Legume plants can establish symbiosis with rhizobia by forming a nitrogen-fixing apparatus that is called nodules. In the nodule formation process, many membrane transport events are involved, but the understanding of those transport systems as well as transporter molecules involved is still very limited. As an approach to clarify the transport systems in nodulation and symbiotic nitrogen fixation, we chose Multidrug And Toxic compound Extrusion (MATE)-type transporters to characterize in detail. In general, MATE-type proteins transport organic substances of wide variety with an electrochemical gradient of H+ or Na+ ions across the membrane.
We have identified a MATE protein gene in Lotus japonicus, LjMATE1, which would be potentially responsive to the nodule formation. LjMATE1 is strongly up-regulated during the nodulation in underground parts of L. japonicus. Reporter gene study showed that LjMATE1 was expressed in inner cortex of nodule. For the transport experiments, this MATE gene is expressed in yeast cells that are subjected to cellular transport assay.

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Inoculation experiments of endophytic bacterium Azospirillum sp., an isolate from surface-sterilized stems of field-grown rice, were conducted in pots under a greenhouse condition, and in paddy fields. Inoculation with Azospirillum significantly enhanced the growth of newly generated leaves and shoot biomass under a greenhouse condition. When rice seedlings were treated with the bacterial suspension (1 x 10⁸ CFU/ml), followed by transplanting in paddy fields, the stem numbers and seed yield were markedly increased during the rice growth. These results indicated that Azospirillum sp. is a promising bacterial inoculant for plant growth promotion and agricultural practice.

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Agriculturally important grasses contain numerous diazotrophic bacteria, the interactions of which are speculated to have some other benefits to the host plants. In this study, we analyzed the effects of a bacterial endophyte, Azospirillum sp., on disease resistance in host rice plants. Rice plants (Oryza sativa cv. Nipponbare) were inoculated with Azospirillum sp. exhibited enhanced resistance against diseases caused by the virulent rice blast fungus Magnaporthe oryzae and by the virulent bacterial pathogen Xanthomonas oryzae. In Arabidopsis, plants (Col-0) were inoculated with Azospirillum sp. also exhibited enhanced resistance against virulent bacterial pathogen, Pseudomonas syringae pv. tomato DC3000. These results indicate the possibility that strain is able to induce disease resistance in rice and Arabidopsis. Furthermore, we studied the effect of colonization on the growth of Arabidopsis.

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Plant hormones play pivotal signaling roles in plant-pathogen interactions. Abscisic acid (ABA) has been shown in many studies to negatively regulate plant defense responses. However, its molecular mechanism still remains poorly understood. Here we report that ABA interacts antagonistically with rice salicylic acid (SA) signaling pathway in rice-M. grisea interaction.
ABA markedly suppressed the SA/benzothiadiazole (BTH)-induced transcriptional upregulation of WRKY45 and OsNPR1, the two key components of the SA signaling pathway in rice, with concurrent development of severe disease symptoms. Such effects of ABA were largely negated by overexpression of OsNPR1 or WRKY45. Endogenous SA levels were not altered in rice by ABA application. These results indicate that ABA acts downstream of SA and upstream of WRKY45 and OsNPR1 in the pathway. Furthermore, we found that M. grisea accumulates ABA in both hyphae (mycelium) and conidia, with higher levels detected in the latter. These results imply that M. grisea uses its own ABA to increase local ABA levels in host rice cells at infection sites, thereby suppressing the rice SA signaling pathway to avoid defense responses.

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MAPK cascades play an important role in defense signaling after recognition of elicitor signals. We have isolated an elicitor-responsive MAPKK, OsMKK4 in rice, and demonstrated that a constitutive active mutant, OsMKK4DD, activates OsMPK3 and OsMPK6. This MAPK cascade induces the expression of multiple defense-related genes, cell death, diterpenoid phytoalexin biosynthesis, and phenylpropanoid biosynthesis. Using osmpk6 knock-out mutant, we demonstrated that OsMPK6 is required for the induction of cell death and regulation of gene expression by induction of OsMKK4DD. On the other hand, regulatory machinery connecting OsMKK4-OsMPK3/OsMPK6 cascade to these defense-related responses remain elusive. We are now analyzing the relationship of OsMKK4-OsMPK3/OsMPK6 cascade and several transcription factors, which possibly regulate cell death, diterpenoid phytoalexin biosynthesis or phenylpropanoid biosynthesis.

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We employed a chemical biology approach to investigate Arabidopsis R-gene mediated defense responses to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (avrRpm1). A high-throughput screening method for HR cell death, using suspension cells, was established and seven compounds that enhance HR were identified from a diverse chemical library. CB_6, one of the isolated potentiators, induced PR1 gene expression in Arabidopsis seedlings, independently of salicylic acid (SA) biosynthesis. CB_6 did not affect endogenous SA levels, suggesting that CB_6 is a partial SA agonist which is not involved in positive feedback regulation of SA. Agonists can be powerful tools for the identification of signaling molecules, as was shown recently by the identification of an abscisic acid receptor, and we therefore started genetic and biochemical analyses using CB_6 to explore SA downstream factors. Twenty eight Arabidopsis sgi (SA agonist insensitive) mutants that exhibited insensitivity to CB_6 were isolated and classified into two groups based on their sensitivity. Four of the CB_6 weak insensitive mutants showed hypersensitivity when grown on media containing 400mM SA, similar to npr1.

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Chemical biology is a research approach to understand living system that uses small organic molecule, which modulate a particular biological process. We are applying chemical biology to elucidate plant disease resistance mechanisms. A high-throughput chemical screen that monitors HR cell death of Arabidopsis cultured cells infected by Pseudomonas syringae pv. tomato DC3000 (avrRpm1), identified seven chemicals which enhance the HR response. These potentiators induced PR1 gene expression when they were applied to Arabidopsis seedlings. Based on their effects on both endogenous SA levels and PR1 inducibility in SA deficient sid2 mutant plants, we categorized the potentiator's putative action points into three groups: 1. Activation of SA biosynthesis, 2. SA mimics and 3. Inhibition of the SA-inactivating pathway. The isolated potentiators CB_8, CB_9 and CB_11 were found to inhibit activity of SAGT, an SA-inactivating enzyme. We analyzed the enzyme activity of UGT74F1, one of the Arabidopsis SAGTs, and the inhibitory effect of the HR potentiators. An inhibitory mechanism will be discussed together with the reaction kinetics of UGT74F1 and the inhibition constants of the potentiators.

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MAPK cascades play pivotal roles in plant immunity. We have isolated potato StWRKY8 as a protein phosphorylated by StMPK1, a potato ortholog of SIPK, in vitro. NbWRKY8 (a homolog of StWRKY8 in Nicotiana benthamiana) was phosphorylated by SIPK, Ntf4 and WIPK in vitro. Alanine scanning mutagenesis indicated that multiple-sites in proline-directed serine (SP) cluster in NbWRKY8, which is conserved in N-terminal region of Group I WRKY, is phosphorylated by SIPK. Immunoblot analyses using anti-phosphopeptide antibodies revealed at least two sites of the SP cluster are phosphorylated by constitutively active mutant of MEK2 or INF1 elicitor in plants. MAPK-mediated phosphorylation of NbWRKY8 enhanced the DNA-binding activity to the W-box sequence and the transcriptional activity. Expression of a phospho-mimicking mutant of NbWRKY8 highly induced the expression of HMGR2 and NADP-ME, downstream genes of NbWRY8. Knock-down of NbWRKY8 increased disease susceptibility to Phytophthora infestans. These results suggest that NbWRKY8 is a physiological substrate of MAPK and positively regulates downstream defense-related genes at the transcriptional level thought phosphorylation of SP cluster.

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SGT1, RAR1 and HSP90 have been well known to have important roles in resistance gene-mediated disease resistance in plants. However, it has been unclear that the roles of these genes on interaction between plants and adapted pathogenic bacteria. In this study, we analyzed the roles of NbSGT1, NbRAR1 and NbHSP90 in interaction between Nicotiana benthamiana and adapted (virulent strain) R. solanacearum (RsOE1-1). In NbRAR1-silenced plants, RsOE1-1 successfully multiplicated in a similar manner to control plants. In contrast, the growth of RsOE1-1 and appearance of bacterial wilt symptom were strikingly suppressed in NbSGT1- and NbHSP90-silenced plants. Moreover, inductions of PR genes were significantly enhanced in NbSGT1- and NbHSP90-silenced plants by inoculation with RsOE1-1 compared to control plants. Therefore NbSGT1 and NbHSP90 might be involved in suppression of immunity in N. benthamiana during adapted R. solanacearum - N. benthamiana interactions.

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To elucidate the molecular mechanisms of disease susceptibility in plants, we identified NbPAP (Nicotiana benthamiana phosphatidic acid phosphatase), which is specifically expressed in Nicotiana plants by inoculation with pathogenic strain of Ralstonia solanacearum (RsOE1-1). In NbPAP-silenced plants, expression of a maker gene for jasmonic acid (JA) signaling PR-4 and generation of reactive oxygen species (ROS) were accelerated by RsOE1-1 inoculation. Silencing of NbPAP reduced growth of RsOE1-1 and appearance of bacterial wilt, suggesting the enhancement of resistance by NbPAP-silencing. The enhanced-resistance was partially compromised by double silencing of NbPAP with either COI1 or RbohB. In contrast, the enhanced-resistance was hardly affected in salicylic acid deficient NahG plant. Moreover, NbPAP-silenced plants also showed highly resistance to pathogenic bacteria Pseudomonas syringae pv. tabaci and P. syringae pv. mellea compared with control plants.
These results indicate that NbPAP acts as a negative regulator of JA- and ROS-signaling in a response to RsOE1-1 infection. Collectively, NbPAP might be a host disease-susceptibility gene for bacterial disease in Nicotiana.

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Inoculation of Arabidopsis ecotype Columbia leaves with Colletotrichum higginsianum results in fungal growth and disease symptoms reminiscent of those induced in other cruciferous plants. We performed map-based cloning and natural variation analysis of 19 Arabidopsis ecotypes to identify a dominant resistance locus against C. higginsianum. We found that the Arabidopsis RCH2 (for recognition of C. higginsianum) locus encodes two NB-LRR proteins, both of which are required for resistance to C. higginsianum in the Arabidopsis ecotype Ws-0. Both proteins are well-characterized R proteins involved in resistance against bacterial pathogens; RRS1 (resistance to Ralstonia solanacearum 1) confers resistance to strain Rs1000 of R. solanacearum and RPS4 to Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 (Pst-avrRps4). Furthermore, we found that both RRS1-Ws and RPS4-Ws genes are required for resistance to Pst-avrRps4 and to Rs1002 R. solanacearum. We therefore demonstrate that a pair of neighboring genes, RRS1-Ws and RPS4-Ws, function cooperatively as a dual R-gene system against at least three distinct pathogens.

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Approximately 21,000 of the Rice-Arabidopsis FOX transgenic Arabidopsis lines have been screened for resistance by dip inoculation with Colletotrichum higginsianum, a fungal pathogen of Arabidopsis. The identities of the overexpressed genes were determined in 112 lines that showed consistent resistance after four independent screens. The rice cDNAs identified by this procedure have been re-transformed into Arabidopsis for overexpression and further screening to confirm the disease resistant phenotypes. At present, at least 24 rice cDNAs endowed resistance to C. higginsianum in re-transformed Arabidopsis. We further investigated disease resistance to Pseudomonas syringae pv. tomato DC3000 in the 112 lines, 18 lines showed high resistance and 24 lines showed moderate resistance, respectively. Ten of the genes that showed resistance to C. higginsianum have been re-introduced and overexpressed in wild-type rice and evaluated for resistance to Magnaporthe grisea. At present, three transgenic rice lines showed resistance to M. grisea. In addition, we will report on the molecular mechanism of disease resistance in Arabidopsis and rice by the genes that endowed resistance.

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Rice WRKY45 is a transcription factor that plays an essential role in BTH-induced disease resistance in rice. Transgenic rice plants overexpressing WRKY45 (WRKY45-ox) exhibit strong resistance to blast and leaf-blight diseases. Whereas constitutive defense responses often cause severe growth defects in plants, WRKY45-ox rice shows only minor growth retardation; thus WRKY45 is expected for its utility in developing multi-disease resistance rice.
To characterize post-translational regulation of WRKY45, we generated transgenic rice overexpressing myc-tagged WRKY45 and analyzed the responses of myc-WRKY45 proteins to phytohormones and inhibitors. Proteasome inhibitor MG132 specifically induced high-level accumulation of myc-WRKY45. Immunoprecipitation assay using anti-polyubiquitin antibody revealed that myc-WRKY45 was polyubiquitinated in vivo. These results suggest that WRKY45 is post-translationally regulated by ubiquitin-proteasome pathway. Furthermore, we found that dephosphorylated myc-WRKY45 was more stable than its phosphorylated form, which suggests that protein phosphorylation is involved in the ubiquitin-proteasome regulation of WRKY45.

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Hypersensitive response (HR) is critical to restrict local spread of pathogens in plant cells. Chloroplasts are photosynthetic organelles that are also involved in the synthesis of stress-induced hormones (JA, SA, ABA) and the generation of reactive oxygen species. In addition, recent works revealed that chloroplasts could quickly recognize the extracellular pathogens via Ca²⁺ signaling. However, the role of chloroplasts in the plant immune response remains elusive. CAS is a plant-specific Ca²⁺-binding protein that is localized in chloroplast thylakoid membranes. Previous studies demonstrated that CAS is essential for generation of cytosolic Ca²⁺ signal and subsequent stomatal closure induced by external Ca²⁺. We examined the role of CAS in plant defense responses in Arabidopsis and Nicotiana benthamiana. The bacterial growth (Pseudomonas syringae pv. tomato DC3000) was significantly suppressed in the CAS-1 knock-out mutants in Arabidopsis. In addition, the R protein-mediated HR-cell death was delayed in the CAS deficient plants of Arabidopsis and N. benthamiana. These findings suggest that chloroplast localized CAS plays a crucial regulatory role in the plant defense responses.

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Systemic acquired resistance (SAR), one of plant defense systems, is induced by pathogen infection through salicylic acid (SA) accumulation. SAR has been practically utilized in rice fields by exploiting the plant activators capable of inducing of SAR. Benzisothiazole (BIT) is a plant activator that induces SAR in rice, tobacco and arabidopsis. In Arabidopsis, environmental stress responses via abscisic acid (ABA) suppresses the SAR induction by BIT. Because of the economical importance of the stable induction of SAR, we have investigated the effects of environmental stresses on SAR in rice. BIT induced the resistance against rice blast fungus, however, which was suppressed by environmental stress. ABA treatment reduced the BIT-induced disease resistance. These data indicated that environmental stress response suppresses SAR development in rice.

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The AtDREB1A gene is an important transcriptional factor that controls many abiotic stress inducible genes and confers abiotic stress tolerance to plants. In our previous study, AtDREB1A enhanced the abiotic stress tolerance in potato. However, the mechanism of abiotic stress tolerance in potato controlled by AtDREB1A was not clear and the orthologous gene in potato had not been reported yet.
To investigate how AtDREB1A improves the abiotic stress tolerance in potato, we tried to identify the abiotic stress- and AtDREB1A-inducible gene using the 22K oligo-DNA microarray. As the result, we identified 47 genes. Fifteen of these genes belonged to stress categories such as dehydrin and heat shock protein. These homologs in AtDREB1A have been reported to be downstream of the AtDREB1A gene. These results suggest that the AtDREB1A acts as a transcriptional factor against abiotic stress in potato and that mechanisms in abiotic stress tolerance controlled by the native transcriptional factor similar to AtDREB1A may exist in potato. In addition, the putative cis-element related to abiotic stress was found in some 5' upstream region of those identified genes.

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The research in my lab is aimed at our basic understanding of the molecular mechanisms controlling cell death in plants. To study of cell death in plants, we have established an Arabidopsis thaliana leaf mesophyll protoplast system to quantitatively examine in stress induced cell death. However, this method is not too efficient for screening of cell death because that invest a good deal of time and effort in preparation for mesophyll protoplasts. Furthermore, it turns out that this step has an effect on cell viability of mesophyll protoplasts. In this report, to improve viability and work saving for preparation mesophyll protoplasts, we show to try and study to suspension cultured cells that Arabidopsis thaliana, Columbia ecotype, T87 suspension cells were obtained from RIKEN BRC.

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In plant behavioral ecology, plant behavior is defined as environmentally induced (e.g. chemical, UV) phenotypic plasticity. Plants' ability to rapidly and reversibly respond to stimuli thereby effecting morphological plastic growth (i.e. variable morphological characteristics over time with respect to stimuli) with increased basal fitness generally defines this unique behavior. In this paper, we described fruit bicoloration behavior in selected plant species found in Panay Island, Philippines. We argued for the best definition of a genuine bicolored fruit display by examining how this signal design fare on Hasson's amplifier effect theory. A genuine bicolored fruit displays two colors in the mature stage (e.g. Dehaasia sp., Clerodendrum sp., etc.) that promote higher fitness of the signaler (e.g. plant) by improving fruit visibility relative to the animal disperser. It also usually involves color contrast between the fruit and the pedicel. Physiological perspectives in the exhibited plant behaviors (e.g. fruit bicoloration) were shown to broaden future research insights in endogenous signals leading to morphological signal design in plant-animal communication study.

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The present research aims to determine the relationship of cytokinin (CK) levels in barley kernels to kernel mass, kernel number, and plant yield. Early studies with barley found a positive relationship between CK content and kernel mass. With the advent of more sensitive and selective mass spectrometric techniques and established methods for extracting and purifying CKs, the roles of CKs in barley kernel development can be more precisely re-examined. To this end, CK profiles across early developmental stages have been generated for kernels of high-yield and low-yield barley varieties. Analysis and subsequent quantification was achieved by liquid chromatography-tandem mass spectrometry. Our results indicate that there is a relationship between concentration in kernels and increased plant yield. Furthermore, we will also discuss the results of an investigation into how CK oxidase might regulate CK levels in barley kernels through the degradation of CKs.

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Chemical biology and chemical genetics are powerful tools to clarify vital phenomenon at the molecular level. These methods are applicable to various organisms, and have advantage of possibility to find new gene function which shows redundancy or lethality by knock-out. However, identification of chemical targets by biochemical approach is technically difficult. To identify the chemical targets by molecular biological approach, we tried a combination of quartz crystal microbalance (QCM) and phage display (PD). First, we tested to detect reported interaction of a chemical inhibitor of brassinosteroid (BR) synthesis, Brz2001, with a BR-synthesis enzyme, AtDWF4. As a result, interaction of biotinylated-Brz2001 with the T7 phage displaying C-terminal of AtDWF4 was detected by QCM. To explore the targets of the chemicals that inhibit reprogramming in Physcomitrella patens, we synthesized biotinylated chemicals and constructed the T7 phage display libraries carrying equalized cDNAs. Now, we are screening these targets by QCM-PD and will discuss our results and efficacy of this method in this meeting.

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Leaf cells are reprogrammed to protonemal apical stem cells within 24 hours after leaf excision in Physcomitrella patens. To characterize the molecular events during this process, we undertook Digital Gene Expression analysis by SOLiD system. Time course expression data for 35,937 annotated genes were obtained from the samples collected during 0 to 24 hours after leaf excision.
After statistical filtering, 3,530 genes were found as the differentially expressed genes. By using self organization map algorithm, they fell into four clusters, increased during the process, decreased during the process, transiently induced within 3 hour, and transiently repressed at 3 hour.
We identified the statistically significant enrichments of specific Gene Ontology terms in them. Gene expression-related genes were enriched in the induced clusters. Stress responsive genes were enriched in the induced, while photosynthesis genes were enriched in the repressed clusters. Some protein kinases, transcription factors, and chromatin-related factors were also regulated distinctively. Furthermore, promoter-reporter analysis for several up-regulated genes revealed their unique spatial expression patterns.

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Liverworts, as basal land plants, are a key group to understand the regulatory mechanisms and the evolution of plant development. Marchantia polymorpha is a common, dioecious liverwort species, and has a haploid-dominant life cycle. We sequenced 30 autosomal PAC clones, which represent approximately 3.3 Mb, and predicted 235 genes, suggesting that the gene density is 0.7 per 10 kb. Considering the genome size of M. polymorpha is 280 Mb, we estimated the total gene number in the genome to be less than 20,000. This is consistent with our prediction based on EST analysis that the gene redundancy would be lower in M. polymorpha than other sequenced land plants, i.e., angiosperms and the moss Physcomitrella patens. Furthermore, few transposable elements and repeats were found in the autosomal regions. We have generated T-DNA-tagged lines using the binary vector pCAMBIA1300 and screened 10,000 lines for morphological mutants under continuous white fluorescent light. We have isolated 22 lines that show various morphological phenotypes, such as formation of callus-like tissue, loss of air-chamber, and loss of gemma formation.

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M. polymorpha is one of emerging models in land plants. However, only two selectable markers (hpt and aadA) for nuclear-genome transformation have been developed to date. To enrich functional genomics tools for liverwort, a novel selectable marker is required. Here, we report that the gene for M. polymorpha acetolactate synthase including mutations (MpmALS) serves as a selectable marker in liverwort. We have identified ESTs encoding ALS of 678 amino acid residues in liverwort. Compared with ALS proteins of other plant species, amino acid residues conferring the sulfonylurea herbicide (chlorsulfuron)-resistance to plants were conserved in liverwort. According to the amino acid substitutions in previous works, we have prepared MpmALS. Amino acid substitutions were introduced at three positions 207 (P to S), 198 (R to S), and 574 (W to L) in MpALS by site-directed mutagenesis. Agrobacterium-mediated transformation was conducted by using mALS as selectable marker under the control of the cauliflower mosaic virus 35S promoter, and transformants were successfully obtained on media containing 1 μM chlorsulfuron. MpmALS should be helpful to conduct functional genomics in liverwort.

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Recently, plant metabolomics is an important research methods for plant omics research. For plant metabolomics research, an integrated database for metabolite and related information (e.g. metabolic pathway) focused on plants is necessary. In this context, it is especially important to construct integrated compound database.
We have developed the GenomeNet database for chemical information to integrate compound/drug databases in the MEXT Integrated Database project. The database provides an integrative search interface of KEGG compound information and other compound/drug databases (e.g. KNApSAcK, PubChem, lipidmaps, lipidbanks). We also develop several tools to analyze chemical structure and reactions, and provide them as GenomeNet tools for chemical analysis. In addition, GenomeNet database for chemical information is highly linked to KEGG databases, which allows users to easily reach pathway and/or genome information from compound search. We introduce current status of GenomeNet database for chemical information and related tools.

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Plant secondary metabolites accumulate through complex chemical modification steps after the biosyntheses of aglycon structures. Those modification processes are finely regulated at the levels of gene expression and biochemical regulation of enzyme proteins. Global metabolic profiling studies unquestionably shed light on the metabolic dynamism comprised of the activities of biosynthesis, accumulation, and hydrolytic release of active moieties from accumulated forms. Such metabolomics information could provide understanding of the biological and physiological significance of diverse secondary metabolisms.
In this study, we developed a quantitative metabolic profiling platform based on mass spectral analyses using GC-MS, LC-ESI-MS, and FT-ICR/MS. As one of typical crops modified to confer desired traits by genetic engineering, we studied a metabolic profiling scheme of potato tuber extracts. In this platform, we studied the accumulation of different glycosylated forms of steroidal alkaloids synthesized from the aglycone through glycosylation reactions, and a total of 78 compounds from primary metabolisms including sugars, amino acids, and nucleic acids were monitored as well.

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Vacuole of plant cells contributes to the storage of various types of metabolites. However, mechanisms of metabolic regulation between cytosol and vacuole have not been clarified yet. To elucidate mechanisms controlling substrate trafficking, we have been analyzing functions of vacuolar transporter candidate genes. One of our targets is a gene encoding vacuolar MATE efflux family protein. We have demonstrated the effect of overexpression of this gene on the metabolite accumulation pattern by using Arabidopsis suspension cultured cells and isolated vacuoles. In this study, we analyzed metabolite accumulation profile of Arabidopsis plant overexpressing this gene. We focused on the analysis of semi-polar metabolites accumulated in the rosette leaves using LC-FTICR-MS. Analysis of data obtained by ESI ionization mode demonstrated that accumulation of flavonol and indol compounds increased in the overexpressor, whereas accumulation of tryptophane and sulfur-containing metabolites decreased. Putative function of this MATE efflux family protein gene is discussed in terms of vacuolar transport. This work was partly supported by CREST, JST.

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Botryococcus braunii produces long-chain liquid hydrocarbon compounds resembling petroleum through photosynthesis. To achieve industrial use of B. braunii for petroleum displacement, the utilization of molecular biological means seems effective. However, the information on the genes of this organism is limited and majority of the nucleotide sequences available to date are those used for phylogenic analysis. Here we obtained global genetic information by EST analysis. Out of 144 strains of B. braunii, three domestic strains (BOT70, BOT22, and BOT88-2) were selected for the EST analysis because of their high growth rates and oil production. For BOT70, we constructed a full-length-enriched cDNA library by the oligo-capping method. The 5' ends of 11,904 randomly chosen clones were sequenced and, therefrom, 2,897 independent genes were retrieved. For BOT22 and BOT88-2, we randomly sequenced 209,429 and 185,936 fragmented cDNAs, respectively. By assembling the sequences, 15,517 and 15,145 indepndent genes were retrieved, respectively. Ongoing analysis is devoted to elucidating the hydrocarbon biosynthesis pathways in B. braunii.

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Species-specific DNA markers (SSMs) are genetic tools essential in investigation of biodiversity and biological interactions in fields. However, developing them is a time-consuming task. We aimed to establish a convenient method for preparation of SSMs that distinguish 3 Brassica sepecies (Brassica rapa, B. napus, and B. juncea) using cDNA micro arrays. Plants were grown for two weeks in a greenhouse. Complimentary DNAs were synthesized from 10μg of total RNA. They were labeled with fluorescent dyes and hybridized to Arabidopsis oligo cDNA array (Affimetrix ATH). We selected 192 genes exhibiting the most significant differences in the signal intensity among the 3 species. We amplified DNA fragments with primers for selected genes using genomic DNA isolated from the Brassica species as a template. The primers were designed according to the nucleotide sequences of Arabidopsis orthologs. We successfully amplified 53 different PCR products for all 3 Brassica species. Forty-three of them have been sequenced and all of them exhibited polymorphic alleles. These data suggested that development of SSMs using DNA arrays is a powerful strategy for construction of SSMs.

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Messenger RNAs with premature stop codons (PTCs) are targeted by RNA quality control systems such as nonsense mediated mRNA decays (NMD). Several recent researches found that it is a common regulatory mechanism of RNA metabolism to generate alternatively spliced mRNAs with PTCs under certain regulation, which are followed by NMD. This mechanism is called RUST (Regulated Unproductive Splicing and Translation). For understanding complex systems of RNA metabolism, studying which genes are subjected by RUST is important. In this study, we developed a novel method to evaluate NMD effects on every intron based on tiling array results. We have performed 18 sets of tiling array experiments to take transcriptome of Arabidopsis thaliana for specific tissues, those under environmental stresses, and those on several developmental stages. We computationally analyzed them and listed introns which possibly retained in mature mRNAs in certain conditions. Besides, in several cases, the genes had small expression values in the conditions where the introns were retained. These results suggested that we successfully found introns which were affected by NMD and RUST.

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RIKEN BioResource Center (BRC) Experimental Plant Division promotes National Bioresource Project (NBRP) as a core center of Arabidopsis/Plant Cultured Cell and DNA. In 2009, available numbers of Arabidopsis FOX lines and EST clones (including full-length clones) from tobacco BY-2 cultured cells have been increased. Mutant and transgenic Arabidopsis, Thellungiella halophila full-length cDNA clones, and transgenic cultured cell lines are now under preparation for distribution to the community. Among them, Arabidopsis FOX lines (deposited from RIKEN PSC) are overexpressors of Arabidopsis full-length cDNA and provided as a seed pool for phenotype screening. Later, this resource will be also provided as individual lines accompanied with the information of transgene for each line. Further information and valuable services for researchers will be presented.

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Plant cultured cells are important biological resources used extensively for cytological, biochemical, and molecular biological studies. RIKEN BioResource Center collects, preserves, and distributes a wide variety of cultured cell lines.
We established novel Arabidopsis thaliana suspension cell cultures derived from leaves of ecotype Columbia, Landsberg erecta, and Wassilewskija. These cell lines displayed different properites, such as cell morphology, ploidy level, growth potential, and chlorophyll content. Furthermore, the suspension cell cultures were successfully preserved in liquid nitrogen and re-established again from cryopreserved stocks. We will provide these cell lines and information about their properties for researchers.
The development of standardized evaluation method is indispensable for characterization and quality control of the cultured cells. In order to examine stability of Arabidopsis cell lines, we are planning to monitor properties of suspension cell cultures and perform transcriptome analysis.

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RIKEN BioResource Center (BRC) takes over the preservation and distribution project of the Sendai Arabidopsis Seed Stock Center (SASSC) and starts distribution of the Arabidopsis natural accession seeds under the National Bio-resource Project (NBRP) supported by Japanese government (http://sassc.epd.brc.riken.jp/sassc/create_search_panel2.php?mode=general). Natural accessions must accumulate mutations that give them advantages to survive under various environmental conditions. Thus Arabidopsis accessions are useful to study the relationship between environmental adaptations and natural variations.
In this study, we try to get phenotype data by focusing on a response to osmotic stress with mannitol. As a result, Arabidopsis natural accessions showed various responses to the mannitol treatment. Since such kind of data is useful for association mapping and/or QTL analysis, we intend to develop new database which contains these phenotype data.

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Cryptomeria japonica D. Don is one of the most important Japanese conifer species. If we can repress pollen production, we will be able to reduce risks of transgene escape from GM C. japonica into native gene pools. We attempt to generate male sterility C. japonica by introducing barnase gene driven by a male strobilus specific promoter from C. japonica.
The seven male strobilus specific genes were isolated using suppression subtractive hybridization (SSH) method. We have isolated promoters of male flower specific genes by TAIL-PCR method. To confirm the tissue specific activity of the promoter regions, the promoter::GUS fusions were introduced into Arabidopsis and C. japonica. The 1009-C47::GUS showed tapetum-specific GUS activity in Arabidopsis and C. japonica. The 1009-C96::GUS lines showed GUS stain in tapetum and guard cell of Arabidopsis. We report that the transgenic Arabidopsis that introduced promoter::barnase construct shows male sterility.
Acknowledgements: This work was supported in part by AFFRC, MAFF, Japan

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Rice was aseptically grown to obtain secreted proteins for comprehensive analysis. Rice plants were grown in a nutrient solution from 1 to 2 weeks after germination, then the culture solution was collected. After root sample was obtained, 400 ml nutrient solution was concentrated to about 100 μl. Sample was purified using 2-D clean-up kit then protein obtained from root and nutrient solution was compared using SDS-PAGE. The band pattern was completely different, thus we considered that those protein in the nutrient solution was selectively secreted from the root. Though we have applied MALDI-TOF-MS after 2D gel separation, only 4 proteins were identified. For the comprehensive analysis of secreted protein, we have applied LC-MS/MS based shotgun sequencing then found a large number of proteins which are similar to pathogen related proteins and so on.

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Choice of Arabidopsis thaliana as a model in mid 1980s made molecular genetic studies of plant developmental processes much easier than before. We started genetic analyses of plant organ development, particularly in axis-dependent tissue differentiation in lateral organs, such as leaves, young floral buds and floral organs. They develop from primordia formed at the fixed position in the peripheral region of shoot apical meristem, and are considered to follow three axes; basal-apical, central-marginal, and adaxial-abaxial. We examined mechanism of the abaxial-side specific gene, FIL, and the adaxial-side specific gene, PHB, and showed that FIL expression is owing to two cis elements, and that PHB expression is controlled by microRNAs which is expressed at the abaxial side, suggesting that the two sides in lateral organ primordia mutually control specific gene expression. In addition, we showed that the marginal cell-specific genes, PRS and WOX1, are involved in the action of leaf margin as a boundary separating tissues specific to the adaxial and the abaxial sides. We are further investigating intercellular signaling factors controlling the axis-dependent development.

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All eukaryotic cells comprise various single membrane-bound organelles, which are interconnected by the trafficking system mediated by vesicular and/or tubular membrane carriers. This system, membrane traffic, plays fundamental roles in various cellular functions including maintenance of organelle identity, transport and retention of organelle resident proteins, and intra- and inter-cellular signaling. RAB GTPases and SNARE molecules are evolutionary conserved key players regulating tethering and fusion steps between donor and target membranes, whose paralogous expansion appears to be closely associated with diversification of organelles and trafficking pathways. We have identified several plant-unique RAB and SNARE molecules, which could be involved in unique functions to plants. Our genetic, biochemical, and cell biological analyses have been revealing their interesting functions, which indicates that land plants have evolved a quite unique mechanism for the regulation of membrane traffic.

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What controls "size" is a fundamental question in biology but intrinsic mechanisms that mediate this control still remain largely unknown. My laboratory is focused on studying the molecular mechanisms that determine cell and organ size in plants. Many cell types in plants increase their nuclear DNA content or ploidy through endoreduplication in which cells replicate their chromosomal DNA without intervening mitoses. Previous studies demonstrate strong correlations between ploidy levels and cell/organ sizes, and we are trying to understand how the endocycle progression is developmentally regulated and how an increase in ploidy modifies plant growth. In this talk I will describe our recent work on a SUMO E3 ligase involved in the endocycle control during the meristem development as well as on a trihelix transcription factor GTL1 required for ploidy-dependent cell growth in trichome development. Based on these studies, I will discuss how the cell cycle progression links with plant growth and development.

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Argonaute (AGO) genes play central roles in RNA silencing. In this study, we analyzed Arabidopsis AGO2 and AGO5. Interestingly, the 5' nucleotide of small RNAs that associated with AGO2 was mainly adenine and that of AGO5 was mainly cytosine. Small RNAs that were abundantly cloned from the AGO2 fraction (miR163-LL and miR390) and from the AGO5 fraction (miR163-UL and miR390*) are derived from the single small RNA duplexes. Furthermore, each strand of the miR163-LL/miR163-UL duplex is selectively sorted to associate with AGO2 or AGO5 in a 5' nucleotide-dependent manner. These results clearly indicate that the mechanism selecting the guide strand is different between AGO proteins even from the identical small RNA duplex. AGO2 localized in the nucleus and in cytoplasm, while AGO5 localized throughout the cytoplasm.

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Recently, large-scale 'omics data' from various plant species has been accumulated in public databases. For example, NCBI provides comprehensive omics data such as UNIGENEs, orthologous groups, gene expressions patterns as well as nucleotide sequences. In model plants, organism-specific databases have been also constructed and maintained, such as RAP-DB, MSU Rice Genome Annotation Project Database, OryzaExpress, SGN, MiBASE, KaFTom. These databases provide highly detailed information of genome annotations, ESTs, full-length cDNAs, DNA markers, Gene Ontology terms, gene expressions, metabolic pathways and so on. Integrated analysis of these large-scale omics data would allow us to understand complex biological systems. Here, we introduce the current status of omics databases and the integrated databases on rice and tomato.

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Lotus japonicus is a typical model legume in Fabaceae with the characteristics of a short life cycle (2-4 months), self-fertility, and relatively simple genome architecture of diploidy (n=6). Mutants in various biological phenomena specific to legumes such as symbiotic nitrogen fixation, and those common to flowering plants such as flower morphogenesis, have been characterized and the genes responsible have been isolated and further studied. The scope of LegumeBase (http://www.legumebase.agr.miyazaki-u.ac.jp/) is the collection, development and conservation of resources of L. japonicus and Glycine max.
In this session, we introduce databases for introduction of bioresources and utilization of genomic information in L. japonicus. Methodological movies illustrating the seeding, cultivating, breeding and seed growing are accessible. Kazusa DNA Research Institute reported structural features of the L. japonicus and root nodule bacteria genome, which are usable for the research of plant-bacterial interaction. We also introduce outlines of utilization of these information and some applications e.g. making phylogenetic tree using MEGA software package.

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Technical advances in imaging procedures generated a variety of information on a large-scale basis in the field of plant organelle research. These large data sets are deposited in databases, and used in further analyses. Among a variety of public databases, image databases exhibit an ability to display organelle dynamics, because organelle dynamics information, which can be interpreted by various parameters including size, number and behavior, is visually exhibited in a single representation. Through image databases, users can grasp organelle dynamics easily compared to text-based description. As image and movie data contain valuable and significant information such as size, length and velocity, image databases contribute to systems biology of plant organelles by combination with other databases that were generated using several 'omics' approaches. In this presentation, we would like to review the image databases, such as our database, PODB2, which are useful for plant organelle research. Moreover, we would like to discuss their potential as image-based tools for systems biology, and as educational aids that accelerate the interest of younger generations in plant biology.

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The three major systems for plant scientific names are: (1) New Engler ,(2) Cronquist, and (3) APG-II (Angiosperm Phylogeny Group). Although APG-II is the academic standard, many research articles often do not follow it. The same can be said to many personally maintained websites for images and encyclopedia of plants. To standardize the naming issue, Metabolomics.JP (http://metabolomics.jp/) provides a portal site for the classification as well as for 7,000 flavonoid structures, 200 crude drug, and external links for images and documents.

Important References for Scientific Names
"The Families and Genera of Vascular Plants" 9-volume Set (Series Ed.: Kubitzki, Klaus) Springer Verlag
"An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG II" THE ANGIOSPERM PHYLOGENY GROUP Botanical Journal of the Linnean Society, 2003, 141, 399-436.

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Vacuole is the largest organelle that occupies about 80% of plant cell volume. Vacuole participates in cellular processes such as development of turgor pressure, accumulation of inorganic ions and metabolites and degradation of discarded proteins. However, the molecular mechanisms underlying the diverse vacuolar functions are not fully understood. To get insight into the molecular basis of the vacuolar functions, we isolated the intact vacuoles from Arabidopsis suspension-cultured cells and conducted the proteomic analysis of vacuolar membrane and vacuolar sap. Several unknown transmembrane proteins were found in the vacuolar membrane and a possibility of several enzyme reactions besides degradative activity was indicated. Vacuolar membrane proteins appeared to be differentially distributed. As observed through immunostaining, V-PPase was distributed evenly whereas V-ATPase was localized to the specific tonoplast regions. V-ATPase was abundant in the detergent resistant membranes (-DRM-) obtained from the vacuoles.
Some novel functions of vacuoles uncovered by proteomic approach will be discussed.

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Plant peroxisomes play important roles in a variety of metabolic reaction such as the photorespiration, the fatty acid β-oxidation, and the glyoxylate cycle. We have identified the novel protein Glycine max PEROXISOMAL ADENINE NUCLEOTIDE CARRIER (GmPNC1) by proteomic analyses of peroxisomal membrane proteins using a blue native/SDS-PAGE technique combined with PMF method. Functional integration of GmPNC1 and its Arabidopsis orthologues, AtPNC1/AtPNC2, into intact Escherichia coli cells revealed these proteins transport ATP across cell membrane. We investigated physiological functions of the PNC in peroxisomal metabolism by analyzing a transgenic Arabidopsis plant in which expressions of Arabidopsis orthologues, AtPNC1 and 2 were suppressed using RNA interference. The pnc1/2 knock-down mutant suppressed the degradation of storage lipids during post-germinative growth and required sucrose for germination. These results suggest that PNC1 contributes to the transport of ATP that is consumed by reactions that generate acyl-CoA for peroxisomal fatty acid β-oxidation during post-germinative growth.

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Nucleus is a highly dynamic organelle and has heterogeneous structure, which is composed of double layered nuclear membrane, nuclear pore and nuclear matrix. Because nuclear architecture provides a physical framework for the genetic activities, it is important to know the mechanisms in which structure of the nucleus is built. To reveal the molecular mechanisms of nuclear biogenesis, we did two comprehensive proteomic analyses of Arabidopsis.
Highly purified nuclei were prepared from cultured cells by using concentrated sucrose buffer. More than 1000 proteins were identified with a mass spectrometer (LTQ-Orbitrap XL). Among them, we found novel nuclear membrane proteins, which are not found in animal and yeast cells.
Nuclear pore complex is a large multi protein complex and is composed of more than 30 types of nucleoporins. To clarify the molecular components of nuclear pore complex, we established high-throughput analytical method for protein complexes. By using this method, we identified plant-specific nucleoporins, which might be involved in various steps of plant development.

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Our previous studies indicated that OsRac1, a small GTP-binding protein, functions as a key regulator in rice innate immunity. The aim of our study is to reveal the defense signaling pathway in rice by applying the proteome analysis. We isolated OsRac1 interacting proteins by affinity chromatography and identified by using mass spectrometry. They included RACK1, a scaffolding protein, Hsp70 and Hsp90, and Sti1 which is a cochaperone for Hsp70/Hsp90 complex. Also, we identified NB-LRR proteins, which are a typical type of resistance proteins for plant innate immunity. We found that these proteins play a role in ROS production for defense signaling and defensome maintenance by stabilization of OsRac1. We named this protein complex Defensome.
We will show the data obtained from proteomic analysis and discuss the mechanism and the role of defensome in rice innate immunity.

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We show three proteome analyses using a highly sensitive nanoLC/MS/MS system to identify novel plant proteins involved in signal responses. In the first example, nuclear and nucleic acids-associated proteome analyses of rice proteins resulted in the identification of about 150 proteins that were likely involved in signal responses. The identified proteins included evolutionarily conserved and glucose responsive WD40 proteins and armadillo/pumilio protein. In the second example, proteome analysis of the phloem sap obtained by the insect laser method resulted in the identification of three FT-like proteins that might act as long-distance signaling molecules. In the third example, comparative proteome analysis using the MS intensity-based and label-free method allowed us to estimate relative abundances for 889 proteins during the greening process of rice seedlings. By the comparison with the results of transcriptome analysis, several proteins appeared to be regulated at the translation step or the degradation process of the proteins. Based on these results, we will discuss the potential of proteomics as a tool for screening novel proteins involved in signal responses in plants.

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Recent studies have revealed various functions for the small ubiquitin-related modifier (SUMO) in diverse biological phenomena, such as regulation of cell division, DNA repair, and transcription, in yeast and animals. In contrast, the characterization of SUMO target proteins in plants is limited, although plant SUMO proteins are involved in many physiological processes, such as stress responses, regulation of flowering time, and defense reactions to pathogen attack.
Here, we reconstituted the Arabidopsis thaliana SUMOylation cascade in E. coli. This system is a rapid and effective method to evaluate the SUMOylation of potential SUMO target proteins. We tested the capability of this system to conjugate the Arabidopsis SUMO isoforms, AtSUMO1, 2, 3, and 5, to a model substrate, AtMYB30, which is an Arabidopsis transcription factor. All four SUMO isoforms tested were able to SUMOylate AtMYB30. Furthermore, SUMOylation sites of AtMYB30 were characterized by LC-MS/MS followed by mutational analysis in combination with this system. The identification of SUMOylation sites enables us to investigate the direct effects of SUMOylation using SUMOylation-defective mutants.

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The studies of anthocyanin proceeded over one century from the beginning of 20th century in Japan. For the mechanism of color appearance, three hypotheses are well known: pH theory proposed by Willstatter, metal-complex theory by Shibata and co-pigment theory by the Robinson's. For metal complex, Hayashi et al. studied in detail the blue pigment commelinin isolated from dayflower; the three dimensional structure was now determined by Japanese researchers. For co-pigmentation, novel anthocyanins, which are very stable in neutral solution, were isolated in succession from the purple flowers of bell flower and Cineraria. These anthocyanins were acylated with above two molecules of aromatic organic acids. Japanese researchers clarified the structures and the mechanism of color stabilization of these polyacyl-anthocyanins by NMR. Concerning pH theory, although the studies using press juice were popular before,the mechanism of pH change is made clear now in cell level using micro technique. Recently,the production of Blue Rose was completed by Santory company in Japan. This gave a dream to anthocyanin researchers. I will describe future directions of anthocyanin studies in this section.

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Almost flower colors of wild and cultivated plants are due to anthocyanins and/or carotenoids. On the other hand, several flower colors are expressed by other pigments. For example, the flower colors of nine family species, such as Portulaca, cactus, cockscomb and Mesembryanthemum belonging to the order Caryophyllales except for the family Caryophyllaceae and Molluginaceae, are due to betalain pigments. Thus, their red purple and yellow flower colors are expressed by betacyanins and betaxanthins, respectively. Anthochlors, chalcones and aurones, contribute to the yellow colors of carnation, Cosmos, Corylopsis, snapdragon and so on. Flavonols substituted 6- or 8-position by hydroxyl group such as gossypetin are named as yellow flavonols and express the yellow colors of some Centaurea and Hibiscus species. When much amount of common flavonol, quercetin, is present in the flowers, e.g. Clematis, it acts as yellow pigment. More recently, it was reported that the deep yellow flower colors of Camellia chrysantha are expressed by common quercetin glycosides together with aluminum ions.

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