The powdery mildew Golovinomyces orontii is an obligate biotrophic fungus that infects Arabidopsis thaliana. For successful infection, powdery mildew has to overcome mutilayer defense system of plants. The conidial germination and appresorial formation are largely affected by the host cuticular components. After invasion, plant hormone salicylic acid (SA) limits powdery mildew growth and proliferation, although exact role of SA in the plant defense response remains to be elucidated. Our detailed morphological analyses revealed that powdery mildew germination and appresorial formation were suppressed on stems compared to leaves, and that this suppression was significantly reduced on wax2/yre/flp1 stems, in which the wax accumulation was reduced and cuticular components were altered. Interestingly, the mutation in WAX2/YRE/FLP1 showed an opposite effect at later infection stages; on wax2/yre/flp1 leaves, mycelial growth and conidiophore formation were retarded. We are currently carrying out further morphological analyses as well as RT-PCR analysis to discuss the multiple roles of WAX2/YRE/FLP1 in the Arabidopsis defense response against powdery mildew.

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We previously reported that bacterial LPS acts as a potent elicitor to induce defense responses in rice cells¹⁾. We also found that the pretreatment of rice cells with a low concentration of LPS induced "primed state" for following chitin elicitation. Recent studies on the latter phenomenon, including simultaneous treatment of rice cells with both elicitors, indicated that the phenomenon should rather be interpreted as a synergistic effect of these elicitors.
To confine the structure required for the biological activities of LPS in rice, we first examined the activity of LOS, a smaller molecule consisting of a defined oligosaccharide unit and Lipid A²⁾. LOS induced defense responses and synergistic effects in rice cells as similar to the whole LPS molecules. Examination of the biological activities of Lipid A and oligosaccharide obtained by mild acid hydrolysis of LOS showed that the Lipid A induced both responses in the rice cells but the remaining portion, oligosaccharide fraction, did not. These results clearly showed that rice cells recognize LPS through the Lipid A structure.
¹⁾Desaki et al., Plant Cell Physiol., 47, 1530 (2006). ²⁾Alba et al., ChemBioChem, 9, 896 (2008).

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Spatiotemporal patterns of cytosolic [Ca²⁺] play a pivotal role as a second messenger in various signaling pathways. However the molecular bases of Ca²⁺ influx and its regulation remain largely unknown. We identified a putative voltage-gated Ca²⁺-permeable channel, OsTPC1, in rice and established its overexpressors as well as its retrotransposon-insertional functional knockout mutants (Ostpc1). OsTPC1 has been suggested to play a role in the regulation of defense responses including activation of a MAP kinase and hypersensitive cell death induced by a xylanase elicitor, TvX in rice cells (Kurusu et al. Plant J. 2005). We here established transgenic cell lines stably expressing apoaequorin and analyzed TvX elicitor-induced cytosolic [Ca²⁺] changes in the wild type and Ostpc1 cells. Moreover, TvX-induced gene expression pattern was compared between the wild type and Ostpc1 cells by oligomicroarray transcriptome analyses. Possible physiological roles of OsTPC1 in elicitor-induced defense responses will be discussed.

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Antimicrobial diterpenoid compounds such as momilactones and phytocassanes are major phytoalexins produced in rice upon pathogen attack. We previously reported that six cytochrome P450 monooxigenase (P450) genes compose a gene cluster with the diperpene cyclase genes for phytocassanes biosynthesis on chromosome 2, and confirmed that CYP71Z7 is involved in the biosyntheses of phytocassanes A, B and D.
Here, we investigated the functions of other P450s, CYP76M5 to CYP76M8, which are located on the phytocassanes biosynthetic gene cluster, by generating their RNAi lines. One of the RNAi lines did not show any production of phytocassanes after elicitor treatment, whereas another line exhibited an ability to produce only phytocassane A, suggesting that these RNAi lines seem to have differential effects on the expression of target P450 genes in the cluster. Expression profiling of these P450 genes in the RNAi lines will help us to know steps catalyzed by these P450s in the biosynthetic pathway for phytocassanes.

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Momilactones and phytocassanes are major phytoalexins in rice. We showed that biosynthetic genes for momilactones are clustered on rice chromosome 4, and that those for phytocassanes are clustered on rice chromosome 2. We also succeeded in identification of a bZIP transcription factor OsTGAP1 that coordinately regulates the expression of all the momilactone biosynthetic genes in the cluster. Here we report that OsTGAP1 is also involved in the expression of the MEP pathway genes (OsDXS3, OsDXR, OsCMS) and phytocassane biosynthetic genes (OsCPS2, OsKSL7). Inductive expression of OsCPS2 and OsKSL7 after treatment with a chitin oligosaccharide elicitor in wild-type cells was weakened in ostgap1 mutant cells. In contrast, OsTGAP1 over-expressing lines exhibited enhanced expression of OsCPS2 and OsKSL7 in response to the elicitor. We also found that the elicitor-induced expression of OsDXS3, OsDXR and OsCMS was significantly suppressed in the ostgap1 mutant cells and that OsTGAP1 over-expressing lines exhibited enhanced expression of these genes. These results indicate that OsTGAP1 is a master transcription factor regulating the inductive production of diterpenoid phytoalexins in rice.

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OsWRKY53 is a chitin oligosaccharide elicitor-induced WRKY gene in rice. Previously we indicated that OsWRKY53 functions as a transcriptional activator, and that overexpression of OsWRKY53 in rice plants resulted in enhanced resistance to the rice blast fungus Magnaporthe oryzae. To identify direct target genes of OsWRKY53, we generated transgenic rice cells which constitutively express an OsWRKY53-HA fusion protein, and we performed ChIP-chip analysis using a custom promoter array which covers promoter regions of rice chitin elicitor-induced genes. As the result of ChIP-chip analysis, we identified several genes as the candidates of the direct target genes of OsWRKY53. These genes included transcription factors and PR protein genes. We further performed transcriptome analysis using rice cells overexpressing a dominant-nagative OsWRKY53 mutant, indicating that the expression levels of the several candidate genes decreased in the transgenic rice cells compared with vector control rice cells. Reporter gene assays to examine if the binding regions of OsWRKY53 function as elicitor-responsive cis-elements of the target genes are now in progress.

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CEBiP (chitin elicitor binding protein) shows high-affinity for chitin elicitor and functions as a biological receptor for this elicitor. However the detailed analysis of the structure-activity relationship of CEBiP has not been accomplished. We noticed that a tobacco BY-2 cell line lacks chitin elicitor responsiveness as well as CEBiP-like binding proteins in the plasma membrane, which makes the cell line as an excellent system for the heterologous expression of CEBiP-like molecules. Actually, the CEBiP protein expressed in the BY-2 cells showed binding characteristics identical to those of rice CEBiP by the analysis of affinity cross-linking assay with biotinylated chitin octasaccharide. The chitin binding activity of CEBiP was eliminated or markedly reduced by deletion of a LysM motif. These results suggest that the expression system and affinity cross-linking assay are useful for the analysis of CEBiP and related chitin receptor candidates.

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Chitin is a major MAMP (Microbe-Associated Molecular Pattern) for various fungi and its fragments, chitin oligosaccharides, are known to induce various defense responses in plant cells. Our recent study showed that CEBiP (chitin elicitor binding protein1)) and OsCERK1 (rice CERK1 homologue2)) play an important role as cell surface receptors for chitin elicitor signaling in rice. Since the co-existence of CEBiP and OsCERK1 are necessary in the effective signal transduction of chitin elicitor in rice, we assumed that the formation of receptor complex consisting of CEBiP and OsCERK1 is important for chitin elicitor signal transduction in rice.
Yeast two-hybrid analyses suggested that CEBiP and OsCERK1 have an ability to form the receptor complex(es) as a hetero- or homo-oligomer through the direct interactions of the extracellular domains. In the rice cells expressing the myc-tagged OsCERK1, it was revealed that OsCERK1 was autophosphorylated and the receptor complex(es) containing CEBiP and OsCERK1 existed in the plasma membrane. Further analyses are in progress. 1) Kaku et al., PNAS, 103, 11086 ('06), 2)Miya et al., PNAS, 104, 19613 ('07).

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Although many receptor-like proteins are believed to exist on the plasma membrane of plant cells, functions of these molecules are largely unknown. One reason for such a situation seems to be the experimental difficulties associated with their biochemical analysis. We tried to develop a new method for the biochemical studies of membrane receptors using a combination of wheat germ cell-free translation system and reconstitution of the in vitro-synthesized protein into liposomes.
We evaluated the validity of this system by using CEBiP(Chitin Elicitor Binding Protein) and CERK1(Chitin Elicitor Receptor Kinase1) as model membrane receptors. At the beginning, CEBiP was partially degraded by endogenous proteases during the expression/reconstitution. However, the addition of GST tag at the N-terminus or protease inhibitors effectively blocked the degradation. CERK1 was successfully synthesized without these treatments. Interestingly, CERK1 seemed to be autophosphorylated when ATP was included in the proteoliposomes. Altogether, these results suggested that receptor-like molecules reconstructed on proteoliposome could be a suitable model system for their biochemical studies.

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Detection of pathogens based on the perception of microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) plays an important role in basal resistance in plants. Chitin oligosaccharide, a representative fungal MAMP, induces defense response in Arabidopsis and rice. By reverse genetic approach we previously identified a novel receptor kinase CERK1 essential for chitin signaling ¹⁾.
To find novel components of chitin elicitor signaling, we screened activation-tagged mutants for those suppressed for chitin response, resulting in the finding of a mutant that lost chitin elicitor responsiveness. In the following generations of the mutant, however, even increased response to the chitin elicitor was observed. This phenomenon suggested a possibility of the overexpression of a negative regulator for defense responses in the original mutant and following silencing. We are evaluating the validity of such a hypothesis and also trying to identify the corresponding gene. ¹⁾Miya et al., PNAS,104, 19613, 2007

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Lipopolysaccharide (LPS), which is a primary constituent of the outer membrane of gram-negative bacteria, is a pathogen-associated molecular pattern. Although LPS causes defense-related responses in plants, none of the molecules consisting of the LPS recognition mechanism have been identified. Previously, we had characterized A. thaliana LPS-binding protein 2 (AtLBP2) and suggested the relationship between AtLBP2 and plant innate immune responses. In this study, we focused the detailed function of AtLBP2 in LPS recognition of plants.
On the other hand, we had reported the LPS-induced A. thaliana seedling growth inhibition. In this study, we showed that LPS recognition (LPS-induced defense responses) was consistent with growth inhibition of seedlings. These results suggest that seedling growth inhibition assay using LPS will be informative to identify LPS receptor protein or the other signaling molecules of plants.

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Phytopathogenic Fusarium species produced a trichothecene family of phytotoxins. Trichothecene-contaminated grains and commodities are frequently reported all over the world. We are studying the mode of action of trichothecenes in Fusarium-susceptible Arabidopsis plants. We reported an AtNFXL1 gene as type A trichothecene, T-2 toxin-inducible gene. The AtNFXL1 gene negatively regulates the T-2 toxin-inducible defense response via a SA biosynthesis. Furthermore, we identified a thionin2.3 as a component of an AtNFXL1 protein complex. Thionin proteins are generally known as antimicrobial proteins. In fact, the thionin2.3 protein expressed in E. coli effectively inhibited the hyphal growth of F. sporotrichioides in media. When leaves of Thi2.3-overexpressed Arabidopsis plants were inoculated with conidia of F. sporotrichioides,their hypal extension was significantly inhibited compared with wild type plants. In addition, disease symptoms were decreased in F. sporotrichioides-inoculated Thi2.3-ox flowers compared with wild type. Thus, the Arabidopsis Thi2.3 protein has an antifugal activity against F. sporotrichioides by inhibition of their hyphal growth.

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Arabidopsis exhibits durable preinvasion resistance against nonadapted fungal pathogens including Colletotrichum species. We have found recently that EDR1 is involved in this cell-periphery resistance. In this study, we investigated the transcript profiles of the edr1 mutant during nonhost defense response by DNA microarray. The analysis revealed that the expression of four plant defensin genes is significantly reduced in the mutant. Real-time PCR analysis showed that the wild-type plant, but not the edr1 mutant, exhibits induced expression of the defensins in response to nonadapted Colletotrichum. The results suggest that EDR1 is required for induced expression of the defensins against the fungal invasion trial. MYC2 transcription factor negatively regulates defensin expression. In contrast to the edr1 mutant, the edr1 myc2mutant exhibited pathogen-induced expression of the defensins with increased resistance against the nonadapted pathogen. Furthermore, constitutive expression of the defensins also partially restored preinvasion resistance in the edr1 mutant. These results strongly suggest that EDR1-mediated induction of plant defensins is involved in nonhost resistance.

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Many living organisms produce small antimicrobial peptides to protect their tissues from infectious microbial agents. Plant defensins are small, highly stable, cysteine-rich antimicrobial proteins that are thought to constitute an important component of plant defense against fungal pathogens. There are a number of such defensins expressed in various plant tissues with differing antifungal activity and spectrum.
We tried to isolate defensin proteins from Brassica juncea seed. The isolated protein was same amino acid sequence with Rs-AFP1 from Raphanus sativus. So we named the protein Bj-AFP1.
In this study, we report the isolation of defensin protein Bj-AFP1 from the seed of Brassica juncea and measurement of anti-microbial assay to several microorganisms.

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Although radiation has been used as the mutagen in mutation breeding, little is known about which types of mutation are induced in plant DNA. In this study, we irradiated rpsL-transgenic Arabidopsis (Arabidopsis/rpsL) with carbon ion beams and γ-rays and analyzed the mutation spectra. The both radiation induced deletion and G:C to A:T transition frequently. In the dry seeds, γ-rays induced shorter deletions than carbon ion beams do. However, the incidence of G:C to T:A and A:T to C:G transversions which result from the formation of 8-oxoguanine, a typical radiation-induced DNA damage, were quite low both in dry seeds and seedlings, contrasting with other organisms. This result suggests that the formation of 8-oxoguanine is low in Arabidopsis compared to other organisms. In an ion beam range, the region that deposits the largest energy when the ion almost stops in the irradiated sample is called Bragg peak region. We also show here the effect of carbon and helium ions around the Bragg peak region on growth and survival of Arabidopsis dry seed.

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Cells degrade their constituents in response to various nutrient starvation stresses. Degradation products are used for the synthesis of new cellular constituents and for obtaining energy. Autophagy, one of the self-degradation mechanisms, has been known to be responsible for the degradation of proteins under nutrient starvation conditions. Cells also degrade phospholipids, the main component of cell membranes, but we have shown that autophagy is not a main contributor to the degradation of phospholipids in cultured tobacco BY-2 cells.
In order to better understand phospholipid degradation, we had BY-2 cells uptake a fatty acid analog labeled by the fluorochrome BODIPY. Then we observed the BODIPY fluorescence by fluorescence microscopy, and measured the fluorescence in the lipid fractions extracted from the cells. The results suggested 80 % of phospholipids are degraded within 24 hours and the degradation products of phospholipids accumulate in vacuoles. We are now trying to identify the degradation product(s) of phospholipids accumulating in vacuoles. In this presentation, we will report the results and discuss the involvement of the central vacuoles in phospholipid degradation.

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Autophagy is one of the mechanisms by which cells degrade their constituents under nutrient starvation conditions. It is mainly executed and controlled by ATG proteins. Autophagy contributes to a nutrient supply under starvation conditions and to the removal of denatured proteins. It has also been reported that autophagy plays a role in various reactions including immunity in mammalian cells. In plant cells, autophagy has been thought to carry out similar functions to mammalian cells. It is also suggested that autophagy has functions peculiar to plants. Although the ATG mutants of Arabidopsis undergo senescence earlier than those of wild type plants, the linking mechanism is not fully understood.
In the present study, to clarify the physiological functions of autophagy in plant cells, we have knocked out the ATG5 gene, an ATG gene essential for autophagy, in the moss Physcomitrella patens. The protonema cells of knockout mutants became yellow earlier than those of wild type plants, when they were cultured on a carbon starvation medium and on a phosphorus starvation medium. The result suggests that defect in autophagy leads to early senescence as has been reported in Arabidopsis.

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A potassium transporter, KUP6 is induced by drought and salinity stress, and abscisic acid (ABA) in Arabidopsis. GUS activity of the KUP6pro:GUS transgenic plants was detected mainly in vascular tissues of root and the localization of KUP6-GFP protein to plasma membrane was observed in 35S:KUP6-GFP transgenic plants by confocal microscopy. We generated kup6kup8 double mutant plants and found that the KUP6 and KUP8 disruption in Arabidopsis resulted in increased formation of the lateral root and root hair. Furthermore, kup6kup8 double mutant showed increased IAA sensitivity and decreased sensitivity to auxin transport inhibitor NPA and ABA in the lateral root growth. KUP6-overexpressing transgenic plants under the control of 35SCaMV promoter showed less transpirational water loss and increased tolerance to drought stress. These data suggested that KUP6 controls both plant growth and stress responses in Arabidopsis.

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Blueberry leaves are rich in antioxidants, and are expected to be useful as a material of functional foods. We have made DNA markers related to polyphenol synthesis genes by cDNA subtraction, and have tried to select cultivars including many polyphenols using DNA markers. It is difficult to isolate RNA from blueberry leaves because the leaves include many polyphenols. We established an efficient method for isolating RNA from blueberry leaves by a modification of the cetyl trimethyl ammonium bromide (CTAB) method. We isolated RNA from blueberry leaves using eight different buffers: borate, HEPES, MES, MOPS, PIPES, TES, Tricine and Tris. We obtained about 80 μg of RNA from 1g of blueberry leaves with a widely used Tris buffer. We obtained more than 200μg of RNA from 1 g of blueberry leaves with a HEPES or a MOPS buffer. The amount of RNA was sufficient for cDNA synthesis. We succeeded at performing cDNA synthesis and increasing the cDNA using polymerase chain reactions. We constructed a number of cDNA subtraction libraries, and have recently isolated cDNA fragments related to polyphenol synthesis genes from these libraries.

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Plants have several mechanisms to adapt various environments. For the purpose of detecting novel RNAs which contribute to environmental responses, we have studied gene expression profiles of Arabidopsis thaliana under some stress conditions using tiling arrays. Then we analyzed the profiles by ARTADE which is a genome wide gene structure prediction program, and found that there were novel antisense RNAs in a lot of gene loci. (Matsui et al.) (2008)PCP 49:1135)
In this study, we tried to verify some detected novel antisense RNAs. We conducted the reverse transcription reaction using strand-specific primers designed every genes and conducted PCR after RNase treatment to inspect existences of sense RNAs and antisense RNAs. Then we examined gene structures by determining the cDNA sequences.
As a result, we successfully confirmed the existence of predicted antisense RNAs. In addition, according to the increased expression of sense RNAs, antisense RNAs tended to come to exist. Furthermore, the antisense RNAs have exon-exon junctions with the same position as their sense RNAs. It is suggested that these antisense RNAs are transcribed from their sense RNAs.

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We identified 7,159 short open reading frames (sORFs) ranging from 90 to 300 bp in the intergenic regions of the Arabidopsis genome. Peptides encoded by sORF are reported to be involved in various functions in several organisms. We developed an agilent custom microarray including sORFs, AGI genes and intergenic transcripts in Arabidopsis to observe sORF expression. The expression of about 1,500 sORFs could be detected in various tissues (rosette leaves, roots, stems, flowers, siliques). Some sORF expressions were confirmed by RT-PCR analysis to validate microarray results. The sORF expression profile was also analyzed during illumination process to identify light-regulated sORFs and we found about 100 light-regulated sORFs. Moreover, we generated sORF overexpression Arabidopsis plants and observed their phenotypes. Some mutants showed altered flower size, hypocotyl length and root length, indicating that sORF can function in Arabidopsis. We will report the expression analysis of sORF and sORF overexpression plants in rice.

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Recent studies have shown that small RNA had important roles on gene expression through DNA methylation, mRNA degradation and inhibition of protein translation. The small RNAs are classified into two large category, miRNA and siRNA, according to their origin. The miRNA was extracted from double stranded region of hairpin structure, however, siRNA was come from the double strand RNA synthesized by RNA dependent RNA polymerase. In spite of the different biogenesis process, both modify the gene expression with complementary sequence. The series of studies on rice mutants deficient in shoot development have revealed that the biogenesis of siRNA, especially ta-siRNA, had a great importance on embryo development. To identify the specific ta-siRNA involved in the shoot development, we have adopted high throughput sequencer (SOLiD system) to the analysis of rice small RNA. In the comparison of small RNA from wild type and mutants defects in shoot development, we found the reduction of the 21nt-length small RNA in the mutants and the wider expression of siRNA from mutant genome. We would like to discuss the whole picture of small RNA brought by the high throughput sequencer.

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Transcription factors (TFs) are master switches of the downstream target genes. Transgenic rice plants overexpressing individual TF cDNAs (TF-OX rice plants) are expected to be highly useful bio-resources for estimating functions of individual TFs and gene-expression networks of the target genes. Approx. 2,500 loci have been found in the rice genome, and about 75% of them are supported by corresponding full-length (FL-) cDNA clones. Initially, the FL-cDNAs with functional protein-coding sequences (CDSs) were selected by inspecting individual CDSs and used as templates to produce TF cDNA Gateway entry clones. Then, by constructing and using the binary Gateway expression plasmids, we are generating TF-OX rice plants. So far, 847 transgenic plants, individually overexpressing 129 TF cDNAs, have been produced. Among them, we observed such altered phenotypes as taller plant height, high tillering, semi dwarf, small grains, and lesion mimic, etc. The latest status on the production of TF-OX transgenic rice plants, interesting phenotypes found in those transgenic plants, and linkage analyses between the altered phenotypes and the TF overexpression will be reported.

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We have developed fifty thousands insertion lines using the endogenous retrotransposon Tos17. Large scale phenotyping for mutant lines shows that half of mutant lines have at least one phenotypic change. However, co-segregation analysis between genotype of Tos17 and phenotype indicates relatively low efficient for tagging by Tos17. We have been analyzing the whole genome sequence of regenerated rice plants from last year to detect cause of the somaclonal variation. We sequenced two lines of regenerated rice at M₄ stage using a next-generation sequencer (Solexa). Coverage of each line is 5-times of rice genome. In the initial read data of the sequencer, about 13% of read have at lease one mismatch to the corresponding rice genomic sequence. Almost mismatches are read error of the sequencer or miss-alignment to repetitive loci. To obtain true mutation, we have developed filter program for Solexa reads. Finally, we obtained from 100 to 200 mutations in regenerated plant. At least, 1 mutation / Mb in the regenerated plant at M₁ stage is estimated. Improvement of our method enables direct identification of mutation or mapping of target loci for phenotype.

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The DNA Data Bank of Japan has been developing a high-throughput read annotation pipeline for the next generation sequencing data. As an example of the annotation pipeline, we report preliminary analysis of genomic variation about two accessions of Oryza longistaminata by a next generation sequencing technology, sequencing by synthesis (illumina Solexa, illumina Inc., San Diego, CA). At the sequencing, approximately 12 mega reads were generated with the paired-end design, where the read length was 75 bp. The generated reads were aligned to O. sativa (Nipponbare) reference genome by using the MAQ program (Li et al., Genome Res, 2008). The results showed that genome coverage for O.longistaminata against Nipponbare was around 40% and the maximum error rate by read position was 2%. Further, we attempted de novo assembly and SNP detection in the preliminary analysis.

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Similarities of gene expression pattern have been used widely to classify gene functions, because genes in the same, or related, biological function often show similar expression patterns. Large-scale gene expression data from various experimental conditions have an important role to evaluate the statistical significance in terms of genome-wide gene expressions. Such data can facilitate the identification of genes with similar expression patterns. In addition, it allows us to construct gene expression network.
To elucidate gene functions and their transcription factors in rice, we aimed to identify gene sets which show similar expression patterns and to develop a gene expression network database. From the GEO, we obtained 244 data sets of microarray platform 'Affymetrix Rice Genome Array', normalized, and calculated Pearson's correlation coefficients between each gene (probe) pair. Based on the statistical indices, gene expression network was analyzed and converted into an interactive graphical viewer. It will contribute to simultaneously search the gene expression similarities and functional annotations. All data obtained here are available in our database 'OryzaExpress'.

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Gene expression pattern is one of the important biological information that represents the process that a gene works. Gene expression pattern has been widely used for the hint in predicting gene function. Gene classification based on gene expression pattern is a powerful approach to understand gene function and/or gene regulation for model plants; those of gene expression are comprehensively characterized sufficiently. However, a large-scale data set is required and there is a limitation of valuation of quantitative gene expression pattern in current gene classification methodology using gene expression correlation. Here, we try to use Cosine distance and Euclidean distance as indices for similarity of gene expression pattern to classify rice genes. We calculated the distances between all pair of genes from gene expression pattern of Affymetrix Rice genome array data, which was processed with RMA and searched for similarly expressed genes. As a result, we found similarly expressed gene populations including far more a designated GO term than using Cosine distance alone. We'll also report the advantage in this methodology to compare gene expression.

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Recent advances in sequencing technologies have led to a remarkable increase in the number of sequences of expressed genes. Comparisons of large-scale sequence data from different samples facilitate identification of such as stage- or tissue-specifically expressed genes. In current approaches for discovering genes from sequence (EST) data, genes are categorized with hierarchical clustering analysis on the basis expression parterres estimated from EST assembling. However, the lack of memory with long calculation time is often caused by large-scale clustering analysis. An innovative method to perform expression analysis with common computational system and short calculation time should be developed to face of the novel sequencing technology. We have developed a new statistical method for large-scale gene expression data by using 'correspondence analysis (CA)'. Sample-specifically expressed genes or genes expressed in some samples are readily detected by the developed method. For results from CA, our three-dimensional plot viewer graphically shows similarities of gene expression parterres. We also present software for expression parterres analysis and interactive plot viewer.

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We have analyzed data of tomato (Solanum lycopersicum) cDNA resources from a laboratory-grown miniature tomato cultivar "Micro-Tom" and provided the obtained information from databases.
To construct 'Kazusa Tomato Unigenes (KTUs)', we used Micro-Tom ESTs from nine cDNA libraries and publicly available tomato ESTs. Information of KTUs including functional annotations is available from MiBASE (http://www.pgb.kazusa.or.jp/mibase/). The current version of KTU (KTU3), constructed from 322,795 ESTs, contains 76,276 Unigenes.
We have also determined 13,227 High Throughput cDNAs (HTCs) from four Micro-Tom full-length cDNA libraries. We provide information of HTCs with functional annotations and structural annotations from KaFTom (http://www.pgb.kazusa.or.jp/kaftom/).
We have also developed a seamlessly integrated database 'TOMATOMICS' that combines information provided from MiBASE and KaFTom.

Acknowledgements: This work was partly supported by National Bio-Resource Project (NBRP). This research was also supported by Dr. Satoh (University of Tsukuba) and Dr. Arie (Tokyo University of Agriculture and Technology).

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Tomato is a one of the model plants for Solanaceae species, and is also a benefit crop material for scientists who want to characterize its unique feature such as fruit development. As a part of National BioResourse Project (NBRP) launched in Japan, a total of 10,793 M1 mutagenesis lines of dwarf variety Micro-Tom, consisting of 4,371 and 6,422 lines were generated by EMS-mutagenesis and gamma-ray irradiation, respectively. The M3 seeds we have completed propagating are now available for scientists upon request to NBRP (http://tomato.nbrp.jp/indexEn.html). From our mutagenesis lines, a number of different varieties of mutants defective in organ development have been isolated, categorized based on the plant ontology, and registered in mutant database called TOMATOMA. These mutant recourses are now available upon request through TOMATOMA.
In addition, we constructed the Micro-Tom BAC library consisting of 54,000 clones with an average insert size of 110 kb, covering about 5 times of the haploid genome (930Mb). The 10,8000 genome BAC end sequencing has launched for developing DNA markers, and the determined sequencing information will be available in public.

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We have sequenced full-length cDNAs obtained from tomato, Solanum lycopersicum cv Micro-Tom, and opened sequence information in March 2009 at KaFTom database (http://www.pgb.kazusa.or.jp/kaftom/). Here we report a large-scale analysis of this tomato full-length cDNA (FLcDNA) collection. Sequence analysis of UTRs demonstrated that tomato has longer 5'- and 3'-UTRs than most other plants but rice. A comparison of FLcDNAs with genes of sixteen plants facilitated the identification of tomato genes that are not found in other plants. These tomato-specific genes appeared to be enriched with domains associated with DNA- and RNA-binding functions. Mapping of the FLcDNAs onto tomato genome sequences demonstrated that introns of tomato genes were longer than those of Arabidopsis and rice. According to a comparison of exon sequences between the FLcDNAs and the tomato genome sequences, the frequency of single-nucleotide polymorphisms between Micro-Tom and the genome-sequencing cultivar (Heinz 1706) was estimated to be 0.078 %. This work was supported by National Bioresource Project, Genome program, Enhancing tomato resources by sequencing Micro-Tom full-length cDNA (2008, MEXT, Japan).

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Linkage maps of cultivated tomato were constructed to promote post-genome sequencing studies in tomato. We discovered 5,607 candidate SNPs by comparison of 49,972 contigs that derived from 229,086 ESTs registered in public databases. Out of the 5,607 candidate SNP markers, 1,536 were tested for polymorphism using Illumina GoldenGate system for two mapping populations, namely AMF2, consisting of "Ailsa Craig", "Micro-Tom", and its F₂ progeny, and MMF2, consisting of "M82", "Micro-Tom", and its F₂ progeny. As the results, a total of 646 and 639 SNP markers showed polymorphism in the AMF2 and the MMF2 populations, respectively, and were subjected to linkage analysis. A linkage map of the AMF2 consisted of 991 loci, including 646 SNPs and 345 SSRs, which were previously developed from EST and BAC-end sequences, and covered 1,468 cM. In the MMF2 population, a total 637 SNP loci were located onto 12 linkage groups representing 1,126 cM. The results in this study will contribute the advances in physiology, molecular genetics, and genomics in tomato.

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In order to investigate the whole genetic system of legume species, we have been analyzing the genome of a model legume, Lotus japonicus.
In 2008, we have released the 315 Mb sequences determined by combination of two independent approaches; clone-by-clone sequencing of large insert clones and random sequencing of selected genomic regions. We continued our efforts on sequencing of genome clones, and the sequence information on 460 clones has been accumulated. Thus, we have updated the sequence information by adding these clone sequences. As a result, the total length of the pseudomolecule, which means genome regions covered by genome clone sequences, was increased from 167 Mbp to 201 Mbp, and the total size of finished (phase 3) sequences was increased from 97 Mbp to 139 Mbp. Using the updated genome sequence and predicted gene information, we have carried out comparative genome analysis against Medicago truncatula and soybean. As a result, high level of syntenic relationship was identified within these legume species. The detailed information on the updated sequences and annotation will be available through our web database, "miyakogusa.jp" (http://kazusa.or.jp/lotus/).

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Plastids are cell organelles that have essential biosynthetic and metabolic activities. To understand the basic mechanism of plastid differentiation from chloroplast to chromoplast, we prepared and analyzed proteins from various types of plastid in"Micro-Tom"tomato fruits at four developmental stages (Mature Green, Yellow, Orange, Red) using shotgun proteomics. The plastids were isolated from tomato fruits by Nycodenz density gradient centrifugation. We identified approximately 440 plastid proteins using a LC-MS/MS. The plastid proteins with the greatest numbers are detected at the orange stage. It is thought that plastids at the orange stage are in possession of both chloroplast and chromoplast functions, and have active proteins related to metabolic production including photosynthetic proteins and carotenoids.
When we compared the chromoplast proteome data in "Micro-Tom"with the data in a bell pepper, we found 42 common proteins.
We observed that approximately 50% of the chromoplast proteins are involved in the metabolic process according to GO. We suggest that chromoplast is involved in production and accumulation of various metabolites.

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Flooding injury is one of the major constraints for cultivation of soybean. A proteomic approach was used for clarifying mechanisms of the flooding injury. Two-day-old seedlings were flooded with water for 12 h, and the roots and hypocotyls were used for 2D-DIGE analysis using CyDye fluorescence labeling with the comparative analysis between untreated and flooding treated samples. Out of detected 555 protein spots, significantly changed 17 protein spots were identified by nanoLC-MS/MS. The identification revealed that flooding caused increase of proteins involved in glycolysis and glycolysis related detoxification and decrease of proteins involved in phenylpropanoid metabolism. On the other hand, phosphorylated proteins were analyzed by Pro-Q diamond staining following 2D-PAGE separation. Out of detected 121 phosphoprotein spots, the phosphorylation levels of 16 protein spots were significantly changed and which contain proteins involved in protein translation. These results suggest that induction of glycolysis and glycolysis related detoxification are primary responses and protein phosphorylation is involved in regulation of protein translation in the early response of flooding.

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Prediction of cis-elements is essential to accelerate the study of gene transcription mechanisms. In eukaryotes, especially in higher organisms, transcription mechanisms are extremely complex. Moreover, sequences homologous to cis-elements exist abundantly, rendering the in silico search for novel cis-elements unsuccessful in many cases by existing methods. To predict putative cis-elements in promoters, we developed a highly sensitive in silico method, Microarray Associated Motif Analysis program (MAMA). MAMA extracts sequences which are highly conserved in promoters of highly regulated genes by various stresses, and which exist more frequently near the transcription start site. In this study, MAMA method was applied to some microarray analysis data from Oryza sativa, Arabidopsis thaliana, and Homo sapiens. As a result, known functional cis-elements were included with the highest score among the predicted elements. This result demonstrated that MAMA is more sensitive for finding functional cis-elements than existing methods.

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Cyanidioschyzon merolae is a unicellular red alga found in acidic hot springs. The cell contains one nucleus, one mitochondrion, and one plastid, each with its own genome. C. merolae has been proposed as the primitive phototrophic eukaryote because of its extremely simple cell structure and minimally redundant small genome. C. merolae has therefore been developed as a model organism for investigating the basic architecture of photosynthetic eukaryotes.
The GFP reporter system is a powerful tool for investigating the subcellular localization of the target protein. Here, we show the establishment of GFP reporter system in C. merolae. In this system, we adopted the promoter of apcC gene, encoding phycocyanin-associated rod linker protein in the nucleus genome. ApcC has been predicted to locate in plastid, and the position of its signal sequence is deduced from comparative study. GFP protein expressed from the apcC promoter was clearly localized in cytosol, whereas GFP fused with the N-terminal or the ORF region of ApcC was localized in the plastid, consistent with the bioinformatic prediction. These results indicate that this reporter system is also effective in C. merolae.

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Dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR) catalyzes the stereo-specific reduction of C17-C18 double bond to form chlorophyllide a (Chlide) in the (baterio)chlorophyll biosynthesis. DPOR consists of two separable components, L-protein (BchL dimer) as a reductase component and NB-protein (BchN-BchB heterotetramer) as a catalytic component, which are functional counterparts of Fe protein and MoFe protein of nitrogenase, respectively. Even though the requirement of ATP for DPOR reaction has been demonstrated, it is still unknown how many ATP molecules are hydrolyzed upon Chlide formation. Here we report a stoichiometric analysis of DPOR from Rhodobacter capsulatus. The observed ATP:Chlide (2e^- equivalent) ratio increased in a linear way with increase in the ratio of L-protein to NB-protein (L/NB ratio). The minimum value of ATP/2e^- ratio of 4 was obtained by extrapolation, which is coincident with that of nitrogenase. This result suggested that the ATP-dependent electron transfer mechanism is common between DPOR and nitrogenase.

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Mg-chelatase composed by CHLI, CHLD, and CHLH subunits catalyzes the insertion of Mg²⁺ into protoporphyrin IX, the first step of chlorophyll biosynthesis. CHLH is catalytic subunit that binds substrate or product porphyrin. Recently, CHLH is proposed to be a plastid-localized abscisic acid receptor, and furthermore, mutations in CHLH resulted in gun phenotype that defects in plastid to nuclear signaling. We have previously reported in vitro analysis of porphyrin affinities of mutant CHLH proteins showing gun phenotype. Here, we found CHLH-porphyrin complexes were rapidly degraded upon illumination in vitro. Consistent with dissociation constants of CHLH to porphyrins, CHLH-protoporphyrin IX complex was more sensitive to light degradation, when compared to that of CHLH-Mg-protoporphyrin IX complex that has lower affinity to CHLH. Interestingly, in vivo CHLH protein level of a gun mutant, cch, was much higher that that of wild type, although the gene expression levels were comparable each other. It is therefore likely that binding of porphyrin to CHLH involves post-translational light degradation of CHLH. Physiological significance of the CHLH photo-degradation will be discussed.

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Most of plants are weak in salinity environment, for example, Arabidopsis thaliana does not grow on the medium containing 200mM NaCl. Although a seagrass (Zostera marina) belongs to monocotyledonous plants, it grows in sea, which contains about 500mM NaCl. To search novel genes for salt tolerance in Z. marina, we have constructed the cDNA library and screened it using Escherichia coli (Ohori et al, JSPP2009). Sequence analysis about ten candidate genes for salt tolerance, cyclophilin-like gene was found. It contains a conserved cyclophilin-like domain (CLD), so we named it ZmCyp1. An ena1 knockout strain (Saccharomyces cerevisiae) was established by diploid-knockout method, this strain is sensitive on the medium containing 200mM NaCl. Ectopic expression of ZmCyp1 with ADH promoter confer to grow on the medium containing about 170mM NaCl. We examine the effect of ZmCyp1 over-expression by the 35S promoter in Arabidopsis thaliana.

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A number of eukaryotic mRNAs contain short upstream open reading frames (uORFs) in the 5' untranslated region. Although peptides encoded by uORFs are not functional in many cases, several examples have been reported in which the uORF-encoded peptide has a role in translational regulation of the downstream ORF.
To identify novel Arabidopsis genes whose translation is controlled by a uORF-encoded peptide, we first searched for uORF-containing genes that are predicted to encode transcription factors by using Arabidopsis full-length cDNA database. Then, we tested amino acid sequence-dependency of the effect of the uORFs on translation of the downstream ORFs. From this screen, we found several uORFs that influence downstream translation in an amino acid sequence-dependent manner. Alanine-scanning analysis of some of those uORFs revealed that the C-terminal regions of the uORF peptides that consist of approximately 10 amino acids are important for the translational regulation.

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The plant metabolome is extremely diverse comprising several thousands of chemical compounds. Current approaches for profiling the metabolome are hampered by the fact that no analytical platform can give comprehensive coverage of all biological metabolites. A promising way to work around this problem is to use several complementary analytical platforms in parallel and combine the resulting data sets. However, stitching together data from different platforms poses several theoretical and practical challenges for how to best normalize and summarize the data to obtain a consensus data set. Here we present a novel strategy for solving this task and a freely available software tool that implements our ideas. To validate our method we tested it on an experimental data set obtained using both GC-, and CE-MS and show its performance in comparison with alternative approaches.

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Presence of α-carotene and its derivatives (lutein, siphonaxanthin, etc.) is limited in a part of phototrophic organisms. C-6' between ε-end group and conjugated double bonds of α-carotene is chiral, and it is possible to take (6'R)- and (6'S)-types, but only (6'R)-type is found. We precisely re-examined distribution and chiralities.
Distribution: Florideophycidae, Cryptophyceae, Euglenophyceae, Chlorarachniophyceae, Prasinophyceae, Chlorophyceae, Ulvophyceae, Charophyceae and land plants have these carotenoids, but Glaucophyceae, Bangiophycidae, Heterokontophyta (Bacillariophyceae, Haeophyceae, etc.), Dinophyceae and Haptophyceae have not.
Chirality: All of α-carotene and its derivatives are (6'R)-type. In biosynthesis in land plants, α-carotene is produced from lycopene by lycopene β-cyclase and lycopene ε-cyclase, which have high homology with each other. In enzymatic reaction, it might be possible to produce both (6'R)- and (6'S)-types, thus ε-cyclase might not restrict the chirality. Binding proteins might restrict the chirality.

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Photosystem I and II (PS I and PS II) complexes were purified in an intact form from a primitive red alga, Cyanidioschyzon merolae to investigate the precise reaction mechanisms in eukaryotic photosystems. PS I complex was a monomer composing PsbO and light harvesting chlorophyll binding protein (LHCI), which is similar to PS I complex from higher plants rather than cyanobacterial one. The secondary electron acceptor A1 was MK-4. Different from PS I complex, the polypeptide composition was similar to that in cyanobacterial PS II complex. A novel low molecular mass polypeptide was also identified in PS II complex purifiede from C. merolae. PS II complex was a monomer. The results obtained in this work insidacate the unique architecture of photosystems in C. merolae.

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We isolated photosystem I (PSI) complex using a Synechocystis sp. PCC 6803 strain (J-His), which expresses the His-tagged PsaJ subunit, to analyze lipid content of PSI. Six molecules of lipid (2 molecules of MGDG, 1 molecule of DGDG, 1 molecule of SQDG, 2 molecules of PG) per reaction center were detected. To investigate the role of DGDG in PSI, we generated dgdA /J-His strain, which is defective in the biosynthesis of DGDG. PSI complexes of the J-His and dgdA /J-His strains could be easily purified by Ni²⁺-affinity column chromatography. The purified PSI complexes from dgdA /J-His cells lacked DGDG and contained 10-12 molecules of lipids per reaction center. PSI activities of thylakoid membranes and PSI complexes from dgdA /J-His cells were lower than those of thylakoid membranes and PSI complexes from J-His cells. These results indicate that the lack of DGDG caused decreased activity of PSI complexes, thereby suggesting the important function of DGDG for maintenance of the PSI activity.

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Thin alumina membrane with incorporated mesoporus silica (NAM) has vertically membrane-penetrated silica pores with 50 nm diameter inside macropores formed in alumina membrane. A previous report indicated that catalase introduced into NAM maintained its activity and showed higher stability [1].
We introduced photosystem I (PSI) reaction center complex, which was purified from a thermophilic cyanobacterium Thermosynechococcus(T.) elongatus, into NAM. PSI forms a trimer with a diameter of 21 nm and a height of 9 nm. PSI was adsorbed only inside the silica pores and not adsorbed to the membrane surface. Absorption spectrum and photo-induced activity of PSI were maintained well. Electron paramagnetic resonance (EPR) analysis indicated the orientation of PSI in the pores to be perpendicular to the wall surface of pore to give its membrane normal to be parallel to the NAM membrane normal.
Behavior and reaction of orientated PSI inside nanopores are studied. We discuss the photo-induced charge separation reaction and electron transfer to the mediators inside nanopores.

[1] T. Itoh et al. J. Mol. Catal. B: Enzym. 2009, 57, 183-187.

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Among the variety of chlorophyll species, 3-mono-vinyl (MV) chlorophyll (Chl) a is most commonly used for the photochemistry in photosynthetic organisms. The exceptions to this is a group of marine cyanobacteria, Prochlorococcus spp., which use 3,8-di-vinyl (DV) Chl a for their photochemistry. In the transgenic mutant of a cyanobacterium Synechocystis sp. PCC 6803 whose enzyme in chlorophyll synthesis, i.e. DV reductase, is blocked, it accumulates DV-Chl a instead of MV-Chl a. This transgenic species was sensitive to high light, and isolated photosystem II complexes with DV-Chl a (DV-PS II) were bleached much faster than PS II with MV-Chl a (MV-PS II). Furthermore, the acceptor-side inhibition was observed on the basis of degradation products of the D1 protein. To identify the reasons for this inhibition in DV-PS II, we measured the yield of singlet oxygen at near infrared region in DV-Chl a solution and DV-PS II, because the singlet oxygen plays a key role of acceptor side photoinhibition. We found MV-Chl a in solution was effective on formation and quenching of singlet oxygen than DV-Chl a. Possible implications of the results will be discussed.

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Acaryochloris spp. are unique cyanobacteria that differ from the majority of photosynthetic organisms by having Chl d as the major pigment. We reported the isolation of PS II core complex from A. marina MBIC 11017 and confirmed the special pair comprised Chl d homodimer in 2007. In our previous work, the Chl d content per two pheophytin a was ca. 55; the presence of CP43' may be a primary reason for why a higher level of pigment contents than expected. In this study, we modified a purification procedure and almost completely removed CP43'. In the current PS II core complexes, the number of Chl d and Chl a per two Phe a molecules was estimated to be 29.6 ± 1.2 and 1.9 ± 0.1, respectively. These values are dramatically lower than the previous report. The absorption spectrum of A. marina PS II at 80 K showed the presence of a new short-wavelength component at 672 nm, probably Chl a, which was detected by a decrease in antenna pigments. There are several enigmas in PS II of A. marina (e.g. redox potential of primary acceptor, localization and function of Chl a). This improved preparation will be suitable for further study.

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Cyanidioschyzon merolae is a kind of unicellular primitive red alga, which whole genome was sequenced in 2004. Isolated photosystem II complex (PS II) of C. merolae contained four extrinsic proteins, PsbO, PsbQ, PsbV, and PsbU, such as other primitive red alga, Cyanidium caldarium. Crystal structures of PS II complexes from thermophilic cyanobacteria at atomic resolution have been reported from several laboratories. However, PsbQ subunit is missing in cyanobacteria. In the PS II of C. caldarium, PsbQ can directly bind to PS II intrinsic proteins essentially independent of the presence of other proteins. The localization of PsbQ subunit has been unknown yet. In this study, nearest neighbor relationships between PsbQ and PS II constituent subunits from C. melorae were investigated by means of crosslinking reagents. We obtained several crosslinking products, which detected by antibody analysis. The localization of the PsbQ subunit is now in progress.

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Red alga is one of the eukaryotic algae closely related to cyanobacteria, but its PSII differs from that of cyanobacteria in that the former contains a PsbQ', the fourth extrinsic protein. The crystal structure of PSII from cyanobacteria has been reported at 2.9 angstrom resolution; however, no reports have been published on the structure of any eukaryotic PSII. In order to elucidate the structure of red algal PSII, we have been purifying and crystallizing PSII from a red alga Cyanidium caldarium, and have succeeded in obtaining crystals diffracting to 3.5 angstrom resolution(Adachi H., Umena Y., Enami I., Henmi T.,Kamiya N., Shen J-R.(2009) Biochim. Biophys. Acta. 1787, 121-128). To improve the resolution of the red algal PSII crystals, we modified the purification procedure of PSII and the conditions for post-crystallization dehydration. As a result, crystals diffracting to a 2.9 angstrom resolution was obtained. This resolution would allow us to elucidate more detailed structure of the red algal PSII.

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