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We identified 163 AP2/EREBP (APETALA2/ethylene-responsive element-binding protein) genes in rice. We analyzed gene structures, phylogenies, domain duplication, genome localizations and expression profiles. Conserved amino acid residues and phylogeny construction using the AP2/ERF conserved domain sequence suggest that in rice the OsAP2/EREBP gene family can be classified broadly into four subfamilies [AP2, RAV (related to ABI3/VP1), DREB (dehydration-responsive element-binding protein) and ERF (ethylene-responsive factor)]. The chromosomal localizations of the OsAP2/EREBP genes indicated 20 segmental duplication events involving 40 genes; 58 redundant OsAP2/EREBP genes were involved in tandem duplication events. There were fewer introns after segmental duplication. We investigated expression profiles of this gene family under biotic stresses [infection with rice viruses such as rice stripe virus (RSV), rice tungro spherical virus (RTSV) and rice dwarf virus (RDV, three virus strains S, O and D84)], and various abiotic stresses. Symptoms of virus infection were more severe in RSV infection than in RTSV and RDV infection. Responses to biotic stresses are novel findings and these stresses enhance the ability to identify the best candidate genes for further functional analysis. The genes of subgroup B-5 were not induced under abiotic treatments whereas they were activated by the three RDV strains. None of the genes of subgroups A-3 were differentially expressed by any of the biotic stresses. Our 44K and 22K microarray results suggest that 53 and 52 non-redundant genes in this family were up-regulated in response to biotic and abiotic stresses, respectively. We further examined the stress responsiveness of most genes by reverse transcription-PCR. The study results should be useful in selecting candidate genes from specific subgroups for functional analysis.

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.

The Rice Annotation Project Database (RAP-DB) was created to provide the genome sequence assembly of the International Rice Genome Sequencing Project (IRGSP), manually curated annotation of the sequence, and other genomics information that could be useful for comprehensive understanding of the rice biology. Since the last publication of the RAP-DB, the IRGSP genome has been revised and reassembled. In addition, a large number of rice-expressed sequence tags have been released, and functional genomics resources have been produced worldwide. Thus, we have thoroughly updated our genome annotation by manual curation of all the functional descriptions of rice genes. The latest version of the RAP-DB contains a variety of annotation data as follows: clone positions, structures and functions of 31 439 genes validated by cDNAs, RNA genes detected by massively parallel signature sequencing (MPSS) technology and sequence similarity, flanking sequences of mutant lines, transposable elements, etc. Other annotation data such as Gnomon can be displayed along with those of RAP for comparison. We have also developed a new keyword search system to allow the user to access useful information. The RAP-DB is available at: http://rapdb.dna.affrc.go.jp/ and http://rapdb.lab.nig.ac.jp/.

Transcripts of thionin genes encoding antimicrobial peptides were present at a high level in rice coleoptiles just after germination, and decreased to an undetectable level after about 3 d, but this decline was suppressed by co-treatment with gibberellic acid (GA(3)) and brassinolide (BL). The temporal expression patterns of key enzyme genes for the biosyntheses of gibberellins (GAs) and brassinosteroids (BRs) were correlated with the fluctuation of thionin mRNAs. Jasmonic acid (JA) replaced the effect of GA3 and BL, and its change in endogenous level was parallel to that of the thionin genes. These results strongly suggest that thionin gene expression was positively regulated by JA, whose endogenous level was synergistically regulated by GAs and BRs. In contrast, thionin gene expression in etiolated seedlings remained high while the endogenous level of JA was low, suggesting the presence of another signaling pathway in the dark to maintain the thionin level.

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.

Motivation: Transcription start site selection and alternative splicing greatly contribute to diversifying gene expression. Recent studies have revealed the existence of alternative first exons, but most have involved mammalian genes, and as yet the regulation of usage of alternative first exons has not been clarified, especially in plants., Results: We systematically identified putative alternative first exon transcripts in rice, verified the candidates using RT-PCR, and searched for the promoter elements that might regulate the alternative first exons. As a result, we detected a number of unreported alternative first exons, some of which are regulated in a tissue-specific manner., Supplementary Information: http://www.bioinfo.sfc.keio.ac.jp/research/intron.

Radial expansion of root cells and elongation of root hairs were induced within 3 d of a massive dose (3 kGy) of gamma irradiation to Arabidopsis thaliana. Because treatment with the antioxidant n-propyl gallate before irradiation suppressed these changes, gamma irradiation partially rescued the rhd2 mutant (defective in NADPH oxidase); the superoxide-generating reagent paraquat induced similar root morphogenesis. These responses appeared to be induced by the active oxygen species (AOS) generated by water radiolysis. Ethylene production was induced immediately after gamma irradiation and reached a steady level after about 2 h. Addition of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid partly induced a similar expansion of root cells and elongation of root hairs. Addition of an inhibitor of ethylene biosynthesis, aminoethoxyvinylglycine, before gamma irradiation completely suppressed the formation of abnormal structures. These results suggest that the AOS is involved in the root morphological changes through the ethylene biosynthesis induced by gamma irradiation in Arabidopsis.

We obtained 32K full-length cDNA sequence data from the rice full-length cDNA project and performed a homology search against NCBI GenBank data. We have also searched homologs of Arabidopsis and other plants' genes with the databases. Comparative analysis of calcium ion transport proteins revealed that the genes specific for muscle and nerve calcium signal transduction systems (VDCC, IP3 receptor, ryanodine receptor) are very different in animals and plants. In contrast, Ca elements with basic functions in cell responses (CNGC, iGlu receptor, Ca(2+)ATPase, Ca2+/Na(+)-K+ ion exchanger) are basically conserved between plants and animals. We also performed comparative analyses of calcium ion binding and/or controlling signal transduction proteins. Many genes specific for muscle and nerve tissue do not exist in plants. However, calcium ion signal transduction genes of basic functions of cell homeostasis and responses were well conserved; plants have developed a calcium ion interacting system that is more direct than in animals. Many species of plants have specifically modified calcium ion binding proteins (CPK, CRK), Ca2+/phospholipid-binding domains, and calcium storage proteins.

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.

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.

The Rice Genome Research Project in Japan performs genome sequencing and comprehensive expression profiling, constructs genetic and physical maps, collects full-length cDNAs and generates mutant lines, all aimed at improving the breeding of the rice plant as a food source. The National Institute of Agrobiological Sciences in Tsukuba, Japan, has accumulated numerous rice biological resources and has already successfully produced a high-quality genome sequence, a high-density genetic map with 3000 markers, 30,000 full-length cDNAs, over 700 expression profiles with a 9000 cDNA microarray and 15,000 flanking sequences with Tos17 insertions in about 3765 mutant lines from about 50,000 transposon insertion lines. These resources are available in the public domain. A new unification tool for functional genomics, called Rice PIPELINE, has also been developed for the dynamic collection and compilation of genomics data (genome sequences, full-length cDNAs, gene expression profiles, mutant lines, cis elements) from various databases. The mission of Rice PIPELINE is to provide a unique scientific resource that pools publicly available rice genomic data for search by clone sequence, clone name, GenBank accession number, or keyword. The web-based form of Rice PIPELINE is available at http://cdna01.dna.affrc.go.jp/PIPE/.

The NAC domain was originally characterized from consensus sequences from petunia NAM and from Arabidopsis ATAF1, ATAF2, and CUC2. Genes containing the NAC domain (NAC family genes) are plant-specific transcriptional regulators and are expressed in various developmental stages and tissues. We performed a comprehensive analysis of NAC family genes in Oryza sativa (a monocot) and Arabidopsis thaliana (a dicot). We found 75 predicted NAC proteins in full-length cDNA data sets of O. sativa (28,469 clones) and 105 in putative genes (28,581 sequences) from the A. thaliana genome. NAC domains from both predicted and known NAC family proteins were classified into two groups and 18 subgroups by sequence similarity. There were a few differences in amino acid sequences in the NAC domains between O. sativa and A. thaliana. In addition, we found 13 common sequence motifs from transcriptional activation regions in the C-terminal regions of predicted NAC proteins. These motifs probably diverged having correlations with NAC domain structures. We discuss the relationship between the structure and function of the NAC family proteins in light of our results and the published data. Our results will aid further functional analysis of NAC family genes.

We used an 8987-EST collection to construct a cDNA microarray system with various genomics information (full-length cDNA, expression profile, high accuracy genome sequence, phenotype, genetic map, and physical map) in rice. This array was used as a probe to hybridize target RNAs prepared from normally grown callus of rice and from callus treated for 6 hr or 3 days with the hormones abscisic acid (ABA) or gibberellin (GA). We identified 509 clones, including many clones that had never been annotated as ABA-or GA-responsive. These genes included not only ABA- or GA-responsive genes but also genes responsive to other physiological conditions such as pathogen infection, heat shock, and metal ion stress. Comparison of ABA- and GA-responsive genes revealed antagonistic regulation for these genes by both hormones except for one defense-related gene, thionin. The gene for thionin was up-regulated by both hormone treatments for 3 days. The upstream regions of all the genes that were regulated by both hormones had cis-elements for ABA and GA response. We performed a clustering analysis of genes regulated by both hormones and various expression profiles that showed three notable clusters (seed tissues, low temperature and sugar starvation, and thionin-gene related). A comparison of the cis-elements for hormone response genes between rice and Arabidopsis thaliana, we identified cis-elements for dehydration-stress response or for expression of amylase gene as Arabidopsis gene-specific or rice gene-specific, respectively.

A cDNA encoding a predicted 15-kDa protein was earlier isolated from sugar-induced genes in rice embryos (Oryza sativa L.) by cDNA microarray analysis. Here we report that this cDNA encodes a novel Ca2+-binding protein, named OsSUR1 (for Oryza sativa sugar-up-regulated-1). The recombinant OsSUR1 protein expressed in Escherichia coli had 45Ca2+-binding activity. Northern analysis showed that the OsSUR1 gene was expressed mainly in the internodes of mature plants and in embryos at an early stage of germination. Expression of the OsSUR1 gene was induced by sugars that could serve as substrates of hexokinase, but expression was not repressed by Ca2+ signaling inhibitors, calmodulin antagonists and inhibitors of protein kinase or protein phosphatase. These results suggested that Os-SUR1 gene expression was stimulated by a hexokinase-dependent pathway not mediated by Ca2+.