene family represents one of the largest gene families in higher plants. Accumulating data suggest that PPR proteins play a central and broad role in modulating the expression of organellar genes in plants. Here we report a rice (Oryza sativa) mutant named young seedling albino (ysa) derived from the rice thermo/photoperiod-sensitive genic male-sterile line Pei'ai64S, which is a leading male-sterile line for commercial two-line hybrid rice production. The ysa mutant develops albino leaves before the three-leaf stage, but the mutant gradually turns green and recovers to normal green at the six-leaf stage. Further investigation showed that the change in leaf color in ysa mutant is associated with changes in chlorophyll content and chloroplast development. Map-based cloning revealed that YSA encodes a PPR protein with 16 tandem PPR motifs. YSA is highly expressed in young leaves and stems, and its expression level is regulated by light. We showed that the ysa mutation has no apparent negative effects on several important agronomic traits, such as fertility, stigma extrusion rate, selfed seed-setting rate, hybrid seed-setting rate, and yield heterosis under normal growth conditions. We further demonstrated that ysa can be used as an early marker for efficient identification and elimination of false hybrids in commercial hybrid rice production, resulting in yield increases by up to approximately 537 kg ha(-1). YSA ZEBRA2, encoding a carotenoid isomerase, is involved in photoprotection in rice 2011 Plant Mol Biol State Key Laboratory of Plant Genomics and National Centre for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No. 1 West Beichen Road, 100101 Chaoyang District, Beijing, China. "zebra" mutants have alternating green and chlorotic crossbands on leaf blades and are widely distributed in monocotyledonous crops. Most recently, we cloned the first responsible gene from rice, ZEBRA2, which also leads to the phenotype of rice preharvest sprouting. ZEBRA2, a single-copy gene in the rice genome, encodes a carotenoid isomerase (CRTISO), the key enzyme catalyzing the conversion of cis-lycopene to all-trans lycopene. ZEBRA2 shares high identity with known CRTISOs from other species. Expression analysis via both RT-PCR and ZEBRA2-promoter-beta-glucuronidase (GUS) transgenic rice indicates that ZEBRA2 is predominantly expressed in mesophyll cells of mature leaves where active photosynthesis occurs. Consistent with the alteration in agronomic traits, the zebra2 mutant exhibits decreased photosynthetic rate and chlorophyll content. Mutation of the ZEBRA2 gene results in the accumulation of all-trans-lycopene precursor, prolycopene (7Z,9Z,7'Z,9'Z tetra cis-lycopene), in dark-grown zebra2 tissues. Light-grown zebra2 mutant exhibits the characteristic "zebra" phenotype and decreased level of lutein, the xanthophyll that is essential for efficient chl triplet quenching. More severe phenotype of the zebra2 mutant under high light intensity indicates that "zebra" phenotype might be caused by photooxidative damages. We conclude that ZEBRA2 is involved in photoprotection in rice. OsCRTISO|ZEBRA2 Rice ZFP15 gene encoding for a novel C2H2-type zinc finger protein lacking DLN box, is regulated by spike development but not by abiotic stresses 2005 Mol Biol Rep State Key Laboratory of Crop Genetics and Germplasm Enhancement, Rice Research Institute, Nanjing Agricultural University, Nanjing 210095, China. A novel C2H2-type zinc finger protein gene, ZFP 15, was cloned from rice by RT-PCR approach. The ZFP 15 gene encodes a protein of 144 amino acid residues with a predicted molecular mass of 15 kDa. The ZFP 15 protein comprises two C2H2-type zinc finger domains, a putative nuclear localization signal (NLS) at its N-terminus but the DLN-box identified in all reported plant C2H2-type zinc finger proteins was not found. A homology search revealed that ZFP 15 gene was localized within a cluster of C2H2-type zinc finger genes in BAC clone OJ1754_E06 mapped on chromosome 3. All three members in the cluster encoded proteins showed high identities in amino acids and might contribute to a co-regulation. The RT-PCR assay revealed that ZFP 15 mRNA was not regulated by cold, salt, drought and ABA stresses, though CRT/DRE and ABRE elements were found in the promoter region of ZFP 15 gene. The expression profiling also showed that ZFP 15 mRNA was expressed with a lower level in leaves and roots, but not detected in stems. Besides, ZFP15 was shown to accumulate much more in flowering spike than in immature spike. Thus, ZFP15, as the first characterized C2H2-type zinc finger protein in rice, might play a regulatory role on rice spike development. ZFP15 Expression analysis of rice A20/AN1-type zinc finger genes and characterization of ZFP177 that contributes to temperature stress tolerance 2008 Gene State key laboratory of crop genetics and germplasm enhancement, Nanjing Agricultural University, Nanjing 210095, China. The A20/AN1-type zinc finger protein family is conserved in animals and plants. Using human AWP1 protein as a query, we identified twelve A20/AN1-type zinc finger proteins in japonica rice. Most of these genes were constitutively expressed in leaves, roots, culms and spikes. Through microarray analysis, it was found that four genes (ZFP177, ZFP181, ZFP176, ZFP173), two genes (ZFP181 and ZFP176) and one gene (ZFP157) were significantly induced by cold, drought and H(2)O(2) treatments, respectively. Further expression analysis showed that ZFP177 was responsive to both cold and heat stresses, but down-regulated by salt. The subcellular localization assay indicated that ZFP177 was localized in cytoplasm in tobacco leaf and root cells. Yeast-one hybrid assay showed that ZFP177 lacked trans-activation potential in yeast cells. Overexpression of ZFP177 in tobacco conferred tolerance of transgenic plants to both low and high temperature stresses, but increased sensitivity to salt and drought stresses. Further we found expression levels of some stress-related genes were inhibited in ZFP177 transgenic plants. These results suggested that ZFP177 might play crucial but differential roles in plant responses to various abiotic stresses. ZFP177 A novel rice C2H2-type zinc finger protein lacking DLN-box/EAR-motif plays a role in salt tolerance 2007 Biochim Biophys Acta State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China. A cDNA for the gene ZFP182, encoding a C2H2-type zinc finger protein, was cloned from rice by RT-PCR. ZFP182 codes an 18.2 kDa protein with two C2H2-type zinc finger motifs, one nuclear localization signal and one Leu-rich domain. The DLN-box/EAR-motif, which exists in most of plant C2H2-type zinc finger proteins, does not exist in ZFP182. The expression analysis showed that ZFP182 gene was constitutively expressed in leaves, culms, roots and spikes at the adult rice plants, and markedly induced in the seedlings by cold (4 degrees C), 150 mM NaCl and 0.1 mM ABA treatments. The approximate 1.4 kb promoter region of ZFP182 gene was fused into GUS reporter gene and transformed into tobacco. The histochemical analysis revealed that GUS expression could not be detected in transformed tobacco seedlings under normal conditions, but strongly observed in tobacco leaf discs and the vascular tissue of roots treated with NaCl or KCl. Expression of ZFP182 in transgenic tobacco and overexpression in rice increased plant tolerance to salt stress. These results demonstrated that ZFP182 might be involved in plant responses to salt stress. ZFP182|ZOS3-21 Genome-wide identification of C2H2 zinc-finger gene family in rice and their phylogeny and expression analysis 2007 Plant Mol Biol Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India. Transcription factors regulate gene expression in response to various external and internal cues by activating or suppressing downstream genes in a pathway. In this study, we provide a complete overview of the genes encoding C(2)H(2) zinc-finger transcription factors in rice, describing the gene structure, gene expression, genome localization, and phylogenetic relationship of each member. The genome of Oryza sativa codes for 189 C(2)H(2) zinc-finger transcription factors, which possess two main types of zinc-fingers (named C and Q). The Q-type zinc fingers contain a conserved motif, QALGGH, and are plant specific, whereas C type zinc fingers are found in other organisms as well. A genome-wide microarray based gene expression analysis involving 14 stages of vegetative and reproductive development along with 3 stress conditions has revealed that C(2)H(2) gene family in indica rice could be involved during all the stages of reproductive development from panicle initiation till seed maturation. A total of 39 genes are up-regulated more than 2-fold, in comparison to vegetative stages, during reproductive development of rice, out of which 18 are specific to panicle development and 12 genes are seed-specific. Twenty-six genes have been found to be up-regulated during three abiotic stresses and of these, 14 genes express specifically during the stress conditions analyzed while 12 are also up-regulated during reproductive development, suggesting that some components of the stress response pathways are also involved in reproduction. ZFP182|ZOS3-21 A TFIIIA-type zinc finger protein confers multiple abiotic stress tolerances in transgenic rice (Oryza sativa L.) 2012 Plant Mol Biol State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Agriculture, Nanjing Agricultural University, Nanjing 210095, China. The TFIIIA-type zinc finger transcription factors are involved in plant development and abiotic stress responses. Most TFIIIA-type zinc finger proteins are transcription repressors due to existence of an EAR-motif in their amino acid sequences. In this work, we found that ZFP182, a TFIIIA-type zinc finger protein, forms a homodimer in the nucleus and exhibits trans-activation activity in yeast cells. The deletion analysis indicated that a Leu-rich region at C-terminus is required for the trans-activation. Overexpression of ZFP182 significantly enhanced multiple abiotic stress tolerances, including salt, cold and drought tolerances in transgenic rice. Overexpression of ZFP182 promotes accumulation of compatible osmolytes, such as free proline and soluble sugars, in transgenic rice. ZFP182 activates the expression of OsP5CS encoding pyrroline-5-carboxylate synthetase and OsLEA3 under stress conditions, while OsDREB1A and OsDREB1B were regulated by ZFP182 under both normal and stress conditions. Interestingly, site-directed mutagenesis assay showed that DRE-like elements in ZFP182 promoter are involved in dehydration-induced expression of ZFP182. The yeast two-hybrid assay revealed that ZFP182 interacted with several ribosomal proteins including ZIURP1, an ubiquitin fused to ribosomal protein L40. The in vivo and in vitro interactions of ZFP182 and ZIURP1 were further confirmed by bimolecular fluorescence complementation and His pull-down assays. Our studies provide new clues in the understanding of the mechanisms for TFIIIA-type zinc finger transcription factor mediated stress tolerance and a candidate gene for improving stress tolerance in crops. ZFP182|ZOS3-21 Increased tolerance of rice to cold, drought and oxidative stresses mediated by the overexpression of a gene that encodes the zinc finger protein ZFP245 2009 Biochem Biophys Res Commun State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, Jiangsu Province, China. ZFP245 is a cold- and drought-responsive gene that encodes a zinc finger protein in rice. The ZFP245 protein localizes in the nucleus and exhibits trans-activation activity. Transgenic rice plants overexpressing ZFP245 were generated and found to display high tolerance to cold and drought stresses. The transgenic plants did not exhibit growth retardation, but showed growth sensitivity against exogenous abscisic acid, increased free proline levels and elevated expression of rice pyrroline-5-carboxylatesynthetase and proline transporter genes under stress conditions. Overproduction of ZFP245 enhanced the activities of reactive oxygen species-scavenging enzymes under stress conditions and increased the tolerance of rice seedlings to oxidative stress. Our data suggest that ZFP245 may contribute to the tolerance of rice plants to cold and drought stresses by regulating proline levels and reactive oxygen species-scavenging activities, and therefore may be useful for developing transgenic crops with enhanced tolerance to abiotic stress. ZFP245 Identification of a rice zinc finger protein whose expression is transiently induced by drought, cold but not by salinity and abscisic acid 2005 DNA Seq State key laboratory of crop genetics and germplasm enhancement, Nanjing Agricultural University, Nanjing 210095, P.R. China A C2H2-type zinc finger protein gene ZFP245 was cloned by RT-PCR approach from cold treated rice seedlings. ZFP245 is 712 bp in length and encodes a 24.5 KDa protein, which has 35% identity in amino acid with SCOF-1, a cold-inducible zinc finger protein from soybean. By database search, ZFP245 was mapped on chromosome 7 and clustered together with another C2H2 zinc finger gene. Tissue expression analysis showed that ZFP245 was constitutively expressed in various rice tissues including roots, stems, leaves and spikes. The semi-quantitative-RT-PCR assay revealed ZFP245 was strongly induced after 6 h exposure to cold and drought stresses, and then reduced to the baseline. However, ZFP245 was not regulated by high salt or abscisic acid treatment. The promoter sequence of 1017 nucleotides, upstream of ZFP245, was cloned directly by PCR approach. Sequence analysis revealed a CRT/DRE element was found within the ZFP245 promoter region. Taken together, ZFP245, as the first identified C2H2-type zinc finger protein involved in stress response in monocots probably plays a role as a transcription regulator in plant cold and drought responses through an ABA-independent pathway. ZFP245 Cloning and characterization of RZF71 encoding a C2H2-type zinc 2007 Hereditas National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China. A rice zinc-finger protein gene, RZF71, encoding the C2H2-type zinc-finger transcription factor was isolated from rice (Oryza sativa L. subs. Japonica) by RT-PCR approach. Gene RZF71 encodes a 25 kDa protein with 250 amino acids, which contains two typical C2H2 zinc finger domains. The expression profiling showed that RZF71 was constitutively expressed in roots, culms, leaves, and flowering spikes. The semi-quantitative RT-PCR assay showed RZF71 was strongly induced by high-salinity and 20% PEG6000 treatments, but not regulated by low temperature and ABA (abscisic acid) treatments. Tran-sient expression of the RZF71-GFP protein in onion epidermal cell showed that RZF71 was localized in cell nuclei. These results indicated that the RZF71 may play an important role in rice responses to salt and osmotic stresses as a transcription factor. ZFP252|RZF71 Differential Metal Selectivity and Gene Expression of Two Zinc Transporters from Rice 2003 Plant Physiol Commonwealth Scientific and Industrial Research Organization Plant Industry-Horticulture Unit, Glen Osmond, South Australia 5064, Australia. Zinc is an essential mineral for a wide variety of physiological and biochemical processes. To understand zinc transport in cereals, we identified putative zinc transporters in gene databases. Three full-length cDNAs were identified and characterized from rice (Oryza sativa). Two of the cDNAs partially complemented a yeast (Saccharomyces cerevisiae) mutant deficient in zinc uptake at low concentrations. The two transporters showed many similarities in function but differed in ionic selectivity and pH optimum of activity. Expression patterns also differed between the two genes. One gene was broadly expressed under all conditions, and the other gene was mainly induced by zinc deficiency to higher levels in roots than in leaves. Although the timing of expression differed between the two genes, localization of expression overlapped in roots. Comparisons of the protein sequences, ionic selectivity, and gene expression patterns of the two transporters suggest that they may play different roles in the physiology of the whole plant. OsZIP1,OsZIP3 Effect of magnesium deficiency on antioxidant status and cadmium toxicity in rice seedlings 2011 J Plant Physiol Department of Agronomy, National Taiwan University, Taipei, Taiwan, ROC. Cadmium (Cd) is one of the most toxic heavy metals and inhibits physiological processes of plants. Magnesium (Mg) is known as one of the essential nutrients for plants. Mg deficiency in plants affects metabolic processes. Plants grown in the field may encounter several abiotic stresses, rather than a single stress. Thus, the relationship between Mg nutrition and Cd toxicity is of ecological importance. In this study, effects of Mg deficiency on antioxidant systems and Cd toxicity in rice seedlings were investigated. Mg deficiency significantly decreased Mg concentrations in shoot and roots of rice seedlings. However, fresh weight and dry weight of rice seedlings were not affected by Mg deficiency. The contents of ascorbate and glutathione (GSH), the ratio of GSH/oxidized glutathione, and the activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, and catalase in Mg-deficient leaves were higher than respective control leaves. Cd toxicity was judged by the decrease in biomass production, decrease in chlorophyll, and induction of oxidative stress. Based on these criteria, we demonstrated that Mg deficiency protected rice seedlings from Cd stress. Moreover, chlorophyll destruction by paraquat was higher in detached leaves from Mg-sufficient than Mg-deficient seedlings. Cd concentration was higher in Mg-deficient shoot and roots than their respective control shoot and roots, suggesting that the protective effect of Mg deficiency against Cd toxicity is not due to reduction of Cd uptake. Moreover, we observed that Cd-decreased Fe and Zn contents in Mg-deficient seedlings were more pronounced than that in Mg-sufficient seedlings. Of particular interest is the finding that the increase in OsIRT1, OsZIP1, and OsZIP3 transcripts caused by Cd in Mg-deficient roots was greater than that in control roots. OsZIP3 ZEBRA-NECROSIS, a thylakoid-bound protein, is critical for the photoprotection of developing chloroplasts during early leaf development 2010 Plant J Department of Plant Science, Plant Genomics and Breeding Institute, and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea. The zebra-necrosis (zn) mutant of rice (Oryza sativa) produces transversely green/yellow-striped leaves. The mutant phenotype is formed by unequal impairment of chloroplast biogenesis before emergence from the leaf sheath under alternate light/dark or high/low temperatures (restrictive), but not under constant light and temperature (permissive) conditions. Map-based cloning revealed that ZN encodes a thylakoid-bound protein of unknown function. Virus-induced gene silencing of a ZN homolog in Nicotiana benthamiana causes leaf variegation with sporadic green/yellow sectors, indicating that ZN is essential for chloroplast biogenesis during early leaf development. Necrotic lesions often occur in the yellow sectors as a result of an excessive accumulation of reactive oxygen species (ROS). The phenotypic severity (leaf variegation and necrosis) and ROS levels are positively correlated with an increase in light intensity under restrictive conditions. In the mutant leaves, chlorophyll (Chl) metabolism, ROS scavenging activities, maximum quantum yield of photosystem II (PSII), and structures and functions of the photosynthetic complexes are normal in the Chl-containing cells, suggesting that ROS are mainly generated from the defective plastids of the Chl-free cells. The PSII activity of normal chloroplasts is hypersensitive to photoinhibition because the recovery rates of PSII are much slower. In the PSII repair, the degradation of damaged D1 is not impaired, suggesting a reduced activity of new D1 synthesis, possibly because of higher levels of ROS generated from the Chl-free cells by excess light. Together, we propose that ZN is required for protecting developing chloroplasts, especially during the assembly of thylakoid protein complexes, from incidental light after darkness. ZN,OsZNL Molecular characterization of the Arginine decarboxylase gene family in rice 2010 Transgenic Res Departament de Produccio Vegetal i Ciencia Forestal, ETSEA, Universitat de Lleida, Av. Alcalde Rovira Roure 191, 25198, Lleida, Spain. Arginine decarboxylase (ADC) is a key enzyme in plants that converts arginine into putrescine, an important mediator of abiotic stress tolerance. Adc genes have been isolated from a number of dicotyledonous plants but the oat and rice Adc genes are the only representatives of monocotyledonous species described thus far. Rice has a small family of Adc genes, and OsAdc1 expression has been shown to fluctuate under drought and chilling stress. We identified and characterized a second rice Adc gene (OsAdc2) which encodes a 629-amino-acid protein with a predicted molecular mass of 67 kDa. An unusual feature of the OsAdc2 gene is the presence of an intron and a short upstream open reading frame in the 5'-UTR. Sequence comparisons showed that OsAdc2 is more closely related to the oat Adc gene than to OsAdc1 or to its dicot homologs, and mRNA analysis showed that the two rice genes are also differently regulated. Whereas OsAdc1 is expressed in leaf, root and stem, OsAdc2 expression is restricted to stem tissue. Protein expression was investigated with specific antibodies against ADC1 and ADC2, corroborating the mRNA data. We discuss the expression profiles of OsAdc1 and OsAdc2 and potential functions for the two corresponding proteins. ADCa|OsAdc2 Posttranscriptional control of photosynthetic mRNA decay under stress conditions requires 3' and 5' untranslated regions and correlates with differential polysome association in rice 2012 Plant Physiol School of Biotechnology and Environmental Engineering, Myongji University, Yongin 449-728, Korea. Abiotic stress, including drought, salinity, and temperature extremes, regulates gene expression at the transcriptional and posttranscriptional levels. Expression profiling of total messenger RNAs (mRNAs) from rice (Oryza sativa) leaves grown under stress conditions revealed that the transcript levels of photosynthetic genes are reduced more rapidly than others, a phenomenon referred to as stress-induced mRNA decay (SMD). By comparing RNA polymerase II engagement with the steady-state mRNA level, we show here that SMD is a posttranscriptional event. The SMD of photosynthetic genes was further verified by measuring the half-lives of the small subunit of Rubisco (RbcS1) and Chlorophyll a/b-Binding Protein1 (Cab1) mRNAs during stress conditions in the presence of the transcription inhibitor cordycepin. To discern any correlation between SMD and the process of translation, changes in total and polysome-associated mRNA levels after stress were measured. Total and polysome-associated mRNA levels of two photosynthetic (RbcS1 and Cab1) and two stress-inducible (Dehydration Stress-Inducible Protein1 and Salt-Induced Protein) genes were found to be markedly similar. This demonstrated the importance of polysome association for transcript stability under stress conditions. Microarray experiments performed on total and polysomal mRNAs indicate that approximately half of all mRNAs that undergo SMD remain polysome associated during stress treatments. To delineate the functional determinant(s) of mRNAs responsible for SMD, the RbcS1 and Cab1 transcripts were dissected into several components. The expressions of different combinations of the mRNA components were analyzed under stress conditions, revealing that both 3' and 5' untranslated regions are necessary for SMD. Our results, therefore, suggest that the posttranscriptional control of photosynthetic mRNA decay under stress conditions requires both 3' and 5' untranslated regions and correlates with differential polysome association. ASCAB9-A|CP26|Lhcb5,CP24,SSIIB Biological functions of asparagine synthetase in plants 2010 Plant Science Département Adaptation des Plantes à l’Environnement, IJPB, UMR1318 AgroParis Tech, Institut National de la Recherche Agronomique, Route de St-Cyr, 78026 Versailles cedex, France Ammonium is a form of inorganic nitrogen derived from several metabolic pathways, and is assimilated into glutamine, glutamate, asparagine and carbamoylphosphate. These molecules play important roles in nitrogen assimilation, recycling, transport and storage in plants. Ammonium assimilation into asparagine is catalyzed by ammonia-dependent asparagine synthetase encoded by asnA (EC 6.3.1.1) or glutamine-dependent asparagine synthetase encoded by asnB (EC 6.3.5.4) in prokaryotes and eukaryotes. These organisms display a distinct distribution of these two forms of asparagine synthetase. Gene and primary protein structure for asparagine synthetase-A and -B from prokaryotes and eukaryotes is examined. Using nucleotide sequences, we constructed a phylogenetic tree that distinguished two major classes (classes I and II) for ASN genes from a range of organisms. Only the glutamine-dependent asparagine synthetases-B have been identified, and are encoded by a small multigene family in plants. The isoenzyme encoded by each member of the gene family provides asparagine at specific phases of development. These include the nitrogen mobilization in germinating seeds, nitrogen recycling in vegetative organs in response to stress, and nitrogen remobilization during seed embryogenesis. The expression of genes for asparagine synthetase is regulated by light and metabolites. Genetic and molecular data using mutants and transgenic plants have provided insights into the light perception by the photoreceptors, carbon and nitrogen sensing and signal transduction mechanism in the asn regulation. Global analysis of carbon and nitrogen metabolites supports the impact of asn regulation in the synthesis and transport of asparagine in plants. ASN,OsASN1 Organ and Cellular Localization of Asparagine Synthetase in Rice Plants 2000 Plant and Cell Physiology Department of Applied Plant Science, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555 Japan DNA gel blot analysis suggested that asparagine synthetase (AS; EC 6.3.5.4) occurred as a single gene in rice. A fusion protein consisting of 17 kDa tagged-region from pET32a(+) expression plasmid and 42 kDa N-terminal region of rice AS was first expressed in Escherichia coli. The resulting polypeptide was purified and a mono-specific antibody for rice AS was prepared after affinity-purification with the antigen. Immunoblotting revealed a high content of AS protein in the leaf sheath at the second position from the fully expanded top leaf and in grains at the middle stage of ripening. Accumulation of mRNA for AS was also observed in these organs. During the ripening of the spikelets, the AS protein contents increased during the first 21 days after flowering, then declined rapidly. Immunolocalization analysis revealed signals for AS protein in the companion cells of vascular bundles of leaf sheath and phloem-parenchyma cells, nucellar projection, and nucellar epidermis of dorsal vascular bundles of grains. ASN Regulation of ATG6/Beclin-1 homologs by abiotic stresses and hormones in rice (Oryza sativa L.) 2012 Genetics and Molecular Research State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China. Autophagy, a complex and conserved mechanism, serving as a defense response in all eukaryotic organisms, is regulated by several proteins, among which ATG proteins are the most important due to their involvement in autophagosome formation. ATG6/Beclin-1 proteins, reported to be essential for autophagosome formation and assigned as a conserved domain, were subjected to database searches. We found three homologs in the rice (Oryza sativa) genome. A phylogeny tree was constructed to establish their across species relationship, which divided them into three distinct groups; two for plants, i.e., monocots and dicots, and one for animals. Evolutionary study of this family by critical amino acid conservation analysis revealed significant functional divergence. The finding of important stress-related cis-acting elements in the promoter region of rice ATG6 genes demonstrated their involvement in abiotic stress responses. Furthermore, expression profiling of rice ATG6 genes based on microarray data, as well as by semi-quantitative RT-PCR showed differential expression when subjected to different stresses suggesting the involvement of OsATG6 genes in abiotic stresses (heat, cold and drought) and hormone (abscisic acid) responses. Analysis of co-expressed genes showed that most of them annotated to DNA repair pathways and proteolysis, etc. Collectively, these results suggest the involvement of OsATG6 genes in different stresses, and provide a basis for further functional studies to investigate the biological mechanism of action of these genes under abiotic stresses. OsATG6a,ATG6B,ATG6C Genome-wide identification, classification, and expression analysis of autophagy-associated gene homologues in rice (Oryza sativa L.) 2011 DNA Res Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, PR China. Autophagy is an intracellular degradation process for recycling macromolecules and organelles. It plays important roles in plant development and in response to nutritional demand, stress, and senescence. Organisms from yeast to plants contain many autophagy-associated genes (ATG). In this study, we found that a total of 33 ATG homologues exist in the rice [Oryza sativa L. (Os)] genome, which were classified into 13 ATG subfamilies. Six of them are alternatively spliced genes. Evolutional analysis showed that expansion of 10 OsATG homologues occurred via segmental duplication events and that the occurrence of these OsATG homologues within each subfamily was asynchronous. The Ka/Ks ratios suggested purifying selection for four duplicated OsATG homologues and positive selection for two. Calculating the dates of the duplication events indicated that all duplication events might have occurred after the origin of the grasses, from 21.43 to 66.77 million years ago. Semi-quantitative RT-PCR analysis and mining the digital expression database of rice showed that all 33 OsATG homologues could be detected in at least one cell type of the various tissues under normal or stress growth conditions, but their expression was tightly regulated. The 10 duplicated genes showed expression divergence. The expression of most OsATG homologues was regulated by at least one treatment, including hormones, abiotic and biotic stresses, and nutrient limitation. The identification of OsATG homologues showing constitutive expression or responses to environmental stimuli provides new insights for in-depth characterization of selected genes of importance in rice. OsATG6a,ATG6B,ATG6C Differential expression of three BOR1 genes corresponding to different genomes in response to boron conditions in hexaploid wheat (Triticum aestivum L.) 2013 Plant Cell Physiol Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, 113-8657 Japan. Boron (B) is an essential micronutrient for plants. Efflux-type B transporters, BORs, have been identified in Arabidopsis thaliana and rice. Here we identified BOR1 genes encoding B efflux transporters, from the hexaploid genome of wheat (Triticum aestivum L.). We cloned three genes closely related to OsBOR1 and named them TaBOR1.1, TaBOR1.2 and TaBOR1.3. All three TaBOR1s showed B efflux activities when expressed in tobacco BY-2 cells. TaBOR1-green fluorescent protein (GFP) fusion proteins were expressed in Arabidopsis leaf cells localized in the plasma membrane. The transcript accumulation patterns of the three genes differ in terms of tissue specificity and B nutrition responses. In roots, transcripts for all three genes accumulated abundantly while in shoots, the TaBOR1.2 transcript is the most abundant, followed by those of TaBOR1.1 and TaBOR1.3. Accumulation of TaBOR1.1 transcript is up-regulated under B deficiency conditions in both roots and shoots. In contrast, TaBOR1.2 transcript accumulation significantly increased in roots under excess B conditions. TaBOR1.3 transcript accumulation was reduced under excess B. Taken together, these results demonstrated that TaBOR1s are the B efflux transporters in wheat and, interestingly, the genes on the A, B and D genomes have different expression patterns. BOR2 Identification of novel genes differentially expressed in compatible and incompatible interactions between rice and Pseudomonas avenae 2002 Plant Science Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5, Takayama Ikoma, Nara 630-0101, Japan Establishment of a plant-pathogen interaction involves differential gene expression in both organisms. To isolate rice genes induced during either compatible or incompatible interactions, we performed a comparative analysis of expression patterns in cultured rice cells, inoculated with either compatible or incompatible strains of Pseudomonas avenae, by fluorescence differential display (FDD). Using 52 sets of arbitrary primer combinations, two cDNAs (IAI1 and IAI2) expressed in incompatible strain-inoculated rice cells and one cDNA (CAII) expressed in compatible strain inoculated cells, were identified. A hydropathy profile revealed that the IAI1 protein spans the membrane at a putative transmembrane domain. The deduced amino acid sequence of IAI2 shares considerable homology with Bowman-Birk proteinase inhibitors over the majority of the sequence. The deduced CAII protein is rich in glycine, containing a putative signal peptide with a potential cleavage site, suggesting its membership in the glycine-rich protein (GRP) family. GRPs are thought to be structural cell wall proteins. When a GFP fusion with the CAII gene was introduced into onion cells by bombardment, green fluorescence of the cell wall was observed. These data suggest that CAII is involved in the repair of cell wall injuries by the compatible strain of P. avenae. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved. CAI1,IAI1,IAI2 Imprinted expression of genes and small RNA is associated with localized hypomethylation of the maternal genome in rice endosperm 2013 Proc Natl Acad Sci U S A Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA. Arabidopsis thaliana endosperm, a transient tissue that nourishes the embryo, exhibits extensive localized DNA demethylation on maternally inherited chromosomes. Demethylation mediates parent-of-origin-specific (imprinted) gene expression but is apparently unnecessary for the extensive accumulation of maternally biased small RNA (sRNA) molecules detected in seeds. Endosperm DNA in the distantly related monocots rice and maize is likewise locally hypomethylated, but whether this hypomethylation is generally parent-of-origin specific is unknown. Imprinted expression of sRNA also remains uninvestigated in monocot seeds. Here, we report high-coverage sequencing of the Kitaake rice cultivar that enabled us to show that localized hypomethylation in rice endosperm occurs solely on the maternal genome, preferring regions of high DNA accessibility. Maternally expressed imprinted genes are enriched for hypomethylation at putative promoter regions and transcriptional termini and paternally expressed genes at promoters and gene bodies, mirroring our recent results in A. thaliana. However, unlike in A. thaliana, rice endosperm sRNA populations are dominated by specific strong sRNA-producing loci, and imprinted 24-nt sRNAs are expressed from both parental genomes and correlate with hypomethylation. Overlaps between imprinted sRNA loci and imprinted genes expressed from opposite alleles suggest that sRNAs may regulate genomic imprinting. Whereas sRNAs in seedling tissues primarily originate from small class II (cut-and-paste) transposable elements, those in endosperm are more uniformly derived, including sequences from other transposon classes, as well as genic and intergenic regions. Our data indicate that the endosperm exhibits a unique pattern of sRNA expression and suggest that localized hypomethylation of maternal endosperm DNA is conserved in flowering plants. CAPIP1 Distribution, structure, organ-specific expression, and phylogenic analysis of the pathogenesis-related protein-3 chitinase gene family in rice (Oryza sativa L.) 2006 Genome Laboratory of Plant Breeding, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan. Rice (Oryza sativa L.) pathogenesis-related (PR)-3 chitinases, like other PR proteins, are each coded by one of the genes of a multigene family in the plant genome. We assembled the database information about rice PR-3 chitinase sequences. A total of 12 PR-3 chitinase loci (Cht1 to Cht12) were found deployed in the rice genome. Some of the loci were occupied by 2 or more alleles. For all the loci expect Cht4, Cht5, Cht6, and Cht11, the amino acid sequence was polymorphic between japonica and indica varieties of rice, but glutamic acid acting as a catalytic residue was completely conserved in all the loci expect Cht7. All the genes except Cht7, which was not tested in this study, were transcripted in some organs (leaf, sheath, root, and meristem) of rice plants. These results suggest that chitinase proteins encoded by the genes at these loci have important biological effects, at least antifungal activities, on rice plants. We also proposed a new classification of rice PR-3 chitinases based on their domain structures. This classification was consistent with the results of phylogenetic analysis of rice chitinases. CHIA1;175,RCH10 A rice lectin receptor-like kinase that is involved in innate immune responses also contributes to seed germination 2013 Plant J State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, 430072, China. Seed germination and innate immunity both have significant effects on plant life spans because they control the plant's entry into the ecosystem and provide defenses against various external stresses, respectively. Much ecological evidence has shown that seeds with high vigor are generally more tolerant of various environmental stimuli in the field than those with low vigor. However, there is little genetic evidence linking germination and immunity in plants. Here, we show that the rice lectin receptor-like kinase OslecRK contributes to both seed germination and plant innate immunity. We demonstrate that knocking down the OslecRK gene depresses the expression of alpha-amylase genes, reducing seed viability and thereby decreasing the rate of seed germination. Moreover, it also inhibits the expression of defense genes, and so reduces the resistance of rice plants to fungal and bacterial pathogens as well as herbivorous insects. Yeast two-hybrid and co-immunoprecipitation experiments revealed that OslecRK interacts with an actin-depolymerizing factor (ADF) in vivo via its kinase domain. Moreover, the rice adf mutant exhibited a reduced seed germination rate due to the suppression of alpha-amylase gene expression. This mutant also exhibited depressed immune responses and reduced resistance to biotic stresses. Our results thus provide direct genetic evidence for a common physiological pathway connecting germination and immunity in plants. They also partially explain the common observation that high-vigor seeds often perform well in the field. The dual effects of OslecRK may be indicative of progressive adaptive evolution in rice. CHS Elicitor induced activation of the methylerythritol phosphate pathway toward phytoalexins biosynthesis in rice 2007 Plant Mol Biol Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Tokyo, Japan. Diterpenoid phytoalexins such as momilactones and phytocassanes are produced via geranylgeranyl diphosphate in suspension-cultured rice cells after treatment with a chitin elicitor. We have previously shown that the production of diterpene hydrocarbons leading to phytoalexins and the expression of related biosynthetic genes are activated in suspension-cultured rice cells upon elicitor treatment. To better understand the elicitor-induced activation of phytoalexin biosynthesis, we conducted microarray analysis using suspension-cultured rice cells collected at various times after treatment with chitin elicitor. Hierarchical cluster analysis revealed two types of early-induced expression (EIE-1, EIE-2) nodes and a late-induced expression (LIE) node that includes genes involved in phytoalexins biosynthesis. The LIE node contains genes that may be responsible for the methylerythritol phosphate (MEP) pathway, a plastidic biosynthetic pathway for isopentenyl diphosphate, an early precursor of phytoalexins. The elicitor-induced expression of these putative MEP pathway genes was confirmed by quantitative reverse-transcription PCR. 1-Deoxy-D: -xylulose 5-phosphate synthase (DXS), 1-deoxy-D: -xylulose 5-phosphate reductoisomerase (DXR), and 4-(cytidine 5'-diphospho)-2-C-methyl-D: -erythritol synthase (CMS), which catalyze the first three committed steps in the MEP pathway, were further shown to have enzymatic activities that complement the growth of E. coli mutants disrupted in the corresponding genes. Application of ketoclomazone and fosmidomycin, inhibitors of DXS and DXR, respectively, repressed the accumulation of diterpene-type phytoalexins in suspension cells treated with chitin elicitor. These results suggest that activation of the MEP pathway is required to supply sufficient terpenoid precursors for the production of phytoalexins in infected rice plants. CLA1|OsDXS1|OsDXS2,OsCMK,OsDXR,OsDXS|OsDXS3,OsHDR,OsHDS,OsMCS A novel interaction between calreticulin and ubiquitin-like nuclear protein in rice 2004 Plant and Cell Physiology Japan Society for the Promotion of Science, Tsukuba, Ibaraki, 305-8602 Japan. Calreticulin (CRT), a major Ca2+-sequestering protein, has been implicated in a variety of cellular functions such as Ca2+ storage, signaling and chaperone activity within the cytoplasm and endoplasmic reticulum. To investigate the biological role of CRT in rice, 21 partial cDNAs, encoding proteins that interacted with rice CRT in a yeast two-hybrid interaction-cloning system, were characterized and the nucleotide sequences were found to be identical to each other. A full-length cDNA of 3.5 kb, obtained from rice genomic sequence data and 5' RACE, codes for a novel protein of 966 amino acid residues and was designated as CRTintP (CRT interacting protein). Primary sequence analysis of CRTintP showed no sequence homology with the known functional proteins; however, a potential ubiquitin-like domain at the N-terminal together with a putative leucine zipper, a nuclear localization signal and several sites for serine/threonine kinases were evident. Cellular localization of CRTintP demonstrated its role in directing green fluorescent protein to the nucleus in onion epidermal cells. Northern and immunoblot analysis showed increased expression of CRT and CRTintP in response to cold stress. Co-immunoprecipitation using anti-CRT antibodies confirmed the existence of the CRT-CRTintP complex in vivo in the stressed leaf tissue, suggesting their potential role in regulating stress response. CRTintP Rice cytosine DNA methyltransferases - gene expression profiling during reproductive development and abiotic stress 2009 FEBS J Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, India. DNA methylation affects important developmental processes in both plants and animals. The process of methylation of cytosines at C-5 is catalysed by DNA methyltransferases (MTases), which are highly conserved, both structurally and functionally, in eukaryotes. In this study, we identified and characterized cytosine DNA MTase genes that are activated with the onset of reproductive development in rice. The rice genome (Oryza sativa L. subsp. japonica) encodes a total of 10 genes that contain the highly conserved MTase catalytic domain. These genes have been categorized into subfamilies on the basis of phylogenetic relationships. A microarray-based gene expression profile of all 10 MTases during 22 stages/tissues that included 14 stages of reproductive development and five vegetative tissues together with three stresses, cold, salt and dehydration stress, revealed specific windows of MTase activity during panicle and seed development. The expression of six methylases was specifically/preferentially upregulated with the initiation of floral organs. Significantly, one of the MTases was also activated in young seedlings in response to cold and salt stress. The molecular studies presented here suggest a greater role for these proteins and the epigenetic process in affecting genome activity during reproductive development and stress than was previously anticipated. DNMT2 Novel Plant Ca[IMAGE]-binding Protein Expressed in Response to Abscisic Acid and Osmotic Stress 1996 Journal of Biological Chemistry Institute of Molecular Biology, Copenhagen University, Denmark. A cDNA corresponding to an mRNA which accumulates in germinating rice seeds in response to the phytohormone abscisic acid was isolated by differential hybridization. Northern blotting indicated that the mRNA also accumulates in vegetative tissues in response to treatment with abscisic acid and to osmotic stress. Sequencing identified a major open reading frame encoding a novel protein of 27.4 kDa. The identity of the open reading frame was confirmed by comparing the translation products of cellular, hybrid-selected, and in vitro transcribed RNAs and by immunoprecipitation. Western blotting of cellular extracts indicated that the protein is associated with microsomal or membrane fractions. Data base searches indicated that it contains a conserved Ca(2+)-binding, EF-hand motif and that related proteins are similarly expressed in Arabidopsis thaliana. A fusion protein purified from Escherichia coli containing the putative EF-hand region was shown to bind Ca2+ in blot binding assays. These data identify a novel gene family encoding proteins involved in the response of plants to abscisic acid and osmotic stress. EFA27 Structure, organization and putative function of the genes identified within a 23.9-kb fragment from Arabidopsis thaliana chromosome IV 1997 Gene Laboratoire de Biologie du Développement des Plantes, Institut de Biotechnologie des Plantes, ERS/CNRS 569, Université de Paris-Sud, Orsay, France. In the framework of the complete genome sequencing programme of the crucifer Arabidopsis thaliana, a 23.9-kb fragment from the long arm of chromosome IV has been analysed. This paper presents a methodological approach, integrating computerized predictions, database screening, the sequencing of cognate cDNAs and a PCR-based detection of expression that allows the accumulation of an important amount of information from an anonymous sequence. This work revealed the organization of novel genes and the vestige of a copia-like retrotransposon. The gene AtRH1 encodes the first member of a new subfamily of the plant DEAD box RNA helicases. A recurrent and complete search of dbEST has been used to evaluate the number of different RNA helicases expressed in A. thaliana. On the 18 discriminated members of the family, only a small number seems to be expressed at a relatively high level. The putative gene AtTS1 encodes a novel terpene synthase in A. thaliana, and the genes G14587-5 and G14587-6 encode unknown proteins. This study illustrates most of the situations that could be encountered during the analysis of an anonymous sequence from A. thaliana. eIF-4a|EIF4A Cloning and expression of a ferredoxin gene in rice roots in response to nitrate and ammonium 1998 Plant Science Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan A ferredoxin cDNA was isolated from a λgt 11 cDNA library constructed from nitrate-induced rice (Oryza sativa L.) roots. The root Fd cDNA clone (pFDRR) was 603 bp long which contained 261-bp 5′ region encoding the C-terminal 72 amino acid residues and 342-bp 3′ noncoding region. A genomic clone was isolated from a λEMBL3 rice genomic library using pFDRR as a probe and its nucleotide sequence was identical with that of the cDNA clone in their overlapping region. The root Fd gene contained 1091-bp 5′ upstream, 444-bp coding and 294-bp 3′ noncoding regions. The transcription start site locates 46 bp upstream of the ATG translation initiation codon. In the 5′ flanking region, two GATA elements and a TCCGCGGA-like sequence are located in addition to TATA box-like and CAAT sequences. Homology between the rice root and leaf Fds showed 60%, while the former protein shared much higher identity with other nonphotosynthetic Fds (66–88%). Southern blot analysis using pFDRR and the rice leaf Fd cDNA (pFDRL) as a DNA probe identified their corresponding genomic Fd DNA. Expression of the root Fd gene was induced in response to nitrate and ammonium. Fd Functional characterization of two flap endonuclease-1 homologues in rice 2003 Gene Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Chiba, 278-8510, Noda, Japan. Flap endonuclease-1 (FEN-1) is an important enzyme involved in DNA replication and repair. Previously, we isolated and characterized a complementary DNA (cDNA) from rice (Oryza sativa) encoding a protein which shows homology with the eukaryotic flap endonuclease-1 (FEN-1). In this report, we found that rice (O. sativa L. cv. Nipponbare) possessed two FEN-1 homologues designated as OsFEN-1a and OsFEN-1b. The OsFEN-1a and OsFEN-1b genes were mapped to chromosome 5 and 3, respectively. Both genes contained 17 exons and 16 introns. Alignment of OsFEN-1a protein with OsFEN-1b protein showed a high degree of sequence similarity, particularly around the N and I domains. Northern hybridization and in situ hybridization analysis demonstrated preferential expression of OsFEN-1a and OsFEN-1b in proliferating tissues such as the shoot apical meristem or young leaves. The levels of OsFEN-1a and OsFEN-1b expression were significantly reduced when cell proliferation was temporarily halted by the removal of sucrose from the growth medium. When the growth-halted cells began to regrow following the addition of sucrose to the medium, both OsFEN-1a and OsFEN-1b were again expressed at high level. These results suggested that OsFEN-1a and OsFEN-1b are required for cell proliferation. Functional complementation assay suggested that OsFEN-1a cDNA had the ability to complement Saccharomyces cerevisiae rad27 null mutant. On the other hand, OsFEN-1b cDNA could not complement the rad27 mutant. The roles of OsFEN-1a and OsFEN-1b in plant DNA replication and repair are discussed. OsFEN-1b Differential expression of three flavanone 3-hydroxylase genes in grains and coleoptiles of wheat 2011 Int J Plant Genomics Institute of Plant Science and Resources, Okayama University, Okayama, Kurashiki 710-0046, Japan. Flavonoid pigments are known to accumulate in red grains and coleoptiles of wheat and are synthesized through the flavonoid biosynthetic pathway. Flavanone 3-hydroxylase (F3H) is a key enzyme at a diverging point of the flavonoid pathway leading to production of different pigments: phlobaphene, proanthocyanidin, and anthocyanin. We isolated three F3H genes from wheat and examined a relationship between their expression and tissue pigmentation. Three F3Hs are located on the telomeric region of the long arm of chromosomes 2A, 2B, and 2D, respectively, designated as F3H-A1, F3H-B1, and F3H-D1. The telomeric regions of the long arms of the chromosomes of homoeologous group 2 of wheat showed a syntenic relationship to the telomeric region of the long arm of rice chromosome 4, on which rice F3H gene was also located. All three genes were highly activated in the red grains and coleoptiles and appeared to be controlled by flavonoid regulators in each tissue. FLS The genomic organization of the gene encoding a nitrate-inducible ferredoxin-NADP+ oxidoreductase from rice roots 1995 Biochim Biophys Acta Research Institute for Food Science, Kyoto University, Japan. A genomic clone of the gene encoding a nitrate-inducible ferredoxin-NADP+ oxidoreductase (FNR) from rice (Oryza sativa L.) roots has been isolated and its nucleotide sequence determined. The clone contains 3897 nucleotides of the gene which consists of six exons interrupted by five introns. The transcription start site was determined by primer extension analysis which locates 64 bp upstream of the ATG translation initiation codon. The 5'-flanking region contains canonical TATA- and CAAT-boxes, and a potential Sp1-binding site. Four ATCAA(A/C) and two inverted TTTGAT sequences are localized in the promoter region and a TGTAA motif occurs three times in the 3'-untranslated region. No significant similarity was found when the 5' flanking region was compared with that of the photosynthetic FNR gene. FNR The rice nuclear gene, VIRESCENT 2, is essential for chloroplast development and encodes a novel type of guanylate kinase targeted to plastids and mitochondria 2007 The Plant Journal Department of Biological Sciences, Faculty of Sciences, Kyushu University, Fukuoka 812-8581, Japan. Guanylate kinase (GK) is a critical enzyme in guanine nucleotide metabolism pathways, catalyzing the phosphorylation of (d)GMP to (d)GDP. Here we show that a novel gene, VIRESCENT 2 (V2), encodes a new type of GK (designated pt/mtGK) that is localized in plastids and mitochondria. We initially identified the V2 gene by positional cloning of the rice v2 mutant. The v2 mutant is temperature-sensitive and develops chlorotic leaves at restrictive temperatures. The v2 mutation causes inhibition of chloroplast differentiation; in particular, it disrupts the chloroplast translation machinery during early leaf development [Sugimoto et al. (2004)Plant Cell Physiol. 45, 985]. In the bacterial and animal species studied to date, GK is localized in the cytoplasm and participates in maintenance of the guanine nucleotide pools required for many fundamental cellular processes. Phenotypic analysis of rice seedlings with RNAi knockdown of cytosolic GK (designated cGK) showed that cGK is indispensable for the growth and development of plants, but not for chloroplast development. Thus, rice has two types of GK, as does Arabidopsis, suggesting that higher plants have two types of GK. Our results suggest that, of the two types of GK, only pt/mtGK is essential for chloroplast differentiation. GK1 Cloning of the rice seed α-globulin-encoding gene: sequence similarity of the 5'-flanking region to those of the genes encoding wheat high-molecular-weight glutenin and barley D hordein 1996 Gene Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Chikusa, Nagoya 464-01, Japan A genomic clone encoding the rice endosperm major globulin (alpha-globulin) with an apparent molecular mass of 26 kDa was isolated, and its nucleotide (nt) sequence and transcription start point (tsp) were determined. The tsp was identical to that of the gene encoding the wheat high-molecular-weight (HMW) glutenin subunit. The consensus '-300 element' and an A + T-rich sequence exist upstream from the TATA box in the 5'-flanking region. A nt sequence of about 130 bp in the 5'-flanking region was found to be markedly homologous to those of the genes encoding the wheat HMW glutenin subunit and barley D hordein. GLB1 Structure and expression of a root-specific rice gene 1992 Plant Mol Biol Department of Plant Molecular Biology, Leiden University, Netherlands. Two rice cDNA clones (COS6 and COS9) were isolated, corresponding to genes that were highly expressed in roots from seedlings and mature plants. A genomic clone (GOS9) corresponding to cDNA clone COS9 was isolated and the intron/exon structure was determined by comparing the nucleotide sequences of the mRNA and the genomic clone. 5' ends and 3' ends of the mRNA were determined by primer extension and S1-nuclease mapping respectively. The open reading frame present in GOS9 potentially encodes a protein (14 kDa) that does not show any significant homology to other proteins in databases. GOS9 Organisation and structural evolution of the rice glutathione S -transferase gene family 2004 Molecular Genetics and Genomics Department of Biomolecular Sciences and Biotechnology, University of Milan, Via Celoria 26, 20133, Milano, Italy. Glutathione S-transferases (GSTs) comprise a large family of key defence enzymes against xenobiotic toxicity. Here we describe the comprehensive characterisation of this important multigene family in the model monocot species rice [ Oryza sativa(L.)]. Furthermore, we investigate the molecular evolution of the family based on the analysis of (1) the patterns of within-genome duplication, and (2) the phylogenetic relationships and evolutionary divergence among rice, Arabidopsis, maize and soybean GSTs. By in-silico screening of the EST and genome divisions of the Genbank/EMBL/DDBJ database we have isolated 59 putative genes and two pseudogenes, making this the largest plant GST family characterised to date. Of these, 38 (62%) are represented by genomic and EST sequences and 23 (38%) are known only from their genomic sequences. A preliminary survey of EST collections shows a large degree of variability in gene expression between different tissues and environmental conditions, with a small number of genes (13) accounting for 80% of all ESTs. Rice GSTs are organised in four main phylogenetic classes, with 91% of all rice genes belonging to the two plant-specific classes Tau (40 genes) and Phi (16 genes). Pairwise identity scores range between 17 and 98% for proteins of the same class, and 7 and 21% for interclass comparisons. Rapid evolution by gene duplication is suggested by the discovery of two large clusters of 7 and 23 closely related genes on chromosomes 1 and 10, respectively. A comparison of the complete GST families in two monocot and two dicot species suggests a monophyletic origin for all Theta and Zeta GSTs, and no more than three common ancestors for all Phi and Tau genes. OsGSTT1 Sodium transport and HKT transporters: the rice model 2003 Plant J Departamento de Biotecnologia, Escuela Tecnica Superior de Ingenieros Agronomos, Universidad Politecnica de Madrid, 28040 Madrid, Spain. Na+ uptake in the roots of K+-starved seedlings of barley, rice, and wheat was found to exhibit fast rate, low Km, and high sensitivity to K+. Sunflower plants responded in a similar manner but the uptake was not K+ sensitive. Ba2+ inhibited Na+ uptake, but not K+ uptake in rice roots. This demonstrated that Na+ and K+ uptake are mediated by different transporters, and that K+ blocked but was not transported by the Na+ transporter. The genome of rice cv. Nipponbare contains seven HKT genes, which may encode Na+ transporters, plus two HKT pseudogenes. Yeast expressions of OsHKT1 and OsHKT4 proved that they are Na+ transporters of high and low affinity, respectively, which are sensitive to K+ and Ba2+. Parallel experiments of K+ and Na+ uptake in yeast expressing the wheat or rice HKT1 transporters proved that they were very different; TaHKT1 transported K+ and Na+, and OsHKT1 only Na+. Transcript expressions in shoots of the OsHKT genes were fairly constant and insensitive to changes in the K+ and Na+ concentrations of the nutrient solution. In roots, the expressions were much lower than in shoots, except for OsHKT4 and OsHKT1 in K+-starved plants. We propose that OsHKT transporters are involved in Na+ movements in rice, and that OsHKT1 specifically mediates Na+ uptake in rice roots when the plants are K+ deficient. The incidence of HKT ESTs in several plant species suggests that the rice model with many HKT genes applies to other plants. HKT3|OsHKT2;3 Nomenclature for HKT transporters, key determinants of plant salinity tolerance 2006 Trends Plant Sci CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia. None HKT3|OsHKT2;3 The Arabidopsis HUELLENLOS Gene, Which Is Essential for Normal Ovule Development, Encodes a Mitochondrial Ribosomal Protein 2001 Plant Cell Section of Molecular and Cellular Biology, University of California, 1 Shields Avenue, Davis, California 95616 The HUELLENLOS (HLL) gene participates in patterning and growth of the Arabidopsis ovule. We have isolated the HLL gene and shown that it encodes a protein homologous to the L14 proteins of eubacterial ribosomes. The Arabidopsis genome also includes a highly similar gene, HUELLENLOS PARALOG (HLP), and genes for both cytosolic (L23) and chloroplast ribosome L14 proteins. Phylogenetic analysis shows that HLL and HLP differ significantly from these other two classes of such proteins. HLL and HLP fusions to green fluorescent protein were localized to mitochondria. Ectopic expression of HLP complemented the hll mutant, indicating that HLP and HLL share redundant functions. We conclude that HLL and HLP encode L14 subunits of mitochondrial ribosomes. HLL mRNA was at significantly higher levels than HLP mRNA in pistils, with the opposite pattern in leaves. This differential expression can explain the confinement of effects of hll mutations to gynoecia and ovules. Our elucidation of the nature of HLL shows that metabolic defects can have specific effects on developmental patterning. HLL Isolation of a monocot 3-hydroxy-3-methylglutaryl coenzyme A reductase gene that is elicitor-inducible 1994 Plant Mol Biol Plant Biology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, 92037, La Jolla, CA, USA The rice (Oryza sativa) phytoalexins, momilactones and oryzalexins, are synthesized by the isoprenoid pathway. An early step in this pathway, one that is rate-limiting in mammalian systems, is catalyzed by the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). A gene that encodes this enzyme has been isolated from rice, and found to contain an open reading frame of 1527 bases. The encoded protein sequence of the rice HMGR appears to be conserved with respect to other HMGR proteins, and 1 or 2 membrane-spanning domains characteristic of plant HMGRs are predicted by a hydropathy plot of the amino acid sequence. The protein is truncated at its 5' end, and shows reduced sequence conservation in this region as compared to other plant sequences. The rice genome contains a small family of HMGR genes. The isolated gene, HMGR I, is expressed at low levels in both vegetative and floral organs of rice plants. It is not induced in plants by wounding, but is strongly and rapidly induced in suspension cells by a fungal cell wall elicitor from the pathogen Magnaporthe grisea, causal agent of rice blast disease. This suggests that HMGR I may be important in the induction of rice phytoalexin biosynthesis in response to pathogen attack, and therefore may play a key role as a component of the inducible defense mechanism in rice. I Cloning, expression and subcellular localization of HN1 and HN1L genes, as well as characterization of their orthologs, defining an evolutionarily conserved gene family 2004 Gene State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, 220 Handan Road, Shanghai 200433, PR China. The present work reported the cloning and characterization of two novel human genes--HN1 (hematopoietic- and neurologic-expressed sequence 1) and HN1L (HN1-like gene) which are proposed to be involved in embryo development. HN1 is mapped on chromosome 17q25.2, with two transcripts (1.0 and 1.6 kb in length, respectively) due to alternative splicing. HN1 is expressed abundantly in testis and skeletal muscle among 16 human tissues, and it is localized in the nucleus indicated by GFP fusion expression. Western blot confirmed that HN1 encodes a 16.5-kDa protein. HN1L is on chromosome 16p13.3, with three splicing in the length of 2.0, 4.0 and 4.2 kb, respectively. HN1L is expressed in a variety of tissues such as liver, kidney, prostate, testis and uterus at varying levels. HN1L gene encodes a 20-kDa protein, which is localized in both the nucleus and cytoplasm. Fourteen of HN1 and sixteen of HN1L homologous genes in different species were determined and analyzed by BLAST searches. Silicon analyses of the 14 orthologous proteins of HN1 and 16 orthologous proteins of HN1L revealed that they share great conservation in vertebrate. Additionally, we identified nine pseudogenes of HN1 (six) and HN1L (three) in the genomes of the human, mouse and rat. Based on sequence alignments and phylogenetic analysis, all these homologous genes and pseudogenes were defined as a HN1 gene family. HN1L Sequence analysis, transcriptional and posttranscriptional regulation of the rice vdac family 2003 Biochim Biophys Acta Laboratoire de Physiologie Vegetale, Universite Libre de Bruxelles, Campus Plaine (CP 206/2), B-1050 Brussels, Belgium. The voltage-dependent anion-selective channel (VDAC) is a mitochondrial outer membrane ion channel. Different isoforms exist in plants but information about their specific role remains to be established. Our purpose is to find out the structural features common to three rice VDAC isoforms and to investigate their (post)transcriptional regulation in response to an osmotic stress. Two new cDNAs encoding mitochondrial VDAC from rice (Oryza sativa) were isolated, sequenced and characterized: a phylogenetic reconstruction permitted identification of orthologues in Poaceae and computer-based analyses predicted 18 transmembrane beta-strands, one amphipathic alpha-helix and two different phosphorylation motifs. The expression of three rice vdac genes was investigated. Northern blot analyses indicated that they were expressed in all plant tissues. There was a differential expression of osvdac1 and osvdac3, whereas osvdac2 was homogeneously expressed in all tissues. No change in vdac expression was observed under an osmotic stress. However, a fast-enhanced expression of vdac was observed in roots during the recovery period after stress release. This enhanced expression is not correlated to the amount of VDAC protein detected in roots suggesting a posttranscriptional regulation. ICS1|OsVDAC1 TaCPK2-A, a calcium-dependent protein kinase gene that is required for wheat powdery mildew resistance enhances bacterial blight resistance in transgenic rice 2013 J Exp Bot Triticeae Research Institute, Sichuan Agricultural University, 211 Huimin Road, Wenjiang, Chengdu, Sichuan, 611130, PR China. Calcium-dependent protein kinases (CPKs) are important Ca2+ signalling components involved in complex immune and stress signalling networks; but the knowledge of CPK gene functions in the hexaploid wheat is limited. Previously, TaCPK2 was shown to be inducible by powdery mildew (Blumeria graminis tritici, Bgt) infection in wheat. Here, its functions in disease resistance are characterized further. This study shows the presence of defence-response and cold-response cis-elements on the promoters of the A subgenome homoeologue (TaCPK2-A) and D subgenome homoeologue (TaCPK2-D), respectively. Their expression patterns were then confirmed by quantitative real-time PCR (qRT-PCR) using genome-specific primers, where TaCPK2-A was induced by Bgt treatment while TaCPK2-D mainly responded to cold treatment. Downregulation of TaCPK2-A by virus-induced gene silencing (VIGS) causes loss of resistance to Bgt in resistant wheat lines, indicating that TaCPK2-A is required for powdery mildew resistance. Furthermore, overexpression of TaCPK2-A in rice enhanced bacterial blight (Xanthomonas oryzae pv. oryzae, Xoo) resistance. qRT-PCR analysis showed that overexpression of TaCPK2-A in rice promoted the expression of OsWRKY45-1, a transcription factor involved in both fungal and bacterial resistance by regulating jasmonic acid and salicylic acid signalling genes. The opposite effect was found in wheat TaCPK2-A VIGS plants, where the homologue of OsWRKY45-1 was significantly repressed. These data suggest that modulation of WRKY45-1 and associated defence-response genes by CPK2 genes may be the common mechanism for multiple disease resistance in grass species, which may have undergone subfunctionalization in promoters before the formation of hexaploid wheat. ICS1|OsVDAC1 Characterization of two cDNA clones encoding isozymes of the F1F0-ATPase inhibitor protein of rice mitochondria 2000 Planta Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, JP Two cDNA clones encoding F1F0-ATPase inhibitor proteins, which are loosely associated with the F1 part of the mitochondrial F1F0-ATPase, were characterized from rice (Oryza sativa L. cv. Nipponbare). A Northern hybridization showed that the two genes (designated as IF 1 -1 and IF 1 -2) are transcribed in all the organs examined. However, the steady-state mRNA levels varied among organs. A comparison of the deduced amino acid sequences of the two IF 1 genes and the amino acid sequence of the mature IF1 protein from potato revealed that IF1-1 and IF1-2 have N-terminal extensions with features that are characteristic of a mitochondrial targeting signal. To determine the subcellular localization of the gene products, the IF1-1 or IF1-2 proteins were fused in frame to the green fluorescent protein (GFP) or the fused GFP-beta-glucuronidase, and expressed transiently in onion or dayflower epidermal cells. Localized fluorescence was detected in mitochondria, confirming that the two IF1 proteins are targeted to mitochondria. IF1-1,IF1-2 Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves 2005 Plant Physiol Commonwealth Scientific and Industrial Research Organization, Plant Industry, Horticulture Unit, Glen Osmond, South Australia. Proanthocyanidins (PAs), also called condensed tannins, can protect plants against herbivores and are important quality components of many fruits. Two enzymes, leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), can produce the flavan-3-ol monomers required for formation of PA polymers. We isolated and functionally characterized genes encoding both enzymes from grapevine (Vitis vinifera L. cv Shiraz). ANR was encoded by a single gene, but we found two highly related genes encoding LAR. We measured PA content and expression of genes encoding ANR, LAR, and leucoanthocyanidin dioxygenase in grape berries during development and in grapevine leaves, which accumulated PA throughout leaf expansion. Grape flowers had high levels of PA, and accumulation continued in skin and seeds from fruit set until the onset of ripening. VvANR was expressed throughout early flower and berry development, with expression increasing after fertilization. It was expressed in berry skin and seeds until the onset of ripening, and in expanding leaves. The genes encoding LAR were expressed in developing fruit, particularly in seeds, but had low expression in leaves. The two LAR genes had different patterns of expression in skin and seeds. During grape ripening, PA levels decreased in both skin and seeds, and expression of genes encoding ANR and LAR were no longer detected. The results indicate that PA accumulation occurs early in grape development and is completed when ripening starts. Both ANR and LAR contribute to PA synthesis in fruit, and the tissue and temporal-specific regulation of the genes encoding ANR and LAR determines PA accumulation and composition during grape berry development. LAR Clustered metallothionein genes are co-regulated in rice and ectopic expression of OsMT1e-P confers multiple abiotic stress tolerance in tobacco via ROS scavenging 2012 BMC Plant Biol Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India. BACKGROUND: Metallothioneins (MT) are low molecular weight, cysteine rich metal binding proteins, found across genera and species, but their function(s) in abiotic stress tolerance are not well documented. RESULTS: We have characterized a rice MT gene, OsMT1e-P, isolated from a subtractive library generated from a stressed salinity tolerant rice genotype, Pokkali. Bioinformatics analysis of the rice genome sequence revealed that this gene belongs to a multigenic family, which consists of 13 genes with 15 protein products. OsMT1e-P is located on chromosome XI, away from the majority of other type I genes that are clustered on chromosome XII. Various members of this MT gene cluster showed a tight co-regulation pattern under several abiotic stresses. Sequence analysis revealed the presence of conserved cysteine residues in OsMT1e-P protein. Salinity stress was found to regulate the transcript abundance of OsMT1e-P in a developmental and organ specific manner. Using transgenic approach, we found a positive correlation between ectopic expression of OsMT1e-P and stress tolerance. Our experiments further suggest ROS scavenging to be the possible mechanism for multiple stress tolerance conferred by OsMT1e-P. CONCLUSION: We present an overview of MTs, describing their gene structure, genome localization and expression patterns under salinity and development in rice. We have found that ectopic expression of OsMT1e-P enhances tolerance towards multiple abiotic stresses in transgenic tobacco and the resultant plants could survive and set viable seeds under saline conditions. Taken together, the experiments presented here have indicated that ectopic expression of OsMT1e-P protects against oxidative stress primarily through efficient scavenging of reactive oxygen species. OsMT1f,OsMT1g,MT2A,OsMT2d DNA variation in the metallothionein genes in wild rice Oryza rufipogon: Relationship between DNA sequence polymorphism, codon bias and gene expression 2005 Genes Genet Syst Laboratory of Plant Genetics, Graduate School of Agriculture, Kyoto University This study examines the relationship between DNA sequence variation and level of gene expression in four metallothionein genes from wild rice Oryza rufipogon. The nucleotide diversity was 0.0028 to 0.0117 over the entire coding and non-coding region, and it was negatively correlated with gene expression for three type 2 metallothionein genes. In contrast, codon bias and percent of preferred codons correlated positively with gene expression. These results indicate that the intensity of natural selection depends on the level of gene expression, which in turn shapes the level of nucleotide polymorphism. In addition, significant linkage disequilibria were frequent between the metallothionein genes, although significance was not confirmed after multiple test correction. This result suggests that metallothionein genes expressed at different levels are epistatic with respect to fitness, and that gene expression is an important factor determining level of DNA polymorphism. MT2A Molecular cloning and characterization of two isoforms of cytoplasmic/mitochondrial type NADPH-dependent thioredoxin reductase from rice 2012 Australian Journal of Crop Science Department of Agricultural Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan-84156-83111, Iran An NADP/thioredoxin system, consisting of NADPH, NADPH-dependent thioredoxin reductase (NTR) and thioredoxin (Trx) plays a post-translational regulatory role by reducing disulfide bonds in target proteins involved in various cellular processes. Plants have a complex NTR/Trx system comprising several Trxs and NTR isoforms. Three genes encoding NTR were found in the genome of rice. OsNTRC is chloroplastic type NTR whereas OsNTRA and OsNTRB are cytoplasmic/mitochondrial type NTR. The presence of two isoforms of cytoplasmic/mitochondrial type NTR with high amino acid identity raises the question of their functional specificity. In the present study, we describe isolation and cloning of two cDNAs encoding OsNTRA and OsNTRB. Both isoforms were heterologously expressed in Escherichia coli and purified using affinity chromatography, enabling comparison of their NADPHdependent reduction activities. OsNTRB and OsNTRA exhibited almost similar in vitro activities. OsNTRB was highly expressed in roots and shoots of one, two and three week-old rice seedlings, whereas the expression of OsNTRA in shoots was much higher than roots. Differential expression of OsNTRA in different tissues suggests that this isoform may have different functions in different tissues. NTRA,NTRB A non-destructive screenable marker, OsFAST, for identifying transgenic rice seeds 2011 Plant Signal Behav Department of Botany, Graduate School of Science, Kyoto University, Kyoto, Japan. The production of transgenic plants has contributed greatly to plant research. Previously, an improved method for screening transgenic Arabidopsis thaliana seeds using the FAST (Fluorescence-Accumulating-Seed Technology) method and FAST marker was reported. Arabidopsis seeds containing the FAST marker may be visually screened using a fluorescence stereomicroscope or blue LED handy-type instrument. Although the FAST method was originally designed for Arabidopsis screens, this study endeavors to adapt this method for the screening of other plants. Here, an optimized technology, designated the OsFAST method, is presented as a useful tool for screening transgenic rice seeds. The OsFAST method is based on the expression of the OsFAST-G marker under the control of a seed-embryo-specific promoter, similar to the Arabidopsis FAST-G marker. The OsFAST method provides a simple and non-destructive method for identifying transgenic rice seeds. It is proposed that the FAST method is adaptable to various plant species and will enable a deeper analysis of the floral-dip method. OLE4 The 6-phosphogluconate dehydrogenase genes are responsive to abiotic stresses in rice 2007 J Integr Plant Biol Crop Research Institute, Shandong Academy of Agricultural Sciences, Jinan 250100, China. Glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44) are both key enzymes of the pentose phosphate pathway (PPP). The OsG6PDH1 and Os6PGDH1 genes encoding cytosolic G6PDH and cytosolic 6PGDH were isolated from rice (Oryza sativa L.). We have shown that Os6PGDH1 gene was up-regulated by salt stress. Here we reported the isolation and characterization of Os6PGDH2 from rice, which encode the plastidic counterpart of 6PGDH. Genomic organization analysis indicated that OsG6PDH1 and OsG6PDH2 genes contain multiple introns, whereas two Os6PGDH1 and Os6PGDH2 genes have no introns in their translated regions. In a step towards understanding the functions of the pentose phosphate pathway in plants in response to various abiotic stresses, the expressions of four genes in the rice seedlings treated by drought, cold, high salinity and abscisic acid (ABA) were investigated. The results show that OsG6PDH1 and OsG6PDH2 are not markedly regulated by the ablotic stresses detected. However, the transcript levels of both Os6PGDH1 and Os6PGDH2 are up-regulated in rice seedlings under drought, cold, high salinity and ABA treatments. Meanwhile, the enzyme activities of G6PDH and 6PGDH in the rice seedlings treated by various abiotic stresses were investigated. Like the mRNA expression patterns, G6PDH activity remains constant but the 6PGDH increases steadily during the treatments. Taken together, we suggest that the pentose phosphate pathway may play an important role in rice responses to ablotic stresses and the second key enzyme of PPP, 6PGDH, may function as a regulator controlling the efficiency of the pathway under ablotic stresses. Os6PGDH2 Expression of a microsporocyte-specific gene encoding dihydroflavonol 4-reductase-like protein is developmentally regulated during early microsporogenesis in rice 2005 Sexual Plant Reproduction Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China In an attempt to understand more about the molecular mechanism by which pollen development is regulated and coordinated in rice, we employed a cDNA subtraction strategy to identify genes that differentially expressed between the wild-type, AN-N and its thermosensitive genic male-sterile mutant (TGMS), AN S-1 during microsporogenesis. Eleven clones exhibiting differential expression patterns were isolated and identified. We report here in detail the molecular cloning and characterization of one of the clones, OS-DFR2 which encodes a dihydroflavonol 4-reductase-like protein. RNA gel blot analysis revealed that OS-DFR2 expressed strongly in anthers but no expression was detected in roots, leaves, stems and pistils, indicating that OS-DFR2 was a tissue-specific gene. Furthermore, the expression of OS-DFR2 was developmentally regulated during early microsporogenesis. It accumulated at high levels in anthers undergoing meiosis and reached a maximal value during tetrad formation stage in the wild-type, AN-N. In AN S-1, the expression of OS-DFR2 resembled that of AN-N except its mRNA level is approximately one-fourth of that found in AN-N at the tetrad formation stage. In situ analysis indicated that OS-DFR2 transcript was specifically expressed in microsporocyte of AN-N; whereas OS-DFR2 was present both in microsporocyte and tapetal cells in AN S-1. The significance and possible role played by OS-DFR2 during male gametophyte development in rice is discussed. OS-ALDH,OS-CAD Opaque7 encodes an acyl-activating enzyme-like protein that affects storage protein synthesis in maize endosperm 2011 Genetics Shanghai Key Laboratory of Bioenergy Crops, School of Life Sciences, Shanghai University, Shanghai 200444, China. In maize, a series of seed mutants with starchy endosperm could increase the lysine content by decreased amount of zeins, the main storage proteins in endosperm. Cloning and characterization of these mutants could reveal regulatory mechanisms for zeins accumulation in maize endosperm. Opaque7 (o7) is a classic maize starchy endosperm mutant with large effects on zeins accumulation and high lysine content. In this study, the O7 gene was cloned by map-based cloning and confirmed by transgenic functional complementation and RNAi. The o7-ref allele has a 12-bp in-frame deletion. The four-amino-acid deletion caused low accumulation of o7 protein in vivo. The O7 gene encodes an acyl-activating enzyme with high similarity to AAE3. The opaque phenotype of the o7 mutant was produced by the reduction of protein body size and number caused by a decrease in the alpha-zeins concentrations. Analysis of amino acids and metabolites suggested that the O7 gene might affect amino acid biosynthesis by affecting alpha-ketoglutaric acid and oxaloacetic acid. Transgenic rice seeds containing RNAi constructs targeting the rice ortholog of maize O7 also produced lower amounts of seed proteins and displayed an opaque endosperm phenotype, indicating a conserved biological function of O7 in cereal crops. The cloning of O7 revealed a novel regulatory mechanism for storage protein synthesis and highlighted an effective target for the genetic manipulation of storage protein contents in cereal seeds. OsAAE3 The role of the OsCam1-1 salt stress sensor in ABA accumulation and salt tolerance in rice 2012 Journal of Plant Biology Biological Sciences Program, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok, 10330, Thailand Involvement of the salt-inducible calmodulin gene, OsCam1-1, in abscisic acid (ABA) biosynthesis during salt stress was studied in the ‘Khoa Dawk Mali 105’ (KDML105) rice cultivar (Oryza sativa L.). FL530-IL, an isogenic salt-resistant line derived from the KDML105 cultivar, accumulated a 2.9-fold higher concentration of ABA in the leaves after salt stress treatment than that for KDML105. A twenty-four and a seven- fold higher level of OsCam1-1 transcripts were detected in the leaves of the FL530-IL and KDML105 rice cultivars, respectively, after 30 min of salt stress compared to non-salt-stressed plants. Transgenic rice lines that constitutively over-express the OsCam1-1 gene were found to up-regulate ABA aldehyde oxidase and 9-cis-epoxycarotenoid dioxygenase 3, two genes involved in ABA biosynthesis, and to have a higher ABA content, when compared to the wild type and the control transgenic lines without OsCam1-1 over-expression. In addition, transgenic plants over-expressing OsCam1-1 were more tolerant to salt stress, with, for example, a better ability to maintain their shoot and root mass (as dry weight) during salt stress, than the control plants. These data indicate that OsCam1-1 signaling is likely to play an important role in ABA biosynthesis, and the level of OsCam1-1 gene expression and ABA accumulation probably contribute to salt resistance in rice. OsAAO Divergent roles in Arabidopsis thaliana development and defense of two homologous genes, aberrant growth and death2 and AGD2-LIKE DEFENSE RESPONSE PROTEIN1, encoding novel aminotransferases 2004 Plant Cell Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637. The disease-resistant Arabidopsis thaliana aberrant growth and death2 (agd2-1) mutant has elevated levels of the defense signal salicylic acid (SA), altered leaf morphology, and mild dwarfism. AGD2 and its close homolog ALD1 (for AGD2-LIKE DEFENSE RESPONSE PROTEIN1) encode aminotransferases that act on an overlapping set of amino acids in vitro. However, kinetic parameters indicate that AGD2 and ALD1 may drive the aminotransferase reaction in opposite directions. ALD1-deficient mutants have the opposite phenotypes from agd2-1, showing reduced SA production and increased disease susceptibility. Furthermore, ALD1 transcript levels are elevated in agd2-1 and are induced in the wild type by bacterial pathogen infection. ALD1 is responsible for some of the elevated SA content and a majority of the disease resistance and dwarfism of agd2-1. A complete knockout of AGD2 renders embryos inviable. We suggest that AGD2 synthesizes an important amino acid-derived molecule that promotes development and suppresses defenses, whereas ALD1 generates a related amino acid-derived molecule important for activating defense signaling. OsAGD2,OsALD1 Functional Characterization of an Aldehyde Dehydrogenase Homologue in Rice 2012 Journal of Integrative Agriculture Key Laboratory for Bio-Resource and Eco-Environment, Minstry of Education/College of Life Sciences, Sichuan University, Chengdu 610064, P.R. China The aldehyde dehydrogenase (ALDH) superfamily of NAD(P)+-dependent enzymes, in general, oxidize a wide range of endogenous and exogenous aliphatic and aromatic aldehydes to their corresponding carboxylic acids and play an essential role in detoxification of reactive oxygen species (ROS) accumulated under the stressed conditions. In order to identify genes required for the stresses responses in the grass crop Oryza sativa, a homologue of ALDH gene (OsALDH22) was isolated and characterized. OsALDH22 is conserved in eukaryotes, shares high homology with the orthologs from aldehyde dehydrogenase subfamily ALDH22. The OsALDH22 encodes a protein of 597 amino acids that in plants exhibit high identity with the orthologs from Zea mays, Sorghum bicolor, Hordeum vulgare and Arabidopsis thaliana, respectively, and the conserved amino acid characteristics for ALDHs are present, including the possible NAD+ binding site (F-V-G-S-P-G-V-G), the catalytic site (V-T-L-E-L-G-G-K) and the Cys active site. Semi-quantitative PCR and real-time PCR analysis indicates that OsALDH22 is expressed differentially in different tissues. Various elevated levels of OsALDH22 expression have been detected when the seedlings exposed to abiotic stresses including dehydration, high salinity and abscisic acid (ABA). Transgenic rice plants overexpressing OsALDH22 show elevated stresses tolerance. On the contrary, down-regulation of OsALDH22 in the RNA interference (RNAi) repression transgenic lines manifests declined stresses tolerance. OsALDH22 Biochar but not earthworms enhances rice growth through increased protein turnover 2012 Soil Biology and Biochemistry UPMC – Bioemco (UMR CNRS 7618), 32 avenue Henri Varagnat, 93143 Bondy Cedex, France The aim of this work was to compare the effects of biochar and earthworms on rice growth and to investigate the possible interactions between both. In addition to classic macroscopic variables we also monitored some leaf-level cellular processes involved in protein turnover. Both biochar and earthworms significantly increased shoot biomass production. However, biochar had a higher effect on the number of leaves (+87%) and earthworms on leaf area (+89%). Biochar also significantly increased the leaf turnover. At the cellular level, biochar but not earthworms enhanced protein catabolism by an increase in leaf proteolytic activities. This could be related to the increased expression of three of the six genes tested related to protein catabolism, one serine protease gene OsSP2 (+24%), one aspartic acid protease gene, Oryzasin(+162%) and one cysteine protease gene OsCatB (+257%). Furthermore, biochar also enhanced the expression level of two genes linked to protein anabolism, coding for the small and large subunits of rubisco (+33% and +30%, for rbcS and rbcL, respectively), the most abundant protein in leaves. In conclusion, our data gives evidence that biochar increased rice biomass production through increased leaf protein turnover (both catabolism and anabolism) whereas earthworms also increased rice biomass production but not through changes in the rate of protein turnover. We hypothesize that earthworms increase nitrogen uptake at a low cost for the plant through a simultaneous increase in mineralization rate and root biomass, probably through the release in the soil of plant growth factors. This could allow plants to accumulate more biomass without an increase in nitrogen metabolism at the leaf level, and without having to support the consecutive energy cost that must bear plants in the biochar treatment. OsAP1,OsCatB,OsSP1 Novel bifunctional nucleases, OmBBD and AtBBD1, are involved in abscisic acid-mediated callose deposition in Arabidopsis 2010 Plant Physiol School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea. Screening of the expressed sequence tag library of the wild rice species Oryza minuta revealed an unknown gene that was rapidly and strongly induced in response to attack by a rice fungal pathogen (Magnaporthe oryzae) and an insect (Nilaparvata lugens) and by wounding, abscisic acid (ABA), and methyl jasmonate treatments. Its recombinant protein was identified as a bifunctional nuclease with both RNase and DNase activities in vitro. This gene was designated OmBBD (for O. minuta bifunctional nuclease in basal defense response). Overexpression of OmBBD in an Arabidopsis (Arabidopsis thaliana) model system caused the constitutive expression of the PDF1.2, ABA1, and AtSAC1 genes, which are involved in priming ABA-mediated callose deposition. This activation of defense responses led to an increased resistance against Botrytis cinerea. atbbd1, the knockout mutant of the Arabidopsis ortholog AtBBD1, was susceptible to attack by B. cinerea and had deficient callose deposition. Overexpression of either OmBBD or AtBBD1 in atbbd1 plants complemented the susceptible phenotype of atbbd1 against B. cinerea as well as the deficiency of callose deposition. We suggest that OmBBD and AtBBD1 have a novel regulatory role in ABA-mediated callose deposition. OsBBD1 Identification of a cis-element that mediates multiple pathways of the endoplasmic reticulum stress response in rice 2013 Plant J Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602, Japan. The accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen leads to ER stress. Intracellular signalling pathways are activated to alleviate the stress. The ER stress sensor IRE1 induces the active form of key transcription factors, such as XBP1 in mammals and bZIP50 in Oryza sativa (rice), by mediating the unconventional splicing of their mRNAs. Although the characterization of cis-elements that are recognized by these transcription factors is essential for understanding ER stress responses, such cis-elements remain unidentified in plants. Here, a cis-element named pUPRE-II was identified from promoters of bZIP50-dependent genes using chromatin immunoprecipitation assays and electrophoretic mobility shift assays. The sequence of pUPRE-II (e.g., 5'-GATGACGCGTAC-3' in the OsSAR1 promoter) was found to be flexible and not identical with that of mUPRE, a cis-element that preferentially interacts with mammalian XBP1. Unexpectedly, the transcription factor bZIP60, another ER stress sensor in rice, and a counterpart of mammalian ATF6, also showed strong binding affinity for pUPRE-II without assistance from co-factors. Reporter assays indicated that pUPRE-II significantly contributes to gene expression mediated by bZIP50 or bZIP60 in rice. Although both bZIP50 and bZIP60 bound to pUPRE-II, these transcription factors showed distinct requirements for transcriptional activation. This study provides a missing link between ER stress sensors and stress-responsive genes in rice. Furthermore, the characteristics of pUPRE-II highlight the uniqueness of ER stress-responsive transcription in plants. OsBiP2|Bip2,OsBiP3|BiP3 Ectopic expression of a rice protein phosphatase 2C gene OsBIPP2C2 in tobacco improves disease resistance 2009 Plant Cell Rep State Key Laboratory of Rice Biology, Department of Plant Protection, Institute of Biotechnology, Zhejiang University-Huajiachi Campus, 310029, Hangzhou, Zhejiang, People's Republic of China. Protein phosphatase 2Cs (PP2Cs) have been demonstrated to play critical roles in regulation of plant growth/development, abscisic acid signaling pathway and adaptation to environmental stresses. Here we report the cloning and molecular characterization of a novel rice protein phosphatase 2C gene, OsBIPP2C2 (Oryza sativa L. BTH-induced protein phosphatase 2C 2). OsBIPP2C2 has three alternatively spliced transcripts and the largest transcript OsBIPP2C2a encodes a 380 aa protein containing all 11 conserved catalytic subdomains of PP2Cs. Expression of OsBIPP2C2a was significantly induced by benzothiadiazole (BTH), one of defense-related signal molecules in plants. Expression of OsBIP2C2a was induced by infection with the blast fungus, Magnaporthe grisea, and the pathogen-induced expression of OsBIPP2C2a in BTH-treated rice seedlings was much earlier and stronger than those in water-treated seedlings. Overexpression of OsBIPP2C2a in transgenic tobacco plants resulted in increased disease resistance against tobacco mosaic virus and Phytophthora parasitica var. nicotianae. Importantly, the OsBIPP2C2a-overexpressing transgenic tobacco plants showed constitutive expression of defense-related genes. These results suggest that OsBIPP2C2a may play an important role in disease resistance through activation of defense response. OsBIPP2C1 Genome wide expression analysis of CBS domain containing proteins in Arabidopsis thaliana (L.) Heynh and Oryza sativa L. reveals their developmental and stress regulation 2009 BMC Genomics Centre for Computational Biology and Bioinformatics, School of Information Technology, Jawaharlal Nehru University, New Delhi, India. ritturajhemant@gmail.com BACKGROUND: In Arabidopsis thaliana (L.) Heynh and Oryza sativa L., a large number of genes encode proteins of unknown functions, whose characterization still remains one of the major challenges. With an aim to characterize these unknown proteins having defined features (PDFs) in plants, we have chosen to work on proteins having a cystathionine beta-synthase (CBS) domain. CBS domain as such has no defined function(s) but plays a regulatory role for many enzymes and thus helps in maintaining the intracellular redox balance. Its function as sensor of cellular energy has also been widely suggested. RESULTS: Our analysis has identified 34 CBS domain containing proteins (CDCPs) in Arabidopsis and 59 in Oryza. In most of these proteins, CBS domain coexists with other functional domain(s), which may indicate towards their probable functions. In order to investigate the role(s) of these CDCPs, we have carried out their detailed analysis in whole genomes of Arabidopsis and Oryza, including their classification, nomenclature, sequence analysis, domain analysis, chromosomal locations, phylogenetic relationships and their expression patterns using public databases (MPSS database and microarray data). We have found that the transcript levels of some members of this family are altered in response to various stresses such as salinity, drought, cold, high temperature, UV, wounding and genotoxic stress, in both root and shoot tissues. This data would be helpful in exploring the so far obscure functions of CBS domain and CBS domain-containing proteins in plant stress responses. CONCLUSION: We have identified, classified and suggested the nomenclature of CDCPs in Arabidopsis and Oryza. A comprehensive analysis of expression patterns for CDCPs using the already existing transcriptome profiles and MPSS database reveals that a few CDCPs may have an important role in stress response/tolerance and development in plants, which needs to be validated further through functional genomics. OsCBSIMPDH1|OsCBSIMPDH1a|OsCBSIMPDH1b,OsCBSPPR1,OsCBSSIS1 Identification and functional characterization of cation-chloride cotransporters in plants 2007 Plant J Centro de Genomica, Instituto Valenciano de Investigaciones Agrarias, Ctra. Moncada-Naquera Km. 5, 46113 Moncada, Valencia, Spain. Chloride (Cl(-)) is an essential nutrient and one of the most abundant inorganic anions in plant tissues. We have cloned an Arabidopsis thaliana cDNA encoding for a member of the cation-Cl(-) cotransporter (CCC) family. Deduced plant CCC proteins are highly conserved, and phylogenetic analyses revealed their relationships to the sub-family of animal K(+):Cl(-) cotransporters. In Xenopus laevis oocytes, the A. thaliana CCC protein (At CCC) catalysed the co-ordinated symport of K(+), Na(+) and Cl(-), and this transport activity was inhibited by the 'loop' diuretic bumetanide, a specific inhibitor of vertebrate Na(+):K(+):Cl(-) cotransporters, indicating that At CCC encodes for a bona fide Na(+):K(+):Cl(-) cotransporter. Analysis of At CCC promoter-beta-glucuronidase transgenic Arabidopsis plants revealed preferential expression in the root and shoot vasculature at the xylem/symplast boundary, root tips, trichomes, leaf hydathodes, leaf stipules and anthers. Plants homozygous for two independent T-DNA insertions in the CCC gene exhibited shorter organs such as inflorescence stems, roots, leaves and siliques. The elongation zone of the inflorescence stem of ccc plants often necrosed during bolt emergence, while seed production was strongly impaired. In addition, ccc plants exhibited defective Cl(-) homeostasis under high salinity, as they accumulated higher and lower Cl(-) amounts in shoots and roots, respectively, than the treated wild type, suggesting At CCC involvement in long-distance Cl(-) transport. Compelling evidence is provided on the occurrence of cation-chloride cotransporters in the plant kingdom and their significant role in major plant developmental processes and Cl(-) homeostasis. OsCCC2 Targeted association analysis identified japonica rice varieties achieving Na(+)/K (+) homeostasis without the allelic make-up of the salt tolerant indica variety Nona Bokra 2011 Theor Appl Genet CIRAD, UPR AIVA, 34398 Montpellier, France. Nourollah.ahmadi@cirad.fr During the last decade, a large number of QTLs and candidate genes for rice tolerance to salinity have been reported. Using 124 SNP and 52 SSR markers, we targeted 14 QTLs and 65 candidate genes for association mapping within the European Rice Core collection (ERCC) comprising 180 japonica accessions. Significant differences in phenotypic response to salinity were observed. Nineteen distinct loci significantly associated with one or more phenotypic response traits were detected. Linkage disequilibrium between these loci was extremely low, indicating a random distribution of favourable alleles in the ERCC. Analysis of the function of these loci indicated that all major tolerance mechanisms were present in the ERCC although the useful level of expression of the different mechanisms was scattered among different accessions. Under moderate salinity stress some accessions achieved the same level of control of Na(+) concentration and Na(+)/K(+) equilibrium as the indica reference variety for salinity tolerance Nona Bokra, although without sharing the same alleles at several loci associated with Na(+) concentration. This suggests (a) differences between indica and japonica subspecies in the effect of QTLs and genes involved in salinity tolerance and (b) further potential for the improvement of tolerance to salinity above the tolerance level of Nona Bokra, provided the underlying mechanisms are complementary at the whole plant level. No accession carried all favourable alleles, or showed the best phenotypic responses for all traits measured. At least nine accessions were needed to assemble the favourable alleles and all the best phenotypic responses. An effective strategy for the accumulation of the favourable alleles would be marker-assisted population improvement. OsCLP3 Identification and expression analysis of hypoxia stress inducible CCCH-type zinc finger protein genes in rice 2013 Journal of Plant Biology Plant Molecular Biology Laboratory, Department of Life Science, Sogang University, Seoul, 121-742, Korea Flooding is one of the threatening abiotic stresses in recent global warming. In order to understand flooding-caused low oxygen stress response at molecular level, microarray-linked isolation of the hypoxia inducible genes were conducted. Seventeen genes that were up-regulated by the factor of more than 3 fold, were confirmed as hypoxia inducible. Among them, a CCCH-type zinc finger protein gene, OsCCCH-Zn-1, was further characterized due to its novelty as a hypoxia-inducible zinc finger gene as well as its significant induction by hypoxia stress. OsCCCH-Zn-1 was also up-regulated by submergence, ABA and drought stresses. In the normal growth condition, OsCCCH-Zn-1 was expressed in the flag leaf sheath, highest internode and developing seeds. In rice, at least 12 CCCH-type zinc finger protein genes were retrieved by in silico analysis. Among these, we found that the zinc finger genes OsCCCH-Zn-1, -2, -6 were induced by hypoxia stress. OsCYP450,OsPPi-PFK,OsPRAGL,OsRNAG Cloning and comparative analysis of the gene encoding diacylglycerol acyltransferase from wild type and cultivated soybean 2006 Theor Appl Genet The National Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 100101 Beijing, China. Diacylglycerol acyltransferase (DGAT), as an important enzyme in triacylglycerol synthesis, catalyzes the final acylation of the Kennedy pathway. In the present study, the GmDGAT gene was cloned from Glycine max by using AtDGAT as a query to search against the soybean EST database and the rapid amplification of cDNA ends (RACE) method. Allelic genes were also isolated from 13 soybean accessions and the divergence of the deduced amino acid sequences were compared. The comparison reveals that although GmDGAT is a highly conserved protein, several differences of insertion/deletion were identified in the N-terminal region of the GmDGATs from various soybean accessions. In the C-terminal regions, a single amino acid mutation specific to both G. max and G. soja was also found. The GmDGAT genomic sequences were further cloned and the number and size of exons in the DGAT genomic sequence were very similar among different plant species, whereas the introns were more diverged. These results may have significance in elucidating the genetic diversity of the GmDGAT among the soybean subgenus. OsDGAT Genomic comparison of P-type ATPase ion pumps in Arabidopsis and rice 2003 Plant Physiol Department of Cell Biology, Plant Division, The Scripps Research Institute, La Jolla, California 92037, USA. Members of the P-type ATPase ion pump superfamily are found in all three branches of life. Forty-six P-type ATPase genes were identified in Arabidopsis, the largest number yet identified in any organism. The recent completion of two draft sequences of the rice (Oryza sativa) genome allows for comparison of the full complement of P-type ATPases in two different plant species. Here, we identify a similar number (43) in rice, despite the rice genome being more than three times the size of Arabidopsis. The similarly large families suggest that both dicots and monocots have evolved with a large preexisting repertoire of P-type ATPases. Both Arabidopsis and rice have representative members in all five major subfamilies of P-type ATPases: heavy-metal ATPases (P1B), Ca2+-ATPases (endoplasmic reticulum-type Ca2+-ATPase and autoinhibited Ca2+-ATPase, P2A and P2B), H+-ATPases (autoinhibited H+-ATPase, P3A), putative aminophospholipid ATPases (ALA, P4), and a branch with unknown specificity (P5). The close pairing of similar isoforms in rice and Arabidopsis suggests potential orthologous relationships for all 43 rice P-type ATPases. A phylogenetic comparison of protein sequences and intron positions indicates that the common angiosperm ancestor had at least 23 P-type ATPases. Although little is known about unique and common features of related pumps, clear differences between some members of the calcium pumps indicate that evolutionarily conserved clusters may distinguish pumps with either different subcellular locations or biochemical functions. osECA1 Cloning, characterization, and expression of two cDNA clones for a rice ferulate-5-hydroxylase gene, a cytochrome P450-dependent monooxygenase 2006 Journal of Plant Biology Division of Radiation Application Research, Korea Atomic Energy Research Institute, 580-185, Jeongeup, Korea Ferulate-5-hydroxylase (F5H) is a cytochrome P450-dependent monooxygenase that catalyses the hydroxylation of ferulic acid, coniferaldehyde, and coniferyl alcohol in the pathways leading to sinapic acid and the syringyl unit of lignin. We have isolated twoF5H genes,OsF5HL andOsF5HL2, from a japonica-type rice (Oryza sativa L. cv. llpoombyeo). They are the firstF5H genes experimentally identified in monocotyledonous plants. Phylogenetic analysis indicated that both genes are closely related to dicotF5H genes fromArabidopsis thaliana andBrassica napus. Southern blot analysis showed that these genes exist as single copies in the rice genome. Alignments of theOsF5HL andOsF5HL2 cDNAs to their genomic DNAs revealed thatOsF5HL has an open reading frame (ORF) of 1590 b from four exons, whileOsF5HL2 has an ORF of 1560 b from two exons. Expression ofOsFSHL is highest in young leaves, whereas that ofOsF5HL2 is greatest in mature leaves. In the roots and stems, transcription levels for both genes are markedly low. These data suggest that theOsF5HL andOsF5HL2 genes belong to theCYP84 subfamily and that their expressions are tissue-specific. OsF5HL,OsF5HL2 FLOWERING BHLH transcriptional activators control expression of the photoperiodic flowering regulator CONSTANS in Arabidopsis 2012 Proc Natl Acad Sci U S A Department of Biology, University of Washington, Seattle, WA 98195-1800, USA. Many plants monitor day-length changes throughout the year and use the information to precisely regulate the timing of seasonal flowering for maximum reproductive success. In Arabidopsis thaliana, transcriptional regulation of the CONSTANS (CO) gene and posttranslational regulation of CO protein are crucial mechanisms for proper day-length measurement in photoperiodic flowering. Currently, the CYCLING DOF FACTOR proteins are the only transcription factors known to directly regulate CO gene expression, and the mechanisms that directly activate CO transcription have remained unknown. Here we report the identification of four CO transcriptional activators, named FLOWERING BHLH 1 (FBH1), FBH2, FBH3, and FBH4. All FBH proteins are related basic helix-loop-helix-type transcription factors that preferentially bind to the E-box cis-elements in the CO promoter. Overexpression of all FBH genes drastically elevated CO levels and caused early flowering regardless of photoperiod, whereas CO levels were reduced in the fbh quadruple mutants. In addition, FBH1 is expressed in the vascular tissue and bound near the transcription start site of the CO promoter in vivo. Furthermore, FBH homologs in poplar and rice induced CO expression in Arabidopsis. These results indicate that FBH proteins positively regulate CO transcription for photoperiodic flowering and that this mechanism may be conserved in diverse plant species. Our results suggest that the diurnal CO expression pattern is generated by a concert of redundant functions of positive and negative transcriptional regulators. OsFBH1 Rice OsHOL1 and OsHOL2 proteins have S-adenosyl-L-methionine-dependent methyltransferase activities toward iodide ions 2012 Plant Biotechnology None None OsHOL1,OsHOL2 Arabidopsis HARMLESS TO OZONE LAYER Protein Methylates a Glucosinolate Breakdown Product and Functions in Resistance to Pseudomonas syringae pv. maculicola 2009 Journal of Biological Chemistry Graduate School of Environment and Information Sciences, Yokohama National University, Yokohama, Kanagawa 240-8501, Japan. Almost all of the chlorine-containing gas emitted from natural sources is methyl chloride (CH(3)Cl), which contributes to the destruction of the stratospheric ozone layer. Tropical and subtropical plants emit substantial amounts of CH(3)Cl. A gene involved in CH(3)Cl emission from Arabidopsis was previously identified and designated HARMLESS TO OZONE LAYER (hereafter AtHOL1) based on the mutant phenotype. Our previous studies demonstrated that AtHOL1 and its homologs, AtHOL2 and AtHOL3, have S-adenosyl-l-methionine-dependent methyltransferase activities. However, the physiological functions of AtHOLs have yet to be elucidated. In the present study, our comparative kinetic analyses with possible physiological substrates indicated that all of the AtHOLs have low activities toward chloride. AtHOL1 was highly reactive to thiocyanate (NCS(-)), a pseudohalide, synthesizing methylthiocyanate (CH(3)SCN) with a very high k(cat)/K(m) value. We demonstrated in vivo that substantial amounts of NCS(-) were synthesized upon tissue damage in Arabidopsis and that NCS(-) was largely derived from myrosinase-mediated hydrolysis of glucosinolates. Analyses with the T-DNA insertion Arabidopsis mutants (hol1, hol2, and hol3) revealed that only hol1 showed increased sensitivity to NCS(-) in medium and a concomitant lack of CH(3)SCN synthesis upon tissue damage. Bacterial growth assays indicated that the conversion of NCS(-) into CH(3)SCN dramatically increased antibacterial activities against Arabidopsis pathogens that normally invade the wound site. Furthermore, hol1 seedlings showed an increased susceptibility toward an Arabidopsis pathogen, Pseudomonas syringae pv. maculicola. Here we propose that AtHOL1 is involved in glucosinolate metabolism and defense against phytopathogens. Moreover, CH(3)Cl synthesized by AtHOL1 could be considered a byproduct of NCS(-) metabolism. OsHOL1,OsHOL2 Comparative Analysis of SWIRM Domain-Containing Proteins in Plants 2012 Comp Funct Genomics Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Xingke Road 723, Guangzhou 51065, China. Chromatin-remodeling complexes affect gene expression by using the energy of ATP hydrolysis to locally disrupt or alter the association of histones with DNA. SWIRM (Swi3p, Rsc8p, and Moira) domain is an alpha-helical domain of about 85 residues in chromosomal proteins. SWIRM domain-containing proteins make up large multisubunit complexes by interacting with other chromatin modification factors and may have an important function in plants. However, little is known about SWIRM domain-containing proteins in plants. In this study, 67 SWIRM domain-containing proteins from 6 plant species were identified and analyzed. Plant SWIRM domain proteins can be divided into three distinct types: Swi-type, LSD1-type, and Ada2-type. Generally, the SWIRM domain forms a helix-turn-helix motif commonly found in DNA-binding proteins. The genes encoding SWIRM domain proteins in Oryza sativa are widely expressed, especially in pistils. In addition, OsCHB701 and OsHDMA701 were downregulated by cold stress, whereas OsHDMA701 and OsHDMA702 were significantly induced by heat stress. These observations indicate that SWIRM domain proteins may play an essential role in plant development and plant responses to environmental stress. OsHXA701|HXA701,OsSWI3C|OsCHB705|CHB705 Impaired sucrose induction1 encodes a conserved plant-specific protein that couples carbohydrate availability to gene expression and plant growth 2006 Plant J Department of Cell and Developmental Biology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK. To identify the molecular mechanisms underlying carbohydrate allocation to storage processes, we have isolated mutants in which the sugar induction of starch biosynthetic gene expression was impaired. Here we describe the IMPAIRED SUCROSE INDUCTION1 (ISI1) gene, which encodes a highly conserved plant-specific protein with structural similarities to Arm repeat proteins. ISI1 is predominantly expressed in the phloem of leaves following the sink-to-source transition during leaf development, but is also sugar-inducible in mesophyll cells. Soil-grown isi1 mutants show reduced plant growth and seed set compared to wild-type Arabidopsis. This growth reduction is not due to reduced carbohydrate availability or a defect in sucrose export from mature leaves, suggesting that isi1 mutant plants do not utilize available carbohydrate resources efficiently. ISI1 interacts synergistically with, but is genetically distinct from, the abscisic acid (ABA) signalling pathway controlling sugar responses via ABI4. Our data show that ISI1 couples the availability of carbohydrates to the control of sugar-responsive gene expression and plant growth. OsISI1 Molecular Characterization and Comparative Sequence Analysis of Defense-Related Gene, Oryza rufipogon Receptor-Like Protein Kinase 1 2012 Int J Mol Sci Centre for Research in Biotechnology for Agriculture (CEBAR) and Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, 50603, Malaysia; E-Mails: yeesong0210@yahoo.com (Y.-S.L.); ranga@siswa.um.edu.my (R.G.). Many of the plant leucine rich repeat receptor-like kinases (LRR-RLKs) have been found to regulate signaling during plant defense processes. In this study, we selected and sequenced an LRR-RLK gene, designated as Oryza rufipogon receptor-like protein kinase 1 (OrufRPK1), located within yield QTL yld1.1 from the wild rice Oryza rufipogon (accession IRGC105491). A 2055 bp coding region and two exons were identified. Southern blotting determined OrufRPK1 to be a single copy gene. Sequence comparison with cultivated rice orthologs (OsI219RPK1, OsI9311RPK1 and OsJNipponRPK1, respectively derived from O. sativa ssp. indica cv. MR219, O. sativa ssp. indica cv. 9311 and O. sativa ssp. japonica cv. Nipponbare) revealed the presence of 12 single nucleotide polymorphisms (SNPs) with five non-synonymous substitutions, and 23 insertion/deletion sites. The biological role of the OrufRPK1 as a defense related LRR-RLK is proposed on the basis of cDNA sequence characterization, domain subfamily classification, structural prediction of extra cellular domains, cluster analysis and comparative gene expression. OsJ_RLL1 Sequence and expression analyses of KIX domain proteins suggest their importance in seed development and determination of seed size in rice, and genome stability in Arabidopsis 2013 Mol Genet Genomics National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, 110067, India. jthakur@nipgr.ac.in The KIX domain, which mediates protein-protein interactions, was first discovered as a motif in the large multidomain transcriptional activator histone acetyltransferase p300/CBP. Later, the domain was also found in Mediator subunit MED15, where it interacts with many transcription factors. In both proteins, the KIX domain is a target of activation domains of diverse transcription activators. It was found to be an essential component of several specific gene-activation pathways in fungi and metazoans. Not much is known about KIX domain proteins in plants. This study aims to characterize all the KIX domain proteins encoded by the genomes of Arabidopsis and rice. All identified KIX domain proteins are presented, together with their chromosomal locations, phylogenetic analysis, expression and SNP analyses. KIX domains were found not only in p300/CBP- and MED15-like plant proteins, but also in F-box proteins in rice and DNA helicase in Arabidopsis, suggesting roles of KIX domains in ubiquitin-mediated proteasomal degradation and genome stability. Expression analysis revealed overlapping expression of OsKIX_3, OsKIX_5 and OsKIX_7 in different stages of rice seeds development. Moreover, an association analysis of 136 in silico mined SNP loci in 23 different rice genotypes with grain-length information identified three non-synonymous SNP loci in these three rice genes showing strong association with long- and short-grain differentiation. Interestingly, these SNPs were located within KIX domain encoding sequences. Overall, this study lays a foundation for functional analysis of KIX domain proteins in plants. OsKIX_11,OsKIX_9 Characterization and heterologous expression of laccase cDNAs from xylem tissues of yellow-poplar (Liriodendron tulipifera) 1999 Plant Mol Biol Warnell School of Forest Resources, University of Georgia, Athens 30602, USA. Four closely related cDNA clones encoding laccase isoenzymes from xylem tissues of yellow-poplar (Ltlacc2.1-4) were identified and sequenced. The inferred yellow-poplar laccase gene products were highly related to one another (79-91% at the amino acid level) and showed significant similarity to other blue copper oxidases, especially with respect to the copper-binding domains. The encoded proteins had N-terminal signal sequences and 17-19 potential N-linked glycosylation sites. The mature proteins were predicted to have molecular masses of ca. 61 kDa (unglycosylated) and high isoelectric points (pI 9.3-9.5). The canonical copper ligands were conserved, with the exception of a Leu residue associated with the axial position of the Type-1 cupric ion. The residue at this position has been proposed to influence the redox potential of Type-1 cupric ions. Northern blot analysis revealed that the yellow-poplar laccase genes are differentially expressed in xylem tissues. The genes were verified as encoding active laccases by heterologous expression in tobacco cells and demonstration of laccase activity in extracts from transformed tobacco cell lines. Oslacc Transgenic tobacco with rice zinc-finger gene OsLOL2 exhibits an enhanced resistance against bacterial-wilt 2011 Australasian Plant Pathology Botany Department, Institute of Chemical and Biological Sciences, University of Gujrat, Gujrat, 50700, Pakistan LSD1-related proteins have been found to regulate programmed cell death (PCD) and disease related signaling in the plants. Rice LSD1-like (OsLOL1) gene was involved in regulation of cell death and OsLOL 2 (rice zinc-finger gene) has been reported to regulate plant growth and disease resistance in plants. Bacterial-wilt is a devastating disease caused by Ralstonia solanacearum that needs to be controlled effectively. To achieve this purpose, Agrobacterium-mediated transgenic tobacco plants were generated, which hold OsLOL 2 gene in their genome encoding zinc-finger proteins. Selection of the T2 OsLOL2 transgenic lines was accomplished using molecular techniques, and later on their physiological characterization was done. Southern-blot assay of the selected transformed lines reveals that the transgene has been incorporated into the tobacco genome. On infection with R. solanacearum strains (GMI1000 and RS1000), the selected T2 transgenic tobacco lines showed enhanced resistance against bacterial-wilt when compared to control plants. Moreover, relatively less bacterial proliferation occurred in the infected stem-tissues of the transgenic lines. Owing to smaller bacterial populations, relatively fewer blockages in xylem vessels were observed during microscopy in the transgenic lines as compared to control. OsLOL1 Genetic analysis of the metabolome exemplified using a rice population 2013 Proc Natl Acad Sci U S A National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan) Plant metabolites are crucial for both plant life and human nutrition. Despite recent advance in metabolomics, the genetic control of plant metabolome remains largely unknown. Here, we performed a genetic analysis of the rice metabolome that provided over 2,800 highly resolved metabolic quantitative trait loci for 900 metabolites. Distinct and overlapping accumulation patterns of metabolites were observed and complex genetic regulation of metabolism was revealed in two different tissues. We associated 24 candidate genes to various metabolic quantitative trait loci by data mining, including ones regulating important morphological traits and biological processes. The corresponding pathways were reconstructed by updating in vivo functions of previously identified and newly assigned genes. This study demonstrated a powerful tool and provided a vast amount of high-quality data for understanding the plasticity of plant metabolome, which may help bridge the gap between the genome and phenome. OsMaT-1,OsMaT-2,OsMaT-3 Alteration of nutrient allocation and transporter genes expression in rice under N, P, K, and Mg deficiencies 2011 Acta Physiologiae Plantarum College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, 210095, China Nitrogen (N), phosphorus (P), potassium (K), and magnesium (Mg) have essential physiological functions in plants. Their interactions in plants are not fully understood especially at the molecular level. In this study, we detected the physiological and molecular responses of rice plants at the vegetative growth phase to N, P, K, and Mg starvations. Deficiencies of N and P resulted in accumulation of soluble sugar and starch in the leaves. The root to shoot ratio increased under N and P deficiencies, but decreased under K and Mg deficiencies. In addition, deficiency of either K or Mg resulted in accumulation of the other cation in shoots. Moreover, K starvation decreased both K and soluble sugar contents in the roots pronouncedly. RT-PCR analysis showed that several sugar transporter genes in the leaves orchestrated with sugar accumulation induced by the nutrient shortages. Expression of a high affinity K transporter gene (OsHAK1) and a putative Mg transporter gene (OsMGT) showed opposite down- and up-regulation in the roots by K starvation. These findings suggest that deficiencies of the major nutrients suppressed the export of carbohydrates from source leaves. The regulated sugar and nutrient transporter genes investigated in this study could be used for elucidating the molecular mechanism of plants in their adaptation to varied nutrient supply. OsMHX Genome-wide identification and expression analysis of rice cell cycle genes 2007 Plant Mol Biol Shandong Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Taian, Shandong, 271018, China. Cyclins, cyclin-dependent kinases, and a number of other proteins control the progression of plant cell cycle. Although extensive studies have revealed the roles of some cell cycle regulators and the underlying mechanisms in Arabidopsis, relatively a small number of cell cycle regulators were functionally analyzed in rice. In this study, we describe 41 regulators in the rice genome. Our results indicate that the rice genome contains a less number of the core cell cycle regulators than the Arabidopsis one does, although the rice genome is much larger than the Arabidopsis one. Eight groups of CDKs similar to those in Arabidopsis were identified in the rice genome through phylogenetic analysis, and the corresponding members in the different groups include E2F, CKI, Rb, CKS and Wee. The structures of the core cell regulators were relatively conserved between the rice and Arabidopsis genomes. Furthermore, the expression of the majority of the core cell cycle genes was spatially regulated, and the most closely related ones showed very similar patterns of expression, suggesting functional redundancy and conservation between the highly similar core cell cycle genes in rice and Arabidopsis. Following auxin or cytokinin treatment, the expression of the core cell cycle genes was either upregulated or downregulated, suggesting that auxin and/or cytokinin may directly regulate the expression of the core cell cycle genes. Our results provide basic information to understand the mechanism of cell cycle regulation and the functions of the rice cell cycle genes. OsMSRPK1|Orysa;CDKG;1 Paraquat Resistant1, a Golgi-localized putative transporter protein, is involved in intracellular transport of paraquat 2013 Plant Physiol State Key Laboratory of Plant Physiology and Biochemistry and National Plant Gene Research Center, College of Biological Sciences, China Agricultural University, Beijing 100193, China. Paraquat is one of the most widely used herbicides worldwide. In green plants, paraquat targets the chloroplast by transferring electrons from photosystem I to molecular oxygen to generate toxic reactive oxygen species, which efficiently induce membrane damage and cell death. A number of paraquat-resistant biotypes of weeds and Arabidopsis (Arabidopsis thaliana) mutants have been identified. The herbicide resistance in Arabidopsis is partly attributed to a reduced uptake of paraquat through plasma membrane-localized transporters. However, the biochemical mechanism of paraquat resistance remains poorly understood. Here, we report the identification and characterization of an Arabidopsis paraquat resistant1 (par1) mutant that shows strong resistance to the herbicide without detectable developmental abnormalities. PAR1 encodes a putative l-type amino acid transporter protein localized to the Golgi apparatus. Compared with the wild-type plants, the par1 mutant plants show similar efficiency of paraquat uptake, suggesting that PAR1 is not directly responsible for the intercellular uptake of paraquat. However, the par1 mutation caused a reduction in the accumulation of paraquat in the chloroplast, suggesting that PAR1 is involved in the intracellular transport of paraquat into the chloroplast. We identified a PAR1-like gene, OsPAR1, in rice (Oryza sativa). Whereas the overexpression of OsPAR1 resulted in hypersensitivity to paraquat, the knockdown of its expression using RNA interference conferred paraquat resistance on the transgenic rice plants. These findings reveal a unique mechanism by which paraquat is actively transported into the chloroplast and also provide a practical approach for genetic manipulations of paraquat resistance in crops. OsPAR1 A Calmodulin-Binding Protein from Rice is Essential to Pollen Development 2011 Journal of Plant Biology School of Life Science, Ludong University, Yantai, Shandong, 264025, China A pollen-expressed gene from rice, OsPCBP, that was previously cloned and identified encodes a novel calmodulin-binding protein. Here, we investigated its functioning in pollen development. Overexpression of OsPCBP in transgenic rice plants did not lead to phenotypic changes but did delay anther formation by about 1 week. Cytological observations at different stages revealed that microspores from the OsPCBP double-stranded RNA interference plants developed normally until the binucleate stage, but about half were later aborted. Meanwhile, the anther microstructure of those plants was unaffected. Further examination of OsPCBP expression in transgenic lines via RT-PCR showed that a significant reduction in transcripts was correlated with this abortion phenotype. Our experimental results provide convincing evidence that OsPCBP protein plays an important role during the late stage of pollen development. OsPCBP Plant ABC proteins--a unified nomenclature and updated inventory 2008 Trends Plant Sci Biomathematics and Bioinformatics Department, Rothamsted Research, Harpenden, AL5 2JQ, UK. The ABC superfamily comprises both membrane-bound transporters and soluble proteins involved in a broad range of processes, many of which are of considerable agricultural, biotechnological and medical potential. Completion of the Arabidopsis and rice genome sequences has revealed a particularly large and diverse complement of plant ABC proteins in comparison with other organisms. Forward and reverse genetics, together with heterologous expression, have uncovered many novel roles for plant ABC proteins, but this progress has been accompanied by a confusing proliferation of names for plant ABC genes and their products. A consolidated nomenclature will provide much-needed clarity and a framework for future research. OsPDR20 Molecular and functional analysis of phosphomannomutase (PMM) from higher plants and genetic evidence for the involvement of PMM in ascorbic acid biosynthesis in Arabidopsis and Nicotiana benthamiana 2007 Plant J The State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Phosphomannomutase (PMM) catalyzes the interconversion of mannose-6-phosphate and mannose-1-phosphate. However, systematic molecular and functional investigations on PMM from higher plants have hitherto not been reported. In this work, PMM cDNAs were isolated from Arabidopsis, Nicotiana benthamiana, soybean, tomato, rice and wheat. Amino acid sequence comparisons indicated that plant PMM proteins exhibited significant identity to their fungal and mammalian orthologs. In line with the similarity in primary structure, plant PMM complemented the sec53-6 temperature sensitive mutant of Saccharomyces cerevisiae. Histidine-tagged Arabidopsis PMM (AtPMM) purified from Escherichia coli converted mannose-1-phosphate into mannose-6-phosphate and glucose-1-phosphate into glucose-6-phosphate, with the former reaction being more efficient than the latter one. In Arabidopsis and N. benthamiana, PMM was constitutively expressed in both vegetative and reproductive organs. Reducing the PMM expression level through virus-induced gene silencing caused a substantial decrease in ascorbic acid (AsA) content in N. benthamiana leaves. Conversely, raising the PMM expression level in N. benthamiana using viral-vector-mediated ectopic expression led to a 20-50% increase in AsA content. Consistent with this finding, transgenic expression of an AtPMM-GFP fusion protein in Arabidopsis also increased AsA content by 25-33%. Collectively, this study improves our understanding on the molecular and functional properties of plant PMM and provides genetic evidence on the involvement of PMM in the biosynthesis of AsA in Arabidopsis and N. benthamiana plants. OsPMM Plant DNA polymerase lambda, a DNA repair enzyme that functions in plant meristematic and meiotic tissues 2004 Eur J Biochem Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Japan. Little is known about the functions of DNA polymerase lambda (Pol lambda) recently identified in mammals. From the genomic sequence information of rice and Arabidopsis, we found that Pol lambda may be the only member of the X-family in higher plants. We have succeeded in isolating the cDNA and recombinant protein of Pol lambda in a higher plant, rice (Oryza sativa L. cv. Nipponbare) (OsPol lambda). OsPol lambda had activities of DNA polymerase, terminal deoxyribonucleotidyl transferase and deoxyribose phosphate lyase, a marker enzyme for base excision repair. It also interacted with rice proliferating cell nuclear antigen (OsPCNA) in a pull-down assay. OsPCNA increased the processivity of OsPol lambda. Northern blot analysis showed that the level of OsPol lambda expression correlated with cell proliferation in meristematic and meiotic tissues, and was induced by DNA-damaging treatments. These properties suggest that plant Pol lambda is a DNA repair enzyme which functions in plant meristematic and meiotic tissues, and that it can substitute for Pol beta and terminal deoxyribonucleotidyl transferase. OsPollambda The role of OsPRA1 in vacuolar trafficking by OsRab GTPases in plant system 2009 Plant Science Daegu Arboretum, Daegu 704-310, Republic of Korea PRA1 has been reported as a prenylated Rab acceptor containing GDF activity in human, rat and yeast. Its existence was also proved in plants by sequence analysis, anticipating its important role as a Rab effector, but defined roles of the plant PRA1 homologs remain to be obscure. Here, to get an insight for their role, we performed yeast two-hybrid screen using the OsRab7 as bait and first isolated the OsPRA1, a putative prenylated Rab acceptor, interacting with this protein. Detailed interaction analysis showed that OsPRA1 interacted not only with GDP-bound OsRab7, but also with several other Rab GTPases involved in vacuolar trafficking, in a prenylation-dependent manner. In addition, GFP-fusion analysis demonstrated that OsPRA1 localized to the prevacuolar compartment, and RNA gel blot analysis revealed its significant expression in rice green-aerial tissues such as shoots and mature stems. These results suggest that OsPRA1 may function as a Rab effector for vacuolar trafficking in the plant system. OsPRA1 Alteration of nitrogen metabolism in rice variety 'Nipponbare' induced by alkali stress 2011 Plant and Soil Key laboratory of Molecular Epigenetics of MOE, Northeast Normal University, Changchun, 130024, Jilin Province, China Effects of AS on the nitrogen metabolism of rice variety 'Nipponbare' mainly comprised two mechanisms. Firstly, in roots, AS caused the reduction of NO 3 − content, which caused two harmful consequences, the large downregulation of OsNR1 expression and the subsequent reduction of NH 4 + production in roots. On the other hand, under AS (pH, 9.11), almost all the NH 4 + was changed to NH3, which caused a severe deficiency of NH 4 + surrounding the roots. Both events might cause a severe deficiency of NH 4 + in roots. Under AS, the increased expression of several OsAMT family members in roots might be an adaptative response to the reduction of NH 4 + content in roots or the NH 4 + deficiency in rhizosphere. Also, the down-regulation of OsNADH-GOGAT and OsGS1;2 in roots might be due to NH 4 + deficiency in roots. Secondly, in shoots, AS caused a larger acuumulatiuon of Na+, which possibly affected photorespiration and led to a continuous decrease of NH 4 + production in shoots, and inhibited the expression of OsFd-GOGAT and OsGS2 in chloroplasts. OsProDH Expression of a RING-HC protein from rice improves resistance to Pseudomonas syringae pv. tomato DC3000 in transgenic Arabidopsis thaliana 2007 J Exp Bot Department of Biology, The Chinese University of Hong Kong, Shatin, Hong Kong SAR. A cDNA clone (OsRHC1) was obtained, which encodes a novel RING zinc finger protein sharing similar structural features (multiple transmembrane domains at the N-half; a unique RING zinc finger consensus Cys-X(2)-Cys-X(11)-Cys-X-His-X(3)-Cys-X(2)-Cys-X(6)-Cys-X(2)-Cys at the C terminus) to a group of closely related annotated proteins from both monocots and dicots. OsRHC1 was found to be localized on plasma membrane of rice cells and induced by wounding in rice lines containing Xa loci. Ecotopic expression of the OsRHC1 cDNA from rice (a monocot) in transgenic Arabidopsis thaliana (a dicot) enhanced the defence response toward Pseudomonas syringae pv. tomato DC3000, suggesting that OsRHC1 may confer broad-spectrum disease resistance. The protective effects were neutralized in the presence of MG132 or in an npr1-3 mutation background, indicating that the function of OsRHC1 is dependent on the ubiquitin-mediated protein degradation via the 26S proteasome and the presence of the key defence response regulator NPR1. OsRHC1 Two types of replication protein A in seed plants 2005 FEBS J Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Japan. Replication protein A (RPA), a heterotrimeric protein composed of 70, 32 and 14-kDa subunits, has been shown to be essential for DNA replication, repair, recombination, and transcription. Previously, we found that, in two seed plants, rice and Arabidopsis, there are two different types of RPA70-kDa subunit. Substantial biochemical and genetic characterization of these two subunits, termed OsRPA70a and OsRPA70b or AtRPA70a and AtRPA70b, respectively, is described in this report. Inactivation of AtRPA70a by transfer DNA insertion or RNA interference is lethal, so the complex containing RPA70a may be essential for DNA replication. Transfer DNA insertion and RNAi lines for AtRPA70b are morphologically normal, albeit hypersensitive to certain mutagens, such as UV-B and methyl methanesulfonate, suggesting that RPA70b functions mostly in DNA repair. In two-hybrid, pull-down and coexpression analysis, OsRPA70b was found to interact more selectively than OsRPA70a with OsRPA32. The data suggest that two different types of RPA heterotrimer are present in seed plants, and that there may be additional 32 and 14-kDa subunit homologs that interact with OsRPA70a. Each of the two probable plant RPA complexes may have different roles in DNA metabolism. OsRPA14 A comparative approach expands the protein-protein interaction node of the immune receptor XA21 in wheat and rice 2013 Genome State Key Laboratory of Crop Stress Biology for Arid Areas/College of Agronomy, Northwest A&F University, 3 Taicheng Road, Yangling, Shaanxi 712100, China. The rice (Oryza sativa) OsXA21 receptor kinase is a well-studied immune receptor that initiates a signal transduction pathway leading to resistance to Xanthomonas oryzae pv. oryzae. Two homologs of OsXA21 were identified in wheat (Triticum aestivum): TaXA21-like1 located in a syntenic region with OsXA21, and TaXA21-like2 located in a nonsyntenic region. Proteins encoded by these two wheat genes interact with four wheat orthologs of known OsXA21 interactors. In this study, we screened a wheat yeast-two-hybrid (Y2H) library using the cytosolic portion of TaXA21-like1 as bait to identify additional interactors. Using full-length T. aestivum and T. monococcum proteins and Y2H assays we identified three novel TaXA21-like1 interactors (TaARG, TaPR2, TmSKL1) plus one previously known in rice (TaSGT1). An additional full-length wheat protein (TaCIPK14) interacted with TaXA21-like2 and OsXA21 but not with TaXA21-like1. The interactions of TaXA21-like1 with TmSKL1 and TaSGT1 were also observed in rice protoplasts using bimolecular fluorescence complementation assays. We then cloned the rice homologs of the novel wheat interactors and confirmed that they all interact with OsXA21. This last result suggests that interspecific comparative interactome analyses can be used not only to transfer known interactions from rice to wheat, but also to identify novel interactions in rice. OsSK4|OsSKL1 Two families of sterol methyltransferases are involved in the first and the second methylation steps of plant sterol biosynthesis 1998 Eur J Biochem Institut de Biologie Moleculaire des Plantes, Departement de Biologie Cellulaire et Moleculaire, Strasbourg, France. pierre@medoc.u-strasbg.fr Two methyl transfers are involved in the biosynthesis of 24-methyl and 24-ethyl sterols, which play major roles in plant growth and development. The first methyl transfer applies to cycloartenol, the second to 24-methylene lophenol. About ten cDNA clones encoding S-adenosyl-L-methionine (AdoMet) sterol methyltransferases (SMTs) have been isolated so far from various plants. According to their deduced amino acid sequences, they were classified in two families, smtl and smt2; in addition, smt2 cDNAs were shown to encode a 24-methylene lophenol C24 methyltransferase [Bouvier-Nave, P., Husselstein, T., Desprez, T. & Benveniste, P. (1997) Eur. J. Biochem. 246, 518-529]. We now report the comparison of two cDNAs isolated from Nicotiana tabacum, Ntsmt1-1 which belongs to the first SMT cDNA family and Ntsmt2-1 which belongs to the second. Both cDNAs were expressed in the yeast null mutant erg6, deficient in SMT. Whereas erg6 is devoid of 24-alkyl sterols, erg6 Ntsmt1-1 contained a majority of 24-methylene sterols and erg6 Ntsmt2-1, a majority of 24-ethylidene sterols, indicating distinct functions for the expression products of these cDNAs. In the presence of AdoMet, delipidated microsomes from erg6 Ntsm1-1 efficiently converted cycloartenol into 24-methylene cycloartanol, but did not produce any 24-ethylidene lophenol upon incubation with 24-methylene lophenol. This demonstrates that cDNA Ntsmt1-1 (and most probably the other plant SMT cDNAs of the first family) encode(s) a cycloartenol C24 methyltransferase. In contrast, delipidated microsomes of erg6 Ntsmt2-1 were shown to methylate preferentially 24-methylene lophenol, as expected from an SMT encoded by an smt2 cDNA. In summary, among various cDNAs isolated from N. tabacum, one (Ntsmt1-1) belongs to the first family of plant SMT cDNAs according to its deduced amino acid sequence and was shown to encode a cycloartenol C24 methyltransferase, whereas another (Ntsmt2-1) belongs to the second family and was shown to encode a 24-methylene lophenol C24 methyltransferase. Meanwhile, two cDNAs were isolated from Oriza sativa and shown to belong to smtl and to smt2 families, respectively. These data disclose the coexistence, in a given plant species, of two distinct SMTs, each catalyzing one step of methylation in the sterol biosynthesis pathway. Ossmt1-1,Ossmt2-1 Molecular and Functional Characterization of Sphingosine-1-Phosphate Lyase Homolog from Higher Plants 2007 J Integr Plant Biol State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, the Chinese Academy of Sciences, Beijing 100101, China Sphingosine-1-phosphate lyase (SPL) is involved in degrading the conserved sphingolipid signaling molecule sph-ingosine-1-phosphate. However, molecular studies on plant SPL have not been reported to date. Here, we present bioinformatic, molecular and functional analyses of putative SPL proteins from Arabidopsis thaliana and rice (designated as AtSPL and OsSPL, respectively). Amino acid sequence comparison revealed that plant SPL contained the pyridoxal-dependent decarboxylase domain and the conserved residue that may be involved in substrate catalysis. When expressed in Saccharomyces cerevisiae, AtSPL and OsSPL corrected the hypersensitive phenotype of the yeast dpl1 deletion strain, which is deficient in endogenous SPL activity, to exogenous supplied sphingolipid long chain bases (LCBs), suggesting that plant SPL protein is functional in vivo in degrading phosphorylated LCBs. In Arabidopsis, AtSPL transcripts were detected in roots, stems, leaves, flowers and siliques. In pAtSPL-AtSPL::GUS transgenic lines, the AtSPL::GUS fusion protein was found in a variety of vegetative and reproductive tissues. AtSPL expression level was dynamically regulated during leaf development and senescence, and was steadily and significantly increased in Arabidopsis seedlings treated with the cell death-inducing fungal toxin fumonisin B1. The potential function of SPL in Arabidopsis is discussed. OsSPL1 Cloning and haplotype analysis of TaSTE, which is associated with plant height in bread wheat (Triticum aestivum L.) 2012 Molecular Breeding College of Agricultural, Shenyang Agricultural University, Shenyang, 110866, Liaoning, China Sterol methyltransferase 1 (STE1) catalyzes ∆7 sterol C-5 desaturation in the conversion of 24-methylenecholesterol to campestral in brassinosteroid (BR) biosynthesis, and STE1 has been proven to influence plant architecture by controlling the BR levels in Arabidopsis. In the present study, TaSTE (wheat), the ortholog of AtSTE1 (Arabidopsis), was cloned and mapped to wheat chromosome homologous group 3 in the interval between simple sequence repeats (SSRs) markers CWM48.1 and WMC532, with genetic distances of 17.7 and 7.6 cM, respectively. Both linkage and association analyses revealed that TaSTE-A1 was significantly associated with plant height in bread wheat. In silico expression results showed similar expression patterns among TaSTE, HvSTE1 (barley), OsSTE1 (also known as OsDWF7 in rice), and AtSTE1, suggesting that these orthologs might share common functions among wheat, barley, rice, and Arabidopsis. The haplotype frequencies in wild species, landraces, and modern cultivars indicated that haplotype II (Hap II) was associated with reduced plant height and positively selected during modern breeding improvement. One pair of complementary allele-specific polymerase chain reaction (PCR) markers can distinguish haplotype I (Hap I) and Hap II and could be developed to assist selection in wheat architecture breeding. OsSTE1|OsDWF7 The N-terminal ATPase AT-hook-containing region of the Arabidopsis chromatin-remodeling protein SPLAYED is sufficient for biological activity 2006 Plant J Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA. The SNF2-like chromatin-remodeling ATPase SPLAYED (SYD) was identified as a co-activator of floral homeotic gene expression in Arabidopsis. SYD is also required for meristem maintenance and regulates flowering under a non-inductive photoperiod. SNF2 ATPases are structurally and functionally conserved from yeast to humans. In addition to the conserved protein features, SYD has a large unique C-terminal domain. We show here that SYD is present as two forms in the nucleus, full-length and truncated, with the latter apparently lacking the C-terminal domain. The ratio of the two forms of endogenous SYD differs in juvenile and in adult tissues. Furthermore, an SYD variant lacking the C-terminal domain (SYDDeltaC) rescues the syd null mutant, indicating that the N-terminal ATPase AT-hook-containing region of SYD is sufficient for biological activity. Plants expressing SYDDeltaC show molecular and morphological phenotypes opposite to those of the null mutant, suggesting that the construct results in increased activity. This increased activity is at least in part due to elevated SYD protein levels in these lines. We propose that the C-terminal domain may control SYD accumulation and/or specific activity in the context of the full-length protein. The presence of the C-terminal domain in rice SYD suggests that its role is probably conserved in the two classes of flowering plants. OsSYD|CHR720 Transgenic analysis of a salt-inhibited OsZFP1 gene from rice 2004 Acta Botanica Sinica Chinese Acad Sci, Inst Genet & Dev Biol, Plant Biotechnol Lab, Beijing 100101, Peoples R China OsZFP1 (Oryza sativa zinc finger protein No.1) gene encodes a protein containing three putative Cys(2)/Cys(2)-type zinc-finger domains, and is negatively regulated by salt stress. The plant expression vector carrying the OsZFP1 gene was constructed under the control of 35S promoter in pCAMBIA1300, and then transformed into Arabidopsis plants and rice calli to overexpress OsZFP1 gene. Both the transgenic Arabidopsis plants and the transgenic rice calli were more sensitive to salt treatment than the wild type. This result implies that OsZFP1 gene may function as a repressor, which probably inhibits the expression of some salt-inducible genes. The transgenic Arabidopsis plants also bolted later than wild type plants upon ABA treatment, suggesting that the function of the OsZFP1 gene may be regulated by ABA. OsZFP1|SRZ3 Sequences of two expressed sequence tags (EST) from rice encoding different cap-binding proteins 1996 Gene Department of Chemistry and Biochemistry, University of Texas at Austin 78712, USA. Wheat has been shown to have two forms of the cap-binding protein that participate in the initiation of translation. To identify cap-binding proteins from other higher plant species, the expressed sequence tag (EST) database was searched. Several rice ESTs were identified with similarity to both forms of the wheat cap-binding proteins. Two of the rice ESTs were obtained and the cDNA sequences completed. The deduced amino acid sequences of the rice cap-binding proteins are compared to the wheat cap-binding proteins and cap-binding proteins from Saccharomyces cerevisiae, Drosophila melanogaster, Xenopus laevis and human. p26,p28 Rice OsPAD4 functions differently from Arabidopsis AtPAD4 in host-pathogen interactions 2014 The Plant Journal National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China The extensively studied Arabidopsis phytoalexin deficient 4 (AtPAD4) gene plays an important role in Arabidopsis disease resistance; however, the function of its sequence ortholog in rice is unknown. Here, we show that rice OsPAD4 appears not to be the functional ortholog of AtPAD4 in host-pathogen interactions, and that the OsPAD4 encodes a plasma membrane protein but that AtPAD4 encodes a cytoplasmic and nuclear protein. Suppression of OsPAD4 by RNA interference (RNAi) increased rice susceptibility to the biotrophic pathogen Xanthomonas oryzae pv. oryzae (Xoo), which causes bacteria blight disease in local tissue. OsPAD4-RNAi plants also show compromised wound-induced systemic resistance to Xoo. The increased susceptibility to Xoo was associated with reduced accumulation of jasmonic acid (JA) and phytoalexin momilactone A (MOA). Exogenous application of JA complemented the phenotype of OsPAD4-RNAi plants in response to Xoo. The following results suggest that OsPAD4 functions differently than AtPAD4 in response to pathogen infection. First, OsPAD4 plays an important role in wound-induced systemic resistance, whereas AtPAD4 mediates systemic acquired resistance. Second, OsPAD4-involved defense signaling against Xoo is JA-dependent, but AtPAD4-involved defense signaling against biotrophic pathogens is salicylic acid-dependent. Finally, OsPAD4 is required for the accumulation of terpenoid-type phytoalexin MOA in rice-bacterium interactions, but AtPAD4-mediated resistance is associated with the accumulation of indole-type phytoalexin camalexin. PAD4|OsPAD4 Sucrose-mediated priming of plant defense responses and broad-spectrum disease resistance by overexpression of the maize pathogenesis-related PRms protein in rice plants 2007 Mol Plant Microbe Interact Departamento de Genetica Molecular, Laboratorio de Genetica Molecular Vegetal, Consorcio CSIC-IRTA, Jordi Girona 18, 08034 Barcelona, Spain. Expression of pathogenesis-related (PR) genes is part of the plant's natural defense response against pathogen attack. The PRms gene encodes a fungal-inducible PR protein from maize. Here, we demonstrate that expression of PRms in transgenic rice confers broad-spectrum protection against pathogens, including fungal (Magnaporthe oryzae, Fusarium verticillioides, and Helminthosporium oryzae) and bacterial (Erwinia chrysanthemi) pathogens. The PRms-mediated disease resistance in rice plants is associated with an enhanced capacity to express and activate the natural plant defense mechanisms. Thus, PRms rice plants display a basal level of expression of endogenous defense genes in the absence of the pathogen. PRms plants also exhibit stronger and quicker defense responses during pathogen infection. We also have found that sucrose accumulates at higher levels in leaves of PRms plants. Sucrose responsiveness of rice defense genes correlates with the pathogen-responsive priming of their expression in PRms rice plants. Moreover, pretreatment of rice plants with sucrose enhances resistance to M. oryzae infection. Together, these results support a sucrose-mediated priming of defense responses in PRms rice plants which results in broad-spectrum disease resistance. OsPAL,Sci1|PR6 DNA binding and dimerization specificity and potential targets for the TCP protein family 2002 Plant J Molecular Genetics Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. The TCP domain is a plant-specific DNA binding domain found in proteins from a diverse array of species, including the cycloidea (cyc) and teosinte branched1 (tb1) gene products and the PCF1 and PCF2 proteins. To understand the role in transcriptional regulation of proteins with this domain, we have analysed the DNA binding and dimerization specificity of the TCP protein family using rice PCF proteins, and further evaluated potential targets for the TCP protein. The seven PCF members including five newly isolated proteins, were able to be grouped into two classes, I and II, based on sequence similarity in the TCP domain. Random binding site selection experiments and electrophoretic mobility shift assays (EMSAs) revealed the consensus DNA binding sequences of these two classes to be distinct but overlapping; GGNCCCAC for class I and GTGGNCCC for class II. The TB1 protein from maize, which belongs to class II, had the same specificity as the rice class II proteins, suggesting the conservation of binding specificity between TCP domains from different species. The yeast 2-hybrid assay and EMSA revealed that these proteins tend to form a homodimer or a heterodimer between members of the same class. We searched predicted 5' flanking sequences of Arabidopsis genes for the consensus binding sequences and found that the consensus sites are distributed in the genome at a considerably lower frequency. We further analysed eight promoters containing the class I consensus TCP sites. The transcriptional activities of six promoters were decreased by a mutation of the TCP binding site, which is consistent with the observation that the class I TCP site can confer transactivation function on a heterologous promoter. These results suggest that the two classes of TCP protein are distinct in DNA binding specificity and transcriptional regulation. PCF3 High time for a roll call: gene duplication and phylogenetic relationships of TCP-like genes in monocots 2011 Ann Bot Department of Cell Biology and Plant Biochemistry, University of Regensburg, Regensburg, Germany. mariana.mondragon@biologie.uni-regensburg.de BaCKGROUND AND AIMS: The TCP family is an ancient group of plant developmental transcription factors that regulate cell division in vegetative and reproductive structures and are essential in the establishment of flower zygomorphy. In-depth research on eudicot TCPs has documented their evolutionary and developmental role. This has not happened to the same extent in monocots, although zygomorphy has been critical for the diversification of Orchidaceae and Poaceae, the largest families of this group. Investigating the evolution and function of TCP-like genes in a wider group of monocots requires a detailed phylogenetic analysis of all available sequence information and a system that facilitates comparing genetic and functional information. METHODS: The phylogenetic relationships of TCP-like genes in monocots were investigated by analysing sequences from the genomes of Zea mays, Brachypodium distachyon, Oryza sativa and Sorghum bicolor, as well as EST data from several other monocot species. KEY RESULTS: All available monocot TCP-like sequences are associated in 20 major groups with an average identity >/=64 % and most correspond to well-supported clades of the phylogeny. Their sequence motifs and relationships of orthology were documented and it was found that 67 % of the TCP-like genes of Sorghum, Oryza, Zea and Brachypodium are in microsyntenic regions. This analysis suggests that two rounds of whole genome duplication drove the expansion of TCP-like genes in these species. CONCLUSIONS: A system of classification is proposed where putative or recognized monocot TCP-like genes are assigned to a specific clade of PCF-, CIN- or CYC/tb1-like genes. Specific biases in sequence data of this family that must be tackled when studying its molecular evolution and phylogeny are documented. Finally, the significant retention of duplicated TCP genes from Zea mays is considered in the context of balanced gene drive. PCF3 A major photoperiod-sensitivity gene tagged with RFLP and isozyme markers in rice 1993 Theor Appl Genet USD A-ARS. Dept. of Agronomy and Range Science, University of California, 95616, Davis, CA, USA. Photoperiod-sensitive rice (Oryza sativa L.) cultivars are widely grown in rainfed lowland areas with unfavorable water regimes. A molecular marker for the trait would be useful in genetic and physiological studies and in developing improved photoperiod-sensitive cultivars. Previous genetic studies identified a major gene for photoperiod sensitivity on chromosome 6. We have tested an isozyme marker and several RFLP probes mapping to chromosome 6 in an attempt to identify marker(s) tightly linked to photoperiod sensitivity in tropical rice cultivars. We report here that the isozyme gene Pgi-2 is linked (23.2+/-4.7 cM) to the photoperiod-sensitivity gene in the cultivar GEB-24. Although association of duration with Pgi-2 alleles can be used to detect segregation of the photoperiod sensitivity gene in crosses, it will probably not be useful as a marker in selection because of its loose linkage. In contrast, a gene for photoperiod sensitivity in the cultivar Puang Rai 2 was found to be closely linked to the rice genomic clone RG64. Among 15 F3 lines homozygous for photoperiod insensitivity, no recombinants were detected with RG64. This clone is thus an excellent probe to follow segregation of the major photoperiod-sensitivity gene in rice crosses. PGI2 Catalytic properties of rice alpha-oxygenase. A comparison with mammalian prostaglandin H synthases 2002 J Biol Chem Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan. Long-chain fatty acids can be metabolized to C(n)(-1) aldehydes by alpha-oxidation in plants. The reaction mechanism of the enzyme has not been elucidated. In this study, a complete nucleotide sequence of fatty acid alpha-oxygenase gene in rice plants (Oryza sativa) was isolated. The deduced amino acid sequence showed some similarity with those of mammalian prostaglandin H synthases (PGHSs). The gene was expressed in Escherichia coli and purified to apparently homogeneous state. It showed the highest activity with linoleic acid and predominantly formed 2-hydroperoxide of the fatty acid (C(n)), which is then spontaneously decarboxylated to form corresponding C(n)(-1) aldehyde. With linoleic or linoleic acids as a substrate, rice alpha-oxygenase formed no product having a lambda(max) at approximately 234 nm, which indicated that the enzyme could not oxygenize the pentadiene system in the substrate. The spectroscopic feature of the purified enzyme in its ferrous state is similar to that of mammalian PGHS, whereas that of dithionite-reduced state showed significant difference. Site-directed mutagenesis revealed that His-158, Tyr-380, and Ser-558 were essential for the alpha-oxygenase activity. These residues are conserved in PGHS and known as a heme ligand, a source of a radical species to initiate oxygenation reaction and a residue involved in substrate binding, respectively. This finding suggested that the initial step of the oxygenation reaction in alpha-oxygenase has a high similarity with that of PGHS. The rice alpha-oxygenase activity was inhibited by imidazole but hardly inhibited by nonsteroidal anti-inflammatory drugs, such as aspirin, ibuprofen, and flurbiprofen, which are known as typical PGHS inhibitors. In addition, peroxidase activity could not be detected with alpha-oxygenase when palmitic acid 2-hydroperoxide was used as a substrate. From these findings, the catalytic resemblance between alpha-oxygenase and PGHS seems to be evident, although there still are differences in their substrate recognitions and peroxidation activities. PIOX|RalphaO Organ-specific and hormone-dependent expression of genes for serine carboxypeptidases during development and following germination of rice grains 1994 Plant Physiol Division of Biological Science, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Japan. Several cDNA clones encoding either serine carboxypeptidases or related proteins of Oryza sativa L. were identified, and the abundance of the corresponding mRNA in immature and germinated grains was examined. The deduced amino acid sequence of each cDNA included key sequences, such as a pentapeptide (G-X-S-X-G/A) that is conserved among many serine carboxypeptidases, and the putative protein products were classified as two general and one novel type of cereal serine carboxypeptidases. Two general types exhibited considerable homology to type I and type III carboxypeptidases of cereal plants. The novel type encoded a serine carboxypeptidase-like protein that was very similar to type III carboxypeptidases of barley and wheat but had slight differences in both the N- and the C-terminal sequences. The mRNAs of each of these carboxypeptidases were observed in immature grains, and they decreased during maturation. The abundance of mRNA for each class of carboxypeptidase increased again following germination with the same time course and in a tissue-specific manner. The mRNAs for type I and type III-like carboxypeptidases were abundant in germinated embryos composed of leaf, root, and scutellum, whereas the mRNA for type III carboxypeptidase was conspicuous in endosperm that contained the aleurone layer. Altered amounts of mRNA in deembryonated half-grains in response to phytohormones, such as gibberellic acid and abscisic acid, were only detectable in the case of type III carboxypeptidase. Southern blot analysis using rice genomic DNA revealed the simple organization of each gene for these three classes of carboxypeptidases. R-CPD3L|cbp31 The classical Ubisch bodies carry a sporophytically produced structural protein (RAFTIN) that is essential for pollen development 2003 Proc Natl Acad Sci U S A Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9. Pollen fecundity is crucial to crop productivity and also to biodiversity in general. Pollen development is supported by the tapetum, a metabolically active sporophytic nurse layer that devotes itself to this process. The tapetum in cereals and a vast majority of other plants is of the nonamoeboid type. Unable to reach out to microspores, it secretes nutrients into the anther locule where the microspores reside and develop. Orbicules (Ubisch bodies), studied in various plants since their discovery approximately 140 years ago, are a hallmark of the secretory tapetum. Their significance to tapetal or pollen development has not been established. We have identified in wheat and rice an anther-specific single-copy gene (per haploid genome equivalent) whose suppression in rice by RNA interference nearly eliminated the seed set. The flowers in the transgenics were normal for female functions, but the pollen collapsed and became less viable. Further characterization of the gene product, named RAFTIN, in wheat has shown that it is present in pro-orbicule bodies and it is accumulated in Ubisch bodies. Furthermore, it is targeted to microspore exine. Although the carboxyl portion of RAFTINs shares short, dispersed amino acid sequences (BURP domain) in common with a variety of proteins of disparate biological contexts, the occurrence RAFTIN per se is limited to cereals; neither the Arabidopsis genome nor the vast collection of ESTs suggests any obvious dicot homologs. Furthermore, our results show that RAFTIN is essential for the late phase of pollen development in cereals. RAFTIN1 The rice bZIP transcriptional activator RITA-1 is highly expressed during seed development 1994 Plant Cell Laboratory of Plant Molecular Biology, Rockefeller University, New York, New York 10021-6399. Systematic protein-DNA binding studies have shown that plant basic leucine zipper (bZIP) proteins exhibit a differential binding specificity for ACGT motifs. Here, we show that the rice transcription activator-1 (RITA-1) displays a broad binding specificity for palindromic ACGT elements, being able to bind A-, C-, and G-box but not T-box elements. By using gel mobility shift assays with probes differing in sequences flanking the hexameric core, we identified high-affinity A-, C-, and G-box binding sites. Quantitative and competition DNA binding studies confirmed RITA-1 specificity for these sites. Using rice protoplasts as a transient expression system, we demonstrated that RITA-1 can transactivate reporter genes possessing high-affinity but not low-affinity RITA-1 binding sites. Our results established a direct relationship between in vivo transactivation and in vitro binding activity. Transient expression assays that demonstrated the ability of RITA-1 to transactivate a construct containing rita-1 5' flanking sequences suggest that the factor may be autoregulated. Histochemical analysis of transgenic rice plants showed that a rita-1-beta-glucuronidase transgene is expressed in aleurone and endosperm cells of developing rice seeds. We propose that RITA-1 plays a role in the regulation of rice genes expressed in developing rice seeds. RITA1 Transcript abundance of rml1, encoding a putative GT1-like factor in rice, is up-regulated by Magnaporthe grisea and down-regulated by light 2004 Gene National Center for Gene Research, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 500 Caobao Road, Shanghai 200233, China. We isolated and sequenced both genomic DNA and cDNA clones, which encoded a putative GT1-like protein with 385 amino acids, from cultivated rice (Oryza sativa ssp. indica). This protein shows significant amino acid sequence similarities with trihelix DNA-binding GT-1a/B2F and GT-1 factors that were identified in dicot plants. Northern blotting analysis indicated that the transcript of the rice GT-1 factor in seedling was up-regulated by the rice blast fungus Magnaporthe grisea, down-regulated by various continuous light conditions and expressed rhythmically in light/dark cycles. This GT1-like factor gene was therefore designated as rml1 (rice gene regulated by M. grisea and light). The putative RML1 protein, encoded by this single copy gene, is thus identified as a new member of the plant-specific GT family of transcription factors in rice. rml1|RML1 A rice (Oryza sativa L.) cDNA encodes a protein sequence homologous to the eukaryotic ribosomal 5S RNA-binding protein 1993 Plant Mol Biol Agricultural Biotechnology Institute, Rural Development Administration, Suwon, Republic of Korea. A rice (Oryza sativa L.) cDNA clone coding for the cytoplasmic ribosomal protein L5, which associates with 5 S rRNA for ribosome assembly, was cloned and its nucleotide sequence was determined. The primary structure of rice L5, deduced from the nucleotide sequence, contains 294 amino acids and has intriguing features some of which are also conserved in other eucaryotic homologues. These include: four clusters of basic amino acids, one of which may serve as a nucleolar localization signal; three repeated amino acid sequences; the conservation of glycine residues. This protein was identified as the nuclear-encoded cytoplasmic ribosomal protein L5 of rice by sequence similarity to other eucaryotic ribosomal 5 S RNA-binding proteins of rat, chicken, Xenopus laevis, and Saccharomyces cerevisiae. Rice L5 shares 51 to 62% amino acid sequence identity with the homologues. A group of ribosomal proteins from archaebacteria including Methanococcus vanniellii L18 and Halobacterium cutirubrum L13, which are known to be associated with 5 S rRNA, also related to rice L5 and the other eucaryotic counterparts, suggesting an evolutionary relationship in these ribosomal 5 S RNA-binding proteins. RPL5 The effects of genetic manipulation of putrescine biosynthesis on transcription and activities of the other polyamine biosynthetic enzymes 2007 Physiologia Plantarum Department of Plant Biology, University of New Hampshire, Durham, NH 03824, USA We have studied the effects of overproduction of putrescine (Put) via transgenic expression of a mouse ornithine decarboxylase (ODC) gene on the expression of native genes for four enzymes involved in polyamine biosynthesis in hybrid poplar (Populus nigra×maximowiczii) cells. An examination of the transcript levels of arginine decarboxylase (ADC), ODC, S-adenosylmethionine decarboxylase (SAMDC) and spermidine synthase (SPDS), as well as their enzyme activities (except SPDS), showed that the expression of different members of the SAMDC and SPDS gene families was affected differently in response to alteration of the cellular Put content. It was further observed that there was a strong correlation between transcript levels and the activity of the respective enzyme in the cells. Moreover, there was no feedback inhibition of the expression of the native ODC or the ADC genes or their enzyme activities by increased Put in the cells. SAMDC1 cDNA microarray analysis of rice anther genes under chilling stress at the microsporogenesis stage revealed two genes with DNA transposon Castaway in the 5'-flanking region 2014 Bioscience, Biotechnology and Biochemistry Plant Physiology Laboratory, National Agricultural Research Center for Tohoku Region Rice is most chilling sensitive at the onset of microspore release. Chilling treatment at this stage causes male sterility. The gene expression profile during the microspore development process under chilling stress was revealed using a microarray that included 8,987 rice cDNAs. As many as 160 cDNAs were up- or down-regulated by chilling during the microspore release stage. RT-PCR analysis of 5 genes confirmed the microarray results. We identified 3 novel genes whose expression levels were remarkably changed by chilling in rice anther. A new cis element that includes a DNA transposon Castaway sequence was found in the 5' upstream region of two genes which were conspicuously down-regulated by chilling temperatures in rice anther. SAMDC1 Fine mapping of Rf3 and Rf4 fertility restorer loci of WA-CMS of rice (Oryza sativa L.) and validation of the developed marker system for identification of restorer lines 2012 Euphytica Crop Improvement Section, Directorate of Rice Research, Rajendranagar, Hyderabad, 500030, India The Wild Abortive (WA) system is the major cytoplasmic male sterility (CMS) source for hybrid rice production in indica rice and its fertility restoration is reported to be controlled by two major loci viz. Rf3 on chromosome 1 and Rf4 on chromosome 10. With the availability of the rice genome sequence, an attempt was made to fine map, develop candidate gene based markers for Rf3 and Rf4 and validate the developed marker system in a set of known restorer lines. Using polymorphic markers developed from microsatellite markers and candidate gene based markers from Rf3 and Rf4 loci, local linkage maps were constructed in two mapping populations of ~1,500 F2 progeny from KRH2 (IR58025A/KMR3R) and DRRH2 (IR68897A/DR714-1-2R) hybrids. QTLs and their interactions for fertility restoration in Rf3 and Rf4 loci were identified. The identified QTL in both mapping populations together explained 66–72 % of the phenotypic variance of the trait suggesting their utility in developing a marker system for identification of fertility restorers for WA-CMS. Sequence comparison of the two candidate genes from the Rf3 and Rf4 regions in male sterile (A) and restorer (R) lines showed 2–3 bp indels and a few substitutions in the Rf3 region and indels of 327 and 106 bp in the Rf4 region respectively. The marker system identified in the present study was validated in 212 restorers and 34 maintainers along with earlier reported markers for fertility restoration of WA-CMS. Together DRCG-RF4-14 and DRCG-RF4-8 for the Rf4 locus and DRRM-RF3-5/DRRM-RF3-10 for the Rf3 locus showed a maximum efficiency of 92 % for identification of restorers. SF21 TheSCARECROWgene's role in asymmetric cell divisions in rice plants 2003 The Plant Journal BioScience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan, Asymmetric cell division is one of the most important mechanisms in the diversification of cell function and fate. In Arabidopsis, SCARECROW (SCR) is essential for the asymmetric division of the cortex/endodermis progenitor cell in the root. To learn more about how SCR is involved in asymmetric division, we analyzed the rice SCR (OsSCR) expression. In the root tip, OsSCR expression was observed in the endodermal cell layer and downregulated in the daughter cortex cell after asymmetric division, just as with Arabidopsis SCR. In leaf primordia, expression of OsSCR was observed in stomatal and ligule formation. In stomatal development, OsSCR was specifically expressed in the stomatal cell files before formation of guard mother cells (GMCs), and then, its expression was localized in GMCs, when the first asymmetric division occurred to generate the GMCs. Before the second asymmetric division of subsidiary mother cells (SMCs), localized OsSCR expression was observed in SMCs in the area close to the GMCs. Before these asymmetric divisions, the localization of OsSCR mRNA in GMC-forming cells and SMCs was observed in the area of the daughter GMC and subsidiary cells. OsSCR expression was also observed in the initiation area of ligule formation, and its downregulation occurred in the inner L2 cells generated by asymmetric division. Based on these observations, we proposed that OsSCR is involved not only in the asymmetric division of the cortex/endodermis progenitor cell but also during stomata and ligule formation by establishing the polarization of cytoplasm. OsSHR1 Genetic analysis of starch paste viscosity parameters in glutinous rice (Oryza sativa L.) 2011 Theor Appl Genet Jiangsu Key Laboratory of Crop Genetics and Physiology, Key Laboratory of Plant Functional Genomics, Ministry of Education of China, Agricultural College of Yangzhou University, 12 East Wenhui Road, Jiangsu, 225009, People's Republic of China. Starch paste viscosity plays an important role in estimating the cooking, eating, and processing quality of rice. The inheritance of starch paste viscosity in glutinous rice remains undefined. In the present study, 118 glutinous rice accessions were collected, and the genotypes of 17 starch synthesis-related genes (SSRG) were analyzed by using 43 gene-specific molecular markers. Association analysis indicated that 10 of 17 SSRGs were involved in controlling the rapid visco analyzer (RVA) profile parameters. Among these, the PUL gene was identified to play an important role in control of peak viscosity (PKV), hot paste viscosity (HPV), cool paste viscosity (CPV), breakdown viscosity (BDV), peak time (PeT), and paste temperature (PaT) in glutinous rice. Other SSRGs involved only a few RVA profile parameters. Furthermore, interactions between SSRGs were found being responsible for PeT, PaT, and BDV. Some of the RVA parameters, including PKV, HPV, CPV, CSV, and PaT, were mainly governed by single SSRG, whereas other parameters, such as BDV, SBV, and PeT, were controlled by a few SSRGs, functioning cooperatively. Further, three near-isogenic lines (NIL) of a japonica glutinous cv. Suyunuo as genetic background, with PUL, SSIII-1, and SSIII-2 alleles replaced with those of indica cv. Guichao 2, were employed to verify the genetic effects of the various genes, and the results were consistent with those obtained from the association analysis. These findings indicated that starch paste viscosity in glutinous rice had a complex genetic system, and the PUL gene played an important role in determining the RVA profile parameters in glutinous rice. These results provide important information for potentially improving the quality of glutinous rice. SSIIB,SSIIC Genetic diversity and domestication history of African rice (Oryza glaberrima) as inferred from multiple gene sequences 2011 Theor Appl Genet State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Xiangshan, Beijing, 100093, China. Nucleotide variation in 14 unlinked nuclear genes was investigated in species-wide samples of African rice (Oryza glaberrima) and its wild progenitor (O. barthii). Average estimates of nucleotide diversity were extremely low in both species (theta (sil) = 0.0007 for O. glaberrima; theta (sil) = 0.0024 for O. barthii). About 70% less diversity was found in O. glaberrima than in its progenitor O. barthii. Coalescent simulation indicated that such dramatic reduction of nucleotide diversity in African rice could be explained mainly by a severe bottleneck during its domestication. The progenitor of African rice maintained also low genetic diversity, which may be attributed to small effective population size in O. barthii. Self-pollinating would be another factor leading to the unusually low diversity in both species. Genealogical analyses showed that all O. glaberrima accessions formed a strongly supported cluster with seven O. barthii individuals that were sampled exclusively from the proposed domestication centers of African rice. Population structure and principal component analyses found that the O. glaberrima group was homogeneous with no obvious genetic subdivision, in contrast to the heterogeneous O. barthii cluster. These findings support a single domestication origin of African rice in areas of the Upper Niger and Sahelian Rivers. SSIIC Arbuscular mycorrhiza-specific signaling in rice transcends the common symbiosis signaling pathway 2008 Plant Cell Department of Plant Molecular Biology, University of Lausane, 1015 Lausane, Switzerland. Knowledge about signaling in arbuscular mycorrhizal (AM) symbioses is currently restricted to the common symbiosis (SYM) signaling pathway discovered in legumes. This pathway includes calcium as a second messenger and regulates both AM and rhizobial symbioses. Both monocotyledons and dicotyledons form symbiotic associations with AM fungi, and although they differ markedly in the organization of their root systems, the morphology of colonization is similar. To identify and dissect AM-specific signaling in rice (Oryza sativa), we developed molecular phenotyping tools based on gene expression patterns that monitor various steps of AM colonization. These tools were used to distinguish common SYM-dependent and -independent signaling by examining rice mutants of selected putative legume signaling orthologs predicted to be perturbed both upstream (CASTOR and POLLUX) and downstream (CCAMK and CYCLOPS) of the central, calcium-spiking signal. All four mutants displayed impaired AM interactions and altered AM-specific gene expression patterns, therefore demonstrating functional conservation of SYM signaling between distant plant species. In addition, differential gene expression patterns in the mutants provided evidence for AM-specific but SYM-independent signaling in rice and furthermore for unexpected deviations from the SYM pathway downstream of calcium spiking. SYMRK A plant mutase that interconverts UDP-arabinofuranose and UDP-arabinopyranose 2007 Glycobiology Forestry and Forest Products Research Institute, Tsukuba, Ibaraki, Japan. Plant cell walls constitute the bulk of the earth renewable source of energy and are a component in the diet of humans and herbivores. l-Arabinofuranosyl (Araf) residues are a quantifiably important constituent of these walls. Plants use uridine diphosphate (UDP)-l-arabinofuranose (UDP-Araf) to donate Araf residues in the biosynthesis of Araf-containing polysaccharides, proteoglycans, and glycoproteins. However, little is known about the formation of UDP-Araf. We now describe the purification and partial characterization of a rice UDP-arabinopyranose mutase (UAM) that catalyzes the formation of UDP-Araf from UDP-arabinopyranose (UDP-Arap). The reaction is reversible and at thermodynamic equilibrium the pyranose form is favored over the furanose form (90 : 10). Three related proteins that are encoded by rice gene loci Os03g40270, Os04g56520, and Os07g41360 were identified from partial amino acid sequences of UAM. These proteins have >80% sequence identity with polypeptides that are reversibly glycosylated in the presence of UDP-sugars. The rice mutase and two functionally active recombinant mutases were shown to be reversibly glycosylated in the presence of UDP-Glc. The cofactor, flavin-adenine-dinucleotide (FAD), is required for the catalytic activity of UDP-galactose mutases of prokaryotes, fungi, and protozoa. The plant mutases, which do not require a cofactor, must therefore have a different catalytic mechanism. Putative UAM-encoding genes are present in the green algae Chlamydomonas reinhardtii, the moss Physcomitrella patens, the gymnosperm Pinus taeda (loblolly pine), and in numerous dicots and monocots, indicating that UAMs are widespread in green plants. OsUAM1,OsUAM2,OsUAM3 Down-regulation of UDP-arabinopyranose mutase reduces the proportion of arabinofuranose present in rice cell walls 2011 Phytochemistry Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan. Arabinoxylans may account for up to 25% of the mass of grass cell walls. The interactions of these polysaccharides with themselves and with cellulose and lignin is believed to affect the walls physical properties and increase the walls resistance to biochemical conversion to fermentable sugars. Arabinoxylans have a backbone composed of 1,4-linked beta-D-xylosyl residues, some of which are substituted at O-2 or O-3 with single arabinofuranosyl (Araf) residues. The Araf residues are likely transferred from UDP-Araf to the xylan backbone by arabinofuranosyltransferases. UDP-Araf is itself formed from UDP-arabinopyranose (UDP-Arap) by UDP-arabinopyranose mutase (UAM). In this study, RNA interference (RNAi) was used to suppress UAM expression in rice plants and thereby reduce the amounts of UDP-Araf available for cell wall synthesis. Several of the transgenic plants had reduced proportions of Araf in their walls together with a decrease in the extent of substitution of the xylan backbone, and a reduction of between 25% and 80% in ferulic acid and p-coumaric acid contents of the cell walls. Those transgenic plants with >25% reduction in the amounts of Araf were dwarfed and infertile. OsUAM1,OsUAM2,OsUAM3 UDP-Arabinopyranose Mutase 3 is Required for Pollen Wall Morphogenesis in Rice (Oryza sativa) 2014 Plant Cell Physiol University of Tsukuba, Faculty of Life and Environmental Sciences, Tsukuba, Ibaraki 305-8572, Japan. L-Ara is a one of the main constituents of cell wall polysaccharides including pectic rhamnogalacturonan I (RG-I), glucuronoarabinoxylans (GAX), and other glycoproteins. It is found predominantly in the furanose form rather than in the thermodynamically more stable pyranose form. UDP-arabinofuranose (UDP-Araf), rather than UDP-arabinopyranose (UDP-Arap), is a sugar donor for the biosynthesis of arabinofuranosyl (Araf) residues. UDP-arabinopyranose mutases (UAMs) have been shown to interconvert UDP-L-arabinofuranose (UDP-Araf) and UDP-L-arabinopyranose (UDP-Arap) and are involved in the biosynthesis of polysaccharides including Araf. The UAM gene family has three members in Oryza sativa. Co-expression networks in silico analysis showed that OsUAM3 expression was independent from OsUAM1 and OsUAM2 co-expression networks. OsUAM1 and OsUAM2 were expressed ubiquitously throughout plant development, but OsUAM3 was expressed primarily in reproductive tissue, particularly at the pollen cell wall formation developmental stage. OsUAM3 co-expression networks include pectin catabolic enzymes. To determine the function of OsUAMs in reproductive tissues, we analyzed RNAi-knockdown transformants (OsUAM3-KD) specific for OsUAM3. OsUAM3-KD plants grew normally and showed abnormal phenotypes in reproductive tissues, especially in terms of the pollen cell wall and exine. In addition, we examined modifications of cell wall polysaccharides at the cellular level using antibodies against polysaccharides including Araf. Immunolocalization of arabinan using the LM6 antibody showed low levels of arabinan in OsUAM3-KD pollen grains. Our results suggest that the function of OsUAM3 is important for synthesis of arabinan side chains of rhamnogalacturonan-I and is required for reproductive developmental processes, especially the formation of the cell wall in pollen. OsUAM3 Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR 2006 Biochem Biophys Res Commun Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi 110 021, India. For accurate and reliable gene expression results, normalization of real-time PCR data is required against a control gene, which displays highly uniform expression in living organisms during various phases of development and under different environmental conditions. We assessed the gene expression of 10 frequently used housekeeping genes, including 18S rRNA, 25S rRNA, UBC, UBQ5, UBQ10, ACT11, GAPDH, eEF-1alpha, eIF-4a, and beta-TUB, in a diverse set of 25 rice samples. Their expression varied considerably in different tissue samples analyzed. The expression of UBQ5 and eEF-1alpha was most stable across all the tissue samples examined. However, 18S and 25S rRNA exhibited most stable expression in plants grown under various environmental conditions. Also, a set of two genes was found to be better as control for normalization of the data. The expression of these genes (with more uniform expression) can be used for normalization of real-time PCR results for gene expression studies in a wide variety of samples in rice. UBQ5 Monitoring expression profiles of rice genes under cold, drought, and high-salinity stresses and abscisic acid application using cDNA microarray and RNA gel-blot analyses 2003 Plant Physiol Biological Resources Division, Japan International Research Center for Agricultural Sciences, 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan. To identify cold-, drought-, high-salinity-, and/or abscisic acid (ABA)-inducible genes in rice (Oryza sativa), we prepared a rice cDNA microarray including about 1700 independent cDNAs derived from cDNA libraries prepared from drought-, cold-, and high-salinity-treated rice plants. We confirmed stress-inducible expression of the candidate genes selected by microarray analysis using RNA gel-blot analysis and finally identified a total of 73 genes as stress inducible including 58 novel unreported genes in rice. Among them, 36, 62, 57, and 43 genes were induced by cold, drought, high salinity, and ABA, respectively. We observed a strong association in the expression of stress-responsive genes and found 15 genes that responded to all four treatments. Venn diagram analysis revealed greater cross talk between signaling pathways for drought, ABA, and high-salinity stresses than between signaling pathways for cold and ABA stresses or cold and high-salinity stresses in rice. The rice genome database search enabled us not only to identify possible known cis-acting elements in the promoter regions of several stress-inducible genes but also to expect the existence of novel cis-acting elements involved in stress-responsive gene expression in rice stress-inducible promoters. Comparative analysis of Arabidopsis and rice showed that among the 73 stress-inducible rice genes, 51 already have been reported in Arabidopsis with similar function or gene name. Transcriptome analysis revealed novel stress-inducible genes, suggesting some differences between Arabidopsis and rice in their response to stress. wsi76 XRCC3 promotes homology-directed repair of DNA damage in mammalian cells 1999 Genes Dev Cell Biology Program, Memorial Sloan-Kettering Cancer Center and Cornell University Graduate School of Medical Sciences, New York, New York 10021, USA. Homology-directed repair of DNA damage has recently emerged as a major mechanism for the maintenance of genomic integrity in mammalian cells. The highly conserved strand transferase, Rad51, is expected to be critical for this process. XRCC3 possesses a limited sequence similarity to Rad51 and interacts with it. Using a novel fluorescence-based assay, we demonstrate here that error-free homology-directed repair of DNA double-strand breaks is decreased 25-fold in an XRCC3-deficient hamster cell line and can be restored to wild-type levels through XRCC3 expression. These results establish that XRCC3-mediated homologous recombination can reverse DNA damage that would otherwise be mutagenic or lethal. XRCC3 The rice (Oryza sativa L.) LESION MIMIC RESEMBLING, which encodes an AAA-type ATPase, is implicated in defense response. 2014 Mol Genet Genomics Iwate Biotechnology Research Center, Narita 22-174-4, Kitakami, Iwate, 024-0003, Japan. Lesion mimic mutants (LMMs) provide a useful tool to study defense-related programmed cell death (PCD) in plants. Although a number of LMMs have been identified in multiple species, most of the candidate genes are yet to be isolated. Here, we report the identification and characterization of a novel rice (Oryza sativa L.) lesion mimic resembling (lmr) mutant, and cloning of the corresponding LMR gene. The LMR locus was initially delineated to 1.2 Mb region on chromosome 6, which was further narrowed down to 155-kb using insertions/deletions (INDELs) and cleavage amplified polymorphic sequence markers developed in this study. We sequenced the open reading frames predicted within the candidate genomic region, and identified a G-A base substitution causing a premature translation termination in a gene that encodes an ATPase associated with various cellular activities type (AAA-type) protein. RNA interference transgenic lines with reduced LMR transcripts exhibited the lesion mimic phenotype similar to that of lmr plants. Furthermore, expression of the wild-type LMR in the mutant background complemented the lesion phenotype, confirming that the mutation identified in LMR is responsible for the mutant phenotype. The pathogenesis-related (PR) genes PBZ1 and PR1 were induced in lmr, which also showed enhanced resistance to rice blast (Magnaporthe oryzae) and bacterial blight (Xanthomonas oryzae pv. oryzae), suggesting LMR is a negative regulator of cell death in rice. The identification of lmr and cloning of the corresponding LMR gene provide an additional resource for the study of PCD in plants. LMR Agribiotechnology: Blue-sky rice 2014 Nature Rice is a staple food, but production is not keeping pace with the rise in global population. So scientists are dreaming big and aiming high to change the future for this crucial grain. None Yield: The search for the rice of the future 2014 Nature Scientists are hoping to make the world's most successful crop even better. None Domestication: The birth of rice 2014 Nature From a wild Asian grass to a refined crop that is the staple diet of half the world's population, the domestication of Oryza sativa spans centuries, but the grain's ancestry is hotly contested. None Biotechnology: Against the grain 2014 Nature Golden rice could help to end a nutritional crisis -- but only if researchers can overcome some daunting technical and political hurdles. None Perspective: Time to unleash rice 2014 Nature Corporate inefficiency and government meddling are curbing production of the vital crop in the countries that need it most, says Robert Zeigler. None Rice by the numbers: A good grain 2014 Nature Millions of people around the world rely on rice as the bulk of their daily diet. This snapshot of the crop's production, consumption and trade shows an overall surplus, but population growth in future decades may affect the situation, writes Emily Elert. None Agriculture: The next frontier 2014 Nature Africa's newfound taste for an old grain has experienced problems -- drought, low yields and costly imports. But new projects are driving the continent towards self-sufficiency. None Contamination: The toxic side of rice 2014 Nature Around the world, researchers are looking for ways to rid rice of a troublesome companion. None Bacterial effector modulation of host E3 ligase activity suppresses PAMP-triggered immunity in rice 2014 Nat Commun Kazuya Ishikawa, Koji Yamaguchi, Kazuaki Sakamoto, Satomi Yoshimura, Kento Inoue & Tsutomu Kawasaki OsPUB44 Expression of an endo-(1,3;1,4)-beta-glucanase in response to wounding, methyl jasmonate, abscisic acid and ethephon in rice seedlings 2009 J Plant Physiol National Agricultural Research Center for Hokkaido Region, 1 Hitsujigaoka, Toyohira-ku, Sapporo 062-8555, Japan. We isolated two rice endo-(1,3;1,4)-beta-glucanase genes, denoted OsEGL1 and OsEGL2, which encoded proteins that shared 64% amino acid sequence identity. Both the OsEGL1 and OsEGL2 genes were successfully expressed in Escherichia coli to produce functional proteins. Purified OsEGL1 and OsEGL2 proteins hydrolyzed (1,3;1,4)-beta-glucans, but not (1,3;1,6)-beta-linked or (1,3)-beta-linked glucopolysaccharides nor carboxymethyl cellulose, similar to previously characterized grass endo-(1,3;1,4)-beta-glucanases. RNA blot analysis revealed that the OsEGL1 gene is expressed constitutively not only in young roots of rice seedlings, but also in mature roots of adult rice plants. Little or no expression of the OsEGL2 gene was observed in all tissues or treatments tested, but database and RT-PCR analysis indicated it is expressed in ripening panicle. In rice seedling leaves, OsEGL1 gene expression significantly increased in response to methyl jasmonate, abscisic acid, ethephon and mechanical wounding. Mechanical wounding also increased the leaf elongation rate in rice seedlings by 16% relative to that of control seedlings at day 4 after treatment. The increase in the leaf elongation rate of rice seedlings treated under mechanical wounding was concomitant with an increase in OsEGL1 expression levels in seedling leaves. OsEGL1 2'-Deoxymugineic acid promotes growth of rice (Oryza sativa L.) by orchestrating iron and nitrate uptake processes under high pH conditions 2014 Plant J Division of Integral Biomolecular Function, Bioorganic Research Institute, Suntory Foundation for Life Sciences, 1-1-1 Wakayamadai, Shimamoto, Mishima, Osaka, 618-8503, Japan. Poaceae plants release 2'-deoxymugineic acid (DMA) and related phytosiderophores to chelate iron (Fe), which often exists as insoluble Fe(III) in the rhizosphere, especially under high pH conditions. Although the molecular mechanisms behind the biosynthesis and secretion of DMA have been studied extensively, little is known about whether DMA has biological roles other than chelating Fe in vivo. Here, we demonstrated that hydroponic cultures of rice (Oryza sativa) seedlings show almost complete restoration in shoot height and soil-plant analysis development (SPAD) values after treatment with 3 to 30 M DMA at high pH (pH 8.0), compared to untreated control seedlings at normal pH (pH 5.8). These changes were accompanied by selective accumulation of Fe over other metals. While this enhanced growth was evident under high pH conditions, DMA application also enhanced seedling growth under normal pH conditions in which Fe was fairly accessible. Microarray and qRT-PCR analyses revealed that exogenous DMA application attenuated the increased expression levels of various genes related to Fe transport and accumulation. Surprisingly, despite the preferential utilization of ammonium over nitrate as a nitrogen source by rice, DMA application also increased nitrate reductase activity and the expression of genes encoding high-affinity nitrate transporters and nitrate reductases, all of which were otherwise considerably lower under high pH conditions. These data suggest that exogenous DMA not only plays an important role in facilitating the uptake of environmental Fe, but also orchestrates Fe and nitrate assimilation for optimal growth under high pH conditions. None Multi-gene knockout utilizing off-target mutations of the CRISPR/Cas9 system in rice 2014 Plant Cell Physiol Plant Genome Engineering Research Unit, Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease 9 system (CRISPR/Cas9) has been demonstrated to be a robust genome engineering tool in a variety of organisms including plants. However, it has been shown that the CRISPR/Cas9 system cleaves genomic DNA sequences containing mismatches to the guide RNA strand. We expected that this low specificity could be exploited to induce multi-homeologous and paralogous gene knockouts. In the case of polyploid plants, simultaneous modification of multiple homeologous genes, i.e., genes with similar but not identical DNA sequences, is often needed to obtain a desired phenotype. Even in diploid plants, disruption of multi-paralogous genes, which have functional redundancy, is often needed. To validate the applicability of the CRISPR/Cas9 system to target mutagenesis of paralogous genes in rice, we designed a single-guide RNA (sgRNA) that recognized 20 base-pair (bp) sequences of cyclin dependent kinase (CDK) B2 as an on-target locus. These 20bps possess similarity to other rice CDK genes (CDKA1, CDKA2, CDKB1) with different numbers of mismatches. We analyzed mutations in these 4 CDK genes in plants regenerated from Cas9/sgRNA-transformed calli and revealed that single, double and triple mutants of CDKA2, CDKB1 and CDKB2 can be created by a single sgRNA. cdc2Os-1|CDKA1,cdc2Os-2|CDKA2,CDKB1;1|CDKB1 OsARF16 Is Involved in Cytokinin-Mediated Inhibition of Phosphate Transport and Phosphate Signaling in Rice (Oryza sativa L.) 2014 PLoS One College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China. Plant responses to phytohormone stimuli are the most important biological features for plants to survive in a complex environment. Cytokinin regulates growth and nutrient homeostasis, such as the phosphate (Pi) starvation response and Pi uptake in plants. However, the mechanisms underlying how cytokinin participates in Pi uptake and Pi signaling are largely unknown. In this study, we found that OsARF16 is required for the cytokinin response and is involved in the negative regulation of Pi uptake and Pi signaling by cytokinin. PRINCIPAL FINDINGS: The mutant osarf16 showed an obvious resistance to exogenous cytokinin treatment and the expression level of the OsARF16 gene was considerably up-regulated by cytokinin. Cytokinin (6-BA) application suppressed Pi uptake and the Pi starvation response in wild-type Nipponbare (NIP) and all these responses were compromised in the osarf16 mutant. Our data showed that cytokinin inhibits the transport of Pi from the roots to the shoots and that OsARF16 is involved in this process. The Pi content in the osarf16 mutant was much higher than in NIP under 6-BA treatment. The expressions of PHOSPHATE TRANSPORTER1 (PHT1) genes, phosphate (Pi) starvation-induced (PSI) genes and purple PAPase genes were higher in the osarf16 mutant than in NIP under cytokinin treatment. CONCLUSION: Our results revealed a new biological function for OsARF16 in the cytokinin-mediated inhibition of Pi uptake and Pi signaling in rice. OsARF16 Plasma membrane receptor-like kinase leaf panicle 2 acts downstream of the DROUGHT AND SALT TOLERANCE transcription factor to regulate drought sensitivity in rice 2014 J Exp Bot National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China Drought is a recurring climatic hazard that reduces the crop yields. To avoid the negative effects of drought on crop production, extensive efforts have been devoted to investigating the complex mechanisms of gene expression and signal transduction during drought stress. Receptor-like kinases (RLKs) play important roles in perceiving extracellular stimuli and activating downstream signalling responses. The rice genome contains >1100 RLK genes, of which only two are reported to function in drought stress. A leucine-rich repeat (LRR)-RLK gene named Leaf Panicle 2 (LP2) was previously found to be strongly expressed in leaves and other photosynthetic tissues, but its function remains unclear. In the present study, it was shown that the expression of LP2 was down-regulated by drought and abscisic acid (ABA). Transgenic plants overexpressing LP2 accumulated less H2O2, had more open stomata in leaves, and showed hypersensitivity to drought stress. Further investigation revealed that transcription of LP2 was directly regulated by the zinc finger transcription factor DROUGHT AND SALT TOLERANCE (DST). In addition, LP2 was identified as a functional kinase localized to the plasma membrane and interacted with the drought-responsive aquaporin proteins OsPIP1; 1, OsPIP1; 3, and OsPIP2; 3. Thus, the findings provided evidence that the LRR-RLK LP2, transcriptionally regulated by the drought-related transcription factor DST, served as a negative regulator in drought response. OsPIP2;3 Genome-wide transcriptome profiles of rice hybrids and their parents 2014 Int J Mol Sci China National Rice Research Institute, Hangzhou 310006, China. Heterosis is a widely studied phenomenon in several plant species. However, its genetic basis still remains to be elucidated. In this study, we used RNA-seq data from two rice genotypes and their reciprocal hybrids, and used a combination of transcriptome profiling and allele-specific expression analysis to identify genes that are differentially expressed in the hybrids and their parents or expressed in an allele-specific manner. The differentially expressed genes (DEGs) were identified by a pairwise comparison of the four genotypes. Detailed annotation of DEGs suggested that these genes showed enrichment in some gene ontology categories, and they tend to have tissue-specific expression patterns compared to all genes. A total of 1033 (10.24%) of 10,195 genes with informative single nucleotide polymorphism (SNPs) were identified as ASE genes. These allele-specific expessed (ASE) genes showed a broader expression breadth suggesting that they function in diverse developmental stages. Among 1033 ASE genes, we also identified 45 ASE transcription factors belonging to 17 transcription factor families. These ASE transcription factors may act in trans to regulate gene expression in filial 1 (F1) hybrids. Our analyses provide a comprehensive transcriptome profile of rice hybrids and their parents, and would be a useful resource for the rice research community. None Functional inactivation of UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) induces early leaf senescence and defence responses in rice 2014 J Exp Bot State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Hubei, 430072, China. Plant leaf senescence and defence responses are important biological processes, but the molecular mechanisms involved are not well understood. This study identified a new rice mutant, spotted leaf 29 (spl29). The SPL29 gene was identified by map-based cloning, and SPL29 was confirmed as UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) by enzymatic analysis. The mutant spl29 lacks UAP activity. The biological phenotypes for which UAP is responsible have not previously been reported in plants. The spl29 mutant displayed early leaf senescence, confirmed by chlorophyll loss and photosystem II decline as physiological indicators, chloroplast degradation as a cellular characteristic, and both upregulation of senescence transcription factors and senescence-associated genes, and downregulation of photosynthesis-related genes, as molecular evidence. Defence responses were induced in the spl29 mutant, shown by enhanced resistance to bacterial blight inoculation and upregulation of defence response genes. Reactive oxygen species, including O2 - and H2O2, accumulated in spl29 plants; there was also increased malondialdehyde content. Enhanced superoxide dismutase activity combined with normal catalase activity in spl29 could be responsible for H2O2 accumulation. The plant hormones jasmonic acid and abscisic acid also accumulated in spl29 plants. ROS and plant hormones probably play important roles in early leaf senescence and defence responses in the spl29 mutant. Based on these findings, it is suggested that UAP1 is involved in regulating leaf senescence and defence responses in rice. OsUAP1|SPL29 Genetic mechanisms underlying yield potential in the rice high-yielding cultivar Takanari, based on reciprocal chromosome segment substitution lines 2014 BMC Plant Biol NARO Institute of Crop Science, Tsukuba 305-8518, Ibaraki, Japan BackgroundIncreasing rice yield potential is a major objective in rice breeding programs, given the need for meeting the demands of population growth, especially in Asia. Genetic analysis using genomic information and high-yielding cultivars can facilitate understanding of the genetic mechanisms underlying rice yield potential. Chromosome segment substitution lines (CSSLs) are a powerful tool for the detection and precise mapping of quantitative trait loci (QTLs) that have both large and small effects. In addition, reciprocal CSSLs developed in both parental cultivar backgrounds may be appropriate for evaluating gene activity, as a single factor or in epistatic interactions.ResultsWe developed reciprocal CSSLs derived from a cross between Takanari (one of the most productive indica cultivars) and a leading japonica cultivar, Koshihikari; both the cultivars were developed in Japan. Forty-one CSSLs covered most of the Takanari genome in the Koshihikari background and 39 CSSLs covered the Koshihikari genome in the Takanari background. Using the reciprocal CSSLs, we conducted yield trials under canopy conditions in paddy fields. While no CSSLs significantly exceeded the recurrent parent cultivar in yield, genetic analysis detected 48 and 47 QTLs for yield and its components in the Koshihikari and Takanari backgrounds, respectively. A number of QTLs showed a trade-off, in which the allele with increased sink-size traits (spikelet number per panicle or per square meter) was associated with decreased ripening percentage or 1000-grain weight. These results indicate that increased sink size is not sufficient to increase rice yield in both backgrounds. In addition, most QTLs were detected in either one of the two genetic backgrounds, suggesting that these loci may be under epistatic control with other gene(s).ConclusionsWe demonstrated that the reciprocal CSSLs are a useful tool for understanding the genetic mechanisms underlying yield potential in the high-yielding rice cultivar Takanari. Our results suggest that sink-size QTLs in combination with QTLs for source strength or translocation capacity, as well as careful attention to epistatic interactions, are necessary for increasing rice yield. Thus, our findings provide a foundation for developing rice cultivars with higher yield potential in future breeding programs. None SAD1, an RNA polymerase I subunit A34.5 of rice, interacts with Mediator and controls various aspects of plant development 2014 Plant J Graduate School of Agriculture and Life Sciences, University of Tokyo, Yayoi, Bunkyo, Tokyo, 113-8657, Japan. The DWARF14 (D14) gene of rice functions within the signaling pathway of strigolactones, a group of plant hormones that inhibits shoot branching. We isolated a recessive mutant named super apical dormant (sad1-1) from a suppressor screen of d14-1. The growth of tillers (vegetative shoot branches) is suppressed in both the d14-1 sad1-1 double mutant and the sad1-1 single mutant. In addition, the sad1-1 mutant shows pleiotropic defects throughout development. SAD1 encodes an ortholog of RPA34.5, a subunit of RNA polymerase I (Pol I). Consequently, the level of ribosomal RNA (rRNA) is severely reduced in the sad1-1 mutant. These results indicate that proper ribosome function is a prerequisite for normal development in plants. The Arabidopsis ortholog of SAD1 was previously isolated as a Mediator-interacting protein. Here we show that SAD1 interacts physically with the Mediator complex through a direct binding with OsMED4, a component of the middle module of the Mediator complex in rice. It is known that Mediator interacts with Pol II, which transcribes mRNAs and functions as a central regulator of transcription. This study indicates a novel aspect of Mediator function in Pol I-controlled rRNA transcription. TFIIF2 and RPC53 are counterparts of RPA34.5 in Pol II and Pol III, respectively. We demonstrate that the rice orthologs of these proteins also interact with OsMED4. Our results suggest that interaction with MED4 in the Mediator complex is a common feature of the three types of RNA polymerases. SAD1,OsMED4,SAD1L,OsTFIIF2-1,OsTFIIF2-2,OsRPC53 Analysis of Genetic Diversity and Population Structure of Rice Germplasm from North-Eastern Region of India and Development of a Core Germplasm Set. 2014 PLoS One Division of Genomic Resources, National Bureau of Plant Genetic Resources, New Delhi, 110 012, India. The North-Eastern region (NER) of India, comprising of Arunachal Pradesh, Assam, Manipur, Meghalaya, Mizoram, Nagaland and Tripura, is a hot spot for genetic diversity and the most probable origin of rice. North-east rice collections are known to possess various agronomically important traits like biotic and abiotic stress tolerance, unique grain and cooking quality. The genetic diversity and associated population structure of 6,984 rice accessions, originating from NER, were assessed using 36 genome wide unlinked single nucleotide polymorphism (SNP) markers distributed across the 12 rice chromosomes. All of the 36 SNP loci were polymorphic and bi-allelic, contained five types of base substitutions and together produced nine types of alleles. The polymorphic information content (PIC) ranged from 0.004 for Tripura to 0.375 for Manipur and major allele frequency ranged from 0.50 for Assam to 0.99 for Tripura. Heterozygosity ranged from 0.002 in Nagaland to 0.42 in Mizoram and gene diversity ranged from 0.006 in Arunachal Pradesh to 0.50 in Manipur. The genetic relatedness among the rice accessions was evaluated using an unrooted phylogenetic tree analysis, which grouped all accessions into three major clusters. For determining population structure, populations K=1 to K=20 were tested and population K=3 was present in all the states, with the exception of Meghalaya and Manipur where, K=5 and K=4 populations were present, respectively. Principal Coordinate Analysis (PCoA) showed that accessions were distributed according to their population structure. AMOVA analysis showed that, maximum diversity was partitioned at the individual accession level (73% for Nagaland, 58% for Arunachal Pradesh and 57% for Tripura). Using POWERCORE software, a core set of 701 accessions was obtained, which accounted for approximately 10% of the total NE India collections, representing 99.9% of the allelic diversity. The rice core set developed will be a valuable resource for future genomic studies and crop improvement strategies. None Estimating rice chlorophyll content and leaf nitrogen concentration with a digital still color camera under natural light. 2014 Plant Methods Institute of Soil Science, Chinese Academy of Sciences, 71 East Beijing Road, Nanjing, 210008 PR China. The color of crop leaves is closely correlated with nitrogen (N) status and can be quantified easily with a digital still color camera and image processing software. The establishment of the relationship between image color indices and N status under natural light is important for crop monitoring and N diagnosis in the field. In our study, a digital still color camera was used to take pictures of the canopies of 6 rice (Oryza sativa L.) cultivars with N treatments ranging from 0 to 315kgNha(-1) in the field under sunny and overcast conditions in 2010 and 2011, respectively.Significant correlations were observed between SPAD readings, leaf N concentration (LNC) and 13 image color indices calculated from digital camera images using three color models: RGB, widely used additive color model; HSV, a cylindrical-coordinate similar to the human perception of colors; and the L (*) a (*) b (*) system of the International Commission on Illumination. Among these color indices, the index b (*) , which represents the visual perception of yellow-blue chroma, has the closest linear relationship with SPAD reading and LNC. However, the relationships between LNC and color indices were affected by the developmental phase. Linear regression models were used to predict LNC and SPAD from color indices and phasic development. After that, the models were validated with independent data. Generally, acceptable performance and prediction were found between the color index b (*) , SPAD reading and LNC with different cultivars and sampling dates under different natural light conditions.Our study showed that digital color image analysis could be a simple method of assessing rice N status under natural light conditions for different cultivars and different developmental stages. None Mutational bias is the driving force for shaping the synonymous codon usage pattern of alternatively spliced genes in rice (Oryza sativa L.). 2014 Mol Genet Genomics Department of Agronomy, and the Key Laboratory for Quality Improvement of Agricultural Products of Zhejiang Province, College of Agriculture and Food Science, Zhejiang A & F University, Lin'an, 311300, Zhejiang, China, liuqp@zafu.edu.cn. Alternative splicing plays important roles in diverse aspects of plant development, metabolism, and stress responses. However, the regulatory mechanisms of alternative splicing of genes still remain incompletely elucidated, especially in plants. In this study, the synonymous codon usage pattern of alternatively spliced (AS) genes in rice was firstly explored using the combination of correspondence analysis (CA), internal CA, correlation and ANOVA analyses. The results show that alternatively and non-alternatively spliced (non-AS) genes have similar tendency for overall codon usage, but exhibit significant difference in 58 out of 64 codons. AS and non-AS genes are both under strong purifying selection, but the former ones have significant lower mutation rate and are prone to be enriched towards the chromosomal ends. In the group of AS genes, the variability in synonymous codon usage between genes is mainly due to the variations in GC content, CDS length, as well as gene functions. Mutational bias that accounts for 25.85% of the total codon usage variability plays a major role in shaping the codon usage pattern of AS genes. In contrast, no obvious evidence is found for the contributions of translational selection, AS types, the conservation of AS events, and numbers of AS variants to the codon usage divergence between AS genes. These findings may be useful for further understanding the mechanisms of origination, differentiation and regulation of alternatively spliced genes in plants. None Structure of the OsSERK2 leucine-rich repeat extracellular domain. 2014 Acta Crystallogr D Biol Crystallogr Physical Biosciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA. Somatic embryogenesis receptor kinases (SERKs) are leucine-rich repeat (LRR)-containing integral membrane receptors that are involved in the regulation of development and immune responses in plants. It has recently been shown that rice SERK2 (OsSERK2) is essential for XA21-mediated resistance to the pathogen Xanthomonas oryzae pv. oryzae. OsSERK2 is also required for the BRI1-mediated, FLS2-mediated and EFR-mediated responses to brassinosteroids, flagellin and elongation factor Tu (EF-Tu), respectively. Here, crystal structures of the LRR domains of OsSERK2 and a D128N OsSERK2 mutant, expressed as hagfish variable lymphocyte receptor (VLR) fusions, are reported. These structures suggest that the aspartate mutation does not generate any significant conformational change in the protein, but instead leads to an altered interaction with partner receptors. OsSERK2 Characterization and expression analyses of two plastidic enolase genes in rice. 2014 Biosci Biotechnol Biochem a Graduate School of Agricultural Science , Kobe University , Kobe , Japan. To verify the presence of enolase related to the chloroplastic glycolysis in rice, database search was carried out and identified seven putative enolase genes in the rice genome. Among them, OsEno1 and OsEno3 encode long proteins with N-terminal extensions. GFP protein fusions of these N-terminal extensions were both targeted to plastids of onion epidermal cell. Promoter::GUS analysis showed that OsEno3 was highly expressed in young developing leaves, but its expression was drastically decreased during leaf development and greening. On the other hand, the expression of OsEno1 was low and detected in limited portions such as leaf sheath at the tiller base. Recombinant OsEno1 protein showed enolase activity with a pH optimum at pH 8.0, whereas OsEno3 did not exhibit detectable activity. Although it remains obscure if OsEno3 encodes a functional enolase in vivo, our results demonstrate that the entire glycolytic pathway does not operate in rice chloroplasts. OsEno1,OsEno2,OsEno3,OsEno5,OsEno6,OsEno7 Coordinated regulation of photosynthesis in rice increases yield and tolerance to environmental stress. 2014 Nat Commun Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia 24061, USA. Plants capture solar energy and atmospheric carbon dioxide (CO2) through photosynthesis, which is the primary component of crop yield, and needs to be increased considerably to meet the growing global demand for food. Environmental stresses, which are increasing with climate change, adversely affect photosynthetic carbon metabolism (PCM) and limit yield of cereals such as rice (Oryza sativa) that feeds half the world. To study the regulation of photosynthesis, we developed a rice gene regulatory network and identified a transcription factor HYR (HIGHER YIELD RICE) associated with PCM, which on expression in rice enhances photosynthesis under multiple environmental conditions, determining a morpho-physiological programme leading to higher grain yield under normal, drought and high-temperature stress conditions. We show HYR is a master regulator, directly activating photosynthesis genes, cascades of transcription factors and other downstream genes involved in PCM and yield stability under drought and high-temperature environmental stress conditions. HYR,GASR2 Genome-wide association study of blast resistance in indica rice 2014 BMC Plant Biol BackgroundRice blast disease is one of the most serious and recurrent problems in rice-growing regions worldwide. Most resistance genes were identified by linkage mapping using genetic populations. We extensively examined 16 rice blast strains and a further genome-wide association study based on genotyping 0.8 million single nucleotide polymorphism variants across 366 diverse indica accessions.ResultsTotally, thirty associated loci were identified. The strongest signal (Chr11_6526998, P =1.17 X 10 -17) was located within the gene Os11g0225100, one of the rice Pia-blast resistance gene. Another association signal (Chr11_30606558) was detected around the QTL Pif. Our study identified the gene Os11g0704100, a disease resistance protein containing nucleotide binding site-leucine rich repeat domain, as the main candidate gene of Pif. In order to explore the potential mechanism underlying the blast resistance, we further examined a locus in chromosome 12, which was associated with CH149 (P =7.53 X 10 -15). The genes, Os12g0424700 and Os12g0427000, both described as kinase-like domain containing protein, were presumed to be required for the full function of this locus. Furthermore, we found some association on chromosome 3, in which it has not been reported any loci associated with rice blast resistance. In addition, we identified novel functional candidate genes, which might participate in the resistance regulation.ConclusionsThis work provides the basis of further study of the potential function of these candidate genes. A subset of true associations would be weakly associated with outcome in any given GWAS; therefore, large-scale replication is necessary to confirm our results. Future research will focus on validating the effects of these candidate genes and their functional variants using genetic transformation and transferred DNA insertion mutant screens, to verify that these genes engender resistance to blast disease in rice. None Isolation of a novel lodging resistance QTL gene involved in strigolactone signaling and its pyramiding with a QTL gene involved in another mechanism 2014 Mol Plant Bioscience and Biotechnology Center, Nagoya University, Nagoya, Japan. Lodging has been a major roadblock to attaining increased crop productivity. In an attempt to understand the mechanism for culm strength in rice, we isolated an effective quantitative trait loci (QTL), STRONG CULM3 (SCM3), the causal gene of which is identical to rice TEOSINTE BRANCHED1 (OsTB1), a gene previously reported to positively control strigolactone (SL) signaling. A near-isogenic line (NIL) carrying SCM3 showed enhanced culm strength and increased spikelet number despite the expected decrease in tiller number, indicating that SL also has a positive role in enhancing culm strength and spikelet number. We produced a pyramiding line carrying SCM3 and SCM2, another QTL encoding APO1 involved in panicle development. The NIL-SCM2+SCM3 showed a much stronger culm than NIL-SCM2 and NIL-SCM3 and an increased spikelet number caused by the additive effect of these QTLs. We discuss the importance of utilizing suitable alleles of these STRONG CULM QTLs without inducing detrimental traits for breeding. OsTB1|FC1|SCM3 Fine mapping of the qLOP2 and qPSR2-1 loci associated with chilling stress tolerance of wild rice seedlings. 2014 Theor Appl Genet College of Bioscience and Biotechnology and Jiangsu Key Laboratory of Crop Genetics and Physiology, Yangzhou University, Yangzhou, Jiangsu Province, China, yzxiaoning@163.com. Using leaf osmotic potential and plant survival rate as chilling-tolerant trait indices, we identified two major quantitative trait loci qLOP2 and qPSR2 - 1 (39.3-kb region) and Os02g0677300 as the cold-inducible gene for these loci. Chilling stress tolerance (CST) at the seedling stage is an important trait affecting rice production in temperate climate and high-altitude areas. To identify quantitative trait loci (QTLs) associated with CST, a mapping population consisting of 151 BC2F1 plants was constructed by using chilling-tolerant Dongxiang wild rice (Oryza rufipogon Griff.) as a donor parent and chilling-sensitive indica as a recurrent parent. With leaf osmotic potential (LOP) and plant survival rate (PSR) as chilling-tolerant trait indexes, two major QTLs, qLOP2 (LOD=3.8) and qPSR2-1 (LOD=3.3), were detected on the long arm of chromosome 2 by composite interval mapping method in QTL Cartographer software, which explained 10.1 and 12.3% of the phenotypic variation, respectively. In R/QTL analyzed result, their major effects were also confirmed. Using molecular marker RM318 and RM106, qLOP2 and qPSR2-1 have been introgressed into chilling-sensitive varieties (93-11 and Yuefeng) by marker-assisted selection procedure (MAS), which resulted in 16 BC5F3 BILs that chilling tolerance have significantly enhanced compare with wild-type parents (P<0.01). Therefore, two large segregating populations of 11,326 BC4F2 and 8,642 BC4F3 were developed to fine mapping of qLOP2 and qPSR2-1. Lastly, they were dissected to a 39.3-kb candidate region between marker RM221 and RS8. Expression and sequence analysis results indicated that Os02g0677300 was a cold-inducible gene for these loci. Our study provides novel alleles for improving rice CST by MAS and contributes to the understanding of its molecular mechanisms. None Transcripts of two ent-copalyl diphosphate synthase genes differentially localize in rice plants according to their distinct biological roles 2014 J Exp Bot Department of Bioresource Engineering, Yamagata University, Yamagata 997-8555, Japan Gibberellins (GAs) are diterpenoid phytohormones that regulate various aspects of plant growth. Tetracyclic hydrocarbon ent-kaurene is a biosynthetic intermediate of GAs, and is converted from geranylgeranyl diphosphate, a common precursor of diterpenoids, via ent-copalyl diphosphate (ent-CDP) through successive cyclization reactions catalysed by two distinct diterpene synthases, ent-CDP synthase and ent-kaurene synthase. Rice (Oryza sativa L.) has two ent-CDP synthase genes, OsCPS1 and OsCPS2. It has been thought that OsCPS1 participates in GA biosynthesis, while OsCPS2 participates in the biosynthesis of phytoalexins, phytocassanes A-E, and oryzalexins A-F. It has been shown previously that loss-of-function OsCPS1 mutants display a severe dwarf phenotype caused by GA deficiency despite possessing another ent-CDP synthase gene, OsCPS2. Here, experiments were performed to account for the non-redundant biological function of OsCPS1 and OsCPS2. Quantitative reverse transcription-PCR (qRT-PCR) analysis showed that OsCPS2 transcript levels were drastically lower than those of OsCPS1 in the basal parts, including the meristem of the second-leaf sheaths of rice seedlings. qRT-PCR results using tissue samples prepared by laser microdissection suggested that OsCPS1 transcripts mainly localized in vascular bundle tissues, similar to Arabidopsis CPS, which is responsible for GA biosynthesis, whereas OsCPS2 transcripts mainly localized in epidermal cells that address environmental stressors such as pathogen attack. Furthermore, the OsCPS2 transgene under regulation of the OsCPS1 promoter complemented the dwarf phenotype of an OsCPS1 mutant, oscps1-1. The results indicate that transcripts of the two ent-CDP synthase genes differentially localize in rice plants according to their distinct biological roles, OsCPS1 for growth and OsCPS2 for defence. OsCPS|OsCPS1,OsCPS2|OsCyc2 Biosynthetic relationship between C28-brassinosteroids and C29-brassinosteroids in rice (Oryza sativa) seedlings. 2014 Phytochemistry Department of Life Science, Chung-Ang University, Seoul 156-756, Republic of Korea. A crude enzyme solution was prepared from young rice seedlings, and the metabolism of C29-brassinosteroids identified from the seedlings was examined. When 28-homoteasterone was added as a substrate, 28-homotyphasterol, teasterone, and 26-nor-28-homoteasterone were characterized as enzyme products by GC–MS/SIM analysis. With 28-homotyphasterol, 28-homoteasterone, typhasterol, 28-homocastasterone, and 26-nor-28-homotyphasterol were formed and identified as products. When 28-homocastasterone was used, castasterone and 26-nor-28-homocastasterone were identified as products. Together with the reduced biological activity of C29-brassinosteroids and their metabolites in the rice lamina inclination assay, these metabolic studies suggest a biosynthetic sequence, 28-homoteasterone ↔ 28-homotyphasterol → 28-homocastasterone for C29-brassinosteroid biosynthesis is connected to the biosynthetic sequence teasterone ↔ typhasterol → castasterone for C28-brassinosteroids by C-28 demethylation, i.e., in order to increase biological activity in the rice plant. Additionally, the C29-brassinosteroids seem to bio-degrade their C-26 demethylated C28-brassinosteroid analogs to reduce brassinosteroid activity in planta. In conclusion, the biosynthesis of C29-brassinosteroids is a likely alternative route to the biologically-active brassinosteroid, castasterone, in rice. None Plastid DNA insertions in plant nuclear genomes: the sites, abundance and ages, and a predicted promoter analysis. 2014 Funct Integr Genomics Heilongjiang Provincial Key University Laboratory of Agricultural Functional Genes, College of Life Science, Northeast Agricultural University, Harbin, 150030, China. The transfer of plastid DNA sequences into plant nuclear genomes plays an important role in the genomic evolution of plants. The abundance of nuclear-localized plastid DNA (nupDNA) correlates positively with nuclear genome size, but the genetic content of nupDNA remains unknown. In this mini review, we analyzed the number of nuclear-localized plastid gene fragments in known plant genomic data. Our analysis suggests that nupDNAs are abundant in plant nuclear genomes and can include multiple complete copies of protein-coding plastid genes. Mutated nuclear copies of plastid genes contained synonymous and nonsynonymous substitutions. We estimated the age of the nupDNAs based on the time when each integration occurred, which was calculated by comparing the nucleotide substitution rates of the nupDNAs and their respective plastid genes. These data suggest that there are two distinct age distribution patterns for nupDNAs in plants, and Oryza sativa and Zea mays were found to contain a very high proportion of young nupDNAs. Expressed sequence tags and predicted promoters of nupDNAs were identified, revealing that certain nuclear-localized plastid genes may be functional and that some have undergone positive natural selection pressure. None A novel method for the simultaneous analysis of seven biothiols in rice (Oryza sativa L.) using hydrophilic interaction chromatography coupled with electrospray tandem mass spectrometry. 2014 J Chromatogr B Analyt Technol Biomed Life Sci China National Rice Research Institute, Hangzhou 310006, China; Laboratory of Quality & Safety Risk Assessment for Rice (Hangzhou), Ministry of Agriculture, Hangzhou 310006, China. Analysis of biothiols is still problematic, due to their high polarity, oxidation sensitivity and time-consuming sample preparation. In this paper, a direct, rapid and sensitive method was developed for simultaneous quantification of unbound cysteine (Cys), glutathione (GSH) and phytochelatins (PCs) in rice leaf, stem and root samples by hydrophilic interaction chromatography coupled with electrospray tandem mass spectrometry (HILIC–MS/MS). Homogenized samples were extracted with water containing 50 mM dithiothreitol, without derivatization and further clean-up, and the extracts were injected directly onto an Xbridge Amide-HILIC column (3.5 μm, 150 mm × 2.1 mm i.d.). The best chromatographic separation and MS sensitivity was achieved using a linear gradient elution with 10 mM aqueous ammonium formate and acetonitrile as the mobile phase. In MS/MS mode the detection limit (S/N ≥ 3) of seven biothiols was 3–105 nM. Good linearities were observed (r > 0.995) with linear dynamic range at least over three orders of magnitude. Recoveries for most analytes were within the range of 77–128%, with relative standard deviations less than 18.2%. The intra-day precision (n = 7) was 6.1–11.7%, and the inter-day precision over 15 d (n = 15) was 8.5–16.3% for all biothiols. The optimized HILIC–MS/MS method was applied to study the influence of different cadmium (Cd) concentrations (0, 1 and 50 μM) on contents of Cys, GSH and PC2–6 in rice tissue. With increasing Cd concentrations in nutrient solutions, contents of PC2–4 in rice roots increased but contents of Cys and GSH decreased. Contents of PC2–4 in both rice leafs and stems increased markedly at high dose Cd (50 μM) treatment compared with controls, compared with low Cd concentrations (1 μM). However, both PC5 and PC6 were not detected throughout the stress experiment. None Development of disease-resistant rice using regulatory components of induced disease resistance. 2014 Front Plant Sci Disease Resistant Crops Research Unit, Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences Tsukuba, Japan. Infectious diseases cause huge crop losses annually. In response to pathogen attacks, plants activate defense systems that are mediated through various signaling pathways. The salicylic acid (SA) signaling pathway is the most powerful of these pathways. Several regulatory components of the SA signaling pathway have been identified, and are potential targets for genetic manipulation of plants' disease resistance. However, the resistance associated with these regulatory components is often accompanied by fitness costs; that is, negative effects on plant growth and crop yield. Chemical defense inducers, such as benzothiadiazole and probenazole, act on the SA pathway and induce strong resistance to various pathogens without major fitness costs, owing to their 'priming effect.' Studies on how benzothiadiazole induces disease resistance in rice have identified WRKY45, a key transcription factor in the branched SA pathway, and OsNPR1/NH1. Rice plants overexpressing WRKY45 were extremely resistant to rice blast disease caused by the fungus Magnaporthe oryzae and bacterial leaf blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo), the two major rice diseases. Disease resistance is often accompanied by fitness costs; however, WRKY45 overexpression imposed relatively small fitness costs on rice because of its priming effect. This priming effect was similar to that of chemical defense inducers, although the fitness costs were amplified by some environmental factors. WRKY45 is degraded by the ubiquitin-proteasome system, and the dual role of this degradation partly explains the priming effect. The synergistic interaction between SA and cytokinin signaling that activates WRKY45 also likely contributes to the priming effect. With a main focus on these studies, I review the current knowledge of SA-pathway-dependent defense in rice by comparing it with that in Arabidopsis, and discuss potential strategies to develop disease-resistant rice using signaling components. None SNP-Seek database of SNPs derived from 3000 rice genomes. 2014 Nucleic Acids Res T.T.Chang Genetic Resources Center, IRRI, Los Baos, Laguna 4031, Philippines n.alexandrov@irri.org zhanggengyun@genomics.cn lizhikang@caas.cn. We have identified about 20 million rice SNPs by aligning reads from the 3000 rice genomes project with the Nipponbare genome. The SNPs and allele information are organized into a SNP-Seek system (http://www.oryzasnp.org/iric-portal/), which consists of Oracle database having a total number of rows with SNP genotypes close to 60 billion (20 M SNPs × 3 K rice lines) and web interface for convenient querying. The database allows quick retrieving of SNP alleles for all varieties in a given genome region, finding different alleles from predefined varieties and querying basic passport and morphological phenotypic information about sequenced rice lines. SNPs can be visualized together with the gene structures in JBrowse genome browser. Evolutionary relationships between rice varieties can be explored using phylogenetic trees or multidimensional scaling plots. None Differential expression of microRNAs by arsenate and arsenite stress in natural accessions of rice. 2014 Metallomics CSIR-National Botanical Research Institute, Council of Scientific and Industrial Research (CSIR-NBRI), Rana Pratap Marg, Lucknow-226001, India. prabodht@hotmail.com prabodht@nbri.res.in. Arsenic (As) contamination of rice (Oryza sativa) imposes a serious threat to human health worldwide. Understanding the molecular mechanisms of As transport and accumulation in rice may provide promising solutions to the problem. MicroRNAs (miRNAs) are a novel class of short, endogenous, non-coding small RNA molecules involved in a wide variety of biological processes such as organ polarity, morphogenesis, floral transition, hormone signalling and adaptation to environment. In the past, a few studies led to the identification of differentially expressed miRNAs in rice in response to arsenite (As(iii)) stress. However, studies related to differential miRNA expression involving natural rice accessions exposed to different species of As have not been carried out. Such studies are required to identify As-species responsive miRNAs in different rice accessions. In this study, we have carried out miRNA profiling in contrasting As accumulating rice accessions using miRNA Array. We report identification of differentially expressed miRNAs in contrasting As accumulating rice cultivars in response to As(iii) (25 uM) and As(v) (50 uM) stress. A significant up-regulation in expression was observed among members of the miR396, miR399, miR408, miR528, miR1861, miR2102 and miR2907 families in response to As(iii) and As(v) stress in both cultivars. In addition, members of the miR164, miR171, miR395, miR529, miR820, miR1432 and miR1846 families were down-regulated. The differentially expressed miRNAs were subjected to validation of expression and bioinformatic analyses to predict and categorise the key miRNAs and their target genes involved in As stress. Analysis suggests that As-species and rice accession specific miRNA might be responsible for the differential response of contrasting rice accessions towards As(iii) and As(v) stress. Study of the proximal promoter sequences of the As-responsive miRNAs suggests that these identified miRNAs contain metal-responsive cis-acting motifs and other elicitor and hormonal related motifs. Our study suggests a miRNA-dependent regulatory mechanism during As species-specific stress in different rice accessions. Further analysis based on results obtained will be helpful in dissecting the molecular mechanism behind As responses in different rice accessions. None Metabolic and co-expression network-based analyses associated with nitrate response in rice 2014 BMC Genomics BACKGROUND: Understanding gene expression and metabolic re-programming that occur in response to limiting nitrogen (N) conditions in crop plants is crucial for the ongoing progress towards the development of varieties with improved nitrogen use efficiency (NUE). To unravel new details on the molecular and metabolic responses to N availability in a major food crop, we conducted analyses on a weighted gene co-expression network and metabolic profile data obtained from leaves and roots of rice plants adapted to sufficient and limiting N as well as after shifting them to limiting (reduction) and sufficient (induction) N conditions. RESULTS: A gene co-expression network representing clusters of rice genes with similar expression patterns across four nitrogen conditions and two tissue types was generated. The resulting 18 clusters were analyzed for enrichment of significant gene ontology (GO) terms. Four clusters exhibited significant correlation with limiting and reducing nitrate treatments. Among the identified enriched GO terms, those related to nucleoside/nucleotide, purine and ATP binding, defense response, sugar/carbohydrate binding, protein kinase activities, cell-death and cell wall enzymatic activity are enriched. Although a subset of functional categories are more broadly associated with the response of rice organs to limiting N and N reduction, our analyses suggest that N reduction elicits a response distinguishable from that to adaptation to limiting N, particularly in leaves. This observation is further supported by metabolic profiling which shows that several compounds in leaves change proportionally to the nitrate level (i.e. higher in sufficient N vs. imitimg N) and respond with even higher levels when the nitrate level is reduced. Notably, these compounds are directly involved in N assimilation, transport, and storage (glutamine, asparagine, glutamate and allantoin) and extend to most amino acids. Based on these data, we hypothesize that plants respond by rapidly mobilizing stored vacuolar nitrate when N deficit is perceived, and that the response likely involves phosphorylation signal cascades and transcriptional regulation. CONCLUSIONS: The co-expression network analysis and metabolic profiling performed in rice pinpoint the relevance of signal transduction components and regulation of N mobilization in response to limiting N conditions and deepen our understanding of N responses and N use in crops. None Efficient use of energy in anoxia-tolerant plants with focus on germinating rice seedlings. 2014 New Phytol Department of Biological Sciences, Faculty of Science, Macquarie University, Sydney, 2109, NSW, Australia. I. II. III. IV. V. References SUMMARY: Anoxia tolerance in plants is distinguished by direction of the sparse supply of energy to processes crucial to cell maintenance and sometimes to growth, as in rice seedlings. In anoxic rice coleoptiles energy is used to synthesise proteins, take up K(+) , synthesise cell walls and lipids, and in cell maintenance. Maintenance of electrochemical H(+) gradients across the tonoplast and plasma membrane is crucial for solute compartmentation and thus survival. These gradients sustain some H(+) -solute cotransport and regulate cytoplasmic pH. Pyrophosphate (PPi ), the alternative energy donor to ATP, allows direction of energy to the vacuolar H(+) -PPi ase, sustaining H(+) gradients across the tonoplast. When energy production is critically low, operation of a biochemical pHstat allows H(+) -solute cotransport across plasma membranes to continue for at least for 18h. In active (e.g. growing) cells, PPi produced during substantial polymer synthesis allows conversion of PPi to ATP by PPi -phosphofructokinase (PFK). In quiescent cells with little polymer synthesis and associated PPi formation, the PPi required by the vacuolar H(+) -PPi ase and UDPG pyrophosphorylase involved in sucrose mobilisation via sucrose synthase might be produced by conversion of ATP to PPi through reversible glycolytic enzymes, presumably pyruvate orthophosphate dikinase. These hypotheses need testing with species characterised by contrasting anoxia tolerance. None ALT1, a Snf2 Family Chromatin Remodeling ATPase, Negatively Regulates Alkaline Tolerance through Enhanced Defense against Oxidative Stress in Rice 2014 PLoS One State Key Laboratory of Plant Genomics and National Plant Gene Research Center, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China; University of Chinese Academy of Sciences, Beijing, China. Alkaline salt stress adversely affects rice growth, productivity and grain quality. However, the mechanism underlying this process remains elusive. We characterized here an alkaline tolerant mutant, alt1 in rice. Map-based cloning revealed that alt1 harbors a mutation in a chromatin remodeling ATPase gene. ALT1-RNAi transgenic plants under different genetic background mimicked the alt1 phenotype, exhibiting tolerance to alkaline stress in a transcript dosage-dependent manner. The predicted ALT1 protein belonged to the Ris1 subgroup of the Snf2 family and was localized in the nucleus, and transcription of ALT1 was transiently suppressed after alkaline treatment. Although the absorption of several metal ions maintained well in the mutant under alkaline stress, expression level of the genes involved in metal ions homeostasis was not altered in the alt1 mutant. Classification of differentially expressed abiotic stress related genes, as revealed by microarray analysis, found that the majority (50/78) were involved in ROS production, ROS scavenging, and DNA repair. This finding was further confirmed by that alt1 exhibited lower levels of H2O2 under alkaline stress and tolerance to methyl viologen treatment. Taken together, these results suggest that ALT1 negatively functions in alkaline tolerance mainly through the defense against oxidative damage, and provide a potential two-step strategy for improving the tolerance of rice plants to alkaline stress. ALT1 Whole genome de novo assemblies of three divergent strains of rice, Oryza sativa , document novel gene space of aus and indica 2014 Genome Biol None Control of grain protein contents through SEMIDWARF1 mutant alleles: sd1 increases the grain protein content in Dee-geo-woo-gen but not in Reimei. 2014 Mol Genet Genomics Hokuriku Research Center, NARO Agricultural Research Center, 1-2-1, Inada, Joetsu, Niigata, 943-0193, Japan A new possibility for genetic control of the protein content of rice grains was suggested by the allele differences of the SEMIDWARF1 (SD1) mutation. Two quantitative trait loci-qPROT1 and qPROT12-were found on chromosomes 1 and 12, respectively, using backcrossed inbred lines of Sasanishiki/Habataki//Sasanishiki///Sasanishiki. One of them, qPROT1, increased almost all grain proteins instead of only certain proteins in the recessive Habataki allele. Fine mapping of qPROT1 revealed that two gene candidates-Os01g0883800 and Os01g0883900-were included in this region. Os01g0883800 encoded Gibberellin 20 oxidase 2 as well as SD1, the dwarf gene used in the so-called 'Green Revolution'. Mutant analyses as well as sequencing analysis using the semi-dwarf mutant cultivars Dee-geo-woo-gen and Calrose 76 revealed that the sd1 mutant showed significantly higher grain protein contents than their corresponding wild-type cultivars, strongly suggesting that the high protein contents were caused by sd1 mutation. However, the sd1 mutant Reimei did not have high grain protein contents. It is possible to control the grain protein content and column length separately by selecting for sd1 alleles. From this finding, the genetic control of grain protein content, as well as the column length of rice cultivars, might be possible. This ability might be useful to improve rice nutrition, particularly in areas where the introduction of semi-dwarf cultivars is not advanced. sd1|GA20ox2 The polycomb group gene EMF2B is essential for maintenance of floral meristem determinacy in rice 2014 Plant J Plant Biology Department, University of California Davis, 1 Shields Ave, Davis, CA 95616, USA. Polycomb Repressive Complex 2 (PRC2) represses the transcriptional activity of target genes through trimethylation of lysine 27 of histone H3. The functions of plant PRC2 have been chiefly described in Arabidopsis, but specific functions in other plant species, especially cereals, are still largely unknown. Here we characterize mutants in the rice EMF2B gene, an ortholog of the Arabidopsis EMBRYONIC FLOWER2 (EMF2) gene. Loss of EMF2B in rice results in complete sterility, and mutant flowers have severe floral organ defects and indeterminacy that resemble loss-of-function mutants in E-function floral organ specification genes. Transcriptome analysis identified the E-function genes OsMADS1, OsMADS6 and OsMADS34 as differentially expressed in the emf2b mutant compared with wild type. OsMADS1 and OsMADS6, known to be required for meristem determinacy in rice, have reduced expression in the emf2b mutant, whereas OsMADS34 which interacts genetically with OsMADS1 was ectopically expressed. Chromatin immunoprecipitation for H3K27me3 followed by quantitative (q)RT-PCR showed that all three genes are presumptive targets of PRC2 in the meristem. Therefore, in rice, and possibly other cereals, PRC2 appears to play a major role in floral meristem determinacy through modulation of the expression of E-function genes. OsEMF2b Metabolome-genome-wide association study dissects genetic architecture for generating natural variation in rice secondary metabolism 2014 Plant J RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Japan; Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, Japan. Plants produce structurally diverse secondary (specialized) metabolites to increase their fitness for survival under adverse environments. Several bioactive compounds for new drugs have been identified through screening of plant extracts. In this study, genome-wide association studies (GWAS) were conducted to investigate the genetic architecture behind the natural variation of rice secondary metabolites. GWAS using the metabolome data of 175 rice accessions successfully identified 323 associations among 143 single nucleotide polymorphisms (SNPs) and 89 metabolites. The data analysis highlighted that levels of many metabolites are tightly associated with a small number of strong quantitative trait loci (QTLs). The tight association may be a mechanism generating strains with distinct metabolic composition through the crossing of two different strains. The results indicate that one plant species produces more diverse phytochemicals than previously expected, and plants still contain many useful compounds for human applications. None The receptor kinase CERK1 has dual functions in symbiosis and immunity signalling 2014 Plant J National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China. The establishment of symbiotic interactions between mycorrhizal fungi, rhizobial bacteria and their legume hosts involves a common symbiosis signalling pathway. This signalling pathway is activated by Nod factors produced by rhizobia and these are recognised by the Nod factor receptors NFR1/LYK3 and NFR5/NFP. Mycorrhizal fungi produce lipochitooligosaccharides (LCOs) similar to Nod factors, as well as short-chain chitin oligomers (CO4/5), implying commonalities in signalling during mycorrhizal and rhizobial associations. Here we show that NFR1/LYK3, but not NFR5/NFP, is required for the establishment of the mycorrhizal interaction in legumes. NFR1/LYK3 is necessary for the recognition of mycorrhizal fungi and the activation of the symbiosis signalling pathway leading to induction of calcium oscillations and gene expression. Chitin oligosaccharides also act as microbe associated molecular patterns that promote plant immunity via similar LysM receptor-like kinases. CERK1 in rice has the highest homology to NFR1 and we show that this gene is also necessary for the establishment of the mycorrhizal interaction as well as for resistance to the rice blast fungus. Our results demonstrate that NFR1/LYK3/OsCERK1 represents a common receptor for chitooligosaccharide-based signals produced by mycorrhizal fungi, rhizobial bacteria (in legumes) and fungal pathogens. It would appear that mycorrhizal recognition has been conserved in multiple receptors across plant species, but additional diversification in certain plant species has defined other signals that this class of receptors can perceive. NFR1|LYK3|OsCERK1 Functional reconstitution of a rice aquaporin water channel, PIP1;1, by a micro-batchwise methodology 2014 Plant Physiol Biochem Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari "Aldo Moro", Bari, Italy. Assessing the selectivity, regulation and physiological relevance of aquaporin membrane channels (AQPs) requires structural and functional studies of wild type and modified proteins. In particular, when characterizing their transport properties, reconstitution in isolation from native cellular or membrane processes is of pivotal importance. Here, we describe rapid and efficient incorporation of OsPIP1;1, a rice AQP, in liposomes at analytical scale. PIP1;1 was produced as a histidine-tagged form, 10His-OsPIP1;1, in an Escherichia coli-based expression system. The recombinant protein was purified by affinity chromatography and incorporated into liposomes by a micro-batchwise technology using egg-yolk phospholipids and the non-polar Amberlite resin. PIP1;1 proteoliposomes and control empty liposomes had good size homogeneity as seen by quasi-elastic light scattering and electron microscopy analyses. By stopped-flow light scattering, indicating correct protein folding of the incorporated protein, the osmotic water permeability exhibited by the PIP1;1 proteoliposomes was markedly higher than empty liposomes. Functional reconstitution of OsPIP1;1 was further confirmed by the low Arrhenius activation energy (3.37 kcal/mol) and sensitivity to HgCl2, a known AQP blocker, of the PIP1;1-mediated osmotic water conductance. These results provide a valuable contribution in fully elucidating the regulation and water-conducting property of PIP1;1, an AQP that needs to hetero-multimerize with AQPs of the PIP2 subgroup to reach the native plasma membrane and play its role. The micro-batchwise methodology is suitable for the functional reconstitution of whichever AQPs and other membrane transport proteins. OsPIP1;1|RWC1 Association mapping for important agronomic traits in core collection of rice (Oryza sativa L.) with SSR markers. 2014 PLoS One Fail Mining elite genes within rice landraces is of importance for the improvement of cultivated rice. An association mapping for 12 agronomic traits was carried out using a core collection of rice consisting of 150 landraces (Panel 1) with 274 simple sequence repeat (SSR) markers, and the mapping results were further verified using a Chinese national rice micro-core collection (Panel 2) and a collection from a global molecular breeding program (Panel 3). Our results showed that (1) 76 significant (P<0.05) trait-marker associations were detected using mixed linear model (MLM) within Panel 1 in two years, among which 32% were identical with previously mapped QTLs, and 11 significant associations had >10% explained ratio of genetic variation; (2) A total of seven aforementioned trait-marker associations were verified within Panel 2 and 3 when using a general linear model (GLM) and 55 SSR markers of the 76 significant trait-marker associations. However, no significant trait-marker association was found to be identical within three panels when using the MLM model; (3) several desirable alleles of the loci which showed significant trait-marker associations were identified. The research provided important information for further mining these elite genes within rice landraces and using them for rice breeding. None Traits and QTLs for development of dry direct-seeded rainfed rice varieties. 2014 J Exp Bot Fail The development of rice varieties for dry direct-seeded conditions can be accelerated by selecting suitable traits. In the present investigation, traits hypothesized to be important for direct-seeded conditions in rainfed systems, including seedling emergence, early vegetative vigour, nutrient uptake, nodal root number, and root hair length and density, were characterized to study the genetic control of these traits and their relationship with grain yield under seedling- and reproductive-stage drought stress. Two BC2F4 mapping populations derived from crosses of Aus276, a drought-tolerant aus variety, with MTU1010 and IR64, high-yielding indica mega-varieties, were developed and studied to identify quantitative trait loci (QTLs) that showed large and consistent effects. A total of 26 QTLs associated with 23 traits and 20 QTLs associated with 13 traits were mapped in the Aus276/3*IR64 and Aus276/3*MTU1010 populations, respectively. qGY6.1, qGY10.1, qGY1.1, and qEVV9.1 were found to be effective in both populations under a wide range of conditions. QTLs for several seedling-stage traits co-located with QTLs for grain yield, including early vegetative vigour and root hair length. On chromosome 5, several QTLs for nutrient uptake co-located with QTLs for root hair density and nematode gall rating. Six lines were selected from both populations based on grain yield and the presence of QTLs, and these lines typically showed improved seedling-stage traits (nodal root number, dry shoot weight, and root hair length and density). The co-located QTLs identified here can be used in research aimed at increasing the yield and adaptability of rainfed rice to direct-seeded conditions. None Using metabolomic approaches to explore chemical diversity in rice. 2014 Mol Plant RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa 230-0045, Japan Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology (PRESTO), Kawaguchi, Saitama 332-0012, Japan miyako.kusano@riken.jp. Rice (Oryza sativa) is an excellent resource, it comprises 25% of the total caloric intake of the world's population and rice plants yield many types of bioactive compounds. To determine the number of metabolites in rice and their chemical diversity, the metabolite composition of cultivated rice has been investigated with analytical techniques such as mass spectrometry (MS) and/or nuclear magnetic resonance spectroscopy and rice metabolite databases have been constructed. This review summarizes current knowledge on metabolites in rice including sugars, amino and organic acids, aromatic compounds, and phytohormones detected by gas chromatography-MS, liquid chromatography-MS, and capillary electrophoresis-MS. The biological properties and the activities of polar and non-polar metabolites produced by rice plants are also presented. Challenges in the estimation of the structure(s) of unknown metabolites by metabolomic approaches are introduced and discussed. Lastly, examples are presented of the successful application of metabolite profiling of rice to characterize gene(s) potentially critical for improving its quality by combining metabolite quantitative trait loci analysis, and to identify potential metabolite biomarkers that play a critical role when rice is grown under abiotic stress conditions. None Genome-wide association analyses provide genetic and biochemical insights into natural variation in rice metabolism. 2014 Nat Genet 1] National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China. [2]. Plant metabolites are important to world food security in terms of maintaining sustainable yield and providing food with enriched phytonutrients. Here we report comprehensive profiling of 840 metabolites and a further metabolic genome-wide association study based on ~6.4 million SNPs obtained from 529 diverse accessions of Oryza sativa. We identified hundreds of common variants influencing numerous secondary metabolites with large effects at high resolution. We observed substantial heterogeneity in the natural variation of metabolites and their underlying genetic architectures among different subspecies of rice. Data mining identified 36 candidate genes modulating levels of metabolites that are of potential physiological and nutritional importance. As a proof of concept, we functionally identified or annotated five candidate genes influencing metabolic traits. Our study provides insights into the genetic and biochemical bases of rice metabolome variation and can be used as a powerful complementary tool to classical phenotypic trait mapping for rice improvement. None Overexpression of a NF-YC transcription factor from bermudagrass confers tolerance to drought and salinity in transgenic rice. 2014 Plant Biotechnol J State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China. Nuclear factor Y (NF-Y) is a ubiquitous transcription factor formed by three distinct subunits, namely NF-YA, NF-YB and NF-YC. A stress-responsive cDNA of NF-YC (Cdt-NF-YC1) was isolated from triploid bermudagrass (Cynodon dactylon Cynodon transvaalensis), and its role in abiotic stress tolerance was investigated in this study. Cdt-NF-YC1 transcript was detected in all vegetative tissues with higher levels being observed in roots. Transcription of Cdt-NF-YC1 in leaves was induced by dehydration, salinity, and treatments with abscisic acid (ABA), hydrogen peroxide (H2 O2 ) or nitric oxide (NO), but not altered by cold. The dehydration- or salt-induced transcription of Cdt-NF-YC1 was blocked by inhibitor of ABA synthesis and scavenger of H2 O2 or NO, indicating that ABA, H2 O2 and NO were involved in the dehydration- and salt-induced transcription of Cdt-NF-YC1. Overexpression of Cdt-NF-YC1 resulted in elevated tolerance to drought and salt stress and increased sensitivity to ABA in transgenic rice. Transcript levels of stress/ABA responsive genes (OsLEA3, OsRAB16A, OsLIP9 and OsP5CS1), ABA synthesis and signalling genes (OsNCED3 and OsABI2), and ABA-independent genes (OsDREB1A, OsDREB1B and OsDREB2A) were substantially higher in transgenic rice than in wild-type plants. The results suggested that that Cdt-NF-YC1 is a good candidate gene to increase drought and salinity tolerance in transgenic rice through modulating gene regulation in both ABA-dependent and ABA-independent pathways. None QTLs for tolerance of drought and breeding for tolerance of abiotic and biotic stress: an integrated approach. 2014 PLoS One International Rice Research Institute (IRRI), Los Baos, Laguna, Philippines. The coupling of biotic and abiotic stresses leads to high yield losses in rainfed rice (Oryza sativa L.) growing areas. While several studies target these stresses independently, breeding strategies to combat multiple stresses seldom exist. This study reports an integrated strategy that combines QTL mapping and phenotypic selection to develop rice lines with high grain yield (GY) under drought stress and non-stress conditions, and tolerance of rice blast.A blast-tolerant BC2F3-derived population was developed from the cross of tropical japonica cultivar Moroberekan (blast- and drought-tolerant) and high-yielding indica variety Swarna (blast- and drought-susceptible) through phenotypic selection for blast tolerance at the BC2F2 generation. The population was studied for segregation distortion patterns and QTLs for GY under drought were identified along with study of epistatic interactions for the trait.Segregation distortion, in favour of Moroberekan, was observed at 50 of the 59 loci. Majority of these marker loci co-localized with known QTLs for blast tolerance or NBS-LRR disease resistance genes. Despite the presence of segregation distortion, high variation for DTF, PH and GY was observed and several QTLs were identified under drought stress and non-stress conditions for the three traits. Epistatic interactions were also detected for GY which explained a large proportion of phenotypic variance observed in the population.This strategy allowed us to identify QTLs for GY along with rapid development of high-yielding purelines tolerant to blast and drought with considerably reduced efforts. Apart from this, it also allowed us to study the effects of the selection cycle for blast tolerance. The developed lines were screened at IRRI and in the target environment, and drought and blast tolerant lines with high yield were identified. With tolerance to two major stresses and high yield potential, these lines may provide yield stability in rainfed rice areas. None Comparative analyses of C4 and C3 photosynthesis in developing leaves of maize and rice. 2014 Nat Biotechnol The Donald Danforth Plant Science Center, St. Louis, Missouri, USA. C4 and C3 photosynthesis differ in the efficiency with which they consume water and nitrogen. Engineering traits of the more efficient C4 photosynthesis into C3 crops could substantially increase crop yields in hot, arid conditions. To identify differences between C4 and C3 photosynthetic mechanisms, we profiled metabolites and gene expression in the developing leaves of Zea mays (maize), a C4 plant, and Oryza sativa (rice), a C3 plant, using a statistical method named the unified developmental model (UDM). Candidate cis-regulatory elements and transcription factors that might regulate photosynthesis were identified, together with differences between C4 and C3 nitrogen and carbon metabolism. The UDM algorithms could be applied to analyze and compare development in other species. These data sets together with community viewers to access and mine them provide a resource for photosynthetic research that will inform efforts to engineer improvements in carbon fixation in economically valuable grass crops. None Integrated analysis of miRNA and mRNA expression profiles in response to Cd exposure in rice seedlings. 2014 BMC Genomics Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China. cychen@isa.ac.cn. Independent transcriptome profile analyses of miRNAs or mRNAs under conditions of cadmium (Cd) stress have been widely reported in plants. However, a combined analysis of sRNA sequencing expression data with miRNA target expression data to infer the relative activities of miRNAs that regulate gene expression changes resulting from Cd stress has not been reported in rice. To elucidate the roles played by miRNAs in the regulation of changes in gene expression in response to Cd stress in rice (Oryza sativa L.), we simultaneously characterized changes in the miRNA and mRNA profiles following treatment with Cd.A total of 163 miRNAs and 2,574 mRNAs were identified to be differentially expressed under Cd stress, and the changes in the gene expression profile in the shoot were distinct from those in the root. At the miRNA level, 141 known miRNAs belonging to 48 families, and 39 known miRNAs in 23 families were identified to be differentially expressed in the root and shoot, respectively. In addition, we identified eight new miRNA candidates from the root and five from the shoot that were differentially expressed in response to Cd treatment. For the mRNAs, we identified 1,044 genes in the root and 448 genes in the shoot that were up-regulated, while 572 and 645 genes were down-regulated in the root and shoot, respectively. GO and KEGG enrichment analyses showed that genes encoding secondary, metabolite synthases, signaling molecules, and ABC transporters were significantly enriched in the root, while only ribosomal protein and carotenoid biosynthesis genes were significantly enriched in the shoot. Then 10 known miRNA-mRNA interaction pairs and six new candidate ones, that showed the opposite expression patterns, were identified by aligning our two datasets against online databases and by using the UEA sRNA toolkit respectively.This study is the first to use high throughput DNA sequencing to simultaneously detect changes in miRNA and mRNA expression patterns in the root and shoot in response to Cd treatment. These integrated high-throughput expression data provide a valuable resource to examine global genome expression changes in response to Cd treatment and how these are regulated by miRNAs. None Differential regulation of proteins in rice (Oryza sativa L.) under iron deficiency. 2014 Plant Cell Rep Key Laboratory of Crop Physiology and Ecology in Southern China, College of Agriculture, Ministry of Agricultural University, Nanjing Agricultural University, Nanjing, China. Sixty-three proteins were identified to be differentially accumulated due to iron deficiency in shoot and root. The importance of these proteins alterations on shoot physiology is discussed. Iron (Fe) is an essential micronutrient for plant growth and its accumulation affects the quality of edible plant organs. To investigate the adaptive mechanism of a Chinese rice variety grown under iron deficiency, proteins differentially accumulated in leaves and roots of Yangdao 6, an indica cultivar, under Fe deficiency growth condition, were profiled using a two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS). The accumulations of seventy-three proteins were detected to be increased or decreased upon iron deficiency, and sixty-three of them were successfully identified. Among the sixty-three proteins, a total of forty proteins were identified in rice leaves, and twenty-three proteins were in roots. Most of these proteins are involved in photosynthesis, C metabolism, oxidative stress, Adenosine triphosphate synthesis, cell growth or signal transduction. The results provide a comprehensive way to understand, at the level of proteins, the adaptive mechanism used by rice shoots and roots under iron deficiency. None Statistical Inference of Selection and Divergence of the Rice Blast Resistance Gene Pi-ta 2014 G3 (Bethesda) Department of Mathematical Sciences, University of Nevada, Las Vegas, Nevada. The resistance gene Pi-ta has been effectively used to control rice blast disease, but some populations of cultivated and wild rice have evolved resistance. Insights into the evolutionary processes that led to this resistance during crop domestication may be inferred from the population history of domesticated and wild rice strains. In this study, we applied a recently developed statistical method, time-dependent Poisson random field model, to examine the evolution of the Pi-ta gene in cultivated and weedy rice. Our study suggests that the Pi-ta gene may have more recently introgressed into cultivated rice, indica and japonica, and U.S. weedy rice from the wild species, O. rufipogon. In addition, the Pi-ta gene is under positive selection in japonica, tropical japonica, U.S. cultivars and U.S. weedy rice. We also found that sequences of two domains of the Pi-ta gene, the nucleotide binding site and leucine-rich repeat domain, are highly conserved among all rice accessions examined. Our results provide a valuable analytical tool for understanding the evolution of disease resistance genes in crop plants. Pita|Pi-4a Rapid diversification of five Oryza AA genomes associated with rice adaptation. 2014 Proc Natl Acad Sci U S A Plant Germplasm and Genomics Center, Germplasm Bank of Wild Species in Southwest China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China; University of the Chinese Academy of Sciences, Beijing 100039, China; Comparative genomic analyses among closely related species can greatly enhance our understanding of plant gene and genome evolution. We report de novo-assembled AA-genome sequences for Oryza nivara, Oryza glaberrima, Oryza barthii, Oryza glumaepatula, and Oryza meridionalis. Our analyses reveal massive levels of genomic structural variation, including segmental duplication and rapid gene family turnover, with particularly high instability in defense-related genes. We show, on a genomic scale, how lineage-specific expansion or contraction of gene families has led to their morphological and reproductive diversification, thus enlightening the evolutionary process of speciation and adaptation. Despite strong purifying selective pressures on most Oryza genes, we documented a large number of positively selected genes, especially those genes involved in flower development, reproduction, and resistance-related processes. These diversifying genes are expected to have played key roles in adaptations to their ecological niches in Asia, South America, Africa and Australia. Extensive variation in noncoding RNA gene numbers, function enrichment, and rates of sequence divergence might also help account for the different genetic adaptations of these rice species. Collectively, these resources provide new opportunities for evolutionary genomics, numerous insights into recent speciation, a valuable database of functional variation for crop improvement, and tools for efficient conservation of wild rice germplasm. None Rice cytochrome P450 MAX1 homologs catalyze distinct steps in strigolactone biosynthesis 2014 Nat Chem Biol Laboratory of Plant Physiology, Wageningen University, Wageningen, the Netherlands. Strigolactones (SLs) are a class of phytohormones and rhizosphere signaling compounds with high structural diversity. Three enzymes, carotenoid isomerase DWARF27 and carotenoid cleavage dioxygenases CCD7 and CCD8, were previously shown to convert all-trans-beta-carotene to carlactone (CL), the SL precursor. However, how CL is metabolized to SLs has remained elusive. Here, by reconstituting the SL biosynthetic pathway in Nicotiana benthamiana, we show that a rice homolog of Arabidopsis MORE AXILLARY GROWTH 1 (MAX1), encodes a cytochrome P450 CYP711 subfamily member that acts as a CL oxidase to stereoselectively convert CL into ent-2'-epi-5-deoxystrigol (B-C lactone ring formation), the presumed precursor of rice SLs. A protein encoded by a second rice MAX1 homolog then catalyzes the conversion of ent-2'-epi-5-deoxystrigol to orobanchol. We therefore report that two members of CYP711 enzymes can catalyze two distinct steps in SL biosynthesis, identifying the first enzymes involved in B-C ring closure and a subsequent structural diversification step of SLs. SLB1|OsMAX1 MYB80 homologues in Arabidopsis, cotton and Brassica: regulation and functional conservation in tapetal and pollen development 2014 BMC Plant Biol BackgroundThe Arabidopsis AtMYB80 transcription factor regulates genes involved in pollen development and controls the timing of tapetal programmed cell death (PCD). Downregulation of AtMYB80 expression precedes tapetal degradation. Inhibition of AtMYB80 expression results in complete male sterility. Full-length AtMYB80 homologs have been isolated in wheat, rice, barley and canola (C genome).ResultsThe complete sequences of MYB80 genes from the Brassica. napus (A gene), B. juncea (A gene), B. oleracea (C gene) and the two orthologs from cotton (Gossypium hirsutum) were determined. The deduced amino acid sequences possess a highly conserved MYB domain, 44-amino acid region and 18-amino acid C-terminal sequence. The cotton MYB80 protein can fully restore fertility of the atmyb80 mutant, while removal of the 44 amino acid sequence abolishes its function. Two conserved MYB cis-elements in the AtMYB80 promoter are required for downregulation of MYB80 expression in anthers, apparently via negative auto-regulation. In cotton, tapetal degradation occurs at a slightly earlier stage of anther development than in Arabidopsis, consistent with an earlier increase and subsequent downregulation in GhMYB80 expression. The MYB80 homologs fused with the EAR repressor motif have been shown to induce male sterility in Arabidopsis. Constructs were designed to maximize the level of male sterility.Conclusions MYB80 genes are conserved in structure and function in all monocot and dicot species so far examined. Expression patterns of MYB80 in these species are also highly similar. The reversible male sterility system developed in Arabidopsis by manipulating MYB80 expression should be applicable to all major crops. OsMYB80 OsLOL1, a C2C2-type zinc finger protein, interacts with OsbZIP58 to promote seed germination through the modulation of gibberellin biosynthesis in Oryza sativa 2014 Plant J State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. Seed germination is a key developmental process in the plant life cycle that is influenced by various environmental cues and phytohormones through gene expression and a series of metabolism pathways. In the present study, we investigated a C2C2-type finger protein, OsLOL1, which promotes gibberellin (GA) biosynthesis and affects seed germination in Oryza sativa (rice). We used OsLOL1 antisense and sense transgenic lines to explore OsLOL1 functions. Seed germination timing in antisense plants was restored to wild type when exogenous GA3 was applied. The reduced expression of the GA biosynthesis gene OsKO2 and the accumulation of ent-kaurene were observed during germination in antisense plants. Based on yeast two-hybrid and firefly luciferase complementation analyses, OsLOL1 interacted with the basic leucine zipper protein OsbZIP58. The results from electrophoretic mobility shift and dual-luciferase reporter assays showed that OsbZIP58 binds the G-box cis-element of the OsKO2 promoter and activates LUC reporter gene expression, and that interaction between OsLOL1 and OsbZIP58 activates OsKO2 gene expression. In addition, OsLOL1 decreased SOD1 gene expression and accelerated programmed cell death (PCD) in the aleurone layer of rice grains. These findings demonstrate that the interaction between OsLOL1 and OsbZIP58 influences GA biosynthesis through the activation of OsKO2 via OsbZIP58, thereby stimulating aleurone PCD and seed germination. RISBZ1|OsbZIP58,OsLOL1 Crossover Formation During Rice Meiosis Relies on Interaction of OsMSH4 and OsMSH5. 2014 Genetics State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. MSH4 encodes a MutS protein that plays a specialized role in meiosis. In eukaryotic species, such as budding yeast, mice, Caenorhabditis elegans, and Arabidopsis, msh4 mutants display meiotic defects with a reduced number of chiasmata. Here, we characterized rice MSH4 by map-based cloning. In Osmsh4 mutants, the chiasma frequency was dramatically decreased to 10% of the wild type, but the synaptonemal complex was normally installed. The double mutant analysis showed that in the Osmsh4 Osmsh5 mutant, the reduction of chiasmata was greater than other zmm mutants. This was consistent with the absence of localization for OsZIP4 and OsMER3 in Osmsh4 and suggests an earlier role for OsMSH4 and OsMSH5 than other ZMM proteins where they may be required to stabilize progenitor Holliday junctions. Using yeast two-hybrid and pull-down assays, we verified the direct physical association between OsMSH4 and OsMSH5 and OsMSH5 and HEI10 in plants for the first time. The MSH4-MSH5 heterodimer has been demonstrated in mammals to stabilize the formation of progenitor and double Holliday junctions that may be resolved as crossovers (COs). We propose that OsMSH4 interacts with OsMSH5 to promote formation of the majority of COs in rice. OsMSH4 Brassinosteroid regulates cell elongation by modulating gibberellin metabolism in rice 2014 Plant Cell State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Brassinosteroid (BR) and gibberellin (GA) are two predominant hormones regulating plant cell elongation. A defect in either of these leads to reduced plant growth and dwarfism. However, their relationship remains unknown in rice (Oryza sativa). Here, we demonstrated that BR regulates cell elongation by modulating GA metabolism in rice. Under physiological conditions, BR promotes GA accumulation by regulating the expression of GA metabolic genes to stimulate cell elongation. BR greatly induces the expression of D18/GA3ox-2, one of the GA biosynthetic genes, leading to increased GA1 levels, the bioactive GA in rice seedlings. Consequently, both d18 and loss-of-function GA-signaling mutants have decreased BR sensitivity. When excessive active BR is applied, the hormone mostly induces GA inactivation through upregulation of the GA inactivation gene GA2ox-3 and also represses BR biosynthesis, resulting in decreased hormone levels and growth inhibition. As a feedback mechanism, GA extensively inhibits BR biosynthesis and the BR response. GA treatment decreases the enlarged leaf angles in plants with enhanced BR biosynthesis or signaling. Our results revealed a previously unknown mechanism underlying BR and GA crosstalk depending on tissues and hormone levels, which greatly advances our understanding of hormone actions in crop plants and appears much different from that in Arabidopsis thaliana. d18|OsGA3ox2,GA2OX3 Identification and characterization of three telomere repeat-binding factors in rice. 2008 Biochem Biophys Res Commun Department of Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea. Telomeres consist of nucleoprotein complexes that protect chromosome end structures. Here, we describe three OsTRBF genes, encoding telomere repeat-binding factors of the single Myb histone family in rice. The predicted proteins contain a Myb DNA-binding motif and a linker histone H1/H5 domain in the N-terminal and central regions, respectively. The OsTRBF transcripts were constitutively detected in rice plants grown under greenhouse conditions. Gel retardation assays showed that these OsTRBF proteins bind specifically to the plant double-stranded telomeric sequence, TTTAGGG, with markedly different binding affinities as judged by their respective dissociation constants. Yeast two-hybrid and in vitro pull-down assays indicated that both OsTRBF1 and OsTRBF2 interact with one another to form homo- and hetero-complexes, while OsTRBF3 appeared to act as a monomer. Our results suggest that OsTRBFs play combinatory roles in the function and structure of telomeres in rice. OsTRBF1,OsTRBF2,OsTRBF3 The promoting effects of alginate oligosaccharides on root development in Oryza sativa L. mediated by auxin signaling. 2014 Carbohydr Polym Institute of Plant Nutrition, Agricultural Resources and Environmental Science, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China; Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Electronic address: snowgirl23@126.com. Alginate oligosaccharides (AOS), which are marine oligosaccharides, are involved in regulating plant root growth, but the promotion mechanism for AOS remains unclear. Here, AOS (10-80 mg/L) induced the expression of auxin-related gene (OsYUCCA1, OsYUCCA5, OsIAA11 and OsPIN1) in rice (Oryza sativa L.) tissues to accelerate auxin biosynthesis and transport, and reduced indole-3-acetic acid (IAA) oxidase activity in rice roots. These changes resulted in the increase of 37.8% in IAA concentration in rice roots, thereby inducing the expression of root development-related genes, promoting root growth in a dose-dependent manner, which were inhibited by auxin transport inhibitor 2,3,5-triiodo benzoic acid (TIBA) and calcium-chelating agent ethylene glycol bis (2-aminoethyl) tetraacetic acid (EGTA). AOS also induced calcium signaling generation in rice roots. Those results indicated that auxin mediated AOS regulation of root development, and calcium signaling may act mainly in the upstream of auxin in the regulation of AOS on rice root development. None Efficient Generation of Marker-Free Transgenic Rice Plants Using an Improved Transposon-Mediated Transgene Reintegration Strategy. 2014 Plant Physiol Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences; Marker-free transgenic plants can be developed through transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize Ac/Ds transposable element can be routinely used to generate marker-free transgenic plants. A Ds-based gene of interest (GOI) was linked to green fluorescent protein (GFP) in T-DNA, and a GFP-aided counter-selection against T-DNA was used together with PCR-based positive selection for the GOI to screen marker-free progeny. To test the efficacy of this strategy, we cloned the Bacillus thuringiensis (Bt) ð-endotoxin gene into the Ds elements and transformed transposon vectors into rice cultivars via Agrobacterium. PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to TAIL-PCR to determine Ds(Bt) reintegration positions, RT-PCR and ELISA to detect Bt expression levels, and bioassays to confirm resistance against the striped stem borer Chilo suppressalis (Walker). Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops. None Fast-Tracking Determination of Homozygous Transgenic Lines and Transgene Stacking Using a Reliable Quantitative Real-Time PCR Assay. 2014 Appl Biochem Biotechnol State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China. The selection of homozygous lines is a crucial step in the characterization of newly generated transgenic plants. This is particularly time- and labor-consuming when transgenic stacking is required. Here, we report a fast and accurate method based on quantitative real-time PCR with a rice gene RBE4 as a reference gene for selection of homozygous lines when using multiple transgenic stacking in rice. Use of this method allowed can be used to determine the stacking of up to three transgenes within four generations. Selection accuracy reached 100% for a single locus and 92.3% for two loci. This method confers distinct advantages over current transgenic research methodologies, as it is more accurate, rapid, and reliable. Therefore, this protocol could be used to efficiently select homozygous plants and to expedite time- and labor-consuming processes normally required for multiple transgene stacking. This protocol was standardized for determination of multiple gene stacking in molecular breeding via marker-assisted selection. None Rice and cold stress: methods for its evaluation and summary of cold tolerance-related quantitative trait loci. 2014 Rice (N Y) Hunan Provincial Key Laboratory of Crop Germplasm Innovation and Utilization, College of Biological Science and Technology, College of Agronomy, Hunan Agricultural University, Changsha 410128, Hunan, China ; Department of Anatomy and Neurobiology, Xiangya School of Medicine, Central South University, Changsha 410013, Hunan, China. Cold stress adversely affects rice (Oryza sativa L.) growth and productivity, and has so far determined its geographical distribution. Dissecting cold stress-mediated physiological changes and understanding their genetic causes will facilitate the breeding of rice for cold tolerance. Here, we review recent progress in research on cold stress-mediated physiological traits and metabolites, and indicate their roles in the cold-response network and cold-tolerance evaluation. We also discuss criteria for evaluating cold tolerance and evaluate the scope and shortcomings of each application. Moreover, we summarize research on quantitative trait loci (QTL) related to cold stress at the germination, seedling, and reproductive stages that should provide useful information to accelerate progress in breeding cold-tolerant rice. None A proteomic analysis of rice seed germination as affected by high temperature and ABA treatment. 2014 Physiol Plant Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China. Seed germination is a critical phase in the plant life cycle, but the specific events associated with seed germination are still not fully understood. In this study, we used two-dimensional gel electrophoresis followed by mass spectrometry to investigate the changes in the proteome during imbibition of Oryza sativa seeds at optimal temperature with or without abscisic acid (ABA) and high temperature (germination thermoinhibition) to further identify and quantify key proteins required for seed germination. A total of 121 protein spots showed a significant change in abundance (1.5-fold increase/decrease) during germination under all conditions. Among these proteins, we found seven proteins specifically associated with seed germination including glycosyl hydrolases family 38 protein, granule-bound starch synthase 1, Os03g0842900 (putative steroleosin-B), N-carbamoylputrescine amidase, spermidine synthase 1, tubulin α-1 chain and glutelin type-A; and a total of 20 imbibition response proteins involved in energy metabolism, cell growth, cell defense and storage proteins. High temperature inhibited seed germination by decreasing the abundance of proteins involved in methionine metabolism, amino acid biosynthesis, energy metabolism, reserve degradation, protein folding and stress responses. ABA treatment inhibited germination and decreased the abundance of proteins associated with methionine metabolism, energy production and cell division. Our results show that changes in many biological processes including energy metabolism, protein synthesis and cell defense and rescue occurred as a result of all treatments, while enzymes involved in methionine metabolism and weakening of cell wall specifically accumulated when the seeds germinated at the optimal temperature. None Global transcriptional profiling of a cold-tolerant rice variety under moderate cold stress reveals different cold stress response mechanisms. 2014 Physiol Plant Rice Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, China; Guangdong Provincial Key Laboratory of New Technology in Rice Breeding, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, China. Gene expression profiling under severe cold stress (4°C) has been conducted in plants including rice. However, rice seedlings are frequently exposed to milder cold stresses under natural environments. To understand the responses of rice to milder cold stress, a moderately low temperature (8°C) was used for cold treatment prior to genome-wide profiling of gene expression in a cold-tolerant japonica variety, Lijiangxintuanheigu (LTH). A total of 5557 differentially expressed genes (DEGs) were found at four time points during moderate cold stress. Both the DEGs and differentially expressed transcription factor genes were clustered into two groups based on their expression, suggesting a two-phase response to cold stress and a determinative role of transcription factors in the regulation of stress response. The induction of OsDREB2A under cold stress is reported for the first time in this study. Among the anti-oxidant enzyme genes, glutathione peroxidase (GPX) and glutathione S-transferase (GST) were upregulated, suggesting that the glutathione system may serve as the main reactive oxygen species (ROS) scavenger in LTH. Changes in expression of genes in signal transduction pathways for auxin, abscisic acid (ABA) and salicylic acid (SA) imply their involvement in cold stress responses. The induction of ABA response genes and detection of enriched cis-elements in DEGs suggest that ABA signaling pathway plays a dominant role in the cold stress response. Our results suggest that rice responses to cold stress vary with the specific temperature imposed and the rice genotype. None The non-homologous end-joining pathway is involved in stable transformation in rice. 2014 Front Plant Sci Plant Genome Engineering Research Unit, Agrogenomics Research Center, National Institute of Agrobiological Sciences Tsukuba, Japan. Stable transformation with T-DNA needs the coordinated activities of many proteins derived from both host plant cells and Agrobacterium. In dicot plants, including Arabidopsis, it has been suggested that non-homologous end-joining (NHEJ)-one of the main DNA double-strand break repair pathways-is involved in the T-DNA integration step that is crucial to stable transformation. However, how this pathway is involved remains unclear as results with NHEJ mutants in Arabidopsis have given inconsistent results. Recently, a system for visualization of stable expression of genes located on T-DNA has been established in rice callus. Stable expression was shown to be reduced significantly in NHEJ knock-down rice calli, suggesting strongly that NHEJ is involved in Agrobacterium-mediated stable transformation in rice. Since rice transformation is now efficient and reproducible, rice is a good model plant in which to elucidate the molecular mechanisms of T-DNA integration. None Metabolism of Reactive Oxygen Species in Cytoplasmic Male Sterility of Rice by Marking Upmost Pulvinus Interval. 2014 Appl Biochem Biotechnol Henan Provincial Key Laboratory of Ion Beam Bioengineering, Zhengzhou University, Zhengzhou, 450052, China. Reactive oxygen species (ROS) and malondialdehyde (MDA) in plant cell are thought to be important inducible factors of cell apoptosis if excessively accumulated in cells. To elucidate the metabolic mechanism of MDA production and scavenging in the cytoplasmic male-sterile (CMS) rice, CMS line and maintainer were employed for studying the relationship at different developmental stages by marking upmost pulvinus interval method of experiment. The results showed that the panicles and leaves of the CMS line had a noticeable higher MDA content than those of maintainer line at all five stages that had been investigated (p<0.05). MDA content in the CMS line in the flag leaves of auricle in the distance 0 mm stage (the meiosis stage) was the highest of the five stages. The increase of MDA contents in sterile panicles and leaves had inducible effects on the enzymic activity of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). However, at the abortion peak stage, MDA was excessively accumulated and antioxidant enzymic activity reduced significantly, resulting in the generation and scavenging of MDA out of balance. None Spatio-temporal distribution of phenolamides and the genetics of natural variation of hydroxycinnamoyl spermidine in rice. 2014 Mol Plant National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Phenolamides constitute a diverse class of secondary metabolites that are found ubiquitously in plants and have been implicated to play important role in a wide range of biological processes such as plant development and defense. However, spatio-temporal accumulation patterns of phenolamides in rice, one of the most important crops, are not available so far, and no gene responsible for the phenolamides biosynthesis has been identified in this species. In this report, we report here the comprehensive metabolic profiling and natural variation analysis of phenolamides in a collection of rice germplasm using an LC-MS-based targeted metabolomics method. Spatio-temporal controlled accumulations were observed for most phenolamides, together with their differential accumulations between the two major subspecies of rice. Further metabolic genome-wide association study (mGWAS) in rice leaf and the in vivo metabolic analysis of the transgenic plants identified Os12g27200 and Os12g27254 as two spermidine hydroxycinnamoyl transferases that might be underlying the natural variation of levels of spermidine conjugates in rice. Our work demonstrates 'gene-to-metabolite' analysis by mGWAS provides a useful tool for functional gene identification and omics-based crop genetic improvement. None Variations in CYP78A13 coding region influence grain size and yield in rice. 2014 Plant Cell Environ State Key Laboratory of Plant Genomics, National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China; University of the Chinese Academy of Sciences, Yuquan Road, Beijing, 100039, China. Grain size is one of the most important determinants of crop yield in cereals. Here, we identified a dominant mutant, big grain2 (bg2-D) from our enhancer-trapping population. Genetic analysis and SiteFinding PCR (polymerase chain reaction) revealed that BG2 encodes a cytochrome P450, OsCYP78A13. Sequence search revealed that CYP78A13 has a paralogue Grain Length 3.2 (GL3.2, LOC_Os03g30420) in rice with distinct expression patterns, analysis of transgenic plants harbouring either CYP78A13 or GL3.2 showed that both can promote grain growth. Sequence polymorphism analysis with 1529 rice varieties showed that the nucleotide diversity at CYP78A13 gene body and the 20-kb flanking region in the indica varieties were markedly higher than those in japonica varieties. Further, comparison of the genomic sequence of CYP78A13 in the japonica cultivar Nipponbare and the indica cultivar 9311 showed that there were three InDels in the promoter region and eight SNPs (single nucleotide polymorphism) in its coding sequence. Detailed examination of the transgenic plants with chimaeric constructs suggested that variation in CYP78A13 coding region is responsible for the variation of grain yield. Taken together, our results suggest that the variations in CYP78A13 in the indica varieties hold potential in rice breeding for application of grain yield improvement. GL3.2,GE|CYP78A13 Genome-wide Identification and Functional Analysis of Genes Expressed Ubiquitously in Rice. 2014 Mol Plant Department of Plant Molecular Systems Biotechnology; Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Korea Graduate School of Biotechnology, Kyung Hee University, Yongin 446-701, Korea khjung2010@khu.ac.kr. Genes that are expressed ubiquitously throughout all developmental stages are thought to be necessary for basic biological or cellular functions. Therefore, determining their biological roles is a great challenge. We identified 4,034 of these genes in rice after studying the results of Agilent 44K and Affymetrix meta-anatomical expression profiles. Among the 105 that were characterized by loss-of-function analysis, 79 were classified as members of gene families, with the majority of them being predominantly expressed. Using T-DNA insertional mutants, we examined 43 genes and found that loss-of-expression in six caused developing seed- or seedling-defective phenotypes. Of them, three are singletons without same family members and easily expect the defective phenotypes by the mutations. Phylogenomic analyses integrating genome-wide transcriptome data revealed the functional dominance of three ubiquitously expressed family genes. From them, we investigated the role of Os03g19890, which is involved in ATP generation within the mitochondria during endosperm development. We also created and evaluated functional networks associated with that gene to understand the molecular mechanism. Our study will provide useful strategy for pheonome analysis of ubiquitously expressed genes. None Whole-Genome Analysis of Herbicide-Tolerant Mutant Rice Generated by Agrobacterium-Mediated Gene Targeting. 2014 Plant Cell Physiol Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan. Gene targeting (GT) is a technique used to modify endogenous genes in target genomes precisely via homologous recombination (HR). Although GT plants are produced using genetic transformation techniques, if the difference between the endogenous and the modified gene is limited to point mutations, GT crops can be considered equivalent to non-genetically modified mutant crops generated by conventional mutagenesis techniques. However, it is difficult to guarantee the non-incorporation of DNA fragments from Agrobacterium in GT plants created by Agrobacterium-mediated GT despite screening with conventional Southern blot and/or PCR techniques. Here, we report a comprehensive analysis of herbicide-tolerant rice plants generated by inducing point mutations in the rice ALS gene via Agrobacterium-mediated GT. We performed genome comparative genomic hybridization (CGH) array analysis and whole-genome sequencing to evaluate the molecular composition of GT rice plants. Thus far, no integration of Agrobacterium-derived DNA fragments has been detected in GT rice plants. However, >1,000 single nucleotide polymorphisms (SNPs) and insertion/deletion (InDels) were found in GT plants. Among these mutations, 20-100 variants might have some effect on expression levels and/or protein function. Information about additive mutations should be useful in clearing out unwanted mutations by backcrossing. None OsAUX1 controls lateral root initiation in rice (Oryza sativa L.). 2014 Plant Cell Environ State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan 430072, China. Polar auxin transport, mediated by influx and efflux transporters, controls many aspects of plant growth and development. The auxin influx carriers in Arabidopsis have been shown to control lateral root development and gravitropism, but little is known about these proteins in rice. This paper reports on the functional characterization of OsAUX1. Three OsAUX1 T-DNA insertion mutants and RNAi knockdown transgenic plants reduced lateral root initiation compared to WT plants. OsAUX1 overexpression plants exhibited increased lateral root initiation and OsAUX1 was highly expressed in lateral roots and lateral root primordia. Similarly, the auxin reporter, DR5-GUS, was expressed at lower levels in osaux1 than in the WT plants, which indicated that the auxin levels in the mutant roots had decreased. Exogenous NAA treatment rescued the defective phenotype in osaux1-1 plants, whereas IAA and 2,4-D could not, which suggested that OsAUX1 was a putative auxin influx carrier. The transcript levels of several auxin-signaling genes and cell cycle genes significantly declined in osaux1, hinting that the regulatory role of OsAUX1 may be mediated by auxin-signaling and cell cycle genes. Overall, our results indicated that OsAUX1 was involved in polar auxin transport and functioned to control auxin-mediated lateral root initiation in rice. OsAUX1,OsAUX1 Jasmonates Induce Both Defense Responses and Communication in Monocotyledonous and Dicotyledonous Plants. 2014 Plant Cell Physiol Biotechnology Research Center, The University of Tokyo, Tokyo, 113-8657 Japan. Jasmonic acid (JA) and its derivatives (jasmonates, JAs) are phytohormones with essential roles in plant defense against pathogenesis and herbivorous arthropods. Both the up- and down-regulation of defense responses are dependent on signaling pathways mediated by JAs as well as other stress hormones (e.g. salicylic acid), generally those involving the transcriptional and post-transcriptional regulation of transcription factors via protein modification and epigenetic regulation. In addition to the typical model plant Arabidopsis (a dicotyledon), advances in genetics research have made rice a model monocot in which innovative pest control traits can be introduced and whose JA signaling pathway can be studied. In this review, we introduce the dynamic functions of JAs in plant defense strategy using defensive substances (e.g. indole alkaloids and terpenoid phytoalexins) and airborne signals (e.g. green leaf volatiles and volatile terpenes) in response to biotrophic and necrotrophic pathogens as well as above-ground and below-ground herbivores. We then discuss the important issue of how the mutualism of herbivorous arthropods with viruses or bacteria can cause cross-talk between JA and other phytohormones to counter the defense systems. None OsMYB103L, an R2R3-MYB transcription factor, influences leaf rolling and mechanical strength in rice (Oryza sativa L.). 2014 BMC Plant Biol State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. dyli@genetics.ac.cn. The shape of grass leaves possesses great value in both agronomy and developmental biology research. Leaf rolling is one of the important traits in rice (Oryza sativa L.) breeding. MYB transcription factors are one of the largest gene families and have important roles in plant development, metabolism and stress responses. However, little is known about their functions in rice.In this study, we report the functional characterization of a rice gene, OsMYB103L, which encodes an R2R3-MYB transcription factor. OsMYB103L was localized in the nucleus with transactivation activity. Overexpression of OsMYB103L in rice resulted in a rolled leaf phenotype. Further analyses showed that expression levels of several cellulose synthase genes (CESAs) were significantly increased, as was the cellulose content in OsMYB103L overexpressing lines. Knockdown of OsMYB103L by RNA interference led to a decreased level of cellulose content and reduced mechanical strength in leaves. Meanwhile, the expression levels of several CESA genes were decreased in these knockdown lines.These findings suggest that OsMYB103L may target CESA genes for regulation of cellulose synthesis and could potentially be engineered for desirable leaf shape and mechanical strength in rice. OsMYB103L RCN1/OsABCG5, an ATP-binding cassette (ABC) transporter, is required for hypodermal suberization of roots in rice (Oryza sativa). 2014 Plant J Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka-Kenjyojima, Eiheiji, Fukui, 910-1195, Japan. Suberin is a complex polymer composed of aliphatic and phenolic compounds. It is a constituent of apoplastic plant interfaces. In many plant species, including rice (Oryza sativa), the hypodermis in the outer part of roots forms a suberized cell wall (the Casparian strip and/or suberin lamellae), which inhibits the flow of water and ions and protects against pathogens. To date, there is no genetic evidence that suberin forms an apoplastic transport barrier in the hypodermis. We discovered that a rice reduced culm number1 (rcn1) mutant could not develop roots longer than 100 mm in waterlogged soil. The mutated gene encoded an ATP-binding cassette (ABC) transporter named RCN1/OsABCG5. RCN1/OsABCG5 gene expression in the wild type was increased in most hypodermal and some endodermal roots cells under stagnant deoxygenated conditions. A GFP-RCN1/OsABCG5 fusion protein localized at the plasma membrane of the wild type. Under stagnant deoxygenated conditions, well suberized hypodermis developed in wild types but not in rcn1 mutants. Under stagnant deoxygenated conditions, apoplastic tracers (periodic acid and berberine) were blocked at the hypodermis in the wild type but not in rcn1, indicating that the apoplastic barrier in the mutant was impaired. The amount of the major aliphatic suberin monomers originating from C(28) and C(30) fatty acids or OH fatty acids was much lower in rcn1 than in the wild type. These findings suggest that RCN1/OsABCG5 has a role in the suberization of the hypodermis of rice roots, which contributes to formation of the apoplastic barrier. Rcn1 Retention of OsNMD3 in the cytoplasm disturbs protein synthesis efficiency and affects plant development in rice. 2014 J Exp Bot State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. The ribosome is the basic machinery for translation, and biogenesis of ribosomes involves many coordinated events. However, knowledge about ribosomal dynamics in higher plants is very limited. This study chose a highly conserved trans-factor, the 60S ribosomal subunit nuclear export adaptor NMD3, to characterize the mechanism of ribosome biogenesis in the monocot plant Oryza sativa (rice). O. sativa NMD3 (OsNMD3) shares all the common motifs and shuttles between the nucleus and cytoplasm via CRM1/XPO1. A dominant negative form of OsNMD3 with a truncated nuclear localization sequence (OsNMD3(NLS)) was retained in the cytoplasm, consequently interfering with the release of OsNMD3 from pre-60S particles and disturbing the assembly of ribosome subunits. Analyses of the transactivation activity and cellulose biosynthesis level revealed low protein synthesis efficiency in the transgenic plants compared with the wild-type plants. Pharmaceutical treatments demonstrated structural alterations in ribosomes in the transgenic plants. Moreover, global expression profiles of the wild-type and transgenic plants were investigated using the Illumina RNA sequencing approach. These expression profiles suggested that overexpression of OsNMD3(NLS) affected ribosome biogenesis and certain basic pathways, leading to pleiotropic abnormalities in plant growth. Taken together, these results strongly suggest that OsNMD3 is important for ribosome assembly and the maintenance of normal protein synthesis efficiency. OsNMD3 A novel senescence-associated gene encoding gamma-aminobutyric acid (GABA):pyruvate transaminase is upregulated during rice leaf senescence 2004 Physiologia Plantarum Institute of Botany, Academia Sinica, Nankang, Taipei, Taiwan The gene Osl2, which is specifically upregulated during leaf senescence in rice (Oryza sativa L. cv. Tainong 67), was cloned and functionally characterized. The protein coding region of the gene consists of 19 exons encoding 516 amino acids, with a putative mitochondrial targeting sequence and conserved sequence of the pyridoxal 5'-phosphate-binding domain. The recombinant Osl2 fusion protein over-expressed in Escherichia coli displays pyruvate-dependent gamma-aminobutyric acid (GABA) transaminase (EC 2.6.1.19) activity. Examination of the expression patterns of the Osl2 gene in rice reveals that Osl2-specific transcripts are induced in the senescing leaves. The temporal profile of Osl2 protein accumulation is correlated with that of pyruvate-dependent GABA transaminase activity in rice leaves, with the highest expression level at the S3 senescent stage. The potential role for GABA transaminase during rice leaf senescence is discussed. Osl2 A new gene controlling hybrid sterility in rice (Oryza sativa L.) 2012 Euphytica State Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing, 210095, China Hybrids between rice subspecies indica and japonica display strong heterosis. However, semi-sterility of inter-subspecific hybrids between indica and japonica varieties is a major obstacle for application of hybrid vigor in rice production. Semi-sterility was previously ascribed to allelic interaction at a number of different loci, whereas, wide-compatibility varieties can overcome hybrid sterility. Variety Nekken 2, which is a source of wide compatibility genes, showed sterility when crossed to the Korean variety Yeong Pung. Genetic and cytological analyses revealed that the semi-sterility was caused by partial abortion of the embryo sac. Genome-wide analysis of the backcross population, Nekken 2/Yeong Pung//Nekken 2 identified two independent loci for hybrid sterility on chromosomes 1 and 12, explaining 18.99 and 18.03% of the phenotypic variance, respectively. To confirm this result, another population of the same backcross containing 216 individuals was tested at a different site in a different year. The locus on chromosome 12 was detected again. Based on the study, the stable QTL on chromosome 12 appeared to be different from previously reported genes for this trait, and was designated as S35(t). None Seed size is determined by the combinations of the genes controlling different seed characteristics in rice. 2011 Theor Appl Genet State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, 198 Shiqiao Road, Hangzhou, 310021, People's Republic of China. Rice seed size is an important agronomic trait in determining the yield potential, and four seed size related genes (GS3, GW2, qSW5/GW5 and GIF1) have been cloned in rice so far. However, the relationship among these four genes is still unclear, which will impede the process of gene pyramiding breeding program to some extent. To shade light on the relationship of above four genes, gene expression analysis was performed with GS3-RNAi, GW2-RNAi lines and CSSL of qSW5 at the transcriptional level. The results clearly showed that qSW5 and GW2 positively regulate the expression of GS3. Meanwhile, qSW5 can be down-regulated by repression of GW2 transcription. Additionally, GIF1 expression was found to be positively regulated by qSW5 but negatively by GW2 and GS3. Moreover, the allelic effects of qSW5 and GS3 were detailedly characterized based on a natural population consisting of 180 rice cultivars. It was indicated that mutual interactions exist between the two genes, in which, qSW5 affecting seed length is masked by GS3 alleles, and GS3 affecting seed width is masked by qSW5 alleles. These findings provide more insights into the molecular mechanisms underlying seed size development in rice and are likely to be useful for improving rice grain yield. GW2 XA23 is an executor R protein and confers broad-spectrum disease resistance in rice 2014 Mol Plant National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), Institute of Crop Science, Chinese Academy of Agriculture Sciences (CAAS), Beijing 100081, China. The majority of plant disease resistance (R) genes encode proteins that share common structural features. However, the transcription activator-like effector (TALE) associated executor type R genes show no considerable sequence homology to any known R genes. We adopted a map-based cloning approach and TALE-based technology to isolate and characterize Xa23, a new executor R gene derived from the wild rice (Oryza rufipogon) that confers an extremely broad spectrum of resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). Xa23 encodes a 113-amino acid protein that shares 50% identity to the known executor R protein XA10. The predicted transmembrane helices in XA23 also overlap with those of XA10. Unlike Xa10, however, Xa23 transcription is specifically activated by AvrXa23, a TALE present in all examined Xoo field isolates. Moreover, the susceptible xa23 allele has an identical open reading frame of Xa23, but differs in promoter region by lacking the TALE binding-element (EBE) for AvrXa23. XA23 can trigger strong hypersensitive response in rice, tobacco and tomato. Our results provide the first evidence that plant genomes have an executor R gene family in which members execute their function and spectrum of disease resistance by recognizing the cognate TALEs in pathogen. XA23 Expression of abiotic stress inducible ETHE1-like protein from rice is higher in roots and is regulated by calcium. 2014 Physiol Plant Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India. ETHYLMALONIC ENCEPHALOPATHY PROTEIN 1 (ETHE1) encodes sulfur dioxygenase (SDO) activity regulating sulfide levels in living organisms. It is an essential gene and mutations in ETHE1 leads to ethylmalonic encephalopathy (EE) in humans and embryo lethality in Arabidopsis. At present, very little is known regarding the role of ETHE1 beyond the context of EE and almost nothing is known about factors affecting its regulation in plant systems. In this study, we have identified, cloned and characterized OsETHE1, a gene encoding ETHE1-like protein from Oryza sativa. ETHE1 proteins in general are most similar to glyoxalase II (GLYII) and hence OsETHE1 has been earlier annotated as OsGLYII1, a putative GLYII gene. Here we show that OsETHE1 lacks GLYII activity and is instead an ETHE1 homolog being localized in mitochondria like its human and Arabidopsis counterparts. We have isolated and analyzed 1618 bp OsETHE1 promoter (pOsETHE1) to examine the factors affecting OsETHE1 expression. For this, transcriptional promoter pOsETHE1: 5-bromo-5-chloro-3-indolyl-D-glucuronide (GUS) fusion construct was made and stably transformed into rice. GUS expression pattern of transgenic pOsETHE1:GUS plants reveal a high root-specific expression of OsETHE1. The pOsETHE1 activity was stimulated by Ca(II) and required light for induction. Moreover, pOsETHE1 activity was induced under various abiotic stresses such as heat, salinity and oxidative stress, suggesting a potential role of OsETHE1 in stress response. OsGLYII1 Genome-wide analysis of rice and Arabidopsis identifies two glyoxalase genes that are highly expressed in abiotic stresses. 2011 Funct Integr Genomics Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India. Glyoxalase pathway, ubiquitously found in all organisms from prokaryotes to eukaryotes, consists of glyoxalase I (GLY I) and glyoxalase II (GLY II) enzymes, which detoxify a cytotoxic molecule, methylglyoxal (MG). Increase in MG has been correlated with various diseases in humans and different abiotic stresses in plants. We have previously shown that overproduction of GLY I and/or GLY II enzymes in transgenic plants provide tolerance towards salinity and heavy metal stresses. We have identified nineteen potential GLY I and four GLY II proteins in rice and twenty two GLY I and nine GLY II proteins in Arabidopsis. An analysis of complete set of genes coding for the glyoxalase proteins in these two genomes is presented, including classification and chromosomal distribution. Expression profiling of these genes has been performed in response to multiple abiotic stresses, in different tissues and during various stages of vegetative and reproductive development using publicly available databases (massively parallel signature sequencing and microarray). AtGLYI8, OsGLYI3, and OsGLYI10 expresses constitutively high in seeds while AtGLYI4, AtGLYI7, OsGLYI6, and OsGLYI11 are highly stress inducible. To complement this analyses, qRT-PCR is performed in two contrasting rice genotypes, i.e., IR64 and Pokkali where OsGLYI6 and OsGLYI11 are found to be highly stress inducible. OsGLYII1,OsGLYII1|OsETHE1,OsGLYII1|OsETHE1,GLYI-11|OsGLYI11|GLYI11|OsGLYI11.2|OsGLYI-11.2|Glb33|OsGLYI11.3 Conserved miR164-targeted NAC genes negatively regulate drought resistance in rice. 2014 J Exp Bot National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. MicroRNAs constitute a large group of endogenous small RNAs of ~22 nt that emerge as vital regulators, mainly by targeting mRNAs for post-transcriptional repression. Previous studies have revealed that the miR164 family in Arabidopsis is comprised of three members which guide the cleavage of the mRNAs of five NAC genes to modulate developmental processes. However, the functions of the miR164-targeted NAC genes in crops are poorly deciphered. In this study, the conserved features of six miR164-targeted NAC genes (OMTN1-OMTN6) in rice are described, and evidence is provided that four of them confer a negative regulatory role in drought resistance. OMTN proteins have the characteristics of typical NAC transcriptional factors. The miR164 recognition sites of the OMTN genes are highly conserved in rice germplasms. Deletion of the recognition sites impaired the transactivation activity, indicating that the conserved recognition sites play a crucial role in maintaining the function of the OMTN proteins. The OMTN genes were responsive to abiotic stresses, and showed diverse spatio-temporal expression patterns in rice. Overexpression of OMTN2, OMTN3, OMTN4, and OMTN6 in rice led to negative effects on drought resistance at the reproductive stage. The expression of numerous genes related to stress response, development, and metabolism was altered in OMTN2-, OMTN3-, OMTN4-, and OMTN6-overexpressing plants. Most of the up-regulated genes in the OMTN-overexpressing plants were down-regulated by drought stress. The results suggest that the conserved miR164-targeted NAC genes may be negative regulators of drought tolerance in rice, in addition to their reported roles in development. OMTN5 Cultivar variability of iron uptake mechanisms in rice (Oryza sativa L.). 2014 Plant Physiol Biochem CBQF - Centro de Biotecnologia e Qumica Fina - Laboratrio Associado, Escola Superior de Biotecnologia, Universidade Catlica Portuguesa/Porto, Rua Arquiteto Lobo Vital, Apartado 2511, EC Asprela, 4202-401 Porto, Portugal. Rice (Oryza sativa L.) is the most important staple food in the world. It is rich in genetic diversity and can grow in a wide range of environments. Iron (Fe) deficiency is a major abiotic stress in crop production and in aerobic soils, where Fe forms insoluble complexes, and is not readily available for uptake. To cope with Fe deficiency, plants developed mechanisms for Fe uptake, and although rice was described as a Strategy II plant, recent evidence suggests that it is capable of utilizing mechanisms from both Strategies. The main objective of this work was to compare two cultivars, Bico Branco (japonica) and Nipponbare (tropical japonica), to understand if the regulation of Fe uptake mechanisms could be cultivar (cv.) dependent. Plants of both cultivars were grown under Fe-deficient and -sufficient conditions and physiological and molecular responses to Fe deficiency were evaluated. Bico Branco cv. developed more leaf chlorosis and was more susceptible to Fe deficiency, retaining more nutrients in roots, than Nipponbare cv., which translocated more nutrients to shoots. Nipponbare cv. presented higher levels of Fe reductase activity, which was significantly up-regulated by Fe deficiency, and had higher expression levels of the Strategy I-OsFRO2 gene in roots, while Bico Branco cv. induced more genes involved in Strategy II. These new findings show that rice cultivars have different responses to Fe deficiency and that the induction of Strategy I or II may be rice cultivar-dependent, although the utilization of the reduction mechanisms seems to be an ubiquitous advantage. None A redox-sensitive cysteine residue regulates the kinase activities of OsMPK3 and OsMPK6 in vitro. 2014 Plant Sci National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China. Electronic address: xiegsh@mail.hzau.edu.cn. Two subgroup A rice mitogen-activated protein kinases (MAPKs), OsMPK3 and OsMPK6, have been implicated in multiple stress responses. However, the redox-control of the kinase activity of these proteins remains unknown. Here, immunoprecipitated OsMPK3 and OsMPK6 were initially activated in 15min, and this activation transiently increased in rice seedlings under H2O2 stress. Among the six conserved cysteine residues, only the fourth cysteine residues in the kinase domain VII, Cys(179) and Cys(210), were required for the in vitro kinase activities of OsMPK3 and OsMPK6, respectively. Moreover, the substitution of these specific cysteine residues with serine abrogated in vitro kinase responses to redox conditions. These results suggest a novel redox-control mechanism for the kinase activities of these MAPKs in vivo. OsMPK6|OsMPK4 The roles of histone acetylation in seed performance and plant development. 2014 Plant Physiol Biochem Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. Electronic address: wangzhi@ibcas.ac.cn. Histone acetylation regulates gene transcription by chromatin modifications and plays a crucial role in the plant development and response to environment cues. The homeostasis of histone acetylation is controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs) in different plant tissues and development stages. The vigorous knowledge of the function and co-factors about HATs (e.g. GCN5) and HDACs (e.g. HDA19, HDA6) has been obtained from model plant Arabidopsis. However, understanding individual role of other HATs and HDACs require more work, especially in the major food crops such as rice, maize and wheat. Many co-regulators have been recently identified to function as a component of HAT or HDAC complex in some specific developmental processes. The described findings show a distinctive and interesting epigenetic regulation network composed of HATs, HDACs and co-regulators playing crucial roles in the seed performance, flowering time, plant morphogenesis, plant response to stresses etc. In this review, we summarized the recent progresses and suggested the perspective of histone acetylation research, which might provide us a new window to understand the epigenetic code of plant development and to improve the crop production and quality. None Acceleration of cyclic electron flow in rice plants (Oryza sativa L.) deficient in the PsbS protein of Photosystem II. 2014 Plant Physiol Biochem Department of Integrated Biological Science, Department of Molecular Biology, Pusan National University, Jangjeon-dong, Geumjeong-gu, Busan 609-735, Republic of Korea; Department of Biology, North-Eastern Federal University, 58 Belinsky Str., Yakutsk 677-027 Republic of Sakha (Yakutia), Russian Federation; Institute of Botany, Azerbaijan National Academy of Sciences, Patamdar Shosse 40, Baku, AZ 1073, Azerbaijan. Electronic address: iszulfugarov@pusan.ac.kr. When compared with Photosystem I (PSI) in wild-type (WT) rice plants, PSI in PsbS-knockout (KO) plants that lack the energy-dependent component of nonphotochemical quenching (NPQ) was less sensitive to photoinhibition. Therefore, we investigated the relationship between NPQ and cyclic electron flow (CEF) around PSI as a photoprotective mechanism. Activities of two CEF routes (PGR5-dependent or NDH-dependent) were compared between those genotypes by using both dark-adapted plants and pre-illuminated plants, i.e., those in which the Calvin-Benson cycle is de-activated and activated, respectively. In dark-adapted leaves activity of the PGR5-dependent route was determined as the rate of P700 photooxidation. Activity was higher in the mutants than in the WT. However, no difference was noted when plants of either genotype were pre-illuminated. When the electron transport pathway was switched to the cyclic mode by infiltrating leaf segments with 150mM sorbitol, 40 DCMU, and 2mM hydroxylamine, the rate of P700 oxidation was faster in the mutant. That difference disappeared when leaves were infiltrated with antimycin A to inhibit the PGR5-dependent route. Chlorophyll fluorescence (Fo) was also evaluated. To achieve an Fo level comparable to that of the WT, activation of the NDH-dependent route in the mutant required pre-illumination at a certain dose. Therefore, we propose that, as an alternate pathway for the photoprotection of photosystems in the absence of energy-dependent quenching, this PGR5-dependent route is more highly activated in the PsbS-KO mutants than in the WT. Moreover, that stronger activity is probably responsible for slower activation of the NDH-dependent route in the mutant. OsPsbS|psbS1 An Acidic PATHOGENESIS-RELATED1 Gene of Oryza grandiglumis is Involved in Disease Resistance Response Against Bacterial Infection. 2014 Plant Pathol J National Crop Experiment Station, Rural Development Administration, Suwon 441-100, Korea. Wild rice, Oryza grandiglumis shows hyper-resistance response to pathogen infection. In order to identify genes necessary for defense response in plants, we have carried out a subtractive hybridization coupled with a cDNA macroarray. An acidic PATHOGENESIS-RELATED1 (PR1) gene of the wild rice is highly identical to the acidic PR1 genes of different plant species. The OgPR1a cDNA has an apparent single open reading frame with a predicted molecular mass 40,621 Da and an isoelectic point of 5.14. Both in silico analysis and a transient expression assay in onion epidermal cells revealed that the OgPR1a protein could be localized in intercellular space in plants. The OgPR1a mRNA was strongly transcribed by the exogenous treatment with ethylene and jasmonic acid as well as protein phosphatase inhibitors. Additionally, ectopic expression of the OgPR1a conferred disease resistance on Arabidopsis to the bacterial and fungal infections. None Effect of quantitative trait loci for seed shattering on abscission layer formation in Asian wild rice Oryza rufipogon. 2014 Breed Sci Laboratory of Plant Breeding, Graduate School of Agricultural Science, Kobe University , 1-1 Rokkodai, Nada, Kobe, Hyogo 657-8501 , Japan. Asian cultivated rice Oryza sativa L. was domesticated from its wild ancestor, O. rufipogon. During domestication, the cultivated rice lost its seed-shattering behaviour. Previous studies have shown that two major quantitative trait loci (QTLs; qSH1 and sh4) are responsible for the seed-shattering degree. Here, we produced introgression lines carrying non-functional alleles from O. sativa 'Nipponbare' at the two major QTLs in the genetic background of wild rice O. rufipogon W630, and examined the effects of the two QTLs on seed shattering and abscission layer formation. The introgression lines, with Nipponbare alleles at either or both loci, showed complete or partial abscission layer formation, respectively, indicating that other unknown loci might be involved in enhancing seed shattering in wild rice. We detected a single QTL named qSH3 regulating seed-shattering degree using an F2 population between Nipponbare and the introgression line carrying Nipponbare alleles at the two QTLs. Although we generated an introgression line for qSH3 alone, no effects on seed shattering were observed. However, a significant effect on seed-shattering degree was observed for the introgression line carrying Nipponbare alleles at qSH3 and the two QTLs, suggesting an important role of qSH3 on seed shattering in coordination with the two QTLs. None Genetic analysis of ion-beam induced extremely late heading mutants in rice. 2014 Breed Sci Faculty of Agriculture, Kagoshima University , 1-21-24 Korimoto, Kagoshima, Kagoshima 890-0065 , Japan. Two extremely late heading mutants were induced by ion beam irradiation in rice cultivar 'Taichung 65': KGM26 and KGM27. The F2 populations from the cross between the two mutants and Taichung 65 showed clear 3 early: 1 late segregation, suggesting control of late heading by a recessive gene. The genes identified in KGM26 and KGM27 were respectively designated as FLT1 and FLT2. The two genes were mapped using the crosses between the two mutants and an Indica cultivar 'Kasalath'. FLT1 was located on the distal end of the short arm of chromosome 8. FLT2 was located around the centromere of chromosome 9. FLT1 might share the same locus as EHD3 because their chromosomal location is overlapping. FLT2 is inferred to be a new gene because no gene with a comparable effect to that of this gene was mapped near the centromere of chromosome 9. In crosses with Kasalath, homozygotes of late heading mutant genes showed a large variation of days to heading, suggesting that other genes affected late heading mutant genes. None QTL analysis on rice grain appearance quality, as exemplifying the typical events of transgenic or backcrossing breeding. 2014 Breed Sci National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan) and National Center of Crop Molecular Breeding, Huazhong Agricultural University , Wuhan 430070 , China. Rice grain shape and yield are usually controlled by multiple quantitative trait loci (QTL). This study used a set of F9-10 recombinant inbred lines (RILs) derived from a cross of Huahui 3 (Bt/Xa21) and Zhongguoxiangdao, and detected 27 QTLs on ten rice chromosomes. Among them, twelve QTLs responsive for grain shape/ or yield were mostly reproducibly detected and had not yet been reported before. Interestingly, the two known genes involved in the materials, with one insect-resistant Bt gene, and the other disease-resistant Xa21 gene, were found to closely link the QTLs responsive for grain shape and weight. The Bt fragment insertion was firstly mapped on the chromosome 10 in Huahui 3 and may disrupt grain-related QTLs resulting in weaker yield performance in transgenic plants. The introgression of Xa21 gene by backcrossing from donor material into receptor Minghui 63 may also contain a donor linkage drag which included minor-effect QTL alleles positively affecting grain shape and yield. The QTL analysis on rice grain appearance quality exemplified the typical events of transgenic or backcrossing breeding. The QTL findings in this study will in the future facilitate the gene isolation and breeding application for improvement of rice grain shape and yield. None Transcriptomic analysis of a psammophyte food crop, sand rice (Agriophyllum squarrosum) and identification of candidate genes essential for sand dune adaptation. 2014 BMC Genomics Fail Sand rice (Agriophyllum squarrosum) is an annual desert plant adapted to mobile sand dunes in arid and semi-arid regions of Central Asia. The sand rice seeds have excellent nutrition value and have been historically consumed by local populations in the desert regions of northwest China. Sand rice is a potential food crop resilient to ongoing climate change; however, partly due to the scarcity of genetic information, this species has undergone only little agronomic modifications through classical breeding during recent years.We generate a deep transcriptomic sequencing of sand rice, which uncovers 67,741 unigenes. Phylogenetic analysis based on 221 single-copy genes showed close relationship between sand rice and the recently domesticated crop sugar beet. Transcriptomic comparisons also showed a high level of global sequence conservation between these two species. Conservation of sand rice and sugar beet orthologs assigned to response to salt stress gene ontology term suggests that sand rice is also a potential salt tolerant plant. Furthermore, sand rice is far more tolerant to high temperature. A set of genes likely relevant for resistance to heat stress, was functionally annotated according to expression levels, sequence annotation, and comparisons corresponding transcriptome profiling results in Arabidopsis.The present work provides abundant genomic information for functional dissection of the important traits in sand rice. Future screening the genetic variation among different ecotypes and constructing a draft genome sequence will further facilitate agronomic trait improvement and final domestication of sand rice. None Genetic dissection of ozone tolerance in rice (Oryza sativa L.) by a genome-wide association study. 2014 J Exp Bot Institute of Crop Science and Resource Conservation (INRES), University of Bonn, Karlrobert-Kreiten Strasse 13, 53115 Bonn, Germany. Tropospheric ozone causes various negative effects on plants and affects the yield and quality of agricultural crops. Here, we report a genome-wide association study (GWAS) in rice (Oryza sativa L.) to determine candidate loci associated with ozone tolerance. A diversity panel consisting of 328 accessions representing all subgroups of O. sativa was exposed to ozone stress at 60 nl l(-1) for 7h every day throughout the growth season, or to control conditions. Averaged over all genotypes, ozone significantly affected biomass-related traits (plant height -1.0%, shoot dry weight -15.9%, tiller number -8.3%, grain weight -9.3%, total panicle weight -19.7%, single panicle weight -5.5%) and biochemical/physiological traits (symptom formation, SPAD value -4.4%, foliar lignin content +3.4%). A wide range of genotypic variance in response to ozone stress were observed in all phenotypes. Association mapping based on more than 30 000 single-nucleotide polymorphism (SNP) markers yielded 16 significant markers throughout the genome by applying a significance threshold of P<0.0001. Furthermore, by determining linkage disequilibrium blocks associated with significant SNPs, we gained a total of 195 candidate genes for these traits. The following sequence analysis revealed a number of novel polymorphisms in two candidate genes for the formation of visible leaf symptoms, a RING and an EREBP gene, both of which are involved in cell death and stress defence reactions. This study demonstrated substantial natural variation of responses to ozone in rice and the possibility of using GWAS in elucidating the genetic factors underlying ozone tolerance. None Genetic differentiation revealed by selective loci of drought-responding EST-SSRs between upland and lowland rice in China. 2014 PLoS One Shanghai Agrobiological Gene Center, Shanghai, China. Upland and lowland rice (Oryza sativa L.) represent two of the most important rice ecotypes adapted to ago-ecosystems with contrasting soil-water conditions. Upland rice, domesticated in the water-limited environment, contains valuable drought-resistant characters that can be used in water-saving breeding. Knowledge about the divergence between upland and lowland rice will provide valuable cues for the evolution of drought-resistance in rice. Genetic differentiation between upland and lowland rice was explored by 47 Simple Sequence Repeats (SSRs) located in drought responding expressed sequence tags (ESTs) among 377 rice landraces. The morphological traits of drought-resistance were evaluated in the field experiments. Different outlier loci were detected in the japonica and indica subspecies, respectively. Considerable genetic differentiation between upland and lowland rice on these outlier loci was estimated in japonica (Fst = 0.258) and indica (Fst = 0.127). Furthermore, populations of the upland and lowland ecotypes were clustered separately on these outlier loci. A significant correlation between genetic distance matrices and the dissimilarity matrices of drought-resistant traits was determined, indicating a certain relationship between the upland-lowland rice differentiation and the drought-resistance. Divergent selections occur between upland and lowland rice on the drought-resistance as the Qsts of some drought-resistant traits are significantly higher than the neutral Fst. In addition, the upland- and lowland-preferable alleles responded differently among ecotypes or allelic types under osmotic stress. This shows the evolutionary signature of drought resistance at the gene expression level. The findings of this study can strengthen our understanding of the evolution of drought-resistance in rice with significant implications in the improvement of rice drought-resistance. None Days to heading 7, a major quantitative locus determining photoperiod sensitivity and regional adaptation in rice. 2014 Proc Natl Acad Sci U S A National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, 100081 Beijing, China; National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, 210095 Nanjing, China; Success of modern agriculture relies heavily on breeding of crops with maximal regional adaptability and yield potentials. A major limiting factor for crop cultivation is their flowering time, which is strongly regulated by day length (photoperiod) and temperature. Here we report identification and characterization of Days to heading 7 (DTH7), a major genetic locus underlying photoperiod sensitivity and grain yield in rice. Map-based cloning reveals that DTH7 encodes a pseudo-response regulator protein and its expression is regulated by photoperiod. We show that in long days DTH7 acts downstream of the photoreceptor phytochrome B to repress the expression of Ehd1, an up-regulator of the "florigen" genes (Hd3a and RFT1), leading to delayed flowering. Further, we find that haplotype combinations of DTH7 with Grain number, plant height, and heading date 7 (Ghd7) and DTH8 correlate well with the heading date and grain yield of rice under different photoperiod conditions. Our data provide not only a macroscopic view of the genetic control of photoperiod sensitivity in rice but also a foundation for breeding of rice cultivars better adapted to the target environments using rational design. Ghd7.1|Hd2|OsPRR37|DTH7 The B″ regulatory subunit of protein phosphatase 2A mediates the dephosphorylation of rice retinoblastoma-related protein-1. 2014 Plant Mol Biol Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary. The phosphorylation of plant retinoblastoma-related (RBR) proteins by cyclin-dependent kinases (CDKs) is well documented, but the counteracting phosphatases have not been identified yet. We report here that rice retinoblastoma-related protein-1 (OsRBR1) interacted with the B″ subunit of rice protein phosphatase 2A (OsPP2A B″) and underwent reversible phosphorylation during the cell division cycle. The OsRBR1-OsPP2A B" association required B domain in OsRBR1 and the C-terminal region of OsPP2A B″. We found by immunoprecipitation that OsPP2A B″, OsPP2A catalytic subunit subtype II, PSTAIRE-type CDK and OsRBR1 were in the same protein complex, indicating a physical association between the phosphatase, the kinase and their common substrate. OsPP2A B″ contains three predicted CDK phosphorylation sites: Ser95, Ser102 and Ser119. The in vitro phosphorylation of Ser95 and Ser119 with PSTAIRE-kinases was verified by mass spectrometry. We generated a series of phosphorylation site mutants to mimic the dephosphorylated or phosphorylated states of OsPP2A B″, and confirmed that all of the three predicted sites can be phosphorylated. Yeast two-hybrid experiments suggested that the phosphorylation of OsPP2A B″ promoted the formation of the OsPP2A holoenzyme. A triple phosphorylation mimicking OsPP2A B″ mutant containing holoenzyme showed higher activity in phosphatase assays. Our data collectively show that the phosphatase activity of OsPP2A against OsRBR1 is regulated by the phosphorylation of its B″ regulatory subunit. However, the analysis of the effect of okadaic acid, a phosphatase inhibitor, in rice cell suspension cultures revealed that the dephosphorylation of OsRBR1 was completely inhibited only by high dose (300 nM) of the okadaic acid during the cell cycle progression. Therefore the role of the protein phosphatase 1 should be considered as an additional post translational regulatory component of RBR protein function in higher plants. OsRBR1 Copper suppresses abscisic acid catabolism and catalase activity, and inhibits seed germination of rice. 2014 Plant Cell Physiol Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China. Although copper (Cu) is an essential micronutrient for plants, a slight excess of Cu in soil can be harmful to plants. Unfortunately, Cu contamination is a growing problem all over the world due to human activities, and poses a soil stress to plant development. As one of the most important biological processes, seed germination is sensitive to Cu stress. However, little is known about the mechanism of Cu-induced inhibition of seed germination. In the present study, we investigated the relationship between Cu and ABA which is the predominant regulator of seed germination. Cu at a concentration of 30 M effectively inhibited germination of rice caryopsis. ABA content in germinating seeds under copper stress was also higher than that under control conditions. Quantitative real-time PCR (qRT-PCR) revealed that Cu treatment reduced the expression of OsABA8ox2, a key gene of ABA catabolism in rice seeds. In addition, both malondialdehyde (MDA) and H2O2 contents were increased by Cu stress in the germinating seeds. Antioxidant enzyme assays revealed that only catalase activity was reduced by excess Cu, which was consistent with the mRNA profile of OsCATa during seed germination under Cu stress. Together, our results demonstrate that suppression of ABA catabolism and catalase (CAT) activity by excess Cu leads to the inhibition of seed germination of rice. OsABA8ox2 An alternatively spliced heat shock transcription factor, OsHSFA2dI, functions in the heat stress-induced unfolded protein response in rice. 2014 Plant Biol (Stuttg) State Key Laboratory of Hybrid Rice, Engineering Research Center for Plant Biotechnology and Germplasm Utilization, Ministry of Education, College of Life Sciences, Wuhan University, Wuhan, China. As sessile organisms, plants have evolved a wide range of defence pathways to cope with environmental stress such as heat shock. However, the molecular mechanism of these defence pathways remains unclear in rice. In this study, we found that OsHSFA2d, a heat shock transcriptional factor, encodes two main splice variant proteins, OsHSFA2dI and OsHSFA2dII in rice. Under normal conditions, OsHSFA2dII is the dominant but transcriptionally inactive spliced form. However, when the plant suffers heat stress, OsHSFA2d is alternatively spliced into a transcriptionally active form, OsHSFA2dI, which participates in the heat stress response (HSR). Further study found that this alternative splicing was induced by heat shock rather than photoperiod. We found that OsHSFA2dI is localised to the nucleus, whereas OsHSFA2dII is localised to the nucleus and cytoplasm. Moreover, expression of the unfolded protein response (UNFOLDED PROTEIN RESPONSE) sensors, OsIRE1, OsbZIP39/OsbZIP60 and the UNFOLDED PROTEIN RESPONSE marker OsBiP1, was up-regulated. Interestingly, OsbZIP50 was also alternatively spliced under heat stress, indicating that UNFOLDED PROTEIN RESPONSE signalling pathways were activated by heat stress to re-establish cellular protein homeostasis. We further demonstrated that OsHSFA2dI participated in the unfolded protein response by regulating expression of OsBiP1. This article is protected by copyright. All rights reserved. OsHSFA2d|OsHSF7 Large scale germplasm screening for identification of novel rice blast resistance sources. 2014 Front Plant Sci Plant Biotechnology, Department of Biology, ETH Zurich (Swiss Federal Institute of Technology) Zurich, Switzerland. Rice is a major cereal crop that contributes significantly to global food security. Biotic stresses, including the rice blast fungus, cause severe yield losses that significantly impair rice production worldwide. The rapid genetic evolution of the fungus often overcomes the resistance conferred by major genes after a few years of intensive agricultural use. Therefore, resistance breeding requires continuous efforts of enriching the reservoir of resistance genes/alleles to effectively tackle the disease. Seed banks represent a rich stock of genetic diversity, however, they are still under-explored for identifying novel genes and/or their functional alleles. We conducted a large-scale screen for new rice blast resistance sources in 4246 geographically diverse rice accessions originating from 13 major rice-growing countries. The accessions were selected from a total collection of over 120,000 accessions based on their annotated rice blast resistance information in the International Rice Genebank. A two-step resistance screening protocol was used involving natural infection in a rice uniform blast nursery and subsequent artificial infections with five single rice blast isolates. The nursery-resistant accessions showed varied disease responses when infected with single isolates, suggesting the presence of diverse resistance genes/alleles in this accession collection. In addition, 289 accessions showed broad-spectrum resistance against all five single rice blast isolates. The selected resistant accessions were genotyped for the presence of the Pi2 resistance gene, thereby identifying potential accessions for isolation of allelic variants of this blast resistance gene. Together, the accession collection with broad spectrum and isolate specific blast resistance represent the core material for isolation of previously unknown blast resistance genes and/or their allelic variants that can be deployed in rice breeding programs. None Recurrent parent genome recovery analysis in a marker-assisted backcrossing program of rice (Oryza sativa L.). 2014 C R Biol Laboratory of Food Crops, Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Backcross breeding is the most commonly used method for incorporating a blast resistance gene into a rice cultivar. Linkage between the resistance gene and undesirable units can persist for many generations of backcrossing. Marker-assisted backcrossing (MABC) along with marker-assisted selection (MAS) contributes immensely to overcome the main limitation of the conventional breeding and accelerates recurrent parent genome (RPG) recovery. The MABC approach was employed to incorporate (a) blast resistance gene(s) from the donor parent Pongsu Seribu 1, the blast-resistant local variety in Malaysia, into the genetic background of MR219, a popular high-yielding rice variety that is blast susceptible, to develop a blast-resistant MR219 improved variety. In this perspective, the recurrent parent genome recovery was analyzed in early generations of backcrossing using simple sequence repeat (SSR) markers. Out of 375 SSR markers, 70 markers were found polymorphic between the parents, and these markers were used to evaluate the plants in subsequent generations. Background analysis revealed that the extent of RPG recovery ranged from 75.40% to 91.3% and from 80.40% to 96.70% in BC1F1 and BC2F1 generations, respectively. In this study, the recurrent parent genome content in the selected BC2F2 lines ranged from 92.7% to 97.7%. The average proportion of the recurrent parent in the selected improved line was 95.98%. MAS allowed identification of the plants that are more similar to the recurrent parent for the loci evaluated in backcross generations. The application of MAS with the MABC breeding program accelerated the recovery of the RP genome, reducing the number of generations and the time for incorporating resistance against rice blast. None Identification of quantitative trait loci for phosphorus use efficiency traits in rice using a high density SNP map. 2014 BMC Genet Fail BackgroundSoil phosphorus (P) deficiency is one of the major limiting factors to crop production. The development of crop varieties with improved P use efficiency (PUE) is an important strategy for sustainable agriculture. The objectives of this research were to identify quantitative trait loci (QTLs) linked to PUE traits using a high-density single nucleotide polymorphism (SNP) map and to estimate the epistatic interactions and environmental effects in rice (Oryza sativa L.).ResultsWe conducted a two-year field experiment under low and normal P conditions using a recombinant inbred population of rice derived from Zhenshan 97 and Minghui 63 (indica). We investigated three yield traits, biomass (BIOM), harvest index (HI), and grain yield (Yield), and eight PUE traits: total P uptake (PUP), P harvest index (PHI), grain P use efficiency (gPUE) based on P accumulation in grains, straw P use efficiency (strPUE) based on P accumulation in straw, P use efficiency for biomass (PUEb) and for grain yield (PUEg) based on P accumulation in the whole plant, P translocation (PT), and P translocation efficiency (PTE). Of the 36 QTLs and 24 epistatic interactions identified, 26 QTLs and 12 interactions were detected for PUE traits. The environment affected seven QTLs and three epistatic interactions. Four QTLs (qPHI1 and qPHI2 for PHI, qPUEg2 for PUEg, and qPTE8 for PTE) with strong effects were environmentally independent. By comparing our results with similar QTLs in previous studies, three QTLs for PUE traits (qPUP1 and qPUP10 for PUP, and qPHI6 for PHI) were found across various genetic backgrounds. Seven regions were shared by QTLs for yield and PUE traits.ConclusionMost QTLs linked to PUE traits were different from those linked to yield traits, suggesting different genetic controls underlying these two traits. Those chromosomal regions with large effects that are not affected by different environments are promising for improving P use efficiency. The seven regions shared by QTLs linked to yield and PUE traits imply the possibility of the simultaneous improvement of yield and PUE traits. None OsGL1-3 is Involved in Cuticular Wax Biosynthesis and Tolerance to Water Deficit in Rice. 2015 PLoS One Key Laboratory for Crop Germplasm Innovation and Utilization of Hunan Province, Hunan Agricultural University, Changsha, China; College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, China. Cuticular wax covers aerial organs of plants and functions as the outermost barrier against non-stomatal water loss. We reported here the functional characterization of the Glossy1(GL1)-homologous gene OsGL1-3 in rice using overexpression and RNAi transgenic rice plants. OsGL1-3 gene was ubiquitously expressed at different level in rice plants except root and its expression was up-regulated under ABA and PEG treatments. The transient expression of OsGL1-3-GFP fusion protein indicated that OsGL1-3 is mainly localized in the plasma membrane. Compared to the wild type, overexpression rice plants exhibited stunted growth, more wax crystallization on leaf surface, and significantly increased total cuticular wax load due to the prominent changes of C30-C32 aldehydes and C30 primary alcohols. While the RNAi knockdown mutant of OsGL1-3 exhibited no significant difference in plant height, but less wax crystallization and decreased total cuticular wax accumulation on leaf surface. All these evidences, together with the effects of OsGL1-3 on the expression of some wax synthesis related genes, suggest that OsGL1-3 is involved in cuticular wax biosynthesis. Overexpression of OsGL1-3 decreased chlorophyll leaching and water loss rate whereas increased tolerance to water deficit at both seedling and late-tillering stages, suggesting an important role of OsGL1-3 in drought tolerance. OsGL1-3 Na(+) and Cl(-) ions show additive effects under NaCl stress on induction of oxidative stress and the responsive antioxidative defense in rice. 2014 Protoplasma Department of Biotechnology, Modern College of Arts, Science and Commerce, University of Pune, Ganeshkhind, Pune, 411 016, India. Despite the fact that when subjected to salinity stress most plants accumulate high concentrations of sodium (Na(+)) and chloride (Cl(-)) ions in their tissues, major research has however been focused on the toxic effects of Na(+). Consequently, Cl(-) toxicity mechanisms in annual plants, particularly in inducing oxidative stress, are poorly understood. Here, the extent to which Na(+) and/or Cl(-) ions contribute in inducing oxidative stress and regulating the adaptive antioxidant defense is shown in two Indica rice genotypes differing in their salt tolerance. Equimolar (100 mM) concentrations of Na(+), Cl(-), and NaCl (EC ≈ 10 dS m(-1)) generated free-radical (O2 (•-), (•)OH) and non-radical (H2O2) forms of reactive oxygen species (ROS) and triggered cell death in leaves of 21-day-old hydroponically grown rice seedlings as evident by spectrophotometric quantifications and histochemical visualizations. The magnitude of ROS-mediated oxidative damage was higher in sensitive cultivar, whereas NaCl proved to be most toxic among the treatments. Salt treatments significantly increased activities of antioxidant enzymes and their isozymes including superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, and glutathione reductase. Na(+) and Cl(-) ions showed additive effects under NaCl in activating the antioxidant enzyme machinery, and responses were more pronounced in tolerant cultivar. The expression levels of SodCc2, CatA, and OsPRX1 genes were largely consistent with the activities of their corresponding enzymes. Salt treatments caused an imbalance in non-enzymatic antioxidants ascorbic acid, α-tocopherol, and polyphenols, with greater impacts under NaCl than Na(+) and Cl(-) separately. Results revealed that though Cl(-) was relatively less toxic than its counter-cation, its effects cannot be totally ignored. Both the cultivars responded in the same manner, but the tolerant cultivar maintained lower Na(+)/K(+) and ROS levels coupled with better antioxidant defense under all three salt treatments. None Transcriptional profile of genes involved in ascorbate glutathione cycle in senescing leaves for an early senescence leaf (esl) rice mutant. 2014 J Plant Physiol Institute of Crop Science, Zhejiang University, Hangzhou, Zhejiang 310058, China. To clarify the complex relationship between ascorbate-glutathione (AsA-GSH) cycle and H2O2-induced leaf senescence, the genotype-dependent difference in some senescence-related physiological parameters and the transcript levels and the temporal patterns of genes involved in the AsA-GSH cycle during leaf senescence were investigated using two rice genotypes, namely, the early senescence leaf (esl) mutant and its wild type. Meanwhile, the triggering effect of exogenous H2O2 on the expression of OsAPX genes was examined using detached leaves. The results showed that the esl mutant had higher H2O2 level than its wild type at the initial stage of leaf senescence. At transcriptional level, the association of expression of various genes involved in the AsA-GSH cycle with leaf senescence was isoform dependent. For OsAPXs, the transcripts of two cytosolic OsAPX genes (OsAPX1 and OsAPX2), thylakoid-bound OsAPX8, chloroplastic OsAPX7 and peroxisomal OsAPX4 exhibited remarkable genotype-dependent variation in their expression levels and temporal patterns during leaf senescence, there were significantly increasing transcripts of OsAXP1 and OsAPX7, severely repressed transcripts of OsAPX4 and OsAPX8 for the esl rice at the initial leaf senescence. In contrast, the repressing transcript of OsAPX8 was highly sensitive to the increasing H2O2 level in the senescing rice leaves, while higher H2O2 concentration resulted in the enhancing transcripts of two cytosolic OsAPX genes, OsAPX7 transcript was greatly variable with different H2O2 concentrations and incubating duration, suggesting that the different OsAPXs isoforms played a complementary role in perceiving and scavenging H2O2 accumulation at various H2O2 concentrations during leaf senescence. Higher H2O2 level, increased AsA level, higher activities of APX and glutathione reductase (GR), and relatively stable GSH content during the entire sampling period in the leaves of esl mutant implied that a close interrelationship existed between AsA level and APX activity in the ongoing senescence of rice leaves. The GSH supply in rice leaves was not the limiting factor for the efficient maintenance of AsA-GSH cycle, despite the senescence-related change in GR activity between the two rice genotypes. None Antisense suppression of LOX3 gene expression in rice endosperm enhances seed longevity. 2014 Plant Biotechnol J Rice Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, China; Incubator of National Key Laboratory of Fujian Crop Germplasm Innovation and Molecular Breeding between Fujian and Ministry of Sciences and Technology, Fuzhou, China; Key Laboratory of Germplasm Innovation and Molecular Breeding of Hybrid Rice for South China, Ministry of Agriculture, Fuzhou, China; South-China Base of National Key Laboratory of Hybrid Rice of China, Fuzhou, China; National Engineering Laboratory of Rice, Fuzhou, Fujian, China. Lipid peroxidation plays a major role in seed longevity and viability. In rice grains, lipid peroxidation is catalyzed by the enzyme lipoxygenase 3 (LOX3). Previous reports showed that grain from the rice variety DawDam in which the LOX3 gene was deleted had less stale flavour after grain storage than normal rice. The molecular mechanism by which LOX3 expression is regulated during endosperm development remains unclear. In this study, we expressed a LOX3 antisense construct in transgenic rice (Oryza sativa L.) plants to down-regulate LOX3 expression in rice endosperm. The transgenic plants exhibited a marked decrease in LOX mRNA levels, normal phenotypes and a normal life cycle. We showed that LOX3 activity and its ability to produce 9-hydroperoxyoctadecadienoic acid (9-HPOD) from linoleic acid were significantly lower in transgenic seeds than in wild-type seeds by measuring the ultraviolet absorption of 9-HPOD at 234 nm and by high-performance liquid chromatography. The suppression of LOX3 expression in rice endosperm increased grain storability. The germination rate of TS-91 (antisense LOX3 transgenic line) was much higher than the WT (29% higher after artificial ageing for 21 days, and 40% higher after natural ageing for 12 months). To our knowledge, this is the first report to demonstrate that decreased LOX3 expression can preserve rice grain quality during storage with no impact on grain yield, suggesting potential applications in agricultural production. OsLOX3|LOX3 A nuclear-localized histone-gene binding protein from rice (OsHBP1b) functions in salinity and drought stress tolerance by maintaining chlorophyll content and improving the antioxidant machinery. 2014 J Plant Physiol Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India. Plants have evolved a number of molecular strategies and regulatory mechanisms to cope with abiotic stresses. Among the various key factors/regulators, transcription factors (TFs) play critical role(s) towards regulating the gene expression patterns in response to stress conditions. Altering the expression of the key TFs can greatly influence plant stress tolerance. OsHBP1b (accession no. KM096571) is one such TF belonging to bZIP family, localized within the Saltol QTL, whose expression is induced upon salinity treatment in the rice seedlings. qRT-PCR based expression studies for OsHBP1b in seedlings of contrasting genotypes of rice showed its differential regulation in response to salinity stress. A GFP based in vivo study showed that the OsHBP1b protein is nuclear localized and possesses the trans-activation activity. As compared to the WT tobacco plants, the transgenic plants ectopically expressing OsHBP1b showed better survival and favourable osmotic parameters (such as germination and survival rate, membrane stability, K(+)/Na(+) ratio, lipid peroxidation, electrolyte leakage and proline contents) under salinity and drought stress. Under salinity conditions, the transgenic plants accumulated lower levels of reactive oxygen species as compared to the WT. It was also accompanied by higher activities of antioxidant enzymes (such as ascorbate peroxidase and superoxide dismutase), thereby demonstrating that transgenic plants are physiologically better adapted towards the oxidative damage. Taken together, our findings suggest that OsHBP1b contributes to abiotic stress tolerance through multiple physiological pathways and thus, may serve as a useful 'candidate gene' for improving multiple stress tolerance in crop plants. OsHBP1b Thioredoxin h isoforms from rice are differentially reduced by NADPH/thioredoxin or GSH/glutaredoxin systems. 2014 Int J Biol Macromol Department of Agricultural Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran. Rice (Oryza sativa L.) has multiple potential genes encoding thioredoxin (Trx) h and NADP-thioredoxin reductase (NTR). These NTR and Trx h isoforms, known as cytoplasmic NTR/Trx system along with multiple members of glutaredoxin (Grx) family constitute a complex redox control system in rice. In the present study, we investigated the kinetic parameters of two rice NTRs, OsNTRA and OsNTRB, toward three endogenous Trx h isoforms, OsTrx1, OsTrx20, and OsTrx23. The results showed that in contrast with OsTrx1 and OsTrx23, the isoform OsTrx20 was not reduced by OsNTR isoforms. The kcat/Km values of OsNTRB and OsNTRA toward OsTrx1 was six- and 13-fold higher than those values toward OsTrx23, respectively, suggesting that OsNTR isoforms do not reduce different OsTrx h isoforms, equivalently. Furthermore, the possible reduction of OsTrx isoforms by the glutathione (GSH)/Grx system was investigated through the heterologous expression of a gene encoding OsGrx9, a bicysteinic CPYC Grx found in rice. Whereas OsTrx23 was not reduced by GSH, OsTrx20 and with less efficiently OsTrx1 were reduced by GSH or GSH/Grx. Therefore, it seems that OsTrx1 can be reduced either by OsNTR or GSH/Grx. These data for the first time provides an evidence for cross-talking between NTR/Trx and GSH/Grx systems in rice. None The role of ethylene and ROS in salinity, heavy metal, and flooding responses in rice. 2014 Front Plant Sci Department of Plant Physiology, Faculty of Biology, Philipps University , Marburg, Germany. Plant growth and developmental processes as well as abiotic and biotic stress adaptations are regulated by small endogenous signaling molecules. Among these, phytohormones such as the gaseous alkene ethylene and reactive oxygen species (ROS) play an important role in mediating numerous specific growth or cell death responses. While apoplastic ROS are generated by plasma membrane-located respiratory burst oxidase homolog proteins, intracellular ROS are produced mainly in electron transfer chains of mitochondria and chloroplasts. Ethylene accumulates in plants due to physical entrapment or by enhanced ethylene biosynthesis. A major crop that must endure high salt and heavy metal concentrations upon flooding in regions of Asia is rice. Ethylene and ROS have been identified as the major signals that mediate salinity, chromium, and flooding stress in rice. This mini review focuses on (i) what is known about ethylene and ROS level control during these abiotic stresses in rice, (ii) how the two signals mediate growth or death processes, and (iii) feedback mechanisms that in turn regulate ethylene and ROS signaling. None Transgenic rice expressing a codon-modified synthetic CP4-EPSPS confers tolerance to broad-spectrum herbicide, glyphosate. 2014 Plant Cell Rep National Research Centre on Plant Biotechnology, Lal Bahadur Shastri Building, Pusa Campus, New Delhi, 110012, India. Highly tolerant herbicide-resistant transgenic rice was developed by expressing codon-modified synthetic CP4 - EPSPS . The transformants could tolerate up to 1 % commercial glyphosate and has the potential to be used for DSR (direct-seeded rice). Weed infestation is one of the major biotic stress factors that is responsible for yield loss in direct-seeded rice (DSR). Herbicide-resistant rice has potential to improve the efficiency of weed management under DSR. Hence, the popular indica rice cultivar IR64, was genetically modified using Agrobacterium-mediated transformation with a codon-optimized CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene, with N-terminal chloroplast targeting peptide from Petunia hybrida. Integration of the transgenes in the selected rice plants was confirmed by Southern hybridization and expression by Northern and herbicide tolerance assays. Transgenic plants showed EPSPS enzyme activity even at high concentrations of glyphosate, compared to untransformed control plants. T 0, T 1 and T 2 lines were tested by herbicide bioassay and it was confirmed that the transgenic rice could tolerate up to 1 % of commercial Roundup, which is five times more in dose used to kill weeds under field condition. All together, the transgenic rice plants developed in the present study could be used efficiently to overcome weed menace. None Dynamic quantitative trait locus analysis of seed vigor at three maturity stages in rice. 2014 PLoS One The Laboratory of Seed Science and Technology, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing Jiangsu, PR China. Seed vigor is an important characteristic of seed quality. In this study, one rice population of recombinant inbred lines (RILs) was used to determine the genetic characteristics of seed vigor, including the germination potential, germination rate, germination index and time for 50% of germination, at 4 (early), 5 (middle) and 6 weeks (late) after heading in two years. A total of 24 additive and 9 epistatic quantitative trait loci (QTL) for seed vigor were identified using QTL Cartographer and QTLNetwork program respectively in 2012; while 32 simple sequence repeat (SSR) markers associated with seed vigor were detected using bulked segregant analysis (BSA) in 2013. The additive, epistatic and QTL × development interaction effects regulated the dry maturity developmental process to improve seed vigor in rice. The phenotypic variation explained by each additive, epistatic QTL and QTL × development interaction ranged from 5.86 to 40.67%, 4.64 to 11.28% and 0.01 to 1.17%, respectively. The QTLs were rarely co-localized among the different maturity stages; more QTLs were expressed at the early maturity stage followed by the late and middle stages. Twenty additive QTLs were stably expressed in two years which might play important roles in establishment of seed vigor in different environments. By comparing chromosomal positions of these stably expressed additive QTLs with those previously identified, the regions of QTL for seed vigor are likely to coincide with QTL for grain size, low temperature germinability and seed dormancy; while 5 additive QTL might represent novel genes. Using four selected RILs, three cross combinations of seed vigor for the development of RIL populations were predicted; 19 elite alleles could be pyramided by each combination. None Rare allele of a previously unidentified histone H4 acetyltransferase enhances grain weight, yield, and plant biomass in rice. 2014 Proc Natl Acad Sci U S A Bioscience and Biotechnology Center, jacobsen@ucla.edu songxj@ibcas.ac.cn ashi@agr.nagoya-u.ac.jp. Grain weight is an important crop yield component; however, its underlying regulatory mechanisms are largely unknown. Here, we identify a grain-weight quantitative trait locus (QTL) encoding a new-type GNAT-like protein that harbors intrinsic histone acetyltransferase activity (OsglHAT1). Our genetic and molecular evidences pinpointed the QTL-OsglHAT1's allelic variations to a 1.2-kb region upstream of the gene body, which is consistent with its function as a positive regulator of the traits. Elevated OsglHAT1 expression enhances grain weight and yield by enlarging spikelet hulls via increasing cell number and accelerating grain filling, and increases global acetylation levels of histone H4. OsglHAT1 localizes to the nucleus, where it likely functions through the regulation of transcription. Despite its positive agronomical effects on grain weight, yield, and plant biomass, the rare allele elevating OsglHAT1 expression has so far escaped human selection. Our findings reveal the first example, to our knowledge, of a QTL for a yield component trait being due to a chromatin modifier that has the potential to improve crop high-yield breeding. OsglHAT1 qEMF3, a novel QTL for the early-morning flowering trait from wild rice, Oryza officinalis, to mitigate heat stress damage at flowering in rice, O. sativa. 2014 J Exp Bot NARO Institute of Crop Science, NARO, 2-1-18 Kannondai, Tsukuba, Ibaraki 305-8518, Japan. A decline in rice (Oryza sativa L.) production caused by heat stress is one of the biggest concerns resulting from future climate change. Rice spikelets are most susceptible to heat stress at flowering. The early-morning flowering (EMF) trait mitigates heat-induced spikelet sterility at the flowering stage by escaping heat stress during the daytime. We attempted to develop near-isogenic lines (NILs) for EMF in the indica-type genetic background by exploiting the EMF locus from wild rice, O. officinalis (CC genome). A stable quantitative trait locus (QTL) for flower opening time (FOT) was detected on chromosome 3. A QTL was designated as qEMF3 and it shifted FOT by 1.5-2.0h earlier for cv. Nanjing 11 in temperate Japan and cv. IR64 in the Philippine tropics. NILs for EMF mitigated heat-induced spikelet sterility under elevated temperature conditions completing flower opening before reaching 35°C, a general threshold value leading to spikelet sterility. Quantification of FOT of cultivars popular in the tropics and subtropics did not reveal the EMF trait in any of the cultivars tested, suggesting that qEMF3 has the potential to advance FOT of currently popular cultivars to escape heat stress at flowering under future hotter climates. This is the first report to examine rice with the EMF trait through marker-assisted breeding using wild rice as a genetic resource. None Photoperiodic Flowering: Time Measurement Mechanisms in Leaves. 2014 Annu Rev Plant Biol Department of Biology, University of Washington, Seattle, WA 98195-1800 and Department of Life Sciences, Ajou University, Suwon 443-749, Korea. Many plants use information about changing day length (photoperiod) to align their flowering time with seasonal changes to increase reproductive success. A mechanism for photoperiodic time measurement is present in leaves, and the day-length-specific induction of the FLOWERING LOCUS T (FT) gene, which encodes florigen, is a major final output of the pathway. Here, we summarize the current understanding of the molecular mechanisms by which photoperiodic information is perceived in order to trigger FT expression in Arabidopsis as well as in the primary cereals wheat, barley, and rice. In these plants, the differences in photoperiod are measured by interactions between circadian-clock-regulated components, such as CONSTANS (CO), and light signaling. The interactions happen under certain day-length conditions, as previously predicted by the external coincidence model. In these plants, the coincidence mechanisms are governed by multilayered regulation with numerous conserved as well as unique regulatory components, highlighting the breadth of photoperiodic regulation across plant species. Expected final online publication date for the Annual Review of Plant Biology Volume 66 is April 29, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates. None Chromosomal locations of a gene underlying heat-accelerated brown spot formation and its suppressor genes in rice. 2014 Mol Genet Genomics Agrogenomics Research Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602, Japan, fukuda@affrc.go.jp. Brown spots on mature leaves from the heading to ripening stages in rice are considered to be lesions induced by heat stress. However, there are few studies of lesions that are induced by heat stress rather than by pathogen infections. To understand the genetic background underlying such lesions, we used the chromosome segment substitution line (CSSL) SL518, derived from a distant cross between rice cultivars Koshihikari (japonica) and Nona Bokra (indica). We observed brown spots on mature leaf blades of the CSSL, although the parents barely showed any spots. Spot formation in SL518 was accelerated by high temperature, suggesting that the candidate gene for spot formation is related to heat stress response. Using progeny derived from a cross between SL518 and Koshihikari, we mapped the causative gene, BROWN-SPOTTED LEAF 1 (BSPL1), on chromosome 5. We speculated that one or more Nona Bokra genes suppress spot formation caused by BSPL1 and identified candidate genomic regions on chromosomes 2 and 9 using a cross between a near-isogenic line for BSPL1 and other CSSLs possessing Nona Bokra segments in the Koshihikari genetic background. In conclusion, our data support the concept that multiple genes are complementarily involved in brown spot formation induced by heat stress and will be useful for cloning of the novel gene(s) related to the spot formation. None Photo-biotechnology as a tool to improve agronomic traits in crops. 2014 Biotechnol Adv Subtropical Horticulture Research Institute, Faculty of Biotechnology, Jeju National University, Jeju 690-756, South Korea. Phytochromes are photosensory phosphoproteins with crucial roles in plant developmental responses to light. Functional studies of individual phytochromes have revealed their distinct roles in the plant's life cycle. Given the importance of phytochromes in key plant developmental processes, genetically manipulating phytochrome expression offers a promising approach to crop improvement. Photo-biotechnology refers to the transgenic expression of phytochrome transgenes or variants of such transgenes. Several studies have indicated that crop cultivars can be improved by modulating the expression of phytochrome genes. The improved traits include enhanced yield, improved grass quality, shade-tolerance, and stress resistance. In this review, we discuss the transgenic expression of phytochrome A and its hyperactive mutant (Ser599Ala-PhyA) in selected crops, such as Zoysia japonica (Japanese lawn grass), Agrostis stolonifera (creeping bentgrass), Oryza sativa (rice), Solanum tuberosum (potato), and Ipomea batatas (sweet potato). The transgenic expression of PhyA and its mutant in various plant species imparts biotechnologically useful traits. Here, we highlight recent advances in the field of photo-biotechnology and review the results of studies in which phytochromes or variants of phytochromes were transgenically expressed in various plant species. We conclude that photo-biotechnology offers an excellent platform for developing crops with improved properties. None Growth, physiological adaptation, and gene expression analysis of two Egyptian rice cultivars under salt stress. 2014 Plant Physiol Biochem Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan. Abiotic stressors, such as high salinity, greatly affect plant growth. In an attempt to explore the mechanisms underlying salinity tolerance, physiological parameters of two local Egyptian rice (Oryza sativa L.) cultivars, Sakha 102 and Egyptian Yasmine, were examined under 50 mM NaCl stress for 14 days. The results indicate that Egyptian Yasmine is relatively salt tolerant compared to Sakha 102, and this was evident in its higher dry mass production, lower leaf Na(+) levels, and enhanced water conservation under salt stress conditions. Moreover, Egyptian Yasmine exhibited lower Na(+)/K(+) ratios in all tissues examined under salinity stress. The ability to maintain such traits seemed to differ in the leaves and roots of Egyptian Yasmine, and the root K(+) content was much higher in Egyptian Yasmine than in Sakha 102. In order to understand the basis for these differences, we studied transcript levels of genes encoding Na(+) and K(+) transport proteins in different tissues. In response to salinity stress, Egyptian Yasmine showed induction of expression of some membrane transporter/channel genes that may contribute to Na(+) exclusion from the shoots (OsHKT1;5), limiting excess Na(+) entry into the roots (OsLti6b), K(+) uptake (OsAKT1), and reduced expression of a Na(+) transporter gene (OsHKT2;1). Therefore, the active regulation of genes related to Na(+) transport at the transcription level may be involved in salt tolerance mechanisms of Egyptian Yasmine, and these mechanisms offer the promise of improved salinity stress tolerance in local Egyptian rice genotypes. None Pectin enhances rice (Oryza sativa) root phosphorus remobilization. 2014 J Exp Bot State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences; Zhejiang University, Hangzhou 310058, China. Plants growing in phosphorus (P)-deficient conditions can either increase their exploration of the environment (hence increasing P uptake) or can solubilize and reutilize P from established tissue sources. However, it is currently unclear if P stored in root cell wall can be reutilized. The present study shows that culture of the rice cultivars 'Nipponbare' (Nip) and 'Kasalath' (Kas) in P-deficient conditions results in progressive reductions in root soluble inorganic phosphate (Pi). However, Nip consistently maintains a higher level of soluble Pi and lower relative cell wall P content than does Kas, indicating that more cell wall P is released in Nip than in Kas. P-deficient Nip has a greater pectin and hemicellulose 1 (HC1) content than does P-deficient Kas, consistent with the significant positive relationship between pectin and root-soluble Pi levels amongst multiple rice cultivars. These observations suggest that increased soluble Pi might result from increased pectin content during P starvation. In vitro experiments showed that pectin releases Pi from insoluble FePO4. Furthermore, an Arabidopsis thaliana mutant with reduced pectin levels (qua1-2), has less root soluble Pi and is more sensitive to P deficiency than the wild type (WT) Col-0, whereas NaCl-treated WT plants exhibit both an increased root pectin content and an elevated soluble Pi content during P-starvation. These observations indicate that pectin can facilitate the remobilization of P deposited in the cell wall. This is a previously unknown mechanism for the reutilization of P in P-starved plants. None Uptake kinetics and translocation of selenite and selenate as affected by iron plaque on root surfaces of rice seedlings. 2014 Planta Key Laboratory of Plant-Soil Interactions of the Ministry of Education, College of Resources and Environmental Sciences, China Agricultural University, Beijing, 100193, People's Republic of China, hqcauedu@126.com. Iron plaque on root surfaces greatly influenced selenium uptake and played different roles in selenite and selenate uptake. Iron plaque commonly forms on rice root surfaces under flooded conditions, but little is known about the relationship between iron plaque and selenium (Se) accumulation. Here, we investigate the effects of iron plaque on Se uptake by and translocation within rice (Oryza sativa) seedlings, and the kinetics of selenite and selenate influx into rice roots (with or without iron plaque) were determined in short-term (30 min) experiments. Rice seedlings were planted in nutrient solutions containing different levels of ferrous ion for 3 days and then transplanted into nutrient solutions with selenite or selenate. Se concentrations in iron plaque were positively associated with the amounts of iron plaque in both selenite and selenate treatments and iron plaque had a higher affinity for selenite than selenate. Results showed that iron plaque on root surfaces greatly influenced Se uptake and played different roles in selenite and selenate uptake. The selenite and selenate uptake kinetics results demonstrated that the presence of iron plaque enhanced selenite uptake, but decreased selenate uptake. In addition, root-Se concentrations increased with the increasing amounts of iron plaque, but Se translocation from roots to shoots was reduced with the increasing amounts of iron plaque in the +selenite treatment. Iron plaque significantly influenced selenite uptake and might act as a pool to selenite accumulation in rice plants. However, iron plaque had no significant effect on selenate uptake or even as a barrier to selenate uptake. None Characterization of a panel of Vietnamese rice varieties using DArT and SNP markers for association mapping purposes. 2014 BMC Plant Biol Fail BackgroundThe development of genome-wide association studies (GWAS) in crops has made it possible to mine interesting alleles hidden in gene bank resources. However, only a small fraction of the rice genetic diversity of any given country has been exploited in the studies with worldwide sampling conducted to date. This study presents the development of a panel of rice varieties from Vietnam for GWAS purposes.ResultsThe panel, initially composed of 270 accessions, was characterized for simple agronomic traits (maturity class, grain shape and endosperm type) commonly used to classify rice varieties. We first genotyped the panel using Diversity Array Technology (DArT) markers. We analyzed the panel structure, identified two subpanels corresponding to the indica and japonica sub-species and selected 182 non-redundant accessions. However, the number of usable DArT markers (241 for an initial library of 6444 clones) was too small for GWAS purposes. Therefore, we characterized the panel of 182 accessions with 25,971 markers using genotyping by sequencing. The same indica and japonica subpanels were identified. The indica subpanel was further divided into six populations (I1 to I6) using a model-based approach. The japonica subpanel, which was more highly differentiated, was divided into 4 populations (J1 to J4), including a temperate type (J2). Passport data and phenotypic traits were used to characterize these populations. Some populations were exclusively composed of glutinous types (I3 and J2). Some of the upland rice varieties appeared to belong to indica populations, which is uncommon in this region of the world. Linkage disequilibrium decayed faster in the indica subpanel (r2 below 0.2 at 101 kb) than in the japonica subpanel (r2 below 0.2 at 425 kb), likely because of the strongest differentiation of the japonica subpanel. A matrix adapted for GWAS was built by eliminating the markers with a minor allele frequency below 5% and imputing the missing data. This matrix contained 21,814 markers. A GWAS was conducted on time to flowering to prove the utility of this panel.ConclusionsThis publicly available panel constitutes an important resource giving access to original allelic diversity. It will be used for GWAS on root and panicle traits. None Expression of a cyclophilin OsCyp2-P isolated from a salt-tolerant landrace of rice in tobacco alleviates stress via ion homeostasis and limiting ROS accumulation. 2014 Funct Integr Genomics Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India. Cyclophilins are a set of ubiquitous proteins present in all subcellular compartments, involved in a wide variety of cellular processes. Comparative bioinformatics analysis of the rice and Arabidopsis genomes led us to identify novel putative cyclophilin gene family members in both the genomes not reported previously. We grouped cyclophilin members with similar molecular weight and subtypes together in the phylogenetic tree which indicated their co-evolution in rice and Arabidopsis. We also characterized a rice cyclophilin gene, OsCyp2-P (Os02g0121300), isolated from a salinity-tolerant landrace, Pokkali. Publicly available massively parallel signature sequencing (MPSS) and microarray data, besides our quantitative real time PCR (qRT-PCR) data suggest that transcript abundance of OsCyp2-P is regulated under different stress conditions in a developmental and organ specific manner. Ectopic expression of OsCyp2-P imparted multiple abiotic stress tolerance to transgenic tobacco plants as evidenced by higher root length, shoot length, chlorophyll content, and K(+)/Na(+) ratio under stress conditions. Transgenic plants also showed reduced lipid peroxidase content, electrolyte leakage, and superoxide content under stress conditions suggesting better ion homeostasis than WT plants. Localization studies confirmed that OsCyp2-P is localized in both cytosol and nucleus, indicating its possible interaction with several other proteins. The overall results suggest the explicit role of OsCyp2-P in bestowing multiple abiotic stress tolerance at the whole plant level. OsCyp2-P operates via reactive oxygen species (ROS) scavenging and ion homeostasis and thus is a promising candidate gene for enhancing multiple abiotic stress tolerance in crop plants. OsCYP2|OsCyp2-P DNL1, encodes cellulose synthase-like D4, is a major QTL for plant height and leaf width in rice (Oryza sativa L.). 2014 Biochem Biophys Res Commun State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China. Electronic address: 2012101127@njau.edu.cn. To further understand the genetics characterize of plant height and leaf width, we selected two different rice germplasms in phenotypes, Xian80 and Suyunuo, to construct genetic population. QTL analysis in a 175 F2 population revealed that major QTLs, qPH12 and qLW12.2, explaining 50.00% and 57.08% phenotypic variation for plant height and leaf width, respectively, were located on the same interval of chromosome 12 flanking SSR markers RM519 and RM1103, and was named DNL1 (Dwarf and Narrowed Leaf 1). Using a segregating populations derived from F2 heterozygous individuals, a total of 1363 dwarfism and narrowed-leaf individuals was selected for screening recombinants. By high-resolution linkage analysis in 141 recombination events, DNL1 was narrowed to a 62.39kb region of InDel markers ID12M28 and HF43. The results of ORF analysis in target region and nucleotide sequence alignment indicated that DNL1 encodes cellulose synthase-like D4 protein, and a single nucleotide substitution (C2488T) in dnl1 result in decrease in plant height and leaf width. Bioinformatical analysis demonstrated that a conserved role for OsCSLD4 in the regulation of plant growth and development. Expression analysis for OsCSLDs showed CSLD4 was highly expressed in roots, while other CSLD members had comparatively lower expression levels, however no clearly evidence about CSLD4/DNL1 expression associated with its function. OsCD1|OsCSLD4|NRL1|ND1|sle1|DNL1 Identification of the region of rice 13 kDa prolamin essential for the formation of ER-derived protein bodies using a heterologous expression system. 2014 Biosci Biotechnol Biochem a Laboratory of Genetic Engineering, Graduate School of Life and Environmental Sciences , Kyoto Prefectural University , Kyoto , Japan. Cereal prolamins, which are alcohol-soluble seed storage proteins, can induce ER-derived protein bodies (PBs) in heterologous tissue. Like maize and wheat prolamins, rice prolamins can form ER-derived PBs, but the region of mature polypeptides that is essential for PB formation has not been identified. In this study, we examined the formation mechanisms of ER-derived PB-like structures by expressing rice 13 kDa prolamin-deletion mutants fused to green fluorescent protein (GFP) in heterologous tissues such as yeast. The 13 kDa prolamin-GFP fusion protein was stably accumulated in transgenic yeast and formed an ER-derived PB-like structure. In contrast, rice α-globulin-GFP fusion protein was transported to vacuoles. In addition, the middle and COOH-terminal regions of 13 kDa prolamin formed ER-derived PB-like structures, whereas the NH2-terminal region of 13 kDa prolamin did not form such structures. These results suggest that the middle and COOH-terminal regions of 13 kDa prolamin can be retained and thus can induce ER-derived PB in yeast. None GLUCAN SYNTHASE-LIKE 5 (GSL5) plays an essential role in male fertility by regulating callose metabolism during microsporogenesis in rice. 2014 Plant Cell Physiol Haidian District, Zhongguancun South Street, No. 12, Beijing 100081, China. Callose plays an important role in pollen development in flowering plants. In rice, ten genes encoding putative callose synthases have been identified; however, none of them has been functionally characterized. In this study, a rice Glucan Synthase-Like 5 (GSL5) knock-out mutant was isolated that exhibited a severe reduction in fertility. Pollen viability tests indicated that the pollen of the mutant was abnormal while the embryo sac was normal. Further, GSL5-RNA interference transgenic plants phenocopied the gsl5 mutant. The RNA expression of GSL5 was found to be knocked out in the gsl5 mutant and knocked known in GSL5-RNA interference transgenic plants by real-time RT-PCR analysis. The male sterility of the mutant was due to abnormal microspore development; an analysis of paraffin sections of the mutant anthers at various developmental stages revealed that abnormal microspore development began in late meiosis. Both the knock-out and knock-down of GSL5 caused a lack of callose in the primary cell wall of meiocytes and in the cell plate of tetrads. As a result, the callose wall of the microspores was defective. This was demonstrated by aniline blue staining and an immunogold labeling assay; the microspores could not maintain their shape, leading to premature swelling and even collapsed microspores. These data suggest that the callose synthase encoded by GSL5 plays a vital role in microspore development during late meiosis and is essential for male fertility in rice. GSL5 Genome-wide screening and functional analysis identify a large number of long noncoding RNAs involved in the sexual reproduction of rice. 2014 Genome Biol Fail Long noncoding RNAs (lncRNAs) play important roles in a wide range of biological processes in mammals and plants. However, the systematic examination of lncRNAs in plants lags behind that in mammals. Recently, lncRNAs have been identified in Arabidopsis and wheat; however, no systematic screening of potential lncRNAs has been reported for the rice genome.In this study, we perform whole transcriptome strand-specific RNA sequencing (ssRNA-seq) of samples from rice anthers, pistils, and seeds 5 days after pollination and from shoots 14 days after germination. Using these data, together with 40 available rice RNA-seq datasets, we systematically analyze rice lncRNAs and definitively identify lncRNAs that are involved in the reproductive process. The results show that rice lncRNAs have some different characteristics compared to those of Arabidopsis and mammals and are expressed in a highly tissue-specific or stage-specific manner. We further verify the functions of a set of lncRNAs that are preferentially expressed in reproductive stages and identify several lncRNAs as competing endogenous RNAs (ceRNAs), which sequester miR160 or miR164 in a type of target mimicry. More importantly, one lncRNA, XLOC_057324, is demonstrated to play a role in panicle development and fertility. We also develop a source of rice lncRNA-associated insertional mutants.Genome-wide screening and functional analysis enabled the identification of a set of lncRNAs that are involved in the sexual reproduction of rice. The results also provide a source of lncRNAs and associated insertional mutants in rice. None A mathematical model of phloem sucrose transport as a new tool for designing rice panicle structure for high grain yield. 2014 Plant Cell Physiol Faculty of Environmental Earth Science, Hokkaido University, N10W5, Kita-ku, Sapporo, 060-0810 Japan. Rice (Oryza sativa) is one of the most important food crops in the world. Numerous quantitative trait loci or genes controlling panicle architecture have been identified to increase grain yield. Yet grain yield, defined as the product of the number of well-ripened grains and their weight, is a complex trait that is determined by multiple factors such as source, sink, and translocation capacity. Mechanistic modelling capturing capacities of source, sink, and transport will help in the theoretical design of crop ideotypes that guarantee high grain yield. In this paper, we present a mathematical model simulating sucrose transport and grain growth within a complex phloem network. The model predicts that the optimal panicle structure for high yield shows a simple grain arrangement with few higher-order branches. In addition, numerical analyses revealed that inefficient delivery of carbon to panicles with higher-order branches prevails regardless of source capacity, indicating the importance of designing grain arrangement and phloem structure. Our model highlights the previously unexplored effect of grain arrangement on the yield, and provides numerical solutions for optimal panicle structure under various source and sink capacities. None Rice phenylalanine ammonia-lyase gene OsPAL4 is associated with broad spectrum disease resistance. 2014 Plant Mol Biol Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO, 80523-1177, USA. Most agronomically important traits, including resistance against pathogens, are governed by quantitative trait loci (QTL). QTL-mediated resistance shows promise of being effective and long-lasting against diverse pathogens. Identification of genes controlling QTL-based disease resistance contributes to breeding for cultivars that exhibit high and stable resistance. Several defense response genes have been successfully used as good predictors and contributors to QTL-based resistance against several devastating rice diseases. In this study, we identified and characterized a rice (Oryza sativa) mutant line containing a 750 bp deletion in the second exon of OsPAL4, a member of the phenylalanine ammonia-lyase gene family. OsPAL4 clusters with three additional OsPAL genes that co-localize with QTL for bacterial blight and sheath blight disease resistance on rice chromosome 2. Self-pollination of heterozygous ospal4 mutant lines produced no homozygous progeny, suggesting that homozygosity for the mutation is lethal. The heterozygous ospal4 mutant line exhibited increased susceptibility to three distinct rice diseases, bacterial blight, sheath blight, and rice blast. Mutation of OsPAL4 increased expression of the OsPAL2 gene and decreased the expression of the unlinked OsPAL6 gene. OsPAL2 function is not redundant because the changes in expression did not compensate for loss of disease resistance. OsPAL6 co-localizes with a QTL for rice blast resistance, and is down-regulated in the ospal4 mutant line; this may explain enhanced susceptibility to Magnoporthe oryzae. Overall, these results suggest that OsPAL4 and possibly OsPAL6 are key contributors to resistance governed by QTL and are potential breeding targets for improved broad-spectrum disease resistance in rice. PAL|OsPAL4 Genes associated with thermosensitive genic male sterility in rice identified by comparative expression profiling. 2014 BMC Genomics Fail Rice (Oryza sativa L.) is one of the most important crops. Thermosensitive genic male sterile (TGMS) lines and photoperiod-sensitive genic male sterile (PGMS) lines have been successfully used in hybridization to improve rice yields. However, the molecular mechanisms underlying male sterility transitions in most PGMS/TGMS rice lines are unclear. In the recently developed TGMS-Co27 line, the male sterility is based on co-suppression of a UDP-glucose pyrophosphorylase gene (Ugp1), but further study is needed to fully elucidate the molecular mechanisms involved.Microarray-based transcriptome profiling of meiosis-stage inflorescences of TGMS-Co27 and wild-type Hejiang 19 (H1493) plants grown at high and low temperatures revealed that 15462 probe sets representing 8303 genes were differentially expressed in the two lines, under the two conditions, or both. Environmental factors strongly affected global gene expression. Some genes important for pollen development were strongly repressed in TGMS-Co27 at high temperature. More significantly, series-cluster analysis of differentially expressed genes (DEGs) between TGMS-Co27 plants grown under the two conditions showed that low temperature induced the expression of a gene cluster. This cluster was found to be essential for sterility transition. It includes many meiosis stage-related genes that are probably important for thermosensitive male sterility in TGMS-Co27, inter alia: Arg/Ser-rich domain (RS)-containing zinc finger proteins, polypyrimidine tract-binding proteins (PTBs), DEAD/DEAH box RNA helicases, ZOS (C2H2 zinc finger proteins of Oryza sativa), at least one polyadenylate-binding protein and some other RNA recognition motif (RRM) domain-containing proteins involved in post-transcriptional processes, eukaryotic initiation factor 5B (eIF5B), ribosomal proteins (L37, L1p/L10e, L27 and L24), aminoacyl-tRNA synthetases (ARSs), eukaryotic elongation factor Tu (eEF-Tu) and a peptide chain release factor protein involved in translation. The differential expression of 12 DEGs that are important for pollen development, low temperature responses or TGMS was validated by quantitative RT-PCR (qRT-PCR).Temperature strongly affects global gene expression and may be the common regulator of fertility in PGMS/TGMS rice lines. The identified expression changes reflect perturbations in the transcriptomic regulation of pollen development networks in TGMS-Co27. Findings from this and previous studies indicate that sets of genes involved in post-transcriptional and translation processes are involved in thermosensitive male sterility transitions in TGMS-Co27. None Biofortification of rice with lysine using endogenous histones. 2014 Plant Mol Biol State Key Laboratory of Agrobiotechnology and School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China, gundamwong2003@yahoo.com.hk. Rice is the most consumed cereal grain in the world, but deficient in the essential amino acid lysine. Therefore, people in developing countries with limited food diversity who rely on rice as their major food source may suffer from malnutrition. Biofortification of stable crops by genetic engineering provides a fast and sustainable method to solve this problem. In this study, two endogenous rice lysine-rich histone proteins, RLRH1 and RLRH2, were over-expressed in rice seeds to achieve lysine biofortification. Their protein sequences passed an allergic sequence-based homology test. Their accumulations in rice seeds were raised to a moderate level by the use of a modified rice glutelin 1 promoter with lowered expression strength to avoid the occurrence of physiological abnormalities like unfolded protein response. The expressed proteins were further targeted to protein storage vacuoles for stable storage using a glutelin 1 signal peptide. The lysine content in the transgenic rice seeds was enhanced by up to 35 %, while other essential amino acids remained balanced, meeting the nutritional standards of the World Health Organization. No obvious unfolded protein response was detected. Different degrees of chalkiness, however, were detected in the transgenic seeds, and were positively correlated with both the levels of accumulated protein and lysine enhancement. This study offered a solution to the lysine deficiency in rice, while at the same time addressing concerns about food safety and physiological abnormalities in biofortified crops. None Heterotic groups of tropical indica rice germplasm. 2014 Theor Appl Genet International Rice Research Institute, DAPO Box 7777, 1301, Metro Manila, Philippines. Four heterotic groups were identified for tropical indica rice germplasm to develop hybrid rice in the tropics based on two studies. Heterotic groups are of fundamental importance in hybrid crop breeding. This study investigated hybrid yield, yield heterosis and combining ability within and among groups based on genetic distance derived from single-nucleotide polymorphism markers. The main objectives of the study were to (1) evaluate the magnitude of yield heterosis among marker-based groups, (2) identify possible heterotic groups for tropical indica hybrid rice, and (3) validate heterotic patterns concluded from a previous study. Seventeen rice parents selected from improved indica germplasm from the tropics with high genetic divergence and 136 derived hybrids were evaluated in five environments. The hybrids had more yield than their parents with an average of 24.1 % mid-parent heterosis. Genotype × environment interaction was the major factor affecting variations in yield and yield heterosis, which raised a necessity and a challenge to develop heterotic rice hybrid adapted to different regions and seasons in the tropics. Yield, yield heterosis and combining ability were significantly increased in inter-group than in intra-group hybrids. Four heterotic groups and three promising hybridization patterns, which could be used in tropical hybrid rice breeding, were identified based on marker-based grouping, yield and yield heterosis analyses in the two studies. The study reveals that molecular markers analysis can serve as a basis for assigning germplasm into heterotic groups and to provide guidelines for parental selection in hybrid rice breeding. None Comprehensive Gene Expression Analysis of Rice Aleurone Cells: Probing the Existence of an Alternative Gibberellin Receptor(s). 2014 Plant Physiol Nagoya University. Current gibberellin (GA) research indicates that GA must be perceived in plant nuclei by its cognate receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1). Recognition of GA by GID1 relieves the repression mediated by the DELLA protein, a model known as the GID1-DELLA GA perception system. There have been reports of potential GA-binding proteins in the plasma membrane that perceive GA and induce α-amylase expression in cereal aleurone cells, which is mechanistically different from the GID1-DELLA system. We therefore examined the expression of the rice α-amylase genes (RAmys) in rice (Oryza sativa) mutants impaired in the GA receptor (gid1) and DELLA repressor (slr1), and confirmed their lack of response to GA in gid1 mutants and constitutive expression in slr1 mutants. We also examined the expression of GA-regulated genes by genome-wide microarray and quantitative reverse-transcription PCR analyses, and confirmed that all GA-regulated genes are modulated by the GID1-DELLA system. Furthermore, we studied the regulatory network involved in GA signaling by using a set of mutants defective in genes involved in GA perception and gene expression, namely gid1, slr1, gid2 (GA-related F-box protein mutant), and gamyb (GA-related transacting factor mutant). Almost all GA up-regulated genes were regulated by the four named GA-signaling components. On the other hand, GA down-regulated genes showed different expression patterns with respect to GID2 and GAMYB; e.g. a considerable number of genes are not controlled by GAMYB or GID2 and GAMYB. Based on these observations, we present a comprehensive discussion of the intricate network of GA-regulated genes in rice aleurone cells. None Metabolic trade-offs between biomass synthesis and photosynthate export at different light intensities in a genome-scale metabolic model of rice. 2014 Front Plant Sci Cell Systems Modelling Group, Department of Biological and Medical Science, Oxford Brookes University Oxford, UK. Previously we have used a genome scale model of rice metabolism to describe how metabolism reconfigures at different light intensities in an expanding leaf of rice. Although this established that the metabolism of the leaf was adequately represented, in the model, the scenario was not that of the typical function of the leaf-to provide material for the rest of the plant. Here we extend our analysis to explore the transition to a source leaf as export of photosynthate increases at the expense of making leaf biomass precursors, again as a function of light intensity. In particular we investigate whether, when the leaf is making a smaller range of compounds for export to the phloem, the same changes occur in the interactions between mitochondrial and chloroplast metabolism as seen in biomass synthesis for growth when light intensity increases. Our results show that the same changes occur qualitatively, though there are slight quantitative differences reflecting differences in the energy and redox requirements for the different metabolic outputs. None Comparative proteomic analysis of indica and japonica rice varieties. 2014 Genet Mol Biol Institute of Life Sciences , Jiangsu University , Zhenjiang , PR China . Indica and japonica are two main subspecies of Asian cultivated rice (Oryza sativa L.) that differ clearly in morphological and agronomic traits, in physiological and biochemical characteristics and in their genomic structure. However, the proteins and genes responsible for these differences remain poorly characterized. In this study, proteomic tools, including two-dimensional electrophoresis and mass spectrometry, were used to globally identify proteins that differed between two sequenced rice varieties (93-11 and Nipponbare). In all, 47 proteins that differed significantly between 93-11 and Nipponbare were identified using mass spectrometry and database searches. Interestingly, seven proteins were expressed only in Nipponbare and one protein was expressed specifically in 93-11; these differences were confirmed by quantitative real-time PCR and proteomic analysis of other indica and japonica rice varieties. This is the first report to successfully demonstrate differences in the protein composition of indica and japonica rice varieties and to identify candidate proteins and genes for future investigation of their roles in the differentiation of indica and japonica rice. None Comparison of Plant-Type Phosphoenolpyruvate Carboxylases from Rice: Identification of Two Plant-Specific Regulatory Regions of the Allosteric Enzyme. 2014 Plant Cell Physiol Functional Plant Research Unit, National Institute of Agrobiological Sciences, Kannondai, Tsukuba 305-8602, Japan. Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of primary metabolism in bacteria, algae, and vascular plants, and it undergoes allosteric regulation by various metabolic effectors. Rice (Oryza sativa) has five plant-type PEPCs, four cytosolic and one chloroplastic. We investigated their kinetic properties using recombinant proteins and found that, like most plant-type PEPCs, rice cytosolic isozymes were activated by glucose 6-phosphate and by alkaline pH. In contrast, no such activation was observed for the chloroplastic isozyme, Osppc4. In addition, Osppc4 showed low affinity for the substrate phosphoenolpyruvate (PEP) and very low sensitivities to allosteric inhibitors Asp and Glu. By comparing the isozyme amino acid sequences and three-dimensional structures simulated on the basis of the reported crystal structures, we identified two regions where Osppc4 has unique features that can be expected to affect its kinetic properties. One is the N-terminal extension; replacement of the extension of Osppc2a (cytosolic) with that from Osppc4 reduced the Asp and Glu sensitivities to ~1/10 the wild-type values but left the PEP affinity unaffected. The other is the N-terminal loop, in which a conserved Lys at the N-terminal end is replaced with a Glu-Ala pair in Osppc4. Replacement of the Lys of Osppc2a with Glu-Ala lowered the PEP affinity to 1/4 the wild-type level (down to the Osppc4 level), without affecting inhibitor sensitivity. Both the N-terminal extension and the N-terminal loop are specific to plant-type PEPCs, suggesting that plant-type isozymes acquired these regions so that their activity could be regulated properly at the sites where they function. Osppc4 Upregulation of jasmonate biosynthesis and jasmonate-responsive genes in rice leaves in response to a bacterial pathogen mimic. 2014 Funct Integr Genomics CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, 500 007, Andhra Pradesh, India. Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice, secretes several cell wall degrading enzymes including cellulase (ClsA) and lipase/esterase (LipA). Prior treatment of rice leaves with purified cell wall degrading enzymes such as LipA can confer enhanced resistance against subsequent X. oryzae pv. oryzae infection. To understand LipA-induced rice defense responses, microarray analysis was performed 12 h after enzyme treatment of rice leaves. This reveals that 867 (720 upregulated and 147 downregulated) genes are differentially regulated (≥2-fold). A number of genes involved in defense, stress, signal transduction, and catabolic processes were upregulated while a number of genes involved in photosynthesis and anabolic processes were downregulated. The microarray data also suggested upregulation of jasmonic acid (JA) biosynthetic and JA-responsive genes. Estimation of various phytohormones in LipA-treated rice leaves demonstrated a significant increase in the level of JA-Ile (a known active form of JA) while the levels of other phytohormones were not changed significantly with respect to buffer-treated control. This suggests a role for JA-Ile in cell wall damage induced innate immunity. Furthermore, a comparative analysis of ClsA- and LipA-induced rice genes has identified key rice functions that might be involved in elaboration of damage-associated molecular pattern (DAMP)-induced innate immunity. None Identification of quantitative trait loci conferring blast resistance in Bodao, a japonica rice landrace. 2014 Genet Mol Res State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Agriculture, Nanjing Agricultural University, Nanjing, China. Bodao, a japonica landrace from the Taihu Lake region of China, is highly resistant to most Chinese isolates of Magnaporthe oryzea, a form of rice blast. To effectively dissect the influence of genetics on this blast resistance, a population of 155 recombinant inbred lines (F2:8) derived from a cross of Bodao x Suyunuo was inoculated with 12 blast isolates. Using a quantitative trait locus (QTL) mapping approach, 13 QTL on chromosomes 1, 2, 9, 11, and 12 were detected from Bodao. Five QTL, including qtl11-1-1, qtl11-3-7, qtl11-4-9, qtl12-1-1, and qtl12-2-3, have not been previously reported. The qtl11-3-7 and qtl11-4-9 may be the two main effective QTL and resistant to 7 and 9 isolates, respectively. The results of the present study will be valuable for the fine mapping and cloning of these two new resistance genes. None Fine mapping of a grain weight quantitative trait locus, qGW6, using near isogenic lines derived from Oryza rufipogon IRGC105491 and Oryza sativa cultivar MR219. 2014 Genet Mol Res School of Environmental and Natural Resource Sciences, Faculty Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia meesiing@yahoo.com. Grain weight is a major component of rice grain yield and is controlled by quantitative trait loci. Previously, a rice grain weight quantitative trait locus (qGW6) was detected near marker RM587 on chromosome 6 in a backcross population (BC2F2) derived from a cross between Oryza rufipogon IRGC105491 and O. sativa cv. MR219. Using a BC2F5 population, qGW6 was validated and mapped to a region of 4.8 cM (1.2 Mb) in the interval between RM508 and RM588. Fine mapping using a series of BC4F3 near isogenic lines further narrowed the interval containing qGW6 to 88 kb between markers RM19268 and RM19271.1. According to the Duncan multiple range test, 8 BC4F4 near isogenic lines had significantly higher 100-grain weight (4.8 to 7.5% over MR219) than their recurrent parent, MR219 (P < 0.05). According to the rice genome automated annotation database, there are 20 predicted genes in the 88-kb target region, and 9 of them have known functions. Among the genes with known functions in the target region, in silico gene expression analysis showed that 9 were differentially expressed during the seed development stage(s) from gene expression series GSE6893; however, only 3 of them have known functions. These candidates provide targets for further characterization of qGW6, which will assist in understanding the genetic control of grain weight in rice. None Genome re-sequencing and bioinformatics analysis of a nutraceutical rice. 2014 Mol Genet Genomics Crop Quality Institute, Fujian Agriculture and Forestry University, 15 Shangxiadian Road, Cangshan District, Fuzhou, 350002, China, linjc2011@126.com. The genomes of two rice cultivars, Nipponbare and 93-11, have been well studied. However, there is little available genetic information about nutraceutical rice cultivars. To remedy this situation, the present study aimed to provide a basic genetic landscape of nutraceutical rice. The genome of Black-1, a black pericarp rice containing higher levels of anthocyanins, flavonoids, and a more potent antioxidant capacity, was sequenced at ≥30 × coverage using Solexa sequencing technology. The complete sequences of Black-1 genome shared more consensus sequences with indica cultivar 93-11 than with Nipponbare. With reference to the 93-11 genome, Black-1 contained 675,207 single-nucleotide polymorphisms, 43,130 insertions and deletions (1-5 bp), 1,770 copy number variations, and 10,911 presence/absence variations. These variations were observed to reside preferentially in Myb domains, NB-ARC domains and kinase domains, providing clues to the diversity of biological functions or secondary metabolisms in this cultivar. Intriguingly, 496 unique genes were identified by comparing it with the genomes of these two rice varieties; among the genes, 119 genes participate in the biosynthesis of secondary metabolites. Furthermore, several unique genes were predicted to be involved in the anthocyanins synthesis pathway. The genome-wide landscape of Black-1 uncovered by this study represents a valuable resource for further studies and for breeding nutraceutical rice varieties. None RiceQTLPro: an integrated database for quantitative trait loci marker mapping in rice plant. 2014 Bioinformation Genomics Division, National Academy of Agricultural Science (NAAS), Jeonju 560-500, Korea. The National Agricultural Biotechnology Information Center (NABIC) in South Korea reconstructed a RiceQTLPro database for gene positional analysis and structure prediction of the chromosomes. This database is an integrated web-based system providing information about quantitative trait loci (QTL) markers in rice plant. The RiceQTLPro has the three main features namely, (1) QTL markers list, (2) searching of markers using keyword, and (3) searching of marker position on the rice chromosomes. This updated database provides 112 QTL markers information with 817 polymorphic markers on each of the 12 chromosomes in rice.The database is available for free at http://nabic.rda.go.kr/gere/rice/geneticMap/ None Functional analysis of OsPGIP1 in rice sheath blight resistance. 2014 Plant Mol Biol National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan, People's Republic of China. As one of the most devastating diseases of rice, sheath blight causes severe rice yield loss. However, little progress has been made in rice breeding for sheath blight resistance. It has been reported that polygalacturonase inhibiting proteins can inhibit the degradation of the plant cell wall by polygalacturonases from pathogens. Here, we prokaryotically expressed and purified OsPGIP1 protein, which was verified by Western blot analysis. Activity assay confirmed the inhibitory activity of OsPGIP1 against the PGase from Rhizoctonia solani. In addition, the location of OsPGIP1 was determined by subcellular localization. Subsequently, we overexpressed OsPGIP1 in Zhonghua 11 (Oryza sativa L. ssp. japonica), and applied PCR and Southern blot analysis to identify the positive T0 transgenic plants with single-copy insertions. Germination assay of the seeds from T1 transgenic plants was carried out to select homozygous OsPGIP1 transgenic lines, and the expression levels of OsPGIP1 in these lines were analyzed by quantitative real-time PCR. Field testing of R. solani inoculation showed that the sheath blight resistance of the transgenic rice was significantly improved. Furthermore, the levels of sheath blight resistance were in accordance with the expression levels of OsPGIP1 in the transgenic lines. Our results reveal the functions of OsPGIP1 and its resistance mechanism to rice sheath blight, which will facilitate rice breeding for sheath blight resistance. OsPGIP1 High-throughput transformation pipeline for a Brazilian japonica rice with bar gene selection. 2014 Protoplasma International Center for Tropical Agriculture A.A. 6713, Cali, Colombia, beatadedicova@hotmail.com. The goal of this work was to establish a transformation pipeline for upland Curinga rice (Oryza sativa L. ssp. japonica) with bar gene selection employing bialaphos and phosphinothricin as selection agents. The following genes of interest: AtNCED3, Lsi1, GLU2, LEW2, PLD-alpha, DA1, TOR, AVP1, and Rubisco were cloned into the binary vector p7i2x-Ubi and were transferred into Agrobacterium strain EHA 105. Embryogenic calli derived from the mature embryos were transformed, and transgenic cells and shoots were selected on the medium supplemented with bialaphos or phosphinothricin (PPT) using a stepwise selection scheme. Molecular analyses were established using polymerase chain reaction and Southern blot for the bar gene and the NOS terminator. Overall, 273 putative transgenic plants were analyzed by Southern blot with 134 events identified. In total, 77 events had a single copy of the transgene integrated in the plant genome while 29 events had two copies. We tested backbone integration in 101 transgenic plants from all constructs and found 60 transgenic plants having no additional sequence integrated in the plant genome. The bar gene activity was evaluated by the chlorophenol red test and the leaf painting test using phosphinothricin with several transgenic plants. The majority of T0 plants carrying the single copy of transgene produced T1 seeds in the screen house. None Development of disease-resistant rice by optimized expression of WRKY45. 2014 Plant Biotechnol J Disease Resistant Crops Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. The rice transcription factor WRKY45 plays a central role in the salicylic acid signalling pathway and mediates chemical-induced resistance to multiple pathogens, including Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae. Previously, we reported that rice transformants overexpressing WRKY45 driven by the maize ubiquitin promoter were strongly resistant to both pathogens; however, their growth and yield were negatively affected because of the trade-off between the two conflicting traits. Also, some unknown environmental factor(s) exacerbated this problem. Here, we report the development of transgenic rice lines resistant to both pathogens and with agronomic traits almost comparable to those of wild-type rice. This was achieved by optimizing the promoter driving WRKY45 expression. We isolated 16 constitutive promoters from rice genomic DNA and tested their ability to drive WRKY45 expression. Comparisons among different transformant lines showed that, overall, the strength of WRKY45 expression was positively correlated with disease resistance and negatively correlated with agronomic traits. We conducted field trials to evaluate the growth of transgenic and control lines. The agronomic traits of two lines expressing WRKY45 driven by the OsUbi7 promoter (PO sUbi7 lines) were nearly comparable to those of untransformed rice, and both lines were pathogen resistant. Interestingly, excessive WRKY45 expression rendered rice plants sensitive to low temperature and salinity, and stress sensitivity was correlated with the induction of defence genes by these stresses. These negative effects were barely observed in the PO sUbi7 lines. Moreover, their patterns of defence gene expression were similar to those in plants primed by chemical defence inducers. OsWRKY45|WRKY45 Chloroplast Genome of Novel Rice Germplasm Identified in Northern Australia. 2014 Trop Plant Biol Queensland Alliance for Agriculture and Food Innovation, University of Queensland, Brisbane, Australia. Rice (Oryza sativa L.) was probably domesticated from O. rufipogon in Asia in the last 10,000 years. Relatives of cultivated rice (A genome species of Oryza) are found in South America, Africa, Australia and Asia. These A genome species are the close relatives of cultivated rice and represent the effective gene pool for rice improvement. Members of this group in Northern Australia include, an annual species, O. meridionalis, and two recently distinguished perennial taxa, to one of which the name O. rufipogon has been applied and the other a perennial form of O. meridionalis. Comparison of whole chloroplast genome sequences of these taxa has now been used to determine the relationships between the wild taxa and cultivated rice. The chloroplast genomes of the perennials were both found to be distinguished from O. rufipogon from Asia by 124 or 125 variations and were distinguished from each other by 53 variations. These populations have remained isolated from the overwhelming genetic impact of the large domesticated rice populations in Asia and may be unique descendants of the gene pool from which domesticated rice arose. The conservation of this wild genetic resource may be critical for global food security. None Stress response of OsETHE1 is altered in response to light and dark conditions. 2014 Plant Signal Behav a Plant Molecular Biology Group, International Center for Genetic Engineering and Biotechnology; Aruna Asaf Ali Marg ; New Delhi , India. ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 (ETHE1), encoding sulfur dioxygenase activity is believed to be an important candidate in sulfur metabolism, where it is involved in amino acid catabolism during carbohydrate starvation and embryo development as seen in Arabidopsis thaliana. OsETHE1, an ETHE1-encoding gene from rice, is in fact induced in response to abiotic stresses, condition which affects nutritional status of the plant, reflecting the need for nutrient remobilization. Sulfur reduction and assimilation are believed to be light-dependent processes and so the genes involved in sulfur oxidation must also be investigated for light-dependent regulatory effects. To this end, we show that the stress response of OsETHE1 is dependent on light and that darkness largely suppresses the stress response of this gene. However, the observed regulatory effect is intricate, varying according to the stress imposed; thereby suggesting the involvement of various aspects of signaling in this process. OsGLYII1|OsETHE1 Effect of air desiccation and salt stress factors on in vitro regeneration of rice (Oryza sativa L.). 2014 Plant Signal Behav a Plant Genetic Engineering Lab; Institute of Biological Sciences ; University of Rajshahi ; Rajshahi , Bangladesh. Enhancement of callus induction and its regeneration efficiency through in vitro techniques has been optimized for 2 abiotic stresses (salt and air desiccation) using 3 rice genotypes viz. BR10, BRRI dhan32 and BRRI dhan47. The highest frequency of callus induction was obtained for BRRI dhan32 (64.44%) in MS medium supplemented with 2, 4-D (2.5 mgL(-1)) and Kin (1.0 mgL(-1)). Different concentrations of NaCl (2.9, 5.9, 8.8 and 11.7 gL(-1)) were used and its effect was recorded on the basis of viability of calli (VC), relative growth rate (RGR), tolerance index (TI) and relative water content (RWC). It was observed that in all cases BRRI dhan47 showed highest performance on tolerance to VC (45.33%), RGR (1.03%), TI (0.20%) and RWC (10.23%) with 11.7 gL(-1) NaCl. Plant regeneration capability was recorded after partial air desiccation pretreatment to calli for 15, 30, 45 and 60 h. In this case BRRI dhan32 gave maximum number of regeneration (76.19%) when 4 weeks old calli were desiccated for 45 h. It was observed that air desiccation was 2-3 folds more effective for enhancing green plantlet regeneration compared to controls. Furthermore, desiccated calli also showed the better capability to survive in NaCl induced abiotic stress; and gave 1.9 fold (88.80%) increased regeneration in 11.7 gL(-1) salt level for BRRI dhan47. Analysis of variance (ANOVA) showed that the genotypes, air desiccation and NaCl had significant effect on plant regeneration at P < 0.01. None Genotyping of Endosperms To Determine Seed Dormancy Genes Regulating Germination Through Embryonic, Endospermic or Maternal Tissues in Rice. 2014 G3 (Bethesda) South Dakota State University; xingyou.gu@sdstate.edu. Seed dormancy is imposed by one or more of the embryo, endosperm and maternal tissues that belong to two generations and represent two ploidy levels. Many quantitative trait loci (QTL) have been identified for seed dormancy as measured by gross effects on reduced germination rate or delayed germination in crop or model plants. This research developed an endosperm genotype-based genetic approach to determine specific tissues through which a mapped QTL regulates germination using rice as a model. This approach involves testing germination velocity for partially after-ripened seeds harvested from single plants heterozygous for a tested QTL and genotyping endosperms from individual germinated and non-germinated seeds with a co-dominant DNA marker located on the QTL peak region. Information collected about the QTL includes genotypic frequencies in germinated and/or non-germinated subpopulations; allelic frequency distributions during a germination period; endosperm or embryo genotypic differences in germination velocity; and genotypic frequencies for gametes involved in the double fertilization to form the sampled seeds. Using this approach, the seed dormancy loci SD12, SD1-2, and SD7-1 were determined to regulate germination through the embryo, endosperm and maternal tissues, respectively; SD12 and SD1-2 acted additively on germination velocity in the offspring tissues; and SD12 was also associated with the preferential fertilization of male gametes in rice. This new genetic approach can be used to characterize mapped genes/QTL for tissue-specific functions in endospermic seeds and for marker-assisted selection of QTL alleles before or immediately after germination in crop breeding. None Phosphate transporter OsPht1;8 in rice plays an important role in phosphorus redistribution from source to sink organs and allocation between embryo and endosperm of seeds. 2014 Plant Sci State Key Laboratory of Crop Genetics and Germplasm Enhancement, MOA Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, 210095, China; Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao 266101, China. Phosphorus (P) redistribution from source to sink organs within plant is required for optimizing growth and development under P deficient condition. In this study, we knocked down expression of a phosphate transporter gene OsPht1;8 (OsPT8) selectively in shoot and/or in seed endosperm by RNA-interference using RISBZ1 and GluB-1 promoter (designate these transgenic lines as SSRi and EnSRi), respectively, to characterize the role of OsPT8 in P redistribution of rice. In comparison to wild type (WT) and EnSRi lines, SSRi lines under P deficient condition accumulated more P in old blades and less P in young blades, corresponding to attenuated and enriched transcripts of P-responsive genes in old and young blades, respectively. The ratio of total P in young blades to that in old blades decreased from 2.6 for WT to 0.9-1.2 for SSRi lines. During the grain-filling stage, relative to WT, SSRi lines showed the substantial decrease of total P content in both endosperm and embryo, while EnSRi lines showed 40-50% decrease of total P content in embryo but similar P content in endosperm. Taken together, our results demonstrate that OsPT8 plays a critical role in redistribution of P from source to sink organs and P homeostasis in seeds of rice. OsPht1;8|OsPT8 Genome-Wide Analysis of MicroRNAs and Their Target Genes Related to Leaf Senescence of Rice. 2014 PLoS One College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China. Grain production of rice (Oryza sativa L.) is a top priority in ensuring food security for human beings. One of the approaches to increase yield is to delay leaf senescence and to extend the available time for photosynthesis. MicroRNAs (miRNAs) are key regulators of aging and cellular senescence in eukaryotes. Here, to help understand their biological role in rice leaf senescence, we report identification of miRNAs and their putative target genes by deep sequencing of six small RNA libraries, six RNA-seq libraries and two degradome libraries from the leaves of two super hybrid rice, Nei-2-You 6 (N2Y6, age-resistant rice) and Liang-You-Pei 9 (LYP9, age-sensitive rice). In total 372 known miRNAs, 162 miRNA candidates and 1145 targets were identified. Compared with the expression of miRNAs in the leaves of LYP9, the numbers of miRNAs up-regulated and down-regulated in the leaves of N2Y6 were 47 and 30 at early stage of grain-filling, 21 and 17 at the middle stage, and 11 and 37 at the late stage, respectively. Six miRNA families, osa-miR159, osa-miR160 osa-miR164, osa-miR167, osa-miR172 and osa-miR1848, targeting the genes encoding APETALA2 (AP2), zinc finger proteins, salicylic acid-induced protein 19 (SIP19), auxin response factors (ARF) and NAC transcription factors, respectively, were found to be involved in leaf senescence through phytohormone signaling pathways. These results provided valuable information for understanding the miRNA-mediated leaf senescence of rice, and offered an important foundation for rice breeding. None Analysis of QTLs Associated with the Rice Quality Related Gene by Double Haploid Populations. 2014 Int J Genomics Division of Plant Biosciences, School of Applied Biosciences, College of Agriculture & Life Science, Kyungpook National University, Daegu 702-701, Republic of Korea. We investigated the growth characteristics and analyzed the physicochemical properties of a doubled haploid population derived from a cross between "Cheongcheong" and "Nagdong" to breed a rice variety that tastes good after cooking and to detect quantitative trait loci (QTLs) associated with the taste of cooked rice. The results showed that these compounds also represent a normal distribution. Correlation analysis of the amylose, protein, and lipid contents indicated that each compound is related to the taste of cooked rice. The QTLs related to amylose content were 4 QTLs, protein content was 2 QTLs, and lipid content was 2 QTLs. Four of the QTLs associated with amylose content were detected on chromosomes 7 and 11. The index of coincidence for the QTLs related to amylose, protein, and lipid content was 70%, respectively. These markers showing high percentage of coincidence can be useful to select desirable lines for rice breeding. None Constitutive expression and silencing of a novel seed specific calcium dependent protein kinase gene in rice reveals its role in grain filling. 2014 J Plant Physiol Biotechnology Laboratory, Directorate of Rice Research, Rajendranagar, Hyderabad 500 030, Andhra Pradesh, India. Ca(2+) sensor protein kinases are prevalent in most plant species including rice. They play diverse roles in plant signaling mechanism. Thirty one CDPK genes have been identified in rice and some are functionally characterized. In the present study, the newly identified rice CDPK gene OsCPK31 was functionally validated by overexpression and silencing in Taipei 309 rice cultivar. Spikelets of overexpressing plants showed hard dough stage within 15d after pollination (DAP) with rapid grain filling and early maturation. Scanning electron microscopy of endosperm during starch granule formation confirmed early grain filling. Further, seeds of overexpressing transgenic lines matured early (20-22 DAP) and the average number of maturity days reduced significantly. On the other hand, silencing lines showed more number of unfilled spikelet without any difference in maturity duration. It will be interesting to further decipher the role of OsCPK31 in biological pathways associated with distribution of photosynthetic assimilates during grain filling stage. OsCPK31 Overexpression of the bZIP transcription factor OsbZIP79 suppresses the production of diterpenoid phytoalexin in rice cells. 2014 J Plant Physiol Department of Biosciences, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi 320-8551, Japan; Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Electronic address: miyamoto@nasu.bio.teikyo-u.ac.jp. Phytoalexins are antimicrobial specialised metabolites that are produced by plants in response to pathogen attack. Momilactones and phytocassanes are major diterpenoid phytoalexins in rice that are synthesised from geranylgeranyl diphosphate that is derived from the methylerythritol phosphate (MEP) pathway. We have previously reported that rice cells overexpressing the basic leucine zipper (bZIP) transcription factor OsTGAP1 exhibit a hyperaccumulation of momilactones and phytocassanes, with hyperinductive expression of momilactone and phytocassane biosynthetic genes and MEP pathway genes, upon response to a chitin oligosaccharide elicitor. For a better understanding of OsTGAP1-mediated regulation of diterpenoid phytoalexin production, we identified OsTGAP1-interacting proteins using yeast two-hybrid screening. Among the OsTGAP1-interacting protein candidates, a TGA factor OsbZIP79 was investigated to verify its physical interaction with OsTGAP1 and involvement in the regulation of phytoalexin production. An in vitro pull-down assay demonstrated that OsTGAP1 and OsbZIP79 exhibited a heterodimeric as well as a homodimeric interaction. A bimolecular fluorescence complementation analysis also showed the interaction between OsTGAP1 and OsbZIP79 in vivo. Intriguingly, whereas OsbZIP79 transactivation activity was observed in a transient reporter assay, the overexpression of OsbZIP79 resulted in suppression of the elicitor-inducible expression of diterpenoid phytoalexin biosynthetic genes, and thus caused a decrease in the accumulation of phytoalexin in rice cells. These results suggest that OsbZIP79 functions as a negative regulator of phytoalexin production triggered by a chitin oligosaccharide elicitor in rice cells, although it remains open under which conditions OsbZIP79 can work with OsTGAP1. OsbZIP79 RNA-Seq analysis of differentially expressed genes in rice under varied nitrogen supplies. 2014 Gene State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China; University of Chinese Academy of Sciences, Beijing 100049, China. Ammonium is the main inorganic nitrogen source in paddy soil. Rice (Oryza sativa), an ammonium-preferring and -tolerant grain crop, is a valuable resource for researching ammonium-uptake mechanism and understanding the molecular networks that the plant copes with ammonium variation. To generate a broad survey of early responses affected by varied ammonium supplies in rice, RNA samples were prepared from the roots and shoots of rice plants subjected to nitrogen-free (0mM ammonium), 1mM ammonium and high ammonium (10mM ammonium) for a short period of 4h (1mM ammonium treatment as the control), respectively, and the transcripts were sequenced using the Illumina/HiSeq™ 2000 RNA sequencing (RNA-Seq) platform. By comparative analysis, 394 differentially expressed genes (DEGs) were identified in roots, among which, 143 and 251 DEGs were up- and down-regulated under nitrogen-free condition, respectively. In shoots, 468 (119 up-regulated/349 down-regulated) DEGs were found under such condition. However, with high ammonium treatment, only 63 genes (6 up-regulated/57 down-regulated) in roots and 115 genes in shoots (93 up-regulated/22 down-regulated) were differentially expressed. According to KEGG analysis, when exposed to nitrogen-free condition, DEGs participating in the carbohydrate and amino acid metabolisms were down-regulated (with 1 exception) in roots as well as in shoots, implying reduced carbohydrate and nitrogen metabolisms. Under high ammonium supply, all DEGs associated with carbohydrate and amino acid metabolisms were down-regulated in roots and to the contrary, up-regulated in shoots. Aldehyde dehydrogenase (ALDH, NAD(+)) [EC: 1.2.1.3] seemed to have played an important role in rice shoots under high ammonium condition, analysis results implicated a coordinative regulation of carbohydrate with amino acid metabolisms under nitrogen deficiency as well as the high ammonium conditions during a short period of several hours in rice. Moreover, transcripts with abundance variation might be precious gene resources in responding to different ammonium supplies in rice. None MPIC: A Mitochondrial Protein Import Components Database for Plant and Non-Plant Species. 2014 Plant Cell Physiol Australian Research Council Centre of Excellence in Plant Energy Biology, Bayliss Building M316, University of Western Australia, 35 Stirling Highway, Crawley 6009, Western Australia, Australia monika.murcha@uwa.edu.au. In the 2 billion years since the endosymbiotic event that gave rise to mitochondria, variations in mitochondrial protein import have evolved across different species. With the genomes of an increasing number of plant species sequenced, it is possible to gain novel insights into mitochondrial protein import pathways. We have generated the Mitochondrial Protein Import Components (MPIC) Database (DB; http://www.plantenergy.uwa.edu.au/applications/mpic) providing searchable information on the protein import apparatus of plant and non-plant mitochondria. An in silico analysis was carried out, comparing the mitochondrial protein import apparatus from 24 species representing various lineages from Saccharomyces cerevisiae (yeast) and algae to Homo sapiens (human) and higher plants, including Arabidopsis thaliana (Arabidopsis), Oryza sativa (rice) and other more recently sequenced plant species. Each of these species was extensively searched and manually assembled for analysis in the MPIC DB. The database presents an interactive diagram in a user-friendly manner, allowing users to select their import component of interest. The MPIC DB presents an extensive resource facilitating detailed investigation of the mitochondrial protein import machinery and allowing patterns of conservation and divergence to be recognized that would otherwise have been missed. To demonstrate the usefulness of the MPIC DB, we present a comparative analysis of the mitochondrial protein import machinery in plants and non-plant species, revealing plant-specific features that have evolved. None Plant-PrAS: A Database of Physicochemical and Structural Properties and Novel Functional Regions in Plant Proteomes. 2014 Plant Cell Physiol RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, 230-0045 Japan Department of Biotechnology and Life Sciences, Faculty of Technology, Tokyo University of Agriculture and Technology, Koganei, Tokyo, 184-8588 Japan. Arabidopsis thaliana is an important model species for studies of plant gene functions. Research on Arabidopsis has resulted in the generation of high-quality genome sequences, annotations and related post-genomic studies. The amount of annotation, such as gene-coding regions and structures, is steadily growing in the field of plant research. In contrast to the genomics resource of animals and microorganisms, there are still some difficulties with characterization of some gene functions in plant genomics studies. The acquisition of information on protein structure can help elucidate the corresponding gene function because proteins encoded in the genome possess highly specific structures and functions. In this study, we calculated multiple physicochemical and secondary structural parameters of protein sequences, including length, hydrophobicity, the amount of secondary structure, the number of intrinsically disordered regions (IDRs) and the predicted presence of transmembrane helices and signal peptides, using a total of 208,333 protein sequences from the genomes of six representative plant species, Arabidopsis thaliana, Glycine max (soybean), Populus trichocarpa (poplar), Oryza sativa (rice), Physcomitrella patens (moss) and Cyanidioschyzon merolae (alga). Using the PASS tool and the Rosetta Stone method, we annotated the presence of novel functional regions in 1,732 protein sequences that included unannotated sequences from the Arabidopsis and rice proteomes. These results were organized into the Plant Protein Annotation Suite database (Plant-PrAS), which can be freely accessed online at http://plant-pras.riken.jp/. None Rice transgene flow: its patterns, model and risk management. 2014 Plant Biotechnol J Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China. Progress has been made in a 12 year's systemic study on the rice transgene flow including (i) with experiments conducted at multiple locations and years using up to 21 pollen recipients, we have elucidated the patterns of transgene flow to different types of rice. The frequency to male sterile lines is 10(1) and 10(3) higher than that to O. rufipogon and rice cultivars. Wind speed and direction are the key meteorological factors affecting rice transgene flow. (ii) A regional applicable rice gene flow model is established and used to predict the maximum threshold distances (MTDs) of gene flow during 30 years in 993 major rice producing counties of southern China. The MTD0.1% for rice cultivars is basically ≤5 m in the whole region, despite climate differs significantly at diverse locations and years. This figure is particularly valuable for the commercialization and regulation of transgenic rice. (iii) The long-term fate of transgene integrated into common wild rice was investigated. Results demonstrated that the F1 hybrids of transgenic rice/O. rufipogon gradually disappeared within 3-5 years, and the Bt or bar gene was not detectable in the mixed population, suggesting the O. rufipogon may possess a strong mechanism of exclusiveness for self-protection. (iv) The flowering time isolation and a 2-m-high cloth-screen protection were proved to be effective in reducing transgene flow. We have proposed to use a principle of classification and threshold management for different types of rice. None Comparative transcriptome sequencing of tolerant rice introgression line and its parents in response to drought stress. 2014 BMC Genomics Institute of Crop Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, South Zhong-Guan-Cun Street 12#, Beijing 100081, China. zhouyongli@caas.cn. Rice (Oryza sativa. L) is more sensitive to drought stress than other cereals, and large genotypic variation in drought tolerance (DT) exists within the cultivated rice gene pool and its wild relatives. Selective introgression of DT donor segments into a drought-sensitive (DS) elite recurrent parent by backcrossing is an effective way to improve drought stress tolerance in rice. To dissect the molecular mechanisms underlying DT in rice, deep transcriptome sequencing was used to investigate transcriptome differences among a DT introgression line H471, the DT donor P28, and the drought-sensitive, recurrent parent HHZ under drought stress.The results revealed constitutively differential gene expression before stress and distinct global transcriptome reprogramming among the three genotypes under a time series of drought stress, consistent with their different genotypes and DT phenotypes. A set of genes with higher basal expression in both H471 and P28 compared with HHZ were functionally enriched in oxidoreductase and lyase activities, implying their positive role in intrinsic DT. Gene Ontology analysis indicated that common up-regulated genes in all three genotypes under mild drought stress were enriched in signaling transduction and transcription regulation. Meanwhile, diverse functional categories were characterized for the commonly drought-induced genes in response to severe drought stress. Further comparative transcriptome analysis between H471 and HHZ under drought stress found that introgression caused wide-range gene expression changes; most of the differentially expressed genes (DEGs) in H471 relative to HHZ under drought were beyond the identified introgressed regions, implying that introgression resulted in novel changes in expression. Co-expression analysis of these DEGs represented a complex regulatory network, including the jasmonic acid and gibberellin pathway, involved in drought stress tolerance in H471.Comprehensive gene expression profiles revealed that genotype-specific drought induced genes and genes with higher expression in the DT genotype under normal and drought conditions contribute jointly to DT improvement. The molecular genetic pathways of drought stress tolerance uncovered in this study, as well as the DEGs co-localized with DT-related QTLs and introgressed intervals, will serve as useful resources for further functional dissection of the molecular mechanisms of drought stress response in rice. None Identification and molecular characterization of tissue-preferred rice genes and their upstream regularly sequences on a genome-wide level. 2014 BMC Plant Biol Fail BackgroundGene upstream regularly sequences (URSs) can be used as one of the tools to annotate the biological functions of corresponding genes. In addition, tissue-preferred URSs are frequently used to drive the transgene expression exclusively in targeted tissues during plant transgenesis. Although many rice URSs have been molecularly characterized, it is still necessary and valuable to identify URSs that will benefit plant transformation and aid in analyzing gene function.ResultsIn this study, we identified and characterized root-, seed-, leaf-, and panicle-preferred genes on a genome-wide level in rice. Subsequently, their expression patterns were confirmed through quantitative real-time RT-PCR (qRT-PCR) by randomly selecting 9candidate tissue-preferred genes. In addition, 5 tissue-preferred URSs were characterized by investigating the URS::GUS transgenic plants. Of these URS::GUS analyses, the transgenic plants harboring LOC_Os03g11350 URS::GUS construct showed the GUS activity only in young pollen. In contrast, when LOC_Os10g22450 URS was used to drive the reporter GUS gene, the GUS activity was detected only in mature pollen. Interestingly, the LOC_Os10g34360 URS was found to be vascular bundle preferred and its activities were restricted only to vascular bundles of leaves, roots and florets. In addition, we have also identified two URSs from genes LOC_Os02G15090 and LOC_Os06g31070 expressed in a seed-preferred manner showing the highest expression levels of GUS activities in mature seeds.ConclusionBy genome-wide analysis, we have identified tissue-preferred URSs, five of which were further characterized using transgenic plants harboring URS::GUS constructs. These data might provide some evidence for possible functions of the genes and be a valuable resource for tissue-preferred candidate URSs for plant transgenesis. None Identification of nitrogen, phosphorus, and potassium deficiencies in rice based on static scanning technology and hierarchical identification method. 2014 PLoS One Institute of Applied Remote Sensing & Information Technology, Zhejiang University, Hangzhou, Zhejiang, China. Establishing an accurate, fast, and operable method for diagnosing crop nutrition is very important for crop nutrient management. In this study, static scanning technology was used to collect images of a rice sample's fully expanded top three leaves and corresponding sheathes. From these images, 32 spectral and shape characteristic parameters were extracted using an RGB mean value function and using the Regionprops function in MATLAB. Hierarchical identification was used to identify NPK deficiencies. First, the normal samples and non-normal (NPK deficiencies) samples were identified. Then, N deficiency and PK deficiencies were identified. Finally, P deficiency and K deficiency were identified. In the identification of every hierarchy, SVFS was used to select the optimal characteristic set for different deficiencies in a targeted manner, and Fisher discriminant analysis was used to build the diagnosis model. In the first hierarchy, the selected characteristics were the leaf sheath R, leaf sheath G, leaf sheath B, leaf sheath length, leaf tip R, leaf tip G, leaf area and leaf G. In the second hierarchy, the selected characteristics were the leaf sheath G, leaf sheath B, white region of the leaf sheath, leaf B, and leaf G. In the third hierarchy the selected characteristics were the leaf G, leaf sheath length, leaf area/leaf length, leaf tip G, difference between the 2nd and 3rd leaf lengths, leaf sheath G, and leaf lightness. The results showed that the overall identification accuracies of NPK deficiencies were 86.15, 87.69, 90.00 and 89.23% for the four growth stages. Data from multiple years were used for validation, and the identification accuracies were 83.08, 83.08, 89.23 and 90.77%. None Making sense of hormone-mediated defense networking: from rice to Arabidopsis. 2014 Front Plant Sci Laboratory of Phytopathology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University Ghent, Belgium. Phytohormones are not only essential for plant growth and development but also play central roles in triggering the plant immune signaling network. Historically, research aimed at elucidating the defense-associated role of hormones has tended to focus on the use of experimentally tractable dicot plants such as Arabidopsis thaliana. Emerging from these studies is a picture whereby complex crosstalk and induced hormonal changes mold plant health and disease, with outcomes largely dependent on the lifestyle and infection strategy of invading pathogens. However, recent studies in monocot plants are starting to provide additional important insights into the immune-regulatory roles of hormones, often revealing unique complexities. In this review, we address the latest discoveries dealing with hormone-mediated immunity in rice, one of the most important food crops and an excellent model for molecular genetic studies in monocots. Moreover, we highlight interactions between hormone signaling, rice defense and pathogen virulence, and discuss the differences and similarities with findings in Arabidopsis. Finally, we present a model for hormone defense networking in rice and describe how detailed knowledge of hormone crosstalk mechanisms can be used for engineering durable rice disease resistance. None Comparative in situ analyses of cell wall matrix polysaccharide dynamics in developing rice and wheat grain. 2014 Planta Rothamsted Research, Harpenden, AL5 2JQ, UK. Cell wall polysaccharides of wheat and rice endosperm are an important source of dietary fibre. Monoclonal antibodies specific to cell wall polysaccharides were used to determine polysaccharide dynamics during the development of both wheat and rice grain. Wheat and rice grain present near synchronous developmental processes and significantly different endosperm cell wall compositions, allowing the localisation of these polysaccharides to be related to developmental changes. Arabinoxylan (AX) and mixed-linkage glucan (MLG) have analogous cellular locations in both species, with deposition of AX and MLG coinciding with the start of grain filling. A glucuronoxylan (GUX) epitope was detected in rice, but not wheat endosperm cell walls. Callose has been reported to be associated with the formation of cell wall outgrowths during endosperm cellularisation and xyloglucan is here shown to be a component of these anticlinal extensions, occurring transiently in both species. Pectic homogalacturonan (HG) was abundant in cell walls of maternal tissues of wheat and rice grain, but only detected in endosperm cell walls of rice in an unesterified HG form. A rhamnogalacturonan-I (RG-I) backbone epitope was observed to be temporally regulated in both species, detected in endosperm cell walls from 12 DAA in rice and 20 DAA in wheat grain. Detection of the LM5 galactan epitope showed a clear distinction between wheat and rice, being detected at the earliest stages of development in rice endosperm cell walls, but not detected in wheat endosperm cell walls, only in maternal tissues. In contrast, the LM6 arabinan epitope was detected in both species around 8 DAA and was transient in wheat grain, but persisted in rice until maturity. None Analysis of genetic diversity and population structure of rice germplasm from north-eastern region of India and development of a core germplasm set. 2014 PLoS One Division of Genomic Resources, National Bureau of Plant Genetic Resources, New Delhi, 110 012, India. The North-Eastern region (NER) of India, comprising of Arunachal Pradesh, Assam, Manipur, Meghalaya, Mizoram, Nagaland and Tripura, is a hot spot for genetic diversity and the most probable origin of rice. North-east rice collections are known to possess various agronomically important traits like biotic and abiotic stress tolerance, unique grain and cooking quality. The genetic diversity and associated population structure of 6,984 rice accessions, originating from NER, were assessed using 36 genome wide unlinked single nucleotide polymorphism (SNP) markers distributed across the 12 rice chromosomes. All of the 36 SNP loci were polymorphic and bi-allelic, contained five types of base substitutions and together produced nine types of alleles. The polymorphic information content (PIC) ranged from 0.004 for Tripura to 0.375 for Manipur and major allele frequency ranged from 0.50 for Assam to 0.99 for Tripura. Heterozygosity ranged from 0.002 in Nagaland to 0.42 in Mizoram and gene diversity ranged from 0.006 in Arunachal Pradesh to 0.50 in Manipur. The genetic relatedness among the rice accessions was evaluated using an unrooted phylogenetic tree analysis, which grouped all accessions into three major clusters. For determining population structure, populations K = 1 to K = 20 were tested and population K = 3 was present in all the states, with the exception of Meghalaya and Manipur where, K = 5 and K = 4 populations were present, respectively. Principal Coordinate Analysis (PCoA) showed that accessions were distributed according to their population structure. AMOVA analysis showed that, maximum diversity was partitioned at the individual accession level (73% for Nagaland, 58% for Arunachal Pradesh and 57% for Tripura). Using POWERCORE software, a core set of 701 accessions was obtained, which accounted for approximately 10% of the total NE India collections, representing 99.9% of the allelic diversity. The rice core set developed will be a valuable resource for future genomic studies and crop improvement strategies. None Genetic mechanisms underlying yield potential in the rice high-yielding cultivar Takanari, based on reciprocal chromosome segment substitution lines. 2014 BMC Plant Biol Fail BackgroundIncreasing rice yield potential is a major objective in rice breeding programs, given the need for meeting the demands of population growth, especially in Asia. Genetic analysis using genomic information and high-yielding cultivars can facilitate understanding of the genetic mechanisms underlying rice yield potential. Chromosome segment substitution lines (CSSLs) are a powerful tool for the detection and precise mapping of quantitative trait loci (QTLs) that have both large and small effects. In addition, reciprocal CSSLs developed in both parental cultivar backgrounds may be appropriate for evaluating gene activity, as a single factor or in epistatic interactions.ResultsWe developed reciprocal CSSLs derived from a cross between Takanari (one of the most productive indica cultivars) and a leading japonica cultivar, Koshihikari; both the cultivars were developed in Japan. Forty-one CSSLs covered most of the Takanari genome in the Koshihikari background and 39 CSSLs covered the Koshihikari genome in the Takanari background. Using the reciprocal CSSLs, we conducted yield trials under canopy conditions in paddy fields. While no CSSLs significantly exceeded the recurrent parent cultivar in yield, genetic analysis detected 48 and 47 QTLs for yield and its components in the Koshihikari and Takanari backgrounds, respectively. A number of QTLs showed a trade-off, in which the allele with increased sink-size traits (spikelet number per panicle or per square meter) was associated with decreased ripening percentage or 1000-grain weight. These results indicate that increased sink size is not sufficient to increase rice yield in both backgrounds. In addition, most QTLs were detected in either one of the two genetic backgrounds, suggesting that these loci may be under epistatic control with other gene(s).ConclusionsWe demonstrated that the reciprocal CSSLs are a useful tool for understanding the genetic mechanisms underlying yield potential in the high-yielding rice cultivar Takanari. Our results suggest that sink-size QTLs in combination with QTLs for source strength or translocation capacity, as well as careful attention to epistatic interactions, are necessary for increasing rice yield. Thus, our findings provide a foundation for developing rice cultivars with higher yield potential in future breeding programs. None Genome-wide association study of blast resistance in indica rice. 2014 BMC Plant Biol Fail BackgroundRice blast disease is one of the most serious and recurrent problems in rice-growing regions worldwide. Most resistance genes were identified by linkage mapping using genetic populations. We extensively examined 16 rice blast strains and a further genome-wide association study based on genotyping 0.8 million single nucleotide polymorphism variants across 366 diverse indica accessions.ResultsTotally, thirty associated loci were identified. The strongest signal (Chr11_6526998, P =1.17¿×¿10¿17) was located within the gene Os11g0225100, one of the rice Pia-blast resistance gene. Another association signal (Chr11_30606558) was detected around the QTL Pif. Our study identified the gene Os11g0704100, a disease resistance protein containing nucleotide binding site-leucine rich repeat domain, as the main candidate gene of Pif. In order to explore the potential mechanism underlying the blast resistance, we further examined a locus in chromosome 12, which was associated with CH149 (P =7.53¿×¿10¿15). The genes, Os12g0424700 and Os12g0427000, both described as kinase-like domain containing protein, were presumed to be required for the full function of this locus. Furthermore, we found some association on chromosome 3, in which it has not been reported any loci associated with rice blast resistance. In addition, we identified novel functional candidate genes, which might participate in the resistance regulation.ConclusionsThis work provides the basis of further study of the potential function of these candidate genes. A subset of true associations would be weakly associated with outcome in any given GWAS; therefore, large-scale replication is necessary to confirm our results. Future research will focus on validating the effects of these candidate genes and their functional variants using genetic transformation and transferred DNA insertion mutant screens, to verify that these genes engender resistance to blast disease in rice. None Genome-wide transcriptome profiles of rice hybrids and their parents. 2014 Int J Mol Sci China National Rice Research Institute, Hangzhou 310006, China. ezhiguo@caas.cn. Heterosis is a widely studied phenomenon in several plant species. However, its genetic basis still remains to be elucidated. In this study, we used RNA-seq data from two rice genotypes and their reciprocal hybrids, and used a combination of transcriptome profiling and allele-specific expression analysis to identify genes that are differentially expressed in the hybrids and their parents or expressed in an allele-specific manner. The differentially expressed genes (DEGs) were identified by a pairwise comparison of the four genotypes. Detailed annotation of DEGs suggested that these genes showed enrichment in some gene ontology categories, and they tend to have tissue-specific expression patterns compared to all genes. A total of 1033 (10.24%) of 10,195 genes with informative single nucleotide polymorphism (SNPs) were identified as ASE genes. These allele-specific expessed (ASE) genes showed a broader expression breadth suggesting that they function in diverse developmental stages. Among 1033 ASE genes, we also identified 45 ASE transcription factors belonging to 17 transcription factor families. These ASE transcription factors may act in trans to regulate gene expression in filial 1 (F1) hybrids. Our analyses provide a comprehensive transcriptome profile of rice hybrids and their parents, and would be a useful resource for the rice research community. None Plasma membrane receptor-like kinase leaf panicle 2 acts downstream of the DROUGHT AND SALT TOLERANCE transcription factor to regulate drought sensitivity in rice. 2014 J Exp Bot National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China. Drought is a recurring climatic hazard that reduces the crop yields. To avoid the negative effects of drought on crop production, extensive efforts have been devoted to investigating the complex mechanisms of gene expression and signal transduction during drought stress. Receptor-like kinases (RLKs) play important roles in perceiving extracellular stimuli and activating downstream signalling responses. The rice genome contains >1100 RLK genes, of which only two are reported to function in drought stress. A leucine-rich repeat (LRR)-RLK gene named Leaf Panicle 2 (LP2) was previously found to be strongly expressed in leaves and other photosynthetic tissues, but its function remains unclear. In the present study, it was shown that the expression of LP2 was down-regulated by drought and abscisic acid (ABA). Transgenic plants overexpressing LP2 accumulated less H2O2, had more open stomata in leaves, and showed hypersensitivity to drought stress. Further investigation revealed that transcription of LP2 was directly regulated by the zinc finger transcription factor DROUGHT AND SALT TOLERANCE (DST). In addition, LP2 was identified as a functional kinase localized to the plasma membrane and interacted with the drought-responsive aquaporin proteins OsPIP1; 1, OsPIP1; 3, and OsPIP2; 3. Thus, the findings provided evidence that the LRR-RLK LP2, transcriptionally regulated by the drought-related transcription factor DST, served as a negative regulator in drought response. DST Genome-wide identification and evolutionary analysis of positively selected miRNA genes in domesticated rice. 2014 Mol Genet Genomics Department of Agronomy, College of Agriculture and Food Science, Zhejiang A & F University, Lin'an, Hangzhou, 311300, People's Republic of China, liuqp@zafu.edu.cn. The next-generation sequencing of tens to hundreds of plant genotypes made the uncovering of miRNA genes evolution available at the genome-wide level. Using the combinations of population genetics and evolutionary biology approaches, we have identified 21 miRNA loci having significant negative Tajima's D and Fu and Li's D* and F* values, of which 14 miRNAs (ps-miRNAs) showing clear signatures of positive selection in domesticated rice. The average sequence diversity (π) of the 21 miRNAs in cultivated rice is only 13.8 % of that in their wild progenitors. Interestingly, protein-coding genes immediately flanking these ps-miRNAs are apparently under weaker selective constraints. Totally, the 21 miRNAs are predicted to target 68 mRNA genes, of which 12 targets are estimated to have endured positive selection during rice evolution. In addition, the expression pattern and potential biological functions of ps-miRNAs targets are further investigated by searching published micro-array data and different mutant databases, respectively. We conclude that miRNAs, like protein-coding genes, should be crucial for driving rice evolution. These analyses may deepen our understanding on the miRNA genes evolution and functions during rice domestication. None Meta-analysis of quantitative trait loci for grain yield and component traits under reproductive-stage drought stress in an upland rice population. 2014 Mol Breed Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development, Jl. Tentara Pelajar 3A, Bogor, 16111 Indonesia. A recombinant inbred population developed from a cross between high-yielding lowland rice (Oryza sativa L.) subspecies indica cv. IR64 and upland tropical rice subspecies japonica cv. Cabacu was used to identify quantitative trait loci (QTLs) for grain yield (GY) and component traits under reproductive-stage drought stress. One hundred fifty-four lines were grown in field trials in Indonesia under aerobic conditions by giving surface irrigation to field capacity every 4 days. Water stress was imposed for a period of 15 days during pre-flowering by withholding irrigation at 65 days after seeding. Leaf rolling was scored at the end of the stress period and eight agronomic traits were evaluated after recovery. The population was also evaluated for root pulling force, and a total of 201 single nucleotide polymorphism markers were used to construct the molecular genetic linkage map and QTL mapping. A QTL for GY under drought stress was identified in a region close to the sd1 locus on chromosome 1. QTL meta-analysis across diverse populations showed that this QTL was conserved across genetic backgrounds and co-localized with QTLs for leaf rolling and osmotic adjustment (OA). A QTL for percent seed set and grains per panicle under drought stress was identified on chromosome 8 in the same region as a QTL for OA previously identified in three different populations. None Fine mapping and candidate gene analysis of a major QTL for panicle structure in rice. 2014 Plant Cell Rep Key Laboratory of Plant Functional Genomics of the Ministry of Education, Jiangsu Key Laboratory of Crop Genetics and Physiology College of Agriculture, Yangzhou University, Yangzhou, 225009, China, youlinp@hotmail.com. A gene not only control tiller and plant height, but also regulate panicle structure by QTL dissection in rice. An ideal panicle structure is important for improvement of plant architecture and rice yield. In this study, using recombinant inbred lines (RILs) of PA64s and 93-11, we identified a quantitative trait locus (QTL), designated qPPB3 for primary panicle branch number. With a BC3F2 population derived from a backcross between a resequenced RIL carrying PA64s allele and 93-11, qPPB3 was fine mapped to a 34.6-kb genomic region. Gene prediction analysis identified four putative genes, among which Os03g0203200, a previously reported gene for plant height and tiller number, Dwarf 88 (D88)/Dwarf 14 (D14), had three nucleotide substitutions in 93-11 compared with PA64s. The T to G substitution resulted in one amino acid change from valine in 93-11 to glycine in PA64s. Real-time PCR analysis showed expression level of D88 was higher in 93-11 than PA64s. The expression of APO1 and IPA1 increased, while GN1a and DST decreased in 93-11 compared with PA64s. Therefore, D88/D14 is not only a key regulator for branching, but also affects panicle structure. None OsWRKY42 represses OsMT1d and induces reactive oxygen species and leaf senescence in rice. 2014 Mol Cells Graduate School of Biotechnology and Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Korea. We isolated a rice (Oryza sativa L.) WRKY gene which is highly upregulated in senescent leaves, denoted OsWRKY42. Analysis of OsWRKY42-GFP expression and its effects on transcriptional activation in maize protoplasts suggested that the OsWRKY42 protein functions as a nuclear transcriptional repressor. OsWRKY42-overexpressing (OsWR KY42OX) transgenic rice plants exhibited an early leaf senescence phenotype with accumulation of the reactive oxygen species (ROS) hydrogen peroxide and a reduced chlorophyll content. Expression analysis of ROS producing and scavenging genes revealed that the metallothionein genes clustered on chromosome 12, especially OsMT1d, were strongly repressed in OsWRKY42OX plants. An OsMT1d promoter:LUC construct was found to be repressed by OsWRKY42 overexpression in rice protoplasts. Finally, chromatin immunoprecipitation analysis demonstrated that OsWRKY42 binds to the W-box of the OsMT1d promoter. Our results thus suggest that OsWRKY42 represses OsMT1d-mediated ROS scavenging and thereby promotes leaf senescence in rice. OsWRKY42,OsMT1d OsMADS32 interacts with PI-like proteins and regulates rice flower development. 2014 J Integr Plant Biol State Key Laboratory of Hybrid Rice, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China. OsMADS32 is a monocot specific MIKC(c) type MADS-box gene that plays an important role in regulating rice floral meristem and organs identity, a crucial process for reproductive success and rice yield. However, its underlying mechanism of action remains to be clarified. Here, we characterized a hypomorphic mutant allele of OsMADS32/CFO1, cfo1-3 and identified its function in controlling rice flower development by bioinformatics and protein-protein interaction analysis. The cfo1-3 mutant produces defective flowers, including loss of lodicule identity, formation of ectopic lodicule or hull-like organs and decreased stamen number, mimicking phenotypes related to the mutation of B class genes. Molecular characterization indicated that mis-splicing of OsMADS32 transcripts in the cfo1-3 mutant resulted in an extra eight amino acids in the K-domain of OsMADS32 protein. By yeast two hybrid and bimolecular fluorescence complementation assays, we revealed that the insertion of eight amino acids or deletion of the internal region in the K1 subdomain of OsMADS32 affects the interaction between OsMADS32 with PISTILLATA (PI)-like proteins OsMADS2 and OsMADS4. This work provides new insight into the mechanism by which OsMADS32 regulates rice lodicule and stamen identity, by interaction with two PI-like proteins via its K domain. CFO1|OsMADS32 Post-translational regulation of rice MADS29 function: homodimerization or binary interactions with other seed-expressed MADS proteins modulate its translocation into the nucleus. 2014 J Exp Bot Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India. OsMADS29 is a seed-specific MADS-box transcription factor that affects embryo development and grain filling by maintaining hormone homeostasis and degradation of cells in the nucellus and nucellar projection. Although it has a bipartite nuclear localization signal (NLS) sequence, the transiently expressed OsMADS29 monomer does not localize specifically in the nucleus. Dimerization of the monomers alters the intracellular localization fate of the resulting OsMADS29 homodimer, which then translocates into the nucleus. By generating domain-specific deletions/mutations, we show that two conserved amino acids (lysine(23) and arginine(24)) in the NLS are important for nuclear localization of the OsMADS29 homodimer. Furthermore, the analyses involving interaction of OsMADS29 with 30 seed-expressed rice MADS proteins revealed 19 more MADS-box proteins, including five E-class proteins, which interacted with OsMADS29. Eleven of these complexes were observed to be localized in the nucleus. Deletion analysis revealed that the KC region (K-box and C-terminal domain) plays a pivotal role in homodimerization. These data suggest that the biological function of OsMADS29 may not only be regulated at the level of transcription and translation as reported earlier, but also at the post-translational level by way of the interaction between OsMADS29 monomers, and between OsMADS29 and other MADS-box proteins. OsMADS29 Selection and molecular characterization of a high tocopherol accumulation rice mutant line induced by gamma irradiation. 2014 Mol Biol Rep Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 1266 Sinjeong, Jeongeup, Jeonbuk, 580-185, Republic of Korea. Tocopherols are micronutrients with antioxidant properties. They are synthesized by photosynthetic bacteria and plants, and play important roles in animal and human nutrition. In this study, we isolated a new rice mutant line with elevated tocopherol content (MRXII) from an in vitro mutagenized population induced by gamma irradiation. The mutant exhibited greater seed longevity than the control, indicating a crucial role for tocopherols in maintaining viability during quiescence, and displayed faster seedling growth during the early growth stage. To study the molecular mechanism underlying vitamin E biosynthesis, we examined the expression patterns of seven rice genes encoding vitamin E biosynthetic enzymes. Accumulation levels of the OsVTE2 transcript and OsVTE2 protein in the MRXII mutant were significantly higher than in the control. Sequence analysis revealed that the MRXII mutant harbored a point mutation in the OsVTE2 promoter region, which resulted in the generation of MYB transcription factor-binding cis-element. These results help identify the promoter regions that regulate OsVTE2 transcription, and offer insights into the regulation of tocopherol content. None Understanding the genetic and epigenetic architecture in complex network of rice flowering pathways. 2014 Protein Cell Rice Research Institute, Sichuan Agricultural University, Chengdu, 611130, China. Although the molecular basis of flowering time control is well dissected in the long day (LD) plant Arabidopsis, it is still largely unknown in the short day (SD) plant rice. Rice flowering time (heading date) is an important agronomic trait for season adaption and grain yield, which is affected by both genetic and environmental factors. During the last decade, as the nature of florigen was identified, notable progress has been made on exploration how florigen gene expression is genetically controlled. In Arabidopsis expression of certain key flowering integrators such as FLOWERING LOCUS C (FLC) and FLOWERING LOCUS T (FT) are also epigenetically regulated by various chromatin modifications, however, very little is known in rice on this aspect until very recently. This review summarized the advances of both genetic networks and chromatin modifications in rice flowering time control, attempting to give a complete view of the genetic and epigenetic architecture in complex network of rice flowering pathways. None BAC and RNA sequencing reveal the brown planthopper resistance gene BPH15 in a recombination cold spot that mediates a unique defense mechanism. 2014 BMC Genomics State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China. gche@whu.edu.cn. Brown planthopper (BPH, Nilaparvata lugens Stål), is the most destructive phloem-feeding insect pest of rice (Oryza sativa). The BPH-resistance gene BPH15 has been proved to be effective in controlling the pest and widely applied in rice breeding programs. Nevertheless, molecular mechanism of the resistance remain unclear. In this study, we narrowed down the position of BPH15 on chromosome 4 and investigated the transcriptome of BPH15 rice after BPH attacked.We analyzed 13,000 BC2F2 plants of cross between susceptible rice TN1 and the recombinant inbred line RI93 that carrying the BPH15 gene from original resistant donor B5. BPH15 was mapped to a 0.0269 cM region on chromosome 4, which is 210-kb in the reference genome of Nipponbare. Sequencing bacterial artificial chromosome (BAC) clones that span the BPH15 region revealed that the physical size of BPH15 region in resistant rice B5 is 580-kb, much bigger than the corresponding region in the reference genome of Nipponbare. There were 87 predicted genes in the BPH15 region in resistant rice. The expression profiles of predicted genes were analyzed. Four jacalin-related lectin proteins genes and one LRR protein gene were found constitutively expressed in resistant parent and considered the candidate genes of BPH15. The transcriptomes of resistant BPH15 introgression line and the susceptible recipient line were analyzed using high-throughput RNA sequencing. In total, 2,914 differentially expressed genes (DEGs) were identified. BPH-responsive transcript profiles were distinct between resistant and susceptible plants and between the early stage (6 h after infestation, HAI) and late stage (48 HAI). The key defense mechanism was related to jasmonate signaling, ethylene signaling, receptor kinase, MAPK cascades, Ca(2+) signaling, PR genes, transcription factors, and protein posttranslational modifications.Our work combined BAC and RNA sequencing to identify candidate genes of BPH15 and revealed the resistance mechanism that it mediated. These results increase our understanding of plant-insect interactions and can be used to protect against this destructive agricultural pest. None Responses of super rice (Oryza sativa L.) to different planting methods for grain yield and nitrogen-use efficiency in the single cropping season. 2014 PLoS One China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou, Zhejiang, China. To break the yield ceiling of rice production, a super rice project was developed in 1996 to breed rice varieties with super high yield. A two-year experiment was conducted to evaluate yield and nitrogen (N)-use response of super rice to different planting methods in the single cropping season. A total of 17 rice varieties, including 13 super rice and four non-super checks (CK), were grown under three N levels [0 (N0), 150 (N150), and 225 (N225) kg ha-1] and two planting methods [transplanting (TP) and direct-seeding in wet conditions (WDS)]. Grain yield under WDS (7.69 t ha-1) was generally lower than TP (8.58 t ha-1). However, grain yield under different planting methods was affected by N rates as well as variety groups. In both years, there was no difference in grain yield between super and CK varieties at N150, irrespective of planting methods. However, grain yield difference was dramatic in japonica groups at N225, that is, there was an 11.3% and 14.1% average increase in super rice than in CK varieties in WDS and TP, respectively. This suggests that high N input contributes to narrowing the yield gap in super rice varieties, which also indicates that super rice was bred for high fertility conditions. In the japonica group, more N was accumulated in super rice than in CK at N225, but no difference was found between super and CK varieties at N0 and N150. Similar results were also found for N agronomic efficiency. The results suggest that super rice varieties have an advantage for N-use efficiency when high N is applied. The response of super rice was greater under TP than under WDS. The results suggest that the need to further improve agronomic and other management practices to achieve high yield and N-use efficiency for super rice varieties in WDS. None Molecular characterization of rice sphingosine-1-phosphate lyase gene OsSPL1 and functional analysis of its role in disease resistance response. 2014 Plant Cell Rep State Key Laboratory of Rice Biology, Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang, 310058, People's Republic of China. Our results indicate that overexpression of OsSPL1 in transgenic tobacco plants attenuated disease resistance and facilitated programmed cell death. Long-chain base phosphates including sphingosine-1-phosphate have been shown to act as signaling mediators in regulating programmed cell death (PCD) and stress responses in mammals. In the present study, we characterized a rice gene OsSPL1, encoding a putative sphingosine-1-phosphate lyase that is involved in metabolism of sphingosine-1-phosphate. Expression of OsSPL1 was down-regulated in rice plants after treatments with salicylic acid, benzothiadiazole and 1-amino cyclopropane-1-carboxylic acid, but was induced by infection with a virulent strain of Magnaporthe oryzae, the causal agent of rice blast disease. Transgenic tobacco lines with overexpression of OsSPL1 were generated and analyzed for the possible role of OsSPL1 in disease resistance response and PCD. The OsSPL1-overexpressing tobacco plants displayed increased susceptibility to infection of Pseudomonas syringae pv. tabaci (Pst), the causal agent of wildfire disease, showing severity of disease symptom and bacterial titers in inoculated leaves, and attenuated pathogen-induced expression of PR genes after infection of Pst as compared to the wild-type and vector-transformed plants. Higher level of cell death, as revealed by dead cell staining, leakage of electrolyte and expression of hypersensitive response indicator genes, was observed in the OsSPL1-overexpressing plants after treatment with fumonisin B1, a fungal toxin that induces PCD in plants. Our results suggest that OsSPL1 has different functions in regulating disease resistance response and PCD in plants. OsSPL1 Predicting hybrid performance in rice using genomic best linear unbiased prediction. 2014 Proc Natl Acad Sci U S A Department of Botany and Plant Sciences, University of California, Riverside, CA 92521; and. Genomic selection is an upgrading form of marker-assisted selection for quantitative traits, and it differs from the traditional marker-assisted selection in that markers in the entire genome are used to predict genetic values and the QTL detection step is skipped. Genomic selection holds the promise to be more efficient than the traditional marker-assisted selection for traits controlled by polygenes. Genomic selection for pure breed improvement is based on marker information and thus leads to cost-saving due to early selection before phenotypes are measured. When applied to hybrid breeding, genomic selection is anticipated to be even more efficient because genotypes of hybrids are predetermined by their inbred parents. Hybrid breeding has been an important tool to increase crop productivity. Here we proposed and applied an advanced method to predict hybrid performance, in which a subset of all potential hybrids is used as a training sample to predict trait values of all potential hybrids. The method is called genomic best linear unbiased prediction. The technology applied to hybrids is called genomic hybrid breeding. We used 278 randomly selected hybrids derived from 210 recombinant inbred lines of rice as a training sample and predicted all 21,945 potential hybrids. The average yield of top 100 selection shows a 16% increase compared with the average yield of all potential hybrids. The new strategy of marker-guided prediction of hybrid yields serves as a proof of concept for a new technology that may potentially revolutionize hybrid breeding. None A network perspective on nitrogen metabolism from model to crop plants using integrated 'omics' approaches. 2014 J Exp Bot RIKEN Center for Sustainable Resource Science (CSRS), 1-7-22 Suehirocho, Tsurumi, Yokohama 230-0045, Japan JST, National Bioscience Database Center (NBDC), 5-3, Yonbancho, Chiyoda, Tokyo 102-0081, Japan. Nitrogen (N), as an essential element in amino acids, nucleotides, and proteins, is a key factor in plant growth and development. Omics approaches such as metabolomics and transcriptomics have become a promising way to inspect complex network interactions in N metabolism and can be used for monitoring the uptake and regulation, translocation, and remobilization of N. In this review, the authors highlight recent progress in omics approaches, including transcript profiling using microarrays and deep sequencing, and show recent technical developments in metabolite profiling for N studies. Further, network analysis studies including network inference methods with correlations, information-theoretic measures, and a network concept to examine gene expression clusters in relation to N regulatory systems in plants are introduced, and integrating network inference methods and integrated networks using multiple omics data are discussed. Finally, this review summarizes recent omics application examples using metabolite and/or transcript profiling analysis to elucidate the regulation of N metabolism and signalling and the coordination of N and carbon metabolism in model plants (Arabidopsis and rice), crops (tomato, maize, and legumes), and trees (Populus). None Small RNAs as important regulators for the hybrid vigour of super-hybrid rice. 2014 J Exp Bot State Key Laboratory of Plant Genomics and National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, PR China. Heterosis is an important biological phenomenon; however, the role of small RNA (sRNA) in heterosis of hybrid rice remains poorly described. Here, we performed sRNA profiling of F1 super-hybrid rice LYP9 and its parents using high-throughput sequencing technology, and identified 355 distinct mature microRNAs and trans-acting small interfering RNAs, 69 of which were differentially expressed sRNAs (DES) between the hybrid and the mid-parental value. Among these, 34 DES were predicted to target 176 transcripts, of which 112 encoded 94 transcription factors. Further analysis showed that 67.6% of DES expression levels were negatively correlated with their target mRNAs either in flag leaves or panicles. The target genes of DES were significantly enriched in some important biological processes, including the auxin signalling pathway, in which existed a regulatory network mediated by DES and their targets, closely associated with plant growth and development. Overall, 20.8% of DES and their target genes were significantly enriched in quantitative trait loci of small intervals related to important rice agronomic traits including growth vigour, grain yield, and plant architecture, suggesting that the interaction between sRNAs and their targets contributes to the heterotic phenotypes of hybrid rice. Our findings revealed that sRNAs might play important roles in hybrid vigour of super-hybrid rice by regulating their target genes, especially in controlling the auxin signalling pathway. The above finding provides a novel insight into the molecular mechanism of heterosis. None Salt-stress induced modulation of chlorophyll biosynthesis during de-etiolation of rice seedlings. 2014 Physiol Plant School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India. Chlorophyll biosynthesis in plants is subjected to modulation by various environmental factors. To understand the modulation of the chlorophyll (Chl) biosynthesis during greening process by salt, 100-200 mM NaCl was applied to the roots of etiolated rice seedlings 12 h prior to the transfer to light. Application of 200 mM NaCl to rice seedlings that were grown in light for further 72 h resulted in reduced dry matter production (-58%) and Chl accumulation (-66%). Ionic imbalance due to salinity stress resulted in additional downregulation (41-45%) of seedling dry weight, Chl and carotenoid contents over and above that of similar osmotic stress induced by polyethylene glycol. Downregulation of Chl biosynthesis may be attributed to decreased activities of Chl biosynthetic pathway enzymes, i.e. 5-aminolevulinic acid (ALA) dehydratase (EC-2.4.1.24), porphobilinogen deaminase (EC-4.3.1.8), coproporphyrinogen III oxidase (EC-1.3.3.3), protoporphyrinogen IX oxidase (EC-1.3.3.4), Mg-protoporphyrin IX chelatase (EC-6.6.1.1) and protochlorophyllide oxidoreductase (EC-1.3.33.1). Reduced enzymatic activities were due to downregulation of their protein abundance and/or gene expression in salt-stressed seedlings. The extent of downregulation of ALA biosynthesis nearly matched with that of protochlorophyllide and Chl to prevent the accumulation of highly photosensitive photodynamic tetrapyrroles that generates singlet oxygen under stress conditions. Although, ALA synthesis decreased, the gene/protein expression of glutamyl-tRNA reductase (EC-1.2.1.70) increased suggesting it may play a role in acclimation to salt stress. The similar downregulation of both early and late Chl biosynthesis intermediates in salt-stressed seedlings suggests a regulatory network of genes involved in tetrapyrrole biosynthesis. None OsABCG15 encodes a membrane protein that plays an important role in anther cuticle and pollen exine formation in rice. 2014 Plant Cell Rep College of Agronomy and Biotechnology, Southwest University, Chongqing, 400715, China. An ABC transporter gene ( OsABCG15 ) was proven to be involved in pollen development in rice. The corresponding protein was localized on the plasma membrane using subcellular localization. Wax, cutin, and sporopollenin are important for normal development of the anther cuticle and pollen exine, respectively. Their lipid soluble precursors, which are produced in the tapetum, are then secreted and transferred to the anther and microspore surface for polymerization. However, little is known about the mechanisms underlying the transport of these precursors. Here, we identified and characterized a member of the G subfamily of ATP-binding cassette (ABC) transporters, OsABCG15, which is required for the secretion of these lipid-soluble precursors in rice. Using map-based cloning, we found a spontaneous A-to-C transition in the fourth exon of OsABCG15 that caused an amino acid substitution of Thr-to-Pro in the predicted ATP-binding domain of the protein sequence. This osabcg15 mutant failed to produce any viable pollen and was completely male sterile. Histological analysis indicated that osabcg15 exhibited an undeveloped anther cuticle, enlarged middle layer, abnormal Ubisch body development, tapetum degeneration with a falling apart style, and collapsed pollen grains without detectable exine. OsABCG15 was expressed preferentially in the tapetum, and the fused GFP-OsABCG15 protein was localized to the plasma membrane. Our results suggested that OsABCG15 played an essential role in the formation of the rice anther cuticle and pollen exine. This role may include the secretion of the lipid precursors from the tapetum to facilitate the transfer of precursors to the surface of the anther epidermis as well as to microspores. OsABCG15 The effects of fluctuations in the nutrient supply on the expression of five members of the AGL17 clade of MADS-box genes in rice. 2014 PLoS One Zhejiang Key Lab of Crop Germplasm, Department of Agronomy, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China. The ANR1 MADS-box gene in Arabidopsis is a key gene involved in regulating lateral root development in response to the external nitrate supply. There are five ANR1-like genes in Oryza sativa, OsMADS23, OsMADS25, OsMADS27, OsMADS57 and OsMADS61, all of which belong to the AGL17 clade. Here we have investigated the responsiveness of these genes to fluctuations in nitrogen (N), phosphorus (P) and sulfur (S) mineral nutrient supply. The MADS-box genes have been shown to have a range of responses to the nutrient supply. The expression of OsMADS61 was transiently induced by N deprivation but was not affected by re-supply with various N sources. The expression of OsMADS25 and OsMADS27 was induced by re-supplying with NO3(-) and NH4NO3, but downregulated by NH4(+). The expression of OsMADS57 was significantly downregulated by N starvation and upregulated by 3 h NO3(-) re-supply. OsMADS23 was the only gene that showed no response to either N starvation nor NO3(-) re-supply. OsMADS57 was the only gene not regulated by P fluctuation whereas the expression of OsMADS23, OsMADS25 and OsMADS27 was downregulated by P starvation and P re-supply. In contrast, all five ANR1-related genes were significantly upregulated by S starvation. Our results also indicated that there were interactions among nitrate, sulphate and phosphate transporters in rice. None Mutation of Oryza sativa CORONATINE INSENSITIVE 1b (OsCOI1b) delays leaf senescence. 2014 J Integr Plant Biol Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, 151-921, Korea. Jasmonic acid (JA) functions in plant development, including senescence and immunity. Arabidopsis thaliana CORONATINE INSENSITIVE 1 encodes a JA receptor and functions in the JA-responsive signaling pathway. The Arabidopsis genome harbors a single COI gene, but the rice (Oryza sativa) genome harbors three COI homologs, OsCOI1a, OsCOI1b, and OsCOI2. Thus, it remains unclear whether each OsCOI has distinct, additive, synergistic, or redundant functions in development. Here, we use the oscoi1b-1 knockout mutants to show that OsCOI1b mainly affects leaf senescence under senescence-promoting conditions. oscoi1b-1 mutants stayed green during dark-induced and natural senescence, with substantial retention of chlorophylls and photosynthetic capacity. Furthermore, several senescence-associated genes were downregulated in oscoi1b-1 mutants, including homologs of Arabidopsis thaliana ETHYLENE INSENSITIVE 3 and ORESARA 1, important regulators of leaf senescence. These results suggest that crosstalk between JA signaling and ethylene signaling affects leaf senescence. The Arabidopsis coi1-1 plants containing 35S:OsCOI1a or 35S:OsCOI1b rescued the delayed leaf senescence during dark incubation, suggesting that both OsCOI1a and OsCOI1b are required for promoting leaf senescence in rice. oscoi1b-1 mutants showed significant decreases in spikelet fertility and grain weight, leading to severe reduction of grain yield, indicating that OsCOI1-mediated JA signaling affects spikelet fertility and grain filling. OsCOI1b Molecular genetic dissection of quantitative trait loci regulating rice grain size. 2014 Annu Rev Genet State Key Laboratory of Plant Genomics and National Plant Gene Research Center, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; email: jyli@genetics.ac.cn. Grain size is one of the most important factors determining rice yield. As a quantitative trait, grain size is predominantly and tightly controlled by genetic factors. Several quantitative trait loci (QTLs) for grain size have been molecularly identified and characterized. These QTLs may act in independent genetic pathways and, along with other identified genes for grain size, are mainly involved in the signaling pathways mediated by proteasomal degradation, phytohormones, and G proteins to regulate cell proliferation and cell elongation. Many of these QTLs and genes have been strongly selected for enhanced rice productivity during domestication and breeding. These findings have paved new ways for understanding the molecular basis of grain size and have substantial implications for genetic improvement of crops. None EF8 is involved in photoperiodic flowering pathway and chlorophyll biogenesis in rice. 2014 Plant Cell Rep State Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing, 210095, China, fengzhiming88@163.com. A DTH8/Ghd8 allele suppresses flowering by altering the expression patterns of the 'florigen' genes, but also negatively regulates chlorophyll biogenesis. Flowering time is a critical agronomic trait determining the growing season, regional adaptation and yield potential in rice (Oryza sativa L.). We characterized a mutant named early flowering 8 (ef8) selected from an ethyl methanesulfonate (EMS)-treated population of indica cultivar 93-11. It showed earlier flowering, less grains per main panicle and slightly darker green leaves than the wild-type 93-11 under natural long-day conditions, but was not significantly different from 93-11 under natural short-day conditions. We isolated the Early Flowering 8 (EF8) gene by map-based cloning. EF8 encodes a putative HAP3 subunit of the CCAAT-box-binding transcription factor, which is localized to the nucleus. EF8 was expressed in various tissues, especially in leaves, with a rhythmic expression pattern. Our data showed that EF8 delayed flowering time under long-day conditions by altering the rhythmic expression patterns of 'florigen' genes Hd3a and RFT1. We also found that EF8 negatively regulates the expression of chlorophyll biosynthetic genes to reduce the chlorophyll content. Our data indicate that EF8 plays an important role in rice photoperiodic flowering pathway as well as yield potential and chlorophyll biogenesis and will be an important target for rice breeding programs. Hd5|DTH8|Ghd8|OsHAP3H|LHD1|EF8 RNAi-directed downregulation of betaine aldehyde dehydrogenase 1 (OsBADH1) results in decreased stress tolerance and increased oxidative markers without affecting glycine betaine biosynthesis in rice (Oryza sativa). 2014 Plant Mol Biol Ministry of Education Key Laboratory for Bio-resource and Eco-environment, State Key Laboratory of Hydraulics and Mountain River Engineering, College of Life Science, Sichuan University, Chengdu, China. As an important osmoprotectant, glycine betaine (GB) plays an essential role in resistance to abiotic stress in a variety of organisms, including rice (Oryza sativa L.). However, GB content is too low to be detectable in rice, although rice genome possesses several orthologs coding for betaine aldehyde dehydrogenase (BADH) involved in plant GB biosynthesis. Rice BADH1 (OsBADH1) has been shown to be targeted to peroxisome and its overexpression resulted in increased GB biosynthesis and tolerance to abiotic stress. In this study, we demonstrated a pivotal role of OsBADH1 in stress tolerance without altering GB biosynthesis capacity, using the RNA interference (RNAi) technique. OsBADH1 was ubiquitously expressed in different organs, including roots, stems, leaves and flowers. Transgenic rice lines downregulating OsBADH1 exhibited remarkably reduced tolerance to NaCl, drought and cold stresses. The decrease of stress tolerance occurring in the OsBADH1-RNAi repression lines was associated with an elevated level of malondialdehyde content and hydrogen peroxidation. No GB accumulation was detected in transgene-positive and transgene-negative lines derived from heterozygous transgenic T0 plants. Moreover, transgenic OsBADH1-RNAi repression lines showed significantly reduced seed set and yield. In conclusion, the downregulation of OsBADH1, even though not causing any change of GB content, was accounted for the reduction of ability to dehydrogenate the accumulating metabolism-derived aldehydes and subsequently resulted in decreased stress tolerance and crop productivity. These results suggest that OsBADH1 possesses an enzyme activity to catalyze other aldehydes in addition to betaine aldehyde (the precursor of GB) and thus alleviate their toxic effects under abiotic stresses. BAD1|OsBADH1 Overexpression of OsHMA3 enhances Cd tolerance and expression of Zn transporter genes in rice. 2014 J Exp Bot Institute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki, Japan. As a member of the heavy metal ATPase (HMA) family, OsHMA3 is a tonoplast-localized transporter for Cd in the roots of rice (Oryza sativa). Overexpression of OsHMA3 selectively reduces Cd accumulation in the grain. Further characterization in the present study revealed that overexpression of OsHMA3 also enhances the tolerance to toxic Cd. The growth of both the roots and shoots was similar in the absence of Cd between an OsHMA3-overexpressed line and vector control, but the Cd-inhibited growth was significantly alleviated in the OsHMA3-overexpressed line. The overexpressed line showed higher Cd concentration in the roots, but lower Cd concentration in the shoots compared with the wild-type rice and vector control line, indicating that overexpression of OsHMA3 enhanced vacuolar sequestration of Cd in the roots. The Zn concentration in the roots of the OsHMA3-overexpressed line was constantly higher than that of vector control, but the Zn concentration in the shoots was similar between the overexpressed line and vector control. Five transporter genes belonging to the ZIP family were constitutively up-regulated in the OsHMA3-overexpressed line. These results suggest that shoot Zn level was maintained by up-regulating these genes involved in the Zn uptake/translocation. Taken together, overexpression of OsHMA3 is an efficient way to reduce Cd accumulation in the grain and to enhance Cd tolerance in rice. qCdT7|OsHMA3 The role of a potassium transporter OsHAK5 in potassium acquisition and transport from roots to shoots in rice at low potassium supply levels. 2014 Plant Physiol State Key Laboratory of Crop Genetics and Germplasm Enhancement (T.Y., S.Z., Y.H., F.W., Q.H., G.C., J.C., T.W., L.Y., G.X.) and Key Laboratory of Plant Nutrition and Fertilization in Lower-Middle Reaches of the Yangtze River, Ministry of Agriculture (T.Y., S.Z., Y.H., F.W., Q.H., G.C., J.C., T.W., L.Y., G.X.), Nanjing Agricultural University, Nanjing 210095, China; andR.H. Smith Institute of Plant Sciences and Genetics in Agriculture, R.H. Smith Faculty of Agriculture, Food, and Environment, Hebrew University of Jerusalem, Rehovot 76100, Israel (N.M.). In plants, K transporter (KT)/high affinity K transporter (HAK)/K uptake permease (KUP) is the largest potassium (K) transporter family; however, few of the members have had their physiological functions characterized in planta. Here, we studied OsHAK5 of the KT/HAK/KUP family in rice (Oryza sativa). We determined its cellular and tissue localization and analyzed its functions in rice using both OsHAK5 knockout mutants and overexpression lines in three genetic backgrounds. A beta-glucuronidase reporter driven by the OsHAK5 native promoter indicated OsHAK5 expression in various tissue organs from root to seed, abundantly in root epidermis and stele, the vascular tissues, and mesophyll cells. Net K influx rate in roots and K transport from roots to aerial parts were severely impaired by OsHAK5 knockout but increased by OsHAK5 overexpression in 0.1 and 0.3 mm K external solution. The contribution of OsHAK5 to K mobilization within the rice plant was confirmed further by the change of K concentration in the xylem sap and K distribution in the transgenic lines when K was removed completely from the external solution. Overexpression of OsHAK5 increased the K-sodium concentration ratio in the shoots and salt stress tolerance (shoot growth), while knockout of OsHAK5 decreased the K-sodium concentration ratio in the shoots, resulting in sensitivity to salt stress. Taken together, these results demonstrate that OsHAK5 plays a major role in K acquisition by roots faced with low external K and in K upward transport from roots to shoots in K-deficient rice plants. OsHAK5 Genetic analysis and QTL detection for resistance to white tip disease in rice. 2014 PLoS One Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China. The inheritance of resistance to white tip disease (WTDR) in rice (Oryza sativa L.) was analyzed with an artificial inoculation test in a segregating population derived from the cross between Tetep, a highly resistant variety that was identified in a previous study, and a susceptible cultivar. Three resistance-associated traits, including the number of Aphelenchoides besseyi (A. besseyi) individuals in 100 grains (NA), the loss rate of panicle weight (LRPW) and the loss rate of the total grains per panicle (LRGPP) were analyzed for the detection of the quantitative trait locus (QTL) in the population after construction of a genetic map. Six QTLs distributed on chromosomes 3, 5 and 9 were mapped. qNA3 and qNA9, conferring reproduction number of A. besseyi in the panicle, accounted for 16.91% and 12.54% of the total phenotypic variance, respectively. qDRPW5a and qDRPW5b, associated with yield loss, were located at two adjacent marker intervals on chromosome 5 and explained 14.15% and 14.59% of the total phenotypic variation and possessed LOD values of 3.40 and 3.39, respectively. qDRPW9 was considered as a minor QTL and only explained 1.02% of the phenotypic variation. qLRGPP5 contributed to the loss in the number of grains and explained 10.91% of the phenotypic variation. This study provides useful information for the breeding of resistant cultivars against white tip disease in rice. None Genome duplication improves rice root resistance to salt stress. 2014 Rice (N Y) Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Wuhan 430062, P.R. China. Salinity is a stressful environmental factor that limits the productivity of crop plants, and roots form the major interface between plants and various abiotic stresses. Rice is a salt-sensitive crop and its polyploid shows advantages in terms of stress resistance. The objective of this study was to investigate the effects of genome duplication on rice root resistance to salt stress.Both diploid rice (HN2026-2x and Nipponbare-2x) and their corresponding tetraploid rice (HN2026-4x and Nipponbare-4x) were cultured in half-strength Murashige and Skoog medium with 150 mM NaCl for 3 and 5 days. Accumulations of proline, soluble sugar, malondialdehyde (MDA), Na(+) content, H(+) (proton) flux at root tips, and the microstructure and ultrastructure in rice roots were examined. We found that tetraploid rice showed less root growth inhibition, accumulated higher proline content and lower MDA content, and exhibited a higher frequency of normal epidermal cells than diploid rice. In addition, a protective gap appeared between the cortex and pericycle cells in tetraploid rice. Next, ultrastructural analysis showed that genome duplication improved membrane, organelle, and nuclei stability. Furthermore, Na(+) in tetraploid rice roots significantly decreased while root tip H(+) efflux in tetraploid rice significantly increased.Our results suggest that genome duplication improves root resistance to salt stress, and that enhanced proton transport to the root surface may play a role in reducing Na(+) entrance into the roots. None Gene interactions and genetics of blast resistance and yield attributes in rice (Oryza sativa L.). 2014 J Genet Indian Council of Agricultural Research (ICAR), Rajendranagar, Hyderabad 500 030, India. divyab0005@gmail.com. Blast disease caused by the pathogen Pyricularia oryzae is a serious threat to rice production. Six generations viz., P1, P2, F1, F2, B1 and B2 of a cross between blast susceptible high-yielding rice cultivar ADT 43 and resistant near isogenic line (NIL) CT13432-3R, carrying four blast resistance genes Pi1, Pi2, Pi33 and Pi54 in combination were used to study the nature and magnitude of gene action for disease resistance and yield attributes. The epistatic interaction model was found adequate to explain the gene action in most of the traits. The interaction was complementary for number of productive tillers, economic yield, lesion number, infected leaf area and potential disease incidence but duplicate epistasis was observed for the remaining traits. Among the genotypes tested under epiphytotic conditions, gene pyramided lines were highly resistant to blast compared to individuals with single genes indicating that the nonallelic genes have a complementary effect when present together. The information on genetics of various contributing traits of resistance will further aid plant breeders in choosing appropriate breeding strategy for blast resistance and yield enhancement in rice. None Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. 2014 Nucleic Acids Res Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA. The Cas9/sgRNA of the CRISPR/Cas system has emerged as a robust technology for targeted gene editing in various organisms, including plants, where Cas9/sgRNA-mediated small deletions/insertions at single cleavage sites have been reported in transient and stable transformations, although genetic transmission of edits has been reported only in Arabidopsis and rice. Large chromosomal excision between two remote nuclease-targeted loci has been reported only in a few non-plant species. Here we report in rice Cas9/sgRNA-induced large chromosomal segment deletions, the inheritance of genome edits in multiple generations and construction of a set of facile vectors for high-efficiency, multiplex gene targeting. Four sugar efflux transporter genes were modified in rice at high efficiency; the most efficient system yielding 87-100% editing in T0 transgenic plants, all with di-allelic edits. Furthermore, genetic crosses segregating Cas9/sgRNA transgenes away from edited genes yielded several genome-edited but transgene-free rice plants. We also demonstrated proof-of-efficiency of Cas9/sgRNAs in producing large chromosomal deletions (115-245 kb) involving three different clusters of genes in rice protoplasts and verification of deletions of two clusters in regenerated T0 generation plants. Together, these data demonstrate the power of our Cas9/sgRNA platform for targeted gene/genome editing in rice and other crops, enabling both basic research and agricultural applications. None The role of the large subunit in redox regulation of the rice endosperm ADP-glucose pyrophosphorylase. 2014 FEBS J Institute of Biological Chemistry, Washington State University, Pullman, WA, USA. The starch regulatory enzyme ADP-glucose pyrophosphorylase is activated by 3-phosphoglyceric acid (3-PGA) and inhibited by inorganic phosphate (Pi ). The activity of the plastid-localized enzyme is also subject to fine regulation by redox control in response to changing light and sugar levels. The less active oxidized form of the enzyme contains an inter-subunit disulfide bond formed between the pair of small subunit's Cys12 residues of the heterotetrameric enzyme. Although this cysteine residue is not conserved in the small subunits of cereal endosperm cytosolic AGPases, biochemical studies of the major rice endosperm enzyme indicate that the cytosolic isoform, like the plastidial enzymes, is subject to redox control. Kinetic analysis revealed that the reduced forms of the partially purified native and purified recombinant AGPases have 6- and 3.4-fold, respectively, more affinity to 3-PGA, rendering the enzymes more active at lower 3-PGA concentration than the non-reduced enzyme. Truncation of the large subunit by removal of N-terminal peptide resulted in a decrease in 3-PGA affinity and loss of redox response of the enzyme. Site-directed mutagenesis of the conserved cysteine residues at the N-terminal of the large subunit showed that C47 and C58, but not C12, are essential for proper redox response of the enzyme. Overall, our results show that the major rice endosperm AGPase activity is controlled by a combination of allosteric regulation and redox control, the latter through modification of the large subunit instead of the small subunit as evident in the plastid-localized enzyme. None OsMLO12, encoding seven transmembrane proteins, is involved with pollen hydration in rice. 2014 Plant Reprod Department of Plant Molecular Systems Biotechnology, Crop Biotech Institute, Kyung Hee University, 1732 Deogyeong-daero, Giheung, Yongin, Gyeonggi, Korea. Hydration is the first step in pollen germination. However, the process is not well understood. OsMLO12 is highly expressed in mature pollen grains; plants containing alleles caused by transfer DNA insertions do not produce homozygous progeny. Reciprocal crosses between wild-type and OsMLO12/osmlo12 plants showed that the mutant alleles were not transmitted through the male gametophyte. Microscopic observations revealed that, although mutant grains became mature pollen with three nuclei, they did not germinate in vitro or in vivo due to a failure in hydration. The OsMLO12 protein has seven transmembrane motifs, with an N-terminal extracellular region and a C-terminal cytosolic region. We demonstrated that the C-terminal region mediates a calcium-dependent interaction with calmodulin. Our findings suggest that pollen hydration is regulated by MLO12, possibly through an interaction with calmodulin in the cytosol. OsMLO12 Fine mapping of qHd1, a minor heading date QTL with pleiotropism for yield traits in rice (Oryza sativa L.). 2014 Theor Appl Genet State Key Laboratory of Rice Biology and Chinese National Center for Rice Improvement, China National Rice Research Institute, Hangzhou, 310006, China. A minor QTL for heading date located on the long arm of rice chromosome 1 was delimitated to a 95.0-kb region using near isogenic lines with sequential segregating regions. Heading date and grain yield are two key factors determining the commercial potential of a rice variety. In this study, rice populations with sequential segregating regions were developed and used for mapping a minor QTL for heading date, qHd1. A total of 18 populations in six advanced generations through BC2F6 to BC2F11 were derived from a single BC2F3 plant of the indica rice cross Zhenshan 97 (ZS97)///ZS97//ZS97/Milyang 46. The QTL was delimitated to a 95.0-kb region flanked by RM12102 and RM12108 in the terminal region of the long arm of chromosome 1. Results also showed that qHd1 was not involved in the photoperiodic response, having an additive effect ranging from 2.4 d to 2.9 d observed in near isogenic lines grown in the paddy field and under the controlled conditions of either short day or long day. The QTL had pleiotropic effects on yield traits, with the ZS97 allele delaying heading and increasing the number of spikelets per panicle, the number of grains per panicle and grain yield per plant. The candidate region contains ten annotated genes including two genes with functional information related to the control of heading date. These results lay a foundation for the cloning of qHd1. In addition, this kind of minor QTLs could be of great significance in rice breeding for allowing minor adjustment of heading date and yield traits. None Positive Darwinian selection is a driving force for the diversification of terpenoid biosynthesis in the genus Oryza. 2014 BMC Plant Biol Department of Plant Sciences, University of Tennessee, Knoxville 37996, TN, USA. fengc@utk.edu. Terpenoids constitute the largest class of secondary metabolites made by plants and display vast chemical diversity among and within species. Terpene synthases (TPSs) are the pivotal enzymes for terpenoid biosynthesis that create the basic carbon skeletons of this class. Functional divergence of paralogous and orthologous TPS genes is a major mechanism for the diversification of terpenoid biosynthesis. However, little is known about the evolutionary forces that have shaped the evolution of plant TPS genes leading to terpenoid diversity.The orthologs of Oryza Terpene Synthase 1 (OryzaTPS1), a rice terpene synthase gene involved in indirect defense against insects in Oryza sativa, were cloned from six additional Oryza species. In vitro biochemical analysis showed that the enzymes encoded by these OryzaTPS1 genes functioned either as (E)-beta-caryophyllene synthases (ECS), or (E)-beta-caryophyllene & germacrene A synthases (EGS), or germacrene D & germacrene A synthases (DAS). Because the orthologs of OryzaTPS1 in maize and sorghum function as ECS, the ECS activity was inferred to be ancestral. Molecular evolutionary detected the signature of positive Darwinian selection in five codon substitutions in the evolution from ECS to DAS. Homology-based structure modeling and the biochemical analysis of laboratory-generated protein variants validated the contribution of the five positively selected sites to functional divergence of OryzaTPS1. The changes in the in vitro product spectra of OryzaTPS1 proteins also correlated closely to the changes in in vivo blends of volatile terpenes released from insect-damaged rice plants.In this study, we found that positive Darwinian selection is a driving force for the functional divergence of OryzaTPS1. This finding suggests that the diverged sesquiterpene blend produced by the Oryza species containing DAS may be adaptive, likely in the attraction of the natural enemies of insect herbivores. None In-depth proteomic analysis of rice embryo reveals its important roles in seed germination. 2014 Plant Cell Physiol Key Laboratory of Plant Germplasm Enhancement and Speciality Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China University of Chinese Academy of Sciences, Beijing 100049, China. Seed germination is a complex physiological process that allows the seed embryo to grow and develop into a photosynthetic organism. The two major constituents of rice seed include the embryo and endosperm, with embryo being of much significance despite its small size. In this study, we conducted a systematic proteomic analysis of the embryos dissected from rice seed at different stages of germination through a combination of gel-based and gel-free strategies. In total, 343 differentially expressed proteins were identified. Among them, 191 were decreased and 152 were increased in terms of expression levels. All these proteins could be sorted into 11 functional groups based on MapMan analysis. Some starch biosynthesis-related enzymes such as starch branching enzyme, granule-bound starch synthase 1 and starch synthase increased during the early stage of germination and then decreased at the late stage, which was similar to the expressional patterns of glycolysis-related enzymes. However, tricarboxylic acid cycle-related enzymes only increased at the later stage. It was also found that sucrose might be an important intermediate for the biosynthesis of starch in embryos. Furthermore, gel-based proteomic analysis of the dissected endosperm showed that the biological processes in the endosperm were heavily regulated by the embryo. This study could provide some new insights into the distinct roles of the embryo and endosperm in rice seed germination. None Genome editing in rice and wheat using the CRISPR/Cas system. 2014 Nat Protoc State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology (IGDB), Chinese Academy of Sciences (CAS), Beijing, China. Targeted genome editing nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), are powerful tools for understanding gene function and for developing valuable new traits in plants. The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas system has recently emerged as an alternative nuclease-based method for efficient and versatile genome engineering. In this system, only the 20-nt targeting sequence within the single-guide RNA (sgRNA) needs to be changed to target different genes. The simplicity of the cloning strategy and the few limitations on potential target sites make the CRISPR/Cas system very appealing. Here we describe a stepwise protocol for the selection of target sites, as well as the design, construction, verification and use of sgRNAs for sequence-specific CRISPR/Cas-mediated mutagenesis and gene targeting in rice and wheat. The CRISPR/Cas system provides a straightforward method for rapid gene targeting within 1-2 weeks in protoplasts, and mutated rice plants can be generated within 13-17 weeks. None Identification and validation of functional markers in a global rice collection by association mapping. 2014 Genome a Department of Agronomy and Plant Breeding, Faculty of Agriculture, Shahid Bahonar University of Kerman, P.O. Box 76169-133, Kerman, Iran. Recent results indicate that marker-assisted selection is an effective approach to reduce the cost and to improve the efficacy and accuracy of selection in plant breeding. This study was conducted to identify and validate molecular markers linked to important breeding traits by association mapping. The association was evaluated between 81 molecular markers (STS, SSR, Indel, CAPS, and PCR-based SNP) and 15 morphological traits in a global panel of 100 rice (Oryza sativa) accessions. The population structure analysis identified three main subpopulations. Obvious kinship relationships were also detected between the rice accessions. Association analysis was performed based on the mixed linear model by considering population structure and family relatedness. In addition, the false discovery rate method was used to correct the multiple testing. A total of 47 marker-trait associations were identified, including 22 markers for 14 traits. Among all, the polymorphism at the loci DDR-GL was highly associated with grain characters (grain length, grain width, and length/width ratio). In addition, marker RM3148 was responsible for five important traits simultaneously. Results demonstrated that such informative markers can be very useful for rice breeding programs using marker-assisted selection. Moreover, the diverse populations of rice accessions are a valuable resource for association mapping of morphological traits. None Comparative cytological and transcriptomic analysis of pollen development in autotetraploid and diploid rice. 2014 Plant Reprod State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, 510642, China. Autotetraploid rice has greater genetic variation and higher vigor than diploid rice, but low pollen fertility is one of the major reasons for low yield of autotetraploid rice. Very little is known about the molecular mechanisms of low pollen fertility of autotetraploid rice. In this study, cytological observations and microarray analysis were used to assess the genetic variation during pollen development in autotetraploid and diploid rice. Many abnormal chromosome behaviors, such as mutivalents, lagged chromosomes, asynchronous cell division, and so on, were found during meiosis in autotetraploid. Microsporogenesis and microgametogenesis in autotetraploid rice was similar to diploid rice, but many different kinds of abnormalities, including microspores degeneration, multi-aperture, and abnormal cell walls, were found in autotetraploid rice. Compared with diploid rice, a total of 1,251 genes were differentially expressed in autotetraploid rice in pollen transcriptome, among them 1,011 and 240 genes were up-regulated and down-regulated, respectively. 124 and 6 genes were co-up-regulated and co-down-regulated during three pollen development stages, respectively. These results suggest that polyploidy induced up-regulation for most of the genes during pollen development. Quantitative RT-PCR was done to validate 12 differentially expressed genes selected from functional categories based on the gene ontology analysis. These stably expressed genes not only related to the pollen development genes, but also involved in cell metabolism, cell physiology, binding, catalytic activity, molecular transducer activity, and transcription regulator activity. The present study suggests that differential expression of some key genes may lead to complex gene regulation and abnormal pollen development in autotetraploid rice. None Comparative metabolite profiling of two rice genotypes with contrasting salt stress tolerance at the seedling stage. 2014 PLoS One Institute of Crop Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing, China. Rice is sensitive to salt stress, especially at the seedling stage, with rice varieties differing remarkably in salt tolerance (ST). To understand the physiological mechanisms of ST, we investigated salt stress responses at the metabolite level.Gas chromatography-mass spectrometry was used to profile metabolite changes in the salt-tolerant line FL478 and the sensitive variety IR64 under a salt-stress time series. Additionally, several physiological traits related to ST were investigated.We characterized 92 primary metabolites in the leaves and roots of the two genotypes under stress and control conditions. The metabolites were temporally, tissue-specifically and genotype-dependently regulated under salt stress. Sugars and amino acids (AAs) increased significantly in the leaves and roots of both genotypes, while organic acids (OAs) increased in roots and decreased in leaves. Compared with IR64, FL478 experienced greater increases in sugars and AAs and more pronounced decreases in OAs in both tissues; additionally, the maximum change in sugars and AAs occurred later, while OAs changed earlier. Moreover, less Na+ and higher relative water content were observed in FL478. Eleven metabolites, including AAs and sugars, were specifically increased in FL478 over the course of the treatment.Metabolic responses of rice to salt stress are dynamic and involve many metabolites. The greater ST of FL478 is due to different adaptive reactions at different stress times. At early salt-stress stages, FL478 adapts to stress by decreasing OA levels or by quickly depressing growth; during later stages, more metabolites are accumulated, thereby serving as compatible solutes against osmotic challenge induced by salt stress. None The genetic and molecular origin of natural variation for the fragrance trait in an elite Malaysian aromatic rice through quantitative trait loci mapping using SSR and gene-based markers. 2014 Gene Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Electronic address: fsgolestan@yahoo.ca. MRQ74, a popular aromatic Malaysian landrace, allows for charging considerably higher prices than non-aromatic landraces. Thus, breeding this profitable trait has become a priority for Malaysian rice breeding. Despite many studies on aroma genetics, ambiguities considering its genetic basis remain. It has been observed that identifying quantitative trait loci (QTLs) based on anchor markers, particularly candidate genes controlling a trait of interest, can increase the power of QTL detection. Hence, this study aimed to locate QTLs that influence natural variations in rice scent using microsatellites and candidate gene-based sequence polymorphisms. For this purpose, an F2 mapping population including 189 individual plants was developed by MRQ74 crosses with 'MR84', a non-scented Malaysian accession. Additionally, qualitative and quantitative approaches were applied to obtain a phenotype data framework. Consequently, we identified two QTLs on chromosomes 4 and 8. These QTLs explained from 3.2% to 39.3% of the total fragrance phenotypic variance. In addition, we could resolve linkage group 8 by adding six gene-based primers in the interval harboring the most robust QTL. Hence, we could locate a putative fgr allele in the QTL found on chromosome 8 in the interval RM223-SCU015RM (1.63cM). The identified QTLs represent an important step toward recognition of the rice flavor genetic control mechanism. In addition, this identification will likely accelerate the progress of the use of molecular markers for gene isolation, gene-based cloning, and marker-assisted selection breeding programs aimed at improving rice cultivars. None High-level hemicellulosic arabinose predominately affects lignocellulose crystallinity for genetically enhancing both plant lodging resistance and biomass enzymatic digestibility in rice mutants. 2014 Plant Biotechnol J National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan, China; Biomass and Bioenergy Research Centre, Huazhong Agricultural University, Wuhan, China; College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China. Rice is a major food crop with enormous biomass residue for biofuels. As plant cell wall recalcitrance basically decides a costly biomass process, genetic modification of plant cell walls has been regarded as a promising solution. However, due to structural complexity and functional diversity of plant cell walls, it becomes essential to identify the key factors of cell wall modifications that could not much alter plant growth, but cause an enhancement in biomass enzymatic digestibility. To address this issue, we performed systems biology analyses of a total of 36 distinct cell wall mutants of rice. As a result, cellulose crystallinity (CrI) was examined to be the key factor that negatively determines either the biomass enzymatic saccharification upon various chemical pretreatments or the plant lodging resistance, an integrated agronomic trait in plant growth and grain production. Notably, hemicellulosic arabinose (Ara) was detected to be the major factor that negatively affects cellulose CrI probably through its interlinking with beta-1,4-glucans. In addition, lignin and G monomer also exhibited the positive impact on biomass digestion and lodging resistance. Further characterization of two elite mutants, Osfc17 and Osfc30, showing normal plant growth and high biomass enzymatic digestion in situ and in vitro, revealed the multiple GH9B candidate genes for reducing cellulose CrI and XAT genes for increasing hemicellulosic Ara level. Hence, the results have suggested the potential cell wall modifications for enhancing both biomass enzymatic digestibility and plant lodging resistance by synchronically overexpressing GH9B and XAT genes in rice. None An improved 2b-RAD approach (I2b-RAD) offering genotyping tested by a rice (Oryza sativa L.) F2 population. 2014 BMC Genomics Beijing Genome Institute-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen 518083, China. xuxun@genomics.cn. 2b-RAD (type IIB endonucleases restriction-site associated DNA) approach was invented by Wang in 2012 and proven as a simple and flexible method for genome-wide genotyping. However, there is still plenty of room for improvement for the existent 2b-RAD approach. Firstly, it doesn't include the samples pooling in library preparation as other reduced representation libraries. Secondly, the information of 2b-RAD tags, such as tags numbers and distributions, in most of species are unknown. The purposes of the research are to improve a new 2b-RAD approach which possesses samples pooling, moreover to figure out the characteristic and application potentiality of 2b-RAD tags by bioinformatics analysis.Twelve adapter1 and an adapter2 were designed. A library approach comprising digestion, ligation, pooling, PCR and size selection were established. For saving costs, we used non-phosphorylated adapters and indexed PCR primers. A F2 population of rice (Oryza sativa .L) was genotyped to validate the new approach. On average, 2000332 high quality reads of each sample were obtained with high evenness. Totally 3598 markers containing 3804 SNPs were discovered and the missing rate was 18.9%. A genetic linkage map of 1385 markers was constructed and 92% of the markers' orders in the genetic map were in accordance with the orders in chromosomes. Meanwhile, the bioinformatics simulation in 20 species showed that the BsaXI had the most widespread recognition sites, indicating that 2b-RAD tags had a powerful application potentiality for high density genetic map. Using modified adapters with a fix base in 3'end, 2b-RAD was also fit for QTL studies with low costs.An improved 2b-RAD genotyping approach was established in this research and named as I2b-RAD. The method was a simple, fast, cost-effective and multiplex sequencing library approach. It could be adjusted by selecting different enzymes and adapters to fit for alternative uses including chromosomes assembly, QTL fine mapping and even natural population analysis. None QTLs for tolerance of drought and breeding for tolerance of abiotic and biotic stress: an integrated approach. 2014 PLoS One International Rice Research Institute (IRRI), Los Baños, Laguna, Philippines. The coupling of biotic and abiotic stresses leads to high yield losses in rainfed rice (Oryza sativa L.) growing areas. While several studies target these stresses independently, breeding strategies to combat multiple stresses seldom exist. This study reports an integrated strategy that combines QTL mapping and phenotypic selection to develop rice lines with high grain yield (GY) under drought stress and non-stress conditions, and tolerance of rice blast.A blast-tolerant BC2F3-derived population was developed from the cross of tropical japonica cultivar Moroberekan (blast- and drought-tolerant) and high-yielding indica variety Swarna (blast- and drought-susceptible) through phenotypic selection for blast tolerance at the BC2F2 generation. The population was studied for segregation distortion patterns and QTLs for GY under drought were identified along with study of epistatic interactions for the trait.Segregation distortion, in favour of Moroberekan, was observed at 50 of the 59 loci. Majority of these marker loci co-localized with known QTLs for blast tolerance or NBS-LRR disease resistance genes. Despite the presence of segregation distortion, high variation for DTF, PH and GY was observed and several QTLs were identified under drought stress and non-stress conditions for the three traits. Epistatic interactions were also detected for GY which explained a large proportion of phenotypic variance observed in the population.This strategy allowed us to identify QTLs for GY along with rapid development of high-yielding purelines tolerant to blast and drought with considerably reduced efforts. Apart from this, it also allowed us to study the effects of the selection cycle for blast tolerance. The developed lines were screened at IRRI and in the target environment, and drought and blast tolerant lines with high yield were identified. With tolerance to two major stresses and high yield potential, these lines may provide yield stability in rainfed rice areas. None Rice, Japonica (Oryza sativa L.). 2015 Methods Mol Biol Center for Plant Transformation, Plant Sciences Institute, and Department of Agronomy, Iowa State University, G405 AGRON, 2104 Agronomy Hall, Ames, IA, 50011-1010, USA, mmain@iastate.edu. The importance of rice, as a food crop, is reflected in the extensive global research being conducted in an effort to improve and better understand this particular agronomic plant. In regard to biotechnology, this has led to the development of numerous genetic transformation protocols. Over the years, many of these methods have become increasingly straightforward, rapid, and efficient, thereby making rice valuable as a model crop for scientific research and functional genomics. The focus of this chapter is on one such protocol that uses Agrobacterium-mediated transformation of Oryza sativa L. ssp. Japonica cv. Nipponbare with an emphasis on tissue desiccation. The explants consist of callus derived from mature seeds which are cocultivated on filter paper postinfection. Hygromycin selection is used for the recovery of subsequent genetically engineered events. None Rice, indica (Oryza sativa L.). 2015 Methods Mol Biol Plant Innovation Center, Japan Tobacco Inc., 700 Higashibara, Iwata, Shizuoka, 438-0802, Japan, yuko.hiei@jt.com. Indica varieties, which are generally recalcitrant to tissue culture and transformation, occupy 80 % of rice cultivation area in the world. Therefore, transformation method for indica rice must be improved greatly so that global rice production would take full advantage of cutting-edge biotechnology. An efficient protocol for indica transformation mediated by Agrobacterium tumefaciens is hereby described. Immature embryos collected from plants in a greenhouse are cocultivated with A. tumefaciens after pretreatment with heat and centrifuging. The protocol was successfully tested in many elite indica cultivars such as IR8, IR24, IR58025B, IR64, IR72, Suweon 258, and Nanjing 11, yielding between 5 and 15 of independent transgenic plants per immature embryo. The use of immature embryos is recommended because gene transfer to them could be much more efficient and much less genotype dependent than gene transfer to callus. None Combining high-throughput phenotyping and genome-wide association studies to reveal natural genetic variation in rice. 2014 Nat Commun 1] Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China [2] National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China [3] MoE Key Laboratory for Biomedical Photonics, Department of Biomedical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China [4] College of Engineering, Huazhong Agricultural University, Wuhan 430070, China. Even as the study of plant genomics rapidly develops through the use of high-throughput sequencing techniques, traditional plant phenotyping lags far behind. Here we develop a high-throughput rice phenotyping facility (HRPF) to monitor 13 traditional agronomic traits and 2 newly defined traits during the rice growth period. Using genome-wide association studies (GWAS) of the 15 traits, we identify 141 associated loci, 25 of which contain known genes such as the Green Revolution semi-dwarf gene, SD1. Based on a performance evaluation of the HRPF and GWAS results, we demonstrate that high-throughput phenotyping has the potential to replace traditional phenotyping techniques and can provide valuable gene identification information. The combination of the multifunctional phenotyping tools HRPF and GWAS provides deep insights into the genetic architecture of important traits. None Exogenous application of methyl jasmonate lowers the effect of cadmium-induced oxidative injury in rice seedlings. 2014 Phytochemistry Department of Bioinformatics, MMV, Banaras Hindu University, Varanasi 221 005, India. Rice seedlings grown under 50μM cadmium alone or in combination with 5μMmethyl jasmonate were investigated for Cd-induced oxidative injury at 3, 7 and 10days of treatment. MeJA treatments alone did not have any significant change in antioxidant enzyme activities or levels of H2O2 and O2(-) in roots/shoots, as compared to controls during 3-10days. The Cd-stressed plants When supplemented with exogenous MeJA revealed significant and consistent changes in activities of antioxidant enzymes CAT, SOD, POD and GR paralleled with an increased GSH-pools than that in plants subjected to Cd-stress alone. Synthesis of GSH driven by increasing demand for GSH in response to Cd-induced oxidative stress in rice was evident. Increased activity of LOX under Cd-stress was noted. Results suggest enhanced Cd-tolerance, lowered Cd(2+) uptake, an improved membrane integrity and 'switching on' of the JA-biosynthesis by LOX in the Cd-stressed rice roots/shoots exposed to MeJA. Exposure to MeJA improved antioxidant response and accumulation of antioxidants which perhaps lowered the Cd-induced oxidative stress in rice. It is this switching on/off of the JA-biosynthesis and ROS mediated signal transduction pathway involving glutathione homeostasis via GR which helps MeJA to mitigate Cd-induced oxidative injury in rice. None Photosynthetic diffusional constraints affect yield in drought stressed rice cultivars during flowering. 2014 PLoS One Institute of Agro-Environmental and Forest Biology, National Research Council, Porano, Italy. Global production of rice (Oryza sativa) grain is limited by water availability and the low 'leaf-level' photosynthetic capacity of many cultivars. Oryza sativa is extremely susceptible to water-deficits; therefore, predicted increases in the frequency and duration of drought events, combined with future rises in global temperatures and food demand, necessitate the development of more productive and drought tolerant cultivars. We investigated the underlying physiological, isotopic and morphological responses to water-deficit in seven common varieties of O. sativa, subjected to prolonged drought of varying intensities, for phenotyping purposes in open field conditions. Significant variation was observed in leaf-level photosynthesis rates (A) under both water treatments. Yield and A were influenced by the conductance of the mesophyll layer to CO2 (g(m)) and not by stomatal conductance (g(s)). Mesophyll conductance declined during drought to differing extents among the cultivars; those varieties that maintained g(m) during water-deficit sustained A and yield to a greater extent. However, the variety with the highest g(m) and yield under well-watered conditions (IR55419-04) was distinct from the most effective cultivar under drought (Vandana). Mesophyll conductance most effectively characterises the photosynthetic capacity and yield of O. sativa cultivars under both well-watered and water-deficit conditions; however, the desired attributes of high g(m) during optimal growth conditions and the capacity for g(m) to remain constant during water-deficit may be mutually exclusive. Nonetheless, future genetic and physiological studies aimed at enhancing O. sativa yield and drought stress tolerance should investigate the biochemistry and morphology of the interface between the sub-stomatal pore and mesophyll layer. None Phenotypic expression of blast resistance gene Pi54 is not affected by its chromosomal position. 2014 Plant Cell Rep National Research Centre on Plant Biotechnology, Lal Bahadur Shastri Centre, Pusa Campus, New Delhi, 110 012, India. This is a novel report in which chromosomal position of the rice blast resistance gene Pi54 was not found to affect significantly the resistance phenotype or morphological traits. Blast disease caused by Magnaporthe oryzae is a serious constraint in rice production at global level. Pi54 gene imparts resistance against M. oryzae. Three different transgenic lines containing Pi54 and its orthologue Pi54rh were shown to be resistant to different races of M. oryzae. To determine the chromosomal location of Pi54 gene in transgenic lines, inverse PCR was performed. Our analysis showed that in two transgenic lines, Pi54 gene was integrated on chromosomes 6 and 10 at 12.94 and 22.30 Mb, respectively. Similarly, Pi54rh allele was integrated on chromosome 1 at 16.25 Mb. The Pi54 gene present on chromosome 6 was located in a non-coding region whereas in the other TP-Pi54 line, the gene was introgressed on chromosome 10 in between the coding region of SAP domain gene. The Pi54rh was also located in the non coding region flanked by the retrotransposon genes. These rice lines were evaluated for eight different traits related to seed and plant morphology and agronomic features for two consecutive years. The transgenic lines containing Pi54 gene have higher tiller number, grain weight, epicotyl length, and yield compared to the non-transgenic control. Multivariate correlation analysis shows that blast resistance was positively correlated with the number of tillers; thousand grain weight and epicotyl length. These results will facilitate precise utilization of Pi54 gene and its orthologue in breeding programs for the development of rice cultivars with broad spectrum and durable resistance to M. oryzae. pikh|pi54 An anther development F-box (ADF) protein regulated by tapetum degeneration retardation (TDR) controls rice anther development. 2014 Planta State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Centre, Changsha, 410125, China, lili@hhrrc.ac.cn. In this study, we reported that a F-box protein, OsADF, as one of the direct targets of TDR , plays a critical role in rice tapetum cell development and pollen formation. The tapetum, the innermost sporophytic tissue of anther, plays an important supportive role in male reproduction in flowering plants. After meiosis, tapetal cells undergo programmed cell death (PCD) and provide nutrients for pollen development. Previously we showed that tapetum degeneration retardation (TDR), a basic helix-loop-helix transcription factor, can trigger tapetal PCD and control pollen wall development during anther development. However, the comprehensive regulatory network of TDR remains to be investigated. In this study, we cloned and characterized a panicle-specific expression F-box protein, anther development F-box (OsADF). By qRT-PCR and RNA in situ hybridization, we further confirmed that OsADF expressed specially in tapetal cells from stage 9 to stage 12 during anther development. In consistent with this specific expression pattern, the RNAi transgenic lines of OsADF exhibited abnormal tapetal degeneration and aborted microspores development, which eventually grew pollens with reduced fertility. Furthermore, we demonstrated that the TDR, a key regulator in controlling rice anther development, could regulate directly the expression of OsADF by binding to E-box motifs of its promoter. Therefore, this work highlighted the possible regulatory role of TDR, which regulates tapetal cell development and pollen formation via triggering the possible ADF-mediated proteolysis pathway. OsADF The bifunctional plant receptor, OsCERK1, regulates both chitin-triggered immunity and arbuscular mycorrhizal symbiosis in rice. 2014 Plant Cell Physiol Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa, 214-8571 Japan These authors contributed equally to this work. Plants are constantly exposed to threats from pathogenic microbes and thus developed an innate immune system to protect themselves. On the other hand, many plants also have the ability to establish endosymbiosis with beneficial microbes such as arbuscular mycorrhizal (AM) fungi or rhizobial bacteria, which improves the growth of host plants. How plants evolved these systems managing such opposite plant-microbe interactions is unclear. We show here that knockout (KO) mutants of OsCERK1, a rice receptor kinase essential for chitin signaling, were impaired not only for chitin-triggered defense responses but also for AM symbiosis, indicating the bifunctionality of OsCERK1 in defense and symbiosis. On the other hand, a KO mutant of OsCEBiP, which forms a receptor complex with OsCERK1 and is essential for chitin-triggered immunity, established mycorrhizal symbiosis normally. Therefore, OsCERK1 but not chitin-triggered immunity is required for AM symbiosis. Furthermore, experiments with chimeric receptors showed that the kinase domains of OsCERK1 and homologs from non-leguminous, mycorrhizal plants could trigger nodulation signaling in legume-rhizobium interactions as the kinase domain of Nod factor receptor1 (NFR1), which is essential for triggering the nodulation program in leguminous plants, did. Because leguminous plants are believed to have developed the rhizobial symbiosis on the basis of AM symbiosis, our results suggest that the symbiotic function of ancestral CERK1 in AM symbiosis enabled the molecular evolution to leguminous NFR1 and resulted in the establishment of legume-rhizobia symbiosis. These results also suggest that OsCERK1 and homologs serve as a molecular switch that activates defense or symbiotic responses depending on the infecting microbes. OsCERK1,CEBiP|OsCEBiP,CEBiP|OsCEBiP Overexpression analysis suggests that FON2-LIKE CLE PROTEIN1 is involved in rice leaf development. 2014 Genes Genet Syst Department of Biological Sciences, Graduate School of Science, The University of Tokyo. Peptide signaling plays important roles in various developmental processes of plants. Genes encoding CLE proteins, which are processed into CLE signaling peptides, are required for maintenance of the shoot apical meristem and for vascular differentiation. FON2-LIKE CLE PROTEIN1 (FCP1), a member of the CLE gene family, negatively regulates meristem maintenance in both shoot and root apical meristems of rice (Oryza sativa). Here, we examined the role of FCP1 in leaf development. We found that overexpression of FCP1 affects various aspects of leaf development in shoots regenerated from calli, making it difficult to distinguish between the leaf blade and leaf sheath. Differentiation of tissues such as vascular bundle and sclerenchyma was strongly inhibited by FCP1 overexpression. Spatial expression patterns of developmental genes DROOPING LEAF (DL) and OsPINHEAD1 (OsPNH1) were severely affected in the FCP1-overexpressing shoots. Whereas DL was expressed in the central region of leaf primordia in control shoots, DL expression was expanded throughout the leaf primordia of the FCP1-overexpressing shoots in early developmental stages. By contrast, OsPNH1, which is expressed in provascular and developing vascular tissues in normal seedlings, was strongly repressed by FCP1 overexpression. Taken together, our results suggest that FCP1 is involved in the regulation of cell fate determination during leaf development. FCP1|OsCLE402 Does the upstream region possessing MULE-like sequence in rice upregulate PsbS1 gene expression? 2014 PLoS One Institute for Environmental Science and Technology, Saitama University, Saitama city, Saitama, Japan; Graduate School of Science and Engineering, Saitama University, Saitama City, Saitama, Japan. The genomic nucleotide sequences of japonica rice (Sasanishiki and Nipponbare) contained about 2.7-kb unique region at the point of 0.4-kb upstream of the OsPsbS1 gene. In this study, we found that japonica rice with a few exceptions possessing such DNA sequences [denoted to OsMULE-japonica specific sequence (JSS)] is distinct by the presence of Mutator-like-element (MULE). Such sequence was absent in most of indica cultivars and Oryza glaberrima. In OsMULE-JSS1, we noted the presence of possible target site duplication (TSD; CTTTTCCAG) and about 80-bp terminal inverted repeat (TIR) near TSD. We also found the enhancement ofOsPsbS1 mRNA accumulation by intensified light, which was not associated with the DNA methylation status in OsMULE/JSS. In addition, O. rufipogon, possible ancestor of modern rice cultivars was found to compose PsbS gene of either japonica (minor) or indica (major) type. Transient gene expression assay showed that the japonica type promoter elevated a reporter gene activity than indica type. OsPsbS|psbS1 Salt tolerant SUV3 overexpressing transgenic rice plants conserve physicochemical properties and microbial communities of rhizosphere. 2014 Chemosphere Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India. Key concerns in the ecological evaluation of GM crops are undesirably spread, gene flow, other environmental impacts, and consequences on soil microorganism's biodiversity. Numerous reports have highlighted the effects of transgenic plants on the physiology of non-targeted rhizospheric microbes and the food chain via causing adverse effects. Therefore, there is an urgent need to develop transgenics with insignificant toxic on environmental health. In the present study, SUV3 overexpressing salt tolerant transgenic rice evaluated in New Delhi and Cuttack soil conditions for their effects on physicochemical and biological properties of rhizosphere. Its cultivation does not affect soil properties viz., pH, Eh, organic C, P, K, N, Ca, Mg, S, Na and Fe(2+). Additionally, SUV3 rice plants do not cause any change in the phenotype, species characteristics and antibiotic sensitivity of rhizospheric bacteria. The population and/or number of soil organisms such as bacteria, fungi and nematodes were unchanged in the soil. Also, the activity of bacterial enzymes viz., dehydrogenase, invertase, phenol oxidases, acid phosphatases, ureases and proteases was not significantly affected. Further, plant growth promotion (PGP) functions of bacteria such as siderophore, HCN, salicylic acid, IAA, GA, zeatin, ABA, NH3, phosphorus metabolism, ACC deaminase and iron tolerance were, considerably, not influenced. The present findings suggest ecologically pertinent of salt tolerant SUV3 rice to sustain the health and usual functions of the rhizospheric organisms. OsSUV3|SUV3 Physiological and biochemical characterization of NERICA-L-44: a novel source of heat tolerance at the vegetative and reproductive stages in rice. 2014 Physiol Plant Indian Agricultural Research Institute (PUSA), New Delhi, 110012, India; Crop and Environmental Sciences Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines. The predicted increase in the frequency and magnitude of extreme heat spikes under future climate can reduce rice yields significantly. Rice sensitivity to high temperatures during the reproductive stage is well documented while the same during the vegetative stage is more speculative. Hence, to identify and characterize novel heat-tolerant donors for both the vegetative and reproductive stages, 71 rice accessions, including approximately 75% New Rice for Africa (NERICAs), were phenotyped across field experiments during summer seasons in Delhi, India, and in a controlled environment study at International Rice Research Institute, Philippines. NERICA-L-44 (NL-44) recorded high seedling survival (52%) and superior growth and greater reproductive success exposed to 42.2°C (sd ± 2.3) under field conditions. NL-44 and the heat-tolerant check N22 consistently displayed lower membrane damage and higher antioxidant enzymes activity across leaves and spikelets. NL-44 recorded 50-60% spikelet fertility, while N22 recorded 67-79% under controlled environment temperature of 38°C (sd±1.17), although both had about 87% fertility under extremely hot field conditions. N22 and NL-44, exposed to heat stress (38°C), had similar pollen germination percent and number of pollen tubes reaching the ovary. NL-44 maintained low hydrogen peroxide production and non-photochemical quenching (NPQ) with high photosynthesis while N22 avoided photosystem II damage through high NPQ under high-temperature stress. NL-44 with its reproductive stage resilience to extreme heat stress, better antioxidant scavenging ability in both vegetative tissue and spikelets and superior yield and grain quality is identified as a novel donor for increasing heat tolerance at both the vegetative and reproductive stages in rice. None TOND1 confers tolerance to nitrogen deficiency in rice. 2014 Plant J State Key Laboratory of Plant Physiology and Biochemistry, National Center for Evaluation of Agricultural Wild Plants (Rice), Beijing Key Laboratory of Crop Genetic Improvement, Department of Plant Genetics and Breeding, China Agricultural University, Beijing, 100193, China. Nitrogen (N), the most important mineral nutrient for plants, is critical to agricultural production systems. N deficiency severely affects rice growth and decreases rice yields. However, excessive use of N fertilizer has caused severe pollution to agricultural and ecological environments. The necessity of breeding of crops that require lower input of N fertilizer has been recognized. Here we identified a major quantitative trait locus on chromosome 12, Tolerance Of Nitrogen Deficiency 1 (TOND1), that confers tolerance to N deficiency in the indica cultivar Teqing. Sequence verification of 75 indica and 75 japonica cultivars from 18 countries and regions demonstrated that only 27.3% of cultivars (41 indica cultivars) contain TOND1, whereas 72.7% of cultivars, including the remaining 34 indica cultivars and all 75 japonica cultivars, do not harbor the TOND1 allele. Over-expression of TOND1 increased the tolerance to N deficiency in the TOND1-deficient rice cultivars. The identification of TOND1 provides a molecular basis for breeding rice varieties with improved grain yield despite decreased input of N fertilizers. TOND1 The Matrix Polysaccharide (1;3,1;4)-beta-d-Glucan is Involved in Silicon-Dependent Strengthening of Rice Cell Wall. 2014 Plant Cell Physiol Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai, 980-8578 Japan. Poales [represented by rice (Oryza sativa L.)] in angiosperms and Equisetum (horsetails) in Pteridophytes are two major groups of heavy silicon (Si) accumulators. In rice, Si is polymerized preferentially in the epidermal cell wall, forming Si-cuticle double layers and Si-cellulose double layers beneath the cuticle. This Si layer is thought to exert various beneficial effects on the growth and development of land plants. Although the recent discovery of the influx and efflux transporters of silicic acid has shed some light on the molecular mechanisms of Si uptake and transport in rice, the mechanism underlying the final incorporation of polymerized Si into the cell wall remains elusive. Despite their phylogenetic distance, the cell walls of the two Si accumulators, Poales and Equisetum, share another common component, i.e. (1;3,1;4)-beta-d-glucan, also known as mixed-linkage glucan (MLG), a matrix polysaccharide not found in other plants. Based on this coincidence, a possible correlation between the functions of Si and MLG in the cell wall has been suggested, but no experimental evidence has been obtained in support of this functional correlation. Here, we present an analysis of the correlative action of Si and MLG on the mechanical properties of leaf blades using a transgenic rice line in which the MLG level was reduced by overexpressing EGL1, which encodes (1;3,1;4)-beta-d-glucanase. The reduction in MLG did not affect total Si accumulation, but it significantly altered the Si distribution profile and reduced the Si-dependent mechanical properties of the leaf blades, strongly suggesting a functional correlation between Si and MLG. None Production of superoxide from Photosystem II in a rice ( Oryza sativa L.) mutant lacking PsbS. 2014 BMC Plant Biol Fail BackgroundPsbS is a 22-kDa Photosystem (PS) II protein involved in non-photochemical quenching (NPQ) of chlorophyll fluorescence. Rice (Oryza sativa L.) has two PsbS genes, PsbS1 and PsbS2. However, only inactivation of PsbS1, through a knockout (PsbS1-KO) or in RNAi transgenic plants, results in plants deficient in qE, the energy-dependent component of NPQ.ResultsIn studies presented here, under fluctuating high light, growth of young seedlings lacking PsbS is retarded, and PSII in detached leaves of the mutants is more sensitive to photoinhibitory illumination compared with the wild type. Using both histochemical and fluorescent probes, we determined the levels of reactive oxygen species, including singlet oxygen, superoxide, and hydrogen peroxide, in leaves and thylakoids. The PsbS-deficient plants generated more superoxide and hydrogen peroxide in their chloroplasts. PSII complexes isolated from them produced more superoxide compared with the wild type, and PSII-driven superoxide production was higher in the mutants. However, we could not observe such differences either in isolated PSI complexes or through PSI-driven electron transport. Time-course experiments using isolated thylakoids showed that superoxide production was the initial event, and that production of hydrogen peroxide proceeded from that.ConclusionThese results indicate that at least some of the photoprotection provided by PsbS and qE is mediated by preventing production of superoxide released from PSII under conditions of excess excitation energy. None Cellular localization and detergent dependent oligomerization of rice allene oxide synthase-1. 2014 J Plant Res Department of Molecular Biotechnology and Kumho Life Science Laboratory, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, 500-757, Republic of Korea. Allene oxide synthase-1 from Oryza sativa (OsAOS1) localizes to the chloroplast, but lacks a putative chloroplast targeting sequence typically found in dicot AOS. Here, kinetic parameters and the oligomerization state/subunit composition of OsAOS1 were characterized in vitro in the absence or presence of detergent micelles. The catalytic efficiency (k cat/K m) of OsAOS1 reached a maximum near the critical micelle concentration for polyoxyethylene 10 tridecyl ether. Native gel analysis showed that OsAOS1 exists as a multimer in the absence of detergent micelles. The multimeric form of OsAOS1 was stably cross-linked in the absence of detergents, while only monomeric OsAOS1 was detected in the presence of detergent micelles. Gel filtration analysis indicated that the oligomeric state of OsAOS1 depends strongly on the detergents and that the monomer becomes the predominant form in the presence of detergent micelles. These data suggest that the detergent-dependent oligomeric state of OsAOS1 is an important factor for the regulation of its catalytic efficiency. OsAOS1 Over-expression of OsPTR6 in rice increased plant growth at different nitrogen supplies but decreased nitrogen use efficiency at high ammonium supply. 2014 Plant Sci State Key Laboratory of Crop Genetics and Germplasm Enhancement, MOA Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, PR China. Electronic address: xiaorongfan@njau.edu.cn. Nitrogen (N) plays a critical role in plant growth and productivity and PTR/NRT1 transporters are critical for rice growth. In this study, OsPTR6, a PTR/NRT1 transporter, was over-expressed in the Nipponbare rice cultivar by Agrobacterium tumefaciens transformation using the ubiquitin (Ubi) promoter. Three single-copy T2 generation transgenic lines, named OE1, OE5 and OE6, were produced and subjected to hydroponic growth experiments in different nitrogen treatments. The results showed the plant height and biomass of the over-expression lines were increased, and plant N accumulation and glutamine synthetase (GS) activities were enhanced at 5.0mmol/L NH4(+) and 2.5mmol/L NH4NO3. The expression of OsATM1 genes in over-expression lines showed that the OsPTR6 over expression increased OsAMT1.1, OsATM1.2 and OsAMT1.3 expression at 0.2 and 5.0mmol/L NH4(+) and 2.5mmol/L NH4NO3. However, nitrogen utilisation efficiency (NUE) was decreased at 5.0mmol/LNH4(+). These data suggest that over-expression of the OsPTR6 gene could increase rice growth through increasing ammonium transporter expression and glutamine synthetase activity (GSA), but decreases nitrogen use efficiency under conditions of high ammonium supply. OsPTR6 Overexpression of the OsChI1 gene, encoding a putative laccase precursor, increases tolerance to drought and salinity stress in transgenic Arabidopsis. 2014 Gene Plant Genomics Lab., Department of Applied Plant Sciences, Kangwon National University, Chuncheon 200-713, Republic of Korea. In a previous study, we identified a number of genes induced by chilling using a microarray approach. In order to investigate the molecular mechanism underlying chilling tolerance and possible crosstalk with other abiotic stresses, we selected a rice gene, OsChI1 (Os01g61160), for further analysis. The OsChI1 gene encodes a putative laccase precursor protein. In accordance with our previous results, its transcript is highly accumulated during a 12-day period of chilling treatment. Higher expression of the OsChI1 gene was also detected in roots and tissues at the vegetative and productive stages. In addition, we also observed increased transcript levels of the OsChI1 gene during dehydration and high salinity conditions. Transient expression of OsChI1 proteins tagged with fluorescence protein in rice protoplasts revealed that OsChI1 is localized in the plasma membrane. The Arabidopsis transgenic plants overexpressing OsChI1-EGFP resulted in an increased tolerance to drought and salinity stress. In silico analysis of OsChI1 suggests that several genes coexpressed with OsChI1 in the root during various abiotic stresses, such as chilling, drought and salt stress, may play an important role in the ROS signaling pathway. Potential roles of OsChI1 in response to abiotic stresses are discussed. OsChI1 A substitution mutation in OsCCD7 cosegregates with dwarf and increased tillering phenotype in rice. 2014 J Genet National Research Centre on Plant Biotechnology, LBS Centre, Indian Agricultural Research Institute, Pusa Campus, New Delhi 110 012, India. rpsnrcpb@yahoo.co.in. Dwarf plant height and tillering ability are two of the most important agronomic traits that determine the plant architecture, and have profound influence on grain yield in rice. To understand the molecular mechanism controlling these two traits, an EMS-induced recessive dwarf and increased tillering1 (dit1) mutant was characterized. The mutant showed proportionate reduction in each internode as compared to wild type revealing that it belonged to the category of dn-type of dwarf mutants. Besides, exogenous application of GA3 and 24-epibrassinolide, did not have any effect on the phenotype of the mutant. The gene was mapped on the long arm of chromosome 4, identified through positional candidate approach and verified by cosegregation analysis. It was found to encode carotenoid cleavage dioxygenase7 (CCD7) and identified as an allele of htd1. The mutant carried substitution of two nucleotides CC to AA in the sixth exon of the gene that resulted in substitution of serine by a stop codon in the mutant, and thus formation of a truncated protein, unlike amino acid substitution event in htd1. The new allele will facilitate further functional characterization of this gene, which may lead to unfolding of newer signalling pathways involving plant development and architecture. HTD1|OsCCD7 OsSUV3 transgenic rice maintains higher endogenous levels of plant hormones that mitigates adverse effects of salinity and sustains crop productivity. 2014 Rice (N Y) Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110 067, India. The SUV3 (suppressor of Var 3) gene encodes a DNA and RNA helicase, which is localized in the mitochondria. Plant SUV3 has not yet been characterized in detail. However, the Arabidopsis ortholog of SUV3 (AT4G14790) has been shown to be involved in embryo sac development. Previously, we have reported that rice SUV3 functions as DNA and RNA helicase and provides salinity stress tolerance by maintaining photosynthesis and antioxidant machinery. Here, we report further analysis of the transgenic OsSUV3 rice plants under salt stress.The transgenic OsSUV3 overexpressing rice T1 lines showed significantly higher endogenous content of plant hormones viz., gibberellic acid (GA3), zeatin (Z) and indole-3-acetic acid (IAA) in leaf, stem and root as compared to wild-type (WT), vector control (VC) and antisense (AS) plants under salt (200 mM NaCl) stress condition. A similar trend of endogenous plant hormones profile was also reflected in the T2 generation of OsSUV3 transgenic rice under defined parameters and stress condition.In response to stress, OsSUV3 rice plants maintained plant hormone levels that regulate the expression of several stress-induced genes and reduce adverse effects of salt on plant growth and development and therefore sustains crop productivity. OsSUV3|SUV3 Rice serine/threonine kinase 1 is required for the stimulation of OsNug2 GTPase activity. 2014 J Plant Physiol Department of Molecular Biotechnology, Dong-A University, Busan 604-714, South Korea. Electronic address: jbheo72@dau.ac.kr. Several GTPases are required for ribosome biogenesis and assembly. We recently identified rice (Oryza sativa) nuclear/nucleolar GTPase 2 (OsNug2), a YlqF/YawG family GTPase, as having a role in pre-60S ribosomal subunit maturation. To investigate the potential factors involved in regulating OsNug2 function, yeast two-hybrid screens were performed using OsNug2 as bait. Rice serine/threonine kinase 1 (OsSTK1) was identified as a candidate interacting protein. OsSTK1 appeared to interact with OsNug2 both in vitro and in vivo. OsSTK1 was found to have no effect on the GTP-binding activity of OsNug2; however, the presence of recombinant OsSTK1 in OsNug2 assay reaction mixtures increased OsNug2 GTPase activity. A kinase assay showed that OsSTK1 had weak autophosphorylation activity and strongly phosphorylated serine 209 of OsNug2. Using yeast complementation testing, we identified a GAL::OsNug2(S209N) mutation-harboring yeast strain that exhibited a growth-defective phenotype on galactose medium at 39°C, which was divergent from that of a yeast strain harboring GAL::OsNug2. The intrinsic GTPase activity of OsNug2(S209N), which was found to be similar to that of OsNug2, was not fully enhanced upon weak binding of OsSTK1. Our findings indicate that OsSTK1 functions as a positive regulator of OsNug2 by enhancing OsNug2 GTPase activity. In addition, phosphorylation of OsNug2 serine 209 is essential for its complete function in biological functional pathway. OsSTK1 Nuclear/nucleolar GTPase 2 proteins as a subfamily of YlqF/YawG GTPases function in pre-60S ribosomal subunit maturation of mono- and dicotyledonous plants. 2011 J Biol Chem Division of Applied Life Sciences (BK21), Graduate School of Gyeongsang National University, Jinju 660-701, Korea. The YlqF/YawG families are important GTPases involved in ribosome biogenesis, cell proliferation, or cell growth, however, no plant homologs have yet to be characterized. Here we isolated rice (Oryza sativa) and Arabidopsis nuclear/nucleolar GTPase 2 (OsNug2 and AtNug2, respectively) that belong to the YawG subfamily and characterized them for pre-60S ribosomal subunit maturation. They showed typical intrinsic YlqF/YawG family GTPase activities in bacteria and yeasts with k(cat) values 0.12 ± 0.007 min(-1) (n = 6) and 0.087 ± 0.002 min(-1) (n = 4), respectively, and addition of 60S ribosomal subunits stimulated their activities in vitro. In addition, OsNug2 rescued the lethality of the yeast nug2 null mutant through recovery of 25S pre-rRNA processing. By yeast two-hybrid screening five clones, including a putative one of 60S ribosomal proteins, OsL10a, were isolated. Subcellular localization and pulldown assays resulted in that the N-terminal region of OsNug2 is sufficient for nucleolar/nuclear targeting and association with OsL10a. OsNug2 is physically associated with pre-60S ribosomal complexes highly enriched in the 25S, 5.8S, and 5S rRNA, and its interaction was stimulated by exogenous GTP. Furthermore, the AtNug2 knockdown mutant constructed by the RNAi method showed defective growth on the medium containing cycloheximide. Expression pattern analysis revealed that the distribution of AtNug2 mainly in the meristematic region underlies its potential role in active plant growth. Finally, it is concluded that Nug2/Nog2p GTPase from mono- and didicotyledonous plants is linked to the pre-60S ribosome complex and actively processed 27S into 25S during the ribosomal large subunit maturation process, i.e. prior to export to the cytoplasm. OsNug2 GID1 modulates stomatal response and submergence tolerance involving ABA and GA signaling in rice. 2014 J Integr Plant Biol National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, 430070, China. Plant responses to abiotic stresses are coordinated by arrays of growth and developmental programs. Gibberellic acid (GA) and abscisic acid (ABA) play critical roles in the developmental programs and environmental responses, respectively, through complex signaling and metabolism networks. However, crosstalk between the two phytohormones in stress responses remains largely unknown. In this study, we report that GIBBERELLIN-INSENSITIVE DWARF 1 (GID1), a soluble receptor for GA, regulates stomatal development and patterning in rice (Oryza sativa L.). The gid1 mutant showed impaired biosynthesis of endogenous ABA under drought stress condition, but it exhibited enhanced sensitivity to exogenous ABA. Scanning electron microscope and infrared thermal image analysis indicated an increase in the stomatal conductance in the gid1 mutant under drought condition. Interestingly, the gid1 mutant had increased levels of chlorophyll and carbohydrates under submergence condition, and showed enhanced ROS-scavenging ability and submergence tolerance compared with the wild-type. Further analyses suggested that the function of GID1 in submergence responses is partially dependent on ABA, and GA signaling by GID1 is involved in submergence tolerance by modulating carbohydrate consumption. Taken together, these findings suggest GID1 plays distinct roles in stomatal response and submergence tolerance through both the ABA and GA signaling pathways in rice. GID1|OsGID1 Rice terpene synthase 20 (OsTPS20) plays an important role in producing terpene volatiles in response to abiotic stresses. 2014 Protoplasma Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu-gil, Jeongeup-si, Jeollabuk-do, 580-185, Republic of Korea. This study examined the volatile terpenes produced by rice seedlings in response to oxidative stress induced by various abiotic factors. Solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) analyses revealed that when exposed to UV-B radiation, rice seedlings emitted a bouquet of monoterpene mixtures in a time-dependent manner. The mixtures comprised limonene, sabinene, myrcene, α-terpinene, beta-ocimene, γ-terpinene, and α-terpinolene. Among them, (S)-limonene was the most abundant volatile, discriminated by chiral SPME-GC-MS. The volatile profiles collected from rice plants treated with both γ-irradiation and H2O2 were identical to those observed in the UV-B irradiated plants, thus indicating that the volatile mixtures were specifically produced in response to oxidative stress, particularly in the presence of H2O2. Using a reverse genetics approach, we isolated full-length rice terpene synthase 20 (OsTPS20, 599 amino acids, 69.39 kDa), which was further characterized as an (S)-limonene synthase by removing the N-terminal signal peptide (63 amino acids) of the protein. The recombinant OsTPS20 protein catalyzed the conversion of geranyl diphosphate to (S)-limonene and other minor monoterpenes, essentially covering all of the volatile compounds detected from the plant. Moreover, qRT-PCR revealed that the transcript levels of OsTPS20 were significantly induced in response to oxidative stress, thereby suggesting that OsTPS20 plays a major role in producing terpene volatiles during abiotic stress. Detailed biochemical analyses and the unusual domain characteristics of OsTPS20 are also discussed in this report. OsTPS20 The rice OsAMT1;1 is a proton-independent feedback regulated ammonium transporter. 2014 Plant Cell Rep State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, No. 71, East Beijing Road, Nanjing, 210008, China. Functional identification of a relatively lower affinity ammonium transporter, OsAMT1;1, which is a proton-independent feedback regulated ammonium transporter in rice. Rice genome contains at least 12 ammonium transporters, though their functionality has not been clearly resolved. Here, we demonstrate the functional properties of OsAMT1;1 applying functional complementation and (15)NH4 (+) uptake determination in yeast cells in combination with electrophysiological measurements in Xenopus oocytes. Our results show that OsAMT1;1 is a NH4 (+) transporter with relatively lower affinity to NH4 (+) (110-129 μM in oocytes and yeast cells, respectively). Under our experimental conditions, OsAMT1;1-mediated NH4 (+) uptake or current is not significantly modulated by extra- or intracellular pH gradient, suggesting that this transporter probably functions as a NH4 (+) uniporter. Inhibition of yeast growth or currents elicited from oocytes by ammonium assimilation inhibitor L-methionine sulfoximine indicates that NH4 (+) transport by OsAMT1;1 is likely feedback regulated by accumulation of the substrate. In addition, effects of phosphorylation inhibitors imply that NH4 (+) uptake by OsAMT1;1 is also modulated by tyrosine-specific protein kinase or calcium-regulated serine/threonine-specific protein phosphatase involved phosphorylation processes. OsAMT1;1|OsAMT1.1 Casein Kinases I and 2α Phosphorylate Oryza Sativa Pseudo-Response Regulator 37 (OsPRR37) in Photoperiodic Flowering in Rice. 2014 Mol Cells Department of Plant Science, Plant Genomics and Breeding Institute, and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea. Flowering time (or heading date) is controlled by intrinsic genetic programs in response to environmental cues, such as photoperiod and temperature. Rice, a facultative shortday (SD) plant, flowers early in SD and late in long-day (LD) conditions. Casein kinases (CKs) generally act as positive regulators in many signaling pathways in plants. In rice, Heading date 6 (Hd6) and Hd16 encode CK2α and CKI, respectively, and mainly function to delay flowering time. Additionally, the major LD-dependent floral repressors Hd2/Oryza sativa Pseudo-Response Regulator 37 (OsPRR37; hereafter PRR37) andGhd7 also confer strong photoperiod sensitivity. In floral induction, Hd16 acts upstream of Ghd7 and CKI interacts with and phosphorylates Ghd7. In addition, Hd6 and Hd16 also act upstream of Hd2. However, whether CKI and CK2α directly regulate the function of PRR37 remains unclear. Here, we use in vitro pull-down and in vivo bimolecular fluorescence complementation assays to show that CKI and CK2α interact with PRR37. We further use in vitro kinase assays to show that CKI and CK2α phosphorylate different regions of PRR37. Our results indicate that direct posttranslational modification of PRR37 mediates the genetic interactions between these two protein kinases and PRR37. The significance of CK-mediated phosphorylation for PRR37 and Ghd7 function is discussed. Ghd7.1|Hd2|OsPRR37|DTH7 Functional characterization of a type 3 metallolthionein isoform (OsMTI-3a) from rice. 2014 Int J Biol Macromol Department of Agricultural Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran. Electronic address: a.shahpiri@cc.iut.ac.ir. Metallothioneins (MTs) are low-molecular weight proteins with high Cys content and a high affinity for metals. Plant MTs are classified into four types based on the arrangement of Cys in their amino acid sequences. In the present study, the gene encoding OsMTI-3a, a type 3 MT found in rice, was cloned into pET41a vector. The resulting construct was transformed into the Escherichia coli strain Rosetta (DE3). Following the induction with isopropyl beta-d-1-thiogalactopyranoside, the OsMTI-3a was expressed as glutathione-S-transferase (GST)-tagged fusion protein. In comparison to control strain, the cells expressing GST-OsMTI-3a accumulated more Cd(2+), Ni(2+) and Zn(2+) when they were grown in the medium containing CdCl2, NiCl2 or ZnSO4. The recombinant GST-OsMTI-3a was purified using affinity chromatography. The UV absorption spectra recorded after the reconstitution of the apo-protein with different metals confirmed that GST-OsMTI-3a was able to form complexes with Cd(2+), Ni(2+), and Zn(2+). The reaction of the protein-metal complexes with 5-5-dithiobis (2-nitrobenzoic) revealed that the order of affinity of GST-OsMTI-3a toward different metals was Ni(2+)≥Cd(2+)>Zn(2+)>Cu(2+). OsMTI-3a A rice DEAD-box RNA helicase protein, OsRH17, suppresses 16S ribosomal RNA maturation in Escherichia coli. 2014 Gene State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310006, China; Key Laboratory of Crop Physiology, Ecology and Genetic Breeding, Ministry of Education, Jiangxi Agricultural University, Nanchang 330045, China. DEAD-box proteins comprise a large protein family. These proteins function in all types of processes in RNA metabolism and are highly conserved among eukaryotes. However, the precise functions of DEAD-box proteins in rice physiology and development remain unclear. In this study, we identified a rice DEAD-box protein, OsRH17, that contains a DEAD domain and all of the common conserved motifs of DEAD-box RNA helicases. OsRH17 was specifically expressed in pollen and differentiated callus and upregulated by application of the plant hormones naphthyl acetic acid (NAA) and abscisic acid (ABA). The OsRH17:GFP fusion protein was localized to the nucleus. Tiny amounts of OsRH17 and partial fragments (N-427 and C-167) were detected when they were expressed in Escherichia coli, a prokaryote. Growth of the host cells was suppressed in E. coli by OsRH17, N-427 or C-167, and this suppression was independent of the concentration of the NaCl in the medium. Expression analysis of rRNAs in E. coli revealed that the 16S rRNA precursor accumulated in transgenic E. coli cells, and the relative growth rate was inversely proportional to the levels of pre-16S rRNA accumulation. Results suggested that OsRH17 may play a role in ribosomal biogenesis and suppress 16S rRNA maturation in E. coli. No visible phenotype was observed in transgenic yeast and rice (overexpressing OsRH17, N-427, and C-167, as well as OsRH17 knockdown), and even in some abiotic and biotic stresses, which could be due to the redundancy in rice under normal conditions. OsRH17 Altered cell wall properties are responsible for ammonium-reduced aluminum accumulation in rice roots. 2014 Plant Cell Environ State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China; University of Chinese Academy of Sciences, Beijing 100049, China. The phytotoxicity of aluminum (Al) ions can be alleviated by ammonium (NH4 (+) ) in rice and this effect has been attributed to the decreased Al accumulation in the roots. Here, the effects of different nitrogen forms on cell wall properties were compared in two rice cultivars differing in Al tolerance. An in vitro Al-binding assay revealed that neither NH4 (+) nor NO3 (-) altered the Al-binding capacity of cell walls, which were extracted from plants not previously exposed to N sources. However, cell walls extracted from NH4 (+) -supplied roots displayed lower Al-binding capacity than those from NO3 (-) -supplied roots when grown in non-buffered solutions. Fourier-transform infrared microspectroscopy analysis revealed that, compared with NO3 (-) -supplied roots, NH4 (+) -supplied roots possessed fewer Al-binding groups (-OH and COO-) and lower contents of pectin and hemicellulose. However, when grown in pH-buffered solutions, these differences in the cell wall properties were not observed. Further analysis showed that the Al-binding capacity and properties of cell walls were also altered by pHs alone. Taken together, our results indicate that the NH4 (+) -reduced Al accumulation was attributed to the altered cell wall properties triggered by pH decrease due to NH4 (+) uptake rather than direct competition for the cell wall binding sites between Al(3+) and NH4 (+) . None Cis-12-oxo-phytodienoic acid stimulates rice defense response to a piercing-sucking insect. 2014 Mol Plant Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. The brown planthopper (BPH, Nilaparvata lugens) is a destructive, monophagous, piercing-sucking insect pest of rice. Previous studies indicated that jasmonic acid (JA) positively regulates rice defense against chewing insect pests but negatively regulates it against the piercing-sucking insect of BPH. We here demonstrated that overexpression of allene oxide cyclase (AOC) but not OPR3 (cis-12-oxo-phytodienoic acid (OPDA) reductase 3, an enzyme adjacent to AOC in the JA synthetic pathway) significantly increased rice resistance to BPH, mainly by reducing the feeding activity and survival rate. Further analysis revealed that plant response to BPH under AOC overexpression was independent of the JA pathway and that significantly higher OPDA levels stimulated rice resistance to BPH. Microarray analysis identified multiple candidate resistance-related genes under AOC overexpression. OPDA treatment stimulated the resistance of radish seedlings to green peach aphid Myzus persicae, another piercing-sucking insect. These results imply that rice resistance to chewing insects and to sucking insects can be enhanced simultaneously through AOC-mediated increases of JA and OPDA and provide direct evidence of the potential application of OPDA in stimulating plant defense responses to piercing-sucking insect pests in agriculture. None Identification of rice Di19 family reveals OsDi19-4 involved in drought resistance. 2014 Plant Cell Rep State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, 430072, China. The OsDi19 proteins functioned as transcription factors and played crucial roles in response to abiotic stress. Overexpression of OsDi19-4 in rice increased drought tolerance by enhancing ROS-scavenging activity. Many transcription factors play crucial roles in plant responses to abiotic stress. Here, comprehensive sequence analysis suggested that the drought-induced 19 (Di19) gene family in rice genome contain seven members, and these proteins contained a well-conserved zinc-finger Di19 domain. Most OsDi19 proteins were mainly targeted to the nucleus and have transactivation activity in yeast. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that most OsDi19 proteins could form protein dimers. Expression analysis demonstrated that the OsDi19 genes were differentially and abundantly expressed in vegetative tissues, but expressed little in reproductive tissues and some of the OsDi19 genes were markedly induced by abiotic stresses and hormones in qRT-PCR analysis and microarray data. Overexpression of one stress-responsive gene, OsDi19-4, in rice resulted in significantly increased tolerance to drought stress compared with the wild type plants. Moreover, obviously increased ROS-scavenging ability was detected in the OsDi19-4-overexpressing plants under normal and drought stress conditions. These results suggested that the increased stress tolerance of OsDi19-4-overexpressing plants may be attributable to the enhanced ROS-scavenging activity. Taken together, these studies provide a detailed overview of the rice Di19 gene family, and suggest that the OsDi19 family may play crucial roles in the plant response to abiotic stress. OsDi19-4 A role for TRIANGULAR HULL1 in fine-tuning spikelet morphogenesis in rice. 2014 Genes Genet Syst Department of Biological Sciences, Graduate School of Science, The University of Tokyo. The lemma and palea, which enclose the pistil, stamens, and lodicules, are the most conspicuous organs in the rice spikelet. We isolated a mutant line (ng6569) in which the lemma and palea were narrower than those of the wild type, and found that the mutant had a defect in TRIANGULAR HULL1 (TH1), which encodes a nuclear protein with an ALOG domain. Detailed morphological analysis indicated that the th1 mutation caused a reduction in the size of tubercles, which are convex structures on the surface of the lemma and palea. This reduction was more pronounced in the apical region of the lemma than in the basal region, resulting in the formation of a beak-like spikelet. By contrast, the number of tubercle rows and their spatial distribution on the lemma were not affected in the th1 mutant. Thus, the TH1 gene seems to be involved in fine-tuning the morphogenesis of the lemma and palea. In situ hybridization analysis revealed that TH1 was highly expressed in the primordia of the lemma and palea, but only weakly expressed in the primordia of the sterile lemma and rudimentary glume. We then examined the effect of th1 mutation on the lemma-like structure formed in the long sterile lemma/glume1 (g1) and extra glume1 (eg1) mutants. The result showed that the th1 mutation strongly affected the morphology of the extra lemma of eg1, but had no significant effect on the transformed lemma of g1. BLS1|BSG1|OsG1L6|TH1 The mitogen-activated protein kinase cascade MKK1-MPK4 mediates salt signaling in rice. 2014 Plant Sci College of Life Sciences, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, PR China. Mitogen-activated protein kinase (MAPK) pathways have been implicated in signal transduction of both biotic and abiotic stresses in plants. In this study, we found that the transcript of a rice (Oryza sativa) MAPKK (OsMKK1) was markedly increased by salt stress. By examining the survival rate and Na(+) content in shoot, we found that OsMKK1-knockout (osmkk1) mutant was more sensitive to salt stress than the wild type. OsMKK1 activity in the wild-type seedlings and protoplasts was increased by salt stress. Yeast two-hybrid and in vitro and in vivo kinase assays revealed that OsMKK1 targeted OsMPK4. OsMPK4 activity was increased by salt, which was impaired in osmkk1 plants. In contrast, overexpression of OsMKK1 increased OsMPK4 activity in protoplasts. By comparing the transcription factors levels between WT and osmkk1 mutant, OsMKK1 was necessary for salt-induced increase in OsDREB2B and OsMYBS3. Taken together, the data suggest that OsMKK1 and OsMPK4 constitute a signaling pathway that regulates salt stress tolerance in rice. OsMKK1 Rice GROWTH UNDER DROUGHT KINASE is required for drought tolerance and grain yield under normal and drought stress conditions. 2014 Plant Physiol Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, Arkansas 72701 (R.V., S.B., A.P.); andVirginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia 24061 (A.K., A.P.). Rice (Oryza sativa) is the primary food source for more than one-half of the world's population. Because rice cultivation is dependent on water availability, drought during flowering severely affects grain yield. Here, we show that the function of a drought-inducible receptor-like cytoplasmic kinase, named GROWTH UNDER DROUGHT KINASE (GUDK), is required for grain yield under drought and well-watered conditions. Loss-of-function gudk mutant lines exhibit sensitivity to salinity, osmotic stress, and abscisic acid treatment at the seedling stage, and a reduction in photosynthesis and plant biomass under controlled drought stress at the vegetative stage. The gudk mutants interestingly showed a significant reduction in grain yield, both under normal well-watered conditions and under drought stress at the reproductive stage. Phosphoproteome profiling of the mutant followed by in vitro assays identified the transcription factor APETALA2/ETHYLENE RESPONSE FACTOR OsAP37 as a phosphorylation target of GUDK. The involvement of OsAP37 in regulating grain yield under drought through activation of several stress genes was previously shown. Our transactivation assays confirmed that GUDK is required for activation of stress genes by OsAP37. We propose that GUDK mediates drought stress signaling through phosphorylation and activation of OsAP37, resulting in transcriptional activation of stress-regulated genes, which impart tolerance and improve yield under drought. Our study reveals insights around drought stress signaling mediated by receptor-like cytoplasmic kinases, and also identifies a primary regulator of grain yield in rice that offers the opportunity to improve and stabilize rice grain yield under normal and drought stress conditions. GUDK A down-regulated epi-allele of the genomes uncoupled 4 gene generates a xantha marker trait in rice. 2014 Theor Appl Genet National Key Laboratory of Rice Biology, Institute of Crop Sciences, Zhejiang University, 310029, Hangzhou, China. A gamma-ray-induced xantha trait is epigenetically controlled by the genomes uncoupled 4 gene with enhanced promoter segment methylation and down-regulated expression in rice. For easy testing and to increase varietal purity, a xantha mutation (xnt), which turns plants yellow and makes them visually distinguishable from normal green rice, has been generated and bred into male sterile lines for hybrid rice production. The xnt locus was previously fine mapped to a ~100-kb interval on chromosome 11, but its identity was unknown. In this study, xnt was further narrowed down to a 57-kb fragment carrying eight opening reading frames (ORFs). All eight ORFs had identical genomic sequences and all but ORF2 (g enomes uncoupled 4, OsGUN4) had similar transcript abundance in the xantha mutant Huangyu B (HYB) and its parental variety Longtefu B (LTB). The expression of OsGUN4, however, was significantly reduced in HYB compared with LTB in terms of both transcript abundance (0.2% that of LTB) and expressed protein level (barely detectable in HYB but greater than the heat shock protein reference in LTB). Therefore, OsGUN4 was identified as the candidate gene underlying the xantha trait. The function of OsGUN4 in the xantha phenotype was confirmed by identification and characterization of new allelic OsGUN4 mutations. Comparative bisulfite genomic sequencing of OsGUN4 revealed increased methylation in a promoter region in the mutant, and the correlation between increased methylation and the xantha phenotype was further verified by demethylation treatment. In summary, we have identified an epi-allele of OsGUN4 as the causal gene of the xantha marker trait and revealed that enhanced methylation in its promoter down-regulated its expression in rice. OsGUN4 OsNF-YB1, a rice endosperm-specific gene, is essential for cell proliferation in endosperm development. 2014 Gene College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China. Cell cycle regulators are crucial for normal endosperm development and seed size determination. However, how the cell cycle related genes regulate endosperm development remains unclear. In this study, we reported a rice Nuclear Factor Y (NF-Y) gene OsNF-YB1, which was also identified as an endosperm-specific gene. Transcriptional profiling and promoter analysis revealed that OsNF-YB1 was highly expressed at the early stages of rice endosperm development (5-7 DAP, days after pollination). Repression of OsNF-YB1 resulted in differential expression of the genes in cell cycle pathway, which caused abnormal seeds with defected embryo and endosperm. Basic cytological analysis demonstrated that the reduced endosperm cell numbers disintegrated with the development of those abnormal seeds in OsNF-YB1 RNAi plants. Taken together, these results suggested that the endosperm-specific gene OsNF-YB1 might be a cell cycle regulator and played a role in maintaining the endosperm cell proliferation. OsNF-YB1 Molecular dissection of the response of a rice leucine-rich repeat receptor-like kinase (LRR-RLK) gene to abiotic stresses. 2014 J Plant Physiol Plant Genomics Lab., Department of Applied Plant Sciences, Kangwon National University, Chuncheon 200-713, Republic of Korea. Leucine-rich repeat (LRR) receptor-like kinase (RLK) proteins play key roles in a variety of biological pathways. In a previous study, we analyzed the members of the rice LRR-RLK gene family using in silico analysis. A total of 23 LRR-RLK genes were selected based on the expression patterns of a genome-wide dataset of microarrays. The Oryza sativa gamma-ray induced LRR-RLK1 (OsGIRL1) gene was highly induced by gamma irradiation. Therefore, we studied its expression pattern in response to various different abiotic and phytohormone treatments. OsGIRL1 was induced on exposure to abiotic stresses such as salt, osmotic, and heat, salicylic acid (SA), and abscisic acid (ABA), but exhibited downregulation in response to jasmonic acid (JA) treatment. The OsGIRL1 protein was clearly localized at the plasma membrane. The truncated proteins harboring juxtamembrane and kinase domains (or only harboring a kinase domain) exhibited strong autophosphorylation. The biological function of OsGIRL1 was investigated via heterologous overexpression of this gene in Arabidopsis plants subjected to gamma-ray irradiation, salt stress, osmotic stress, and heat stress. A hypersensitive response was observed in response to salt stress and heat stress, whereas a hyposensitive response was observed in response to gamma-ray treatment and osmotic stress. These results provide critical insights into the molecular functions of the rice LRR-RLK genes as receptors of external signals. OsGIRL1 Gα modulates salt-induced cellular senescence and cell division in rice and maize. 2014 J Exp Bot Department of Biology at the University of North Carolina, Chapel Hill, NC, 27599-3280, USA. The plant G-protein network, comprising Gα, Gbeta, and Gγ core subunits, regulates development, senses sugar, and mediates biotic and abiotic stress responses. Here, we report G-protein signalling in the salt stress response using two crop models, rice and maize. Loss-of-function mutations in the corresponding genes encoding the Gα subunit attenuate growth inhibition and cellular senescence caused by sodium chloride (NaCl). Gα null mutations conferred reduced leaf senescence, chlorophyll degradation, and cytoplasm electrolyte leakage under NaCl stress. Sodium accumulated in both wild-type and Gα-mutant shoots to the same levels, suggesting that Gα signalling controls cell death in leaves rather than sodium exclusion in roots. Growth inhibition is probably initiated by osmotic change around root cells, because KCl and MgSO4 also suppressed seedling growth equally as well as NaCl. NaCl lowered rates of cell division and elongation in the wild-type leaf sheath to the level of the Gα-null mutants; however there was no NaCl-induced decrease in cell division in the Gα mutant, implying that the osmotic phase of salt stress suppresses cell proliferation through the inhibition of Gα-coupled signalling. These results reveal two distinct functions of Gα in NaCl stress in these grasses: attenuation of leaf senescence caused by sodium toxicity in leaves, and cell cycle regulation by osmotic/ionic stress. None Overexpression of a rice heme activator protein gene (OsHAP2E) confers resistance to pathogens, salinity and drought, and increases photosynthesis and tiller number. 2014 Plant Biotechnol J Faculty of Agriculture, United Graduate School of Agricultural Sciences, Ehime University, Matsuyama, Ehime, Japan. Heme activator protein (HAP), also known as nuclear factor Y or CCAAT binding factor (HAP/NF-Y/CBF), has important functions in regulating plant growth, development and stress responses. The expression of rice HAP gene (OsHAP2E) was induced by probenazole (PBZ), a chemical inducer of disease resistance. To characterize the gene, the chimeric gene (OsHAP2E::GUS) engineered to carry the structural gene encoding glucuronidase (GUS) driven by the promoter from OsHAP2E was introduced into rice. The transgenic lines of OsHAP2Ein::GUS with the intron showed high GUS activity in the wounds and surrounding tissues. When treated by salicylic acid (SA), isonicotinic acid (INA), abscisic acid (ABA) and hydrogen peroxide (H2 O2 ), the lines showed GUS activity exclusively in vascular tissues and mesophyll cells. This activity was enhanced after inoculation with Magnaporthe oryzae or Xanthomonas oryzae pv. oryzae. The OsHAP2E expression level was also induced after inoculation of rice with M. oryzae and X. oryzae pv. oryzae and after treatment with SA, INA, ABA and H2 O2, respectively. We further produced transgenic rice overexpressing OsHAP2E. These lines conferred resistance to M. oryzae or X. oryzae pv. oryzae and to salinity and drought. Furthermore, they showed a higher photosynthetic rate and an increased number of tillers. Microarray analysis showed up-regulation of defence-related genes. These results suggest that this gene could contribute to conferring biotic and abiotic resistances and increasing photosynthesis and tiller numbers. OsHAP2E The effect of Silicon on photosynthesis and expression of its relevant genes in rice (Oryza sativa L.) under high-zinc stress. 2014 PLoS One Ministry of Agriculture Key Laboratory of Crop Nutrition and Fertilization, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, China. The main objectives of this study were to elucidate the roles of silicon (Si) in alleviating the effects of 2 mM zinc (high Zn) stress on photosynthesis and its related gene expression levels in leaves of rice (Oryza sativa L.) grown hydroponically with high-Zn stress. The results showed that photosynthetic parameters, including net photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO2 concentration, chlorophyll concentration and the chlorophyll fluorescence, were decreased in rice exposed to high-Zn treatment. The leaf chloroplast structure was disordered under high-Zn stress, including uneven swelling, disintegrated and missing thylakoid membranes, and decreased starch granule size and number, which, however, were all counteracted by the addition of 1.5 mM Si. Furthermore, the expression levels of Os08g02630 (PsbY), Os05g48630 (PsaH), Os07g37030 (PetC), Os03g57120 (PetH), Os09g26810 and Os04g38410 decreased in Si-deprived plants under high-Zn stress. Nevertheless, the addition of 1.5 mM Si increased the expression levels of these genes in plants under high-Zn stress at 72 h, and the expression levels were higher in Si-treated plants than in Si-deprived plants. Therefore, we conclude that Si alleviates the Zn-induced damage to photosynthesis in rice. The decline of photosynthesis in Zn-stressed rice was attributed to stomatal limitation, and Si activated and regulated some photosynthesis-related genes in response to high-Zn stress, consequently increasing photosynthesis. None STRIPE2 encodes a putative dCMP deaminase that plays an important role in chloroplast development in rice. 2014 J Genet Genomics National Key Laboratory of Plant Molecular Genetics and National Center of Plant Gene Research, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China. Mutants with abnormal leaf coloration are good genetic materials for understanding the mechanism of chloroplast development and chlorophyll biosynthesis. In this study, a rice mutant st2 (stripe2) with stripe leaves was identified from the gamma-ray irradiated mutant pool. The st2 mutant exhibited decreased accumulation of chlorophyll and aberrant chloroplasts. Genetic analysis indicated that the st2 mutant was controlled by a single recessive locus. The ST2 gene was finely confined to a 27-kb region on chromosome 1 by the map-based cloning strategy and a 5-bp deletion in Os01g0765000 was identified by sequence analysis. The deletion happened in the joint of exon 3 and intron 3 and led to new spliced products of mRNA. Genetic complementation confirmed that Os01g0765000 is the ST2 gene. We found that the ST2 gene was expressed ubiquitously. Subcellular localization assay showed that the ST2 protein was located in mitochondria. ST2 belongs to the cytidine deaminase-like family and possibly functions as the dCMP deaminase, which catalyzes the formation of dUMP from dCMP by deamination. Additionally, exogenous application of dUMP could partially rescue the st2 phenotype. Therefore, our study identified a putative dCMP deaminase as a novel regulator in chloroplast development for the first time. ST2 MicroRNA biogenesis factor DRB1 is a phosphorylation target of mitogen activated protein kinase MPK3 in both rice and Arabidopsis. 2014 FEBS J National Institute of Plant Genome Research, New Delhi, India. MicroRNA (miRNA) biogenesis requires AtDRB1 (double-stranded RNA binding protein)/HYL1 (Hyponastic Leaves1) protein for processing and maturation of miRNA precursors. The AtDRB1/HYL1 protein associates with AtDCL1 (Dicer-Like1) and accurately processes primary-miRNAs (pri-mRNAs) first to precursor-miRNAs (pre-miRNAs) and finally to mature miRNAs. The dephosphorylation of AtDRB1/HYL1 protein is very important for the precise processing of miRNA precursors. The monocot model crop plant Oryza sativa encodes four orthologues of AtDRB1/HYL1 protein, the only one encoded by Arabidopsis thaliana. The present study focuses on the functionality of the O. sativa DRBs as the orthologues of AtDRB1/HYL1 by using RNA binding assays and in planta protein-protein interaction analysis. Further, mitogen-activated protein kinase MPK3 is established as the kinase phosphorylating DRB1 protein in both the model plants, O. sativa and Arabidopsis. MicroRNA microarray analysis in atmpk3 and atmpk6 mutants indicate the importance of AtMPK3 in maintaining the level of miRNAs in the plant. OsDRB1-1,OsDRB1-2 A gene cluster encoding lectin receptor kinases confers broad-spectrum and durable insect resistance in rice 2014 Nature Biotechnology National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing, China. The brown planthopper (BPH) is the most destructive pest of rice (Oryza sativa) and a substantial threat to rice production, causing losses of billions of dollars annually1, 2. Breeding of resistant cultivars is currently hampered by the rapid breakdown of BPH resistance2. Thus, there is an urgent need to identify more effective BPH-resistance genes. Here, we report molecular cloning and characterization of Bph3, a locus in rice identified more than 30 years ago that confers resistance to BPH. We show that Bph3 is a cluster of three genes encoding plasma membrane-Clocalized lectin receptor kinases (OsLecRK1-OsLecRK3). Introgression of Bph3 into susceptible rice varieties by transgenic or marker-assisted selection strategies significantly enhanced resistance to both the BPH and the white back planthopper. Our results suggest that these lectin receptor kinase genes function together to confer broad-spectrum and durable insect resistance and provide a resource for molecular breeding of insect-resistant rice cultivars. OslecRK|OsLecRK1,OsLecRK2,OsLecRK3,OsLecRK4 Lack of cytosolic glutamine synthetase1;2 in vascular tissues of axillary buds causes severe reduction in their outgrowth and disorder of metabolic balance in rice seedlings. 2014 Plant J Graduate School of Agriculture Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555, Japan. The development and elongation of active tillers in rice was severely reduced by a lack of cytosolic glutamine synthetase1;2 (GS1;2), and, to a lesser extent, lack of NADH-glutamate synthase1 in knockout mutants. In situ hybridization using the basal part of wild-type seedlings clearly showed that expression of OsGS1;2 was detected in the phloem companion cells of the nodal vascular anastomoses and large vascular bundles of axillary buds. Accumulation of lignin, visualized using phloroglucin HCl, was also observed in these tissues. The lack of GS1;2 resulted in reduced accumulation of lignin. Re-introduction into the mutants of OsGS1;2 cDNA under the control of its own promoter successfully restored the outgrowth of tillers and lignin deposition to wild-type levels. Transcriptomic analysis using a 5 mm basal region of rice shoots showed that the GS1;2 mutants accumulated reduced amounts of mRNAs for carbon and nitrogen metabolism, including C1 unit transfer in lignin synthesis. Although a high content of strigolactone in rice roots is known to reduce active tiller number, the reduction of outgrowth of axillary buds observed in the GS1;2 mutants was independent of the level of strigolactone. Thus metabolic disorder caused by the lack of GS1;2 resulted in a severe reduction in the outgrowth of axillary buds and lignin deposition. GLN1;2|GS1;2|OsGS1;2 Higher sterol content regulated by CYP51 with concomitant lower phospholipid contents in membranes is a common strategy for aluminium tolerance in several plant species. 2014 J Exp Bot Faculty of Agriculture, Yamagata University, Tsuruoka 997-8555, Japan wagatuma@tds1.tr.yamagata-u.ac.jp. Several studies have shown that differences in lipid composition and in the lipid biosynthetic pathway affect the aluminium (Al) tolerance of plants, but little is known about the molecular mechanisms underlying these differences. Phospholipids create a negative charge at the surface of the plasma membrane and enhance Al sensitivity as a result of the accumulation of positively charged Al(3+) ions. The phospholipids will be balanced by other electrically neutral lipids, such as sterols. In the present research, Al tolerance was compared among pea (Pisum sativum) genotypes. Compared with Al-tolerant genotypes, the Al-sensitive genotype accumulated more Al in the root tip, had a less intact plasma membrane, and showed a lower expression level of PsCYP51, which encodes obtusifoliol-14α-demethylase (OBT 14DM), a key sterol biosynthetic enzyme. The ratio of phospholipids to sterols was higher in the sensitive genotype than in the tolerant genotypes, suggesting that the sterol biosynthetic pathway plays an important role in Al tolerance. Consistent with this idea, a transgenic Arabidopsis thaliana line with knocked-down AtCYP51 expression showed an Al-sensitive phenotype. Uniconazole-P, an inhibitor of OBT 14DM, suppressed the Al tolerance of Al-tolerant genotypes of maize (Zea mays), sorghum (Sorghum bicolor), rice (Oryza sativa), wheat (Triticum aestivum), and triticale (×Triticosecale Wittmark cv. Currency). These results suggest that increased sterol content, regulated by CYP51, with concomitant lower phospholipid content in the root tip, results in lower negativity of the plasma membrane. This appears to be a common strategy for Al tolerance among several plant species. None Identification of CROWN ROOTLESS1-regulated genes in rice reveals specific and conserved elements of postembryonic root formation. 2014 New Phytol Université Montpellier 2, Montpellier, France. In monocotyledons, the root system is mostly composed of postembryonic shoot-borne roots called crown roots. In rice (Oryza sativa), auxin promotes crown root initiation via the LOB-domain transcription factor (LBD) transcription factor CROWN ROOTLESS1 (CRL1); however, the gene regulatory network downstream of CRL1 remains largely unknown. We tested CRL1 transcriptional activity in yeast and in planta, identified CRL1-regulated genes using an inducible gene expression system and a transcriptome analysis, and used in situ hybridization to demonstrate coexpression of a sample of CRL1-regulated genes with CRL1 in crown root primordia. We show that CRL1 positively regulates 277 genes, including key genes involved in meristem patterning (such as QUIESCENT-CENTER SPECIFIC HOMEOBOX; QHB), cell proliferation and hormone homeostasis. Many genes are homologous to Arabidopsis genes involved in lateral root formation, but about a quarter are rice-specific. Our study reveals that several genes acting downstream of LBD transcription factors controlling postembryonic root formation are conserved between monocots and dicots. It also provides evidence that specific genes are involved in the formation of shoot-derived roots in rice. CRL1|ARL1 Identification of target genes of the bZIP transcription factor OsTGAP1, whose overexpression causes elicitor-induced hyperaccumulation of diterpenoid phytoalexins in rice cells. 2014 PLoS One Department of Biosciences, Teikyo University, Utsunomiya, Tochigi, Japan; Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Tokyo, Japan. Phytoalexins are specialised antimicrobial metabolites that are produced by plants in response to pathogen attack. Momilactones and phytocassanes are the major diterpenoid phytoalexins in rice and are synthesised from geranylgeranyl diphosphate, which is derived from the methylerythritol phosphate (MEP) pathway. The hyperaccumulation of momilactones and phytocassanes due to the hyperinductive expression of the relevant biosynthetic genes and the MEP pathway gene OsDXS3 in OsTGAP1-overexpressing (OsTGAP1ox) rice cells has previously been shown to be stimulated by the chitin oligosaccharide elicitor. In this study, to clarify the mechanisms of the elicitor-stimulated coordinated hyperinduction of these phytoalexin biosynthetic genes in OsTGAP1ox cells, transcriptome analysis and chromatin immunoprecipitation with next-generation sequencing were performed, resulting in the identification of 122 OsTGAP1 target genes. Transcriptome analysis revealed that nearly all of the momilactone and phytocassane biosynthetic genes, which are clustered on chromosomes 4 and 2, respectively, and the MEP pathway genes were hyperinductively expressed in the elicitor-stimulated OsTGAP1ox cells. Unexpectedly, none of the clustered genes was included among the OsTGAP1 target genes, suggesting that OsTGAP1 did not directly regulate the expression of these biosynthetic genes through binding to each promoter region. Interestingly, however, several OsTGAP1-binding regions were found in the intergenic regions among and near the cluster regions. Concerning the MEP pathway genes, only OsDXS3, which encodes a key enzyme of the MEP pathway, possessed an OsTGAP1-binding region in its upstream region. A subsequent transactivation assay further confirmed the direct regulation of OsDXS3 expression by OsTGAP1, but other MEP pathway genes were not included among the OsTGAP1 target genes. Collectively, these results suggest that OsTGAP1 participates in the enhanced accumulation of diterpenoid phytoalexins, primarily through mechanisms other than the direct transcriptional regulation of the genes involved in the biosynthetic pathway of these phytoalexins. OsTGAP1|OsbZIP37 The miR156-SPL9-DFR pathway coordinates the relationship between development and abiotic stress tolerance in plants. 2014 Plant J National Key Laboratory of Plant Molecular Genetics and Collaborative Innovation Center for Genetics and Development, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032, China. Young organisms have relatively strong resistance to diseases and adverse conditions. When confronted with adversity, the process of development is delayed in plants. This phenomenon is thought to result from the rebalancing of energy, which helps plants to coordinate the relationship between development and stress tolerance; however, the molecular mechanism underlying this phenomenon remains mysterious. In this study, we found that miR156 integrates environmental signals to ensure timely flowering, thus enabling the completion of breeding. Under stress conditions, miR156 is induced to maintain the plant in the juvenile state for a relatively long period of time, whereas under favorable conditions, miR156 is suppressed to accelerate the developmental transition. Blocking the miR156 signaling pathway in Arabidopsis thaliana with 35S::MIM156 (via target mimicry) increased the sensitivity of the plant to stress treatment, whereas overexpression of miR156 increased stress tolerance. In fact, this mechanism is also conserved in Oryza sativa (rice). We also identified downstream genes of miR156, i.e. SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9) and DIHYDROFLAVONOL-4-REDUCTASE (DFR), which take part in this process by influencing the metabolism of anthocyanin. Our results uncover a molecular mechanism for plant adaptation to the environment through the miR156-SPLs-DFR pathway, which coordinates development and abiotic stress tolerance. None Overexpression of the JAZ factors with mutated jas domains causes pleiotropic defects in rice spikelet development. 2014 Plant Signal Behav a Bioscience and Biotechnology Center ; Nagoya University ; Chikusa , Nagoya , Japan. In a determinate meristem, such as a floral meristem, a genetically determined number of organs are produced before the meristem is terminated. In rice, iterative formation of organs during flower development with defects in meristem determinacy, classically called 'proliferation', is caused by several mutations and observed in dependence on environmental conditions. Here we report that overexpression of several JAZ proteins, key factors in jasmonate signaling, with mutations in the Jas domains causes an increase in the numbers of organs in florets, aberrant patterns of organ formation and repetitious organ production in spikelets. Our results imply that JAZ factors modulate mechanisms that regulate meristem functions during spikelet development. None Combinations of Hd2 and Hd4 genes determine rice adaptability to Heilongjiang Province, northern limit of China. 2014 J Integr Plant Biol Northeast Institute of Geography and Agroecology, Key Laboratory of Soybean Molecular Design Breeding, Chinese Academy of Sciences, Harbin 150081, China; Graduate University of Chinese Academy of Sciences, Beijing 100049, China. Heading date is a key trait in rice domestication and adaption, and a number of quantitative trait loci (QTLs) have been identified. The rice cultivars (Oryza sativa L.) in the Heilongjiang Province, the northernmost region of China, have to flower extremely early to fulfill their life cycle. However, the critical genes or different gene combinations controlling early flowering in this region have not been determined. QTL and candidate gene analysis revealed that Hd2/Ghd7.1/OsPRR37 plays a major role in controlling rice distribution in Heilongjiang. Further association analysis with a collection of rice cultivars demonstrated that other three major QTL genes (Hd4/Ghd7, Hd5/DTH8/Ghd8, and Hd1) also participate in regulating heading date under natural long day (LD) conditions. Hd2/Ghd7.1/OsPRR37 and Hd4/Ghd7 are two major QTLs and function additively. With the northward rice cultivation, the Hd2/Ghd7.1/OsPRR37 and Hd4/Ghd7 haplotypes became non-functional alleles. Hd1 might be non-functional in most of Heilongjiang rice varieties, implying that recessive hd1 were selected during local rice breeding. Non-functional Hd5/DTH8/Ghd8 is very rare, but constitutes a potential target for breeding extremely early flowering cultivars. Our results indicated that diverse genetic combinations of Hd1, Hd2, Hd4, and Hd5 determined the different distribution of rice varieties in this northernmost province of China. None Alterations in SiRNA and MiRNA Expression Profiles Detected by Deep Sequencing of Transgenic Rice with SiRNA-Mediated Viral Resistance. 2015 PLoS One Department of Biology, Miami University, Oxford, Ohio, United States of America. RNA-mediated gene silencing has been demonstrated to serve as a defensive mechanism against viral pathogens by plants. It is known that specifically expressed endogenous siRNAs and miRNAs are involved in the self-defense process during viral infection. However, research has been rarely devoted to the endogenous siRNA and miRNA expression changes under viral infection if the resistance has already been genetically engineered in plants. Aiming to gain a deeper understanding of the RNA-mediated gene silencing defense process in plants, the expression profiles of siRNAs and miRNAs before and after viral infection in both wild type and transgenic anti-Rice stripe virus (RSV) rice plants were examined by small RNA high-throughput sequencing. Our research confirms that the newly generated siRNAs, which are derived from the engineered inverted repeat construct, is the major contributor of the viral resistance in rice. Further analysis suggests the accuracy of siRNA biogenesis might be affected when siRNAs machinery is excessively used in the transgenic plants. In addition, the expression levels of many known miRNAs are dramatically changed due to RSV infection on both wild type and transgenic rice plants, indicating potential function of those miRNAs involved in plant-virus interacting process. None Spatiotemporal distribution of phenolamides and the genetics of natural variation of hydroxycinnamoyl spermidine in rice. 2014 Mol Plant National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Phenolamides constitute a diverse class of secondary metabolites that are found ubiquitously in plants and have been implicated to play an important role in a wide range of biological processes, such as plant development and defense. However, spatiotemporal accumulation patterns of phenolamides in rice, one of the most important crops, are not available, and no gene responsible for phenolamide biosynthesis has been identified in this species. In this study, we report the comprehensive metabolic profiling and natural variation analysis of phenolamides in a collection of rice germplasm using a liquid chromatography-mass spectrometry-based targeted metabolomics method. Spatiotemporal controlled accumulations were observed for most phenolamides, together with their differential accumulations between the two major subspecies of rice. Further metabolic genome-wide association study (mGWAS) in rice leaf and in vivo metabolic analysis of the transgenic plants identified Os12g27220 and Os12g27254 as two spermidine hydroxycinnamoyl transferases that might underlie the natural variation of levels of spermidine conjugates in rice. Our work demonstrates that gene-to-metabolite analysis by mGWAS provides a useful tool for functional gene identification and omics-based crop genetic improvement. None Using Metabolomic Approaches to Explore Chemical Diversity in Rice. 2014 Mol Plant RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa 230-0045, Japan; Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan; Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan. Electronic address: miyako.kusano@riken.jp. Rice (Oryza sativa) is an excellent resource; it comprises 25% of the total caloric intake of the world's population, and rice plants yield many types of bioactive compounds. To determine the number of metabolites in rice and their chemical diversity, the metabolite composition of cultivated rice has been investigated with analytical techniques such as mass spectrometry (MS) and/or nuclear magnetic resonance spectroscopy and rice metabolite databases have been constructed. This review summarizes current knowledge on metabolites in rice including sugars, amino and organic acids, aromatic compounds, and phytohormones detected by gas chromatography-MS, liquid chromatography-MS, and capillary electrophoresis-MS. The biological properties and the activities of polar and nonpolar metabolites produced by rice plants are also presented. Challenges in the estimation of the structure(s) of unknown metabolites by metabolomic approaches are introduced and discussed. Lastly, examples are presented of the successful application of metabolite profiling of rice to characterize the gene(s) that are potentially critical for improving its quality by combining metabolite quantitative trait loci analysis and to identify potential metabolite biomarkers that play a critical role when rice is grown under abiotic stress conditions. None OsARF16, a transcription factor regulating auxin redistribution, is required for iron deficiency response in rice (Oryza sativa L.). 2014 Plant Sci College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China. Electronic address: shencj@hznu.edu.cn. Plant response to iron deficiency is the most important feature for survival in Fe-limited soils. Several phytohormones, including auxin, are involved in iron uptake and homeostasis. However, the mechanisms behind how auxin participates in the iron deficiency response in rice are largely unknown. We found that OsARF16 was involved in the iron deficiency response and the induction of iron deficiency response genes. Most Fe-deficient symptoms could be partially restored in the osarf16 mutant, including dwarfism, photosynthesis decline, a reduction in iron content and root system architecture (RSA) regulation. OsARF16 expression was induced in the roots and shoots by Fe deprivation. Restoration of the phenotype could also be mimicked by 1-NOA, an auxin influx inhibitor. Furthermore, the qRT-PCR data indicated that the induction of Fe-deficiency response genes by iron deficiency was more compromised in the osarf16 mutant than in Nipponbare. In conclusion, osarf16, an auxin insensitive mutant, was involved in iron deficiency response in rice. Our results reveal a new biological function for OsARF16 and provide important information on how ARF-medicated auxin signaling affects iron signaling and the iron deficiency response. This work may help us to improve production or increased Fe nutrition of rice to iron deficiency by regulating auxin signaling. OsARF16 Phosphate acquisition efficiency and phosphate starvation tolerance locus (PSTOL1) in rice. 2014 J Genet Department of Genetics, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia 741 252, India. somnathbhat@yahoo.com. Phosphate availability is a major factor limiting tillering, grain filling vis-á-vis productivity of rice. Rice is often cultivated in soil like red and lateritic or acid, with low soluble phosphate content. To identify the best genotype suitable for these types of soils, P acquisition efficiency was estimated from 108 genotypes. Gobindabhog, Tulaipanji, Radhunipagal and Raghusail accumulated almost equal amounts of phosphate even when they were grown on P-sufficient soil. Here, we have reported the presence as well as the expression of a previously characterized rice gene, phosphate starvation tolerance locus (PSTOL1) in a set of selected genotypes. Two of four genotypes did not show any detectable expression but carried the gene. One mega cultivar, Swarna did not possess this gene but showed high P-deficiency tolerance ability. Increase of root biomass, not length, in P-limiting situations might be considered as one of the selecting criteria at the seedling stage. Neither the presence of PSTOL1 gene nor its closely-linked SSR RM1261, showed any association with P-deficiency tolerance among the 108 genotypes. Not only this, but the presence of PSTOL1 in recombinant inbred line (RIL) developed from a cross between Gobindabhog and Satabdi, also did not show any linkage with P-deficiency tolerance ability. Thus, before considering PSTOL1 gene in MAB, its expression and role in P-deficiency tolerance in the donor parent must be ascertained. PSTOL1 Mapping and introgression of QTL for yield and related traits in two backcross populations derived from Oryza sativa cv. Swarna and two accessions of O. nivara. 2014 J Genet Directorate of Rice Research, Rajendranagar, Hyderabad 500 030, India. m.swamy@irri.org. Advanced backcross QTL (AB-QTL) analysis was carried out in two Oryza nivara-derived BC2F2 populations. For nine traits, we identified 28 QTL in population 1 and 26 QTL in population 2. The two most significant yield-enhancing QTL, yldp9.1 and yldp2.1 showed an additive effect of 16 and 7 g per plant in population 1, while yld2.1 and yld11.1 showed an additive effect of 11 and 10 g per plant in population 2. At least one O. nivara-derived QTL with a phenotypic variance of >15% was detected for seven traits in population 1 and three traits in population 2. The O. nivara-derived QTL ph1.1, nt12.1, nsp1.1, nfg1.1, bm11.1, yld2.1 and yld11.1 were conserved at the same chromosomal locations in both populations. Two major QTL clusters were detected at the marker intervals RM488-RM431 and RM6-RM535 on chromosomes 1 and 2, respectively. The colocation of O. nivara-derived yield QTL with yield meta-QTL on chromosomes 1, 2 and 9 indicates their accuracy and consistency. The major-effect QTL reported in this study are useful for marker-assisted breeding and are also suitable for further fine mapping and candidate gene identification. None Gene pyramiding enhances durable blast disease resistance in rice. 2015 Sci Rep National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. Effective control of blast, a devastating fungal disease of rice, would increase and stabilize worldwide food production. Resistance mediated by quantitative trait loci (QTLs), which usually have smaller individual effects than R-genes but confer broad-spectrum or non-race-specific resistance, is a promising alternative to less durable race-specific resistance for crop improvement, yet evidence that validates the impact of QTL combinations (pyramids) on the durability of plant disease resistance has been lacking. Here, we developed near-isogenic experimental lines representing all possible combinations of four QTL alleles from a durably resistant cultivar. These lines enabled us to evaluate the QTLs singly and in combination in a homogeneous genetic background. We present evidence that pyramiding QTL alleles, each controlling a different response to M. oryzae, confers strong, non-race-specific, environmentally stable resistance to blast disease. Our results suggest that this robust defence system provides durable resistance, thus avoiding an evolutionary "arms race" between a crop and its pathogen. None AEF1/MPR25 is implicated in RNA editing of plastid atpF and mitochondrial nad5 and also promotes atpF splicing in Arabidopsis and rice. 2015 Plant J Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, 35 Stirling Highway, Crawley, 6009, WA, Australia. RNA editing is an essential mechanism that modifies target cytidines to uridine in both mitochondrial and plastid mRNA. Target sites are recognised by pentatricopeptide repeat (PPR) proteins. Using bioinformatics predictions based on the code describing sequence recognition by PPR proteins, we have identified an Arabidopsis editing factor required for the editing of atpF in plastids. A loss of function mutation in ATPF EDITING FACTOR 1 (AEF1, AT3G22150) results in severe variegation, presumably due to decreased plastid ATP synthase levels. Loss of editing at the atpF site is coupled with a large decrease in splicing of the atpF transcript, even though the editing site is within an exon and 53 nucleotides distant from the splice site. The rice orthologue of AEF1, MPR25, has been reported to be required for editing of a site in mitochondrial nad5 transcripts and we confirm that editing of the same site is affected in the Arabidopsis aef1 mutant. We also show that splicing of chloroplast atpF transcripts is affected in the rice mpr25 mutant. AEF1 is thus highly unusual for an RNA editing specificity factor in having functions in both organelles. This article is protected by copyright. All rights reserved. MPR25 Application of microRNA gene resources in the improvement of agronomic traits in rice. 2015 Plant Biotechnol J Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory for Biocontrol, Sun Yat-sen University, Guangzhou, China. microRNAs (miRNAs) are important nonprotein-coding genes that are involved in almost all biological processes, including cell differentiation and fate determination, developmental regulation, and immune responses. Investigations have shown that some miRNAs can highly affect plant agricultural traits, including virus resistance, nematode resistance, drought and salinity tolerance, heavy metal detoxification, biomass yield, grain yield, fruit development and flower development. Therefore, these miRNAs are considered a newly identified gene resource for the genetic improvement of crops. In this review, we will summarize the recent findings of the rice miRNA-directed regulatory network, which controls agronomic traits such as yield, quality and stress tolerance, and explore the outlook for the uses of these miRNA-associated traits in rice biotechnology. None Decreased photosynthesis in the erect panicle 3 (ep3) mutant of rice is associated with reduced stomatal conductance and attenuated guard cell development. 2015 J Exp Bot Plant and Crop Sciences Division, School of Biosciences, University of Nottingham, Nottingham LE12 5RD, UK. The ERECT PANICLE 3 gene of rice encodes a peptide that exhibits more than 50% sequence identity with the Arabidopsis F-box protein HAWAIIAN SKIRT (HWS). Ectopic expression of the Os02g15950 coding sequence, driven by the HWS (At3g61950) promoter, rescued the hws-1 flower phenotype in Arabidopsis confirming that EP3 is a functional orthologue of HWS. In addition to displaying an erect inflorescence phenotype, loss-of-function mutants of Os02g15950 exhibited a decrease in leaf photosynthetic capacity and stomatal conductance. Analysis of a range of physiological and anatomical features related to leaf photosynthesis revealed no alteration in Rubisco content and no notable changes in mesophyll size or arrangement. However, both ep3 mutant plants and transgenic lines that have a T-DNA insertion within the Os02g15950 (EP3) gene exhibit smaller stomatal guard cells compared with their wild-type controls. This anatomical characteristic may account for the observed decrease in leaf photosynthesis and provides evidence that EP3 plays a role in regulating stomatal guard cell development. EP3|LP Primary metabolism plays a central role in molding silicon-inducible brown spot resistance in rice. 2015 Mol Plant Pathol Laboratory of Phytopathology, Faculty of Bioscience Engineering, Ghent University, B-9000 Ghent, Belgium. Over the past decades, a multitude of studies have shown the ability of silicon (Si) to protect various plants against a range of microbial pathogens exhibiting different lifestyles and infection strategies. Despite this relative wealth of knowledge, understanding of the action mechanism of Si is still in its infancy, which hinders its widespread application for agricultural purposes. In an attempt to further elucidate the molecular underpinnings of Si-induced disease resistance, we studied the transcriptome of control and Si-treated rice plants infected with the necrotrophic brown spot fungus Cochliobolus miyabeanus. Analysis of brown-spot infected control plants suggested that C. miyabeanus represses plant photosynthetic processes and nitrate reduction in order to trigger premature senescence and cause disease. In Si-treated plants, however, these pathogen-induced metabolic alterations are strongly impaired, suggesting that Si alleviates stress imposed by the pathogen. Interestingly, Si also significantly increased photorespiration rates in brown spot-infected plants. Even though photorespiration is often considered a wasteful process, recent studies indicate that this metabolic bypass also enhances resistance during abiotic stress and pathogen attack by protecting the plant's photosynthetic machinery. In view of these findings, our results favor a scenario whereby Si enhances brown spot resistance by counteracting C. miyabeanus-induced senescence and cell death via increased photorespiration. Moreover, our results shed light onto the mechanistic basis of Si-afforded disease control and support the view that in addition to activating plant immune responses, Si also can reduce disease severity by interfering with pathogen virulence strategies. None Abscisic acid and the key enzymes and genes in sucrose-to-starch conversion in rice spikelets in response to soil drying during grain filling. 2015 Planta Jiangsu Key Laboratory of Crop Genetics and Physiology, Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, 225009, China. Abscisic acid mediates the effect of post-anthesis soil drying on grain filling through regulating the activities of key enzymes and expressions of genes involved in sucrose-to-starch conversion in rice spikelets. This study investigated if abscisic acid (ABA) would mediate the effect of post-anthesis soil drying on grain filling through regulating the key enzymes in sucrose-to-starch conversion in rice (Oryza sativa L.) spikelets. Two rice cultivars were field-grown. Three treatments, well-watered (WW), moderate soil drying (MD), and severe soil drying (SD), were imposed from 6 days after full heading until maturity. When compared with those under the WW, grain filling rate, grain weight, and sink activity, in terms of the activities and gene expression levels of sucrose synthase, ADP glucose pyrophosphorylase, starch synthase, and starch branching enzyme, in inferior spikelets were substantially increased under the MD, whereas they were markedly decreased in both superior and inferior spikelets under the SD. The two cultivars showed the same tendencies. Both MD and SD increased ABA content and expression levels of its biosynthesis genes in spikelets, with more increase under the SD than the MD. ABA content was significantly correlated with grain filling rate and sink activities under both WW and MD, while the correlations were not significant under the SD. Application of a low concentration ABA to WW plants imitated the results under the MD, and applying with a high concentration ABA showed the effect of the SD. The results suggest that ABA plays a vital role in grain filling through regulating sink activity and functions in a dose-dependent manner. An elevated ABA level under the MD enhances, whereas a too high level of ABA under the SD decreases, sink activity. None Three CCT domain-containing genes were identified to regulate heading date by candidate gene-based association mapping and transformation in rice. 2015 Sci Rep National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. CCT domain-containing genes generally control flowering in plants. Currently, only six of the 41 CCT family genes have been confirmed to control flowering in rice. To efficiently identify more heading date-related genes from the CCT family, we compared the positions of heading date QTLs and CCT genes and found that 25 CCT family genes were located in the QTL regions. Association mapping showed that a total of 19 CCT family genes were associated with the heading date. Five of the seven associated genes within QTL regions and two of four associated genes outside of the QTL regions were confirmed to regulate heading date by transformation. None of the seven non-associated genes outside of the QTL regions regulates heading date. Obviously, combination of candidate gene-based association mapping with linkage analysis could improve the identification of functional genes. Three novel CCT family genes, including one non-associated (OsCCT01) and two associated genes (OsCCT11 and OsCCT19) regulated the heading date. The overexpression of OsCCT01 delayed flowering through suppressing the expression of Ehd1, Hd3a and RFT1 under both long day and short day conditions. Potential functions in regulating heading date of some untested CCT family genes were discussed. OsCCT01,OsCCT11,OsCCT19 Infection of Ustilaginoidea virens intercepts rice seed formation but activates grain-filling-related genes 2014 J Integr Plant Biol Rice Research Institute, Sichuan Agricultural University, Chengdu, 611130, China. Rice false smut has become an increasingly serious disease in rice production worldwide. The typical feature of this disease is that the fungal pathogen Ustilaginoidea virens (Uv) specifically infects rice flower and forms false smut balls, the ustiloxins-containing ball-like fungal colony, of which the size is usually several times larger than that of a mature rice seed. However, the underlying mechanisms of Uv-rice interaction are poorly understood. Here, we applied time-course microscopic and transcriptional approaches to investigate rice responses to Uv infection. The results demonstrated that flower-opening process and expression of associated transcription factors, including ARF6 and ARF8, were inhibited in Uv-infected spikelets. The ovaries in infected spikelets were interrupted in fertilization and thus were unable to set seeds. However, a number of grain-filling-related genes, including seed storage protein genes, starch anabolism genes and endosperm-specific transcription factors (RISBZ1 and RPBF), were highly transcribed as if the ovaries were fertilized. In addition, critical defense-related genes like NPR1 and PR1 were down-regulated by Uv infection. Our data imply that Uv might hijack host nutrient reservoir by activation of grain-filling network for the need of growth and formation of false smut balls. None Regulation of inflorescence branch development in rice through a novel pathway involving the pentatricopeptide repeat protein sped1-D 2014 Genetics Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Panicle type has a direct bearing on rice yield. Here, we characterized a rice clustered-spikelet mutant, sped1-D, with shortened pedicels and/or secondary branches, which exhibits decreased pollen fertility. We cloned sped1-D and found that it encodes a pentatricopeptide repeat protein. We investigated the global expression profiles of wild-type, 9311, and sped1-D plants using Illumina RNA sequencing. The expression of several GID1L2 family members was downregulated in the sped1-D mutant, suggesting that the gibberellin (GA) pathway is involved in the elongation of pedicels and/or secondary branches. When we overexpressed one GID1L2, AK070299, in sped1-D plants, the panicle phenotype was restored to varying degrees. In addition, we analyzed the expression of genes that function in floral meristems and found that RFL and WOX3 were severely downregulated in sped1-D. These results suggest that sped1-D may prompt the shortening of pedicels and secondary branches by blocking the action of GID1L2, RFL, and Wox3. Moreover, overexpression of sped1-D in Arabidopsis resulted in the shortening of pedicels and clusters of siliques, which indicates that the function of sped1-D is highly conserved in monocotyledonous and dicotyledonous plants. Sequence data from this article have been deposited with the miRBase Data Libraries under accession no. MI0003201. sped1-D A glutathione responsive rice glyoxalase II, OsGLYII-2, functions in salinity adaptation by maintaining better photosynthesis efficiency and anti-oxidant pool 2014 Plant J Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India. OsGLYII2 Expression of abiotic stress inducible ETHE1-like protein from rice is higher in roots and is regulated by calcium 2014 Physiol Plant Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India. ETHYLMALONIC ENCEPHALOPATHY PROTEIN 1 (ETHE1) encodes sulfur dioxygenase (SDO) activity regulating sulfide levels in living organisms. It is an essential gene and mutations in ETHE1 leads to ethylmalonic encephalopathy (EE) in humans and embryo lethality in Arabidopsis. At present, very little is known regarding the role of ETHE1 beyond the context of EE and almost nothing is known about factors affecting its regulation in plant systems. In this study, we have identified, cloned and characterized OsETHE1, a gene encoding ETHE1-like protein from Oryza sativa. ETHE1 proteins in general are most similar to glyoxalase II (GLYII) and hence OsETHE1 has been earlier annotated as OsGLYII1, a putative GLYII gene. Here we show that OsETHE1 lacks GLYII activity and is instead an ETHE1 homolog being localized in mitochondria like its human and Arabidopsis counterparts. We have isolated and analyzed 1618 bp OsETHE1 promoter (pOsETHE1) to examine the factors affecting OsETHE1 expression. For this, transcriptional promoter pOsETHE1: 5-bromo-5-chloro-3-indolyl-D-glucuronide (GUS) fusion construct was made and stably transformed into rice. GUS expression pattern of transgenic pOsETHE1:GUS plants reveal a high root-specific expression of OsETHE1. The pOsETHE1 activity was stimulated by Ca(II) and required light for induction. Moreover, pOsETHE1 activity was induced under various abiotic stresses such as heat, salinity and oxidative stress, suggesting a potential role of OsETHE1 in stress response. OsGLYII3 A plausible mechanism, based upon Short-Root movement, for regulating the number of cortex cell layers in roots 2014 Proc Natl Acad Sci U S A Department of Biology, University of Pennsylvania, Philadelphia, PA 19104 Formation of specialized cells and tissues at defined times and in specific positions is essential for the development of multicellular organisms. Often this developmental precision is achieved through intercellular signaling networks, which establish patterns of differential gene expression and ultimately the specification of distinct cell fates. Here we address the question of how the Short-root (SHR) proteins from Arabidopsis thaliana (AtSHR), Brachypodium distachyon (BdSHR), and Oryza sativa (OsSHR1 and OsSHR2) function in patterning the root ground tissue. We find that all of the SHR proteins function as mobile signals in A. thaliana and all of the SHR homologs physically interact with the AtSHR binding protein, Scarecow (SCR). Unlike AtSHR, movement of the SHR homologs was not limited to the endodermis. Instead, the SHR proteins moved multiple cell layers and determined the number of cortex, not endodermal, cell layers formed in the root. Our results in A. thaliana are consistent with a mechanism by which the regulated movement of the SHR transcription factor determines the number of cortex cell layers produced in the roots of B. distachyon and O. sativa. These data also provide a new model for ground tissue patterning in A. thaliana in which the ability to form a functional endodermis is spatially limited independently of SHR. OsSHR2 RNase Z(S1) processes UbL40 mRNAs and controls thermosensitive genic male sterility in rice 2014 Nat Commun State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources; Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions; College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. Thermosensitive genic male-sterile (TGMS) lines, which are male-sterile at restrictive (high) temperatures but male-fertile at permissive (low) temperatures, have been widely used in breeding two-line hybrid rice (Oryza sativa L.). Here we find that mutation of thermosensitive genic male sterile 5 (tms5) in rice causes the TGMS trait through a loss of RNase Z(S1) function. We show that RNase Z(S1) processes the mRNAs of three ubiquitin fusion ribosomal protein L40 (UbL40) genes into multiple fragments in vitro and in vivo. In tms5 mutants, high temperature results in increased levels of UbL40 mRNAs. Overaccumulation of UbL40 mRNAs causes defective pollen production and male sterility. Our results uncover a novel mechanism of RNase Z(S1)-mediated UbL40 mRNA regulation and shows that loss of this regulation produces TGMS in rice, a finding with potential applications in hybrid crop breeding. UbL404,UbL402,UbL401,TMS5 The Wall-associated Kinase gene family in rice genomes. 2014 Plant Sci Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul, Brazil; Centro de Biotecnologia e Programa de Pós-Graduação em Biologia Celular e Molecular, Universidade Federal do Rio Grande do Sul, Brazil. The environment is a dynamic system in which life forms adapt. Wall-Associated Kinases (WAK) are a subfamily of receptor-like kinases associated with the cell wall. These genes have been suggested as sensors of the extracellular environment and triggers of intracellular signals. They belong to the ePK superfamily with or without a conserved arginine before the catalytic subdomain VIB, which characterizes RD and non-RD WAKs. WAK is a large subfamily in rice. We performed an extensive comparison of WAK genes from A. thaliana (AtWAK), O. sativa japonica and indica subspecies (OsWAK). Phylogenetic studies and WAK domain characterization allowed for the identification of two distinct groups of WAK genes in Arabidopsis and rice. One group corresponds to a cluster containing only OsWAKs that most likely expanded after the monocot-dicot separation, which evolved into a non-RD kinase class. The other group comprises classical RD-kinases with both AtWAK and OsWAK representatives. Clusterization analysis using extracellular and kinase domains demonstrated putative functional redundancy for some genes, but also highlighted genes that could recognize similar extracellular stimuli and activate different cascades. The gene expression pattern of WAKs in response to cold suggests differences in the regulation of the OsWAK genes in the indica and japonica subspecies. Our results also confirm the hypothesis of functional diversification between A. thaliana and O. sativa WAK genes. Furthermore, we propose that plant WAKs constitute two evolutionarily related but independent subfamilies: WAK-RD and WAK-nonRD. Recognition of this structural division will further provide insights to understanding WAK functions and regulations. None Proteomic and transcriptomic analysis of rice tranglutaminase and chloroplast-related proteins. 2014 Plant Sci Molecular Genetics Department, Centre for Research in Agricultural Genomics: CRAG (Consorci CSIC-IRTA-UAB-UB), Campus UAB, Bellaterra, 08193 Barcelona, Spain. Electronic address: nefer@costarricense.cr. The recently cloned rice transglutaminase gene (tgo) is the second plant transglutaminase identified to date (Campos et al. Plant Sci. 205-206 (2013) 97-110). Similarly to its counterpart in maize (tgz), this rice TGase was localized in the chloroplast, although in this case not exclusively. To further characterise plastidial tgo functionality, proteomic and transcriptomic studies were carried out to identify possible TGO-related proteins. Some LHCII antenna proteins were identified as TGO related using an in vitro proteomic approach, as well as ATPase and some PSII core proteins by mass spectrometry. To study the relationship between TGO and other plastidial proteins, a transcriptomic in vivo Dynamic Array (Fluidigm™) was used to analyse the mRNA expression of 30 plastidial genes with respect to that of tgo, in rice plants subjected to different periods of continuous illumination. The results indicated a gene-dependent tendency in the expression pattern that was related to tgo expression and to the illumination cycle. For certain genes, including tgo, significant differences between treatments, principally at the initiation and/or at the end of the illumination period, connected with the day/night cycling of gene expression, were observed. The tgo expression was especially related to plastidial proteins involved in photoprotection and the thylakoid electrochemical gradient. None Creation of fragrant rice by targeted knockout of the OsBADH2 gene using TALEN technology. 2015 Plant Biotechnol J State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. Fragrant rice is favoured worldwide because of its agreeable scent. The presence of a defective badh2 allele encoding betaine aldehyde dehydrogenase (BADH2) results in the synthesis of 2-acetyl-1-pyrroline (2AP), which is a major fragrance compound. Here, transcription activator-like effector nucleases (TALENs) were engineered to target and disrupt the OsBADH2 gene. Six heterozygous mutants (30%) were recovered from 20 transgenic hygromycin-resistant lines. Sanger sequencing confirmed that these lines had various indel mutations at the TALEN target site. All six transmitted the BADH2 mutations to the T1 generation; and four T1 mutant lines tested also efficiently transmitted the mutations to the T2 generation. Mutant plants carrying only the desired DNA sequence change but not the TALEN transgene were obtained by segregation in the T1 and T2 generations. The 2AP content of rice grains of the T1 lines with homozygous mutations increased from 0 to 0.35-0.75 mg/kg, which was similar to the content of a positive control variety harbouring the badh2-E7 mutation. We also simultaneously introduced three different pairs of TALENs targeting three separate rice genes into rice cells by bombardment and obtained lines with mutations in one, two and all three genes. These results indicate that targeted mutagenesis using TALENs is a useful approach to creating important agronomic traits. OsBADH2|fgr Improving rice tolerance to potassium deficiency by enhancing OsHAK16p:WOX11-controlled root development. 2015 Plant Biotechnol J State Key Laboratory of Crop Genetics and Germplasm Enhancement, MOA Key Laboratory of Plant Nutrition and Fertilization in Lower-Middle Reaches of the Yangtze River, Nanjing Agricultural University, Nanjing, China. Potassium (K) deficiency in plants confines root growth and decreases root-to-shoot ratio, thus limiting root K acquisition in culture medium. A WUSCHEL-related homeobox (WOX) gene, WOX11, has been reported as an integrator of auxin and cytokinin signalling that regulates root cell proliferation. Here, we report that ectopic expression of WOX11 gene driven by the promoter of OsHAK16 encoding a low-K-enhanced K transporter led to an extensive root system and adventitious roots and more effective tiller numbers in rice. The WOX11-regulated root and shoot phenotypes in the OsHAK16p:WOX11 transgenic lines were supported by K-deficiency-enhanced expression of several RR genes encoding type-A cytokinin-responsive regulators, PIN genes encoding auxin transporters and Aux/IAA genes. In comparison with WT, the transgenic lines showed increases in root biomass, root activity and K concentrations in the whole plants, and higher soluble sugar concentrations in roots particularly under low K supply condition. The improvement of sugar partitioning to the roots by the expression of OsHAK16p:WOX11 was further indicated by increasing the expression of OsSUT1 and OsSUT4 genes in leaf blades and several OsMSTs genes in roots. Expression of OsHAK16p:WOX11 in the rice grown in moderate K-deficient soil increased total K uptake by 72% and grain yield by 24%-32%. The results suggest that enlarging root growth and development by the expression of WOX11 in roots could provide a useful option for increasing K acquisition efficiency and cereal crop productivity in low K soil. WOX11 Positive-negative-selection-mediated gene targeting in rice. 2015 Front Plant Sci Laboratory of Plant Molecular Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology Ikoma, Japan. Gene targeting (GT) refers to the designed modification of genomic sequence(s) through homologous recombination (HR). GT is a powerful tool both for the study of gene function and for molecular breeding. However, in transformation of higher plants, non-homologous end joining (NHEJ) occurs overwhelmingly in somatic cells, masking HR-mediated GT. Positive-negative selection (PNS) is an approach for finding HR-mediated GT events because it can eliminate NHEJ effectively by expression of a negative-selection marker gene. In rice-a major crop worldwide-reproducible PNS-mediated GT of endogenous genes has now been successfully achieved. The procedure is based on strong PNS using diphtheria toxin A-fragment as a negative marker, and has succeeded in the directed modification of several endogenous rice genes in various ways. In addition to gene knock-outs and knock-ins, a nucleotide substitution in a target gene was also achieved recently. This review presents a summary of the development of the rice PNS system, highlighting its advantages. Different types of gene modification and gene editing aimed at developing new plant breeding technology based on PNS are discussed. None Rice stripe virus affects the viability of its vector offspring by changing developmental gene expression in embryos. 2015 Sci Rep Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences; Jiangsu Technical Service Center of Diagnosis and Detection for Plant Virus Diseases, Nanjing 210014, People's Republic of China. Plant viruses may affect the viability and development process of their herbivore vectors. Small brown planthopper (SBPH) is main vector of Rice stripe virus (RSV), which causes serious rice stripe disease. Here, we reported the effects of RSV on SBPH offspring by crossing experiments between viruliferous and non-viruliferous strains. The life parameters of offspring from different cross combinations were compared. The hatchability of F1 progeny from viruliferous parents decreased significantly, and viruliferous rate was completely controlled by viruliferous maternal parent. To better elucidate the underlying biological mechanisms, the morphology of eggs, viral propagation and distribution in the eggs and expression profile of embryonic development genes were investigated. The results indicated that RSV replicated and accumulated in SBPH eggs resulting in developmental stunt or delay of partial eggs; in addition, RSV was only able to infect ovum but not sperm. According to the expression profile, expression of 13 developmental genes was regulated in the eggs from viruliferous parents, in which two important regulatory genes (Ls-Dorsal and Ls-CPO) were most significantly down-regulated. In general, RSV exerts an adverse effect on SBPH, which is unfavourable for the expansion of viruliferous populations. The viewpoint is also supported by systematic monitoring of SBPH viruliferous rate. None Rice RING E3 ligase may negatively regulate gamma-ray response to mediate the degradation of photosynthesis-related proteins. 2015 Planta Plant Genomics Lab, Department of Applied Plant Sciences, Kangwon National University, Chuncheon, 200-713, Republic of Korea. In this study, our findings regarding the regulation of GA irradiation-induced OsGIRP1 in relation to the levels of photosynthesis-related proteins such as OsrbcL1 and OsrbcS1 and hypersensitive responses of overexpressing plants to GR irradiation provide insight into the molecular functions of OsGIRP1 as a negative regulator in response to the stress of radiation. The RING (Really Interesting New Gene) finger proteins are known to play crucial roles in various abiotic stresses in plants. Here, we report on RING finger E3 ligase, Oryza sativa gamma rays-induced RING finger protein1 gene (OsGIRP1), which is highly induced by gamma rays (GR) irradiation. In vitro ubiquitination assay demonstrated that a single amino acid substitution (OsGIRP1(C196A)) of the RING domain showed no E3 ligase activity, supporting the notion that the activity of most E3s is specified by a RING domain. We isolated at least 6 substrate proteins of OsGIRP1, including 2 Rubisco subunits, OsrbcL1 and OsrbcSl, via yeast two-hybridization and bimolecular fluorescence complementation assays. OsGIRP1 and its partner proteins were targeted to the cytosol and the cytosol or chloroplasts, respectively; however, florescence signals of the complexes with OsGIPR1 were observed in the cytosol. Protein degradation in cell extracts showed that OsGIRP1 mediates proteolysis of 2 substrates, OsrbcS1 and OsrbcL1, via the 26S proteasome degradation pathway. The Arabidopsis plants overexpressing OsGIRP1 clearly exhibited increased sensitivity to GR irradiation. These results might suggest that OsGIRP1 acts as a negative regulator of GR response to mediate the degradation of photosynthesis-related proteins. OsMSR1,Os4NPP1,OsDNLZ1,OsrbcL1 beta-aminobutyric acid-induced resistance against root-knot nematodes in rice is based on increased basal defence. 2015 Mol Plant Microbe Interact Ghent university, Molecular biotechnology, GENT, Belgium ; ji.hongli@ugent.be. The non-protein amino acid beta-aminobutyric acid (BABA) is known to protect plants against various pathogens. The mode of action is relatively diverse and specific in different plant-pathogen systems. To extend the analysis of the mode of action of BABA to plant parasitic nematodes in monocot plants, we evaluated the effect of BABA against the root-knot nematode (RKN) Meloidogyne graminicola in rice. BABA treatment of rice plants inhibited nematode penetration and resulted in delayed nematode and giant cell development. BABA-induced resistance (BABA-IR) was still functional in mutants or transgenics defective in salicylic acid (SA) biosynthesis and response, or abscisic acid (ABA) response. Pharmacological inhibition of jasmonic acid (JA) and ethylene (ET) biosynthesis indicated that BABA-IR against rice RKN likely occurs independent of JA and ET. However, histochemical and biochemical quantification in combination with quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) data suggest that BABA protects rice against RKN through the activation of basal defence mechanisms of the plant such as reactive oxygen species (ROS) accumulation, lignin formation and callose deposition. None Alteration of osa-miR156e expression affects rice plant architecture and strigolactones (SLs) pathway. 2015 Plant Cell Rep National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, 430070, China, chenzhihui75@sina.com. Overexpressing osa - miR156e in rice produced a bushy mutant and osa - miR156e regulation of tillering may do this through the strigolactones (SLs) pathway. Appropriate downregulation of osa - miR156 expression contributed to the improvement of plant architecture. Tillering is one of the main determinants for rice architecture and yield. In this study, a bushy mutant of rice was identified with increased tiller number, reduced plant height, prolonged heading date, low seed setting, and small panicle size due to a T-DNA insertion which essentially elevated the expression of osa-miR156e. Transgenic plants with constitutive expression of osa-miR156e also had the bushy phenotype, which showed osa-miR156 may control apical dominance and tiller outgrowth via regulating the strigolactones signaling pathway. Furthermore, the extent of impaired morphology was correlated with the expression level of osa-miR156e. In an attempt to genetically improve rice architecture, ectopic expression of osa-miR156e under the GAL4-UAS system or OsTB1 promoter was conducted. According to agronomic trait analysis, pTB1:osa-miR156e transgenic plants significantly improved the grain yield per plant compared to plants overexpressing osa-miR156e, even though the yield was still inferior to the wild type, making it a very interesting albeit negative result. Our results suggested that osa-miR156 could serve as a potential tool for modifying rice plant architecture through genetic manipulation of the osa-miR156 expression level. None Maternal control of seed size in plants. 2015 J Exp Bot State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Seed size is a key determinant of evolutionary fitness, and is also one of the most important components of seed yield. In angiosperms, seed development begins with double fertilization, which leads to the formation of a diploid embryo and a triploid endosperm. The outermost layer of the seed is the seed coat, which differentiates from maternal integuments. Therefore, the size of a seed is determined by the co-ordinated growth of the embryo, endosperm, and maternal tissue. Recent studies have identified several factors that act maternally or zygotically to regulate seed size, and revealed possible mechanisms that underlie seed size control in Arabidopsis and rice. In this review, we summarize current research progress in maternal control of seed size and discuss the roles of several newly identified regulators in maternal regulation of seed growth. None Novel roles of hydrogen peroxide (H2 O2 ) in regulating pectin synthesis and demethylesterification in the cell wall of rice (Oryza sativa) root tips. 2015 New Phytol State Key Laboratory of Rice Biology, China National Rice Research Institute, 359 Tiyuchang Road, Hangzhou, 310006, China; College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, 310018, China. Hydrogen peroxide (H2 O2 ) has been reported to increase lignin formation, enhance cell wall rigidification, restrict cell expansion and inhibit root elongation. However, our results showed that it not only inhibited rice (Oryza sativa) root elongation, but also increased root diameter. No study has reported how and why H2 O2 increases cell expansion and root diameter. Exogenous H2 O2 and its scavenger 4-hydroxy-Tempo were applied to confirm the roles of H2 O2 . Immunofluorescence, fluorescence probe, ruthenium red staining, histological section and spectrophotometry were used to monitor changes in the degree of pectin methylesterification, pectin content, pectin methylesterase (PME) activity and H2 O2 content. Exogenous H2 O2 inhibited root elongation, but increased cell expansion and root diameter significantly. H2 O2 not only increased the region of pectin synthesis and pectin content in root tips, but also increased PME activity and pectin demethylesterification. The scavenger 4-hydroxy-Tempo reduced root H2 O2 content and recovered H2 O2 -induced increases in cell expansion and root diameter by inhibiting pectin synthesis, PME activity and pectin demethylesterification. H2 O2 plays a novel role in the regulation of pectin synthesis, PME activity and pectin demethylesterification. H2 O2 increases cell expansion and root diameter by increasing pectin content and demethylesterification. None A hemicellulose-bound form of silicon with potential to improve the mechanical properties and regeneration of the cell wall of rice. 2015 New Phytol College of Resources and Environment, Huazhong Agricultural University, Wuhan, 430070, China. Silicon (Si) plays a large number of diverse roles in plants, but the structural and chemical mechanisms operating at the single-cell level remain unclear. We isolate the cell walls from suspension-cultured individual cells of rice (Oryza sativa) and fractionate them into three main fractions including cellulose (C), hemicellulose (HC) and pectin (P). We find that most of the Si is in HC as determined by inductively coupled plasma-mass spectrometry (ICP-MS), where Si may covalently crosslink the HC polysacchrides confirmed by X-ray photoelectron spectroscopy (XPS). The HC-bound form of Si could improve both the mechanical property and regeneration of the cell walls investigated by a combination of atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). This study provides further evidence that HC could be the major ligand bound to Si, which broadens our understanding of the chemical nature of 'anomalous' Si in plant cell walls. None Strigolactones, a Novel Carotenoid-Derived Plant Hormone. 2015 Annu Rev Plant Biol Center for Desert Agriculture, Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Kingdom of Saudi Arabia; email: salim.babili@kaust.edu.sa. Strigolactones (SLs) are carotenoid-derived plant hormones and signaling molecules. When released into the soil, SLs indicate the presence of a host to symbiotic fungi and root parasitic plants. In planta, they regulate several developmental processes that adapt plant architecture to nutrient availability. Highly branched/tillered mutants in Arabidopsis, pea, and rice have enabled the identification of four SL biosynthetic enzymes: a cis/trans-carotene isomerase, two carotenoid cleavage dioxygenases, and a cytochrome P450 (MAX1). In vitro and in vivo enzyme assays and analysis of mutants have shown that the pathway involves a combination of new reactions leading to carlactone, which is converted by a rice MAX1 homolog into an SL parent molecule with the typical tricyclic lactone moiety. In this review, we focus on SL biosynthesis, describe the hormonal and environmental factors that determine this process, and discuss SL transport and downstream signaling as well as the role of SLs in regulating plant development. Expected final online publication date for the Annual Review of Plant Biology Volume 66 is April 29, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates. None Fine mapping of RBG2, a quantitative trait locus for resistance to Burkholderia glumae, on rice chromosome 1. 2015 Mol Breed National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602 Japan. Bacterial grain rot (BGR), caused by the bacterial pathogen Burkholderia glumae, is a destructive disease of rice. At anthesis, rice panicles are attacked by the pathogen, and the infection causes unfilled or aborted grains, reducing grain yield and quality. Thus, increasing the level of BGR resistance is an important objective for rice breeding. A quantitative trait locus (QTL) on rice chromosome 1 that controls BGR resistance was previously detected in backcross inbred lines (BILs) derived from a cross between Kele, a resistant traditional lowland cultivar (indica) that originated in India, and Hitomebore, a susceptible modern lowland cultivar (temperate japonica) from Japan. Further genetic analyses using a BC3F6 population derived from a cross between a resistant BIL (BC2F5) and Hitomebore confirmed that a QTL for BGR resistance was located on the long arm of chromosome 1. To define more precisely the chromosomal region underlying this QTL, we identified nine BC2F6 plants in which recombination occurred near the QTL. Substitution mapping using homozygous recombinant and nonrecombinant plants demonstrated that the QTL, here designated as Resistance to Burkholderia glumae 2 (RBG2), was located in a 502-kb interval defined by simple sequence repeat markers RM1216 and RM11727. None Nonuniform gene expression pattern detected along the longitudinal axis in the matured rice leaf. 2015 Sci Rep Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun 130024, China. Rice (Oryza sativa) is a staple crop that supports half the world's population and an important monocot model system. Monocot leaf matures in a basipetal manner, and has a well-defined developmental gradient along the longitudinal axis. However, little is known about its transcriptional dynamics after leaf maturation. In this study, we have reconstructed a high spatial resolution transcriptome for the matured rice leaf by sectioning the leaf into seven 3-cm fragments. We have performed strand-specific Illumina sequencing to generate gene expression profiles for each fragment. We found that the matured leaf contains a longitudinal gene expression gradient, with 6.97% (2,603) of the expressed genes showing differentially expression along the seven sections. The leaf transcriptome showed a gradual transition from accumulating transcripts related to primary cell wall and basic cellular metabolism at the base to those involved in photosynthesis and energy production in the middle, and catabolic metabolism process toward the tip. None Rice OsFLS2-mediated perception of bacterial flagellins is evaded by Xanthomonas oryzae pvs. oryzae and oryzicola. 2015 Mol Plant Department of Plant Pathology, China Agricultural University, Beijing 100193, China; Key Laboratory of Plant Pathology, Ministry of Agriculture, China Agricultural University, Beijing 100193, China. Bacterial flagellins are often recognized by the receptor kinase FLAGELLIN SENSITIVE2 (FLS2) and activate MAMP-triggered immunity in dicotyledonous plants. However, the capacity of monocotyledonous rice to recognize flagellins of key rice pathogens and its biological relevance remain poorly understood. We demonstrated that ectopically expressed OsFLS2 in Arabidopsis senses the eliciting flg22 peptide and in vitro purified Acidovorax avenae (Aa) flagellin in an expression level-dependent manner, but does not recognize purified flagellins or derivative flg22(Xo) peptides of Xanthomonas oryzae pvs. oryzae (Xoo) and oryzicola (Xoc). Consistently, the flg22 peptide and purified Aa flagellin, but not Xoo/Xoc flagellins, induce various immune responses such as defense gene induction and MAPK activation in rice. Perception of flagellin by rice does induce strong resistance to Xoo infection, as shown after pretreatment of rice leaves with Aa flagellin. OsFLS2 was found to exhibit different perception specificities or sensitivities to the flg22 region from AtFLS2. In additional work, post-translational modification of Xoc flagellin was altered by deletion of glycosyltransferase-encoding rbfC but this had little effect on Xoc motility and did not detectably reduce virulence on rice. Deletion of flagellin-encoding fliC from Xoo/Xoc blocked swimming motility but also did not significantly alter Xoo/Xoc virulence. The results suggest that Xoo/Xoc carry flg22-region amino acid changes that allow motility while evading the ancient flagellin detection system in rice, which retains recognition capacity for other bacterial pathogens. OsFLS2 Ectopic expression of myo-inositol 3-phosphate synthase induces a wide range of metabolic changes and confers salt tolerance in rice. 2014 Plant Sci Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan. Salt stress is an important factor that limits crop production worldwide. The salt tolerance of plants is a complex biological process mediated by changes in gene expression and metabolite composition. The enzyme myo-inositol 3-phosphate synthase (MIPS; EC 5.5.1.4) catalyzes the first step of myo-inositol biosynthesis, and overexpression of the MIPS gene enhances salt stress tolerance in several plant species. In this study, we performed metabolite profiling of both MIPS-overexpressing and wild-type rice. The enhanced salt stress tolerance of MIPS-overexpressing plants was clear based on growth and the metabolites under salt stress. We found that constitutive overexpression of the rice MIPS gene resulted in a wide range of metabolic changes. This study demonstrates for the first time that overexpression of the MIPS gene increases various metabolites responsible for protecting plants from abiotic stress. Activation of both basal metabolism, such as glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle, and inositol metabolism is induced in MIPS-overexpressing plants. We discuss the relationship between the metabolic changes and the improved salt tolerance observed in transgenic rice. None OsJAZ9 acts as a transcriptional regulator in jasmonate signaling and modulates salt stress tolerance in rice. 2014 Plant Sci National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. The basic functions of plant-specific TIFY proteins as transcriptional regulators have been reported in plants. Some TIFY genes are responsive to abiotic stresses, but the functions of these genes in stress tolerance have seldom been reported. OsJAZ9 is a member of the JAZ subfamily which belongs to the TIFY gene family in rice (Oryza sativa). Suppression of OsJAZ9 resulted in reduced salt tolerance. The altered salt tolerance was mainly due to changes in ion (especially K(+)) homeostasis, which was supported by the altered expression levels of several ion transporter genes. The OsJAZ9-suppression rice plants showed increased sensitivity to jasmonic acid (JA) treatment. OsJAZ9 interacts with OsCOI1a, a component of the SCF(COI1) E3 ubiquitin ligase complex, in a coronatine-dependent manner, suggesting that OsJAZ9 is involved in the regulation of JA signaling. OsJAZ9 interacts with several bHLH transcription factors including OsbHLH062 via the Jas domain. OsbHLH062 can bind to an E-box in the promoters of the ion transporter genes such as OsHAK21, and most of these ion transporter genes are responsive to JA treatment. We found that OsJAZ9 can also interact with OsNINJA, a rice homolog of the Arabidopsis thaliana transcriptional repressor NINJA in JA signaling. Both OsJAZ9 and OsNINJA (Novel Interactor of JAZ) repressed OsbHLH062-mediated transcription activation. These results together suggest that OsJAZ9 acts as a transcriptional regulator by forming a transcriptional regulation complex with OsNINJA and OsbHLH to fine tune the expression of JA-responsive genes involved in salt stress tolerance in rice. OsJAZ9|OsTIFY11a,OsbHLH1|OsbHLH062 Isolation of a Novel Lodging Resistance QTL Gene Involved in Strigolactone Signaling and Its Pyramiding with a QTL Gene Involved in Another Mechanism. 2014 Mol Plant Bioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi 464-8601, Japan. Lodging has been a major roadblock to attaining increased crop productivity. In an attempt to understand the mechanism for culm strength in rice, we isolated an effective quantitative trait locus (QTL), STRONG CULM3 (SCM3), the causal gene of which is identical to rice TEOSINTE BRANCHED1 (OsTB1), a gene previously reported to positively control strigolactone (SL) signaling. A near-isogenic line (NIL) carrying SCM3 showed enhanced culm strength and increased spikelet number despite the expected decrease in tiller number, indicating that SL also has a positive role in enhancing culm strength and spikelet number. We produced a pyramiding line carrying SCM3 and SCM2, another QTL encoding APO1 involved in panicle development. The NIL-SCM2+SCM3 showed a much stronger culm than NIL-SCM2 and NIL-SCM3 and an increased spikelet number caused by the additive effect of these QTLs. We discuss the importance of utilizing suitable alleles of these STRONG CULM QTLs without inducing detrimental traits for breeding. None CD2-1, the C-terminal region of flagellin, modulates the induction of immune responses in rice. 2015 Mol Plant Microbe Interact Nagahama Institute of Bio-Science and Technology, Graduate School of Biosciences, Nagahama, Shiga, Japan ; b106061@nagahama-i-bio.ac.jp. Flagellin from the rice avirulent N1141 strain of Acidovorax avenae, functions as a pathogen-associated molecular pattern (PAMP) and induces PAMP-triggered immunity (PTI) in rice. To study the recognition mechanism of flagellin in rice, we attempted to define the region(s) of the flagellin protein required to activate the PTI response. Based on domain classification, we produced four fragments of N1141 flagellin: N-terminal D0, D1 and D2 domains (ND0-2), N-terminal D2, D3, and C-terminal D2 domains (ND2-CD2), C-terminal D2, D1, and D0 domains (CD2-0), and C-terminal D2 and D1 domains (CD2-1). The C-terminal CD2-1 and CD2-0 fragments induced PTI responses in cultured rice cells. Synthetic flg22, which is sufficient to produce the flagellin response in Arabidopsis, and the N-terminal flagellin fragments containing flg22 region elicited very weak immune responses in rice. OsFLS2, the rice ortholog of AtFLS2 which mediates flg22 recognition, was not involved in CD2-0 or CD2-1 recognition in rice. In addition, CD2-0 triggered resistance to coinfection with pathogenic bacteria. Taken together, these data suggest that rice mainly recognizes flagellin CD2-1 by a receptor distinct from OsFLS2, and that this epitope recognition leads to PTI responses. OsFLS2 Genome-Wide Identification and Functional Analysis of Genes Expressed Ubiquitously in Rice. 2014 Mol Plant Department of Plant Molecular Systems Biotechnology & Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Korea; Graduate School of Biotechnology, Kyung Hee University, Yongin 446-701, Korea. Electronic address: khjung2010@khu.ac.kr. Genes that are expressed ubiquitously throughout all developmental stages are thought to be necessary for basic biological or cellular functions. Therefore, determining their biological roles is a great challenge. We identified 4034 of these genes in rice after studying the results of Agilent 44K and Affymetrix meta-anatomical expression profiles. Among 105 genes that were characterized by loss-of-function analysis, 79 were classified as members of gene families, the majority of which were predominantly expressed. Using T-DNA insertional mutants, we examined 43 genes and found that loss of expression of six genes caused developing seed- or seedling-defective phenotypes. Of these, three are singletons without similar family members and defective phenotypes are expected from mutations. Phylogenomic analyses integrating genome-wide transcriptome data revealed the functional dominance of three ubiquitously expressed family genes. Among them, we investigated the function of Os03g19890, which is involved in ATP generation within the mitochondria during endosperm development. We also created and evaluated functional networks associated with this gene to understand the molecular mechanism. Our study provides a useful strategy for pheonome analysis of ubiquitously expressed genes in rice. None Characterization of rice small heat shock proteins targeted to different cellular organelles. 2015 Cell Stress Chaperones Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, 560012, India. Small heat shock proteins (sHSPs) are a family of ATP-independent molecular chaperones which prevent cellular protein aggregation by binding to misfolded proteins. sHSPs form large oligomers that undergo drastic rearrangement/dissociation in order to execute their chaperone activity in protecting substrates from stress. Substrate-binding sites on sHSPs have been predominantly mapped on their intrinsically disordered N-terminal arms. This region is highly variable in sequence and length across species, and has been implicated in both oligomer formation and in mediating chaperone activity. Here, we present our results on the functional and structural characterization of five sHSPs in rice, each differing in their subcellular localisation, viz., cytoplasm, nucleus, chloroplast, mitochondria and peroxisome. We performed activity assays and dynamic light scattering studies to highlight differences in the chaperone activity and quaternary assembly of sHSPs targeted to various organelles. By cloning constructs that differ in the length and sequence of the tag in the N-terminal region, we have probed the sensitivity of sHSP oligomer assembly and chaperone activity to the length and amino acid composition of the N-terminus. In particular, we have shown that the incorporation of an N-terminal tag has significant consequences on sHSP quaternary structure. None The WRKY45-2-WRKY13-WRKY42 Transcriptional Regulatory Cascade Is Required for Rice Resistance to Fungal Pathogen. 2015 Plant Physiol Huazhong Agricultural University. Blast caused by fungal Magnaporthe oryzae is a devastating disease of rice worldwide, and this fungus also infects barley. At least 11 rice WRKY transcription factors have been reported to regulate rice response to M. oryzae either positively or negatively. However, the relationships of these WRKYs in the rice defense signaling pathway against M. oryzae are unknown. Previous studies have revealed that rice WRKY13 (as a transcriptional repressor) and WRKY45-2 enhance resistance to M. oryzae. Here we show that rice WRKY42, functioning as a transcriptional repressor, suppresses resistance to M. oryzae. WRKY42-RNA interference (RNAi) and WRKY42-overexpressing (oe) plants showed increased resistance and susceptibility to M. oryzae, accompanied by increased or reduced jasmonate acid (JA) content, respectively, compared with wild-type plants. JA pretreatment enhanced the resistance of WRKY42-oe plants to M. oryzae. WRKY13 directly suppressed WRKY42. WRKY45-2, functioning as a transcriptional activator, directly activated WRKY13. In addition, WRKY13 directly suppressed WRKY45-2 by feedback regulation. The WRKY13-RNAi WRKY45-2-oe and WRKY13-oe WRKY42-oe double transgenic lines showed increased susceptibility to M. oryzae compared with WRKY45-2-oe and WRKY13-oe plants, respectively. These results suggest that the three WRKYs form a sequential transcriptional regulatory cascade. WRKY42 may negatively regulate rice response to M. oryzae by suppressing JA signaling-related genes, and WRKY45-2 transcriptionally activates WRKY13, whose encoding protein in turn transcriptionally suppresses WRKY42 to regulate rice resistance to M. oryzae. OsWRKY13|WRKY13,OsWRKY42|WRKY42 Genome-wide association mapping of salinity tolerance in rice (Oryza sativa). 2015 DNA Res National Research Centre on Plant Biotechnology, New Delhi 110012, India. Salinity tolerance in rice is highly desirable to sustain production in areas rendered saline due to various reasons. It is a complex quantitative trait having different components, which can be dissected effectively by genome-wide association study (GWAS). Here, we implemented GWAS to identify loci controlling salinity tolerance in rice. A custom-designed array based on 6,000 single nucleotide polymorphisms (SNPs) in as many stress-responsive genes, distributed at an average physical interval of <100 kb on 12 rice chromosomes, was used to genotype 220 rice accessions using Infinium high-throughput assay. Genetic association was analysed with 12 different traits recorded on these accessions under field conditions at reproductive stage. We identified 20 SNPs (loci) significantly associated with Na(+)/K(+) ratio, and 44 SNPs with other traits observed under stress condition. The loci identified for various salinity indices through GWAS explained 5-18% of the phenotypic variance. The region harbouring Saltol, a major quantitative trait loci (QTLs) on chromosome 1 in rice, which is known to control salinity tolerance at seedling stage, was detected as a major association with Na(+)/K(+) ratio measured at reproductive stage in our study. In addition to Saltol, we also found GWAS peaks representing new QTLs on chromosomes 4, 6 and 7. The current association mapping panel contained mostly indica accessions that can serve as source of novel salt tolerance genes and alleles. The gene-based SNP array used in this study was found cost-effective and efficient in unveiling genomic regions/candidate genes regulating salinity stress tolerance in rice. None Induced jasmonate signaling leads to contrasting effects on root damage and herbivore performance. 2015 Plant Physiol MPI-CE; Induced defenses play a key role in plant resistance against leaf-feeders. Yet, very little is known about the signals that are involved in defending plants against root-feeders and how they are influenced by abiotic factors. We investigated these aspects for the interaction between rice (Oryza sativa) and two root-feeding insects, the generalist cucumber beetle (Diabrotica balteata) and the more specialized rice water weevil (Lissorhoptrus oryzophilus). Rice plants responded to root-attack by increasing the production of jasmonic acid (JA) and abscisic acid (ABA), while, in contrast to herbivore-attacked leaves, salicylic acid (SA) and ethylene (ET) levels remained unchanged. The JA response was decoupled from flooding and remained constant over different soil moisture levels. Exogenous application of methyl JA (MeJA) to the roots markedly decreased the performance of both root herbivores, while ABA and the ET precursor 1-aminocyclopropane-1-carboxylic acid (ACC) did not have any effect. JA-deficient transgenic asLOX and mutant allene oxide cyclase (AOC) "hebiba" plants lost more root biomass under attack from both root herbivores. Surprisingly, herbivore weight gain was decreased markedly in asLOX but not in hebiba mutant plants, despite the higher root biomass removal. This effect was correlated with an herbivore-induced reduction of sucrose pools in asLOX roots. Taken together, our experiments demonstrate that jasmonates are induced signals that protect rice roots from herbivores under varying abiotic conditions and that boosting jasmonate responses can strongly enhance rice resistance against root pests. Furthermore, we demonstrate that a rice 13-lipoxygenase regulates root primary metabolites and specifically improves root herbivore growth. None Asparagine synthetase1, but not asparagine synthetase2, is responsible for the biosynthesis of asparagine following the supply of ammonium to rice roots. 2015 Plant Cell Physiol Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555, Japan. Asparagine is synthesized from glutamine by the reaction of asparagine synthetase (AS) and is the major nitrogen form in both xylem and phloem sap in rice (Oryza sativa L.). There are two genes encoding AS, OsAS1 and OsAS2, in rice, but the functions of individual AS isoenzymes are largely unknown. Cell-type and NH4 (+) inducible expression of OsAS1 as well as analyses of knockout mutants were carried out in this study to characterize AS1. OsAS1 was mainly expressed in the roots with in situ hybridization showing that the corresponding mRNA was specifically accumulated in the three cell layers of root surface (epidermis, exodermis, and sclerenchyma) in an NH4 (+)-dependent manner. Conversely, OsAS2 mRNA was abundant in leaf blades and sheathes of rice. Although OsAS2 mRNA was detectable in the roots, its content decreased when NH4 (+) was supplied. Retrotransposon-mediated knockout mutants lacking AS1 showed slight stimulation of shoot length and slight reduction in root length at the seedling stage. On the other hand, the mutation caused an approximately 80-90% reduction in free asparagine content in both roots and xylem sap. These results suggest that AS1 is responsible for the synthesis of asparagine in rice roots following the supply of NH4 (+). Characteristics of the NH4 (+)-dependent increase and the root-surface cell-specific expression of OsAS1 gene are very similar to our previous results on cytosolic glutamine synthetase1;2 and NADH-glutamate synthase1 in rice roots. Thus, AS1 is apparently coupled with the primary assimilation of NH4 (+) in rice roots. OsASN1|OsAS1,OsAS2 Gene knockout of glutathione reductase 3 results in increased sensitivity to salt stress in rice. 2015 Plant Mol Biol Department of Agricultural Chemistry, National Taiwan University, Taipei, 10617, Taiwan. Glutathione reductase (GR) is one of important antioxidant enzymes in plants. This enzyme catalyzes the reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) with the accompanying oxidation of NADPH. Previously, we showed that salt-stress-responsive GR3 is a functional protein localized in chloroplasts and mitochondria in rice. To learn more about the role of GR3 in salt-stress tolerance, we investigated the response to 100 mM NaCl treatment in wild-type rice (WT); GR3 knockout mutant of rice (gr3); and the functional gr3-complementation line (C1). Rice GR3 was primarily expressed in roots at the seedling stage and ubiquitously expressed in all tissues except the sheath at heading stage. GR3 promoter-GUS was expressed in the vascular cylinder and cortex of root tissues in rice seedlings, vascular tissue of nodes, embryo and aleurone layer of seeds, and young flowers. Under both normal and salt-stress conditions, total GR activity was decreased by 20 % in gr3. Oxidative stress, indicated by malondialdehyde content, was greater in gr3 than the WT under salt stress. As compared with the WT, gr3 was sensitive to salt and methyl viologen; it showed inhibited growth, decreased maximal efficiency of photosystem II, decreased GSH and GSSG contents, and the ratio of GSH to GSSG. Conversely, the gr3-complementation line C1 rescued the tolerance to methyl viologen and salinity and recovered the growth and physiological damage caused by salinity. These results reveal that GR3 plays an important role in salt stress tolerance by regulating the GSH redox state in rice. OsGR3,OsGR3|GR3 Elevated levels of CYP94 family gene expression alleviate the jasmonate response and enhance salt tolerance in rice. 2015 Plant Cell Physiol Bioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan. A plant hormone jasmonate and its conjugates (JAs) have important roles in growth control, leaf senescence and defence responses against insects and microbial attacks. JA biosynthesis is induced by several stresses, including mechanical wounding, pathogen attacks, drought and salinity stresses. However, the roles of JAs under abiotic stress conditions are unclear. Here we report that increased expression of the cytochrome P450 family gene CYP94C2b enhanced viability of rice plants under saline conditions. This gene encodes an enzyme closely related to CYP94C1 that catalyses conversion of bioactive jasmonate-isoleucine (JA-Ile) into 12OH-JA-Ile and 12COOH-JA-Ile. Inactivation of JA was facilitated in a rice line with enhanced CYP94C2b expression, and responses to exogenous JA and wounding were alleviated. Moreover, salt stress-induced leaf senescence but not natural senescence was delayed in the transgenic rice. These results suggest that bioactive JAs have a negative effect on viability under salt stress conditions and demonstrate that manipulating JA metabolism confers enhanced salt tolerance in rice. CYP94C2a Transcriptome-based analysis of mitogen-activated protein kinase cascades in the rice response to Xanthomonas oryzae infection. 2015 Rice (N Y) National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, 430070 China. Mitogen-activated protein (MAP) kinase cascades, with each cascade consisting of a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a MAP kinase (MAPK), play important roles in dicot plant responses to pathogen infection. However, no single MAP kinase cascade has been identified in rice, and the functions of MAP kinase cascades in rice - pathogen interactions are unknown.To explore the contribution of MAP kinase cascade in rice in response to Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight, one of the devastating diseases of rice worldwide, we performed a comprehensive expression analysis of rice MAP kinase cascade genes. We transcriptionally analyzed all the 74 MAPKKK genes, 8 MAPKK, and 17 MAPK genes in two pairs of susceptible and resistant rice lines, with each pair having the same genetic background, to determine the rice response to Xoo infection. The expression of a large number of MAP kinase cascade genes changed in response to infection, and some of the genes also showed different expression in resistant and susceptible reactions. In addition, some MAPKKK genes co-expressed with MAPKK and/or MAPK genes, and MAPKK genes co-expressed with MAPK genes.These results provide a new perspective regarding the putative roles of rice MAP kinase gene candicates and potential cascade targets for further characterization in rice-pathogen interactions. None Comparative proteomics analysis reveals the mechanism of fertility alternation of thermo-sensitive genic male sterile rice lines under low temperature inducement. 2015 Proteomics State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, P R China. Thermo-sensitive genic male sterile (TGMS) rice line has made great economical contributions in rice production. However, the fertility of TGMS rice line during hybrid seed production is frequently influenced by low temperature, thus leading to its fertility/sterility alteration and hybrid seed production failure. To understand the mechanism of fertility alternation under low temperature inducement, the extracted proteins from young panicles of two TGMS rice lines at the fertility alternation sensitivity stage were analyzed by 2-DE. 83 protein spots were found to be significantly changed in abundance, and identified by MALDI-TOF/TOF MS. The identified proteins were involved in 16 metabolic pathways and cellular processes. The young panicles of TGMS rice line Zhu 1S possessed the lower ROS-scavenging, IAA level, soluble protein and sugar contents as well as the faster anther wall disintegration than those of TGMS rice line Zhun S. All these major differences might result in that the former is more stable in fertility than the latter. Based on the majority of the 83 identified proteins, together with microstructural, physiological and biochemical results, a possible fertile alteration mechanism in the young panicles of TGMS rice line under low temperature inducement was proposed. Such a result will help us in breeding of TGMS rice lines and production of hybrid seed. This article is protected by copyright. All rights reserved. None Stable mitotic inheritance of rice minichromosomes in cell suspension cultures. 2015 Plant Cell Rep School of Life Sciences, State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Sha Tin, New Territories, Hong Kong. Suspension cell cultures of rice minichromosomes were established. The minichromosomes in suspension cultured cells were mitotically stable and had active gene expression, thus have the potential to be used as gene expression vectors to produce valuable bioactive products. The plant artificial chromosome (PAC) is a novel vector for plant genetic engineering to produce genetically modified crops with multiple transgenes, or to produce valuable bioactive products through the expression of multiple genes or biochemical pathways as a bioreactor. PAC is mainly constructed by engineered minichromosomes through telomere-mediated chromosome truncations. We have constructed rice minichromosomes in a previous study. Thus, the understanding of rice minichromosome inheritance under different culture conditions has potential importance for their utility in future studies and applications. In this study, we performed suspension cultures of three rice minichromosome-containing cell lines, 1004-111, 1008-100 and 1004-011. Two cell lines, 1004-111 and 1008-100, showed typical S growth pattern consisting of a lag phase, an active growing exponential phase and a stationary phase, whereas cell line 1004-011 grew very slowly and eventually died. Both 1004-111 and 1008-100 minichromosomes were stably transmitted in cell suspension cultures without any abnormality. Foreign gene expression was verified from 1004-111 and 1008-100 minichromosomes in suspension cultures. The stable mitotic inheritance of minichromosomes and gene expression from them indicated that rice minichromosomes could be maintained and propagated in cell suspension cultures. This study tested key parameters for suspension cultures of rice cell lines with minichromosomes, and proved in concept the potential for industrial use of PAC vectors as bioreactors. OsSHM1 A Key ABA Catabolic Gene, OsABA8ox3, Is Involved in Drought Stress Resistance in Rice. 2015 PLoS One College of Life Sciences, South China Agricultural University, Guangzhou, China. Expressions of ABA biosynthesis genes and catabolism genes are generally co-regulated in plant development and responses to environmental stress. Up-regulation of OsNCED3 gene, a key gene in ABA biosynthesis, has been suggested as a way to enhance plant drought resistance but little is known for the role of ABA catabolic genes during drought stress. In this study, we found that OsABA8ox3 was the most highly expressed gene of the OsABA8ox family in rice leaves. Expression of OsABA8ox3 was promptly induced by rehydration after PEG-mimic dehydration, a tendency opposite to the changes of ABA level. We therefore constructed rice OsABA8ox3 silencing (RNA interference, RNAi) and overexpression plants. There were no obvious phenotype differences between the transgenic seedlings and wild type under normal condition. However, OsABA8ox3 RNAi lines showed significant improvement in drought stress tolerance while the overexpression seedlings were hypersensitive to drought stress when compared with wild type in terms of plant survival rates after 10 days of unwatering. Enzyme activity analysis indicated that OsABA8ox3 RNAi plants had higher superoxide dismutase (SOD) and catalase (CAT) activities and less malondialdehyde (MDA) content than those of wild type when the plants were exposed to dehydration treatment, indicating a better anti-oxidative stress capability and less membrane damage. DNA microarray and real-time PCR analysis under dehydration treatment revealed that expressions of a group of stress/drought-related genes, i.e. LEA genes, were enhanced with higher transcript levels in OsABA8ox3 RNAi transgenic seedlings. We therefore conclude that that OsABA8ox3 gene plays an important role in controlling ABA level and drought stress resistance in rice. OsABA8ox3 CURVED CHIMERIC PALEA 1 encoding an EMF1-like protein maintains epigenetic repression of OsMADS58 in rice palea development. 2015 Plant J National Key Laboratory of Plant Molecular Genetics and National Center for Plant Gene Research (Shanghai), Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China. Floral organ specification is controlled by various MADS-box genes in both dicots and monocots, whose expression is often subjected to both genetic and epigenetic regulation in Arabidopsis thaliana. However, little is known about the role of epigenetic modification of MADS-box genes during rice flower development. Here, we report the characterization of a rice gene, CURVED CHIMERIC PALEA 1 (CCP1) that functions in palea development. Mutation in CCP1 resulted in abnormal palea with ectopic stigmatic tissues and other pleiotropic phenotypes. We found that OsMADS58, a C-class gene responsible for carpel morphogenesis, was ectopically expressed in the ccp1palea, indicating that the ccp1 palea was misspecified and partially acquired carpel-like identity. Constitutive expression of OsMADS58 in the wild-type rice plants caused morphological abnormality of palea similar to that of ccp1, whereas OsMADS58 knockdown by RNAi in ccp1 could rescue the abnormal phenotype of mutant palea, suggesting that the repression of OsMADS58 expression by CCP1 is critical for palea development. Map-based cloning revealed that CCP1 encodes a putative plant-specific EMBRYONIC FLOWER1 (EMF1)-like protein. Chromatin immunoprecipitation assay showed that the level of the H3K27me3 at the OsMADS58 locus was greatly reduced in ccp1compared with that in the wild type. Taken together, our results show that CCP1 plays an important role in palea development through maintaining H3K27me3-mediated epigenetic silence of the carpel identity-specifying gene OsMADS58, shedding light on the epigenetic mechanism in floral organ development. This article is protected by copyright. All rights reserved. CCP1 PHIV-RootCell: a supervised image analysis tool for rice root anatomical parameter quantification. 2015 Front Plant Sci CIRAD, UMR AGAP Montpellier, France ; Plateforme Histocytologie et Imagerie Cellulaire Végétale, INRA-CIRAD Montpellier, France. We developed the PHIV-RootCell software to quantify anatomical traits of rice roots transverse section images. Combined with an efficient root sample processing method for image acquisition, this program permits supervised measurements of areas (those of whole root section, stele, cortex, and central metaxylem vessels), number of cell layers and number of cells per cell layer. The PHIV-RootCell toolset runs under ImageJ, an independent operating system that has a license-free status. To demonstrate the usefulness of PHIV-RootCell, we conducted a genetic diversity study and an analysis of salt stress responses of root anatomical parameters in rice (Oryza sativa L.). Using 16 cultivars, we showed that we could discriminate between some of the varieties even at the 6 day-olds stage, and that tropical japonica varieties had larger root sections due to an increase in cell number. We observed, as described previously, that root sections become enlarged under salt stress. However, our results show an increase in cell number in ground tissues (endodermis and cortex) but a decrease in external (peripheral) tissues (sclerenchyma, exodermis, and epidermis). Thus, the PHIV-RootCell program is a user-friendly tool that will be helpful for future genetic and physiological studies that investigate root anatomical trait variations. None A hemicellulose-bound form of silicon inhibits cadmium ion uptake in rice (Oryza sativa) cells. 2015 New Phytol College of Resources and Environment, Huazhong Agricultural University, Wuhan, 430070, China. Silicon (Si) alleviates cadmium (Cd) toxicity in rice (Oryza sativa). However, the chemical mechanisms at the single-cell level are poorly understood. Here, a suspension of rice cells exposed to Cd and/or Si treatments was investigated using a combination of plant cell nutritional, molecular biological, and physical techniques including in situ noninvasive microtest technology (NMT), polymerase chain reaction (PCR), inductively coupled plasma mass spectroscopy (ICP-MS), and atomic force microscopy (AFM) in Kelvin probe mode (KPFM). We found that Si-accumulating cells had a significantly reduced net Cd(2+) influx, compared with that in Si-limited cells. PCR analyses of the expression levels of Cd and Si transporters in rice cells showed that, when the Si concentration in the medium was increased, expression of the Si transporter gene Low silicon rice 1 (Lsi1) was up-regulated, whereas expression of the gene encoding the transporter involved in the transport of Cd, Natural resistance-associated macrophage protein 5 (Nramp5), was down-regulated. ICP-MS results revealed that 64% of the total Si in the cell walls was bound to hemicellulose constituents following the fractionation of the cell walls, and consequently inhibited Cd uptake. Furthermore, AFM in KPFM demonstrated that the heterogeneity of the wall surface potential was higher in cells cultured in the presence of Si than in those cultured in its absence, and was homogenized after the addition of Cd. These results suggest that a hemicellulose-bound form of Si with net negative charges is responsible for inhibition of Cd uptake in rice cells by a mechanism of [Si-hemicellulose matrix]Cd complexation and subsequent co-deposition. None Genetic engineering of the Xa10 promoter for broad-spectrum and durable resistance to Xanthomonas oryzae pv. oryzae. 2015 Plant Biotechnol J Temasek Life Sciences Laboratory, National University of Singapore, Singapore, Singapore; Department of Biological Sciences, National University of Singapore, Singapore, Singapore. Many pathovars of plant pathogenic bacteria Xanthomonas species inject transcription activator-like (TAL) effectors into plant host cells to promote disease susceptibility or trigger disease resistance. The rice TAL effector-dependent disease resistance gene Xa10 confers narrow-spectrum race-specific resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight disease in rice. To generate broad-spectrum and durable resistance to Xoo, we developed a modified Xa10 gene, designated as Xa10(E5) . Xa10(E5) has an EBE-amended promoter containing 5 tandemly arranged EBEs each responding specifically to a corresponding virulent or avirulent TAL effector and a stable transgenic rice line containing Xa10(E5) was generated in the cultivar Nipponbare. The Xa10(E5) gene was specifically induced by Xoo strains that harbour the corresponding TAL effectors and conferred TAL effector-dependent resistance to the pathogens at all developmental stages of rice. Further disease evaluation demonstrated that the Xa10(E5) gene in either Nipponbare or 9311 genetic backgrounds provided broad-spectrum disease resistance to 27 of the 28 Xoo strains collected from 11 countries. The development of Xa10(E5) and transgenic rice lines provides new genetic materials for molecular breeding of rice for broad-spectrum and durable disease resistance to bacterial blight. XA10 Mechanisms of growth and patterns of gene expression in oxygen-deprived rice coleoptiles. 2015 Plant J Department of Botany, School of Life Science, La Trobe University, Victoria, 3086, Australia. Coleoptiles of rice (Oryza sativa) seedlings grown under water commonly elongate up to 1 mm h(-1) to reach the atmosphere. We analysed this highly specialised phenomenon, first by measuring epidermal cell lengths along the coleoptile axis to infer elongation rates. This revealed a cohort of cells in the basal zone that elongated rapidly following emergence from the embryo, reaching 200 μm within 12 h. After filming coleoptiles in vivo for a day, kinematic analysis was applied. Eight time-sliced 'segments' were defined by their emergence from the embryo at four-hourly intervals, revealing a mathematically simple growth model. Each segment entering the coleoptile from the embryo elongated at a constant velocity, resulting in accelerating growth for the entire organ. Consistent with the epidermal cell lengths, relative rates of elongation (mm mm(-1) h(-1) ) were ten-fold greater in the small, newly emerged basal segments than the older distal tip segments. This steep axial gradient defined two contrasting growth zones (bases vs tips) in which we measured: 1) ATP production, protein, RNA and DNA content and 2) the global transcriptome under steady-state normoxia, hypoxia (3% O2 ) and anoxia. The transcriptome revealed tip-specific induction of genes encoding TCP transcription factors, RNA helicase, ribosomal proteins and proteins involved in protein folding, whilst FBOX domain-containing proteins in the ubiquitin E3-SCF complex were induced specifically in bases under low oxygen. We ascribed sustained elongation under hypoxia to hypoxia-specific responses such as controlled suppression of photosystem components and induction of RNA binding/splicing functions, indicating strategic allocation of energy to cell extension. This article is protected by copyright. All rights reserved. None Expression and functional analysis of the plant-specific histone deacetylase HDT701 in rice. 2015 Front Plant Sci Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, South China Botanical Garden, Chinese Academy of Sciences Guangzhou, China ; University of Chinese Academy of Sciences, Chinese Academy of Sciences Beijing, China. Reversible histone acetylation and deacetylation at the N-terminus of histone tails play a crucial role in regulating eukaryotic gene activity. Acetylation of core histones is associated with gene activation, whereas deacetylation of histone is often correlated with gene repression. The level of histone acetylation is antagonistically catalyzed by histone acetyltransferases citation(HATs) and histone deacetylases (HDACs). In this work, we examined the subcellular localization, expression pattern and function of HDT701, a member of the plant-specific HD2-type histone deacetylase in rice. HDT701 is localized at the subcellular level in the nucleus. Histochemical GUS-staining analysis revealed that HDT701 is constitutively expressed throughout the life cycle of rice. Overexpression of HDT701 in rice decreases ABA, salt and osmotic stress resistance during seed germination. Delayed seed germination of HDT701 overexpression lines is associated with decreased histone H4 acetylation and down-regulated expression of GA biosynthetic genes. Moreover, overexpression of HDT701 in rice enhances salt and osmotic stress resistance during the seedling stage. Taken together, our findings suggested that HDT701 may play an important role in regulating seed germination in response to abiotic stresses in rice. OsHDT1|HDT701 Genomic analysis of hybrid rice varieties reveals numerous superior alleles that contribute to heterosis. 2015 Nat Commun National Center for Gene Research, Collaborative Innovation Center for Genetics and Development, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200233, China. Exploitation of heterosis is one of the most important applications of genetics in agriculture. However, the genetic mechanisms of heterosis are only partly understood, and a global view of heterosis from a representative number of hybrid combinations is lacking. Here we develop an integrated genomic approach to construct a genome map for 1,495 elite hybrid rice varieties and their inbred parental lines. We investigate 38 agronomic traits and identify 130 associated loci. In-depth analyses of the effects of heterozygous genotypes reveal that there are only a few loci with strong overdominance effects in hybrids, but a strong correlation is observed between the yield and the number of superior alleles. While most parental inbred lines have only a small number of superior alleles, high-yielding hybrid varieties have several. We conclude that the accumulation of numerous rare superior alleles with positive dominance is an important contributor to the heterotic phenomena. None Characterization of a Null Allelic Mutant of the Rice NAL1 Gene Reveals Its Role in Regulating Cell Division. 2015 PLoS One College of Life Sciences, Qingdao Agricultural University, Qingdao, China; National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, China. Leaf morphology is closely associated with cell division. In rice, mutations in Narrow leaf 1 (NAL1) show narrow leaf phenotypes. Previous studies have shown that NAL1 plays a role in regulating vein patterning and increasing grain yield in indica cultivars, but its role in leaf growth and development remains unknown. In this report, we characterized two allelic mutants of NARROW LEAF1 (NAL1), nal1-2 and nal1-3, both of which showed a 50% reduction in leaf width and length, as well as a dwarf culm. Longitudinal and transverse histological analyses of leaves and internodes revealed that cell division was suppressed in the anticlinal orientation but enhanced in the periclinal orientation in the mutants, while cell size remained unaltered. In addition to defects in cell proliferation, the mutants showed abnormal midrib in leaves. Map-based cloning revealed that nal1-2 is a null allelic mutant of NAL1 since both the whole promoter and a 404-bp fragment in the first exon of NAL1 were deleted, and that a 6-bp fragment was deleted in the mutant nal1-3. We demonstrated that NAL1 functions in the regulation of cell division as early as during leaf primordia initiation. The altered transcript level of G1- and S-phase-specific genes suggested that NAL1 affects cell cycle regulation. Heterogenous expression of NAL1 in fission yeast (Schizosaccharomyces pombe) further supported that NAL1 affects cell division. These results suggest that NAL1 controls leaf width and plant height through its effects on cell division. NAL1|qFLW4 The RhoGAP SPIN6 Associates with SPL11 and OsRac1 and Negatively Regulates Programmed Cell Death and Innate Immunity in Rice. 2015 PLoS Pathog State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China; Hunan Provincial Key Laboratory of Crop Germplasm Innovation and Utilization and College of Agronomy, Hunan Agricultural University, Changsha, Hunan, China. The ubiquitin proteasome system in plants plays important roles in plant-microbe interactions and in immune responses to pathogens. We previously demonstrated that the rice U-box E3 ligase SPL11 and its Arabidopsis ortholog PUB13 negatively regulate programmed cell death (PCD) and defense response. However, the components involved in the SPL11/PUB13-mediated PCD and immune signaling pathway remain unknown. In this study, we report that SPL11-interacting Protein 6 (SPIN6) is a Rho GTPase-activating protein (RhoGAP) that interacts with SPL11 in vitro and in vivo. SPL11 ubiquitinates SPIN6 in vitro and degrades SPIN6 in vivo via the 26S proteasome-dependent pathway. Both RNAi silencing in transgenic rice and knockout of Spin6 in a T-DNA insertion mutant lead to PCD and increased resistance to the rice blast pathogen Magnaporthe oryzae and the bacterial blight pathogen Xanthomonas oryzae pv. oryzae. The levels of reactive oxygen species and defense-related gene expression are significantly elevated in both the Spin6 RNAi and mutant plants. Strikingly, SPIN6 interacts with the small GTPase OsRac1, catalyze the GTP-bound OsRac1 into the GDP-bound state in vitro and has GAP activity towards OsRac1 in rice cells. Together, our results demonstrate that the RhoGAP SPIN6 acts as a linkage between a U-box E3 ligase-mediated ubiquitination pathway and a small GTPase-associated defensome system for plant immunity. SPIN6 Genetic manipulation of a high-affinity PHR1 target cis-element to improve phosphorous uptake in Oryza sativa L. 2015 Plant Mol Biol State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, 310058, China, ruanwenyuan@zju.edu.cn. Phosphorus (P) is an essential macronutrient for crop development and production. Phosphate starvation response 1 (PHR1) acts as the central regulator for Pi-signaling and Pi-homeostasis in plants by binding to the cis-element PHR1 binding sequence (P1BS; GNATATNC). However, how phosphate starvation-induced gene expression is regulated remains obscure. In this work, we investigated the DNA binding affinity of the PHR1 ortholog OsPHR2 to its downstream target genes in Oryza sativa (rice). We confirmed that a combination of P1BS and P1BS-like motifs are essential for stable binding by OsPHR2. Furthermore, we report that variations in P1BS motif bases affected the binding affinity of OsPHR2 and that the highest affinity motif was GaATATtC (designated the A-T-type P1BS). We also found that a combination of two A-T-type P1BS elements in tandem, namely HA-P1BS, was very efficient for binding of OsPHR2. Using the cis-regulator HA-P1BS, we modified the promoters of Transporter Traffic Facilitator 1 (PHF1), a key factor controlling endoplasmic reticulum-exit of phosphate transporters to the plasma membrane, for efficient uptake of phosphorous in an energetically neutral way. Transgenic plants with the modified promoters showed significantly enhanced tolerance to low phosphate stress in both solution and soil conditions, which provides a new strategy for crop improvement to enhance tolerance of nutrient deficiency. OsPHR1|PHR1 An appropriate concentration of arginine is required for normal root growth in rice. 2014 Plant Signal Behav Institute of Plant Science and Resources; Okayama University; Kurashiki, Japan. Plant roots play an important role in uptake of water and nutrients, support of above-ground part and environmental sensing, but the molecular mechanisms underlying the root development are poorly understood in rice. We found that a gene (OsASL1) encoding argininosuccinate lyase is involved in normal root development of rice. OsASL1 cleaves argininosuccinate to arginine and fumarate reversibly, the last step in the arginine biosynthetic pathway. Here, we further characterized OsASL1 in terms of expression pattern, subcellular localization, and arginine effect on the root growth. A detailed expression analysis revealed that 2 transcripts of OsASL1, OsASL1.1 and OsASL1.2, showed different expression patterns; OsASL1.1 was expressed in most organs throughout the whole growth period, whereas OsASL1.2 was mainly expressed in the roots. In contrast to plastid-localized OsASL1.1, OsASL1.2 was localized to the cytosol and nucleus. The short-root phenotype of the mutant was not rescued by exogenous addition of the sodium nitroprusside, a nitric oxide donor, but rescued by an appropriate concentration of Arg. Our results indicate that the subcellular localization was determined by the N terminus of OsASL1 and that appropriate concentration of Arg is required for normal root elongation in rice. OsASL1 Toward deciphering the genome-wide transcriptional responses of rice to phosphate starvation and recovery. 2014 Plant Signal Behav a Australian Research Council Centre of Excellence in Plant Energy Biology; The University of Western Australia; Perth, WA Australia. Phosphate (Pi) limitation is one of the major factors negatively impacting crop yield worldwide. Next generation sequencing (NGS) was used to profile the transcriptomes of rice (Oryza sativa) roots and shoots after phosphate starvation and recovery, shedding further light on the complex and dynamic mechanisms involved in Pi homeostasis. The use of NGS also enabled the identification of previously not annotated loci and novel isoforms of genes that are specifically induced by Pi starvation. Furthermore, phosphate re-feeding was observed to have a unique response with a variety of transcription factors and kinases induced in a transient manner. Expression profiles of miRNAs were also assessed upon long-term Pi starvation in roots and shoots revealing several novel miRNAs associated with Pi starvation. Altogether, this study provides key findings regarding Pi homeostasis in plants that will provide a valuable resource for research aimed at generating crops with increased Pi acquisition/use efficiency. None POLYAMINE OXIDASE 1 from rice (Oryza sativa) is a functional ortholog of Arabidopsis POLYAMINE OXIDASE 5. 2014 Plant Signal Behav Graduate School of Life Sciences; Tohoku University; Sendai, Miyagi, Japan. POLYAMINE OXIDASE 1 (OsPAO1), from rice (Oryza sativa), and POLYAMINE OXIDASE 5 (AtPAO5), from Arabidopsis (Arabidopsis thaliana), are enzymes sharing high identity at the amino acid level and with similar characteristics, such as polyamine specificity and pH preference; furthermore, both proteins localize to the cytosol. A loss-of-function Arabidopsis mutant, Atpao5-2, was hypersensitive to low doses of exogenous thermospermine but this phenotype could be rescued by introduction of the wild-type AtPAO5 gene. Introduction of OsPAO1, under the control of a constitutive promoter, into Atpao5-2 mutants also restored normal thermospermine sensitivity, allowing growth in the presence of low levels of thermospermine, along with a concomitant decrease in thermospermine content in plants. By contrast, introduction of OsPAO3, which encodes a peroxisome-localized polyamine oxidase, into Atpao5-2 plants could not rescue any of the mutant phenotypes in the presence of thermospermine. These results suggest that OsPAO1 is the functional ortholog of AtPAO5. OsPAO1|PAO1 Tissue-specific transcriptional profiling of iron-deficient and cadmium-stressed rice using laser capture microdissection. 2014 Plant Signal Behav a Departments of Global Agricultural Sciences and Applied Biological Chemistry; Graduate School of Agricultural and Life Sciences; University of Tokyo; Bunkyo-ku, Tokyo, Japan. Several metals are essential nutrients for plants. However, they become toxic at high levels and deleteriously affect crop yield and quality. We recently reported the spatial gene expression profiles of iron (Fe)-deficient and cadmium (Cd)-stressed rice using laser microdissection and microarray analysis. The roots of Fe-deficient and Cd-stressed rice were separated into the vascular bundle (VB), cortex (Cor), and epidermis plus exodermis (EP). In addition, vascular bundles from new and old leaves at the lowest node, which are important for metal distribution, were analyzed separately (newDC and oldDC, respectively). Genes expressed in a tissue-specific manner in the VB, Cor, EP, newDC, and oldDC formed large clusters. The genes upregulated in all of the VB, Cor, and EP by Fe deficiency formed a substantial cluster that was smaller than the tissue-specific clusters. Significant numbers of genes expressed in newDC or oldDC were also expressed in VB in roots, suggesting that vascular bundles in the lowest nodes and roots have a partially common function. The expression patterns of transporter families involved in metal homeostasis were investigated, and members of each family were either expressed differentially in each tissue or showed different responses to Fe deficiency. One potassium transporter gene, OsHAK22, was upregulated by Fe deficiency in VB, Cor, and EP, suggesting that OsHAK22 is involved in potassium transport associated with mugineic acids secretion. None Multiple roles of plant volatiles in jasmonate-induced defense response in rice. 2014 Plant Signal Behav a Faculty of Agriculture and Gene Research Center; Kagawa University; Miki; Kagawa, Japan. The plant hormone jasmonic acid (JA) has a crucial role in defense responses against pathogens in rice. We recently reported that some volatile compounds accumulate in response to JA treatment, and that the monoterpene linalool plays an important role in JA-induced resistance to rice bacterial blight caused by Xanthomonas oryzae pv oryzae (Xoo) in rice. One of the JA-responsive volatiles, (E,E)-2,4-heptadienal, has both antibacterial and antifungal activity against Xoo, and the rice fungal pathogen Magnaporthe oryzae. In addition, (E,E)-2,4-heptadienal was toxic to rice plants. These phenomena were not observed when linalool was treated. These results indicate that accumulation of the (E,E)-2,4-heptadienal in response to JA is a double-edged sword, but it is essential for survival against pathogen attacks in rice. None Physiological response of rice (Oryza sativa L.) genotypes to elevated nitrogen applied under field conditions. 2014 Plant Signal Behav a Department of Plant Physiology; GB Pant University of Agriculture & Technology; Uttarakhand, India. Field experiment was conducted at G.B.P.U.A.T. Pantnagar, Uttarakhand, India in rainy season of 2008 and 2009 to study the impacts of increased nitrogen doses on growth dynamics, biomass partitioning, chaffy grain and nitrogen use efficiency in 4 rice genotypes viz., Vasumati, Tulsi, Kasturi and Krishna Hamsa. Four doses (N0, N50, N100 and N200 kg N ha(-1)) of nitrogen in the form of urea were applied in 3 split. Increased trend in growth dynamics during active tillering and flowering stage, and biomass partitioning at the time of active tillering and flowering stage was observed with respect to nitrogen doses. Chaffy grain number and chaffy grain weight per 5 panicles was significantly increased with enhancing nitrogen doses and was highest for Vasumati. Nitrogen use efficiency (NUE) was increased up to N100 kg N ha(-1) and it was declined with rising nitrogen doses (N200 kg N ha(-1)). The highest values for NUE was achieved by rice genotype Krishna Hamsa whereas lowest by Vasumati. In addition to this, a significant correlation between nitrogen doses and growth dynamics, biomass partitioning and chaffy grain was observed. These findings suggest that growth dynamics, biomass partitioning, chaffy grain could be enhanced by the input of high rate of nitrogen fertilizer but not nitrogen use efficiency. Therefore, this study is useful to screen most N efficient genotypes which can be strongly suggested to rice growers to enhance crop yield irrespective of use of high dose of N fertilizers. None qAC2, a novel QTL that interacts with Wx and controls the low amylose content in rice (Oryza sativa L.). 2015 Theor Appl Genet Rice Breeding Research Team, NARO Institute of Crop Science, 2-1-18, Kannondai, Tsukuba, Ibaraki, 305-8518, Japan. This manuscript reports the fine mapping of a novel QTL, qAC2 controlling the low amylose in rice. The action mechanism of the qAC2 is also investigated by the analysis of genetic interactions to Wx (a) , Wx (b) , du1, du2 and du3. Amylose content of the rice (Oryza sativa L.) endosperm greatly affects starch properties and eating quality of cooked rice. Seeds of japonica rice cultivar Kuiku162 have low amylose content (AC) and good eating quality. Our analysis revealed a novel QTL, designated as qAC2 that contributed to the low AC of Kuiku162. qAC2 was fine mapped within a 74.9-kb region between two insertion and deletion markers, KID3001 and KID5101, on the long arm of chromosome 2. Seven genes are predicted in this region, but none of them is known to be related to the regulation of AC. The AC of a near-isogenic line (NIL110) carrying qAC2 (Kuiku) , the Kuiku162 allele of qAC2, in the genetic background of japonica cultivar Itadaki was lower by 1.1 % points than that of Itadaki. The chain length distributions of amylopectin were similar in NIL110 and Itadaki; therefore, the low AC of NIL110 was caused by a decrease in the actual AC, but not by a difference in the amylopectin structure. The interaction analyses revealed that qAC2 (Kuiku) has epistatic interaction with Wx (a) . The qAC2 (Kuiku) has epistatic interactions with two loci, du1 and du2, on Wx (b) , whereas the genetic effect of qAC2 (Kuiku) has additive to that of du3 on Wx (b) . Thus, similar to du1 and du2, qAC2 may have a function related to Wx (b) mRNA splicing. None Punctual Transcriptional Regulation by the Rice Circadian Clock under Fluctuating Field Conditions. 2015 Plant Cell Functional Plant Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. Plant circadian clocks that oscillate autonomously with a roughly 24-h period are entrained by fluctuating light and temperature and globally regulate downstream genes in the field. However, it remains unknown how punctual internal time produced by the circadian clock in the field is and how it is affected by environmental fluctuations due to weather or daylength. Using hundreds of samples of field-grown rice (Oryza sativa) leaves, we developed a statistical model for the expression of circadian clock-related genes integrating diurnally entrained circadian clock with phase setting by light, both responses to light and temperature gated by the circadian clock. We show that expression of individual genes was strongly affected by temperature. However, internal time estimated from expression of multiple genes, which may reflect transcriptional regulation of downstream genes, is punctual to 22 min and not affected by weather, daylength, or plant developmental age in the field. We also revealed perturbed progression of internal time under controlled environment or in a mutant of the circadian clock gene GIGANTEA. Thus, we demonstrated that the circadian clock is a regulatory network of multiple genes that retains accurate physical time of day by integrating the perturbations on individual genes under fluctuating environments in the field. None Optimization of recombinant expression enables discovery of novel cytochrome P450 activity in rice diterpenoid biosynthesis. 2015 Appl Microbiol Biotechnol Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, 50011, USA. The oxygenation reactions catalyzed by cytochromes P450 (CYPs) play critical roles in plant natural products biosynthesis. At the same time, CYPs are one of most challenging enzymes to functionally characterize due to the difficulty of recombinantly expressing these membrane-associated monooxygenases. In the course of investigating rice diterpenoid biosynthesis, we have developed a synthetic biology approach for functional expression of relevant CYPs in Escherichia coli. In certain cases, activity was observed for only one of two closely related paralogs although it seems clear that related reactions are required for production of the known diterpenoids. Here, we report that optimization of the recombinant expression system enabled characterization of not only these previously recalcitrant CYPs, but also discovery of additional activity relevant to rice diterpenoid biosynthesis. Of particular interest, CYP701A8 was found to catalyze 3beta-hydroxylation of syn-pimaradiene, which is presumably relevant to momilactone biosynthesis, while CYP71Z6 & 7 were found to catalyze multiple reactions, with CYP71Z6 catalyzing the production of 2α,3α-dihydroxy-ent-isokaurene via 2α-hydroxy-ent-isokaurene, and CYP71Z7 catalyzing the production of 3α-hydroxy-ent-cassadien-2-one via 2α-hydroxy-ent-cassadiene and ent-cassadien-2-one, which may be relevant to oryzadione and phytocassane biosynthesis, respectively. OsKOS1|OsKOL4|CYP701A8 Brassinosteroids Promote Development of Rice Pollen Grains and Seeds by Triggering Expression of CSA, a MYB Domain Protein. 2015 Plant J Joint International Research Laboratory of Metabolic & Developmental Sciences, Shanghai Jiao Tong University-University of Adelaide Joint Centre for Agriculture and Health, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China. Transport of photoassimilates from leaf tissues (source regions) to the sink organs is essential for plant development. Here, we show that a phytohormone, the brassinosteroids (BRs) promotes pollen and seed development in rice by directly promoting expression of Carbon Starved Anther (CSA) which encodes a MYB domain protein. Over-expression of the BR-synthesis gene D11 or a BR-signaling factor OsBZR1 results in higher sugar accumulation in developing anthers and seeds, as well as higher grain yield compared with control non-transgenic plants. Conversely, knockdown of D11 or OsBZR1 expression causes defective pollen maturation and reduced seed size and weight, with less accumulation of starch in comparison with the control. Mechanically, OsBZR1 directly promotes CSA expression and CSA directly triggers expression of sugar partitioning and metabolic genes during pollen and seed development. These findings provide insight into how BRs enhance plant reproduction and grain yield in an important agricultural crop. This article is protected by copyright. All rights reserved. CSA OsNAC2 encoding a NAC transcription factor affects plant height through mediating the gibberellic acid pathway in rice. 2015 Plant J State Key Laboratory of Genetic Engineering, Institute of Genetics, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, 200433, China. Plant height and flowering time are key agronomic traits affecting yield in rice (Oryza sativa). In this study, we investigated the functions in rice growth and development of OsNAC2, encoding a NAC transcription factor in rice. Transgenic plants that constitutively expressed OsNAC2 had shorter internodes, shorter spikelets, and were more insensitive to gibberellic acid (GA3 ). In addition, the levels of GAs decreased in OsNAC2 overexpression plants, but increased in OsNAC2 knockdown plants, compared with the wild type. Moreover, flowering was delayed for approximately 5 days in transgenic lines. The transcription of Hd3a, a flowering-time related gene, was suppressed in transgenic lines. In addition, transgenic Arabidopsis plants expressing OsNAC2 were also more insensitive to GA3 . The expression levels of GA biosynthetic genes OsKO2 and OsKAO were repressed. The expression of OsSLRL, encoding a repressor in the GA signal pathway, and OsEATB, which encodes a repressor of GA biosynthesis, were both enhanced. Western blotting indicated that DELLA also accumulated at the protein level. Dual-luciferase reporter analyses, yeast one-hybrid assays and ChIP-qPCR suggested that OsNAC2 directly interacted with the promoter of OsEATB and OsKO2. Taken together, we proposed that OsNAC2 is a negative regulator of the plant height and flowering time, which acts by directly regulating key genes of the GA pathway in rice. This article is protected by copyright. All rights reserved. OsNAC2|OsTIL1|OMTN2 Aequorin-based luminescence imaging reveals differential calcium signalling responses to salt and reactive oxygen species in rice roots. 2015 J Exp Bot College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China. It is well established that both salt and reactive oxygen species (ROS) stresses are able to increase the concentration of cytosolic free Ca(2+) ([Ca(2+)]i), which is caused by the flux of calcium (Ca(2+)). However, the differences between these two processes are largely unknown. Here, we introduced recombinant aequorin into rice (Oryza sativa) and examined the change in [Ca(2+)]i in response to salt and ROS stresses. The transgenic rice harbouring aequorin showed strong luminescence in roots when treated with exogenous Ca(2+). Considering the histological differences in roots between rice and Arabidopsis, we reappraised the discharging solution, and suggested that the percentage of ethanol should be 25%. Different concentrations of NaCl induced immediate [Ca(2+)]i spikes with the same durations and phases. In contrast, H2O2 induced delayed [Ca(2+)]i spikes with different peaks according to the concentrations of H2O2. According to the Ca(2+) inhibitor research, we also showed that the sources of Ca(2+) induced by NaCl and H2O2 are different. Furthermore, we evaluated the contribution of [Ca(2+)]i responses in the NaCl- and H2O2-induced gene expressions respectively, and present a Ca(2+)- and H2O2-mediated molecular signalling model for the initial response to NaCl in rice. None Transgenic expression of an unedited mitochondrial orfB gene product from wild abortive (WA) cytoplasm of rice (Oryza sativa L.) generates male sterility in fertile rice lines. 2015 Planta Advanced Laboratory for Plant Genetic Engineering, Indian Institute of Technology, Kharagpur, 721302, India. Over-expression of the unedited mitochondrial orfB gene product generates male sterility in fertile indica rice lines in a dose-dependent manner. Cytoplasmic male sterility (CMS) and nuclear-controlled fertility restoration are widespread developmental features in plant reproductive systems. In self-pollinated crop plants, these processes often provide useful tools to exploit hybrid vigour. The wild abortive CMS has been employed in the majority of the "three-line" hybrid rice production since 1970s. In the present study, we provide experimental evidence for a positive functional relationship between the 1.1-kb unedited orfB gene transcript, and its translated product in the mitochondria with male sterility. The generation of the 1.1-kb unedited orfB gene transcripts increased during flowering, resulting in low ATP synthase activity in sterile plants. Following insertion of the unedited orfB gene into the genome of male-fertile plants, the plants became male sterile in a dose-dependent manner with concomitant reduction of ATPase activity of F1F0-ATP synthase (complex V). Fertility of the transgenic lines and normal activity of ATP synthase were restored by down-regulation of the unedited orfB gene expression through RNAi-mediated silencing. The genetic elements deciphered in this study could further be tested for their use in hybrid rice development. None Identification of rice cornichon as a possible cargo receptor for the Golgi-localized sodium transporter OsHKT1;3. 2015 J Exp Bot Instituto de Biotecnología, Universidad Nacional de Autónoma de México, Cuernavaca, Morelos, 62250, México Department of Plant Biology, Carnegie Institution for Science, Stanford, CA 94305, USA. Membrane proteins are synthesized and folded in the endoplasmic reticulum (ER), and continue their path to their site of residence along the secretory pathway. The COPII system has been identified as a key player for selecting and directing the fate of membrane and secretory cargo proteins. Selection of cargo proteins within the COPII vesicles is achieved by cargo receptors. The cornichon cargo receptor belongs to a conserved protein family found in eukaryotes that has been demonstrated to participate in the selection of integral membrane proteins as cargo for their correct targeting. Here it is demonstrated at the cellular level that rice cornichon OsCNIH1 interacts with OsHKT1;3 and, in yeast cells, enables the expression of the sodium transporter to the Golgi apparatus. Physical and functional HKT-cornichon interactions are confirmed by the mating-based split ubiquitin system, bimolecular fluorescence complementation, and Xenopus oocyte and yeast expression systems. The interaction between the two proteins occurs in the ER of plant cells and their co-expression in oocytes leads to the sequestration of the transporter in the ER. In the yeast cornichon mutant erv14, OsHKT1;3 is mistargeted, preventing the toxic effects of sodium transport in the cell observed in wild-type cells or in the erv14 mutant that co-expressed OsHKT1;3 with either OsCNIH1 or Erv14p. Identification and characterization of rice cornichon as a possible cargo receptor opens up the opportunity to improve our knowledge on membrane protein targeting in plant cells. OsHKT1;3 Genetic architecture of natural variation in rice chlorophyll content revealed by genome wide association study. 2015 Mol Plant National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China. Chlorophyll content is one of the most important physiological traits as it is closely related to leaf photosynthesis and crop yield potential. So far, few genes have been reported to be involved in natural variation of chlorophyll content in rice (Oryza sativa), and the extents of variation explored are very limited. We conducted a genome wide association study (GWAS) using a diverse worldwide collection of 529 O. sativa accessions. A total of 46 significant association loci were identified. Three F2 mapping populations with parents selected from the association panel were tested for validation of GWAS signals. We clearly demonstrated that Grain number, plant height, and heading date7 (Ghd7) was a major locus for natural variation of chlorophyll content at heading stage by combining evidences from near-isogenic lines and transgenic plants. The enhanced expression of Ghd7 decreased chlorophyll content, mainly through down-regulating the expression of genes involved in the biosynthesis of chlorophyll and chloroplast. In addition, Narrow leaf1 (NAL1) corresponded to one significant association region repeatedly detected in two years. We revealed high-degree polymorphism in 5'UTR and four non-synonymous SNPs in the coding region of NAL1, and observed diverse effects of the major haplotypes. The loci or candidate genes identified would help fine-tune and optimize antenna size of canopies in rice breeding. Ghd7 qRT9, a quantitative trait locus controlling root thickness and root length in upland rice. 2015 J Exp Bot Collaborative Innovation Center of Henan Grain Crops, Rice Engineer Center in Henan Province, Henan Agricultural University, Zhengzhou, 450002, China. Breeding for strong root systems is an important strategy for improving drought avoidance in rice. To clone genes responsible for strong root traits, an upland rice introgression line IL392 with thicker and longer roots than the background parent lowland rice Yuefu was selected. A quantitative trait locus (QTL), qRT9, controlling root thickness and root length was detected under hydroponic culture using 203 F2:3 populations derived from a cross between Yuefu and IL392. The qRT9 locus explained 32.5% and 28.1% of the variance for root thickness and root length, respectively. Using 3185 F2 plants, qRT9 was ultimately narrowed down to an 11.5kb region by substitution mapping. One putative basic helix-loop-helix (bHLH) transcription factor gene, LOC_Os09g28210 (named OsbHLH120), is annotated in this region. Sequences of OsbHLH120 in 11 upland rice and 13 lowland rice indicated that a single nucleotide polymorphism (SNP) at position 82 and an insertion/deletion (Indel) at position 628-642 cause amino acid changes and are conserved between upland rice and lowland rice. Phenotypic analysis indicated that the two polymorphisms were significantly associated with root thickness and root length under hydroponic culture. Quantitative real-time PCR showed that OsbHLH120 was strongly induced by polyethylene glycol (PEG), salt, and abscisic acid, but higher expression was present in IL392 roots than in Yuefu under PEG and salt stress. The successfully isolated locus, qRT9, enriches our knowledge of the genetic basis for drought avoidance and provides an opportunity for breeding drought avoidance varieties by utilizing valuable genes in the upland rice germplasm. qRT9 A Targeted Metabolomics Approach toward Understanding Metabolic Variations in Rice under Pesticide Stress. 2015 Anal Biochem Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran. Diazinon insecticide is widely applied throughout rice (Oryza sativa L.) fields in Iran. However, concerns are now being raised about its potential adverse impacts on rice fields. In this study, a time-course metabolic change in rice plants was investigated after diazinon treatment using gas chromatography-mass spectrometry (GC-MS) and subsequently, statistical strategy of Random Forest (RF) was performed in order to find the stress-associated effects. According to the results, a wide range of metabolites were dynamically varied as a result of the plant response to diazinon such as biosynthesis and metabolism of sugars, amino acids, organic acids and Phenylpropanoids, all correlating with the exposure time. Plant response was involved in multiple metabolic pathways, most of which were correlated with the exposure time. In this study, RF was explored as a potential multivariate method for GC/MS analysis of metabolomics data of rice (Oryza sativa L.) plants under diazinon stress; more than 31 metabolites were quantitatively determined and time-course metabolic response of the plant during different days after treatment was measured. Results demonstrated RF as a potential multivariate method for gas chromatography-mass spectrometry analysis of changes in plant metabolome under insecticide stress. None Differential expression of GS5 regulates grain size in rice. 2015 J Exp Bot National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China. Grain weight is a major determinant of grain yield. GS5 is a positive regulator of grain size such that grain width, filling, and weight are correlated with its expression level. Previous work suggested that polymorphisms of GS5 in the promoter region might be responsible for the variation in grain size. In this study, two single nucleotide polymorphisms (SNPs) between the wide-grain allele GS5-1 and the narrow-grain allele GS5-2 in the upstream region of the gene that were responsible for the differential expression in developing young panicles were identified. These two polymorphs altered the responses of the GS5 alleles to abscisic acid (ABA) treatments, resulting in higher expression of GS5-1 than of GS5-2 in developing young panicles. It was also shown that SNPs in light-responsive elements of the promoter altered the response to light induction, leading to higher expression of GS5-2 than GS5-1 in leaves. Enhanced expression of GS5 competitively inhibits the interaction between OsBAK1-7 and OsMSBP1 by occupying the extracellular leucine-rich repeat (LRR) domain of OsBAK1-7, thus preventing OsBAK1-7 from endocytosis caused by interacting with OsMSBP1, providing an explanation for the positive association between grain size and GS5 expression. These results advanced our understanding of the molecular mechanism by which GS5 controls grain size. GS5 Identification and analysis of the mechanism underlying heat-inducible expression of rice aconitase 1. 2015 Plant Sci Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, China; Institute of Technical Biology and Agriculture Engineering, Chinese Academy of Sciences, Hefei 230031, China. Respiratory metabolism is an important though poorly understood facet of plant adaptation to stress. Posttranslational modification of aconitase, a component of the tricarboxylic acid cycle (TCA), may be involved in stress tolerance. However, such stress-related transcriptional regulation and its mechanism remain unknown. In this study, we found that expression of the rice Aconitase gene OsACO1 is induced in a time-dependent manner by heat but not other typical abiotic stresses. To analyze the transcriptional regulation mechanism underlying the response to heat, the OsACO1 promoter (POsACO1) was isolated and characterized in transgenic rice. Using qualitative and quantitative analyses, we found that the expression of the GUS reporter gene responded to heat in different tissues and at different stages of development when driven by POsACO1. A series of 5' distal deletions of POsACO1 was generated to delineate the region responsible for heat-induced gene expression. Transient expression analyses in tobacco leaves identified a 322-bp minimal region between -1386 and -1065 as being essential and sufficient for heat-induced expression by POsACO1. We screened for known heat response-related cis-elements in this 322-bp region; however, sequences correlating with heat-induced gene expression were not identified in POsACO1. Therefore, truncations and successive mutagenesis analyses were performed in this 322-bp region. By comparing the activities of promoter fragments and their derivatives, our results indicated that the heat response element resided in a 9-bp region between -1132 and -1124, a sequence that contains a W-box motif. Additional site-directed mutagenesis analyses eliminated the heat response activity of POsACO1 via the W-box element, and an electrophoretic mobility shift assay (EMSA) indicated the binding of POsACO1 by factors in the nuclear extracts of heat-stressed rice seedlings in a W-box-dependent manner. Our results illustrate the expression pattern of a key component of the TCA response to abiotic stress and establish a putative regulatory pathway in the transcriptional modulation of rice respiratory metabolism genes in response to heat. OsACO1 Three novel alleles of FLOURY ENDOSPERM2 (FLO2) confer dull grains with low amylose content in rice. 2014 Plant Sci Department of Agronomy, Chiayi Agricultural Experiment Station, Chiayi, Taiwan. Rice is a major food source for much of the world, and expanding our knowledge of genes conferring specific rice grain attributes will benefit both farmer and consumer. Here we present novel dull grain mutants with a low amylose content (AC) derived from mutagenesis of Oryza sativa, ssp. japonica cv. Taikeng 8 (TK8). Positional cloning of the gene conferring the dull grain phenotype revealed a point mutation located at the acceptor splice site of intron 11 of FLOURY ENDOSPERM2 (FLO2), encoding a tetratricopeptide repeat domain (TPR)-containing protein. Three novel flo2 alleles were identified herein, which surprisingly conferred dull rather than floury grains. The allelic diversity of flo2 perturbed the expression of starch synthesis-related genes including OsAGPL2, OsAGPS2b, OsGBSSI, OsBEI, OsBEIIb, OsISA1, and OsPUL. The effect of the flo2 mutations on the physicochemical properties of the grain included a low breakdown, setback, and consistency of rice, indicating a good elasticity and soft texture of cooked rice grains. The effects of FLO2, combined with the genetic background of the germplasm and environmental effects, resulted in a variety of different amylose content levels, grain appearance, and physicochemical properties of rice, providing a host of useful information to future grain-quality research and breeding. FLO2 OsSEC24, a functional SEC24-like protein in rice, improves tolerance to iron deficiency and high pH by enhancing H(+) secretion mediated by PM-H(+)-ATPase. 2015 Plant Sci College of Life Sciences, Capital Normal University, No. 105 Xisanhuan North Street, Beijing 100048, China. Iron is abundant in the soil, but its low solubility in neutral or alkaline soils limits its uptake. Plants can rely on rhizosphere acidification to increase iron solubility. OsSEC27p was previously found to be a highly up-regulated gene in iron-deficient rice roots. Here, pH-dependent complementation assays using yeast mutants sec24Δ/SEC24 and sec27Δ/SEC27 showed that OsSEC27 could functionally complement SEC24 but not SEC27 in yeast; thus, it was renamed as OsSEC24. We found that OsSEC24-transgenic tobacco plants increased the length and number of roots under iron deficiency at pH 8.0. To explore how OsSEC24 confers tolerance to iron deficiency, we utilized transgenic tobacco, rice and rice protoplasts. H(+) flux measurements using Non-invasive Micro-test Technology (NMT) indicated that the transgenic OsSEC24 tobacco and rice enhanced H(+) efflux under iron deficiency. Conversely, the application of plasma membrane PM-H(+)-ATPase inhibitor vanadate elucidated that H(+) secretion increased by OsSEC24 was mediated by PM-H(+)-ATPase. OsPMA2 was used as a representative of iron deficiency-responsive PM-H(+)-ATPases in rice root via RT-PCR analysis. In transgenic rice protoplasts OsPMA2 was packaged into OsSEC24 vesicles after export from the ER through confocal-microscopy observation. Together, OsSEC24 vesicles, along with PM-H(+)-ATPases stimulate roots formation under iron deficiency by enhancing rhizosphere acidification. OsSEC24 Recent advances in the dissection of drought-stress regulatory networks and strategies for development of drought-tolerant transgenic rice plants. 2015 Front Plant Sci Laboratory of Plant Molecular Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo Japan. Advances have been made in the development of drought-tolerant transgenic plants, including cereals. Rice, one of the most important cereals, is considered to be a critical target for improving drought tolerance, as present-day rice cultivation requires large quantities of water and as drought-tolerant rice plants should be able to grow in small amounts of water. Numerous transgenic rice plants showing enhanced drought tolerance have been developed to date. Such genetically engineered plants have generally been developed using genes encoding proteins that control drought regulatory networks. These proteins include transcription factors, protein kinases, receptor-like kinases, enzymes related to osmoprotectant or plant hormone synthesis, and other regulatory or functional proteins. Of the drought-tolerant transgenic rice plants described in this review, approximately one-third show decreased plant height under non-stressed conditions or in response to abscisic acid treatment. In cereal crops, plant height is a very important agronomic trait directly affecting yield, although the improvement of lodging resistance should also be taken into consideration. Understanding the regulatory mechanisms of plant growth reduction under drought stress conditions holds promise for developing transgenic plants that produce high yields under drought stress conditions. Plant growth rates are reduced more rapidly than photosynthetic activity under drought conditions, implying that plants actively reduce growth in response to drought stress. In this review, we summarize studies on molecular regulatory networks involved in response to drought stress. In a separate section, we highlight progress in the development of transgenic drought-tolerant rice plants, with special attention paid to field trial investigations. None Interpreting lemma and palea homologies: a point of view from rice floral mutants. 2015 Front Plant Sci Rice Biotechnology Research Project, Rice Research Division, National Agriculture and Food Research Organization (NARO) Institute of Crop Science , Tsukuba, Ibaraki, Japan. In contrast to eudicot flowers which typically exhibit sepals and petals at their periphery, the flowers of grasses are distinguished by the presence of characteristic outer organs. In place of sepals, grasses have evolved the lemma and the palea, two bract-like structures that partially or fully enclose the inner reproductive organs. With little morphological similarities to sepals, whether the lemma and palea are part of the perianth or non-floral organs has been a longstanding debate. In recent years, comparative studies of floral mutants as well as the availability of whole genome sequences in many plant species have provided strong arguments in favor of the hypothesis of lemma and palea being modified sepals. In rice, a feature of the palea is the bending of its lateral region into a hook-shaped marginal structure. This allows the palea to lock into the facing lemma region, forming a close-fitting lemma-palea enclosure. In this article, we focus on the rice lemma and palea and review some of the key transcription factors involved in their development and functional specialization. Alternative interpretations of these organs are also addressed. None Hd3a promotes lateral branching in rice. 2015 Plant J Laboratory of Plant Molecular Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192, Japan. Accumulating evidence indicates that FLOWERING LOCUS T (FT) protein is the mobile floral signal florigen. A rice FT homolog, Hd3a, is transported from the phloem to shoot apical cells where it interacts with 14-3-3 proteins and the transcription factor OsFD1 to form a florigen activation complex (FAC) that activates a rice homolog of the floral identity gene APETALLA1. Recent studies showed that florigen has roles in plant development beyond flowering; however, the exact nature of these roles is not well understood. It is not clear whether FT is transported to organs outside the shoot apex and whether FAC formation is required for processes other than flowering. We show here that Hd3a protein accumulated in axillary meristems to promote branching and that FAC formation was required. Analysis of transgenic plants revealed that Hd3a promotes branching through lateral bud outgrowth. Hd3a protein produced in the phloem reached the axillary meristem in the lateral bud, and its transport was required for promotion of branching. Moreover, mutant Hd3a proteins defective in FAC formation but competent in transport failed to promote branching. Finally, we show that Hd3a promotes branching independently from strigolactone and FC1, a transcription factor that inhibits branching in rice. Together, these results suggest that Hd3a functions as a mobile signal for branching in rice. This article is protected by copyright. All rights reserved. Hd3a Loss-of-function mutation of rice SLAC7 decreases chloroplast stability and induces a photoprotection mechanism in rice. 2015 J Integr Plant Biol National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China. Plants absorb sunlight to power the photochemical reactions of photosynthesis, which can potentially damage the photosynthetic machinery. However, the mechanism that protects chloroplasts from the damage remains unclear. In this work, we demonstrated that rice (Oryza sativa L.) SLAC7 is a generally expressed membrane protein. Loss-of-function of SLAC7 caused continuous damage to the chloroplasts of mutant leaves under normal light conditions. Ion leakage indicators related to leaf damage such as H2 O2 and ABA levels were significantly higher in slac7-1 than in the wild type. Consistently, the photosynthesis efficiency and Fv/Fm ratio of slac7-1 were significantly decreased (similar to photoinhibition). In response to chloroplast damage, slac7-1 altered its leaf morphology (curled or fused leaf) by the synergy between plant hormones and transcriptional factors to decrease the absorption of light, suggesting that a photoprotection mechanism for chloroplast damage was activated in slac7-1. When grown in dark conditions, slac7-1 displayed a normal phenotype. SLAC7 under the control of AtSLAC1 promoter could partially complement the phenotypes of Arabidopsis slac1 mutants, indicating a partial conservation of SLAC protein functions. These results suggest that SLAC7 is essential for maintaining the chloroplast stability in rice. SLAC7 Rice immune regulator, OsPti1a, is specifically phosphorylated at the plasma membrane. 2015 Plant Signal Behav a Plant proteomics research unit , RIKEN center for Sustainable Resource Science , 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045 , Japan; OsPti1a (Pto-interacting protein 1a) has important roles in the regulation of immune responses in rice. Phosphorylation of a conserved threonine in OsPti1a is necessary to activate defense responses; however, the regulatory mechanism of OsPti1a-mediated immune responses is still obscure. Recently, we revealed that OsPti1a forms protein complex(es) at the plasma membrane and this localization is required for its function. Here, we show that membrane-localized OsPti1a was selectively phosphorylated. Additionally, phosphorylation was not required for the localization of OsPti1a at the membrane. These results suggest that OsPti1a protein is selectively regulated by its phosphorylation after OsPti1a localizes to the plasma membrane. OsPti1a Reduced ABA Accumulation in the Root System is Caused by ABA exudation in Upland Rice (Oryza sativa L. var. Gaoshan1) and These Enhanced Drought Adaptation. 2015 Plant Cell Physiol CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, National Center for Nanoscience and Technology of China Beijing 100190, China Department of Biology, Hong Kong Baptist University, Hong Kong, China. Lowland rice (Nipponbare) and upland rice (Gaoshan 1) that are comparable under normal and moderate drought conditions showed dramatic differences in severe drought conditions, including natural-occurring long-term drought and simulated rapid water deficits. We focused on their root response and found that enhanced tolerance of upland rice to severe drought conditions was mainly due to the lower level of ABA in its roots than that in the lowland rice. We firstly excluded the effect of ABA biosynthesis and catabolism on root-accumulated ABA levels in both rice by monitoring the expressions of four OsNCED genes and two OsABA8ox genes. Next, we excluded the impact of the aerial parts on roots by suppressing leaf-biosynthesized ABA with fluridone and NDGA (nordihydroguaiaretic acid) and measuring ABA level in detached roots. Instead, we proved that upland rice had the ability to export considerably more root-sourced ABA than lowland rice under severe drought, which improved ABA-dependent drought adaptation. The investigation of apoplastic pH in root cell and root anatomy evidenced that ABA leakage in the root system of upland rice was related to high apoplastic pH and the absence of Casparian bands in the sclerenchyma layer. Finally, taking some genes as examples, we predicted that different ABA levels in rice roots stimulated distinct ABA perception and signaling cascades, which influenced its response to water stress. None Ethylene Signaling in Rice and Arabidopsis: Conserved and Diverged Aspects. 2015 Mol Plant State Key Lab of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Ethylene as a gas phytohormone plays significant roles in the whole life cycle of plants, ranging from growth and development to stress responses. A linear ethylene signaling pathway has been established in the dicotyledonous model plant Arabidopsis. However, the ethylene signaling mechanism in monocotyledonous plants such as rice is largely unclear. In this review, we compare the ethylene response phenotypes of dark-grown seedlings of Arabidopsis, rice, and other monocotyledonous plants (maize, wheat, sorghum, and Brachypodium distachyon) and pinpoint that rice has a distinct phenotype of root inhibition but coleoptile promotion in etiolated seedlings upon ethylene treatment. We further summarize the homologous genes of Arabidopsis ethylene signaling components in these monocotyledonous plants and discuss recent progress. Although conserved in most aspects, ethylene signaling in rice has evolved new features compared with that in Arabidopsis. These analyses provide novel insights into the understanding of ethylene signaling in the dicotyledonous Arabidopsis and monocotyledonous plants, particularly rice. Further characterization of rice ethylene-responsive mutants and their corresponding genes will help us better understand the whole picture of ethylene signaling mechanisms in plants. None Loss of floral repressor function adapts rice to higher latitudes in Europe. 2015 J Exp Bot University of Milan, Department of Biosciences, Via Celoria 26, 20133 Milan, Italy. The capacity to discriminate variations in day length allows plants to align flowering with the most favourable season of the year. This capacity has been altered by artificial selection when cultivated varieties became adapted to environments different from those of initial domestication. Rice flowering is promoted by short days when HEADING DATE 1 (Hd1) and EARLY HEADING DATE 1 (Ehd1) induce the expression of florigenic proteins encoded by HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1). Repressors of flowering antagonize such induction under long days, maintaining vegetative growth and delaying flowering. To what extent artificial selection of long day repressor loci has contributed to expand rice cultivation to Europe is currently unclear. This study demonstrates that European varieties activate both Hd3a and RFT1 expression regardless of day length and their induction is caused by loss-of-function mutations at major long day floral repressors. However, their contribution to flowering time control varies between locations. Pyramiding of mutations is frequently observed in European germplasm, but single mutations are sufficient to adapt rice to flower at higher latitudes. Expression of Ehd1 is increased in varieties showing reduced or null Hd1 expression under natural long days, as well as in single hd1 mutants in isogenic backgrounds. These data indicate that loss of repressor genes has been a key strategy to expand rice cultivation to Europe, and that Ehd1 is a central node integrating floral repressive signals. None Variations between the photosynthetic properties of elite and landrace Chinese rice cultivars revealed by simultaneous measurements of 820nm transmission signal and chlorophyll a fluorescence induction. 2015 J Plant Physiol CAS Key Laboratory of Computational Biology, Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. The difference between the photosynthetic properties of elite and landrace Chinese rice cultivars was studied, using chlorophyll a fluorescence induction (mostly a monitor of Photosystem II activity) and I820 transmission signal (mostly a monitor of Photosystem I activity) to identify potential photosynthetic features differentiating these two groups, which show different degrees of artificial selection and grain yields. A higher fluorescence (related to PSII) IP rise phase and a lower P700(+) (related to PSI) accumulation were observed in the elite cultivars as compared to the landraces. Using these data, together with simulation data from a kinetic model of fluorescence induction, we show that the high IP rise phase and the low P700(+) accumulation can be a result of transient block on electron transfer and traffic jam on the electron acceptor side of PSI under a high [NADPH]/[NADP(+)] ratio. Considering that the ferredoxin NADP(+) reductase (FNR) transcript levels of XS134 (a representative elite cultivars) remains unaffected during the first few minutes of light/dark transition compared to Q4145 (a representative landrace cultivars), which shows a strong decline during the same time range, we propose that the FNR of elite cultivars may take more time to be inactivated in darkness. During this time the FNR enzyme can continue to reduce NADP(+) molecules, leading to initially high [NADPH]/[NADP(+)] ratio during OJIP transient. These data suggested a potential artificial selection of FNR during the breeding process of these examined elite rice cultivars. None Genetic analysis of seed-shattering genes in rice using an F3:4 population derived from an Oryza sativa x Oryza rufipogon cross. 2015 Genet Mol Res Department of Plant Resources, College of Industrial Science, Kongju National University, Yesan, Korea. Seed shattering of wild plant species is thought to be an adaptive trait to facilitate seed dispersal. For rice breeding, seed shatter-ing is an important trait for improving breeding strategies, particularly when developing lines use interspecific hybrids and introgression of genes from wild species. We developed F3:4 recombinant inbred lines from an interspecific cross between Oryza sativa cv. Ilpoombyeo and Oryza rufipogon. In this study, we genetically analyzed known shat-tering-related loci using the F3:4 population of O. sativa/O. rufipogon. CACTA-AG190 was significantly associated with the shattering trait CACTA-TD according to bulked segregant analysis results, and was found in the qSH-1 region of chromosome 1. Fine genetic mapping of the flanking regions around qSH-1 based on CACTA-AG190 revealed multiple-sequence variations. The highest limit of detection based on quantitative trait locus analysis was observed between shaap-7715 and a 518-bp insertion site. Two other quantitative trait locus analyses of seed-shattering-related loci, qSH-4 and sh-h, were performed using simple sequence repeat and allele-pecific single nucleotide polymor-phism markers. Our results can be applied for rice-breeding research, such as marker-assisted selection between cultivated and wild rice. None Natural variation of rice blast resistance gene Pi-d2. 2015 Genet Mol Res Agricultural Environment and Resources Research Institute, Yunnan Academy of Agricultural Sciences, Kunming, Yunnan Province, China. Studying natural variation in rice resistance genes of cultivated and wild rice relatives can predict resistance stability to rice blast fungus. In the present study, the protein coding regions of the rice R gene Pi-d2 in 35 rice accessions, including Oryza sativa L. subsp. indica Kato (Aus), indica (IND), temperate japonica (TEJ), tropical japonica (TRJ), aromatic (ARO); subgroups of Oryza sativa; 6 accessions of wild rice varieties; O. nivara; and O. rufipogon were analyzed. A total of 13 nucleotide differences were found in the open reading frames (ORFs) of Pi-d2. Translation of these ORFs revealed 9 variants; 3 were novel Pi-d2 variants. Variants H2 and H5 were identified in accessions of cultivated rice and O. nivara, H1, H3, H4, H6, and H8 were only identified in cultivated rice. H2 and H5 were the common types of IND and O. nivara, H8 was the common type of TRJ and AUS, H6 was the specific type of AUS, and H3 was the specific type of ARO. H7 and H9 were specific haplotypes of O. nivara and O. rufipogon, respectively. These findings demonstrate that Pi-d2 variants are useful indicators for each subgroup, and Pi-d2 is an ancient gene that predates speciation of rice subgroups. OsPK10|Pid2 COLD1 Confers Chilling Tolerance in Rice. 2015 Cell Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. Rice is sensitive to cold and can be grown only in certain climate zones. Human selection of japonica rice has extended its growth zone to regions with lower temperature, while the molecular basis of this adaptation remains unknown. Here, we identify the quantitative trait locus COLD1 that confers chilling tolerance in japonica rice. Overexpression of COLD1(jap) significantly enhances chilling tolerance, whereas rice lines with deficiency or downregulation of COLD1(jap) are sensitive to cold. COLD1 encodes a regulator of G-protein signaling that localizes on plasma membrane and endoplasmic reticulum (ER). It interacts with the G-protein α subunit to activate the Ca(2+) channel for sensing low temperature and to accelerate G-protein GTPase activity. We further identify that a SNP in COLD1, SNP2, originated from Chinese Oryza rufipogon, is responsible for the ability of COLD(jap/ind) to confer chilling tolerance, supporting the importance of COLD1 in plant adaptation. COLD1 Reduction of pyruvate orthophosphate dikinase activity is associated with high temperature-induced chalkiness in rice grains. 2015 Plant Physiol Biochem MOA Key Laboratory of Crop Ecophysiology and Farming System in the Middle Reaches of the Yangtze River, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China. Global warming affects both rice (Oryza sativa) yields and grain quality. Rice chalkiness due to high temperature during grain filling would lower the grain quality. The biochemical and molecular mechanisms responsible for the increased occurrence of chalkiness under high temperature are not fully understood. Previous research suggested that cytosolic pyruvate orthophosphate dikinase (cyPPDK, EC 2.7.9.1) in rice modulates carbon metabolism. The objective of this study was to determine the relationship between cyPPDK and high temperature-induced chalkiness. High temperature treatments were applied during the grain filling of two rice cultivars (9311 and TXZ-25) which had different sensitivity of chalkiness to high temperature. Chalkiness was increased significantly under high temperature treatment, especially for TXZ-25. A shortened grain filling duration and a decreased grain weight in both cultivars were caused by high temperature treatment. A reduction in PPDK activities due to high temperature was observed during the middle and late grain filling periods, accompanied by down regulated cyPPDK mRNA and protein levels. The temperature effects on the developmental regulation of PPDK activity were confirmed at transcription, translation and post-translational levels. PPDK activities were insensitive to variation in PPDK levels, suggesting the rapid phosphorylation mechanism of this protein. The two varieties showed similar responses to the high temperature treatment in both PPDK activities and chalkiness. We concluded that high temperature-induced chalkiness was associated with the reduction of PPDK activity. None Activation of Symbiosis Signaling by Arbuscular Mycorrhizal Fungi in Legumes and Rice. 2015 Plant Cell John Innes Centre, Norwich NR4 7UH, United Kingdom. Establishment of arbuscular mycorrhizal interactions involves plant recognition of diffusible signals from the fungus, including lipochitooligosaccharides (LCOs) and chitooligosaccharides (COs). Nitrogen-fixing rhizobial bacteria that associate with leguminous plants also signal to their hosts via LCOs, the so-called Nod factors. Here, we have assessed the induction of symbiotic signaling by the arbuscular mycorrhizal (Myc) fungal-produced LCOs and COs in legumes and rice (Oryza sativa). We show that Myc-LCOs and tetra-acetyl chitotetraose (CO4) activate the common symbiosis signaling pathway, with resultant calcium oscillations in root epidermal cells of Medicago truncatula and Lotus japonicus. The nature of the calcium oscillations is similar for LCOs produced by rhizobial bacteria and by mycorrhizal fungi; however, Myc-LCOs activate distinct gene expression. Calcium oscillations were activated in rice atrichoblasts by CO4, but not the Myc-LCOs, whereas a mix of CO4 and Myc-LCOs activated calcium oscillations in rice trichoblasts. In contrast, stimulation of lateral root emergence occurred following treatment with Myc-LCOs, but not CO4, in M. truncatula, whereas both Myc-LCOs and CO4 were active in rice. Our work indicates that legumes and non-legumes differ in their perception of Myc-LCO and CO signals, suggesting that different plant species respond to different components in the mix of signals produced by arbuscular mycorrhizal fungi. None The Rice CK2 Kinase Regulates Trafficking of Phosphate Transporters in Response to Phosphate Levels. 2015 Plant Cell State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China. Phosphate transporters (PTs) mediate phosphorus uptake and are regulated at the transcriptional and posttranslational levels. In one key mechanism of posttranslational regulation, phosphorylation of PTs affects their trafficking from the endoplasmic reticulum (ER) to the plasma membrane. However, the kinase(s) mediating PT phosphorylation and the mechanism leading to ER retention of phosphorylated PTs remain unclear. In this study, we identified a rice (Oryza sativa) kinase subunit, CK2beta3, which interacts with PT2 and PT8 in a yeast two-hybrid screen. Also, the CK2α3/beta3 holoenzyme phosphorylates PT8 under phosphate-sufficient conditions. This phosphorylation inhibited the interaction of PT8 with PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1, a key cofactor regulating the exit of PTs from the ER to the plasma membrane. Additionally, phosphorus starvation promoted CK2beta3 degradation, relieving the negative regulation of PT phosphorus-insufficient conditions. In accordance, transgenic expression of a nonphosphorylatable version of OsPT8 resulted in elevated levels of that protein at the plasma membrane and enhanced phosphorus accumulation and plant growth under various phosphorus regimes. Taken together, these results indicate that CK2α3/beta3 negatively regulates PTs and phosphorus status regulates CK2α3/beta3. CK2alpha2,CK2alpha3,CK2beta1,CK2beta3 Low pH-Induced Changes of Antioxidant Enzyme and ATPase Activities in the Roots of Rice (Oryza sativa L.) Seedlings. 2015 PLoS One State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou, Zhejiang, 310006, P. R. China. Soil acidification is the main problem in the current rice production. Here, the effects of low pH on the root growth, reactive oxygen species metabolism, plasma membrane functions, and the transcript levels of the related genes were investigated in rice seedlings (Oryza sativa L.) in a hydroponic system at pH 3.5, 4.5, and 5.5. There were two hybrid rice cultivars in this trial, including Yongyou 12 (YY12, a japonica hybrid) and Zhongzheyou 1 (ZZY1, an indica hybrid). Higher H+ activity markedly decreased root length, the proportion of fine roots, and dry matter production, but induced a significant accumulation of hydrogen peroxide (H2O2), and led to serious lipid peroxidation in the roots of the two varieties. The transcript levels of copper/zinc superoxide dismutase 1 (Cu/Zn SOD1), copper/zinc superoxide dismutase 2 (Cu/Zn SOD2), catalase A (CATA) and catalase B (CATB) genes in YY12 and ZZY1 roots were significantly down-regulated after low pH exposure for two weeks. Meanwhile, a significant decrease was observed in the expression of the P-type Ca2+-ATPases in roots at pH 3.5. The activities of antioxidant enzymes (SOD, CAT) and plasma membrane (PM) Ca2+-ATPase in the two varieties were dramatically inhibited by strong rhizosphere acidification. However, the expression levels of ascorbate peroxidase 1 (APX1) and PM H+-ATPase isoform 7 were up-regulated under H+ stress compared with the control. Significantly higher activities of APX and PM H+-ATPase could contribute to the adaptation of rice roots to low pH. None CO2 Responsive CCT protein, CRCT Is a Positive Regulator of Starch Synthesis in Vegetative Organs of Rice. 2015 Plant Physiol Kobe University fukayama@people.kobe-u.ac.jp. A novel CO2 Responsive protein (CRCT) containing a CONSTANS, CONSTANS-like and TOC1 (CCT) domain but not Zinc finger motif is described which is up-regulated under elevated CO2 in rice (Oryza sativa). The expression of CRCT showed diurnal oscillation peaked at the end of light period and was also increased by sugars such as glucose and sucrose. Promoter GUS analysis showed that CRCT was highly expressed in the phloem of various tissues such as leaf blade and leaf sheath. Overexpression or RNAi knockdown of CRCT had no appreciable effect on plant growth and photosynthesis except that tiller angle was significantly increased by the overexpression. More importantly, starch content in leaf sheath which serves as a temporary storage organ for photoassimilates, was markedly increased in overexpression lines and decreased in knockdown lines. The expressions of several genes related to starch synthesis such as ADP-glucose pyrophospholylase and α-glucan phospholylase were significantly changed in transgenic lines and positively correlated with the expression levels of CRCT. Given these observations, we suggest that CRCT is a positive regulator of starch accumulation in vegetative tissues, regulating coordinated expression of starch synthesis genes in response to the levels of photoassimilates. NRR|CRCT Whole-Genome Analysis Revealed the Positively Selected Genes during the Differentiation of indica and Temperate japonica Rice. 2015 PLoS One Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fujian Agriculture & Forestry University, Fuzhou, China; College of Crop Science, Fujian Agriculture & Forestry University, Fuzhou, China. To investigate the selective pressures acting on the protein-coding genes during the differentiation of indica and japonica, all of the possible orthologous genes between the Nipponbare and 93-11 genomes were identified and compared with each other. Among these genes, 8,530 pairs had identical sequences, and 27,384 pairs shared more than 90% sequence identity. Only 2,678 pairs of genes displaying a Ka/Ks ratio significantly greater than one were revealed, and most of these genes contained only nonsynonymous sites. The genes without synonymous site were further analyzed with the SNP data of 1529 O. sativa and O. rufipogon accessions, and 1068 genes were identified to be under positive selection during the differentiation of indica and temperate japonica. The positively selected genes (PSGs) are unevenly distributed on 12 chromosomes, and the proteins encoded by the PSGs are dominant with binding, transferase and hydrolase activities, and especially enriched in the plant responses to stimuli, biological regulations, and transport processes. Meanwhile, the most PSGs of the known function and/or expression were involved in the regulation of biotic/abiotic stresses. The evidence of pervasive positive selection suggested that many factors drove the differentiation of indica and japonica, which has already started in wild rice but is much lower than in cultivated rice. Lower differentiation and less PSGs revealed between the Or-It and Or-IIIt wild rice groups implied that artificial selection provides greater contribution on the differentiation than natural selection. In addition, the phylogenetic tree constructed with positively selected sites showed that the japonica varieties exhibited more diversity than indica on differentiation, and Or-III of O. rufipogon exhibited more than Or-I. None Molecular Screening of Blast Resistance Genes in Rice using SSR Markers. 2015 Plant Pathol J Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi-221005, India. Rice Blast is the most devastating disease causing major yield losses in every year worldwide. It had been proved that using resistant rice varieties would be the most effective way to control this disease. Molecular screening and genetic diversities of major rice blast resistance genes were determined in 192 rice germplasm accessions using simple sequence repeat (SSR) markers. The genetic frequencies of the 10 major rice blast resistance genes varied from 19.79% to 54.69%. Seven accessions IC337593, IC346002, IC346004, IC346813, IC356117, IC356422 and IC383441 had maximum eight blast resistance gene, while FR13B, Hourakani, Kala Rata 1-24, Lemont, Brown Gora, IR87756-20-2-2-3, IC282418, IC356419, PKSLGR-1 and PKSLGR-39 had seven blast resistance genes. Twenty accessions possessed six genes, 36 accessions had five genes, 41 accessions had four genes, 38 accessions had three genes, 26 accessions had two genes, 13 accessions had single R gene and only one accession IC438644 does not possess any one blast resistant gene. Out of 192 accessions only 17 accessions harboured 7 to 8 blast resistance genes. None Stress-induced Oryza sativa BAT1 dual helicase exhibits unique bipolar translocation. 2015 Protoplasma International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India, narendra@icgeb.res.in. HLA-B associated transcript 1 (BAT1) protein, also named as spliceosome RNA helicase UAP56, is a member of the DExD/H-box family of helicases. However, regulation under stress, biochemical properties, and functions of plant homologue of BAT1 are poorly understood. Here, we report the purification and detailed biochemical characterization of the Oryza sativa homologue of BAT1 (OsBAT1/UAP56) protein (52 kDa) and regulation of its transcript under abiotic stress. OsBAT1 transcript levels are enhanced in rice seedlings in response to abiotic stress including salt stress and abscisic acid. Purified OsBAT1 protein exhibits the DNA- and RNA-dependent ATPase, RNA helicase, and DNA- and RNA-binding activities. Interestingly OsBAT1 also exhibits unique DNA helicase activity, which has not been reported so far in any BAT1 homologue. Moreover, OsBAT1 translocates in both the 3' to 5' and 5' to 3' directions, which is also a unique property. The K m value for OsBAT1 DNA helicase is 0.9753 nM and for RNA helicase is 1.7536 nM, respectively. This study demonstrates several unique characteristics of OsBAT1 especially its ability to unwind both DNA and RNA duplexes; bipolar translocation and its transcript upregulation under abiotic stresses indicate that it is a multifunctional protein. Overall, this study represents significant contribution in advancing our knowledge regarding functions of OsBAT1 in RNA and DNA metabolism and its putative role in abiotic stress signaling in plants. OsAIP1|OsBAT1 miRNA plays a role in the antagonistic effect of selenium on arsenic stress in rice seedlings. 2015 Metallomics Department of Biotechnology, Jamia Millia Islamia, Jamia Nagar, New Delhi, 110025, India. meetu_gpt@yahoo.com. MicroRNAs (miRNAs), the small non-coding RNAs, have been implicated in various biological processes including adaptation during environmental stress. The present work explores the involvement of miRNA during arsenic (As) and selenium (Se) treatment in rice seedlings. Arsenic is a heavy metalloid causing severe adverse effects on the growth and development of plants while Se is another metalloid and an essential micro-nutrient when present in appropriate amounts. It was observed that the presence of Se along with As mitigated the adverse effects of As on seedling germination, root-shoot growth, total chlorophyll and protein contents. The measurement of stress indicators such as proline, cysteine and MDA also indicated similar effects. Analysis of the miRNA profile using microarrays under As, Se and As + Se treatments showed differential regulation of at least 46 miRNAs in rice seedlings compared to untreated control. 18 of these miRNAs showed differential regulation among different treatments. Furthermore the microarray data were validated using real time PCR. The target genes of a few of these miRNAs showed inverse transcript accumulation. The possible role of miR395 and miR398 in the antagonistic effect on the adverse response of As in the presence of Se in rice seedlings is discussed. None Brassinosteroids are involved in Fe homeostasis in rice (Oryza sativa L.). 2015 J Exp Bot State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, P. R. China. Brassinosteroids (BRs) are steroid hormones that modulate numerous physiological processes in plants. However, few studies have focused on the involvement of BRs in sensing and responding to the stress of mineral nutrient deficiency. In the present study, we evaluated the roles of BRs in the response of rice (Oryza sativa) to iron (Fe) deficiency during Fe uptake, transport, and translocation. Exogenous application of 24-epibrassinolide (EBR) to wild-type (WT) plants exaggerated leaf symptoms of Fe deficiency and suppressed growth. EBR increased and decreased Fe concentrations in roots and shoots, respectively, under both Fe-deficient and Fe-sufficient conditions. Transcripts involved in Fe homeostasis, including OsIRT1, OsYSL15, OsYSL2, OsNAS1, and OsNAS2, were enhanced by EBR under Fe-deficient conditions. EBR depressed expression of OsNAS1, OsNAS2, and OsYSL2 in shoots, and inhibited Fe transport and translocation via the phloem. Rice mutant d2-1, which is defective in BR biosynthesis, was more tolerant to Fe deficiency than the WT, and accumulated greater amounts of Fe in roots than the WT under Fe-sufficient conditions. A greater upregulation of OsIRT1, OsYSL15, OsYSL2, OsNAS1, and OsNAS2 in the d2-1 mutant compared to the WT was found under Fe-sufficient conditions, while expression of these genes in the d2-1 mutant was lower than in the WT under Fe-deficient conditions. The greater tolerance of the d2-1 mutant could be partly mitigated by exogenous application of EBR. These novel findings highlight the important role of BR in mediating the response of strategy II plants to Fe deficiency by regulating Fe uptake and translocation in rice. None A Mutation in GIANT CHLOROPLAST Encoding a PARC6 Homologue Affects Spikelet Fertility in Rice. 2015 Plant Cell Physiol Institute of Plant Science and Resources (IPSR), Okayama University, Kurashiki, Okayama 710-0046 Japan. Chloroplasts are not generated de novo but proliferate from a preexisting population of plastids present in meristematic cells. Chloroplast division is executed by the coordinated action of at least two molecular machineries: internal machinery located on the stromal side of the inner envelope membrane and external machinery located on the cytosolic side of the outer envelope membrane. To date, molecular studies of chloroplast division in higher plants have been limited to several species such as Arabidopsis. To elucidate chloroplast division in rice, we performed forward genetics and isolated a mutant displaying large chloroplasts among an EMS mutagenized Oryza sativa spp japonica Nipponbare population. Using a map-based approach, this mutation, termed giant chloroplast (gic), was allocated in a gene that encodes a protein that is homologous to Paralog of ARC6 (PARC6), which is known to play a role in chloroplast division. GIC is unique in that it has a long C-terminal extension that is not present in other PARC6 homologues. Characterization of gic phenotypes in a rice field showed that gic exhibited defective growth in seed setting, suggesting that the gic mutant negatively affects the reproductive stage. This report is the first describing chloroplast division mutant in monocotyledons and its effect on plant development. GIC Knock-down of stress inducible OsSRFP1 encoding an E3 ubiquitin ligase with transcriptional activation activity confers abiotic stress tolerance through enhancing antioxidant protection in rice. 2015 Plant Mol Biol State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, 210095, China. E3 ubiquitin ligases are involved in a variety of physiological processes. This study demonstrated the function of a previously unknown rice RING finger E3 ligase, Oryza sativa Stress-related RING Finger Protein 1 (OsSRFP1) in stress responses in rice. OsSRFP1 was ubiquitously expressed in various rice organs, with the higher expression levels in roots, panicles and culm nodes. The transcript of OsSRFP1 was induced by cold, dehydration, salt, H2O2 and abscisic acid treatments. Interestingly, the OsSRFP1-overexpressing plants were less tolerant to salt, cold and oxidative stresses than wild type plants; while the RNA interference silencing of OsSRFP1 plants were more tolerant than wild type without yield penalty. Compared with the wild type, amounts of free proline and activities of antioxidant enzymes were increased in the RNAi plants but decreased in the overexpression plants under cold stress, which were inversely correlated with the malondialdehyde and hydrogen peroxide (H2O2) levels in the tested lines. Microarray analysis showed that expression of numerous genes involving in ROS homeostasis was altered in the OsSRFP1-overexpressing plants under normal and cold conditions. In vitro ubiquitination assays showed that OsSRFP1 possessed E3 ubiquitin ligase activity and the intact RING domain was essential for the activity. Moreover, OsSRFP1 might function in transcriptional regulation with nuclear localization. Taken together, our results demonstrate that OsSRFP1 may have dual functions in post-translational and transcriptional regulations in modulating abiotic stress responses in rice, at least in part, by negatively regulating antioxidant enzymes-mediated reactive oxygen species removal. OsSRFP1 Designing climate-resilient rice with ideal grain quality suited for high-temperature stress. 2015 J Exp Bot Grain Quality and Nutrition Center, International Rice Research Institute, DAPO Box 7777, Metro Manila 1301, Philippines n.sreenivasulu@irri.org. To ensure rice food security, the target outputs of future rice breeding programmes should focus on developing climate-resilient rice varieties with emphasis on increased head rice yield coupled with superior grain quality. This challenge is made greater by a world that is increasingly becoming warmer. Such environmental changes dramatically impact head rice and milling yield as well as increasing chalkiness because of impairment in starch accumulation and other storage biosynthetic pathways in the grain. This review highlights the knowledge gained through gene discovery via quantitative trait locus (QTL) cloning and structural-functional genomic strategies to reduce chalk, increase head rice yield, and develop stable lines with optimum grain quality in challenging environments. The newly discovered genes and the knowledge gained on the influence of specific alleles related to stability of grain quality attributes provide a robust platform for marker-assisted selection in breeding to design heat-tolerant rice varieties with superior grain quality. Using the chalkiness trait in rice as a case study, we demonstrate here that the emerging field of systems genetics can help fast-track the identification of novel alleles and gene targets that can be pyramided for the development of environmentally robust rice varieties that possess improved grain quality. None Priming effect of abscisic acid on alkaline stress tolerance in rice (Oryza sativa L.) seedlings. 2015 Plant Physiol Biochem Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun, Jilin, 130102, China; University of Chinese Academy of Sciences, Beijing, 100049, China. Saline-alkaline stress is characterized by high salinity and high alkalinity (high pH); alkaline stress has been shown to be the primary factor inhibiting rice seedling growth. In this study, we investigated the potential priming effect of abscisic acid (ABA) on tolerance of rice seedlings to alkaline stress simulated by Na2CO3. Seedlings were pretreated with ABA at concentrations of 0 (control), 10, and 50 μM by root-drench for 24 h and then transferred to a Na2CO3 solution that did not contain ABA. Compared to control treatment, pretreatment with ABA substantially improved the survival rate of rice seedlings and increased biomass accumulation after 7 days under the alkaline condition. ABA application at 10 μM also alleviated the inhibitory effects of alkaline stress on the total root length and root surface area. Physiologically, ABA increased relative water content (RWC) and decreased cell membrane injury degree (MI) and Na(+)/K(+) ratios. In contrast, fluridone (an ABA biosynthesis inhibitor) decreased the RWC and increased MI in shoots under the alkaline conditions. These data suggest that ABA has a potent priming effect on the adaptive response to alkaline stress in rice and may be useful for improving rice growth in saline-alkaline paddy fields. None Genome-wide analysis of DNA methylation in photoperiod- and thermo-sensitive male sterile rice Peiai 64S. 2015 BMC Genomics State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, 430072, People's Republic of China, hujh2010@163.com. Epigenetic modifications play important roles in the regulation of plant development. DNA methylation is an important epigenetic modification that dynamically regulates gene expression during developmental processes. However, little studies have been reported about the methylation profiles of photoperiod- and thermo-sensitive genic male sterile (PTGMS) rice during the fertility transition.In this study, using methylated DNA immunoprecipitation sequencing (MeDIP-seq), the global DNA methylation patterns were compared in the rice PTGMS line PA64S under two different environments (different temperatures and day lengths). The profiling of the DNA methylation under two different phenotypes (sterility and fertility) revealed that hypermethylation was observed in PA64S (sterility), and 1258 differentially methylated regions (DMRs) were found between PA64S (sterility) and PA64S (fertility). Twenty differentially methylated genes of them were further validated through bisulfite sequencing, and four of these genes were analyzed by qRT-PCR. Especially, a differentially methylated gene (LOC_Os08g38210), which encoded transcription factor BIM2, is a component of brassinosteroid signaling in rice. The hypermethylated BIM2 gene may suppress some downstream genes in brassinosteroid signaling pathway, and thus affect the male fertility in PA64S.The results presented here indicated that hypermethylation was observed in PA64S (sterility). Gene Ontology (GO) analysis and KEGG analysis revealed that flavone and flavonol biosynthrsis, circadian rhythm, photosynthesis and oxidative phosphorylation pathways were involved in sterility-fertility transition of PA64S. None Transcriptome changes in rice (Oryza sativa L.) in response to high night temperature stress at the early milky stage. 2015 BMC Genomics Key Laboratory of Crop Physiology, Ecology and Genetic Breeding (Jiangxi Agricultural University), Ministry of Education, Jiangxi Province, 330045, China, jlliao514815@163.com. Rice yield and quality are adversely affected by high temperatures, especially at night; high nighttime temperatures are more harmful to grain weight than high daytime temperatures. Unfortunately, global temperatures are consistently increasing at an alarming rate and the minimum nighttime temperature has increased three times as much as the corresponding maximum daytime temperature over the past few decades.We analyzed the transcriptome profiles for rice grain from heat-tolerant and -sensitive lines in response to high night temperatures at the early milky stage using the Illumina Sequencing method. The analysis results for the sequencing data indicated that 35 transcripts showed different expressions between heat-tolerant and -sensitive rice, and RT-qPCR analyses confirmed the expression patterns of selected transcripts. Functional analysis of the differentially expressed transcripts indicated that 21 genes have functional annotation and their functions are mainly involved in oxidation-reduction (6 genes), metabolic (7 genes), transport (4 genes), transcript regulation (2 genes), defense response (1 gene) and photosynthetic (1 gene) processes. Based on the functional annotation of the differentially expressed genes, the possible process that regulates these differentially expressed transcripts in rice grain responding to high night temperature stress at the early milky stage was further analyzed. This analysis indicated that high night temperature stress disrupts electron transport in the mitochondria, which leads to changes in the concentration of hydrogen ions in the mitochondrial and cellular matrix and influences the activity of enzymes involved in TCA and its secondary metabolism in plant cells.Using Illumina sequencing technology, the differences between the transcriptomes of heat-tolerant and -sensitive rice lines in response to high night temperature stress at the early milky stage was described here for the first time. The candidate transcripts may provide genetic resources that may be useful in the improvement of heat-tolerant characters of rice. The model proposed here is based on differences in expression and transcription between two rice lines. In addition, the model may support future studies on the molecular mechanisms underlying plant responses to high night temperatures. None Epigenetic regulation of rice flowering and reproduction. 2015 Front Plant Sci State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, International Associated Laboratory of CNRS-Fudan-HUNAU on Plant Epigenome Research, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University Shanghai, China ; CNRS, Institut de Biologie Moléculaire des Plantes, Université de Strasbourg Strasbourg, France. Current understanding of the epigenetic regulator roles in plant growth and development has largely derived from studies in the dicotyledonous model plant Arabidopsis thaliana. Rice (Oryza sativa) is one of the most important food crops in the world and has more recently becoming a monocotyledonous model plant in functional genomics research. During the past few years, an increasing number of studies have reported the impact of DNA methylation, non-coding RNAs and histone modifications on transcription regulation, flowering time control, and reproduction in rice. Here, we review these studies to provide an updated complete view about chromatin modifiers characterized in rice and in particular on their roles in epigenetic regulation of flowering time, reproduction, and seed development. None In Silico Simulation Modeling Reveals the Importance of the Casparian Strip for Efficient Silicon Uptake in Rice Roots. 2015 Plant Cell Physiol Ecosystem Informatics Division, National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba, 305-8604 Japan sakuraigen@affrc.go.jp. Silicon (Si) uptake by the roots is mediated by two different transporters, Lsi1 (passive) and Lsi2 (active), in rice (Oryza sativa). Both transporters are polarly localized in the plasma membranes of exodermal (outer) and endodermal (inner) cells with Casparian strips. However, it is unknown how rice is able to take up large amounts of Si compared with other plants, and why rice Si transporters have a characteristic cellular localization pattern. To answer these questions, we simulated Si uptake by rice roots by developing a mathematical model based on a simple diffusion equation that also accounts for active transport by Lsi2. In this model, we calibrated the model parameters using in vivo experimental data on the Si concentrations in the xylem sap and a Monte Carlo method. In our simulation experiments, we compared the Si uptake between roots with various transporter and Casparian strip locations and estimated the Si transport efficiency of roots with different localization patterns and quantities of the Lsi transporters. We found that the Si uptake by roots that lacked Casparian strips was lower than that of normal roots. This suggests that the double-layer structure of the Casparian strips is an important factor in the high Si uptake by rice. We also found that among various possible localization patterns, the most efficient one was that of the wild-type rice; this may explain the high Si uptake capacity of rice. None FT-like proteins induce transposon silencing in the shoot apex during floral induction in rice. 2015 Proc Natl Acad Sci U S A Plant Global Education Project and Laboratory of Plant Molecular Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan; and. Floral induction is a crucial developmental step in higher plants. Florigen, a mobile floral activator that is synthesized in the leaf and transported to the shoot apex, was recently identified as a protein encoded by FLOWERING LOCUS T (FT) and its orthologs; the rice florigen is Heading date 3a (Hd3a) protein. The 14-3-3 proteins mediate the interaction of Hd3a with the transcription factor OsFD1 to form a ternary structure called the florigen activation complex on the promoter of OsMADS15, a rice APETALA1 ortholog. However, crucial information, including the spatiotemporal overlap among FT-like proteins and the components of florigen activation complex and downstream genes, remains unclear. Here, we confirm that Hd3a coexists, in the same regions of the rice shoot apex, with the other components of the florigen activation complex and its transcriptional targets. Unexpectedly, however, RNA-sequencing analysis of shoot apex from wild-type and RNA-interference plants depleted of florigen activity revealed that 4,379 transposable elements (TEs; 58% of all classifiable rice TEs) were expressed collectively in the vegetative and reproductive shoot apex. Furthermore, in the reproductive shoot apex, 214 TEs were silenced by florigen. Our results suggest a link between floral induction and regulation of TEs. Hd3a A T-DNA insertion mutant Osmtd1 aas altered in architecture by upregulating MicroRNA156f in rice. 2015 J Integr Plant Biol Hunan Provincial Key Laboratory of Phytohormones and Growth Development, Hunan Provincial Key Laboratory for Crop Germplasm Innovation and Utilization, Hunan Agricultural University, Changsha, 410128, China. The plant architecture is an important factor for crop production. Some members of microRNA156 (miR156) and their target genes SQUAMOSA Promoter-Binding Protein-Like (SPL) were identified to play essential roles in the establishment of plant architecture. However, the roles and regulation of miR156 is not well understood yet. Here, we identified a T-DNA insertion mutant Osmtd1 (Oryza sativa multi-tillering and dwarf mutant). Osmtd1 produced more tillers and displayed short stature phenotype. We determined that the dramatic morphological changes were caused by a single T-DNA insertion in Osmtd1. Further analysis revealed that the T-DNA insertion was located in the gene Os08g34258 encoding a putative inhibitor I family protein. Os08g34258 was knocked out and OsmiR156f was significantly upregulated in Osmtd1. Overexpression of Os08g34258 in Osmtd1 complemented the defects of the mutant architecture, while overexpression of OsmiR156f in wild-type rice phenocopied Osmtd1. We showed that the expression of OsSPL3, OsSPL12 and OsSPL14 was significantly down-regulated in Osmtd1 or OsmiR156f overexpressed lines, indicating that OsSPL3, OsSPL12 and OsSPL14 were possibly direct target genes of OsmiR156f. Our results suggested that OsmiR156f controlled plant architecture by mediating plant stature and tiller outgrowth and might be regulated by an unknown protease inhibitor I family protein. Osmtd1 Selenium and auxin mitigates arsenic stress in rice (Oryza sativa L.) by combining the role of stress indicators, modulators and genotoxicity assay. 2015 J Hazard Mater Ecotoxicogenomics Lab Department of Biotechnology, Jamia Millia Islamia, New Delhi-25, India. Arsenic (As) is known to disrupt the biological function in plants by inhibiting their growth and developmental process, while selenium (Se) is an essential micronutrient within the appropriate amount. Phytohormone auxin on the other hand is an established growth regulator and plays a significant role in stress management. Present study is designed to see the effect of Se and auxin on morphological and biochemical characteristics and, on the genotoxicity in rice plants under As stress. The observations indicated that seedlings supplemented only with As showed inhibition in the growth parameters, however, co-application of Se and auxin improved growth of rice seedlings, level of stress indicators, (chlorophyll, protein, MDA content) and modulators (cysteine, proline) as compared the individual treatment of As. Genomic template stability calculated through changes in RAPD profile showed consistent results when compared with the indicator and modulator parameters. Altered DNA profile showed varying degrees of polymorphism, highest in roots of As treated seedlings and lowest in roots of Se+auxin and As+Se treated seedlings. Altogether, this study conclude that application of Se and auxin alone or in combination were more effective in lowering the As induced stress in rice. None The role of thionins in rice defence against root pathogens. 2015 Mol Plant Pathol Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000, Ghent, Belgium; Institute of Plant Protection, Sichuan Academy of Agricultural Sciences, Jingjusi road 20, 610066, Chengdu, China. Thionins are antimicrobial peptides that are involved in plant defence. Here, we present an in-depth analysis of the role of rice thionin genes in defence responses against two root pathogens: the root-knot nematode Meloidogyne graminicola and the oomycete Pythium graminicola. The expression of rice thionin genes was observed to be differently regulated by defence-related hormones, while all analyzed genes were consistently down-regulated in Meloidogyne graminicola-induced galls, at least until 7 days post inoculation (dpi). Transgenic lines of Oryza sativa cv. Nipponbare overproducing OsTHI7 reveal decreased susceptibility to M. graminicola infection and Pythium graminicola colonization. Taken together, these results demonstrate the role of rice thionin genes in rice defence against two of the most damaging root pathogens attacking rice. None Physiological mechanisms contributing to the QTL-combination effects on improved performance of IR64 rice NILs under drought. 2015 J Exp Bot International Rice Research Institute, Los Baños, Laguna 4031, Philippines a.henry@irri.org. Characterizing the physiological mechanisms behind major-effect drought-yield quantitative trait loci (QTLs) can provide an understanding of the function of the QTLs - as well as plant responses to drought in general. In this study, we characterized rice (Oryza sativa L.) genotypes with QTLs derived from drought-tolerant traditional variety AdaySel that were introgressed into drought-susceptible high-yielding variety IR64, one of the most popular megavarieties in South Asian rainfed lowland systems. Of the different combinations of the four QTLs evaluated, genotypes with two QTLs (qDTY 2.2 + qDTY 4.1 ) showed the greatest degree of improvement under drought compared with IR64 in terms of yield, canopy temperature, and normalized difference vegetation index (NDVI). Furthermore, qDTY 2.2 and qDTY 4.1 showed a potential for complementarity in that they were each most effective under different severities of drought stress. Multiple drought-response mechanisms were observed to be conferred in the genotypes with the two-QTL combination: higher root hydraulic conductivity and in some cases greater root growth at depth. As evidenced by multiple leaf water status and plant growth indicators, these traits affected transpiration but not transpiration efficiency or harvest index. The results from this study highlight the complex interactions among major-effect drought-yield QTLs and the drought-response traits they confer, and the need to evaluate the optimal combinations of QTLs that complement each other when present in a common genetic background. None A gene expression profiling of early rice stamen development that reveals inhibition of photosynthetic genesby OsMADS58. 2015 Mol Plant State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, China. Stamen is a unique plant organ where germ cells or microsporocytes that commit to meiosis are initiated from somatic cells during its early developmental process. While genes determining stamen identity is known according to ABC model of floral development, little information is available about how these genes affect germ cell initiation.By using Affymetrix "GeneChip® Rice Genome Array" assessing 51279 transcripts, we established a dynamic gene expression profile (GEP) of early stamen developmental process of rice (Oryza sativa). Systematic analysis of the GEP data not only demonstrated the quality of the GEP, but also revealed new expression patterns of developmentally important genes including meiosis-, tapetum-, and phytohormone-related. Following the finding of systematic low expression of nuclear genes encoding photosynthetic proteins in early rice stamen, we found that a C-class MADS box protein OsMADS58 can bind many nuclear coded photosystem and other light reaction genes based on ChIP-seq analysis. When the expression level of OsMADS58 is downregulated, e.g. in osmads58 mutant, the expression levels of OsMADS58 target genes increase, more prochloroplasts are observed, and more ROS signal is detected in the mutant anthers. These findings revealed a novel link between stamenidentity determination and hypoxia status establishment. OSMADS58 A new finely mapped Oryza australiensis-derived QTL in rice confers resistance to brown planthopper. 2015 Gene National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, PR China. Brown planthopper (BPH) is the most destructive pest of rice in Asia. The BPH resistance in the introgression line IR65482-17-511-5-7 (IR65482-17) is derived from the wild rice species Oryza australiensis. An F2:3 population from a cross between Zhenshan 97 (ZS97) and IR65482-17 was used to map three quantitative trait loci (QTLs) for seedling resistance and feeding rate to BPH. The loci were distributed on chromosomes 2, 4 and 12. The QTL qBph4.2 on chromosome 4 had the largest effect, and contributed 36-44% of the phenotypic variance with a LOD score of 19-29. To validate the effect of qBph4.2, two near-isogenic lines (NILs) containing the qBph4.2 locus in the backgrounds of ZS97 and 9311 were developed by marker-assisted backcrossing (MABC). BPH bioassays showed that lines homozygous for the IR65482-17 allele (NIL+) of qBph4.2 tented to have significantly higher seedling resistance to BPH than those homozygous for the ZS97 or 9311 alleles (NIL-). Resistance was associated with a lower feeding rate by the insect. qBph4.2 was delimited to a ~300kb (0.04cM) region flanked by markers RM261 and S1, and co-segregating with XC4-27. This study will facilitate map-based cloning and marker-assisted selection of the gene, and permits further studies of gene function and resistance mechanisms in rice: BPH interaction. None Genomic selection and association mapping in rice (Oryza sativa): effect of trait genetic architecture, training population composition, marker number and statistical model on accuracy of rice genomic selection in elite, tropical rice breeding lines. 2015 PLoS Genet Department of Plant Breeding and Genetics, Cornell University, Ithaca, New York, United States of America. Genomic Selection (GS) is a new breeding method in which genome-wide markers are used to predict the breeding value of individuals in a breeding population. GS has been shown to improve breeding efficiency in dairy cattle and several crop plant species, and here we evaluate for the first time its efficacy for breeding inbred lines of rice. We performed a genome-wide association study (GWAS) in conjunction with five-fold GS cross-validation on a population of 363 elite breeding lines from the International Rice Research Institute's (IRRI) irrigated rice breeding program and herein report the GS results. The population was genotyped with 73,147 markers using genotyping-by-sequencing. The training population, statistical method used to build the GS model, number of markers, and trait were varied to determine their effect on prediction accuracy. For all three traits, genomic prediction models outperformed prediction based on pedigree records alone. Prediction accuracies ranged from 0.31 and 0.34 for grain yield and plant height to 0.63 for flowering time. Analyses using subsets of the full marker set suggest that using one marker every 0.2 cM is sufficient for genomic selection in this collection of rice breeding materials. RR-BLUP was the best performing statistical method for grain yield where no large effect QTL were detected by GWAS, while for flowering time, where a single very large effect QTL was detected, the non-GS multiple linear regression method outperformed GS models. For plant height, in which four mid-sized QTL were identified by GWAS, random forest produced the most consistently accurate GS models. Our results suggest that GS, informed by GWAS interpretations of genetic architecture and population structure, could become an effective tool for increasing the efficiency of rice breeding as the costs of genotyping continue to decline. None Homotypic clustering of OsMYB4 binding site motifs in promoters of the rice genome and cellular-level implications on sheath blight disease resistance. 2015 Gene School of Bio Sciences and Technology, VIT University, Vellore 632014, India. The promoter regions (1kb upstream sequences) of 45,836 annotated genes of rice were analyzed for the presence of OsMYB4 binding sites using a Perl program algorithm. Based on the homotypic clustering concept, 113 promoters were found to have more than 4 binding site motifs. Among the downstream genes of these promoters, five genes which are known to have a role in disease resistance were selected and the binding capacity of OsMYB4 protein in the promoter regions was analyzed by docking studies. Expression level of these genes was analyzed by RT-PCR in Rhizoctonia solani infected rice seedlings. Upon pathogen challenge, higher expression of aminotransferase, ankyrin and WRKY 12 genes was observed corresponding to higher expression of Osmyb4. Over-expression of Osmyb4 cDNA in rice leaf tissues by agro-infection failed to result in similar over-expression of aminotransferase, ankyrin and WRKY 12 as expected. Although the role of OsMYB4 in sheath blight resistance was found to be definitive based on our initial results, artificial over-expression of this TF was observed to be insufficient in regulating the disease resistance related genes. Osmyb4 Viral-inducible Argonaute18 confers broad-spectrum virus resistance in rice by sequestering a host microRNA. 2015 Elife State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China. Viral pathogens are a major threat to rice production worldwide. Although RNA interference (RNAi) is known to mediate antiviral immunity in plant and animal models, the mechanism of antiviral RNAi in rice and other economically important crops is poorly understood. Here, we report that rice resistance to evolutionarily diverse viruses requires Argonaute18 (AGO18). Genetic studies reveal that the antiviral function of AGO18 depends on its activity to sequester microRNA168 (miR168) to alleviate repression of rice AGO1 essential for antiviral RNAi. Expression of miR168-resistant AGO1a in ago18 background rescues or increases rice antiviral activity. Notably, stable transgenic expression of AGO18 confers broad-spectrum virus resistance in rice. Our findings uncover a novel cooperative antiviral activity of two distinct AGO proteins and suggest a new strategy for the control of viral diseases in rice. AGO18 Fine mapping of a major quantitative trait locus, qLG-9, that controls seed longevity in rice (Oryza sativa L.). 2015 Theor Appl Genet Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, Miyagi, 980-8577, Japan, sasaki@isas.a.u-tokyo.ac.jp. We fine-mapped a quantitative trait locus, qLG - 9, for seed longevity detected between Japonica-type and Indica-type cultivars. qLG - 9 was mapped in a 30-kb interval of the Nipponbare genome sequence. A quantitative trait locus, qLG-9, for seed longevity in rice has previously been detected on chromosome 9 by using backcross inbred lines derived from a cross between Japonica-type (Nipponbare) and Indica-type (Kasalath) cultivars. In the present study, the chromosomal location of qLG-9 was precisely determined by fine-scale mapping. Firstly, allelic difference in qLG-9 was verified by QTL analysis of an F2 population derived from a cross between Nipponbare and NKSL-1, in which a segment of Kasalath chromosome 9 was substituted in Nipponbare genetic background. Then, we selected F2 plants in which recombination had occurred near qLG-9 and performed F3 progeny testing on these plants to determine the genotype classes of qLG-9. Eventually, qLG-9 was mapped in a 30-kb interval (defined by two markers, CAPSb and CHPa12) of the Nipponbare genome sequence. This allowed us to nominate positional candidate genes of qLG-9. Additionally, we developed near-isogenic lines (NIL) for qLG-9 by marker-assisted selection. qLG-9 NIL showed significantly higher seed longevity than isogenic control of Nipponbare. These results will facilitate cloning of the gene(s) underlying qLG-9 as well as marker-assisted transfer of desirable genes for seed longevity improvement in rice. None OsSDS is essential for DSB formation in rice meiosis. 2015 Front Plant Sci Ministry of Education Key Laboratory of Agriculture Biodiversity for Plant Disease Management, Yunnan Agricultural University Kunming, China. SDS is a meiosis specific cyclin-like protein and required for DMC1 mediated double-strand break (DSB) repairing in Arabidopsis. Here, we found its rice homolog, OsSDS, is essential for meiotic DSB formation. The Ossds mutant is normal in vegetative growth but both male and female gametes are inviable. The Ossds meiocytes exhibit severe defects in homologous pairing and synapsis. No γH2AX immunosignals in Ossds meiocytes together with the suppression of chromosome fragmentation in Ossds-1 Osrad51c, both provide strong evidences that OsSDS is essential for meiotic DSB formation. Immunostaining investigations revealed that meiotic chromosome axes are normally formed but both SC installation and localization of recombination elements are failed in Ossds. We suspected that this cyclin protein has been differentiated pretty much between monocots and dicots on its function in meiosis. SDS|OsSDS Comprehensive Analysis Suggests Overlapping Expression of Rice ONAC Transcription Factors in Abiotic and Biotic Stress Responses. 2015 Int J Mol Sci National Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang 310029, China. slj.1226@163.com. NAC (NAM/ATAF/CUC) transcription factors comprise a large plant-specific gene family that contains more than 149 members in rice. Extensive studies have revealed that NAC transcription factors not only play important roles in plant growth and development, but also have functions in regulation of responses to biotic and abiotic stresses. However, biological functions for most of the members in the NAC family remain unknown. In this study, microarray data analyses revealed that a total of 63 ONAC genes exhibited overlapping expression patterns in rice under various abiotic (salt, drought, and cold) and biotic (infection by fungal, bacterial, viral pathogens, and parasitic plants) stresses. Thirty-eight ONAC genes exhibited overlapping expression in response to any two abiotic stresses, among which 16 of 30 selected ONAC genes were upregulated in response to exogenous ABA. Sixty-five ONAC genes showed overlapping expression patterns in response to any two biotic stresses. Results from the present study suggested that members of the ONAC genes with overlapping expression pattern may have pleiotropic biological functions in regulation of defense response against different abiotic and biotic stresses, which provide clues for further functional analysis of the ONAC genes in stress tolerance and pathogen resistance. None Natural allelic diversity in OsDREB1F gene in the Indian wild rice germplasm led to ascertain its association with drought tolerance. 2015 Plant Cell Rep National Research Center on Plant Biotechnology, IARI, New Delhi, 110012, India. Three coding SNPs and one haplotype identified in the OsDREB1F gene have potential to be associated with drought tolerance in rice. Drought is a serious constraint to rice production worldwide, that can be addressed by deployment of drought tolerant genes. OsDREB1F, one of the most potent drought tolerance transcription activator genes, was re-sequenced for allele mining and association study in a set of 136 wild rice accessions and four cultivated rice. This analysis led to identify 22 SNPs with eight haplotypes based on allelic variations in the accessions used. The nucleotide variation-based neutrality tests suggested that the OsDREB1F gene has been subjected to purifying selection in the studied set of rice germplasm. Six different OsDREB1F protein variants were identified on the basis of translated amino acid residues amongst the orthologues. Five protein variants were truncated due to deletions in coding region and found susceptible to drought stress. Association study revealed that three coding SNPs of this gene were significantly associated with drought tolerance. One OsDREB1F variant in the activation domain of OsDREB1F gene which led to conversion of aspartate amino acid to glutamate was found to be associated with drought tolerance. Three-dimensional homology modeling assisted to understand the functional significance of this identified potential allele for drought tolerance in rice. The natural allelic variants mined in the OsDREB1F gene can be further used in translational genomics for improving the water use efficiency in rice. OsDREB1F|RCBF2 Deciphering and prediction of plant dynamics under field conditions. 2015 Curr Opin Plant Biol Functional Plant Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan. Electronic address: tizawa@nias.affrc.go.jp. Elucidation of plant dynamics under fluctuating natural environments is a challenging goal in plant physiology. Recently, using a computer statistics integrating a series of transcriptome data of field-grown rice leaves during an entire crop season and several corresponding environmental data such as solar radiation and ambient temperature, most parts of transcriptome have been modeled. This reveals the detailed contributions of developmental timing, circadian clocks and each environmental factor to transcriptome dynamics in the field and can predict transcriptome dynamics under given environments. Furthermore, some traits such as flowering time in natural environments have been shown to be predicted by mathematical models based on gene-networks parameterized with data obtained in the laboratory, and phenology models refined by knowledge of molecular genetics. New molecular physiology is beginning in plant science. None Comparative Performance of Hybrid and Elite Inbred Rice Varieties with respect to Their Source-Sink Relationship. 2015 ScientificWorldJournal Department of Agricultural Botany, Sher-e-Bangla Agricultural University, Dhaka 1207, Bangladesh. Hybrid rice varieties have higher yield potential over inbred varieties. This improvement is not always translated to the grain yield and its physiological causes are still unclear. In order to clarify it, two field experiments were conducted including two popular indica hybrids (BRRI hybrid dhan2 and Heera2) and one elite inbred (BRRI dhan45) rice varieties. Leaf area index, chlorophyll status, and photosynthetic rate of flag leaf, postheading crop growth rate, shoot reserve translocation, source-sink relation and yield, and its attributes of each variety were comprehensively analyzed. Both hybrid varieties outyielded the inbred. However, the hybrids and inbred varieties exhibited statistically identical yield in late planting. Both hybrids accumulated higher amount of biomass before heading and exhibited greater remobilization of assimilates to the grain in early plantings compared to the inbred variety. Filled grain (%) declined significantly at delayed planting in the hybrids compared to elite inbred due to increased temperature impaired-inefficient transport of assimilates. Flag leaf photosynthesis parameters were higher in the hybrid varieties than those of the inbred variety. Results suggest that greater remobilization of shoot reserves to the grain rendered higher yield of hybrid rice varieties. None Automated characterization of flowering dynamics in rice using field-acquired time-series RGB images. 2015 Plant Methods Institute for Sustainable Agro-ecosystem Services, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1. Midori-cho, Nishi-Tokyo, Tokyo 188-0002 Japan. Flowering (spikelet anthesis) is one of the most important phenotypic characteristics of paddy rice, and researchers expend efforts to observe flowering timing. Observing flowering is very time-consuming and labor-intensive, because it is still visually performed by humans. An image-based method that automatically detects the flowering of paddy rice is highly desirable. However, varying illumination, diversity of appearance of the flowering parts of the panicles, shape deformation, partial occlusion, and complex background make the development of such a method challenging.We developed a method for detecting flowering panicles of rice in RGB images using scale-invariant feature transform descriptors, bag of visual words, and a machine learning method, support vector machine. Applying the method to time-series images, we estimated the number of flowering panicles and the diurnal peak of flowering on each day. The method accurately detected the flowering parts of panicles during the flowering period and quantified the daily and diurnal flowering pattern.A powerful method for automatically detecting flowering panicles of paddy rice in time-series RGB images taken under natural field conditions is described. The method can automatically count flowering panicles. In application to time-series images, the proposed method can well quantify the daily amount and the diurnal changes of flowering during the flowering period and identify daily peaks of flowering. None Development and GBS-genotyping of introgression lines (ILs) using two wild species of rice, O. meridionalis and O. rufipogon, in a common recurrent parent, O. sativa cv. Curinga. 2015 Mol Breed Department of Plant Breeding and Genetics, Cornell University, 162 Emerson Hall, Ithaca, NY 14853-1901 USA. Two populations of interspecific introgression lines (ILs) in a common recurrent parent were developed for use in pre-breeding and QTL mapping. The ILs were derived from crosses between cv Curinga, a tropical japonica upland cultivar, and two different wild donors, Oryza meridionalis Ng. accession (W2112) and Oryza rufipogon Griff. accession (IRGC 105491). The lines were genotyped using genotyping-by-sequencing (GBS) and SSRs. The 32 Curinga/O. meridionalis ILs contain 76.73 % of the donor genome in individual introgressed segments, and each line has an average of 94.9 % recurrent parent genome. The 48 Curinga/O. rufipogon ILs collectively contain 97.6 % of the donor genome with an average of 89.9 % recurrent parent genome per line. To confirm that these populations were segregating for traits of interest, they were phenotyped for pericarp color in the greenhouse and for four agronomic traits-days to flowering, plant height, number of tillers, and number of panicles-in an upland field environment. Seeds from these IL libraries and the accompanying GBS datasets are publicly available and represent valuable genetic resources for exploring the genetics and breeding potential of rice wild relatives. None Involvement of OsPht1;4 in phosphate acquisition, and mobilization facilitates embryo development in rice. 2015 Plant J State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Ministry of Agriculture, Nanjing Agricultural University, 210095, China. Phosphate (Pi) transporters mediate acquisition, and transportation of Pi within plants. Here, we investigated the functions of OsPht1;4 (OsPT4), one of the thirteen members of the Pht1 family in rice. qRT-PCR analysis revealed strong expression of OsPT4 in roots and embryos, and OsPT4 promoter analysis using reporter genes confirmed these findings. Analysis using rice protoplasts showed that OsPT4 was localized to plasma membrane. OsPT4 complemented yeast mutant defective in Pi uptake, and also facilitated elevated accumulation of Pi in Xenopus oocytes. Further, OsPT4-genetically modified (GM) rice lines were generated by knockout/down, and overexpression of OsPT4. Pi concentrations in roots, and shoots were significantly lower, and higher in knockout/down, and overexpressing plants, respectively, compared to wild-type under different Pi regimes. (33) Pi uptake translocation assays corroborated altered acquisition, and mobilization of Pi in OsPT4-GM plants. The effects of altered expression levels of OsPT4 in GM plants were also evident on concentration of Pi, and size of embryo, and several attributes related to seed development. Overall, our results suggest that OsPT4 encodes a plasma membrane-localized Pi transporter facilitating acquisition, and mobilization of Pi, and also plays an important role in development of embryo in rice. This article is protected by copyright. All rights reserved. OsPht1;4|OsPT4 Differential regulation of genes by retrotransposons in rice promoters. 2015 Plant Mol Biol Department of Biological Sciences, Michigan Technological University, Houghton, MI, 49931, USA. Rice genome harbors genes and promoters with retrotransposon insertions. There is very little information about their function. The effect of retrotransposon insertions in four rice promoter regions on gene regulation, was investigated using promoter-reporter gene constructs with and without retrotransposons. Differences in expression levels of gus and egfp reporter genes in forward orientation and rfp in reverse orientation were evaluated in rice plants with transient expression employing quantitative RT-PCR analysis, histochemical GUS staining, and eGFP and RFP fluorescent microscopy. The presence of SINE in the promoter 1 (P1) resulted in higher expression levels of the reporter genes, whereas the presence of LINE in P2 or gypsy LTR retrotransposon in P3 reduced expression of the reporter genes. Furthermore, the SINE in P1 acts as an enhancer in contrast with the LINE in P2 and the gypsy LTR retrotransposon in P3 which act as silencers. CTAA and CGG motifs in these retrotransposons are the likely candidates for the downregulation compared to TCTT motif (SINE) which is a candidate for the upregulation of gene expression. The effect of retrotransposons on gene regulation correlated with the earlier investigation of conservation patterns of these four retrotransposon insertions in several rice accessions implying their evolutionary significance. None MONOCULM 3, an Ortholog of WUSCHEL in Rice, Is Required for Tiller Bud Formation. 2015 J Genet Genomics State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. WUSCHEL (WUS) plays an essential role for the maintenance of meristem activity in dicots, but its function is still elusive in monocots. We isolated a new monoculm mutant, monoculm 3 (moc3), in which a point mutation causes the premature termination of rice O. sativa WUS (OsWUS). Morphological observation revealed that the formation of tiller buds was disrupted in moc3. MOC3 was localized in the nuclear and could interact with TOPLESS-RELATED PROTEINS (TPRs). The expression of MOC3 was induced by cytokinins and defection of MOC3 affected the expression of several two-component cytokinin response regulators, OsRRs and ORRs. Our results suggest that MOC3 is required for the formation of axillary buds and has a complex relationship with cytokinins. MOC3 Overexpressing CYP71Z2 Enhances Resistance to Bacterial Blight by Suppressing Auxin Biosynthesis in Rice. 2015 PLoS One Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement/Jiangsu High Quality Rice R&D Center, Nanjing 210014, China. The hormone auxin plays an important role not only in the growth and development of rice, but also in its defense responses. We've previously shown that the P450 gene CYP71Z2 enhances disease resistance to pathogens through regulation of phytoalexin biosynthesis in rice, though it remains unclear if auxin is involved in this process or not.The expression of CYP71Z2 was induced by Xanthomonas oryzae pv. oryzae (Xoo) inoculation was analyzed by qRT-PCR, with GUS histochemical staining showing that CYP71Z2 expression was limited to roots, blades and nodes. Overexpression of CYP71Z2 in rice durably and stably increased resistance to Xoo, though no significant difference in disease resistance was detected between CYP71Z2-RNA interference (RNAi) rice and wild-type. Moreover, IAA concentration was determined using the HPLC/electrospray ionization/tandem mass spectrometry system. The accumulation of IAA was significantly reduced in CYP71Z2-overexpressing rice regardless of whether plants were inoculated or not, whereas it was unaffected in CYP71Z2-RNAi rice. Furthermore, the expression of genes related to IAA, expansin and SA/JA signaling pathways was suppressed in CYP71Z2-overexpressing rice with or without inoculation.These results suggest that CYP71Z2-mediated resistance to Xoo may be via suppression of IAA signaling in rice. Our studies also provide comprehensive insight into molecular mechanism of resistance to Xoo mediated by IAA in rice. Moreover, an available approach for understanding the P450 gene functions in interaction between rice and pathogens has been provided. Oscyp71Z2|CYP71Z2 Genome-Wide Association Mapping for Yield and Other Agronomic Traits in an Elite Breeding Population of Tropical Rice (Oryza sativa). 2015 PLoS One International Rice Research Institute, Los Baños, Philippines. Genome-wide association mapping studies (GWAS) are frequently used to detect QTL in diverse collections of crop germplasm, based on historic recombination events and linkage disequilibrium across the genome. Generally, diversity panels genotyped with high density SNP panels are utilized in order to assay a wide range of alleles and haplotypes and to monitor recombination breakpoints across the genome. By contrast, GWAS have not generally been performed in breeding populations. In this study we performed association mapping for 19 agronomic traits including yield and yield components in a breeding population of elite irrigated tropical rice breeding lines so that the results would be more directly applicable to breeding than those from a diversity panel. The population was genotyped with 71,710 SNPs using genotyping-by-sequencing (GBS), and GWAS performed with the explicit goal of expediting selection in the breeding program. Using this breeding panel we identified 52 QTL for 11 agronomic traits, including large effect QTLs for flowering time and grain length/grain width/grain-length-breadth ratio. We also identified haplotypes that can be used to select plants in our population for short stature (plant height), early flowering time, and high yield, and thus demonstrate the utility of association mapping in breeding populations for informing breeding decisions. We conclude by exploring how the newly identified significant SNPs and insights into the genetic architecture of these quantitative traits can be leveraged to build genomic-assisted selection models. None Nitric oxide generated by nitrate reductase increases nitrogen uptake capacity by inducing lateral root formation and inorganic nitrogen uptake under partial nitrate nutrition in rice. 2015 J Exp Bot State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China. Increasing evidence shows that partial nitrate nutrition (PNN) can be attributed to improved plant growth and nitrogen-use efficiency (NUE) in rice. Nitric oxide (NO) is a signalling molecule involved in many physiological processes during plant development and nitrogen (N) assimilation. It remains unclear whether molecular NO improves NUE through PNN. Two rice cultivars (cvs Nanguang and Elio), with high and low NUE, respectively, were used in the analysis of NO production, nitrate reductase (NR) activity, lateral root (LR) density, and (15)N uptake under PNN, with or without NO production donor and inhibitors. PNN increased NO accumulation in cv. Nanguang possibly through the NIA2-dependent NR pathway. PNN-mediated NO increases contributed to LR initiation, (15)NH4 (+)/(15)NO3 (-) influx into the root, and levels of ammonium and nitrate transporters in cv. Nanguang but not cv. Elio. Further results revealed marked and specific induction of LR initiation and (15)NH4 (+)/(15)NO3 (-) influx into the roots of plants supplied with NH4 (+)+sodium nitroprusside (SNP) relative to those supplied with NH4 (+) alone, and considerable inhibition upon the application of cPTIO or tungstate (NR inhibitor) in addition to PNN, which is in agreement with the change in NO fluorescence in the two rice cultivars. The findings suggest that NO generated by the NR pathway plays a pivotal role in improving the N acquisition capacity by increasing LR initiation and the inorganic N uptake rate, which may represent a strategy for rice plants to adapt to a fluctuating nitrate supply and increase NUE. None The OsSec18 complex interacts with P0(P1-P2)2 to regulate vacuolar morphology in rice endosperm cell 2015 BMC Plant Biol State Key Laboratory of Hybrid Rice and College of Life Sciences, Wuhan University, Luojia Hill, Wuhan, Hubei Province, 430072, China In the present study, we investigated the function of OsSec18 in rice and found that OsSec18 complements the temperature-sensitive phenotype and interferes with vacuolar morphogenesis in yeast. Overexpression of OsSec18 in rice decreased the plant height and 1000-grain weight and altered the morphology of the protein bodies. Further examination revealed that OsSec18 presented as a 290-kDa complex in rice endosperm cells. Moreover, Os60sP0 was identified a component of this complex, demonstrating that the OsSec18 complex contains another complex of P0(P1-P2)2 in rice endosperm cells. Furthermore, we determined that the N-terminus of OsSec18 can interact with the N- and C-termini of Os60sP0, whereas the C-terminus of OsSec18 can only interact with the C-terminus of Os60sP0. OsSec18 Identification of genomic regions and the isoamylase gene for reduced grain chalkiness in rice. 2015 PLoS One National Key Laboratory of Crop Genetic Improvement, Wuhan, China; College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China. Grain chalkiness is an important grain quality related to starch granules in the endosperm. A high percentage of grain chalkiness is a major problem because it diminishes grain quality in rice. Here, we report quantitative trait loci identification for grain chalkiness using high-throughput single nucleotide polymorphism genotyping of a chromosomal segment substitution line population in which each line carried one or a few introduced japonica cultivar Nipponbare segments in the genetic background of the indica cultivar ZS97. Ten quantitative trait loci regions were commonly identified for the percentage of grain chalkiness and the degree of endosperm chalkiness. The allelic effects at nine of these quantitative trait loci reduced grain chalkiness. Furthermore, a quantitative trait locus (qPGC8-2) on chromosome 8 was validated in a chromosomal segment substitution line-derived segregation population, and had a stable effect on chalkiness in a multiple-environment evaluation of the near-isogenic lines. Residing on the qPGC8-2 region, the isoamylase gene (ISA1) was preferentially expressed in the endosperm and revealed some nucleotide polymorphisms between two varieties, Nipponbare and ZS97. Transgenic lines with suppression of ISA1 by RNA interference produced grains with 20% more chalkiness than the control. The results support that the gene may underlie qPGC8-2 for grain chalkiness. The multiple-environment trials of the near-isogenic lines also show that combination of the favorable alleles such as the ISA1 gene for low chalkiness and the GS3 gene for long grains considerably improved grain quality of ZS97, which proves useful for grain quality improvement in rice breeding programs. None Genetic analysis of cold tolerance at the germination and booting stages in rice by association mapping. 2015 PLoS One Key Laboratory of Crop Heterosis and Utilization, Ministry of Education, Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing, 100193, China; Rice Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, Guangxi, 530005, China. Low temperature affects the rice plants at all stages of growth. It can cause severe seedling injury and male sterility resulting in severe yield losses. Using a mini core collection of 174 Chinese rice accessions and 273 SSR markers we investigated cold tolerance at the germination and booting stages, as well as the underlying genetic bases, by association mapping. Two distinct populations, corresponding to subspecies indica and japonica showed evident differences in cold tolerance and its genetic basis. Both subspecies were sensitive to cold stress at both growth stages. However, japonica was more tolerant than indica at all stages as measured by seedling survival and seed setting. There was a low correlation in cold tolerance between the germination and booting stages. Fifty one quantitative trait loci (QTLs) for cold tolerance were dispersed across all 12 chromosomes; 22 detected at the germination stage and 33 at the booting stage. Eight QTLs were identified by at least two of four measures. About 46% of the QTLs represented new loci. The only QTL shared between indica and japonica for the same measure was qLTSSvR6-2 for SSvR. This implied a complicated mechanism of old tolerance between the two subspecies. According to the relative genotypic effect (RGE) of each genotype for each QTL, we detected 18 positive genotypes and 21 negative genotypes in indica, and 19 positive genotypes and 24 negative genotypes in japonica. In general, the negative effects were much stronger than the positive effects in both subspecies. Markers for QTL with positive effects in one subspecies were shown to be effective for selection of cold tolerance in that subspecies, but not in the other subspecies. QTL with strong negative effects on cold tolerance should be avoided during MAS breeding so as to not cancel the effect of favorable QTL at other loci. None Time-Course Association Mapping of the Grain-Filling Rate in Rice (Oryza sativa L.). 2015 PLoS One State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China. Detecting quantity trait locus (QTLs) and elite alleles that are associated with grain-filling rate (GFR) in rice is essential for promoting the utilization of hybrid japonica rice and improving rice yield. Ninety-five varieties including 58 landraces and 37 elite varieties from the core germplasm collection were genotyped with 263 simple sequence repeat (SSR) markers. The GFR of the 95 varieties was evaluated at five stages, 7, 14, 21, 28 and 35 days after flowering (DAF) both in 2011 and 2012. We found abundant phenotypic and genetic diversity in the studied population. A population structure analysis identified seven subpopulations. A linkage disequilibrium (LD) analysis indicated that the levels of LD ranged from 60.3 cM to 84.8 cM and artificial selection had enhanced the LD. A time-course association analysis detected 31 marker-GFR associations involving 24 SSR markers located on chromosomes 1, 2, 3, 4, 5, 6, 8, 9, 11 and 12 of rice at five stages. The elite alleles for high GFR at each stage were detected. Fifteen excellent parental combinations were predicted, and the best parental combination 'Nannongjing62401×Laolaihong' could theoretically increase 4.086 mg grain-1 d-1 at the five stages. Our results demonstrate that the time-course association mapping for GFR in rice could detect elite alleles at different filling stages and that these elite alleles could be used to improve the GFR via pyramiding breeding. None Functions for rice RFL in vegetative axillary meristem specification and outgrowth. 2015 J Exp Bot Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India. Axillary meristems (AMs) are secondary shoot meristems whose outgrowth determines plant architecture. In rice, AMs form tillers, and tillering mutants reveal an interplay between transcription factors and the phytohormones auxin and strigolactone as some factors that underpin this developmental process. Previous studies showed that knockdown of the transcription factor gene RFL reduced tillering and caused a very large decrease in panicle branching. Here, the relationship between RFL, AM initiation, and outgrowth was examined. We show that RFL promotes AM specification through its effects on LAX1 and CUC genes, as their expression was modulated on RFL knockdown, on induction of RFL:GR fusion protein, and by a repressive RFL-EAR fusion protein. Further, we report reduced expression of auxin transporter genes OsPIN1 and OsPIN3 in the culm of RFL knockdown transgenic plants. Additionally, subtle change in the spatial pattern of IR4 DR5:GFP auxin reporter was observed, which hints at compromised auxin transport on RFL knockdown. The relationship between RFL, strigolactone signalling, and bud outgrowth was studied by transcript analyses and by the tillering phenotype of transgenic plants knocked down for both RFL and D3. These data suggest indirect RFL-strigolactone links that may affect tillering. Further, we show expression modulation of the auxin transporter gene OsPIN3 upon RFL:GR protein induction and by the repressive RFL-EAR protein. These modified forms of RFL had only indirect effects on OsPIN1. Together, we have found that RFL regulates the LAX1 and CUC genes during AM specification, and positively influences the outgrowth of AMs though its effects on auxin transport. RFL|APO2 Autophagy supports biomass production and nitrogen use efficiency at the vegetative stage in rice. 2015 Plant Physiol Tohoku University. Much of the nitrogen in leaves is distributed to chloroplasts, mainly in photosynthetic proteins. During leaf senescence, chloroplastic proteins including Rubisco are rapidly degraded, and the released nitrogen is remobilized and reused in newly developing tissues. Autophagy facilitates the degradation of intracellular components for nutrient recycling in all eukaryotes and recent studies have revealed critical roles for autophagy in Rubisco degradation and nitrogen remobilization into seeds in Arabidopsis thaliana. Here, we examined the function of autophagy in vegetative growth and nitrogen usage in a cereal plant, rice (Oryza sativa). An autophagy-disrupted rice mutant, Osatg7-1, showed reduced biomass production and nitrogen use efficiency compared to the wild type. While Osatg7-1 showed early visible leaf senescence, the nitrogen concentration remained high in the senescent leaves. (15) N pulse-chase analysis revealed suppression of nitrogen remobilization during leaf senescence in Osatg7-1. Accordingly, the reduction of nitrogen available for newly developing tissues in Osatg7-1 likely led its reduced leaf area and tillers. The limited leaf growth in Osatg7-1 decreased the photosynthetic capacity of the plant. Much of the nitrogen remaining in senescent leaves of Osatg7-1 was in soluble proteins, and the Rubisco concentration in senescing leaves of Osatg7-1 was about 2.5 times higher than in the wild type. Transmission electron micrographs showed a cytosolic fraction rich with organelles in senescent leaves of Osatg7-1. Our results suggest that autophagy contributes to efficient nitrogen remobilization at the whole-plant level by facilitating protein degradation for nitrogen recycling in senescent leaves. None Quantitative Proteomic Analysis of the Rice (Oryza sativa L.) Salt Response. 2015 PLoS One State key laboratory of crop genetics and germplasm enhancement, Nanjing Agricultural University, Nanjing, 210095, China; Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing, 210095, China. Salt stress is one of most serious limiting factors for crop growth and production. An isobaric Tags for Relative and Absolute Quantitation (iTRAQ) approach was used to analyze proteomic changes in rice shoots under salt stress in this study. A total of 56 proteins were significantly altered and 16 of them were enriched in the pathways of photosynthesis, antioxidant and oxidative phosphorylation. Among these 16 proteins, peroxiredoxin Q and photosystem I subunit D were up-regulated, while thioredoxin M-like, thioredoxin x, thioredoxin peroxidase, glutathione S-transferase F3, PSI subunit H, light-harvesting antenna complex I subunits, chloroplast chaperonin, vacuolar ATP synthase subunit H, and ATP synthase delta chain were down-regulated. Moreover, physiological data including total antioxidant capacity, peroxiredoxin activity, chlorophyll a/b content, glutathione S-transferase activity, reduced glutathione content and ATPase activity were consistent with changes in the levels of these proteins. The levels of the mRNAs encoding these proteins were also analyzed by real-time quantitative reverse transcription PCR, and approximately 86% of the results were consistent with the iTRAQ data. Importantly, our data suggest the important role of PSI in balancing energy supply and ROS generation under salt stress. This study provides information for an improved understanding of the function of photosynthesis and PSI in the salt-stress response of rice. None Genome-wide association study of rice (Oryza sativa L.) leaf traits with a high-throughput leaf scorer. 2015 J Exp Bot National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, PR China College of Engineering, Huazhong Agricultural University, Wuhan 430070, PR China Agricultural Bioinformatics Key Laboratory of Hubei Province, Huazhong Agricultural University, Wuhan 430070, PR China. Leaves are the plant's solar panel and food factory, and leaf traits are always key issues to investigate in plant research. Traditional methods for leaf trait measurement are time-consuming. In this work, an engineering prototype has been established for high-throughput leaf scoring (HLS) of a large number of Oryza sativa accessions. The mean absolute per cent of errors in traditional measurements versus HLS were below 5% for leaf number, area, shape, and colour. Moreover, HLS can measure up to 30 leaves per minute. To demonstrate the usefulness of HLS in dissecting the genetic bases of leaf traits, a genome-wide association study (GWAS) was performed for 29 leaf traits related to leaf size, shape, and colour at three growth stages using HLS on a panel of 533 rice accessions. Nine associated loci contained known leaf-related genes, such as Nal1 for controlling the leaf width. In addition, a total of 73, 123, and 177 new loci were detected for traits associated with leaf size, colour, and shape, respectively. In summary, after evaluating the performance with a large number of rice accessions, the combination of GWAS and high-throughput leaf phenotyping (HLS) has proven a valuable strategy to identify the genetic loci controlling rice leaf traits. None MutMap accelerates breeding of a salt-tolerant rice cultivar 2015 Nat Biotechnol Iwate Biotechnology Research Center, Kitakami, Iwate, Japan. OsRR22|HST1 A cytochrome P450, OsDSS1, is involved in growth and drought stress responses in rice (Oryza sativa L.). 2015 Plant Mol Biol Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate, 024-0003, Japan, olimt@ibrc.or.jp. Cytochrome P450s are among the largest protein coding gene families in plant genomes. However, majority of the genes remain uncharacterized. Here, we report the characterization of dss1, a rice mutant showing dwarfism and reduced grain size. The dss1 phenotype is caused by a non-synonymous point mutation we identified in DSS1, which is member of a P450 gene cluster located on rice chromosome 3 and corresponds to the previously reported CYP96B4/SD37 gene. Phenotypes of several dwarf mutants characterized in rice are associated with defects in the biosynthesis or perception of the phytohormones gibberellins (GAs) and brassinosteroids (BRs). However, both GA and BR failed to rescue the dss1 phenotype. Hormone profiling revealed the accumulation of abscisic acid (ABA) and ABA metabolites, as well as significant reductions in GA19 and GA53 levels, precursors of the bioactive GA1, in the mutant. The dss1 contents of cytokinin and auxins were not significantly different from wild-type plants. Consistent with the accumulation of ABA and metabolites, germination and early growth was delayed in dss1, which also exhibited an enhanced tolerance to drought. Additionally, expressions of members of the DSS1/CYP96B gene cluster were regulated by drought stress and exogenous ABA. RNA-seq-based transcriptome profiling revealed, among others, that cell wall-related genes and genes involved in lipid metabolism were up- and down-regulated in dss1, respectively. Taken together, these findings suggest that DSS1 mediates growth and stress responses in rice by fine-tuning GA-to-ABA balance, and might as well play a role in lipid metabolism. OsDSS1 OsKinesin-13A Is an Active Microtubule Depolymerase Involved in Glume Length Regulation via Affecting Cell Elongation. 2015 Sci Rep Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. Grain size is an important trait influencing both the yield and quality of rice and its major determinant is glume size. However, how glume size is regulated remains largely unknown. Here, we report the characterization of OsKinesin-13A, which regulates cell elongation and glume length in rice. The mutant of OsKinesin-13A, sar1, displayed length reduction in grains and other organs including internodes, leaves and roots. The grain phenotype in sar1 was directly caused by reduction in glume length, which in turn restricted caryopsis size. Histological results revealed that length decrease in sar1 organs resulted from abnormalities in cell elongation. The orientation of cellulose microfibrils was defective in sar1. Consistently, sar1 showed reduced transverse orientation of cortical microtubules. Further observations demonstrated that microtubule turnover was decreased in sar1. OsKinesin-13A was shown to be an active microtubule depolymerase and mainly distributed on vesicles derived from the Golgi apparatus and destined for the cell surface. Thus, our results suggest that OsKinesin-13A utilizes its microtubule depolymerization activity to promote microtubule turnover, which may not only influence transverse orientation of cortical microtubules but also facilitate vesicle transport from the Golgi apparatus to the cell surface, and thus affects cellulose microfibril orientation and cell elongation. SRS3|OsKINESIN-13A Comprehensive analysis of regulatory elements of the promoters of rice sulphate transporter gene family and functional characterisation of OsSul1;1 promoter under different metal stress. 2015 Plant Signal Behav a CSIR-National Botanical Research Institute (CSIR-NBRI) , Rana Pratap Marg, Lucknow 226001 , India. Adverse environmental conditions including heavy metal stress impose severe effects on the plant growth and development limiting productivity and yield. Studies demonstrated that changes in genome-wide expression modulate various biochemical processes and molecular components in response to heavy metal stress in plants. Some of the key components involved in such a regulation are the transcription initiation machinery, nucleotide sequence of promoters and presence of cis-acting elements. Therefore, identification of the putative cis-acting DNA sequences involved in gene regulation and functional characterization of promoters are important steps in understanding response of plants to heavy metal stress. In this study, comprehensive analysis of the proximal promoters of members of rice sulphate transporter gene family which is an essential component of stress response has been carried out. Analysis suggests presence of various common stress related cis-acting elements in the promoters of members of this gene family. In addition, transcriptional regulation of the arsenic-responsive high affinity sulphate transporter, OsSul1;1, has been studied through development of Arabidopsis transgenic lines expressing reporter gene encoding beta-glucuronidase under the control of OsSul1;1 promoter. Analysis of the transgenic lines suggests differential response of the OsSul1;1 promoter to various heavy metals as well as other abiotic stresses. None Rice ubiquitin ligase EL5 prevents root meristematic cell death under high nitrogen conditions and interacts with a cytosolic GAPDH. 2015 Plant Signal Behav a National Institute of Agrobiological Sciences ; Tsukuba , 305-8602 Japan ; Root formation in rice transformants overexpressing mutated EL5 (mEL5) was severely inhibited because of meristematic cell death. Cell death was caused by nitrogen sources, particularly nitrate forms, in the culture medium. Nitrite treatment increased the cytokinin contents in roots, but mEL5 contained more cytokinins than non-transformants. Transcriptome profiling showed overlaps between nitrite-responsive genes in non-transformants and genes with altered expression in untreated mEL5. These results indicate that impairment of EL5 function activates nitrogen signaling despite the absence of a nitrogen source. Physical interaction between the EL5 C-terminal region and a cytosolic glyceraldehyde-3-phosphate dehydrogenase, OsGapC2, was demonstrated in vitro and in vivo. Elucidation of the role of glyceraldehyde-3-phosphate dehydrogenase in oxidative cell death in plants is expected in future. EL5 Nucleotide Diversity Analysis of Three Major Bacterial Blight Resistance Genes in Rice. 2015 PLoS One Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Prof. C. R. Rao Road, Gachibowli, Hyderabad, 500046, India. Nucleotide sequence polymorphisms among R gene alleles influence the process of co-evolutionary interaction between host and pathogen by shaping the response of host plants towards invading pathogens. Here, we present the DNA sequence polymorphisms and diversities present among natural alleles of three rice bacterial blight resistance genes, Xa21, Xa26 and xa5. The diversity was examined across different wild relatives and cultivars of Oryza species. Functional significance of selected alleles was evaluated through semi-quantitative reverse transcription polymerase chain reaction and real time PCR. The greatest nucleotide diversity and singleton variable sites (SVS) were present in Xa26 (π = 0.01958; SVS = 182) followed by xa5 and Xa21 alleles. The highest frequency of single nucleotide polymorphisms were observed in Xa21 alleles and least in xa5. Transition bias was observed in all the genes and 'G' to 'A' transitions were more favored than other form of transitions. Neutrality tests failed to show the presence of selection at these loci, though negative Tajima's D values indicate the presence of a rare form of polymorphisms. At the interspecies level, O. nivara exhibited more diversity than O. sativa. We have also identified two nearly identical resistant alleles of xa5 and two sequentially identical alleles of Xa21. The alleles of xa5 showed basal levels of expression while Xa21 alleles were functionally not expressed. None Iron- and manganese-assisted cadmium tolerance in Oryza sativa L.: lowering of rhizotoxicity next to functional photosynthesis. 2015 Planta Department of Plant Sciences, University of Hyderabad, Hyderabad, 500046, Telangana, India. Cadmium toxicity is alleviated by iron and manganese supplements because of reduction in cadmium accumulation and upholding of redox regulation that prevent cadmium-inducible damage to root growth and photosynthesis. Cadmium toxicity in Oryza sativa L. MTU 7029 was investigated in the presence of different concentrations of the micronutrients Fe and Mn. It had been observed that these micronutrients reduce Cd uptake and minimize Cd-inducible rhizotoxicity. The photosynthetic electron transport chain, which is the hub of Fe containing metalloproteins, was severely affected by Cd and resulted in reduced bioproductivity under Cd stress. However, exogenous Fe restored the photosynthetic electron transport. Thus, due to the maintenance of the photosynthetic electron transport, the Cd tolerance was improved during Fe supplement. Both antioxidant enzymes and non-enzymatic antioxidant metabolites were found to play important roles in the alleviation of Cd stress under Fe or Mn supplement. It is concluded that the presence of excess Fe and Mn protects rice plants from Cd stress. None Dissecting root proteome of transgenic rice cultivars unravels metabolic alterations and accumulation of novel stress responsive proteins under drought stress. 2015 Plant Sci Laboratory of Translational Research on Transgenic Crops, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700019, West Bengal, India. Generation of drought tolerant rice plants by overexpressing Arabidopsis DREB1A is a significant development for abiotic stress research. However, the metabolic network regulated in the drought tolerant transgenic rice plants is poorly understood. In this research study, we have demonstrated the comparative proteome analysis between the roots of wild type and transgenic DREB1A overexpressing homozygous plants under drought stress condition. After 7d of dehydration stress at reproductive stage, the plants were re-watered for 24h. The roots were collected separately from wild type and transgenic plants grown under water, drought stress and re-watering conditions and total proteins were analyzed by two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS). Among the large number of differentially accumulated spots, 30, 27 and 20 spots were successfully identified as differentially expressed proteins in three different conditions respectively. The major class of identified proteins belongs to carbohydrate and energy metabolism category while stress and defense related proteins are especially up-accumulated under drought stress in both the plants. A novel protein, R40C1 was reported to be up-accumulated in roots of transgenic plants which may play an important role in generation of drought tolerant plants. Protein-protein interaction helps to identify the network of drought stress signaling pathways. None The enhanced drought tolerance of rice plants under ammonium is related to aquaporin (AQP). 2015 Plant Sci Jiangsu Key Lab for Organic Waste Utilization and National Engineering Research Center for Organic-based Fertilizers, Nanjing Agricultural University, 210095 China. Previously, we demonstrated that drought resistance in rice seedlings was increased by ammonium (NH4(+)) treatment, but not by nitrate (NO3(-)) treatment, and that the change was associated with root development. To study the effects of different forms of nitrogen on water uptake and root growth under drought conditions, we subjected two rice cultivars (cv. 'Shanyou 63' hybrid indica and cv. 'Yangdao 6' indica, China) to polyethylene glycol-induced drought stress in a glasshouse using hydroponic culture. Under drought conditions, NH4(+) significantly stimulated root growth compared to NO3(-), as indicated by the root length, surface area, volume, and numbers of lateral roots and root tips. Drought stress decreased the root elongation rate in both cultivars when they were supplied with NO3(-), while the rate was unaffected in the presence of NH4(+). Drought stress significantly increased root protoplast water permeability, root hydraulic conductivity, and the expression of root aquaporin (AQP) plasma intrinsic protein (PIP) genes in rice plants supplied with NH4(+); these changes were not observed in plants supplied with NO3(-). Additionally, ethylene, which is involved in the regulation of root growth, accumulated in rice roots supplied with NO3(-) under conditions of drought stress. We conclude that the increase in AQP expression and/or activity enhanced the root water uptake ability and the drought tolerance of rice plants supplied with NH4(+). None Development of genome-wide insertion/deletion markers in rice based on graphic pipeline platform. 2015 J Integr Plant Biol State Key Laboratory of Hybrid Rice, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China. DNA markers play important roles in plant breeding and genetics. Insertion/Deletion (InDel) marker is one kind of co-dominant DNA markers widely used since low cost and high precision. However, canonical way of searching for InDel markers is time-consuming and labor-intensive. We developed an end-to-end computational solution (InDel Markers Development Platform, IMDP) to identify genome-wide InDel markers under a graphic pipeline environment. IMDP constitutes assembled genome sequences alignment pipeline (AGA-pipe) and next-generation re-sequencing data mapping pipeline (NGS-pipe). With AGA-pipe we are able to identify 12,944 markers between the genome of rice cultivars Nipponbare and 93-11. Using NGS-pipe, we reported 34,794 InDels from re-sequencing data of rice cultivars Wu-Yun-Geng7 and Guang-Lu-Ai4. Combining AGA-pipe and NGS-pipe, we developed 205,659 InDels in 8 japonica and 9 indica cultivars and 2,681 InDels showed subgroup-specific pattern. PCR analysis of subgroup-specific markers indicated that the precision reached 90% (86 of 95). Finally, to make them available to public, we have integrated the InDels/markers information into a website (Rice InDel Marker Database, RIMD, http://202.120.45.71/). The application of IMDP in rice will facilitate efficiency for development of genome-wide InDel markers, in addition it can be used in other species with reference genome sequences and NGS data. None Unique miRNome during anthesis in drought-tolerant indica rice var. Nagina 22. 2015 Planta Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Marg, New Delhi, 110021, India. Drought-tolerant rice variety, Nagina 22 (N22), has a unique spikelet miRNome during anthesis stage drought as well as transition from heading to anthesis. Molecular characterization of genetic diversity of rice is essential to understand the evolution and molecular basis of various agronomically important traits such as drought tolerance. miRNAs play an important role in regulating plant development as well as stress response such as drought. In this study, we characterized the yet unexplored dynamics of the spikelet miRNA population during developmental transition from 'heading' to 'anthesis' as well as anthesis stage drought stress in a drought-tolerant indica rice variety, N22. A significant proportion of miRNA population (~20 %) in N22 spikelets is modulated during transition from heading to anthesis indicating a unique miRNome at anthesis, a developmental stage highly sensitive to stress (drought/heat). Based on the analysis of degradome data, majority of differentially regulated miRNAs appear to regulate transcription factors, some of which are implicated in regulation of development and fertilization. Similarly, drought during anthesis leads to a global change in miRNA expression pattern including those which regulate ROS homeostasis. It was possible to identify several miRNAs that were not reported to be drought responsive in earlier studies. Interestingly, a significant proportion of the drought-regulated miRNAs co-localize within QTLs related to drought tolerance and associated traits. Comparison of the expression profiles between N22 and Pusa Basmati 1 (drought sensitive) identified miRNAs with variety-specific expression patterns during phase transition (miR164, miR396, miR812, and miR1881) as well as drought stress (miR1881) indicating an evolution of a distinct and variety-specific regulatory mechanism. The promoters of these miRNAs contain LREs (light-responsive elements) and are induced by dark treatment. It was also possible to identify 4 novel miRNAs including an intronic miRNA that was conserved in both rice varieties. None Genome-wide analysis of DNA methylation in photoperiod- and thermo-sensitive male sterile rice Peiai 64S. 2015 BMC Genomics State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, 430072, People's Republic of China. hujh2010@163.com. Epigenetic modifications play important roles in the regulation of plant development. DNA methylation is an important epigenetic modification that dynamically regulates gene expression during developmental processes. However, little studies have been reported about the methylation profiles of photoperiod- and thermo-sensitive genic male sterile (PTGMS) rice during the fertility transition.In this study, using methylated DNA immunoprecipitation sequencing (MeDIP-seq), the global DNA methylation patterns were compared in the rice PTGMS line PA64S under two different environments (different temperatures and day lengths). The profiling of the DNA methylation under two different phenotypes (sterility and fertility) revealed that hypermethylation was observed in PA64S (sterility), and 1258 differentially methylated regions (DMRs) were found between PA64S (sterility) and PA64S (fertility). Twenty differentially methylated genes of them were further validated through bisulfite sequencing, and four of these genes were analyzed by qRT-PCR. Especially, a differentially methylated gene (LOC_Os08g38210), which encoded transcription factor BIM2, is a component of brassinosteroid signaling in rice. The hypermethylated BIM2 gene may suppress some downstream genes in brassinosteroid signaling pathway, and thus affect the male fertility in PA64S.The results presented here indicated that hypermethylation was observed in PA64S (sterility). Gene Ontology (GO) analysis and KEGG analysis revealed that flavone and flavonol biosynthrsis, circadian rhythm, photosynthesis and oxidative phosphorylation pathways were involved in sterility-fertility transition of PA64S. None Global epigenomic analysis indicates that Epialleles contribute to Allele-specific expression via Allele-specific histone modifications in hybrid rice. 2015 BMC Genomics State Key Laboratory of Hybrid Rice and College of Life Sciences, Wuhan University, Luojia Hill, Wuhan, 430072, , Hubei Province, China. zhibin.guo@whu.edu.cn. For heterozygous genes, alleles on the chromatin from two different parents exhibit histone modification variations known as allele-specific histone modifications (ASHMs). The regulation of allele-specific gene expression (ASE) by ASHMs has been reported in animals. However, to date, the regulation of ASE by ASHM genes remains poorly understood in higher plants.We used chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) to investigate the global ASHM profiles of trimethylation on histone H3 lysine 27 (H3K27me3) and histone H3 lysine 36 (H3K36me3) in two rice F1 hybrids. A total of 522 to 550 allele-specific H3K27me3 genes and 428 to 494 allele-specific H3K36me3 genes were detected in GL × 93-11 and GL × TQ, accounting for 11.09% and 26.13% of the total analyzed genes, respectively. The epialleles between parents were highly related to ASHMs. Further analysis indicated that 52.48% to 70.40% of the epialleles were faithfully inherited by the F1 hybrid and contributed to 33.18% to 46.55% of the ASHM genes. Importantly, 66.67% to 82.69% of monoallelic expression genes contained the H3K36me3 modification. Further studies demonstrated a significant positive correlation of ASE with allele-specific H3K36me3 but not with H3K27me3, indicating that ASHM-H3K36me3 primarily regulates ASE in this study.Our results demonstrate that epialleles from parents can be inherited by the F1 to produce ASHMs in the F1 hybrid. Our findings indicate that ASHM-H3K36me3, rather than H3K27me3, mainly regulates ASE in hybrid rice. None Rice tissue-specific promoters and condition-dependent promoters for effective translational application. 2015 J Integr Plant Biol Department of Plant Molecular Systems Biotechnology & Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Korea. Rice (Oryza sativa) is one of the most important staple food crops for more than half of the world's population. The demand is increasing for food security because of population growth and environmental challenges triggered by climate changes. This scenario has led to more interest in developing crops with greater productivity and sustainability. The process of genetic transformation, a major tool for crop improvement, utilizes promoters as one of its key elements. Those promoters are generally divided into three types: constitutive, spatio-temporal, and condition-dependent. Transcriptional control of a constitutive promoter often leads to reduced plant growth, due to a negative effect of accumulated molecules during cellular functions or energy consumption. To maximize the effect of a transgene on transgenic plants, it is better to use condition-dependent or tissue-specific promoters. However, until now, those types have not been as widely applied in crop biotechnology. In this review, we introduce and discuss four groups of tissue-specific promoters (50 promoters in total) and six groups of condition-dependent promoters (27 promoters). These promoters can be utilized to fine-tune desirable agronomic traits and develop crops with tolerance to various stresses, enhanced nutritional value, and advanced productivity. None Variation and impact of drought-stress patterns across upland rice target population of environments in Brazil. 2015 J Exp Bot Embrapa Rice & Beans Rodovia GO-462 km 12 Zona Rural, 75375-000 Santo Antônio de Goiás, GO, Brazil alexandre.heinemann@embrapa.br. The upland rice (UR) cropped area in Brazil has decreased in the last decade. Importantly, a portion of this decrease can be attributed to the current UR breeding programme strategy, according to which direct grain yield selection is targeted primarily to the most favourable areas. New strategies for more-efficient crop breeding under non-optimal conditions are needed for Brazil's UR regions. Such strategies should include a classification of spatio-temporal yield variations in environmental groups, as well as a determination of prevalent drought types and their characteristics (duration, intensity, phenological timing, and physiological effects) within those environmental groups. This study used a process-based crop model to support the Brazilian UR breeding programme in their efforts to adopt a new strategy that accounts for the varying range of environments where UR is currently cultivated. Crop simulations based on a commonly grown cultivar (BRS Primavera) and statistical analyses of simulated yield suggested that the target population of environments can be divided into three groups of environments: a highly favorable environment (HFE, 19% of area), a favorable environment (FE, 44%), and least favourable environment (LFE, 37%). Stress-free conditions dominated the HFE group (69% likelihood) and reproductive stress dominated the LFE group (68% likelihood), whereas reproductive and terminal drought stress were found to be almost equally likely to occur in the FE group. For the best and worst environments, we propose specific adaptation focused on the representative stress, while for the FE, wide adaptation to drought is suggested. 'Weighted selection' is also a possible strategy for the FE and LFE environment groups. None Development of salinity tolerance in rice by constitutive-overexpression of genes involved in the regulation of programmed cell death. 2015 Front Plant Sci Centre for Tropical Crops and Biocommodities, Queensland University of Technology Brisbane, QLD, Australia. Environmental factors contribute to over 70% of crop yield losses worldwide. Of these drought and salinity are the most significant causes of crop yield reduction. Rice is an important staple crop that feeds more than half of the world's population. However among the agronomically important cereals rice is the most sensitive to salinity. In the present study we show that exogenous expression of anti-apoptotic genes from diverse origins, AtBAG4 (Arabidopsis), Hsp70 (Citrus tristeza virus) and p35 (Baculovirus), significantly improves salinity tolerance in rice at the whole plant level. Physiological, biochemical and agronomical analyses of transgenic rice expressing each of the anti-apoptotic genes subjected to salinity treatment demonstrated traits associated with tolerant varieties including, improved photosynthesis, membrane integrity, ion and ROS maintenance systems, growth rate, and yield components. Moreover, FTIR analysis showed that the chemical composition of salinity-treated transgenic plants is reminiscent of non-treated, unstressed controls. In contrast, wild type and vector control plants displayed hallmark features of stress, including pectin degradation upon subjection to salinity treatment. Interestingly, despite their diverse origins, transgenic plants expressing the anti-apoptotic genes assessed in this study displayed similar physiological and biochemical characteristics during salinity treatment thus providing further evidence that cell death pathways are conserved across broad evolutionary kingdoms. Our results reveal that anti-apoptotic genes facilitate maintenance of metabolic activity at the whole plant level to create favorable conditions for cellular survival. It is these conditions that are crucial and conducive to the plants ability to tolerate/adapt to extreme environments. None Development and evaluation of near-isogenic lines for major blast resistance gene(s) in Basmati rice. 2015 Theor Appl Genet Division of Genetics, Indian Agricultural Research Institute, New Delhi, 110012, India. A set of NILs carrying major blast resistance genes in a Basmati rice variety has been developed. Also, the efficacy of pyramids over monogenic NILs against rice blast pathogen Magnaporthe oryzae has been demonstrated. Productivity and quality of Basmati rice is severely affected by rice blast disease. Major genes and QTLs conferring resistance to blast have been reported only in non-Basmati rice germplasm. Here, we report incorporation of seven blast resistance genes from the donor lines DHMASQ164-2a (Pi54, Pi1, Pita), IRBLz5-CA (Pi2), IRBLb-B (Pib), IRBL5-M (Pi5) and IRBL9-W (Pi9) into the genetic background of an elite Basmati rice variety Pusa Basmati 1 (PB1). A total of 36 near-isogenic lines (NILs) comprising of 14 monogenic, 16 two-gene pyramids and six three-gene pyramids were developed through marker-assisted backcross breeding (MABB). Foreground, recombinant and background selection was used to identify the plants with target gene(s), minimize the linkage drag and increase the recurrent parent genome (RPG) recovery (93.5-98.6 %), respectively, in the NILs. Comparative analysis performed using 50,051 SNPs and 500 SSR markers revealed that the SNPs provided better insight into the RPG recovery. Most of the monogenic NILs showed comparable performance in yield and quality, concomitantly, Pusa1637-18-7-6-20 (Pi9), was significantly superior in yield and stable across four different environments as compared to recurrent parent (RP) PB1. Further, among the pyramids, Pusa1930-12-6 (Pi2+Pi5) showed significantly higher yield and Pusa1633-7-8-53-6-8 (Pi54+Pi1+Pita) was superior in cooking quality as compared to RP PB1. The NILs carrying gene Pi9 were found to be the most effective against the concoction of virulent races predominant in the hotspot locations for blast disease. Conversely, when analyzed under artificial inoculation, three-gene pyramids expressed enhanced resistance as compared to the two-gene and monogenic NILs. None Genetic basis of sRNA quantitative variation analyzed using an experimental population derived from an elite rice hybrid. 2015 Elife National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China. We performed a genetic analysis of sRNA abundance in flag leaf from an immortalized F2 (IMF2) population in rice. We identified 53,613,739 unique sRNAs and 165,797 sRNA expression traits (s-traits). A total of 66,649 s-traits mapped 40,049 local-sQTLs and 30,809 distant-sQTLs. By defining 80,362 sRNA clusters, 22,263 sRNA cluster QTLs (scQTLs) were recovered for 20,249 of all the 50,139 sRNA cluster expression traits (sc-traits). The expression levels for most of s-traits from the same genes or the same sRNA clusters were slightly positively correlated. While genetic co-regulation between sRNAs from the same mother genes and between sRNAs and their mother genes was observed for a portion of the sRNAs, most of the sRNAs and their mother genes showed little co-regulation. Some sRNA biogenesis genes were located in distant-sQTL hotspots and showed correspondence with specific length classes of sRNAs suggesting their important roles in the regulation and biogenesis of the sRNAs. None Whole-Genome Resequencing and Transcriptomic Analysis to Identify Genes Involved in Leaf-Color Diversity in Ornamental Rice Plants. 2015 PLoS One Genomics Division, National Academy of Agricultural Science (NAAS), Rural Development Administration (RDA), Jeonju, Korea. Rice field art is a large-scale art form in which people design rice fields using various kinds of ornamental rice plants with different leaf colors. Leaf color-related genes play an important role in the study of chlorophyll biosynthesis, chloroplast structure and function, and anthocyanin biosynthesis. Despite the role of different metabolites in the traditional relationship between leaf and color, comprehensive color-specific metabolite studies of ornamental rice have been limited. We performed whole-genome resequencing and transcriptomic analysis of regulatory patterns and genetic diversity among different rice cultivars to discover new genetic mechanisms that promote enhanced levels of various leaf colors. We resequenced the genomes of 10 rice leaf-color accessions to an average of 40× reads depth and >95% coverage and performed 30 RNA-seq experiments using the 10 rice accessions sampled at three developmental stages. The sequencing results yielded a total of 1,814 × 106 reads and identified an average of 713,114 SNPs per rice accession. Based on our analysis of the DNA variation and gene expression, we selected 47 candidate genes. We used an integrated analysis of the whole-genome resequencing data and the RNA-seq data to divide the candidate genes into two groups: genes related to macronutrient (i.e., magnesium and sulfur) transport and genes related to flavonoid pathways, including anthocyanidin biosynthesis. We verified the candidate genes with quantitative real-time PCR using transgenic T-DNA insertion mutants. Our study demonstrates the potential of integrated screening methods combined with genetic-variation and transcriptomic data to isolate genes involved in complex biosynthetic networks and pathways. None Interactive effects of nitric oxide and glutathione in mitigating copper toxicity of rice (Oryza sativa L.) seedlings. 2015 Plant Signal Behav a Laboratory of Plant Stress Responses; Department of Applied Biological Science; Faculty of Agriculture ; Kagawa University ; Kagawa , Japan. Nitric oxide (NO) and glutathione (GSH) are 2 vital components of the antioxidant system that play diverse roles in plant responses to abiotic stresses. Recently, we have reported that exogenous supply of both these molecules reduced copper (Cu) toxicity in rice seedlings. Individual as well as co-treatment of sodium nitroprusside (SNP: a NO donor) and GSH with Cu significantly mitigated the adverse effects of Cu, evident in the reduced level of oxidative markers such as H2O2, superoxide (O2(·-)), malondialdehyde (MDA), and proline (Pro). GSH content and most of the antioxidative and glyoxalase enzymes were up-regulated upon Cu stress, indicating their responses were co-related with the level of stress. Our results indicated that direct ROS scavenging, reduced Cu uptake, and the balanced antioxidative and glyoxalase systems, at least in part, successfully executed NO- and GSH-mediated alleviation of Cu toxicity in rice seedlings. In addition, the combined effect of adding SNP and GSH together was more efficient than the effect of adding them individually. Here, we are speculating that 1) GSH and Pro could be used as potential markers for copper stress, and 2) adding SNP and GSH might produce S-nitrosoglutathione (GSNO) which could be a source of bioactive NO and may affect many regulatory processes involved in Cu-stress tolerance. We further note that the combined effect of adding SNP and GSH was pronounced in inhibiting the uptake and translocation of Cu in rice seedlings. None Identifying and confirming quantitative trait loci associated with heat tolerance at flowering stage in different rice populations. 2015 BMC Genet International Rice Research Institute, DAPO Box 7777, Metro Manila, 1301, Philippines. c.ye@irri.org. Climate change is affecting rice production in many countries. Developing new rice varieties with heat tolerance is an essential way to sustain rice production in future global warming. We have previously reported four quantitative trait loci (QTLs) responsible for rice spikelet fertility under high temperature at flowering stage from an IR64/N22 population. To further explore additional QTL from other varieties, two bi-parental F2 populations and one three-way F2 population derived from heat tolerant variety Giza178 were used for indentifying and confirming QTLs for heat tolerance at flowering stage.Four QTLs (qHTSF1.2, qHTSF2.1, qHTSF3.1 and qHTSF4.1) were identified in the IR64/Giza178 population, and two other QTLs (qHTSF6.1 and qHTSF11.2) were identified in the Milyang23/Giza178 population. To confirm the identified QTLs, another three-way-cross population derived from IR64//Milyang23/Giza178 was genotyped using 6K SNP chips. Five QTLs were identified in the three-way-cross population, and three of those QTLs (qHTSF1.2, qHTSF4.1 and qHTSF6.1) were overlapped with the QTLs identified in the bi-parental populations. The tolerance alleles of these QTLs were from the tolerant parent Giza178 except for qHTSF3.1. The QTL on chromosome 4 (qHTSF4.1) is the same QTL previously identified in the IR64/N22 population.The results from different populations suggest that heat tolerance in rice at flowering stage is controlled by several QTLs with small effects and stronger heat tolerance could be attained through pyramiding validated heat tolerance QTLs. QTL qHTSF4.1 was consistently detected across different genetic backgrounds and could be an important source for enhancing heat tolerance in rice at flowering stage. Polymorphic SNP markers in these QTL regions can be used for future fine mapping and developing SNP chips for marker-assisted breeding. None OsKASI, a beta-ketoacyl-[acyl carrier protein] synthase I, is involved in root development in rice (Oryza sativa L.). 2015 Planta College of Science and Technology, Ningbo University, Ningbo, 315211, People's Republic of China. The involvement of OsKASI in FA synthesis is found to play a critical role in root development of rice. The root system plays important roles in plant nutrient and water acquisition. However, mechanisms of root development and molecular regulation in rice are still poorly understood. Here, we characterized a rice (Oryza sativa L.) mutant with shortened roots due to a defect in cell elongation. Map-based cloning revealed that the mutation occurred in a putative 3-oxoacyl-synthase, an ortholog of beta-ketoacyl-[acyl carrier protein] synthase I (KASI) in Arabidopsis, thus designated as OsKASI. OsKASI was found to be ubiquitously expressed in various tissues throughout the plant and OsKASI protein was localized in the plastid. In addition, OsKASI deficiency resulted in reduced fertility and a remarkable change in fatty acid (FA) composition and contents in roots and seeds. Our results demonstrate that involvement of OsKASI in FA synthesis is required for root development in rice. OsKASI Iron deficiency regulated OsOPT7 is essential for iron homeostasis in rice. 2015 Plant Mol Biol Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan. The molecular mechanism of iron (Fe) uptake and transport in plants are well-characterized; however, many components of Fe homeostasis remain unclear. We cloned iron-deficiency-regulated oligopeptide transporter 7 (OsOPT7) from rice. OsOPT7 localized to the plasma membrane and did not transport Fe(III)-DMA or Fe(II)-NA and GSH in Xenopus laevis oocytes. Furthermore OsOPT7 did not complement the growth of yeast fet3fet4 mutant. OsOPT7 was specifically upregulated in response to Fe-deficiency. Promoter GUS analysis revealed that OsOPT7 expresses in root tips, root vascular tissue and shoots as well as during seed development. Microarray analysis of OsOPT7 knockout 1 (opt7-1) revealed the upregulation of Fe-deficiency-responsive genes in plants grown under Fe-sufficient conditions, despite the high Fe and ferritin concentrations in shoot tissue indicating that Fe may not be available for physiological functions. Plants overexpressing OsOPT7 do not exhibit any phenotype and do not accumulate more Fe compared to wild type plants. These results indicate that OsOPT7 may be involved in Fe transport in rice. OPT|OsOPT7 Screening of rice (Oryza sativa L.) OsPR1b-interacting factors and their roles in resisting bacterial blight. 2015 Genet Mol Res Institute of Grassland Science, Northeast Normal University, Key Laboratory of Vegetation Ecology, Ministry of Education, Northeast Normal University, Changchun, China. PR genes, a type of genetic marker, are constitutively expressed at background levels, while being easily inducible by pathogenic bacteria. By using a yeast two-hybrid technique, four rice (Oryza sativa L.) OsPR1b-interacting factors were screened. Homozygous plants overexpressing OsPR1b were prepared by transgenic technology. We postulated that OsPR1b may participate in the resistance signaling pathway of rice. Of simultaneous treatments with hormones and pathogenic bacteria, exogenously applying JA and ET significantly increased the expression level of OsPR1b genes in seedlings. Compared with the control group that was inoculated with water, inoculation with a mixture of water and pathogenic bacteria hardly affected the expression level of OsPR1b gene, while cotreatment with SA and pathogenic bacteria slightly upregulated the expression level. However, cotreatment with JA or ET and pathogenic bacteria managed to significantly upregulate the expression level of the OsPR1b gene by 4.8 or 5.7 fold. PR genes, which are sensitive, are prone to many unknown factors during expression, and the detailed regulatory mechanisms in rice still require in-depth studies. OsPR1b Opportunities of marker-assisted selection for rice fragrance through marker-trait association analysis of microsatellites and gene-based markers. 2015 Plant Biol (Stuttg) Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia. Developing fragrant rice through marker-assisted/aided selection (MAS) is an economical and profitable approach worldwide for the enrichment of an elite genetic background with a pleasant aroma. The PCR-based DNA markers that distinguish the alleles of major fragrance genes in rice have been synthesized to develop rice scent biofortification through MAS. Thus, the present study examined the aroma biofortification potential of these co-dominant markers in a germplasm panel of 189 F2 progenies developed from crosses between a non-aromatic variety (MR84) and a highly aromatic but low-yielding variety (MRQ74) to determine the most influential diagnostic markers for fragrance biofortification. The SSRs and functional DNA markers RM5633 (on chromosome 4), RM515, RM223, L06, NKSbad2, FMbadh2-E7, BADEX7-5, Aro7, and SCU015RM (on chromosome 8) were highly associated with the 2AP (2-acetyl-1-pyrroline) content across the population. The alleles traced via these markers were also in high linkage disequilibrium (R(2) >0.70) and explained for approximately 12.1, 27.05, 27.05, 27.05, 25.42, 25.42, 20.53, 20.43, and 20.18% of the total phenotypic variation observed for these biomarkers, respectively. F2 plants harboring the favorable alleles of these effective markers produced higher levels of fragrance. Hence, these rice plants can be used as donor parents to increase the development of fragrance-biofortified tropical rice varieties adapted to growing conditions and consumer preferences, thus contributing to the global rice market. This article is protected by copyright. All rights reserved. None Evaluation of reference genes for RT-qPCR studies in the leaves of rice seedlings under salt stress. 2015 Genet Mol Res Departamento de Botânica, Universidade Federal de Pelotas, Pelotas, RS, Brasil gabriela.moraes.freitas@gmail.com. To obtain accurate and reliable results for the expression of genes of interest using quantitative real-time polymerase chain reaction (RT-qPCR) techniques, it is necessary to normalize the data by comparing them to constitutive genes that exhibit uniform expression levels under experimental conditions. In this study, the stability of expression was evaluated for the following ten candidate reference genes in rice leaves (Oryza sativa L.) from the BRS Bojuru and BRS Ligeirinho genotypes that were subjected to salt stress (150 mM): actin 11 (ACT11), beta-tubulin (beta-TUB), eukaryote elongation factor 1-α (Eef-1), eukaryotic initiation factor 4-α (eIF-4-α), E2 ubiquitin-conjugating enzyme (UBC-E2), ubiquitin 5 (UBQ5), ubiquitin 10 (UBQ10), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TIP41-like, and cyclophilin. The stability of expression for the aforementioned genes was then compared to that of three LTP genes using UBQ10, Eef-1, and eIF-4-α as references. After analyzing the expression levels using analysis of variance tests, the results indicated that UBQ10 was the most stable in all treatments (M = 0.404 and SV = 0.327). Furthermore, the eIF-4-α, TIP41-like, and cyclophilin genes exhibited the highest total coefficient of variation (CV = 269, 169.2, 179.2, respectively), which signifies that they exhibited the least stable expression. The expression levels of each candidate gene (LTP7, LTP10, and LTP13) were in contrast to the reference genes. Therefore, we concluded that UBQ10 is the best reference gene for RT-qPCR reactions under the experimental conditions. The expression analysis of LTP7, LTP10, and LTP13 confirmed the importance of validating reference genes to achieve accurate RT-qPCR results. None TALEN-mediated targeted mutagenesis produces a large variety of heritable mutations in rice. 2015 Plant Biotechnol J Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, China. CRISPR/Cas9 and TALEN are currently the two systems of choice for genome editing. We have studied the efficiency of the TALEN system in rice as well as the nature and inheritability of TALEN-induced mutations and found important features of this technology. The N287C230 TALEN backbone resulted in low mutation rates (0-6.6%), but truncations in its C-terminal domain dramatically increased efficiency to 25%. In most transgenic T0 plants, TALEN produced a single prevalent mutation accompanied by a variety of low-frequency mutations. For each independent T0 plant, the prevalent mutation was present in most tissues within a single tiller as well as in all tillers examined, suggesting that TALEN-induced mutations occurred very early in the development of the shoot apical meristem. Multigenerational analysis showed that TALEN-induced mutations were stably transmitted to the T1 and T2 populations in a normal Mendelian fashion. In our study, the vast majority of TALEN-induced mutations (~81%) affected multiple bases and ~70% of them were deletions. Our results contrast with published reports for the CRISPR/Cas9 system in rice, in which the predominant mutations affected single bases and deletions accounted for only 3.3% of the overall mutations. None Multiple cold resistance loci confer the high cold tolerance adaptation of Dongxiang wild rice (Oryza rufipogon) to its high-latitude habitat. 2015 Theor Appl Genet Key Laboratory of Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, China. Dongxiang wild rice is phylogenetically close to temperate japonica and contains multiple cold resistance loci conferring its adaptation to high-latitude habitat. Understanding the nature of adaptation in wild populations will benefit crop breeding in the development of climate-resilient crop varieties. Dongxiang wild rice (DXWR), the northernmost common wild rice known, possesses a high degree of cold tolerance and can survive overwintering in its native habitat. However, to date, it is still unclear how DXWR evolved to cope with low-temperature environment, resulting in limited application of DXWR in rice breeding programs. In this study, we carried out both QTL mapping and phylogenetic analysis to discern the genetic mechanism underlying the strong cold resistance. Through a combination of interval mapping and single locus analysis in two genetic populations, at least 13 QTLs for seedling cold tolerance were identified in DXWR. A phylogenetic study using both genome-wide InDel markers and markers associated with cold tolerance loci reveals that DXWR belongs to the Or-III group, which is most closely related to cold-tolerant Japonica rice rather than to the Indica cultivars that are predominant in the habitat where DXWR grows. Our study paves the way toward an understanding of the nature of adaptation to a northern habitat in O. rufipogon. The QTLs identified in DXWR in this study will be useful for molecular breeding of cold-tolerant rice. None The rice ALS3 encoding a novel pentatricopeptide repeat protein is required for chloroplast development and seedling growth. 2015 Rice (N Y) Development Center of Plant Germplasm Resources, College of Life and Environment Sciences, Shanghai Normal University, Shanghai, 200234 China. Pentatricopeptide repeat (PPR) proteins play essential roles in modulating the expression of organelle genes and have expanded greatly in higher plants. However, molecular mechanisms of most rice PPR genes remain unclear.In this study, a new rice PPR mutant, asl3 (albino seedling lethality3) exhibits an albino lethal phenotype at the seedling stage. This albino phenotype was associated with altered photosynthetic-pigment and chloroplast development. Map-based cloning showed that ASL3 encodes a novel rice PPR protein with 10 tandem PPR motifs, which localizes to the chloroplast. ASL3 showed tissue-specific expression, as it was highly expressed in the chlorenchyma, but expressed at much lower levels in roots and panicles. RNAi of ASL3 confirmed that ASL3 plays an essential role in the early development and chloroplast development in rice. Moreover, expression analysis revealed that the asl3 mutation severely affected the transcriptional levels of important genes associated with plastid translation machinery and photosynthesis, which may impair photosynthesis and finally led to the seedling death in asl3 mutant. These results evidenced the important role of ASL3 in the early development of rice, especially chloroplast development.The ASL3 gene encoded a novel chloroplast-targeted PPR protein with 10 tandem PPR motifs in rice. Disruption of the ASL3 would lead to a defective chloroplast and seedling lethality, and affected expression levels of genes associated with chloroplast development and photosynthesis at early leaf stage of rice. ALS3 Expression and Purification of OsVDAC4. 2015 Methods Enzymol National Centre for Biological Sciences, TIFR, Bangalore, India. The voltage-dependent anion channel (VDAC), a major component of the mitochondrial outer membrane, has emerged as an important player in cell function, survival, and death signaling. VDAC function is modulated by its interaction with proteins such as hexokinase, adenine nucleotide translocator, and apoptotic proteins like Bax. Monitoring the activity of VDAC and its modulation in the complex cellular milieu is fraught with complications. Minimizing the number of components in the study is one approach to teasing apart various aspects of its function. In this chapter, we have described detailed protocols for the purification of a rice VDAC isoform, OsVDAC4 after overexpression in a bacterial system. The protein is solubilized with LDAO and then reconstituted into liposomes or planar bilayers to verify its competence to fold into a functionally active form. None Bottlenecks in carotenoid biosynthesis and accumulation in rice endosperm are influenced by the precursor-product balance. 2015 Plant Biotechnol J Department of Plant Production and Forestry Science, ETSEA, University of Lleida-Agrotecnio Center, Lleida, Spain. The profile of secondary metabolites in plants reflects the balance of biosynthesis, degradation and storage, including the availability of precursors and products that affect the metabolic equilibrium. We investigated the impact of the precursor-product balance on the carotenoid pathway in the endosperm of intact rice plants because this tissue does not normally accumulate carotenoids, allowing us to control each component of the pathway. We generated transgenic plants expressing the maize phytoene synthase gene (ZmPSY1) and the bacterial phytoene desaturase gene (PaCRTI), which are sufficient to produce beta-carotene in the presence of endogenous lycopene beta-cyclase. We combined this mini-pathway with the Arabidopsis thaliana genes AtDXS (encoding 1-deoxy-D-xylulose 5-phosphate synthase, which supplies metabolic precursors) or AtOR (the ORANGE gene, which promotes the formation of a metabolic sink). Analysis of the resulting transgenic plants suggested that the supply of isoprenoid precursors from the MEP pathway is one of the key factors limiting carotenoid accumulation in the endosperm and that the overexpression of AtOR increased the accumulation of carotenoids in part by up-regulating a series of endogenous carotenogenic genes. The identification of metabolic bottlenecks in the pathway will help to refine strategies for the creation of engineered plants with specific carotenoid profiles. None Unraveling the Intricate Nexus of Molecular Mechanisms Governing Rice Root Development: OsMPK3/6 and Auxin-Cytokinin Interplay. 2015 PLoS One National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, 110067, India. The root system is an imperative component of a plant, involved in water and nutrient acquisition from the soil. Any subtle change in the root system may lead to drastic changes in plant productivity. Both auxin and cytokinin are implicated in regulating various root developmental aspects. One of the major signaling cascades facilitating various hormonal and developmental allocations is the Mitogen Activated Protein Kinase (MAPK) cascade. Innumerable efforts have been made to unravel the complex nexus involved in rice root development. In spite of a plethora of studies, a comprehensive study aiming to decipher the plausible cross-talk of MAPK signaling module with auxin and cytokinin signaling components in rice is missing. In the present study, extensive phenomics analysis of different stages of rice roots; transcript profiling by qRT-PCR of entire gene family of MAPK, MAPKK and PIN genes; as well as protein level and activity of potential MAPKs was investigated using western and immuno kinase assays both on auxin and cytokinin treatment. The above study led to the identification of various novel rice root specific phenotypic traits by using GiA roots software framework. High expression profile of OsMPK3/6, OsMKK4/5 and OsPIN 1b/9 and their marked transcript level modulation in response to both auxin and cytokinin was observed. Finally, the protein levels and activity assay further substantiated our present findings. Thus, OsMPK3/6-OsMKK4/5 module is elucidated as the putative, key player in auxin-cytokinin interaction augmenting their role by differentially regulating the expression patterns of OsPIN 1b/9 in root development in rice. None MRG702, a reader protein of H3K4me3 and H3K36me3, is involved in brassinosteroid-regulated growth and flowering time control in rice. 2015 Plant Physiol Fudan University; The methylation of histone H3 lysine 36 (H3K36) plays critical roles in brassinosteroid (BR)-related processes, and is involved in controlling flowering time in rice. Although enzymes that catalyze this methylation reaction have been described, little is known about the recognition mechanisms to decipher H3K36 methylation information in rice. In this study, biochemical characterizations showed that MRG702 binds to tri-methylated H3K4 and H3K36 in vitro. Similar to the loss-of-function mutants of the rice H3K36 methyltransferase gene SDG725, the MRG702 knock-down mutants displayed typical BR-deficient mutant and late-flowering phenotypes. Gene transcription analyses showed that MRG702 knock-down resulted in the down-regulation of BR-related genes, including D11, BRI1, and BU1, and several flowering genes including Ehd1, Ehd2/OsID1/RID1, Ehd3,OsMADS50, Hd3a, and RFT1. A binding analysis showed that MRG702 directly binds to the chromatin at target gene loci. This binding is dependent on the level of H3K36me2/me3, which is mediated by SDG725. Together, our results demonstrate that MRG702 acts as a reader protein of H3K4me3 and H3K36me3 and deciphers the H3K36 methylation information set by SDG725. Therefore, the role of MRG702 in the BR pathway and in controlling flowering time in rice is to function as a reader protein to decipher methylation information. MRG702,MRG701 Rice PCR1 influences grain weight and Zn accumulation in grains. 2015 Plant Cell Environ POSTECH-UZH Cooperative Laboratory, Department of Integrative Bioscience and Biotechnology, Pohang University of Science and Technology, Pohang, 790-784, Korea. Proteins containing a placenta-specific 8 domain (PLAC8) function as major organ size regulators in Solanum lycopersicum and Zea may, and putative metal ion transporters in Arabidopsis thaliana, Oryza sativa, and Brassica juncea. However, it is unknown how PLAC8 domain-containing proteins fulfill such diverse roles. Here, we found that plant cadmium resistance 1 (PCR1) influences both zinc (Zn) accumulation and grain weight in rice. OsPCR1-knockout and -knockdown lines produced lighter grains than the wild type, while OsPCR1-overexpression lines produced heavier grains. Furthermore, the grains of OsPCR1-knockdown lines exhibited substantially higher Zn and lower cadmium (Cd) concentrations than the control, as did yeast heterologously expressing OsPCR1. Through sequence analysis, we showed that the amino acid sequence of japonica-type PCR1 was distinct from that of indica-type and wild rice accessions. This difference was correlated with distinct Zn-related phenotypes. Japonica-type PCR1 had a shorter N-terminus than did PCR1 in the other rice types, and yeast heterologously expressing japonica-type PCR1 was more sensitive to Zn than was yeast expressing indica-type PCR1. Furthermore, japonica-type grains accumulated less Zn than did indica-type grains. Our study suggests that rice PCR1 maintains metal ion homeostasis and grain weight and might have been selected for during domestication. OsPCR1 Gene Editing by Co-Transformation of TALEN and Chimeric RNA/DNA Oligonucleotides on the Rice OsEPSPS Gene and the Inheritance of Mutations. 2015 PLoS One Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang Province, China. Although several site-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas, have emerged as powerful tools for targeted gene editing in many organisms, to date, gene targeting (GT) in plants remains a formidable challenge. In the present study, we attempted to substitute a single base in situ on the rice OsEPSPS gene by co-transformation of TALEN with chimeric RNA/DNA oligonucleotides (COs), including different strand composition such as RNA/DNA (C1) or DNA/RNA (C2) but contained the same target base to be substituted. In contrast to zero GT event obtained by the co-transformation of TALEN with homologous recombination plasmid (HRP), we obtained one mutant showing target base substitution although accompanied by undesired deletion of 12 bases downstream the target site from the co-transformation of TALEN and C1. In addition to this typical event, we also obtained 16 mutants with different length of base deletions around the target site among 105 calli lines derived from transformation of TALEN alone (4/19) as well as co-transformation of TELAN with either HRP (5/30) or C1 (2/25) or C2 (5/31). Further analysis demonstrated that the homozygous gene-edited mutants without foreign gene insertion could be obtained in one generation. The induced mutations in transgenic generation were also capable to pass to the next generation stably. However, the genotypes of mutants did not segregate normally in T1 population, probably due to lethal mutations. Phenotypic assessments in T1 generation showed that the heterozygous plants with either one or three bases deletion on target sequence, called d1 and d3, were more sensitive to glyphosate and the heterozygous d1 plants had significantly lower seed-setting rate than wild-type. None MicroRNA399 is involved in multiple nutrient starvation responses in rice. 2015 Front Plant Sci State Key Laboratory of Plant Genomics, National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences Beijing, China. The increasing evidences have revealed that microRNAs (miRNAs) play significant role in nutrient stress response. Previously, miR399 was documented to be induced by phosphorus (P) starvation and involved in regulating P starvation responses. To further investigate the function of miR399 in rice (Oryza sativa L.), we performed GeneChip analysis with OsmiR399 over-expressing plants. Interestingly, our results showed that, besides P starvation responsive genes, the expression of a number of genes involved in iron (Fe), potassium (K), sodium (Na), and calcium (Ca) absorption was dramatically up-regulated in OsmiR399 over-expressing plants. Consistently, the concentrations of Fe, K, Na, and Ca were also increased in OsmiR399 over-expressing plants. The expression of OsmiR399 was also up-regulated by these nutrient starvations, respectively. Moreover, the loss-of-function of LTN1, the down-stream target of OsmiR399, also resulted in the increase of multiple metal elements and the up-regulation of the absorption related genes. These results indicated that OsmiR399 participates in the regulation of multiple nutrient starvation responses, which also gives new view on understanding the interaction among different nutrients mediated by miR399. None Rice FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (OsFKF1) promotes flowering independent of photoperiod. 2015 Plant Cell Environ Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea. In the facultative long-day (LD) plant Arabidopsis thaliana, FLAVIN-BINDING, KELCH REPEAT, F-BOX1 (FKF1) is activated by blue light and promotes flowering through the transcriptional and post-transcriptional regulation of CONSTANS under inductive LD conditions. By contrast, the facultative short day (SD) plant rice (Oryza sativa) flowers early under inductive SD and late under non-inductive LD conditions; the regulatory function of OsFKF1 remains elusive. Here we show that osfkf1 mutants flower late under SD, LD, and natural LD conditions. Transcriptional analysis revealed that OsFKF1 up-regulates expression of the floral activator Ehd2 and down-regulates expression of the floral repressor Ghd7; these regulators up- and down-regulate Ehd1 expression, respectively. Moreover, OsFKF1 can upregulate Ehd1 expression under blue light treatment, without affecting the expression of Ehd2 and Ghd7. In contrast to the LD-specific floral activator Arabidopsis FKF1, OsFKF1 likely acts as an autonomous floral activator because it promotes flowering independent of photoperiod, probably via its distinct roles in controlling expression of rice-specific genes including Ehd2, Ghd7, and Ehd1. Like Arabidopsis FKF1, which interacts with GI and CDF1, OsFKF1 also interacts with OsGI and OsCDF1 (also termed OsDOF12). Thus, we have identified similar and distinct roles of FKF1 in Arabidopsis and rice. FKF1|OsFKF1 The E3 ligase OsPUB15 interacts with the receptor-like kinase PID2 and regulates plant cell death and innate immunity. 2015 BMC Plant Biol Fail Rice blast disease is one of the most destructive diseases of rice worldwide. We previously cloned the rice blast resistance gene Pid2, which encodes a transmembrane receptor-like kinase containing an extracellular B-lectin domain and an intracellular serine/threonine kinase domain. However, little is known about Pid2-mediated signaling.Here we report the functional characterization of the U-box/ARM repeat protein OsPUB15 as one of the PID2-binding proteins. We found that OsPUB15 physically interacted with the kinase domain of PID2 (PID2K) in vitro and in vivo and the ARM repeat domain of OsPUB15 was essential for the interaction. In vitro biochemical assays indicated that PID2K possessed kinase activity and was able to phosphorylate OsPUB15. We also found that the phosphorylated form of OsPUB15 possessed E3 ligase activity. Expression pattern analyses revealed that OsPUB15 was constitutively expressed and its encoded protein OsPUB15 was localized in cytosol. Transgenic rice plants over-expressing OsPUB15 at early stage displayed cell death lesions spontaneously in association with a constitutive activation of plant basal defense responses, including excessive accumulation of hydrogen peroxide, up-regulated expression of pathogenesis-related genes and enhanced resistance to blast strains. We also observed that, along with plant growth, the cell death lesions kept spreading over the whole seedlings quickly resulting in a seedling lethal phenotype.These results reveal that the E3 ligase OsPUB15 interacts directly with the receptor-like kinase PID2 and regulates plant cell death and blast disease resistance. OsPUB15 The OsSec18 complex interacts with P0(P1-P2)2 to regulate vacuolar morphology in rice endosperm cell. 2015 BMC Plant Biol Fail Sec18p/N-ethylmaleimide-sensitive factor (NSF) is a conserved eukaryotic ATPase, which primarily functions in vesicle membrane fusion from yeast to human. However, the function of the OsSec18 gene, a homologue of NSF in rice, remains unknown.In the present study, we investigated the function of OsSec18 in rice and found that OsSec18 complements the temperature-sensitive phenotype and interferes with vacuolar morphogenesis in yeast. Overexpression of OsSec18 in rice decreased the plant height and 1000-grain weight and altered the morphology of the protein bodies. Further examination revealed that OsSec18 presented as a 290-kDa complex in rice endosperm cells. Moreover, Os60sP0 was identified a component of this complex, demonstrating that the OsSec18 complex contains another complex of P0(P1-P2)2 in rice endosperm cells. Furthermore, we determined that the N-terminus of OsSec18 can interact with the N- and C-termini of Os60sP0, whereas the C-terminus of OsSec18 can only interact with the C-terminus of Os60sP0.Our results revealed that the OsSec18 regulates vacuolar morphology in both yeast and rice endosperm cell and the OsSec18 interacts with P0(P1-P2)2 complex in rice endosperm cell. OsSec18 Characterization and fine mapping of a female fertility associated gene Ff1(t) in rice. 2015 J Genet Key Laboratory of Agriculture Responding to Climate Change, Jiangxi Agricultural University, Nanchang 30045, People's Republic of China. shen_xh20000913@126.com. Female-sterile line can be used as a pollinator which has a great potential for hybrid seeds production. However, reports on female fertility are fewer than male fertility. Here, we characterized a recessive female fertility weakening mutant ff1(t) from rice. The spikelet fertility was seriously affected in the mutant. Reciprocal crosses and pollen vitality assay suggest that the decreased fertility was caused by the defective female gametophytes. Further investigation indicated that the mutant ovary development was inhibited before fertilization and failed swelling after flowering. Genetic analysis and fine mapping showed that the mutant was controlled by a single recessive gene, residing on a 16.8 kb region on the long arm of chromosome 1. The gene annotation indicated that there was only one putative gene encoding lysine decarboxylase-like protein in this region, which was allelic to LOG. Further, the sequence analysis was carried out and a substitution at the splice site of intron 2 / exon 3 was revealed in ff1(t) mutant, resulting in the change of reading frame. The finding of novel allele of LOG locus will facilitate the understanding of the mechanisms of female gametophyte development. None QTLs underlying natural variation of root growth angle among rice cultivars with the same functional allele of DEEPER ROOTING 1. 2015 Rice (N Y) National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602 Japan. The functional allele of the rice gene DEEPER ROOTING 1 (DRO1) increases the root growth angle (RGA). However, wide natural variation in RGA is observed among rice cultivars with the functional DRO1 allele. To elucidate genetic factors related to such variation, we quantitatively measured RGA using the basket method and analyzed quantitative trait loci (QTLs) for RGA in three F2 mapping populations derived from crosses between the large RGA-type cultivar Kinandang Patong and each of three accessions with varying RGA: Momiroman has small RGA and was used to produce the MoK-F2 population; Yumeaoba has intermediate RGA (YuK-F2 population); Tachisugata has large RGA (TaK-F2 population). All four accessions belong to the same haplotype group of functional DRO1 allele.We detected the following statistically significant QTLs: one QTL on chromosome 4 in MoK-F2, three QTLs on chromosomes 2, 4, and 6 in YuK-F2, and one QTL on chromosome 2 in TaK-F2. Among them, the two QTLs on chromosome 4 were located near DRO2, which has been previously reported as a major QTL for RGA, whereas the two major QTLs for RGA on chromosomes 2 (DRO4) and 6 (DRO5) were novel. With the LOD threshold reduced to 3.0, several minor QTLs for RGA were also detected in each population.Natural variation in RGA in rice cultivars carrying functional DRO1 alleles may be controlled by a few major QTLs and by several additional minor QTLs. None Allelic effects on starch structure and properties of six starch biosynthetic genes in a rice recombinant inbred line population. 2015 Rice (N Y) CSIRO Agriculture Flagship, GPO Box 1600, Canberra, ACT 2601 Australia ; College of Medicine, Biology and Environment, Australian National University, Canberra, ACT 0200 Australia. The genetic diversity of six starch biosynthetic genes (Wx, SSI, SSIIa, SBEI, SBEIIa and SBEIIb) in indica and japonica rices opens an opportunity to produce a new variety with more favourable grain starch quality. However, there is limited information about the effects of these six gene allele combinations on starch structure and properties. A recombinant inbred line population from a cross between indica and japonica varieties offers opportunities to combine specific alleles of the six genes.The allelic (indica vs japonica) effects of six starch biosynthetic genes on starch structure, functional properties, and abundance of granule bound proteins in rice grains were investigated in a common genetic background using a recombinant inbred line population. The indica Wx (Wxi) allele played a major role while indica SSI (SSIi), japonica SSIIa (SSIIaj) and indica SBEI (SBEIi) alleles had minor roles on the increase of amylose content. SSIIaj and japonica SBEIIb (SBEIIbj) alleles had a major and a minor role on high ratio of ∑DP ≤ 10 to ∑DP ≤ 24 fractions (RCL10/24), respectively. Both major alleles (Wxi and SSIIaj) reduced peak viscosity (PV), onset, peak and end gelatinization temperatures (GTs) of amylopectin, and increased amylose-lipid complex dissociation enthalpy compared with their counterpart-alleles, respectively. SBEIIai and SBEIIbj decreased PV, whereas SSIi and SBEIIbj decreased FV. SBEIi reduced setback viscosity and gelatinization enthalpy. RCL10/24 of chain length distribution in amylopectin is negatively correlated with PV and BD of paste property and GTs of thermal properties. We also report RILs with superior starch properties combining Wxi, SSIj, SSIIaj, SBEIi and SBEIIbj alleles. Additionally, a clear relation is drawn to starch biosynthetic gene alleles, starch structure, properties, and abundance of granule bound starch biosynthetic enzymes inside starch granules.Rice Wxi and SSIIaj alleles play major roles, while SSIi, SBEIi, SBEIIai and SBEIIbj alleles have minor roles in the determination of starch properties between indica and japonica rice through starch structural modification. The combination of these alleles is a key factor for starch quality improvement in rice breeding programs. RCL10/24 value is critical for starch structure and property determination. None Abiotic stress and genome dynamics: specific genes and transposable elements response to iron excess in rice. 2015 Rice (N Y) Plant Genomics and Breeding Center, Eliseu Maciel School of Agronomy, Federal University of Pelotas, 96010-610 Pelotas, RS Brazil ; Present address: Universidade Tecnológica Federal do Paraná, Campus Pato Branco, 85503-390 Pato Branco, PR Brazil. Iron toxicity is a root related abiotic stress, occurring frequently in flooded soils. It can affect the yield of rice in lowland production systems. This toxicity is associated with high concentrations of reduced iron (Fe(2+)) in the soil solution. Although the first interface of the element is in the roots, the consequences of an excessive uptake can be observed in several rice tissues. In an original attempt to find both genes and transposable elements involved in the response to an iron toxicity stress, we used a microarray approach to study the transcriptional responses of rice leaves of cv. Nipponbare (Oryza sativa L. ssp. japonica) to iron excess in nutrient solution.A large number of genes were significantly up- or down-regulated in leaves under the treatment. We analyzed the gene ontology and metabolic pathways of genes involved in the response to this stress and the cis-regulatory elements (CREs) present in the promoter region of up-regulated genes. The majority of genes act in the pathways of lipid metabolic process, carbohydrate metabolism, biosynthesis of secondary metabolites and plant hormones. We also found genes involved in iron acquisition and mobilization, transport of cations and regulatory mechanisms for iron responses, and in oxidative stress and reactive oxygen species detoxification. Promoter regions of 27% of genes up-regulated present at least one significant occurrence of an ABA-responsive CRE. Furthermore, and for the first time, we were able to show that iron stress triggers the up-regulation of many LTR-retrotransposons. We have established a complete inventory of transposable elements transcriptionally activated under iron excess and the CREs which are present in their LTRs.The short-term response of Nipponbare seedlings to iron excess, includes activation of genes involved in iron homeostasis, in particular transporters, transcription factors and ROS detoxification in the leaves, but also many transposable elements. Our data led to the identification of CREs which are associated with both genes and LTR-retrotransposons up-regulated under iron excess. Our results strengthen the idea that LTR-retrotransposons participate in the transcriptional response to stress and could thus confer an adaptive advantage for the plant. None Multiple tissue-specific expression of rice seed-shattering gene SH4 regulated by its promoter pSH4. 2015 Rice (N Y) Ministry of Education Key Laboratory for Biodiversity and Ecological Engineering, Institute of Biodiversity Science, Fudan University, Songhu Road 2005, Shanghai, 200436 China. Rice seed shattering is an important domestication syndrome encoded by a gene named as SH4. The coding region of SH4 has been well studied regarding its function and roles in evolution. However, its promoter has not been identified, which limited our understanding of the detailed regulatory mechanisms of this gene. It is therefore critical to characterize the promoter and study its expression pattern.We analyzed the 5' upstream sequences of this gene and identified a ~2.6 kb fragment with typical promoter features, which was designated as pSH4. The promoter contained a number of cis-acting elements related to abscisic acid (ABA) and a CpG island that were characteristics of multiple tissue-specific expression. We isolated and ligated pSH4 to the beta-glucuronidase (GUS) reporter gene, and transformed it into a japonica rice cultivar to determine the multiple expression pattern of SH4. Histochemical location and fluorescence analyses of GUS activity of transgenic plants indicated multiple tissue-specific expression of pSH4 in the seed-pedicel junction region of mature panicles (with highest level), stems, coleoptiles of germinated seeds, and scutella of mature seeds.The multiple tissue-specific expression pSH4 is categorized as a spatiotemporal promoter that drives the expression of the SH4 gene in different rice tissues, in addition to the seed-pedicel junction region. Our findings suggest that SH4 may have additional functions in the growth and development of rice, apart from its major role in seed shattering. sh4|SHA1 Molecular breeding of thermo-sensitive genic male sterile (TGMS) lines of rice for blast resistance using Pi2 gene. 2015 Rice (N Y) National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, 430070 China. Blast disease caused by the fungal pathogen Magnaporthe oryzae is one of the big problems in rice production in China, especially for high yield hybrid varieties made from a two-line system in which thermo-sensitive genic male sterile (TGMS) lines are used. In this study, we report the introgression of a rice blast resistance gene Pi2 from VE6219 into C815S, an elite rice TGMS line, leading to the development of blast resistant TGMS lines through marker assisted selection (MAS) and phenotypic selection approaches.Four new TGMS lines with blast resistance gene Pi2 were developed from C815S (an elite TGMS line susceptible to the blast, used as recurrent parent) and VE6219 (a blast resistant line harboring Pi2, used as donor parent). The pathogenicity assays inoculated with 53 blast prevalent isolates in glasshouse showed that the blast resistant frequency of the four TGMS lines was 94.3%-98.1% that is equivalent to blast resistant donor parent VE6219. The field evaluation of the new lines and hybrids made from them at a blast epidemic site also showed high resistant levels against the blast. The genetic background of the newly developed TGMS lines were examined using a whole-genome single nucleotide polymorphism (SNP) array (RICE6K) that turned out more than 83% of the genomic markers were derived from the recurrent parent. The critical temperature points of fertility-sterility alteration of the new TGMS lines were between 22°C and 23°C of daily mean temperature, which is similar to that of C815S. The complete male sterility under natural growth conditions at Wuhan last more than 80 days. Their agronomic and grain quality traits meet the requirement for two-line hybrid rice production.The broad-spectrum and durable rice blast resistant gene Pi2 was introgressed into the elite TGMS line C815S background. The newly developed TGMS lines can be practically used for two-line hybrid rice breeding and must play an important role in sustainable rice production in China. Pi9|Piz-t|Pi2 A QTL for root growth angle on rice chromosome 7 is involved in the genetic pathway of DEEPER ROOTING 1. 2015 Rice (N Y) National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602 Japan. Root growth angle (RGA) is an important trait that influences the ability of rice to avoid drought stress. DEEPER ROOTING 1 (DRO1), which is a major quantitative trait locus (QTL) for RGA, is responsible for the difference in RGA between the shallow-rooting cultivar IR64 and the deep-rooting cultivar Kinandang Patong. However, the RGA differences between these cultivars cannot be fully explained by DRO1. The objective of this study was to identify new QTLs for RGA explaining the difference in RGA between these cultivars.By crossing IR64 (which has a non-functional allele of DRO1) with Kinandang Patong (which has a functional allele of DRO1), we developed 26 chromosome segment substitution lines (CSSLs) that carried a particular chromosome segment from Kinandang Patong in the IR64 genetic background. Using these CSSLs, we found only one chromosomal region that was related to RGA: on chromosome 9, which includes DRO1. Using an F2 population derived from a cross between Kinandang Patong and the Dro1-NIL (near isogenic line), which had a functional DRO1 allele in the IR64 genetic background, we identified a new QTL for RGA (DRO3) on the long arm of chromosome 7.DRO3 may only affect RGA in plants with a functional DRO1 allele, suggesting that DRO3 is involved in the DRO1 genetic pathway. None Pseudo-backcrossing design for rapidly pyramiding multiple traits into a preferential rice variety. 2015 Rice (N Y) Rice Science Center, Kasetsart University, Kamphaeng Saen, Nakhon Pathom 73140 Thailand ; Interdisciplinary Graduate Program in Genetic Engineering, Kasetsart University, Chatuchak, Bangkok 10900 Thailand. Pyramiding multiple genes into a desirable genetic background can take years to accomplish. In this paper, a pseudo-backcrossing scheme was designed to shorten the backcrossing cycle needed. PinK3, an aromatic and potentially high-yielding rice variety-although one that is intolerant to flash flooding (Sub) and susceptible to bacterial leaf blight (BB), leaf-neck blast (BL) and the brown planthopper (BPH)-was used as a genetic basis for significant improvements through gene pyramiding.Four resistance donors with five target genes (Sub1A-C, xa5, Xa21, TPS and SSIIa) and three QTLs (qBph3, qBL1 and qBL11) were backcrossed individually using markers into the pseudo-recurrent parent 'PinK3' via one cycle of backcrossing followed by two cycles of pseudo-backcrossing and three selfings with rigorous foreground marker-assisted selection. In total, 29 pseudo-backcross inbred lines (BILs) were developed. Genome composition was surveyed using 61 simple sequence repeats (SSRs), 35 of which were located on six carrier chromosomes, with the remainder located on six non-carrier chromosomes. The recurrent genome content (%RGC) and donor genome content (%DGC), which were based on the physical positions of BC1F2, ranged from 69.99 to 88.98% and 11.02 to 30.01%, respectively. For the pseudo-BC3F3BILs, the %RGC and %DGC ranged from 74.50 to 81.30% and 18.70 to 25.50%, respectively. These results indicated that without direct background selection, no further increases in %RGC were obtained during pseudo-backcrossing, whereas rigorous foreground marker-assisted selection tended to reduce linkage drag during pseudo-backcrossing. The evaluation of new traits in selected pseudo-BC3F3BILs indicated significant improvements in resistance to BB, BL, BPH and Sub compared with PinK3, as well as significant improvements in grain yield (21-68%) over the donors, although yield was 7-26% lower than in 'PinK3'. All pyramided lines were aromatic and exhibited improved starch profiles, rendering them suitable for industrial food applications.Results show that our new pyramiding platform, which is based on marker-assisted pseudo-backcrossing, can fix five target genes and three QTLs into a high-yielding pseudo-recurrent background within seven breeding cycles in four years. This multiple pseudo-backcrossing platform decreases the time required to generate new rice varieties exhibiting complex, durable resistance to biotic and abiotic stresses in backgrounds with desirable qualities. None Current advance methods for the identification of blast resistance genes in rice. 2015 C R Biol Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; Department of Plant Breeding and Genetics, Faculty of Crop Production, Sindh Agriculture University Tandojam, Sindh, Pakistan. Rice blast caused by Magnaporthe oryzae is one of the most devastating diseases of rice around the world and crop losses due to blast are considerably high. Many blast resistant rice varieties have been developed by classical plant breeding and adopted by farmers in various rice-growing countries. However, the variability in the pathogenicity of the blast fungus according to environment made blast disease a major concern for farmers, which remains a threat to the rice industry. With the utilization of molecular techniques, plant breeders have improved rice production systems and minimized yield losses. In this article, we have summarized the current advanced molecular techniques used for controlling blast disease. With the advent of new technologies like marker-assisted selection, molecular mapping, map-based cloning, marker-assisted backcrossing and allele mining, breeders have identified more than 100 Pi loci and 350 QTL in rice genome responsible for blast disease. These Pi genes and QTLs can be introgressed into a blast-susceptible cultivar through marker-assisted backcross breeding. These molecular techniques provide timesaving, environment friendly and labour-cost-saving ways to control blast disease. The knowledge of host-plant interactions in the frame of blast disease will lead to develop resistant varieties in the future. None Axillary Meristem Formation in Rice Requires the WUSCHEL Ortholog TILLERS ABSENT1. 2015 Plant Cell Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan Department of Integrated Bioscience, Graduate School of Frontier Science, University of Tokyo, Kashiwa-shi, Chiba, Tokyo 277-8562, Japan. Axillary shoot formation is a key determinant of plant architecture. Formation of the axillary shoot is regulated by initiation of the axillary meristem or outgrowth of the axillary bud. Here, we show that rice (Oryza sativa) TILLERS ABSENT1 (TAB1; also known as Os WUS), an ortholog of Arabidopsis thaliana WUS, is required to initiate axillary meristem development. We found that formation of the axillary meristem in rice proceeds via a transient state, which we term the premeristem, characterized by the expression of OSH1, a marker of indeterminate cells in the shoot apical meristem. In the tab1-1 (wus-1) mutant, however, formation of the axillary meristem is arrested at various stages of the premeristem zone, and OSH1 expression is highly reduced. TAB1/WUS is expressed in the premeristem zone, where it shows a partially overlapping pattern with OSH1. It is likely, therefore, that TAB1 plays an important role in maintaining the premeristem zone and in promoting the formation of the axillary meristem by promoting OSH1 expression. Temporal expression patterns of WUSCHEL-RELATED HOMEOBOX4 (WOX4) indicate that WOX4 is likely to regulate meristem maintenance instead of TAB1 after establishment of the axillary meristem. Lastly, we show that the prophyll, the first leaf in the secondary axis, is formed from the premeristem zone and not from the axillary meristem. MOC3|OsWUS Ethylene Responses in Rice Roots and Coleoptiles Are Differentially Regulated by a Carotenoid Isomerase-Mediated Abscisic Acid Pathway. 2015 Plant Cell State Key Lab of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China University of Chinese Academy of Sciences, Beijing 100049, China. Ethylene and abscisic acid (ABA) act synergistically or antagonistically to regulate plant growth and development. ABA is derived from the carotenoid biosynthesis pathway. Here, we analyzed the interplay among ethylene, carotenoid biogenesis, and ABA in rice (Oryza sativa) using the rice ethylene response mutant mhz5, which displays a reduced ethylene response in roots but an enhanced ethylene response in coleoptiles. We found that MHZ5 encodes a carotenoid isomerase and that the mutation in mhz5 blocks carotenoid biosynthesis, reduces ABA accumulation, and promotes ethylene production in etiolated seedlings. ABA can largely rescue the ethylene response of the mhz5 mutant. Ethylene induces MHZ5 expression, the production of neoxanthin, an ABA biosynthesis precursor, and ABA accumulation in roots. MHZ5 overexpression results in enhanced ethylene sensitivity in roots and reduced ethylene sensitivity in coleoptiles. Mutation or overexpression of MHZ5 also alters the expression of ethylene-responsive genes. Genetic studies revealed that the MHZ5-mediated ABA pathway acts downstream of ethylene signaling to inhibit root growth. The MHZ5-mediated ABA pathway likely acts upstream but negatively regulates ethylene signaling to control coleoptile growth. Our study reveals novel interactions among ethylene, carotenogenesis, and ABA and provides insight into improvements in agronomic traits and adaptive growth through the manipulation of these pathways in rice. OsCRTISO|ZEBRA2|MHZ5 Characterization and immunomodulatory activity of rice hull polysaccharides. 2015 Carbohydr Polym School of Medicine, Department of Pharmacology, China Medical University, Taichung, Taiwan. Rice hulls (Oryza sativa) are high in carbohydrate content and have been utilized as dietary fiber. The immunomodulatory bioactivity of rice hull polysaccharides (RHPS) has rarely been reported. This study demonstrated the structural characteristics and immunomodulating of RHPS. The RHPS were fractioned using DEAE-650M column, producing one neutral and 3 acidic polysaccharide fractions. RHPS were examined using HPAEC-PAD, HP-SEC, NMR and GC-MS for structural characteristics. The results showed that RHPS consisted of arabinose, galactose, glucose, mannose, and xylose in ratios of 10:44.8:29.8:9.3:6.1 and comprised (1→3)-Gal as backbone, and its average molecular weight was 77kDa. The presence of type II arabinogalactan (AGII) was confirmed through LM2-ELISA and Yariv gel diffusion showed the RHPS had AGII features. This study examined the immunomodulatory effects of orally administering RHPS in vivo. The RHPS increased the cytotoxicity of splenic natural killer cells, macrophage phagocytosis, and cytokine inductions. This is the first study to demonstrate the structural characteristics of an active polysaccharide from rice hulls and its immunopharmacological effects in vivo. None Metabolic and transcriptomic signatures of rice floral organs reveal sugar starvation as a factor in reproductive failure under heat and drought stress. 2015 Plant Cell Environ Max-Planck-Institute of Molecular Plant Physiology, Am Mühlenberg 1, D-14476 Potsdam, Germany. Heat and drought stress are projected to become major challenges to sustain rice (Oryza sativa L.) yields with global climate change. Both stresses lead to yield losses when they coincide with flowering. A significant knowledge gap exists in the mechanistic understanding of the responses of rice floral organs that determine reproductive success under stress. Our work connects the metabolomic and transcriptomic changes in anthers, pistils before pollination and pollinated pistils in a heat tolerant (N22) and a heat sensitive (Moroberekan) cultivar. Systematic analysis of the floral organs revealed contrasts in metabolic profiles across anthers and pistils. Constitutive metabolic markers were identified that can define reproductive success in rice under stress. Six out of nine candidate metabolites identified by intersection analysis of stressed anthers were differentially accumulated in N22 compared to Moroberekan under non-stress conditions. Sugar metabolism was identified to be the crucial metabolic and transcriptional component that differentiated floral organ tolerance or susceptibility to stress. While susceptible Moroberekan specifically showed high expression of the Carbon Starved Anthers (CSA) gene under combined heat and drought, tolerant N22 responded with high expression of genes encoding a sugar transporter (MST8) and a cell wall invertase (INV4) as markers of high sink strength. None Genome-Wide Distribution, Organisation and Functional Characterization of Disease Resistance and Defence Response Genes across Rice Species. 2015 PLoS One National Research Center on Plant Biotechnology, Pusa Campus, New Delhi, 110012, India; School of Life Sciences, Devi Ahilya University, Khandwa Road, Indore, 452017, India. The resistance (R) genes and defense response (DR) genes have become very important resources for the development of disease resistant cultivars. In the present investigation, genome-wide identification, expression, phylogenetic and synteny analysis was done for R and DR-genes across three species of rice viz: Oryza sativa ssp indica cv 93-11, Oryza sativa ssp japonica and wild rice species, Oryza brachyantha. We used the in silico approach to identify and map 786 R -genes and 167 DR-genes, 672 R-genes and 142 DR-genes, 251 R-genes and 86 DR-genes in the japonica, indica and O. brachyanth a genomes, respectively. Our analysis showed that 60.5% and 55.6% of the R-genes are tandemly repeated within clusters and distributed over all the rice chromosomes in indica and japonica genomes, respectively. The phylogenetic analysis along with motif distribution shows high degree of conservation of R- and DR-genes in clusters. In silico expression analysis of R-genes and DR-genes showed more than 85% were expressed genes showing corresponding EST matches in the databases. This study gave special emphasis on mechanisms of gene evolution and duplication for R and DR genes across species. Analysis of paralogs across rice species indicated 17% and 4.38% R-genes, 29% and 11.63% DR-genes duplication in indica and Oryza brachyantha, as compared to 20% and 26% duplication of R-genes and DR-genes in japonica respectively. We found that during the course of duplication only 9.5% of R- and DR-genes changed their function and rest of the genes have maintained their identity. Syntenic relationship across three genomes inferred that more orthology is shared between indica and japonica genomes as compared to brachyantha genome. Genome wide identification of R-genes and DR-genes in the rice genome will help in allele mining and functional validation of these genes, and to understand molecular mechanism of disease resistance and their evolution in rice and related species. None Characterization of the Ubiquitin-Conjugating Enzyme Gene Family in Rice and Evaluation of Expression Profiles under Abiotic Stresses and Hormone Treatments. 2015 PLoS One China National Rice Research Institute, 359 Tiyuchang Road, Hangzhou, 310006, China. Ubiquitin-conjugating enzyme E2s (UBCs), which catalyze the transfer of ubiquitin to substrate or E3 ligases, are key enzymes in ubiquitination modifications of target proteins. However, little is known about the knowledge of UBC gene family in rice. In this study, a total of 39 UBC encoding genes, which all contained an UBC domain with a cysteine active site, were identified in the rice genome. These were classified into fifteen distinct subfamilies based upon their sequence similarity and phylogenetic relationships. A subset of 19 OsUBC genes exhibited chromosomal duplication; 4 and 15 OsUBC genes were tandemly and segmentally duplicated, respectively. Comprehensive analyses were performed to investigate the expression profiles of OsUBC genes in various stages of vegetative and reproductive development using data from EST, Microarrays, MPSS, and real-time PCR. Many OsUBC genes exhibited abundant and tissue-specific expression patterns. Moreover, 14 OsUBCs were found to be differentially expressed under treatments with drought, or salt stresses. The expression analysis after treatments with IAA, 6-BA, GA and ABA indicated that almost all OsUBC genes were responsive to at least two of the four hormones. Several genes were significantly down-regulated under all of the hormone treatments, and most of the genes reduced by 6-BA were also reduced by GA. This study will facilitate further studies of the OsUBC gene family and provide useful clues for functional validation of OsUBCs in rice. None The Tyrosine Aminomutase TAM1 Is Required for beta-Tyrosine Biosynthesis in Rice. 2015 Plant Cell Boyce Thompson Institute for Plant Research, Ithaca, New York 14853. Non-protein amino acids, often isomers of the standard 20 protein amino acids, have defense-related functions in many plant species. A targeted search for jasmonate-induced metabolites in cultivated rice (Oryza sativa) identified (R)-beta-tyrosine, an isomer of the common amino acid (S)-α-tyrosine in the seeds, leaves, roots, and root exudates of the Nipponbare cultivar. Assays with 119 diverse cultivars showed a distinct presence/absence polymorphism, with beta-tyrosine being most prevalent in temperate japonica cultivars. Genetic mapping identified a candidate gene on chromosome 12, which was confirmed to encode a tyrosine aminomutase (TAM1) by transient expression in Nicotiana benthamiana and in vitro enzyme assays. A point mutation in TAM1 eliminated beta-tyrosine production in Nipponbare. Rice cultivars that do not produce beta-tyrosine have a chromosome 12 deletion that encompasses TAM1. Although beta-tyrosine accumulation was induced by the plant defense signaling molecule jasmonic acid, bioassays with hemipteran and lepidopteran herbivores showed no negative effects at physiologically relevant beta-tyrosine concentrations. In contrast, root growth of Arabidopsis thaliana and other tested dicot plants was inhibited by concentrations as low as 1 μM. As beta-tyrosine is exuded into hydroponic medium at higher concentrations, it may contribute to the allelopathic potential of rice. TAM1 Transgenic Expression of the Dicotyledonous Pattern Recognition Receptor EFR in Rice Leads to Ligand-Dependent Activation of Defense Responses. 2015 PLoS Pathog Department of Plant Pathology and the Genome Center, University of California, Davis, Davis, California, United States of America; Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America. Plant plasma membrane localized pattern recognition receptors (PRRs) detect extracellular pathogen-associated molecules. PRRs such as Arabidopsis EFR and rice XA21 are taxonomically restricted and are absent from most plant genomes. Here we show that rice plants expressing EFR or the chimeric receptor EFR::XA21, containing the EFR ectodomain and the XA21 intracellular domain, sense both Escherichia coli- and Xanthomonas oryzae pv. oryzae (Xoo)-derived elf18 peptides at sub-nanomolar concentrations. Treatment of EFR and EFR::XA21 rice leaf tissue with elf18 leads to MAP kinase activation, reactive oxygen production and defense gene expression. Although expression of EFR does not lead to robust enhanced resistance to fully virulent Xoo isolates, it does lead to quantitatively enhanced resistance to weakly virulent Xoo isolates. EFR interacts with OsSERK2 and the XA21 binding protein 24 (XB24), two key components of the rice XA21-mediated immune response. Rice-EFR plants silenced for OsSERK2, or overexpressing rice XB24 are compromised in elf18-induced reactive oxygen production and defense gene expression indicating that these proteins are also important for EFR-mediated signaling in transgenic rice. Taken together, our results demonstrate the potential feasibility of enhancing disease resistance in rice and possibly other monocotyledonous crop species by expression of dicotyledonous PRRs. Our results also suggest that Arabidopsis EFR utilizes at least a subset of the known endogenous rice XA21 signaling components. None Quantitative trait locus analysis and fine mapping of the qPL6 locus for panicle length in rice. 2015 Theor Appl Genet National Key Laboratory of Plant Genetics and National Centre of Plant Gene, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai, 200032, China. Two QTLs were identified to control panicle length in rice backcross lines, and one QTL qPL6 was finely mapped with potential in high yield breeding. Panicle length (PL) is the key determinant of panicle architecture in rice, and strongly affects yield components, such as grain number per panicle. However, this trait has not been well studied genetically nor its contribution to yield improvement. In this study, we performed quantitative trait locus (QTL) analysis for PL in four backcross populations derived from the cross of Nipponbare (japonica) and WS3 (indica), a new plant type (NPT) variety. Two QTLs were identified on chromosome 6 and 8, designated as qPL6 and qPL8, respectively. Near-isogenic lines (NILs) were developed to evaluate their contribution to important agronomic traits. We found that qPL6 and qPL8 had additive effects on PL trait. For the qPL6 locus, the WS3 allele also increased panicle primary and secondary branches and grain number per panicle. Moreover, this allele conferred wide and strong culms, a character of lodging resistance. By analyzing key recombinants in two steps, the qPL6 locus was finely mapped to a 25-kb interval, and 3 candidate genes were identified. According to the single nucleotide polymorphisms (SNPs) within candidate genes, 5 dCaps markers were designed and used to get haplotypes of 96 modern Chinese varieties, which proved that qPL6 locus is differentiated between indica and temperate japonica varieties. Taken together, the superior qPL6 allele can be applied in rice breeding programs for large sink size, particularly for japonica varieties that originally lack the allele. None Downregulation of Rice DWARF 14 LIKE Suppress Mesocotyl Elongation via a Strigolactone Independent Pathway in the Dark. 2015 J Genet Genomics Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Strigolactones (SLs) are a class of plant hormones that control plant development in response to environmental conditions. In rice, mesocotyl elongation is regulated by SLs in the dark, while mesocotyls are longer in SL deficient or insensitive mutants. SLs are perceived by DWARF14 (D14), which is a member of a small gene family. In this study, we examined the function of another D14 family gene in rice, D14 LIKE (D14L), focusing on mesocotyl growth. The mesocotyls of D14L RNAi lines are longer than those of WT in the dark. This phenotype is enhanced when the D14L RNAi lines are combined with the d14 mutation, suggesting that D14 and D14L work independently to inhibit mesocotyl elongation. This phenotype is alleviated by the exogenous supply of GR24, a synthetic SL, suggesting that D14L is not necessary for SL signaling. D14L mRNA is predominantly expressed in vascular bundles and crown root primordia. Our results suggest that D14L and D14 confer their effects via an SL independent pathway and an SL signaling pathway respectively. D14L Physiological, anatomical and transcriptional alterations in a rice mutant leading to enhanced water stress tolerance. 2015 AoB Plants National Research Centre on Plant Biotechnology, IARI, New Delhi, India Department of Botany, North Orissa University, Baripada, Odisha, India. Water stress is one of the most severe constraints to crop productivity. Plants display a variety of physiological and biochemical responses both at cellular and whole organism level upon sensing water stress. Leaf rolling, stomatal closure, deeper root penetration, higher relative water content and better osmotic adjustment are some of the mechanisms plants employ to overcome water stress. In the current study, we report a mutant, ewst1 with enhanced water stress tolerance, identified from the EMS induced mutant population of rice variety Nagina22, by field screening followed by withdrawal of irrigation in pots and hydroponics (PEG 6000). Though ewst1 was morphologically similar to the wild type (WT) for 35 of the 38 morphological descriptors (except chalky endosperm/expression of white core, decorticated grain colour and grain weight), it showed enhanced germination in PEG infused medium. It exhibited increased maximum root length without increase in its root weight, root volume and total root number on crown as compared to the WT under stress in PVC tube experiment. It also showed better performance for various physiological parameters such as relative water content, cell membrane stability and chlorophyll concentration upon water stress in pot experiment. Alterations in number of xylem, phloem, dimension of central metaxylem and number of closed stomata were observed in ewst1 as compared to the WT in root anatomy and stomatal microscopy studies. Comparative genomewide transcriptome analysis identified genes related to exocytosis, secondary metabolites, tryptophan biosynthesis, protein phosphorylation and other signaling pathways to be playing a role in enhanced response to water stress in ewst1. Possible involvement of a candidate gene with respect to the observed morpho-physiological and transcriptional changes and its role in stress tolerance are discussed. The mutant identified and characterized in this study will be useful for further dissection of water stress tolerance in rice. None Spikelet-specific variation in ethylene production and constitutive expression of ethylene receptors and signal transducers during grain filling of compact- and lax-panicle rice (Oryza sativa) cultivars. 2015 J Plant Physiol Environmental Biotechnology Laboratory, Institute of Life Sciences, Bhubaneswar 751023, Odisha, India. Grain yields in modern super rice cultivars do not always meet the expectations because many spikelets are located on secondary branches in closely packed homogeneous distribution in these plants, and they do not fill properly. The factors limiting grain filling of such spikelets, especially in the lower panicle branches, are elusive. Two long-duration rice cultivars differing in panicle density, Mahalaxmi (compact) and Upahar (lax), were cultivated in an open field plot. Grain filling, ethylene production and constitutive expression of ethylene receptors and ethylene signal transducers in apical and basal spikelets of the panicle were compared during the early post-anthesis stage, which is the most critical period for grain development. In another experiment, a similar assessment was made for the medium-duration cultivars compact-panicle OR-1918 and lax-panicle Lalat. Grain weight of the apical spikelets was always higher than that of the basal spikelets. This gradient of grain weight was wide in the compact-panicle cultivars and narrow in the lax-panicle cultivars. Compared to apical spikelets, the basal spikelets produced more ethylene at anthesis and retained the capacity for post-anthesis expression of ethylene receptors and ethylene signal transducers longer. High ethylene production enhanced the expression of the RSR1 gene, but reduced expression of the GBSS1 gene. Ethylene inhibited the partitioning of assimilates of developing grains resulting in low starch biosynthesis and high accumulation of soluble carbohydrates. It is concluded that an increase in grain/spikelet density in rice panicles reduces apical dominance to the detriment of grain filling by production of ethylene and/or enhanced perception of the ethylene signal. Ethylene could be a second messenger for apical dominance in grain filling. The manipulation of the ethylene signal would possibly improve rice grain yield. None Rapid Identification of Major QTLs Associated with Rice Grain Weight and Their Utilization. 2015 PLoS One Institute of Nuclear Agricultural Sciences, College of Agriculture and Biotechnology, Zhejiang University, Huajiachi Campus, Hangzhou, 310029, P. R. China. To uncover the genetics of rice grain weight, we constructed an RIL population derived from a cross between a large grain accession M201 and a small size variety JY293. Specific Locus Amplified Fragment Sequencing (SLAF-Seq) technology was used to genotype two bulked DNA pools made from individual DNA of the heaviest 30 lines and the lightest 30 lines according to the 1000 grain weight (TGW). Bulked segregant analysis (BSA) was used to identify SLAFs strongly associated with TGW. Two marker-intensive regions at 24,600,000-24,850,000 bp and 25,000,000-25,350,000 bp on chromosome 3 were identified tightly related to the TGW. Then a linkage map of chromosome 3 was constructed with SSR markers and some SLAF derived single nucleotide polymorphisms (SNPs). Quantitative trait locus (QTL) mapping for TGW, grain length, grain width, and grain thickness revealed one major QTL in the second hot-region and two other minor QTLs for grain weight. These three QTLs displayed hierarchical effects on grain length and grain weight in order of qTGW3.2 (qGL3) qTGW3.1 (GS3) qTGW3.3. Multiple comparisons of means among the eight combinations of 3 QTLs revealed that the lines with two of three QTLs deriving from M201 displayed a large grain weight phenotype (TGW 40.2g, average data of three years) and lines with both qTGW3.1 and qTGW3.3 alleles from M201 (42.5g) had similar grain weight to the qTGW3.2 (40.8g) alone. Two strategies with similar effectiveness were proposed to improve grain weight by marker-assisted selection (MAS). One is to introduce the novel qTGW3.2 allele alone, and the other is to pyramid qTGW3.1 and qTGW3.3 alleles together. One new allele of GS3 (39 bp deletion in intron 1) and two SNPs in coding sequence of qGL3 identified in this study from M201 are useful in pyramiding elite alleles for molecular breeding for improvement of rice yield. None A new approach for annotation of transposable elements using small RNA mapping. 2015 Nucleic Acids Res Center for Applied Genetic Technologies. University of Georgia, 111, Riverbend Dr., Athens, GA 30602, USA. Transposable elements (TEs) are mobile genomic DNA sequences found in most organisms. They so densely populate the genomes of many eukaryotic species that they are often the major constituents. With the rapid generation of many plant genome sequencing projects over the past few decades, there is an urgent need for improved TE annotation as a prerequisite for genome-wide studies. Analogous to the use of RNA-seq for gene annotation, we propose a new method for de novo TE annotation that uses as a guide 24 nt-siRNAs that are a part of TE silencing pathways. We use this new approach, called TASR (for Transposon Annotation using Small RNAs), for de novo annotation of TEs in Arabidopsis, rice and soybean and demonstrate that this strategy can be successfully applied for de novo TE annotation in plants. Executable PERL is available for download from: http://tasr-pipeline.sourceforge.net/. None RiceNet v2: an improved network prioritization server for rice genes. 2015 Nucleic Acids Res Department of Biotechnology, College of Life Sciences and Biotechnology, Yonsei University, Seoul, Korea. Rice is the most important staple food crop and a model grass for studies of bioenergy crops. We previously published a genome-scale functional network server called RiceNet, constructed by integrating diverse genomics data and demonstrated the use of the network in genetic dissection of rice biotic stress responses and its usefulness for other grass species. Since the initial construction of the network, there has been a significant increase in the amount of publicly available rice genomics data. Here, we present an updated network prioritization server for Oryza sativa ssp. japonica, RiceNet v2 (http://www.inetbio.org/ricenet), which provides a network of 25 765 genes (70.1% of the coding genome) and 1 775 000 co-functional links. Ricenet v2 also provides two complementary methods for network prioritization based on: (i) network direct neighborhood and (ii) context-associated hubs. RiceNet v2 can use genes of the related subspecies O. sativa ssp. indica and the reference plant Arabidopsis for versatility in generating hypotheses. We demonstrate that RiceNet v2 effectively identifies candidate genes involved in rice root/shoot development and defense responses, demonstrating its usefulness for the grass research community. None Establishment of a prediction model for the miRNA-based heading date characteristics of rice in the booting stage. 2015 Genet Mol Res Department of Plant Industry, National Pingtung University of Science & Technology, Pingtung, Taiwan. Rice (Oryza sativa L.) is one of the most important food crops in the world. In Taiwan, due to the warm climate, there are two harvests annually. However, the yield and quality of rice can vary between each crop season in any given year. Previous reports have shown that microRNAs (miRNAs) play a crucial role in many developmental and physiological processes in plants. In this study, the heading date characteristics of 167 rice cultivars from the second crop season were recorded, and 27 rice cultivars were selected for preliminary microarray analysis. A total of 14 miRNAs from different heading date characteristics in 21 cultivars were selected based on significant differences in their expression profiles. Using a correlation analysis between the heading date and selected miRNA expression obtained from real-time polymerase chain reaction (PCR) assays, we developed a heading date prediction model. The model includes nine miRNA genes with corresponding R2 values of 0.8. To confirm the model, a real-time PCR analysis was performed on an additional 27 rice cultivars and we found the model predicted the heading date with accuracy. Therefore, the developed prediction may be useful in further studies aimed at confirming the reliability of the use of miRNA in molecular breeding and to increase the selection efficiency of rice cultivars and breeding. None A comparative study of stress-related gene expression under single stress and intercross stress in rice. 2015 Genet Mol Res State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou, China. Plant hormones play important roles in the crosstalk between biotic and abiotic stresses in rice throughout its entire growth period. However, these interactions are not completely understood. In this study, the physiological performance of rice seedlings under a single stress and a sequential combination of various stresses (intercross stress) was determined. We found that catalase, superoxide dismutase, and peroxidase activities and malondialdehyde were highly regulated by intercross stresses. Furthermore, the expression levels of pathogenesis-related genes and drought stress-related genes under various treatments were analyzed. We demonstrated that under drought-disease intercross stress, the expression levels of the PR4, PAL, and Cht-1 genes were significantly upregulated, while under salt-disease intercross stress, the expression levels of the PR1a, PBZ1, Gns1, and Cht-1 genes underwent significant changes. Regardless of the type of intercross stress, the expression of LOX-RLL was significantly affected. We also showed that the expression of drought stress-related genes OsSKIPa, OsNADPH1, JRC0594, and OsGL1-2 was significantly regulated, suggesting that these genes play important roles in the interaction between biotic and abiotic stresses. We, therefore, conclude that the interactions between various types of biotic and abiotic stresses vary in a complex pattern and would require further in-depth investigation. None Genome-wide polymorphisms between the parents of an elite hybrid rice and the development of a novel set of PCR-based InDel markers. 2015 Genet Mol Res Chinese National Center for Rice Improvement/State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou, China. Genome-wide re-sequencing of the Zhenshan 97 (ZS97) and Milyang 46 (MY46) parents of an elite three-line hybrid rice developed in China resulted in the generation of 9.91 G bases of data with an effective sequencing depth of 11.66x and 11.51x, respectively. Detection of genome-wide DNA polymorphisms, single nucleotide polymorphisms (SNPs), short insertions/deletions (InDels; 1-5 bp), and structural variations (SVs), which is an invaluable variation resource for genetic research and molecular marker-assisted breeding, was conducted by comparing whole-genome re-sequencing data. A total of 364,488 SNPs, 61,181 InDels and 6298 SVs were detected in ZS97 and 364,179 SNPs, 61,984 InDels and 6408 SVs were detected in MY46 compared to the 9311 reference sequence. Synteny analysis of the variation revealed a total of 77,013 identical and 181,737 different SNPs and 15,021 identical and 1205 different InDels between ZS97 and MY46, respectively. A total of 180 InDels 3-8 bp in length between ZS97 and MY46 were selected for experimental validation; 160 polymerase chain reaction products were efficiently separated on 6% non-denaturing polyacrylamide gels. Identification of genome-wide variation among the parents of the elite hybrid as well as the set of 160 polymerase chain reaction-based InDel markers will facilitate future genetic studies and the molecular breeding of hybrid rice. None Down-regulation of lipoxygenase gene reduces degradation of carotenoids of golden rice during storage. 2015 Planta Laboratory of Translational Research on Transgenic Crops, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, West Bengal, India. Down-regulation of lipoxygenase enzyme activity reduces degradation of carotenoids of bio-fortified rice seeds which would be an effective tool to reduce huge post-harvest and economic losses of bio-fortified rice seeds during storage. Bio-fortified provitamin A-enriched rice line (golden rice) expressing higher amounts of beta-carotene in the rice endosperm provides vitamin A for human health. However, it is already reported that degradation of carotenoids during storage is a major problem. The gene responsible for degradation of carotenoids during storage has remained largely unexplored till now. In our previous study, it has been shown that r9-LOX1 gene is responsible for rice seed quality deterioration. In the present study, we attempted to investigate if r9-LOX1 gene has any role in degradation of carotenoids in rice seeds during storage. To establish our hypothesis, the endogenous lipoxygenase (LOX) activity of high-carotenoid golden indica rice seed was silenced by RNAi technology using aleurone layer and embryo-specific Oleosin-18 promoter. To check the storage stability, LOX enzyme down-regulated high-carotenoid T3 transgenic rice seeds were subjected to artificial aging treatment. The results obtained from biochemical assays (MDA, ROS) also indicated that after artificial aging, the deterioration of LOX-RNAi lines was considerably lower compared to beta-carotene-enriched transgenic rice which had higher LOX activity in comparison to LOX-RNAi lines. Furthermore, it was also observed by HPLC analysis that down-regulation of LOX gene activity decreases co-oxidation of beta-carotene in LOX-RNAi golden rice seeds as compared to the beta-carotene-enriched transgenic rice, after artificial aging treatment. Therefore, our study substantially establishes and verifies that LOX is a key enzyme for catalyzing co-oxidation of beta-carotene and has a significant role in deterioration of beta-carotene levels in the carotenoid-enriched golden rice. None Regulation of Histone Methylation and Reprogramming of Gene Expression in the Rice Inflorescence Meristem. 2015 Plant Cell National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, 430070 Wuhan, China. Rice inflorescence meristem (IM) activity is essential for panicle development and grain production. How chromatin and epigenetic mechanisms regulate IM activity remains unclear. Genome-wide analysis revealed that in addition to genes involved in the vegetative to reproductive transition, many metabolic and protein synthetic genes were activated in IM compared with shoot apical meristem and that a change in the H3K27me3/H3K4me3 ratio was an important factor for the differential expression of many genes. Thousands of genes gained or lost H3K27me3 in IM, and downregulation of the H3K27 methyltransferase gene SET DOMAIN GROUP 711 (SDG711) or mutation of the H3K4 demethylase gene JMJ703 eliminated the increase of H3K27me3 in many genes. SDG711-mediated H3K27me3 repressed several important genes involved in IM activity and many genes that are silent in the IM but activated during floral organogenesis or other developmental stages. SDG711 overexpression augmented IM activity and increased panicle size; suppression of SDG711 by RNA interference had the opposite effect. Double knockdown/knockout of SDG711 and JMJ703 further reduced panicle size. These results suggest that SDG711 and JMJ703 have agonistic functions in reprogramming the H3K27me3/H3K4me3 ratio and modulating gene expression in the IM. JMJ703 The rice transcription factor OsWRKY47 is a positive regulator of the response to water deficit stress. 2015 Plant Mol Biol Instituto de Agrobiotecnología del Litoral (CONICET-UNL), Colectora ruta 168 km 0, Paraje El Pozo, 3000, Santa Fe, Argentina. OsWRKY47 is a divergent rice transcription factor belonging to the group II of the WRKY family. A transcriptomic analysis of the drought response of transgenic rice plants expressing P SARK ::IPT, validated by qPCR, indicated that OsWRKY47 expression was induced under drought stress in P SARK ::IPT plants. A PCR-assisted site selection assay (SELEX) of recombinant OsWRKY47 protein showed that the preferred sequence bound in vitro is (G/T)TTGACT. Bioinformatics analyses identified a number of gene targets of OsWRKY47; among these two genes encode a Calmodulin binding protein and a Cys-rich secretory protein. Using Oswrk47 knockout mutants and transgenic rice overexpressing OsWRKY47 we show that the transcription of these putative targets were regulated by OsWRKY47. Phenotypic analysis carried out with transgenic rice plants showed that Oswrky47 mutants displayed higher sensitivity to drought and reduced yield, while plants overexpressing OsWRKY47 were more tolerant. OsWRKY47 Fine-mapping and validating qHTSF4.1 to increase spikelet fertility under heat stress at flowering in rice. 2015 Theor Appl Genet International Rice Research Institute, DAPO Box 7777, 1301, Metro Manila, Philippines, c.ye@irri.org. This study fine mapped and validated a QTL on rice chromosome 4 that increases spikelet fertility under high temperature (over 37 °C) at the flowering stage. Climate change has a negative effect on crop production and food security. Understanding the genetic mechanism of heat tolerance and developing heat-tolerant varieties is essential to cope with future global warming. Previously, we reported on a QTL (qHTSF4.1) from an IR64/N22 population responsible for rice spikelet fertility under high-temperature stress at the flowering stage. To further fine map and validate the effect of qHTSF4.1, PCR-based SNP markers were developed and used to genotype BC2F2, BC3F2, BC3F3, and BC5F2 populations from the same cross. The interval of the QTL was narrowed down to about 1.2 Mb; however, further recombination was not identified even with a large BC5F2 population that was subsequently developed and screened. The sequence in the QTL region is highly conserved and a large number of genes in the same gene family were observed to be clustered in the region. The QTL qHTSF4.1 consistently increased spikelet fertility in all of the backcross populations. This was confirmed using 24 rice varieties. Most of the rice varieties with the QTL showed a certain degree of heat tolerance under high-temperature conditions. In a BC5F2 population with clean background of IR64, QTL qHTSF4.1 increased spikelet fertility by about 15 %. It could be an important source for enhancing heat tolerance in rice at the flowering stage. PCR-based SNP markers developed in this study can be used for QTL introgression and for pyramiding with other agronomically important QTLs/genes through marker-assisted selection. None Molecular characterization and application of a novel cytoplasmic male sterility-associated mitochondrial sequence in rice. 2015 BMC Genet Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China, College of Life Science, South-Central University for Nationalities, Wuhan, 430074, China. yanptan@mail.scuec.edu.cn. Cytoplasmic male sterility (CMS) is a maternally inherited inability to produce functional pollen found in numerous flowering plant species. CMS is associated with mitochondrial DNA mutation, novel chimeric open reading frames (ORFs), and rearrangement of coding and noncoding regions of the mitochondrial genome.BLAST (Basic Local Alignment Search Tool) analysis indicated that L-sp1, a new sequence-characterized amplified region, is non-homologous to atp6-orfH79 (or atp6-orf79) and WA352 cloned CMS-associated genes. L-sp1 was found in 11 of 102 wild rice accessions belonging to four AA genome species: Oryza rufipogon, Oryza nivara, Oryza glumaepatula, and Oryza meridionalis. Using L-sp1, two new CMS lines were developed, from either low natural fertility plants or sterile plants, by backcrossing BC1F1 with Yuetai B. Northern blot and RT-PCR revealed that L-sp1 was only expressed in the anthers of w1/YTB, w2/YTB, w1/YTB//YTB, and w2/YTB//YTB when in the same cytoplasm background.L-sp1 is a single-copy chimeric CMS-associated gene found in the mitochondrial genome. It can be expressed in anthers with the same specific cytoplasm background, and will be a useful molecular marker for the development and marker-assisted selection of new CMS lines. None Transcriptome changes in rice (Oryza sativa L.) in response to high night temperature stress at the early milky stage. 2015 BMC Genomics Key Laboratory of Crop Physiology, Ecology and Genetic Breeding (Jiangxi Agricultural University), Ministry of Education, Jiangxi Province, 330045, China. jlliao514815@163.com. Rice yield and quality are adversely affected by high temperatures, especially at night; high nighttime temperatures are more harmful to grain weight than high daytime temperatures. Unfortunately, global temperatures are consistently increasing at an alarming rate and the minimum nighttime temperature has increased three times as much as the corresponding maximum daytime temperature over the past few decades.We analyzed the transcriptome profiles for rice grain from heat-tolerant and -sensitive lines in response to high night temperatures at the early milky stage using the Illumina Sequencing method. The analysis results for the sequencing data indicated that 35 transcripts showed different expressions between heat-tolerant and -sensitive rice, and RT-qPCR analyses confirmed the expression patterns of selected transcripts. Functional analysis of the differentially expressed transcripts indicated that 21 genes have functional annotation and their functions are mainly involved in oxidation-reduction (6 genes), metabolic (7 genes), transport (4 genes), transcript regulation (2 genes), defense response (1 gene) and photosynthetic (1 gene) processes. Based on the functional annotation of the differentially expressed genes, the possible process that regulates these differentially expressed transcripts in rice grain responding to high night temperature stress at the early milky stage was further analyzed. This analysis indicated that high night temperature stress disrupts electron transport in the mitochondria, which leads to changes in the concentration of hydrogen ions in the mitochondrial and cellular matrix and influences the activity of enzymes involved in TCA and its secondary metabolism in plant cells.Using Illumina sequencing technology, the differences between the transcriptomes of heat-tolerant and -sensitive rice lines in response to high night temperature stress at the early milky stage was described here for the first time. The candidate transcripts may provide genetic resources that may be useful in the improvement of heat-tolerant characters of rice. The model proposed here is based on differences in expression and transcription between two rice lines. In addition, the model may support future studies on the molecular mechanisms underlying plant responses to high night temperatures. None OsTCP19 influences developmental and abiotic stress signaling by modulating ABI4-mediated pathways. 2015 Sci Rep National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi. 110067, India. Class-I TCP transcription factors are plant-specific developmental regulators. In this study, the role of one such rice gene, OsTCP19, in water-deficit and salt stress response was explored. Besides a general upregulation by abiotic stresses, this transcript was more abundant in tolerant than sensitive rice genotypes during early hours of stress. Stress, tissue and genotype-dependent retention of a small in-frame intron in this transcript was also observed. Overexpression of OsTCP19 in Arabidopsis caused upregulation of IAA3, ABI3 and ABI4 and downregulation of LOX2, and led to developmental abnormalities like fewer lateral root formation. Moreover, decrease in water loss and reactive oxygen species, and hyperaccumulation of lipid droplets in the transgenics contributed to better stress tolerance both during seedling establishment and in mature plants. OsTCP19 was also shown to directly regulate a rice triacylglycerol biosynthesis gene in transient assays. Genes similar to those up- or downregulated in the transgenics were accordingly found to coexpress positively and negatively with OsTCP19 in Rice Oligonucleotide Array Database. Interactions of OsTCP19 with OsABI4 and OsULT1 further suggest its function in modulation of abscisic acid pathways and chromatin structure. Thus, OsTCP19 appears to be an important node in cell signaling which crosslinks stress and developmental pathways. OsTCP19,OsABI4,OsULT1 Combination Patterns of Major R Genes Determine the Level of Resistance to the M. oryzae in Rice (Oryza sativa L.). 2015 PLoS One Lixiahe Agricultural Research Institute of Jiangsu Province, Yangzhou, 225007, P.R. China; Key Laboratory of Plant Functional Genomics, Ministry of Education, Yangzhou University, Yangzhou, 225009, P.R. China. Rice blast caused by Magnaporthe oryzae is the most devastating disease of rice and poses a serious threat to world food security. In this study, the distribution and effectiveness of 18 R genes in 277 accessions were investigated based on pathogenicity assays and molecular markers. The results showed that most of the accessions exhibited some degree of resistance (resistance frequency, RF >50%). Accordingly, most of the accessions were observed to harbor two or more R genes, and the number of R genes harbored in accessions was significantly positively correlated with RF. Some R genes were demonstrated to be specifically distributed in the genomes of rice sub-species, such as Pigm, Pi9, Pi5 and Pi1, which were only detected in indica-type accessions, and Pik and Piz, which were just harbored in japonica-type accessions. By analyzing the relationship between R genes and RF using a multiple stepwise regression model, the R genes Pid3, Pi5, Pi9, Pi54, Pigm and Pit were found to show the main effects against M. oryzae in indica-type accessions, while Pita, Pb1, Pik, Pizt and Pia were indicated to exhibit the main effects against M. oryzae in japonica-type accessions. Principal component analysis (PCA) and cluster analysis revealed that combination patterns of major R genes were the main factors determining the resistance of rice varieties to M. oryzae, such as 'Pi9+Pi54', 'Pid3+Pigm', 'Pi5+Pid3+Pigm', 'Pi5+Pi54+Pid3+Pigm', 'Pi5+Pid3' and 'Pi5+Pit+Pid3' in indica-type accessions and 'Pik+Pib', 'Pik+Pita', 'Pik+Pb1', 'Pizt+Pia' and 'Pizt+Pita' in japonica-type accessions, which were able to confer effective resistance against M. oryzae. The above results provide good theoretical support for the rational utilization of combinations of major R genes in developing rice cultivars with broad-spectrum resistance. None Transcriptome profiling of the spl5 mutant reveals that SPL5 has a negative role in the biosynthesis of serotonin for rice disease resistance. 2015 Rice (N Y) College of Chemistry & Life Sciences, Zhejiang Normal University, Jinhua, 321004 China. Rice mutant, spl5 (spotted leaf 5), has spontaneous hypersensitive-like lesions on its leaves and shows enhanced resistance to pathogens, indicating that SPL5 plays a role in programmed cell death (PCD) and disease resistance. To understand the molecular mechanism of SPL5 gene, we investigated the transcriptome profiles of the spl5 mutant leaves with few lesions (FL) and leaves with many lesions (ML) compared to the wild-type (WT) leaves respectively by microarray.The data from microarray revealed that 243 and 896 candidate genes (Fold change ≥ 3.0) were up- or down-regulated in the spl5-FL and spl5-ML, respectively, and a large number of these genes involved in biotic defense responses or reactive oxygen species (ROS) metabolism. Interestingly, according to our microarray and real-time PCR assays, the expressions of a transcription factor OsWRKY14 and genes responsible for the biosynthesis of serotonin, anthranilate synthase (AS), indole-3-glycerolphosphate synthase (IGPS), tryptophan synthase (TS) and tryptophan decarboxylase (TDC) were significantly up-regulated in the spl5 mutant. It has been reported previously that TS and TDC expressions are regulated by OsWRKY14 in rice, which raises the possibility that OsWRKY14 regulates serotonin production through the up-regulation of TS and TDC. Our HPLC analysis further confirmed that serotonin levels were higher in the leaves of spl5 mutant than that in WT.Since the serotonin plays a critical role in inducing disease-resistance, the increased serotonin level may contribute, at least partly, to the disease resistance in spl5. The SPL5 gene may act as a negative regulatory factor activating the serotonin metabolic pathway, and these results might provide a new insight into the spl5-induced defense response mechanisms in plants. spl5|SF3b3 Profiling the expression domains of a rice-specific microRNA under stress. 2015 Front Plant Sci Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology , New Delhi, India. Plant microRNAs (miRs) have emerged as important regulators of gene expression under normal as well as stressful environments. Rice is an important cereal crop whose productivity is compromised due to various abiotic stress factors such as salt, heat and drought. In the present study, we have investigated the role of rice-specific Osa-miR820, in indica rice cultivars showing contrasting response to salt stress. The dissection of expression patterns indicated that the miR is present in all the tissues but is enriched in the anther tissues. In salinity, the miR levels are up-regulated in the leaf tissues but down-regulated in the root tissues. To map the deregulation under salt stress comprehensive time kinetics of expression was performed in the leaf and root tissues. The reproductive stages were also analyzed under salt stress. It emerged that a common regulatory scheme for Osa-miR820 expression is present in the salt-susceptible Pusa Basmati 1 and salt-tolerant Pokkali varieties, although there is a variation in the levels of the miR and its target transcript, OsDRM2. The regulation of Osa-miR820 and its target were also studied under other abiotic stresses. This study thus captures the window for the miR-target correlation and the putative role of this regulation is discussed. This will help in gaining useful insights on the role of species specific miRs in plant development and abiotic stress response. None Fine Mapping and Candidate Gene Analysis of qSTL3, a Stigma Length-Conditioning Locus in Rice (Oryza sativa L.). 2015 PLoS One State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, 210095, China. The efficiency of hybrid seed production can be improved by increasing the percentage of exserted stigma, which is closely related to the stigma length in rice. In the chromosome segment substitute line (CSSL) population derived from Nipponbare (recipient) and Kasalath (donor), a single CSSL (SSSL14) was found to show a longer stigma length than that of Nipponbare. The difference in stigma length between Nipponbare and SSSL14 was controlled by one locus (qSTL3). Using 7,917 individuals from the SSSL14/Nipponbare F2 population, the qSTL3 locus was delimited to a 19.8-kb region in the middle of the short arm of chromosome 3. Within the 19.8-kb chromosome region, three annotated genes (LOC_Os03g14850, LOC_Os03g14860 and LOC_Os03g14880) were found in the rice genome annotation database. According to gene sequence alignments in LOC_Os03g14850, a transition of G (Nipponbare) to A (Kasalath) was detected at the 474-bp site in CDS. The transition created a stop codon, leading to a deletion of 28 amino acids in the deduced peptide sequence in Kasalath. A T-DNA insertion mutant (05Z11CN28) of LOC_Os03g14850 showed a longer stigma length than that of wild type (Zhonghua 11), validating that LOC_Os03g14850 is the gene controlling stigma length. However, the Kasalath allele of LOC_Os03g14850 is unique because all of the alleles were the same as that of Nipponbare at the 474-bp site in the CDS of LOC_Os03g14850 among the investigated accessions with different stigma lengths. A gene-specific InDel marker LQ30 was developed for improving stigma length during rice hybrid breeding by marker-assisted selection. OsMADS72|qSTL3 OsIAA6, a member of the rice Aux/IAA gene family, is involved in drought tolerance and tiller outgrowth. 2015 Plant Sci Crop Biotechnology Institute, GreenBio Science and Technology, Seoul National University, Pyeongchang 232-916, Republic of Korea. Electronic address: harin0723@snu.ac.kr. Auxin signaling is a fundamental part of many plant growth processes and stress responses and operates through Aux/IAA protein degradation and the transmission of the signal via auxin response factors (ARFs). A total of 31 Aux/IAA genes have been identified in rice (Oryza sativa), some of which are induced by drought stress. However, the mechanistic link between Aux/IAA expression and drought responses is not well understood. In this study we found that the rice Aux/IAA gene OsIAA6 is highly induced by drought stress and that its overexpression in transgenic rice improved drought tolerance, likely via the regulation of auxin biosynthesis genes. We observed that OsIAA6 was specifically expressed in the axillary meristem of the basal stem, which is the tissue that gives rise to tillers. A knock-down mutant of OsIAA6 showed abnormal tiller outgrowth, apparently due to the regulation of the auxin transporter OsPIN1 and the rice tillering inhibitor OsTB1. Our results confirm that the OsIAA6 gene is involved in drought stress responses and the control of tiller outgrowth. IAA6|OsIAA6 An evolutionarily conserved gene, FUWA, plays a role in determining panicle architecture, grain shape and weight in rice. 2015 Plant J National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, 100081, Beijing, China. Plant breeding relies on creation of novel allelic combinations for desired traits. Identification and utilization of beneficial alleles, rare alleles and evolutionarily conserved genes in the germplasm (and thus could be called "hidden" genes) provide an effective approach to achieve this goal. Here we show that a chemical induced null mutation in an evolutionarily conserved gene, FUWA, alters multiple important agronomic traits in rice, including panicle architecture, grain shape and weight. FUWA encodes a NHL domain containing protein, with a preferential expression in meristems of root, shoot apical and inflorescence, where it restricts excessive cell division. Sequence analysis revealed that FUWA has undergone a bottleneck effect and become fixed in landraces and modern cultivars during domestication and breeding. We further confirm a highly conserved role of FUWA homologues in determining panicle architecture and grain development in rice, maize and sorghum through genetic transformation. Strikingly, knocking-down FUWA transcription level by RNA interference leads to an erect panicle and an increased grain size in both indica and japonica genetic backgrounds. This study illustrates an approach of creating new germplasm with improved agronomic traits for crop breeding through tapping into evolutionary conserved genes. This article is protected by copyright. All rights reserved. FUWA Salicylic acid modulates arsenic toxicity by reducing its root to shoot translocation in rice (Oryza sativa L.). 2015 Front Plant Sci Division of Plant Ecology and Environmental Science, Department of Environmental Science, Council of Scientific and Industrial Research - National Botanical Research Institute Lucknow, India. Arsenic (As) is posing serious health concerns in South East Asia where rice, an efficient accumulator of As, is prominent crop. Salicylic acid (SA) is an important signaling molecule and plays a crucial role in resistance against biotic and abiotic stress in plants. In present study, ameliorative effect of SA against arsenate (As(V)) toxicity has been investigated in rice (Oryza sativa L.). Arsenate stress hampered the plant growth in terms of root, shoots length, and biomass as well as it enhanced the level of H2O2 and MDA in dose dependent manner in shoot. Exogenous application of SA, reverted the growth, and oxidative stress caused by As(V) and significantly decreased As translocation to the shoots. Level of As in shoot was positively correlated with the expression of OsLsi2, efflux transporter responsible for root to shoot translocation of As in the form of arsenite (As(III)). SA also overcame As(V) induced oxidative stress and modulated the activities of antioxidant enzymes in a differential manner in shoots. As treatment hampered the translocation of Fe in the shoot which was compensated by the SA treatment. The level of Fe in root and shoot was positively correlated with the transcript level of transporters responsible for the accumulation of Fe, OsNRAMP5, and OsFRDL1, in the root and shoot, respectively. Co-application of SA was more effective than pre-treatment for reducing As accumulation as well as imposed toxicity. None CARMO: A Comprehensive Annotation Platform for Functional Exploration of Rice Multi-Omics Data. 2015 Plant J National Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032, China. High-throughput technology is gradually becoming a powerful tool for routine research in rice. Interpretation of biological significance from the huge amount of data is a critical but nontrivial task, especially for rice, whose gene annotations rely heavily on sequence similarity instead of direct experimental evidence. Here we present CARMO, which provides multiple web-based analysis tools for in-depth mining and visualization of rice multi-omics data. The central idea is systematic integration of 1,819 samples from omics studies and diverse sources of functional evidence (15,401 terms), which are further organized into gene sets and higher-level gene modules. In this way, the high-throughput data can easily be compared across studies and platforms, and notably, integration of multiple types of evidence provides biological interpretation from the level of modules with high confidence. In addition, the functions and pathways for thousands of genes lacking description or validation are deduced from genes with concerted expression from the constructed co-expression network or gene modules. Overall, CARMO provides comprehensive annotations for transcriptomic data sets, epi-genomic modification sites, SNPs from genome re-sequencing, and the large gene lists derived from these omics studies. Well-organized results, as well as multiple tools for interactive visualization, are available through a user-friendly web interface. Finally, we illustrate how CARMO extracts biological insight with four examples, demonstrating CARMO as a highly useful resource for intensive data mining and hypothesis generation from rice multi-omics data. CARMO is freely available online (http://bioinfo.sibs.ac.cn/carmo). This article is protected by copyright. All rights reserved. None Single base substitution in OsCDC48 is responsible for premature senescence and death phenotype in rice. 2015 J Integr Plant Biol State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China. A premature senescence and death 128 (psd128) mutant was isolated from an EMS-induced rice IR64 mutant bank. The premature senescence phenotype appeared at the six-leaf stage and the plant died at the early heading stage. psd128 exhibited impaired chloroplast development with significantly reduced photosynthetic ability, chlorophyll and carotenoid contents, root vigor, soluble protein content and increased malonaldehyde content. Furthermore, the expression of senescence-related genes was significantly altered in psd128. The mutant trait was controlled by a single recessive nuclear gene. Using map-based strategy, the mutation Oryza sativa cell division cycle 48 (OsCDC48) was isolated and predicted to encode a putative AAA-type ATPase with 809 amino-acid residuals. A single base substitution at position C2347T in psd128 resulted in a premature stop codon. Functional complementation could rescue the mutant phenotype. In addition, RNA interference resulted in the premature senescence and death phenotype. OsCDC48 was expressed constitutively in the root, stem, leaf and panicle. Subcellular analysis indicated that OsCDC48:YFP fusion proteins were located both in the cytoplasm and nucleus. OsCDC48 was highly conserved with more than 90% identity in the protein levels among plant species. Our results indicated that the impaired function of OsCDC48 was responsible for the premature senescence and death phenotype. OsCDC48 Salt Tolerant and Sensitive Rice Varieties Display Differential Methylome Flexibility under Salt Stress. 2015 PLoS One Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Genomics of Plant Stress. Av. da República, 2780-157 Oeiras, Portugal. DNA methylation has been referred as an important player in plant genomic responses to environmental stresses but correlations between the methylome plasticity and specific traits of interest are still far from being understood. In this study, we inspected global DNA methylation levels in salt tolerant and sensitive rice varieties upon salt stress imposition. Global DNA methylation was quantified using the 5-methylcytosine (5mC) antibody and an ELISA-based technique, which is an affordable and quite pioneer assay in plants, and in situ imaging of methylation sites in interphase nuclei of tissue sections. Variations of global DNA methylation levels in response to salt stress were tissue- and genotype-dependent. We show a connection between a higher ability of DNA methylation adjustment levels and salt stress tolerance. The salt-tolerant rice variety Pokkali was remarkable in its ability to quickly relax DNA methylation in response to salt stress. In spite of the same tendency for reduction of global methylation under salinity, in the salt-sensitive rice variety IR29 such reduction was not statistically supported. In 'Pokkali', the salt stress-induced demethylation may be linked to active demethylation due to increased expression of DNA demethylases under salt stress. In 'IR29', the induction of both DNA demethylases and methyltransferases may explain the lower plasticity of DNA methylation. We further show that mutations for epigenetic regulators affected specific phenotypic parameters related to salinity tolerance, such as the root length and biomass. This work emphasizes the role of differential methylome flexibility between salt tolerant and salt sensitive rice varieties as an important player in salt stress tolerance, reinforcing the need to better understand the connection between epigenetic networks and plant responses to environmental stresses. None Accumulation of long-lived mRNAs associated with germination in embryos during seed development of rice. 2015 J Exp Bot Department of Plant Production, United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan. Mature dry seeds contain translatable mRNAs called long-lived mRNAs. Early studies have shown that protein synthesis during the initial phase of seed germination occurs from long-lived mRNAs, without de novo transcription. However, the gene expression systems that generate long-lived mRNAs in seeds are not well understood. To examine the accumulation of long-lived mRNAs in developing rice embryos, germination tests using the transcriptional inhibitor actinomycin D (Act D) were performed with the Japonica rice cultivar Nipponbare. Although over 70% of embryos at 10 days after flowering (DAF) germinated in the absence of the inhibitor, germination was remarkably impaired in embryos treated with Act D. In contrast, more than 70% of embryos at 20, 25, 30 and 40 DAF germinated in the presence of Act D. The same results were obtained when another cultivar, Koshihikari, was used, indicating that the long-lived mRNAs required for germination predominantly accumulate in embryos between 10 and 20 DAF during seed development. RNA-Seq identified 529 long-lived mRNA candidates, encoding proteins such as ABA, calcium ion and phospholipid signalling-related proteins, and HSP DNA J, increased from 10 to 20 DAF and were highly abundant in 40 DAF embryos of Nipponbare and Koshihikari. We also revealed that these long-lived mRNA candidates are clearly up-regulated in 10 DAF germinating embryos after imbibition, suggesting that the accumulation of these mRNAs in embryos is indispensable for the induction of germination. The findings presented here may facilitate in overcoming irregular seed germination or producing more vigorous seedlings. None Manipulation of the rice L-galactose pathway: evaluation of the effects of transgene overexpression on ascorbate accumulation and abiotic stress tolerance. 2015 PLoS One Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, Jiangsu, China; Agricultural Science Institute of Coastal Region of Jiangsu, Yancheng 224002, Jiangsu, China. Ascorbic acid (AsA) is the most abundant water-soluble antioxidant in plants, and it plays a crucial role in plant growth, development and abiotic stress tolerance. In the present study, six key Arabidopsis or rapeseed genes involved in AsA biosynthesis were constitutively overexpressed in an elite Japonica rice cultivar. These genes encoded the GDP-mannose pyrophosphorylase (GMP), GDP-mannose-3',5'-epimerase (GME), GDP-L-galactose phosphorylase (GGP), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH), and L-galactono-1,4-lactone dehydrogenase (GalLDH). The effects of transgene expression on rice leaf AsA accumulation were carefully evaluated. In homozygous transgenic seedlings, AtGGP transgenic lines had the highest AsA contents (2.55-fold greater than the empty vector transgenic control), followed by the AtGME and AtGDH transgenic lines. Moreover, with the exception of the AtGPP lines, the increased AsA content also provoked an increase in the redox state (AsA/DHA ratio). To evaluate salt tolerance, AtGGP and AtGME transgenic seedlings were exposed to salt stress for one week. The relative plant height, root length and fresh weight growth rates were significantly higher for the transgenic lines compared with the control plants. Altogether, our results suggest that GGP may be a key rate-limiting step in rice AsA biosynthesis, and the plants with elevated AsA contents demonstrated enhanced tolerance for salt stress. None Interactions of OsMADS1 with Floral Homeotic Genes in Rice Flower Development. 2015 Mol Plant State Key Laboratory of Hybrid Rice, Shanghai Jiao Tong University-University of Adelaide Joint Centre for Agriculture and Health, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 20040, China. During reproductive development, rice plants develop unique flower organs which determine the final grain yield. OsMADS1, one of SEPALLATA-like MADS-box genes, has been unraveled to play critical roles in rice floral organ identity specification and floral meristem determinacy. However, molecular mechanisms underlying interactions of OsMADS1 with other floral homeotic genes in regulating flower development remains largely elusive. In this work, we studied the genetic interactions of OsMADS1 with B-, C-, and D-class genes along with physical interactions among their proteins, and provided some important evidence to further support the neofunctionalization of two rice C-class genes. The physical and genetic interaction of OsMADS1 and OsMADS3 is essential for floral meristem activity maintenance and organ identity specification; while OsMADS1 physically and genetically interacts with OsMADS58 in regulating floral meristem determinacy and suppressing spikelet meristem reversion. Gene expression profiling further identified that OsMADS1 regulates expression of OsMADS17, and affects other genes involved in floral identity and hormone signaling. In brief, this work provides new insights about the physical and regulatory interaction network of OsMADS1 with other floral homeotic genes in rice floral organ identity specification and meristem determinacy. OsMADS1|LHS1|AFO Marker-assisted breeding of a LOX-3-null rice line with improved storability and resistance to preharvest sprouting. 2015 Theor Appl Genet NARO, Institute of Crop Science, 2-1-18 Kannondai, Tsukuba, Ibaraki, 305-8518, Japan, suzuyasu@affrc.go.jp. Breakage of the tight linkage between rice seed lipoxygenase - 3 and easy preharvest sprouting trait led to breeding of lines with few stale flavors after long storage and desirable preharvest sprouting resistance. Lipoxygenase-3 (LOX-3) is involved in the production of volatile constituents in stored rice, and the development of stale flavor is delayed in LOX-3 null rice. In the process of breeding new LOX-3-null lines with long storability, we found a close association between LOX-3 and preharvest sprouting resistance. To determine whether this relationship was due to the tight linkage of two genes or the pleiotropic effect of LOX-3, we performed marker-assisted selection using a BC3F3 population derived from crosses between LOX-3-present/preharvest sprouting-resistant lines and LOX-3-null/preharvest susceptible lines. In one individual, a recombination event occurred 13 kb downstream of LOX-3 (RM15750) and a significant quantitative trait locus, namely qPHS3, for easy preharvest sprouting trait (LOD = 10.4) was detected in an 842-kb region between RM15711 and RM15768. Using BC3F4 and BC3F5 populations, we succeeded in selecting LOX-3-absent and preharvest sprouting-resistant lines with only a 393-kb introgressed chromosome segment from the donor line for LOX-3-null at the LOX-3 locus on chromosome 3. This result indicated that the LOX-3 gene and the locus affecting preharvest sprouting are distinct. The selected line was named 'Hokuriku 244'. Sensory testing of rice grains with and without LOX-3 confirmed that stale flavor production in LOX-3-null rice during storage was lower than in normal LOX-3 rice. These results indicated that rice varieties with little stale flavor after long storage and preharvest sprouting resistance had been selected. OsLOX3|LOX3 A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. 2015 Mol Plant State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangzhou 510642, China; College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edited 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homologous end-joining mechanism followed by homologous recombination-based repair. We also obtained uniform biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T0 rice and T1Arabidopsis plants by simultaneous targeting of multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement. None Two linked genes on rice chromosome 2 for F1 pollen sterility in a hybrid between Oryza sativa and O. glumaepatula. 2014 Breed Sci Plant Breeding Laboratory, Faculty of Agriculture, Kyushu University , 6-10-1, Hakozaki, Higashi, Fukuoka 812-8581 , Japan. Hybrid incompatibility plays an important role in establishment of post-zygotic reproductive isolation. To unveil genetic basis of hybrid incompatibilities between diverged species of genus Oryza AA genome species, we conducted genetic dissection of hybrid sterility loci, S22(t), which had been identified in backcross progeny derived from Oryza sativa ssp. japonica (recurrent parent) and South American wild rice O. glumaepatula near the end of the short arm of chromosome 2. The S22(t) region was found to be composed of two loci, designated S22A and S22B, that independently induce F1 pollen sterility. Pollen grains containing either of the sterile alleles (S22A-glum (s) or S22B-glum (s) ) were sterile if produced on a heterozygous plant. No transmission of the S22A-glum (s) allele via pollen was observed, whereas a low frequency of transmission of S22B-glum (s) was observed. Cytological analysis showed that the sterile pollen grains caused by S22A could reach the bicellular or tricellular stage, and the nearly-sterile pollen grains caused by S22B could reach the tricellular stage. Our genetic analysis showed repulsion linkage effect is possible to induce strong reproductive barrier by high pollen sterility based on recombination value and transmission ratio of hybrid sterility gene to the progeny was influenced by frequency of competitors on fertilization. None Identification of a novel gene (Apq1) from the indica rice cultivar 'Habataki' that improves the quality of grains produced under high temperature stress. 2014 Breed Sci Toyama Prefectural Agricultural, Forestry & Fisheries Research Center , 1124-1 Yoshioka, Toyama, Toyama 939-8153 , Japan. The appearance of brown rice grown under high temperature conditions is an important characteristic for improvement in Japanese rice breeding programs. We performed a QTL analysis of the appearance quality of brown rice using chromosome segment substitution lines of the indica cultivar 'Habataki' in the 'Koshihikari' genetic background. A line carrying a 'Habataki' segment on chromosome 7 showed a high percentage of perfect grains produced under high temperature conditions during the ripening period. To verify the role of this segment, and to narrow down the region containing the useful allele, substitution mapping was performed using multiple paired lines. As a result, the chromosomal location of a gene that we named Appearance quality of brown rice 1 (Apq1) was delimited to a 48-kb region. In addition, we developed an Apq1-near isogenic line (NIL) to evaluate the effect of Apq1 on various agronomic traits. Under high temperature conditions during the ripening period, the Apq1-NIL produced significantly higher percentages of perfect grains than 'Koshihikari'. Other agronomic traits, including yield and palatability, were similar between the Apq1-NIL and 'Koshihikari'. Therefore, the 'Habataki' allele of Apq1 will be useful in breeding programs aimed at improving the quality of grains ripened under high temperature conditions. None CLUSTERED PRIMARY BRANCH 1, a new allele of DWARF11, controls panicle architecture and seed size in rice. 2015 Plant Biotechnol J Department of Plant Genetics and Breeding, National Center for Evaluation of Agricultural Wild Plants (Rice), Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing, China. Panicle architecture and seed size are important agronomic traits that directly determine grain yield in rice (Oryza sativa L.). Although a number of key genes controlling panicle architecture and seed size have been cloned and characterized in recent years, their genetic and molecular mechanisms remain unclear. In this study, we identified a mutant that produced panicles with fascicled primary branching and reduced seeds in size. We isolated the underlying CLUSTERED PRIMARY BRANCH 1 (CPB1) gene, a new allele of DWARF11 (D11) encoding a cytochrome P450 protein involved in brassinosteroid (BR) biosynthesis pathway. Genetic transformation experiments confirmed that a His360Leu amino acid substitution residing in the highly conserved region of CPB1/D11 was responsible for the panicle architecture and seed size changes in the cpb1 mutants. Overexpression of CPB1/D11 under the background of cpb1 mutant not only rescued normal panicle architecture and plant height, but also had a larger leaf angle and seed size than the controls. Furthermore, the CPB1/D11 transgenic plants driven by panicle-specific promoters can enlarge seed size and enhance grain yield without affecting other favourable agronomic traits. These results demonstrated that the specific mutation in CPB1/D11 influenced development of panicle architecture and seed size, and manipulation of CPB1/D11 expression using the panicle-specific promoter could be used to increase seed size, leading to grain yield improvement in rice. D11|CPB1|CYP724B1 Unmasking Novel Loci for Internal Phosphorus Utilization Efficiency in Rice Germplasm through Genome-Wide Association Analysis. 2015 PLoS One Crop, Livestock and Environment Division, Japan International Research Centre for Agricultural Science, Tsukuba, Ibaraki, Japan. Depletion of non-renewable rock phosphate reserves and phosphorus (P) fertilizer price increases has renewed interest in breeding P-efficient varieties. Internal P utilization efficiency (PUE) is of prime interest because there has been no progress to date in breeding for high PUE. We characterized the genotypic variation for PUE present within the rice gene pool by using a hydroponic system that assured equal plant P uptake, followed by mapping of loci controlling PUE via Genome-Wide Association Studies (GWAS). Loci associated with PUE were mapped on chromosomes 1, 4, 11 and 12. The highest PUE was associated with a minor indica-specific haplotype on chromosome 1 and a rare aus-specific haplotype on chromosome 11. Comparative variant and expression analysis for genes contained within the chromosome 1 haplotype identified high priority candidate genes. Differences in coding regions and expression patterns between genotypes of contrasting haplotypes, suggested functional alterations for two predicted nucleic acid-interacting proteins that are likely causative for the observed differences in PUE. The loci reported here are the first identified for PUE in any crop that is not confounded by differential P uptake among genotypes. Importantly, modern rice varieties lacked haplotypes associated with superior PUE, and would thus benefit from targeted introgressions of these loci from traditional donors to improve plant growth in phosphorus-limited cropping systems. None Ozone-Induced Rice Grain Yield Loss Is Triggered via a Change in Panicle Morphology That Is Controlled by ABERRANT PANICLE ORGANIZATION 1 Gene. 2015 PLoS One Center for Environmental Biology and Ecosystem, National Institute for Environmental Studies, Tsukuba, Ibaraki, 305-8506, Japan; Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8577, Japan. Rice grain yield is predicted to decrease in the future because of an increase in tropospheric ozone concentration. However, the underlying mechanisms are unclear. Here, we investigated the responses to ozone of two rice (Oryza Sativa L.) cultivars, Sasanishiki and Habataki. Sasanishiki showed ozone-induced leaf injury, but no grain yield loss. By contrast, Habataki showed grain yield loss with minimal leaf injury. A QTL associated with grain yield loss caused by ozone was identified in Sasanishiki/Habataki chromosome segment substitution lines and included the ABERRANT PANICLE ORGANIZATION 1 (APO1) gene. The Habataki allele of the APO1 locus in a near-isogenic line also resulted in grain yield loss upon ozone exposure, suggesting APO1 involvement in ozone-induced yield loss. Only a few differences in the APO1 amino acid sequences were detected between the cultivars, but the APO1 transcript level was oppositely regulated by ozone exposure: i.e., it increased in Sasanishiki and decreased in Habataki. Interestingly, the levels of some phytohormones (jasmonic acid, jasmonoyl-L-isoleucine, and abscisic acid) known to be involved in attenuation of ozone-induced leaf injury tended to decrease in Sasanishiki but to increase in Habataki upon ozone exposure. These data indicate that ozone-induced grain yield loss in Habataki is caused by a reduction in the APO1 transcript level through an increase in the levels of phytohormones that reduce leaf damage. APO1|OsAPO1|SCM2 Whole genome sequencing of elite rice cultivars as a comprehensive information resource for marker assisted selection. 2015 PLoS One Agrobiodiversity research area, International Center for Tropical Agriculture, Cali, Colombia. Current advances in sequencing technologies and bioinformatics revealed the genomic background of rice, a staple food for the poor people, and provided the basis to develop large genomic variation databases for thousands of cultivars. Proper analysis of this massive resource is expected to give novel insights into the structure, function, and evolution of the rice genome, and to aid the development of rice varieties through marker assisted selection or genomic selection. In this work we present sequencing and bioinformatics analyses of 104 rice varieties belonging to the major subspecies of Oryza sativa. We identified repetitive elements and recurrent copy number variation covering about 200 Mbp of the rice genome. Genotyping of over 18 million polymorphic locations within O. sativa allowed us to reconstruct the individual haplotype patterns shaping the genomic background of elite varieties used by farmers throughout the Americas. Based on a reconstruction of the alleles for the gene GBSSI, we could identify novel genetic markers for selection of varieties with high amylose content. We expect that both the analysis methods and the genomic information described here would be of great use for the rice research community and for other groups carrying on similar sequencing efforts in other crops. None Rapid responses of mesophyll conductance to changes of CO2 concentration, temperature and irradiance are affected by N supplements in rice. 2015 Plant Cell Environ National Key Laboratory of Crop Genetic Improvement, MOA Key Laboratory of Crop Ecophysiology and Farming System in the Middle Reaches of the Yangtze River, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China. Photosynthesis in C3 plants is significantly limited by mesophyll conductance (gm ), which can vary with leaf anatomical traits and nitrogen (N) supplements. Several studies have investigated the response of gm to N supplements, however none examined the implications of N supplements on the response of gm to rapid environmental changes. Here we investigated the effect of N supplement on gm and the response of gm to change of CO2 , temperature and irradiance in rice. High N supplement (HN) increased mesophyll cell wall surface area and chloroplast surface area exposed to intercellular airspace per leaf area, and reduced cell wall thickness. These changes resulted in increased gm . The gm of leaves with HN was more sensitive to changes in CO2 concentration, temperature, and irradiance. The difference in leaf structural features between low N supplement and HN indicates that a rapid change in gm is related to the regulation of diffusion through biological membranes rather than leaf structural features. These results will contribute to an understanding of the determinants of gm response to rapid changes in environmental factors. None The role of nodes in arsenic storage and distribution in rice. 2015 J Exp Bot Department of Sustainable Soils and Grassland Systems, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK. Knowledge of arsenic (As) accumulation in rice (Oryza sativa L.) is important for minimizing As transfer to the food chain. The aim of this study was to investigate the role of rice nodes in As storage and distribution. Synchrotron μX-ray fluorescence (μ-XRF) was used to map As distribution in the top node and internode of a lsi2 mutant defective in silicon/arsenite efflux carrier and its wild-type (WT) grown in soil. Lsi2 expression in different tissues during grain filling was investigated by quantitative RT-PCR. Arsenite or dimethylarsinic acid (DMA) was supplied to excised panicles to investigate the roles of Lsi2 and phytochelatins (PC) in As distribution. μ-XRF mapping revealed As storage in the phloem of different vascular bundles in the top node and internode. Soil-grown plants of lsi2 had markedly decreased As accumulation in the phloem compared with the WT. Lsi2 was strongly expressed, not only in the roots but also in the nodes. When excised panicles were exposed to As(III), the lsi2 mutant distributed more As to the node and flag leaf but less As to the grain compared with the WT, while there was no significant difference in DMA distribution. Inhibition of PC synthesis by l-buthionine-sulphoximine decreased As(III) deposition in the top node but increased As accumulation in the grain and flag leaf. The results suggest that rice nodes serve as a filter restricting As(III) distribution to the grain. Furthermore, Lsi2 plays a role in As(III) distribution in rice nodes and phytochelatins are important compounds for As(III) storage in the nodes. None Disruption of the rice nitrate transporter OsNPF2.2 hinders root-to-shoot nitrate transport and vascular development. 2015 Sci Rep Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement &Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China. Plants have evolved to express some members of the nitrate transporter 1/peptide transporter family (NPF) to uptake and transport nitrate. However, little is known of the physiological and functional roles of this family in rice (Oryza sativa L.). Here, we characterized the vascular specific transporter OsNPF2.2. Functional analysis using cDNA-injected Xenopus laevis oocytes revealed that OsNPF2.2 is a low-affinity, pH-dependent nitrate transporter. Use of a green fluorescent protein tagged OsNPF2.2 showed that the transporter is located in the plasma membrane in the rice protoplast. Expression analysis showed that OsNPF2.2 is nitrate inducible and is mainly expressed in parenchyma cells around the xylem. Disruption of OsNPF2.2 increased nitrate concentration in the shoot xylem exudate when nitrate was supplied after a deprivation period; this result suggests that OsNPF2.2 may participate in unloading nitrate from the xylem. Under steady-state nitrate supply, the osnpf2.2 mutants maintained high levels of nitrate in the roots and low shoot:root nitrate ratios; this observation suggests that OsNPF2.2 is involved in root-to-shoot nitrate transport. Mutation of OsNPF2.2 also caused abnormal vasculature and retarded plant growth and development. Our findings demonstrate that OsNPF2.2 can unload nitrate from the xylem to affect the root-to-shoot nitrate transport and plant development. OsNPF2.2 OsAGSW1, an ABC1-like kinase gene, is involved in the regulation of grain size and weight in rice. 2015 J Exp Bot Guangdong Key Lab of Biotechnology for Plant Development, College of Life Science, South China Normal University, Guangzhou 510631, China Vegetable Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, Guangdong, 510640, China. Grain shape and weight are two determining agronomic traits of rice yield. ABC1 (Activity of bc1 complex) is a newly found atypical kinase in plants. Here, we report on an ABC1 protein kinase gene, OsAGSW1 (ABC1-like kinase related to Grain size and Weight). Expression of OsAGSW1-GFP in rice revealed that OsAGSW1 is localized to the chloroplasts in rice. Analysis of OsAGSW1 promoter::beta-glucuronidase transgenic rice indicated that this gene was highly expressed in vascular bundles in shoot, hull and caryopsis. Furthermore, OsAGSW1-RNAi and overexpressed transgenic rice lines were generated. Stable transgenic lines overexpressing OsAGSW1 exhibited a phenotype with a significant increase in grain size, grain weight, grain filling rate and 1000-grain weight compared with the wild-type and RNAi transgenic plants. Microscopy analysis showed that spikelet hulls just before heading were different in the OsAGSW1-overexpressed plants compared with wild-type and OsAGSW1 RNAi rice. Further cytological analysis showed that the number of external parenchyma cells in rice hulls of OsAGSW1-overexpressed plants increased, leading to wider and longer spikelet hulls than those of the wild-type and OsAGSW1-RNAi plants. The vascular cross-sectional area in lemma, carpopodium and ovules also strikingly increased and area of both xylem and phloem were enlarged in the OsAGSW1-overexpressed plants. Thus, our results demonstrated that OsAGSW1 plays an important role in seed shape and size of rice by regulating the number of external parenchyma cells and the development of vascular bundles, providing a new insight into the functions of ABC1 genes in plants. OsAGSW1 Transcriptome diversity among rice root types during asymbiosis and interaction with arbuscular mycorrhizal fungi. 2015 Proc Natl Acad Sci U S A Department of Plant Molecular Biology, University of Lausanne, 1015 Lausanne, Switzerland; Faculty of Biology, Genetics, University of Munich, 82152 Martinsried, Germany; caroline.gutjahr@lmu.de up220@cam.ac.uk. Root systems consist of different root types (RTs) with distinct developmental and functional characteristics. RTs may be individually reprogrammed in response to their microenvironment to maximize adaptive plasticity. Molecular understanding of such specific remodeling-although crucial for crop improvement-is limited. Here, RT-specific transcriptomes of adult rice crown, large and fine lateral roots were assessed, revealing molecular evidence for functional diversity among individual RTs. Of the three rice RTs, crown roots displayed a significant enrichment of transcripts associated with phytohormones and secondary cell wall (SCW) metabolism, whereas lateral RTs showed a greater accumulation of transcripts related to mineral transport. In nature, arbuscular mycorrhizal (AM) symbiosis represents the default state of most root systems and is known to modify root system architecture. Rice RTs become heterogeneously colonized by AM fungi, with large laterals preferentially entering into the association. However, RT-specific transcriptional responses to AM symbiosis were quantitatively most pronounced for crown roots despite their modest physical engagement in the interaction. Furthermore, colonized crown roots adopted an expression profile more related to mycorrhizal large lateral than to noncolonized crown roots, suggesting a fundamental reprogramming of crown root character. Among these changes, a significant reduction in SCW transcripts was observed that was correlated with an alteration of SCW composition as determined by mass spectrometry. The combined change in SCW, hormone- and transport-related transcript profiles across the RTs indicates a previously overlooked switch of functional relationships among RTs during AM symbiosis, with a potential impact on root system architecture and functioning. None Role of rice cytosolic hexokinase OsHXK7 in sugar signaling and metabolism. 2015 J Integr Plant Biol Crop Biotech Institute & Graduate School of Biotechnology, Kyung Hee University, Yongin 446-701, Korea. We characterized the function of the rice cytosolic hexokinase OsHXK7 (Oryza sativa Hexokinase7), which is highly upregulated when seeds germinate under O2 -deficient conditions. According to transient expression assays that used the promoter:luciferase fusion construct, OsHXK7 enhanced the glucose (Glc)-dependent repression of a rice α-amylase gene (RAmy3D) in the mesophyll protoplasts of maize, but its catalytically inactive mutant alleles did not. Consistently, the expression of OsHXK7, but not its catalytically inactive alleles, complemented the Arabidopsis glucose insensitive2-1 (gin2-1) mutant, thereby resulting in the wild type characteristics of Glc-dependent repression, seedling development, and plant growth. Interestingly, OsHXK7-mediated Glc-dependent repression was abolished in the O2 -deficient mesophyll protoplasts of maize. This result provides compelling evidence that OsHXK7 functions in sugar signaling via a glycolysis-dependent manner under normal conditions, but its signaling role is suppressed when O2 is deficient. The germination of two null OsHXK7 mutants, oshxk7-1 and oshxk7-2, was affected by O2 deficiency, but overexpression enhanced germination in rice. This result suggests the distinct role that OsHXK7 plays in sugar metabolism and efficient germination by enforcing glycolysis-mediated fermentation in O2 -deficient rice. OsHXK7 Regulation of OsmiR156h through Alternative Polyadenylation Improves Grain Yield in Rice. 2015 PLoS One Institute of Technical Biology and Agriculture Engineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, China; The State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. Substantial increases in grain yield of cereal crops are required to feed a growing human population. Here we show that a natural variant of SEMIDWARF AND HIGH-TILLERING (SDT) increases harvest index and grain productivity in rice. Gain-of-function sdt mutation has a shortened polyadenylation tail on the OsmiR156h microRNA precursor, which cause the up-regulation of OsmiR156h. The plants carrying the semidominant sdt allele exhibit reduced plant height, enhanced lodging resistance, increased tiller numbers per plant, and resulting in an increased grain yield. We also show that combining the sdt allele with the OsSPL14WFP allele can be effective in simultaneously improving tillering capacity and panicle branching, thereby leading to higher harvest index and grain yield. Most importantly, pyramiding of the sdt allele and the green revolution gene sd1 enhances grain yield by about 20% in hybrid rice breeding. Our results suggest that the manipulation of the polyadenylation status of OsmiR156 represents a novel strategy for improving the yield potential of rice over what is currently achievable. SDT Effect of carbohydrates and night temperature on night respiration in rice. 2015 J Exp Bot CIRAD, UMR AGAP, F-34398 Montpellier, France. Global warming causes night temperature (NT) to increase faster than day temperature in the tropics. According to crop growth models, respiration incurs a loss of 40-60% of photosynthate. The thermal sensitivity of night respiration (R n) will thus reduce biomass. Instantaneous and acclimated effects of NT on R n of leaves and seedlings of two rice cultivars having a variable level of carbohydrates, induced by exposure to different light intensity on the previous day, were investigated. Experiments were conducted in a greenhouse and growth chambers, with R n measured on the youngest fully expanded leaves or whole seedlings. Dry weight-based R n was 2.6-fold greater for seedlings than for leaves. Leaf R n was linearly related to starch (positive intercept) and soluble sugar concentration (zero intercept). Increased NT caused higher R n at a given carbohydrate concentration. The change of R n at NT increasing from 21 °C to 31 °C was 2.4-fold for the instantaneous response but 1.2- to 1.7-fold after acclimation. The maintenance component of R n (R m'), estimated by assimilate starvation, averaged 28% in seedlings and 34% in leaves, with no significant thermal effect on this ratio. The acclimated effect of increased NT on R m' across experiments was 1.5-fold for a 10 °C increase in NT. No cultivar differences were observed in R n or R m' responses. The results suggest that the commonly used Q10=2 rule overestimates thermal response of respiration, and R n largely depends on assimilate resources. None Genetic architecture of variation in heading date among Asian rice accessions. 2015 BMC Plant Biol National Institute of Agrobiological Sciences, 2-1-2 Kannondai, 305-8602, Tsukuba, Ibaraki, Japan. horikiyo@affrc.go.jp. Heading date, a crucial factor determining regional and seasonal adaptation in rice (Oryza sativa L.), has been a major selection target in breeding programs. Although considerable progress has been made in our understanding of the molecular regulation of heading date in rice during last two decades, the previously isolated genes and identified quantitative trait loci (QTLs) cannot fully explain the natural variation for heading date in diverse rice accessions.To genetically dissect naturally occurring variation in rice heading date, we collected QTLs in advanced-backcross populations derived from multiple crosses of the japonica rice accession Koshihikari (as a common parental line) with 11 diverse rice accessions (5 indica, 3 aus, and 3 japonica) that originate from various regions of Asia. QTL analyses of over 14,000 backcrossed individuals revealed 255 QTLs distributed widely across the rice genome. Among the detected QTLs, 128 QTLs corresponded to genomic positions of heading date genes identified by previous studies, such as Hd1, Hd6, Hd3a, Ghd7, DTH8, and RFT1. The other 127 QTLs were detected in different chromosomal regions than heading date genes.Our results indicate that advanced-backcross progeny allowed us to detect and confirm QTLs with relatively small additive effects, and the natural variation in rice heading date could result from combinations of large- and small-effect QTLs. We also found differences in the genetic architecture of heading date (flowering time) among maize, Arabidopsis, and rice. None The Phosphate Transporter Gene OsPht1;4 Is Involved in Phosphate Homeostasis in Rice. 2015 PLoS One National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, 430070, P.R. China. A total of 13 phosphate transporters in rice (Oryza sative) have been identified as belonging to the Pht1 family, which mediates inorganic phosphate (Pi) uptake and transport. We report the biological property and physiological role of OsPht1;4 (OsPT4). Overexpressing OsPT4 resulted in significant higher Pi accumulation in roots, straw and brown rice, and suppression of OsPT4 caused decreased Pi concentration in straw and brown rice. Expression of the beta-glucuronidase reporter gene driven by the OsPT4 promoter showed that OsPT4 is expressed in roots, leaves, ligules, stamens, and caryopses under sufficient Pi conditions, consistent with the expression profile showing that OsPT4 has high expression in roots and flag leaves. The transcript level of OsPT4 increased significantly both in shoots and roots with a long time Pi starvation. OsPT4 encoded a plasma membrane-localized protein and was able to complement the function of the Pi transporter gene PHO84 in yeast. We concluded that OsPT4 is a functional Pi-influx transporter involved in Pi absorption in rice that might play a role in Pi translocation. This study will enrich our understanding about the physiological function of rice Pht1 family genes. OsPht1;4|OsPT4 Identification of QTLs and possible candidate genes conferring sheath blight resistance in rice (Oryza sativa L.). 2015 Springerplus Institute of Biotechnology, Acharya N G Ranga Agricultural University, Hyderabad, 500030 India. Sheath blight, caused by the pathogenic fungus Rhizoctonia solani Kühn, is one of the most devastating diseases in rice. Breeders have always faced challenges in acquiring reliable and absolute resistance to this disease in existing rice germplasm. In this context, 40 rice germplasm including eight wild, four landraces, twenty- six cultivated and two advanced breeding lines were screened utilizing the colonized bits of typha. Except Tetep and ARC10531 which expressed moderate level of resistance to the disease, none could be found to be authentically resistant. In order to map the quantitative trait loci (QTLs) governing the sheath blight resistance, two mapping populations (F2 and BC1F2) were developed from the cross BPT-5204/ARC10531. Utilizing composite interval mapping analysis, 9 QTLs mapped to five different chromosomes were identified with phenotypic variance ranging from 8.40 to 21.76%. Two SSR markers namely RM336 and RM205 were found to be closely associated with the major QTLs qshb7.3 and qshb9.2 respectively and were attested as well in BC1F2 population by bulk segregant analysis approach. A hypothetical beta 1-3 glucanase with other 31 candidate genes were identified in silico utilizing rice database RAP-DB within the identified QTL region qshb9.2. A detailed insight into these candidate genes will facilitate at molecular level the intricate nature of sheath blight, a step forward towards functional genomics. None Amylopectin biosynthetic enzymes from developing rice seed form enzymatically active protein complexes. 2015 J Exp Bot Department of Biological Production, Akita Prefectural University, 241-438 Kaidobata-Nishi, Shimoshinjo-Nakano, Akita city, Akita 010-0195, Japan. Amylopectin is a highly branched, organized cluster of glucose polymers, and the major component of rice starch. Synthesis of amylopectin requires fine co-ordination between elongation of glucose polymers by soluble starch synthases (SSs), generation of branches by branching enzymes (BEs), and removal of misplaced branches by debranching enzymes (DBEs). Among the various isozymes having a role in amylopectin biosynthesis, limited numbers of SS and BE isozymes have been demonstrated to interact via protein-protein interactions in maize and wheat amyloplasts. This study investigated whether protein-protein interactions are also found in rice endosperm, as well as exploring differences between species. Gel permeation chromatography of developing rice endosperm extracts revealed that all 10 starch biosynthetic enzymes analysed were present at larger molecular weights than their respective monomeric sizes. SSIIa, SSIIIa, SSIVb, BEI, BEIIb, and PUL co-eluted at mass sizes >700kDa, and SSI, SSIIa, BEIIb, ISA1, PUL, and Pho1 co-eluted at 200-400kDa. Zymogram analyses showed that SSI, SSIIIa, BEI, BEIIa, BEIIb, ISA1, PUL, and Pho1 eluted in high molecular weight fractions were active. Comprehensive co-immunoprecipitation analyses revealed associations of SSs-BEs, and, among BE isozymes, BEIIa-Pho1, and pullulanase-type DBE-BEI interactions. Blue-native-PAGE zymogram analyses confirmed the glucan-synthesizing activity of protein complexes. These results suggest that some rice starch biosynthetic isozymes are physically associated with each other and form active protein complexes. Detailed analyses of these complexes will shed light on the mechanisms controlling the unique branch and cluster structure of amylopectin, and the physicochemical properties of starch. None OsHrd3 is necessary for maintaining the quality of endoplasmic reticulum-derived protein bodies in rice endosperm. 2015 J Exp Bot Functional Transgenic Crops Research Unit, Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. Large amounts of seed storage proteins (SSPs) are produced in the maturing endosperm of rice seeds. Rice SSPs are synthesized as secretory proteins on the rough endoplasmic reticulum (ER), and are transported and deposited into protein complexes called protein bodies (PB-I and PB-II). Due to the high production of SSPs, unfolded SSPs may be generated during this process. However, it was previously unclear how such unfolded proteins are selected among synthesized products and removed from the ER to maintain protein quality in the endosperm. Since Hrd3/SEL1L recognizes unfolded proteins in yeast and mammalian protein quality control systems, the role of OsHrd3 in protein quality control in rice endosperm was investigated. Co-immunoprecipitation experiments demonstrated that OsHrd3 interacts with components of the Hrd1 ubiquitin ligase complex such as OsOS-9 and OsHrd1 in rice protoplasts. Endosperm-specific suppression of OsHrd3 in transgenic rice reduced the levels of polyubiquitinated proteins and resulted in unfolded protein responses (UPRs) in the endosperm, suggesting that OsHrd3-mediated polyubiquitination plays an important role in ER quality control. It was found that a cysteine-rich 13kDa prolamin, RM1, was polyubiquitinated in wild-type (WT) seeds but not in OsHrd3 knockdown (KD) seeds. RM1 formed aberrant aggregates that were deposited abnormally in OsHrd3 KD seeds, resulting in deformed PB-I. Therefore, the quality of protein bodies is maintained by polyubiquitination of unfolded SSPs through the Hrd1 ubiquitin ligase system in rice endosperm. OsHrd3,OsHrd1,OsOS9 The OsHKT1;1 Transporter is Involved in Salt Tolerance and Regulated by an MYB-Type Transcription Factor. 2015 Plant Physiol CITY: Nanjing POSTAL_CODE: 210095 China [CN] Nanjing Agricultural University whzhang@njau.edu.cn. Sodium transporters play key roles in plant tolerance to salt stress. Here, we report that a member of the High-Affinity K+ Transporter (HKT) family, OsHKT1;1, in rice (Oryza sativa L. cv. Nipponbare) plays an important role in reducing Na+ accumulation in shoots to cope with salt stress. The oshkt1;1 mutant plants displayed hypersensitivity to salt stress. They contained less Na+ in the phloem sap and accumulated more Na+ in the shoots compared to the wild type. OsHKT1;1 was expressed mainly in the phloem of leaf blades and up-regulated in response to salt stress. Using a yeast one-hybrid approach, a novel MYB coiled-coil type transcription factor, OsMYBc, was found to bind to the OsHKT1;1 promoter. In planta chromatin immunoprecipitation and in vitro electrophoresis mobility shift assays demonstrated that OsMYBc binds to AAANATNC(C/T) fragments within the OsHKT1;1 promoter. Mutation of the OsMYBc-binding nucleotides resulted in a decrease in promoter activity of OsHKT1;1. Knockout of OsMYBc resulted in a reduction in NaCl-induced expression of OsHKT1;1 and salt sensitivity. Taken together, these results suggest that OsHKT1;1 has a role in controlling Na+ concentration and preventing sodium toxicity in leaf blades, and is regulated by the OsMYBc transcription factor. OsHKT1;1|OsHKT4 Functional architecture of two exclusively late stage pollen-specific promoters in rice (Oryza sativa L.). 2015 Plant Mol Biol Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, 20 Nanxincun, Xiangshan, Beijing, 100093, China. Late stage pollen-specific promoters are important tools in crop molecular breeding. Several such promoters, and their functional motifs, have been well characterized in dicotyledonous plants such as tomato and tobacco. However, knowledge about the functional architecture of such promoters is limited in the monocotyledonous plant rice. Here, pollen-late-stage-promoter 1 (PLP1) and pollen-late-stage-promoter 2 (PLP2) were characterized using a stable transformation system in rice. Histochemical staining showed that the two promoters exclusively drive GUS expression in late-stage pollen grains in rice. 5' deletion analysis revealed that four regions, including the -1159 to -720 and the -352 to -156 regions of PLP1 and the -740 to -557 and the -557 to -339 regions of PLP2, are important in maintaining the activity and specificity of these promoters. Motif mutation analysis indicated that 'AGAAA' and 'CAAT' motifs in the -740 to -557 region of PLP2 act as enhancers in the promoter. Gain of function experiments indicated that the novel TA-rich motif 'TACATAA' and 'TATTCAT' in the core region of the PLP1 and PLP2 promoters is necessary, but not sufficient, for pollen-specific expression in rice. Our results provide evidence that the enhancer motif 'AGAAA' is conserved in the pollen-specific promoters of both monocots and eudicots, but that some functional architecture characteristics are different. None OsTCTP, encoding a translationally controlled tumor protein, plays an important role in mercury tolerance in rice. 2015 BMC Plant Biol State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, 310058, China. zhqwang@zju.edu.cn. Mercury (Hg) is not only a threat to public health but also a growth risk factor to plants, as it is readily accumulated by higher plants. Accumulation of Hg in plants disrupts many cellular-level functions and inhibits growth and development; however, the detoxification and tolerance mechanisms of plants to Hg stress are still not fully understood. Exposure to toxic Hg also occurs in some crops cultivated under anoxic conditions, such as rice (Oryza sativa L.), a model organism and one of the most important cultivated plants worldwide. In this study, we functionally characterized a rice translationally controlled tumor protein gene (Os11g43900, OsTCTP) involved in Hg stress tolerance.OsTCTP was ubiquitously expressed in all examined plant tissues, especially in actively dividing and differentiating tissues, such as roots and nodes. OsTCTP was found to localize both the cytosol and the nucleus. OsTCTP was induced by mercuric chloride, cupric sulfate, abscisic acid, and hydrogen peroxide at the protein level in a time-dependent manner. Overexpression of OsTCTP potentiated the activities of several antioxidant enzymes, reduced the Hg-induced H2O2 levels, and promoted Hg tolerance in rice, whereas knockdown of OsTCTP produced opposite effects. And overexpression of OsTCTP did not prevent Hg absorption and accumulation in rice. We also demonstrated that Asn 48 and Asn 97 of OsTCTP amino acids were not the potential N-glycosylation sites.Our results suggest that OsTCTP is capable of decreasing the Hg-induced reactive oxygen species (ROS), therefore, reducing the damage of ROS and enhancing the tolerance of rice plants to Hg stress. Thus, OsTCTP is a valuable gene for genetic engineering to improve rice performance under Hg contaminated paddy soils. OsTCTP A Gibberellin-Mediated DELLA-NAC Signaling Cascade Regulates Cellulose Synthesis in Rice. 2015 Plant Cell State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Cellulose, which can be converted into numerous industrial products, has important impacts on the global economy. It has long been known that cellulose synthesis in plants is tightly regulated by various phytohormones. However, the underlying mechanism of cellulose synthesis regulation remains elusive. Here, we show that in rice (Oryza sativa), gibberellin (GA) signals promote cellulose synthesis by relieving the interaction between SLENDER RICE1 (SLR1), a DELLA repressor of GA signaling, and NACs, the top-layer transcription factors for secondary wall formation. Mutations in GA-related genes and physiological treatments altered the transcription of CELLULOSE SYNTHASE genes (CESAs) and the cellulose level. Multiple experiments demonstrated that transcription factors NAC29/31 and MYB61 are CESA regulators in rice; NAC29/31 directly regulates MYB61, which in turn activates CESA expression. This hierarchical regulation pathway is blocked by SLR1-NAC29/31 interactions. Based on the results of anatomical analysis and GA content examination in developing rice internodes, this signaling cascade was found to be modulated by varied endogenous GA levels and to be required for internode development. Genetic and gene expression analyses were further performed in Arabidopsis thaliana GA-related mutants. Altogether, our findings reveal a conserved mechanism by which GA regulates secondary wall cellulose synthesis in land plants and provide a strategy for manipulating cellulose production and plant growth. SLR1|OsGAI,MYB61 Cytoplasmic-genetic male sterility gene provides direct evidence for some hybrid rice recently evolving into weedy rice. 2015 Sci Rep Weed Research Laboratory of Nanjing Agricultural University, No.1 Weigang, Xuanwu District, Nanjing 210095, China. Weedy rice infests paddy fields worldwide at an alarmingly increasing rate. There is substantial evidence indicating that many weedy rice forms originated from or are closely related to cultivated rice. There is suspicion that the outbreak of weedy rice in China may be related to widely grown hybrid rice due to its heterosis and the diversity of its progeny, but this notion remains unsupported by direct evidence. We screened weedy rice accessions by both genetic and molecular marker tests for the cytoplasmic male sterility (CMS) genes (Wild abortive, WA, and Boro type, BT) most widely used in the production of indica and japonica three-line hybrid rice as a diagnostic trait of direct parenthood. Sixteen weedy rice accessions of the 358 tested (4.5%) contained the CMS-WA gene; none contained the CMS-BT gene. These 16 accessions represent weedy rices recently evolved from maternal hybrid rice derivatives, given the primarily maternal inheritance of this trait. Our results provide key direct evidence that hybrid rice can be involved in the evolution of some weedy rice accessions, but is not a primary factor in the recent outbreak of weedy rice in China. None Variation in NRT1.1B contributes to nitrate-use divergence between rice subspecies. 2015 Nat Genet State Key Laboratory of Plant Genomics, National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. Asian cultivated rice (Oryza sativa L.) consists of two main subspecies, indica and japonica. Indica has higher nitrate-absorption activity than japonica, but the molecular mechanisms underlying that activity remain elusive. Here we show that variation in a nitrate-transporter gene, NRT1.1B (OsNPF6.5), may contribute to this divergence in nitrate use. Phylogenetic analysis revealed that NRT1.1B diverges between indica and japonica. NRT1.1B-indica variation was associated with enhanced nitrate uptake and root-to-shoot transport and upregulated expression of nitrate-responsive genes. The selection signature of NRT1.1B-indica suggests that nitrate-use divergence occurred during rice domestication. Notably, field tests with near-isogenic and transgenic lines confirmed that the japonica variety carrying the NRT1.1B-indica allele had significantly improved grain yield and nitrogen-use efficiency (NUE) compared to the variety without that allele. Our results show that variation in NRT1.1B largely explains nitrate-use divergence between indica and japonica and that NRT1.1B-indica can potentially improve the NUE of japonica. OsNRT1.3|NRT1.1B|OsNPF6.5 The Stable Level of Glutamine synthetase 2 Plays an Important Role in Rice Growth and in Carbon-Nitrogen Metabolic Balance. 2015 Int J Mol Sci Microelement Research Center, Key Laboratory of Arable Land Conservation (Middle and Lower Reaches of Yangtze River), Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, China. baoaili19870212@163.com. Glutamine synthetase 2 (GS2) is a key enzyme involved in the ammonium metabolism in plant leaves. In our previous study, we obtained GS2-cosuppressed plants, which displayed a normal growth phenotype at the seedling stage, while at the tillering stage they showed a chlorosis phenotype. In this study, to investigate the chlorosis mechanism, we systematically analyzed the plant growth, carbon-nitrogen metabolism and gene expressions between the GS2-cosuppressed rice and wild-type plants. The results revealed that the GS2-cosuppressed plants exhibited a poor plant growth phenotype and a poor nitrogen transport ability, which led to nitrogen accumulation and a decline in the carbon/nitrogen ratio in the stems. Interestingly, there was a higher concentration of soluble proteins and a lower concentration of carbohydrates in the GS2-cosuppressed plants at the seedling stage, while a contrasting result was displayed at the tillering stage. The analysis of the metabolic profile showed a significant increase of sugars and organic acids. Additionally, gene expression patterns were different in root and leaf of GS2-cosuppressed plants between the seedling and tillering stage. These results indicated the important role of a stable level of GS2 transcription during normal rice development and the importance of the carbon-nitrogen metabolic balance in rice growth. None Leaf Lateral Asymmetry in Morphological and Physiological Traits of Rice Plant. 2015 PLoS One National Key Laboratory of Crop Genetic Improvement, Ministry of Agriculture Key Laboratory of Crop Ecophysiology and Farming System in the Middle Reaches of the Yangtze River, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, China. Leaf lateral asymmetry in width and thickness has been reported previously in rice. However, the differences between the wide and narrow sides of leaf blade in other leaf morphological and physiological traits were not known. This study was conducted to quantify leaf lateral asymmetry in leaf width, leaf thickness, specific leaf weight (SLW), leaf nitrogen (N) concentration based on dry weight (Nw) and leaf area (Na), and chlorophyll meter reading (SPAD). Leaf morphological and physiological traits of the two lateral halves of the top three leaves at heading stage were measured on 23 rice varieties grown in three growing seasons in two locations. Leaf lateral asymmetry was observed in leaf width, leaf thickness, Nw, Na, and SPAD, but not in SLW. On average, the leaf width of the wide side was about 17% higher than that of the narrow side. The wide side had higher leaf thickness than the narrow side whereas the narrow side had higher Nw, Na, and SPAD than the wide side. We conclude that the narrow side of leaf blade maintained higher leaf N status than the wide side based on all N-related parameters, which implies a possibility of leaf lateral asymmetry in photosynthetic rate in rice plant. None Extensive sequence variation in rice blast resistance gene Pi54 makes it broad spectrum in nature. 2015 Front Plant Sci National Research Centre on Plant Biotechnology, Pusa Campus New Delhi, India ; Department of Biotechnology, Himachal Pradesh University Shimla, India. Rice blast resistant gene, Pi54 cloned from rice line, Tetep, is effective against diverse isolates of Magnaporthe oryzae. In this study, we prospected the allelic variants of the dominant blast resistance gene from a set of 92 rice lines to determine the nucleotide diversity, pattern of its molecular evolution, phylogenetic relationships and evolutionary dynamics, and to develop allele specific markers. High quality sequences were generated for homologs of Pi54 gene. Using comparative sequence analysis, InDels of variable sizes in all the alleles were observed. Profiling of the selected sites of SNP (Single Nucleotide Polymorphism) and amino acids (N sites ≥ 10) exhibited constant frequency distribution of mutational and substitutional sites between the resistance and susceptible rice lines, respectively. A total of 50 new haplotypes based on the nucleotide polymorphism was also identified. A unique haplotype (H_3) was found to be linked to all the resistant alleles isolated from indica rice lines. Unique leucine zipper and tyrosine sulfation sites were identified in the predicted Pi54 proteins. Selection signals were observed in entire coding sequence of resistance alleles, as compared to LRR domains for susceptible alleles. This is a maiden report of extensive variability of Pi54 alleles in different landraces and cultivated varieties, possibly, attributing broad-spectrum resistance to Magnaporthe oryzae. The sequence variation in two consensus region: 163 and 144 bp were used for the development of allele specific DNA markers. Validated markers can be used for the selection and identification of better allele(s) and their introgression in commercial rice cultivars employing marker assisted selection. pikh|pi54 ABA-induced CCCH tandem zinc finger protein OsC3H47 decreases ABA sensitivity and promotes drought tolerance in Oryza sativa. 2015 Biochem Biophys Res Commun Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China; College of Horticulture, South China Agricultural University, Guangzhou 510642, China. Water deficit causes multiple negative impacts on plants, such as reactive oxygen species (ROS) accumulation, abscisic acid (ABA) induction, stomatal closure, and decreased photosynthesis. Here, we characterized OsC3H47, which belongs to CCCH zinc-finger families, as a drought-stress response gene. It can be strongly induced by NaCl, PEG, ABA, and drought conditions. Overexpression of OsC3H47 significantly enhanced tolerance to drought and salt stresses in rice seedlings, which indicates that OsC3H47 plays important roles in post-stress recovery. However, overexpression of OsC3H47 reduced the ABA sensitivity of rice seedlings. This suggests that OsC3H47 is a newly discovered gene that can control rice drought-stress response, and it may play an important role in ABA feedback and post-transcription processes. OsC3H47 Cross-species network analysis uncovers conserved nitrogen-regulated network modules in rice. 2015 Plant Physiol Instituto de Ingenieria Genetica y Biologia Molecular (INGEBI-CONICET); In this study, we used a cross-species network approach to uncover nitrogen-regulated network modules conserved across a model and a crop species. By translating gene "network knowledge" from the data-rich model Arabidopsis (Arabidopsis thaliana) to a crop (Oryza sativa), we identified evolutionarily conserved N-regulatory modules as targets for translational studies to improve N-use efficiency in transgenic plants. To uncover such conserved N-regulatory network modules, we first generated a N-regulatory network based solely on rice (O. sativa) transcriptome and gene interaction data. Next, we enhanced the "network knowledge" in the rice N-regulatory network using transcriptome and gene interaction data from Arabidopsis and new data from Arabidopsis and rice plants exposed to the same N-treatment conditions. This cross-species network analysis uncovered a set of N-regulated transcription factors (TFs) predicted to target the same genes and network modules in both species. Supernode analysis of the TFs and their targets in these conserved network modules uncovered genes directly related to nitrogen use (e.g. N-assimilation) and to other shared biological processes indirectly related to nitrogen. This cross-species network approach was validated with members of two TF families in the supernode network, bZIP-TGA and HRS1/HHO family, have recently been experimentally validated to mediate the N-response in Arabidopsis. None The potassium transporter OsHAK21 functions in the maintenance of ion homeostasis and tolerance to salt stress in rice. 2015 Plant Cell Environ State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, People's Republic of China. The intracellular potassium (K(+) ) homeostasis, which is crucial for plant survival in saline environments, is modulated by K(+) channels and transporters. Some members of the High-Affinity K(+) transporter (HAK) family are believed to function in the regulation of plant salt tolerance, but the physiological mechanisms remain unclear. Here, we report a significant inducement of OsHAK21 expression by high salinity treatment, and provide genetic evidence of the involvement of OsHAK21 in rice salt tolerance. Disruption of OsHAK21 rendered plants sensitive to salt stress. Compared to the wild type, oshak21 accumulated less K(+) and considerably more Na(+) in both shoots and roots, and had a significantly lower K(+) net uptake rate but higher Na(+) uptake rate. Our analyses of subcellular localizations and expression patterns showed that OsHAK21 was localized in the plasma membrane, and expressed in xylem parenchyma and individual endodermal cells (putative passage cells). Further functional characterizations of OsHAK21 in K(+) uptake-deficient yeast and Arabidopsis revealed that OsHAK21 possesses K(+) transporter activity. These results demonstrate that OsHAK21 may mediate K(+) absorption by the plasma membrane, and play crucial roles in the maintenance of the Na(+) /K(+) homeostasis in rice under salt stress. OsHAK21 Rice potassium transporter OsHAK1 is essential for maintaining potassium mediated growth and functions in salt tolerance over low and high potassium concentration ranges. 2015 Plant Cell Environ State Key Laboratory of Crop Genetics and Germplasm Enhancement, MOA Key Laboratory of Plant Nutrition and Fertilization in Lower-Middle Reaches of the Yangtze River, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, P.R. China. Potassium (K) absorption and translocation in plants rely on multiple K transporters for adapting varied K supply and saline conditions. Here, we report the expression patterns and physiological roles of OsHAK1, a member belonging to KT/KUP/HAK gene family in rice (Oryza sativa L). Expression of OsHAK1 is up-regulated by K deficiency or salt stress in various tissues, particularly in the root and shoot apical meristem, the epidermises and steles of root, and vascular bundles of shoot. Both oshak1 knockout mutants in comparison to their respective Dongjin or Manan wild types showed dramatically reduction in K concentration and stunted root and shoot growth. Knockout of OsHAK1 reduced the K absorption rate of unit root surface area by ∼50-55% and ∼30%, and total K uptake by ∼80% and ∼65% at 0.05-0.1mM and 1mM K supply level, respectively. The root net high affinity K uptake of oshak1 mutants was sensitive to salt stress, but not to ammonium supply. Over-expression of OsHAK1 in rice increased K uptake and K/Na ratio. The positive relationship between K concentration and shoot biomass in the mutants suggests that OsHAK1 plays an essential role in K-mediated rice growth and salt tolerance over low and high K concentration ranges. OsHAK1 Brassinosteroid insensitive 1-associated kinase 1 (OsI-BAK1) is associated with grain filling and leaf development in rice. 2015 J Plant Physiol Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia. Brassinosteroid Insensitive 1 (BRI1)-Associated Kinase I (BAK1) has been reported to interact with BRI1 for brassinosteroid (BR) perception and signal transduction that regulate plant growth and development. The aim of this study is to investigate the functions of a rice OsBAK1 homologue, designated as OsI-BAK1, which is highly expressed after heading. Silencing of OsI-BAK1 in rice plants produced a high number of undeveloped green and unfilled grains compared to the untransformed plants. Histological analyses demonstrated that embryos were either absent or retarded in their development in these unfilled rice grains of OsI-BAK1 RNAi plants. Down regulation of OsI-BAK1 caused a reduction in cell number and enlargement in leaf bulliform cells. Furthermore, transgenic rice plants overexpressing OsI-BAK1 were demonstrated to have corrugated and twisted leaves probably due to increased cell number that caused abnormal bulliform cell structure which were enlarged and plugged deep into leaf epidermis. The current findings suggest that OsI-BAK1 may play an important role in the developmental processes of rice grain filling and leaf cell including the bulliform cells. OsI-BAK1 Ethylene Biosynthesis is Promoted by Very-Long-Chain Fatty Acids during Lysigenous Aerenchyma Formation in Rice Roots. 2015 Plant Physiol CITY: Nagoya Japan [JP] Nagoya University. In rice (Oryza sativa L.) roots, lysigenous aerenchyma, which is created by programmed cell death and lysis of cortical cells, is constitutively formed under aerobic conditions, and its formation is further induced under oxygen-deficient conditions. Ethylene is involved in the induction of aerenchyma formation. reduced culm number 1 (rcn1) is a rice mutant in which the gene encoding the ATP-binding cassette transporter RCN1/OsABCG5 is defective. Here, we report that the induction of aerenchyma formation was reduced in roots of rcn1 grown in stagnant deoxygenated nutrient solution (i.e., under stagnant conditions, which mimic oxygen-deficient conditions in waterlogged soils). 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a key enzyme in ethylene biosynthesis. Stagnant conditions hardly induced the expression of ACS1 in rcn1 roots, resulting in low ethylene production in the roots. Accumulation of saturated very-long-chain fatty acids (VLCFAs) of 24, 26 and 28 carbons was reduced in rcn1 roots. Exogenously supplied VLCFA (26 carbons) increased the expression level of ACS1, and induced aerenchyma formation in rcn1 roots. Moreover, in rice lines in which the gene encoding a fatty acid elongase (CUT1L) was silenced, both ACS1 expression and aerenchyma formation were reduced. Interestingly, the expression of ACS1, CUT1L and RCN1/OsABCG5 was induced predominantly in the outer part of roots (OPR) under stagnant conditions. These results suggest that, in rice under oxygen-deficient conditions, VLCFAs increase ethylene production by promoting ACC biosynthesis in the OPR, which, in turn, induces aerenchyma formation in the root cortex. Rcn1|OsABCG5 Effects of different warming patterns on the translocations of cadmium and copper in a soil-rice seedling system. 2015 Environ Sci Pollut Res Int Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, No. 71 East Beijing Road, Nanjing, 210008, China, lqge@issas.ac.cn. Heavy-metal-polluted rice poses potential threats to food security and has received great attention in recent years, while how elevated temperature affects the translocation of heavy metals in soil-rice system is unclear. In this study, potting experiments were conducted in plant growth chambers for 24 days to evaluate the effects of different warming patterns on cadmium (Cd) and copper (Cu) migrations in soil-rice seedling system. Rice seedlings were cultivated under four different day/night temperature patterns: 25/18 °C (CK), 25/23 °C (N5), 30/18 °C (D5), and 30/23 °C (DN5), respectively. Non-contaminated soil (CS), Cd/Cu lightly polluted soil (LS), and highly polluted soil (HS) were chosen for experiments. The results showed that different warming patterns decreased soil pH and elevated available soil Cd/Cu concentrations. The shoot and root biomass were increased by 39.0-320 and 28.6-348 %, respectively. Warming induced significant (p < 0.05) increase of Cd/Cu uptake and translocation in rice seedlings, especially for the Cd concentration in shoot. The Cd concentrations of shoot increased by 5-12 times and up to 8 times for LS and HS, respectively. Meanwhile, the Cd concentration of shoot increased with warming while that of root kept unchanged, indicating that warming promoted cadmium translocation from root to shoot (about -four to nine times of CK), while warming changed the Cu concentration of shoot similarly to that of root and had no significant effects on Cu translocations in rice seedlings. Our study may provide improved understanding for Cd/Cu fates in soil-rice system by warming and imply that heavy metals had the higher environmental risk under the future global warming. None Overexpression of an AP2/ERF Type Transcription Factor OsEREBP1 Confers Biotic and Abiotic Stress Tolerance in Rice. 2015 PLoS One Centre for Cellular and Molecular Biology, Hyderabad, India. AP2/ERF-type transcription factors regulate important functions of plant growth and development as well as responses to environmental stimuli. A rice AP2/ERF transcription factor, OsEREBP1 is a downstream component of a signal transduction pathway in a specific interaction between rice (Oryza sativa) and its bacterial pathogen, Xoo (Xanthomonas oryzae pv. oryzae). Constitutive expression of OsEREBP1 in rice driven by maize ubiquitin promoter did not affect normal plant growth. Microarray analysis revealed that over expression of OsEREBP1 caused increased expression of lipid metabolism related genes such as lipase and chloroplastic lipoxygenase as well as several genes related to jasmonate and abscisic acid biosynthesis. PR genes, transcription regulators and Aldhs (alcohol dehydrogenases) implicated in abiotic stress and submergence tolerance were also upregulated in transgenic plants. Transgenic plants showed increase in endogenous levels of α-linolenate, several jasmonate derivatives and abscisic acid but not salicylic acid. Soluble modified GFP (SmGFP)-tagged OsEREBP1 was localized to plastid nucleoids. Comparative analysis of non-transgenic and OsEREBP1 overexpressing genotypes revealed that OsEREBP1 attenuates disease caused by Xoo and confers drought and submergence tolerance in transgenic rice. Our results suggest that constitutive expression of OsEREBP1 activates the jasmonate and abscisic acid signalling pathways thereby priming the rice plants for enhanced survival under abiotic or biotic stress conditions. OsEREBP1 is thus, a good candidate gene for engineering plants for multiple stress tolerance. OsEREBP1 Proteomic Analysis of Phosphoproteins in the Rice Nucleus during Early Stage of Seed Germination. 2015 J Proteome Res Fail The early stage of seed germination is the first step in the plant life cycle without visible morphological change. To investigate the mechanism controlling the early stage of rice seed germination, a gel-free/label-free nuclear phosphoproteomics was performed. A total of 3,467 phosphopeptides belonging to 102 nuclear phosphoproteins of rice embryos were identified. Protein synthesis related proteins were mainly phosphorylated. During the first 24 h following imbibition, 115 nuclear phosphoproteins were identified and significant changes in phosphorylation level over time were observed in 29 phosphoproteins. Cluster analysis indicated that nucleotide binding proteins and zinc finger CCCH/BED type proteins increased in abundance during the first 12 h of imbibition and then decreased. The in silico protein-protein interactions for 29 nuclear phosphoproteins indicated that Sas10/Utp3 protein, which functions in snoRNA bind and gene silence, was the center of the phosphoproteins network in nuclei. The germination rate of seeds was significantly slow down with phosphatase-inhibitor treatment. The mRNA expression of zinc finger CCCH type protein did not change and zinc finger BED type was up-regulated in rice embryos during early stage of germination with phosphatase-inhibitor treatment. These results suggest that phosphorylation and dephosphorylation of nuclear protein involve in rice seed germination. Furthermore, transcription factors such as zinc finger CCCH/BED type protein might play a key role through nuclear phosphoproteins, and Sas10/Utp3 protein might be interacted with nuclear phosphoproteins in rice embryos to mediate the early stage of seed germination. None Five pectinase gene expressions highly responding to heat stress in rice floral organs revealed by RNA-seq analysis. 2015 Biochem Biophys Res Commun Center of Cooperative Innovation for Crop, Agronomy College, Anhui Agricultural University, Hefei 230036, Anhui, China; Jiangsu Collaboraive Innovation Center for Modern Crop Production, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China. Heat stress hurts rice, and floral organs are mostly sensitive to heat stress. We aimed to unravel molecular responses to heat stress in rice floral organs using Illumina/Solexa sequencing technology for addressing the increasing concern of globle warming. At meiophase of the pollen mother cell (pulvinus flat), the plants were stressed for 3 d at 38 C, and RNA was extracted from the stressed pistil and stamen for RNA-Seq sequencing to build the heat stress transcriptom library. A total of 7178 defferentially expressed genes (DEGs) between the normal and heat stress libraries were significant, 61% up-regulated and 39% down-regulated. The 7178 DEGs were significantly classified to 34 gene ontology (GO) categories, and 11 of the GO categories were significantly enriched. The GO:0016787 for hydrolase activity of molecular function was mostly enriched with the least probability, and included 11 DEGs named Hy1 - Hy11. Expression levels of five DEGs, Hy4 - Hy6 and Hy9 - Hy10 for starch and sucrose metablism via pectinase, increased 12 - 14 times in response to the heat stress. Further investigation of the five DEGs for pectin metabolism and association with reported heat responsive genes may help develop a molecular strategy to remedy heat damage in rice. None XRCC3 is essential for proper double-strand break repair and homologous recombination in rice meiosis. 2015 J Exp Bot Key Laboratory of Plant Functional Genomics of Ministry of Education/Jiangsu Key Laboratory of Crop Genetics and Physiology/ Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China. RAD51 paralogues play important roles in the assembly and stabilization of RAD51 nucleoprotein filaments, which promote homologous pairing and strand exchange reactions in organisms ranging from yeast to vertebrates. XRCC3, a RAD51 paralogue, has been characterized in budding yeast, mouse, and Arabidopsis. In the present study, XRCC3 in rice was identified and characterized. The rice xrcc3 mutant exhibited normal vegetative growth but complete male and female sterility. Cytological investigations revealed that homologous pairing and synapsis were severely disrupted in the mutant. Meiotic chromosomes were frequently entangled from diplotene to metaphase I, resulting in chromosome fragmentation at anaphase I. The immunostaining signals from γH2AX were regular, implying that double-strand break (DSB) formation was normal in xrcc3 meiocytes. However, COM1 was not detected on early prophase I chromosomes, suggesting that the DSB end-processing system was destroyed in the mutant. Moreover, abnormal chromosome localization of RAD51C, DMC1, ZEP1, ZIP4, and MER3 was observed in xrcc3. Taken together, the results suggest that XRCC3 plays critical roles in both DSB repair and homologous chromosome recombination during rice meiosis. XRCC3 Phenotypic and genetic dissection of component traits for early vigour in rice using plant growth modelling, sugar content analyses and association mapping. 2015 J Exp Bot CIAT, Agrobiodiversity, AA 6713, Cali, Colombia m.c.rebolledo@cgiar.org. Early vigour of rice, defined as seedling capacity to accumulate shoot dry weight (SDW) rapidly, is a complex trait. It depends on a genotype propensity to assimilate, store, and/or use non-structural carbohydrates (NSC) for producing large and/or numerous leaves, involving physiological trade-offs in the expression of component traits and, possibly, physiological and genetic linkages. This study explores a plant-model-assisted phenotyping approach to dissect the genetic architecture of rice early vigour, applying the Genome Wide Association Study (GWAS) to morphological and NSC measurements, as well as fitted parameters for the functional-structural plant model, Ecomeristem. Leaf size, number, SDW, and source-leaf NSC concentration were measured on a panel of 123 japonica accessions. The data were used to estimate Ecomeristem genotypic parameters driving organ appearance rate, size, and carbon dynamics. GWAS was performed based on 12 221 single-nucleotide polymorphisms (SNP). Twenty-three associations were detected at P <1×10(-4) and 64 at P <5×10(-4). Associations for NSC and model parameters revealed new regions related to early vigour that had greater significance than morphological traits, providing additional information on the genetic control of early vigour. Plant model parameters were used to characterize physiological and genetic trade-offs among component traits. Twelve associations were related to loci for cloned genes, with nine related to organogenesis, plant height, cell size or cell number. The potential use of these associations as markers for breeding is discussed. None Quantitative trait locus mapping of deep rooting by linkage and association analysis in rice. 2015 J Exp Bot Shanghai Agrobiological Gene Center, No. 2901, Beidi Road, Minhang District, Shanghai 201106, PR China Fudan University, No. 220, Handan Road, Yangpu District, Shanghai 200433, PR China. Deep rooting is a very important trait for plants' drought avoidance, and it is usually represented by the ratio of deep rooting (RDR). Three sets of rice populations were used to determine the genetic base for RDR. A linkage mapping population with 180 recombinant inbred lines and an association mapping population containing 237 rice varieties were used to identify genes linked to RDR. Six quantitative trait loci (QTLs) of RDR were identified as being located on chromosomes 1, 2, 4, 7, and 10. Using 1 019 883 single-nucleotide polymorphisms (SNPs), a genome-wide association study of the RDR was performed. Forty-eight significant SNPs of the RDR were identified and formed a clear peak on the short arm of chromosome 1 in a Manhattan plot. Compared with the shallow-rooting group and the whole collection, the deep-rooting group had selective sweep regions on chromosomes 1 and 2, especially in the major QTL region on chromosome 2. Seven of the nine candidate SNPs identified by association mapping were verified in two RDR extreme groups. The findings from this study will be beneficial to rice drought-resistance research and breeding. None Genetic shift in local rice populations during rice breeding programs in the northern limit of rice cultivation in the world. 2015 Theor Appl Genet NARO Hokkaido Agricultural Research Center, National Agricultural Research Organization, Sapporo, Hokkaido, 062-8555, Japan, kfujino@affrc.go.jp. The rapid accumulation of pre-existing mutations may play major roles in the establishment and shaping of adaptability for local regions in current rice breeding programs. The cultivated rice, Oryza sativa L., which originated from tropical regions, is now grown worldwide due to the concerted efforts of breeding programs. However, the process of establishing local populations and their origins remain unclear. In the present study, we characterized DNA polymorphisms in the rice variety KITAAKE from Hokkaido, one of the northern limits of rice cultivation in the world. Indel polymorphisms were attributed to transposable element-like insertions, tandem duplications, and non-TE deletions as the original mutation events in the NIPPONBARE and KITAAKE genomes. The allele frequencies of the KITAAKE alleles markedly shifted to the current variety types among the local population from Hokkaido in the last two decades. The KITAAKE alleles widely distributed throughout wild rice and cultivated rice over the world. These have accumulated in the local population from Hokkaido via Japanese landraces as the ancestral population of Hokkaido. These results strongly suggested that combinations of pre-existing mutations played a role in the establishment of adaptability. This approach using the re-sequencing of local varieties in unique environmental conditions will be useful as a genetic resource in plant breeding programs in local regions. None Natural alleles of a proteasome α2 subunit gene contribute to thermotolerance and adaptation of African rice. 2015 Nat Genet National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. Global warming threatens many aspects of human life, for example, by reducing crop yields. Breeding heat-tolerant crops using genes conferring thermotolerance is a fundamental way to help deal with this challenge. Here we identify a major quantitative trait locus (QTL) for thermotolerance in African rice (Oryza glaberrima), Thermo-tolerance 1 (TT1), which encodes an α2 subunit of the 26S proteasome involved in the degradation of ubiquitinated proteins. Ubiquitylome analysis indicated that OgTT1 protects cells from heat stress through more efficient elimination of cytotoxic denatured proteins and more effective maintenance of heat-response processes than achieved with OsTT1. Variation in TT1 has been selected for on the basis of climatic temperature and has had an important role in local adaptati