Complementation of the tomato HWS gene with its Arabidopsis counterpart demonstrates conservation of the gene function between both species.

The HAWAIIAN SKIRT (HWS) gene was originally described in Arabidopsis for the characteristic fusion of sepals in the mutant. A tomato line mutated in the putative ortholog gene was isolated in a previous study. The tomato hws-1 mutant showed facultative parthenocarpy and produced fruits with elevated Brix, revealing the gene as a hopeful resource for crop improvement. To confirm the orthology relationship between the Arabidopsis and tomato HWS genes, the hws-1 mutant was complemented with either the tomato wild-type genomic fragment or the Arabidopsis sequence of the gene. In both complementation experiments, defective phenotypes of hws-1 are rescued, albeit to different extents. Recovery of these phenotypes, which include parthenocarpic fruit production, increased Brix, loss of leaflet serration, alteration of bud and petal shape, firmly establishes SlHWS as an ortholog of the originally described HWS in Arabidopsis. This work indicates that the function of HWS is likely to be conserved in a wide range of plant species.

Rapid turnover of antimicrobial-type cysteine-rich protein genes in closely related Oryza genomes.

Defensive and reproductive protein genes undergo rapid evolution. Small, cysteine-rich secreted peptides (CRPs) act as antimicrobial agents and function in plant intercellular signaling and are over-represented among reproductively expressed proteins. Because of their roles in defense, reproduction and development and their presence in multigene families, CRP variation can have major consequences for plant phenotypic and functional diversification. We surveyed the CRP genes of six closely related Oryza genomes comprising Oryza sativa ssp. japonica and ssp. indica, Oryza glaberrima and three accessions of Oryza rufipogon to observe patterns of evolution in these gene families and the effects of variation on their gene expression. These Oryza genomes, like other plant genomes, have accumulated large reservoirs of CRP sequences, comprising 26 groups totaling between 676 and 843 genes, in contrast to antimicrobial CRPs in animal genomes. Despite the close evolutionary relationships between the genomes, we observed rapid changes in number and structure among CRP gene families. Many CRP sequences are in gene clusters generated by local duplications, have undergone rapid turnover and are more likely to be silent or specifically expressed. By contrast, conserved CRP genes are more likely to be highly and broadly expressed. Variable CRP genes created by repeated duplication, gene modification and inactivation can gain new functions and expression patterns in newly evolved gene copies. For the CRP proteins, the process of gain/loss by deletion or duplication at gene clusters seems to be an important mechanism in evolution of the gene families, which also contributes to their expression evolution.

Rice expression atlas in reproductive development.

Gene expression throughout the reproductive process in rice (Oryza sativa) beginning with primordia development through pollination/fertilization to zygote formation was analyzed. We analyzed 25 stages/organs of rice reproductive development including early microsporogenesis stages with 57,381 probe sets, and identified around 26,000 expressed probe sets in each stage. Fine dissection of 25 reproductive stages/organs combined with detailed microarray profiling revealed dramatic, coordinated and finely tuned changes in gene expression. A decrease in expressed genes in the pollen maturation process was observed in a similar way with Arabidopsis and maize. An almost equal number of ab initio predicted genes and cloned genes which appeared or disappeared coordinated with developmental stage progression. A large number of organ-/stage-specific genes were identified; notably 2,593 probe sets for developing anther, including 932 probe sets corresponding to ab initio predicted genes. Analysis of cell cycle-related genes revealed that several cyclin-dependent kinases (CDKs), cyclins and components of SCF E3 ubiquitin ligase complexes were expressed specifically in reproductive organs. Cell wall biosynthesis or degradation protein genes and transcription factor genes expressed specifically in reproductive stages were also newly identified. Rice genes homologous to reproduction-related genes in other plants showed expression profiles both consistent and inconsistent with their predicted functions. The rice reproductive expression atlas is likely to be the most extensive and most comprehensive data set available, indispensable for unraveling functions of many specific genes in plant reproductive processes that have not yet been thoroughly analyzed.

Development of a novel data mining tool to find cis-elements in rice gene promoter regions.

BACKGROUND: Information on more than 35 000 full-length Oryza sativa cDNAs, together with associated microarray gene expression data collected under various treatment conditions, has made it feasible to identify motifs that are conserved in gene promoters and may act as cis-regulatory elements with key roles under the various conditions. RESULTS: We have developed a novel tool that searches for cis-element candidates in the upstream, downstream, or coding regions of differentially regulated genes. The tool first lists cis-element candidates by motif searching based on the supposition that if there are cis-elements playing important roles in the regulation of a given set of genes, they will be statistically overrepresented and will be conserved. Then it evaluates the likelihood scores of the listed candidate motifs by association rule analysis. This strategy depends on the idea that motifs overrepresented in the promoter region could play specific roles in the regulation of expression of these genes. The tool is designed so that any biological researchers can use it easily at the publicly accessible Internet site http://hpc.irri.cgiar.org/tool/nias/ces. We evaluated the accuracy and utility of the tool by using a dataset of auxin-inducible genes that have well-studied cis-elements. The test showed the effectiveness of the tool in identifying significant relationships between cis-element candidates and related sets of genes. CONCLUSION: The tool lists possible cis-element motifs corresponding to genes of interest, and it will contribute to the deeper understanding of gene regulatory mechanisms in plants.

Comparative molecular biological analysis of membrane transport genes in organisms.

Comparative analyses of membrane transport genes revealed many differences in the features of transport homeostasis in eight diverse organisms, ranging from bacteria to animals and plants. In bacteria, membrane-transport systems depend mainly on single genes encoding proteins involved in an ATP-dependent pump and secondary transport proteins that use H(+) as a co-transport molecule. Animals are especially divergent in their channel genes, and plants have larger numbers of P-type ATPase and secondary active transporters than do other organisms. The secondary transporter genes have diverged evolutionarily in both animals and plants for different co-transporter molecules. Animals use Na(+) ions for the formation of concentration gradients across plasma membranes, dependent on secondary active transporters and on membrane voltages that in turn are dependent on ion transport regulation systems. Plants use H(+) ions pooled in vacuoles and the apoplast to transport various substances; these proton gradients are also dependent on secondary active transporters. We also compared the numbers of membrane transporter genes in Arabidopsis and rice. Although many transporter genes are similar in these plants, Arabidopsis has a more diverse array of genes for multi-efflux transport and for response to stress signals, and rice has more secondary transporter genes for carbohydrate and nutrient transport.

Gene organization in rice revealed by full-length cDNA mapping and gene expression analysis through microarray.

Rice (Oryza sativa L.) is a model organism for the functional genomics of monocotyledonous plants since the genome size is considerably smaller than those of other monocotyledonous plants. Although highly accurate genome sequences of indica and japonica rice are available, additional resources such as full-length complementary DNA (FL-cDNA) sequences are also indispensable for comprehensive analyses of gene structure and function. We cross-referenced 28.5K individual loci in the rice genome defined by mapping of 578K FL-cDNA clones with the 56K loci predicted in the TIGR genome assembly. Based on the annotation status and the presence of corresponding cDNA clones, genes were classified into 23K annotated expressed (AE) genes, 33K annotated non-expressed (ANE) genes, and 5.5K non-annotated expressed (NAE) genes. We developed a 60mer oligo-array for analysis of gene expression from each locus. Analysis of gene structures and expression levels revealed that the general features of gene structure and expression of NAE and ANE genes were considerably different from those of AE genes. The results also suggested that the cloning efficiency of rice FL-cDNA is associated with the transcription activity of the corresponding genetic locus, although other factors may also have an effect. Comparison of the coverage of FL-cDNA among gene families suggested that FL-cDNA from genes encoding rice- or eukaryote-specific domains, and those involved in regulatory functions were difficult to produce in bacterial cells. Collectively, these results indicate that rice genes can be divided into distinct groups based on transcription activity and gene structure, and that the coverage bias of FL-cDNA clones exists due to the incompatibility of certain eukaryotic genes in bacteria.

Combining in silico mapping and arraying: an approach to identifying common candidate genes for submergence tolerance and resistance to bacterial leaf blight in rice.

Several genes/QTLs governing resistance/tolerance to abiotic and biotic stresses have been reported and mapped in rice. A QTL for submergence tolerance was found to be co-located with a major QTL for broad-spectrum bacterial leaf blight (bs-blb) resistance on the long arm of chromosome 5 in indica cultivars FR13A and IET8585. Using the Nipponbare (japonica) and 93-11 (indica) genome sequences, we identified, in silico, candidate genes in the chromosomal region [Kottapalli et al. (2006)]. Transcriptional profiling of FR13A and IET8585 using a rice 22K oligo array validated the above findings. Based on in silico analysis and arraying we observed that both cultivars respond to the above stresses through a common signaling system involving protein kinases, adenosine mono phosphate kinase, leucine rich repeat, PDZ/DHR/GLGF, and response regulator receiver protein. The combined approaches suggest that transcription factor EREBP on long arm of chromosome 5 regulates both submergence tolerance and blb resistance. Pyruvate decarboxylase and alcohol dehydrogenase, co-located in the same region, are candidate downstream genes for submergence tolerance at the seedling stage, and t-snare for bs-blb resistance. We also detected up-regulation of novel defense/stress-related genes including those encoding fumaryl aceto acetate (FAA) hydrolase, scramblase, and galactose oxidase, in response to the imposed stresses.

Microarray analysis of genes that respond to gamma-irradiation in Arabidopsis.

To reveal the signal transduction mechanism of the response to stress in the form of active oxygen species, we used a microarray system to analyze gene expression patterns 2 or 24 h after gamma-irradiation of Arabidopsis. gamma-Irradiation induces several signal transduction and metabolite genes. By analysis of cis-elements located on the promoter region of the gamma-responsive genes, we have also found several cis-elements related to various signal transduction systems. We also analyzed the pleiotropic mutant ttg1-1, which has a dramatically altered physiological response to gamma-irradiation. By comparing the gene expression patterns of wild-type (Ler) and ttg1-1 mutant plants after gamma-irradiation, we identified various TTG1-regulated gamma-response genes. Analysis of the cis-elements in the promoter region of the gamma-responsive genes also revealed that the many transcription factors interacting with TTG1 protein (WD40 protein) are related to the gamma-responsive gene expression.

Radial expansion of root cells and elongation of root hairs of Arabidopsis thaliana induced by massive doses of gamma irradiation.

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.

Comparative analysis of plant and animal calcium signal transduction element using plant full-length cDNA data.

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.

Collection, mapping, and annotation of over 28,000 cDNA clones from japonica rice.

We collected and completely sequenced 28,469 full-length complementary DNA clones from Oryza sativa L. ssp. japonica cv. Nipponbare. Through homology searches of publicly available sequence data, we assigned tentative protein functions to 21,596 clones (75.86%). Mapping of the cDNA clones to genomic DNA revealed that there are 19,000 to 20,500 transcription units in the rice genome. Protein informatics analysis against the InterPro database revealed the existence of proteins presented in rice but not in Arabidopsis. Sixty-four percent of our cDNAs are homologous to Arabidopsis proteins.

Levels of active oxygen species are controlled by ascorbic acid and anthocyanin in Arabidopsis.

Stabilization of the levels of active oxygen species (AOS) is important to the survival of organisms. To clarify the system controlling levels of AOS in plants, this study used an electron spin resonance (ESR) method to directly measure superoxide radical (O(2)(.-)) scavenging activities in the wild-type Arabidopsis thaliana (Col and Ler ecotypes), two anthocyanin mutants (tt3 and ttg1), and an ascorbic acid mutant (vtc1). Under ordinary growth conditions, Arabidopsis contained superoxide-scavenging activity (SOSA) of approximately 300-500 SOD units/g of fresh weight. The ESR pattern indicated that most (40-50%) of this activity was due to ascorbic acid. For the analysis of SOSA under conditions of oxidative stress, synthesis of AOS was induced by gamma-irradiation. The radical scavenging activity in irradiated plants increased approximately 10-fold following an associated increase in the accumulation of ascorbic acid and anthocyanin. The accumulation of ascorbic acid and anthocyanin was suppressed by treatment with an antioxidant before irradiation and was induced by treatment with a radical-generating reagent. The contributions of ascorbic acid and anthocyanin to the total superoxide radical scavenging activity differed among ecotypes. In the Ler ecotype, ascorbic acid accumulated at twice the level of that in the Col ecotype, and induction of anthocyanin was half that in Col. To confirm the activity of ascorbic acid and anthocyanin against AOS stress, the viability of the wild type and mutants (tt2, tt3,tt5, ttg1, and vtc1) was examined after gamma-irradiation. Only the plants in which ascorbic acid and anthocyanin were induced had the ability to grow and flower.

Comprehensive analysis of NAC family genes in Oryza sativa and Arabidopsis thaliana.

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.