Nearly 4 billion hectares of forest covers the earth's surface, roughly 30 percent of its total land area, and its 5 percent is forest plantation. Forests are expected as sustainable bioresource substitutional of fossil fuels and are noteworthy for the role of CO2 fixation. Especially, Eucalyptus species constitute the most widely planted hardwood trees in the world and are most profitable for pulp production due to the fast growing and higher content of cellulose. Eucalyptus species are focused on as biomass energy source and Eucalyptus genome is currently being sequenced.
We selected hundreds of elite candidate trees (Eucalyptus globulus) that showed more than 1.5 times of wood volume compared with the biggest surrounding trees or were able to survive in drought areas from the plantation area in Western Australia. Several lines had higher photosynthesis activity under the drought condition in a greenhouse. Furthermore, coline oxidase from soil bacterium, Arthrobactor globiformis were introduced into Eucalyptus hypocotyls and the transgenic plants with higher expression levels were produced. We are evaluating their characters.

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Competition is now accelerating rapidly, with a number of groups around the world investigating various algal species and strains for their potential in oil production.
Botryococcus excretes most of its liquid hydrocarbons from the cells, retaining the oil within the colonial extracellular matrix. The many strains of Botryococcus differ in the amount of lipid production and growth capacity. Several strains with high hydrocarbon productivity and growth rates have been identified and submitted for further research and development. Research so far has more than doubled the ultimate level of biomass growth through the addition of organic waste. Botryococcus is mixotrophic, allowing it to use both light and chemical sources of energy for growth, further enhancing its already considerable potential as a hydrocarbon production system. The current undergoing project is aimed specifically at improving the cost balance of algae-based fuel generation by increasing hydrocarbon production by an order of magnitude.

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All Southeastern Asian countries are facing a shortage of available land for tree plantation. The increased demand for trees that would result from the establishment of a sizeable tree-based bioethanol industry in Southeast Asia would require 1) the development and use of fast-growing tree species as feedstock and 2) more efficient use of existing plantations. One of the tasks of modern plant science is to identify and confirm the plant species whose cell walls are susceptible to complete saccharification. Their lingo-cellulose must be easily degradable into monosaccharides, though, in a particular species that meets the other requirements, we may be able to enhance this quality through genetic engineering, now that the structure and function of wall components are beginning to be understood.
Recalcitrance to saccharification is a major limiting factor of the conversion of lignocellulosic biomass to ethanol. An increase in the sacharification was tested in the xylems of sengon (Paraserianthes falcataria) and mangium (Acacia mangium) by the genetic reduction of xyloglucan.

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Seed dormancy is a poorly understood process. To identify molecular components that regulate seed dormancy, we screened T-DNA insertion lines and identified a mutant designated honsu (hon). HON loss-of-function mutants display deep seed dormancy, whereas HON-overexpressing lines display shallow seed dormancy. HON encodes a seed-specific group A phosphatase 2C (PP2C) and is the major negative regulator of seed dormancy among group A PP2Cs. Like other PP2C family members, HON interacts with PYR1/RCAR11 in the presence of abscisic acid (ABA). Our analysis indicates that HON inhibits ABA signaling and activates gibberellin (GA) signaling, and both of these conditions must be satisfied to promote the release of seed dormancy. However, activation of GA signaling by HON is an indirect effect attributable to weakened ABA signaling. HON mRNA levels are increased in mutants displaying shallow seed dormancy or under conditions that promote the release of seed dormancy, and decreased in mutants displaying deep seed dormancy or under conditions that deepen seed dormancy. Taken together, our results indicate that seeds homeostatically regulate dormancy by adjusting the level of HON.

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Temperature is a main environmental cue that directs the season of germination of winter- and summer-annual seeds. The seeds of winter-annuals, such as Arabidopsis, are dispersed in spring but their germination is suppressed by high temperature during summer. Supraoptimal and suboptimal temperatures often inhibit germination of crop seeds and reduce efficiency of their production severely. We used Arabidopsis as a model, and found that high temperature enhances expression of NCED9, and suppresses germination through the action of ABA in continuous light condition. In red-light pulse condition, however, ABA may not be a main factor since germination of ABA deficient mutant seeds was inhibited by high temperature. PIL5 is shown to be a main germination suppressor in far-red light pulse condition. Analyses of PIL5 loss of function mutant seeds indicated that high temperature suppresses germination through the action of PIL5 in this condition. We also found PIF4, which is shown to work on high temperature response of the seedlings, is another germination suppressor. We will discuss how temperature regulate the action of these germination suppressors.

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An Arabidopsis accession Cvi exhibits relatively strong seed dormancy compared to commonly used accessions such as Col and Ler. Primary dormancy of after-ripened Cvi depends largely on the seed coat; removal of the seed coat induced the growth of embryos (germination) upon imbibition. It is known that cold treatment of the imbibed seeds (stratification) breaks seed dormancy, however, we found that long-term cold imbibition of Cvi seeds induced secondary dormancy. Interestingly, the secondary dormant seeds did not germinate even after the removal of the seed coat. Inhibition of ABA biosynthesis by fluridone partially prevented the secondary dormancy induction, suggesting that ABA is involved in this response. However, it appears that ABA levels do not always correlate with seed dormancy status if seed dormancy and ABA levels were compared among accessions. To understand the molecular mechanisms that regulate seed dormancy status and its relationship with endogenous ABA levels, now we are conducting QTL mapping using an embryo-dormant accession that contain elevated endogenous ABA levels.

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Cereals crops, rice, wheat, and maize, are the most important food. Seed germination under undesirable environmental conditions will contribute to stable cereal productions and to the enlargement of the cultivation areas for the demands of people around the world. However, they have different characters on the anatomy of seed. To understand the molecular bases of seed germination in cereals, identification of genes controlling seed germination is necessary. Using a genomics approach, a novel gene controlling seed germination in rice, qLTG3-1, has been identified. qLTG3-1 is a major quantitative trait locus (QTL) controlling tolerance to low-temperature at seed germination stage, termed low-temperature germinability. This QTL encodes a protein of unknown function and is strongly expressed in the embryo prior to and during seed germination. Expression of qLTG3-1 was tightly associated with vacuolation of the tissues covering the embryo. I will discuss the role of qLTG3-1 on seed germination in rice and other cereals.

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Seed dormancy is an important agronomic trait for rice and other cereal crops because it is associated with pre-harvest sprouting, which reduces seed quality. Seed dormancy is a typical complex trait determined by a series of quantitative trait loci (QTLs). We attempted to isolate QTLs from a variety of chromosome segment substitution lines (CSSLs) derived from crosses between japonica/japonica, japonica/indica, and japonica/wild rice. Previously, we reported five QTLs related to seed dormancy from "Kasalath". Furthermore, we detected QTLs from additional CSSLs, and wild rice introgression lines. Among them, we reported the molecular cloning of a major QTL designated as "Seed Dormancy 4" (Sdr4). In addition, we performed fine-scale mapping of Sdr7, the QTL on "Habataki" chromosome 1L, using more than 4600 F2 plants, thereby delimiting the candidate region to 24.5 kb. The corresponding genomic sequence of "Habataki" contained one specific gene to the "Habataki" genomes, was an ortholog of the Arabidopsis dormancy-related gene.
We present the data from QTL findings, evaluation, fine mapping for isolation, and functional analysis for understanding the rice seed dormancy network.

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Development of higher plants once interrupted through seed formation resumes by seed germination. Gene expression programs operating after the onset of germination are drastically altered relative to those operating during embryogenesis; the programs conferring seed characters are terminated, and those for postgerminative development are initiated. Mutants of the seed maturation master regulators, LEC1, LEC2 and FUS3, are broadly impaired with seed maturation-related events. In addition, the mutants show heterochronic characters as exemplified by true leaf-like cotyledons, and SAM and RAM activation during seed development. Our transcriptome analysis indicates that a wide range of genes expressed during postgerminative development are up-regulated in the mutant embryos. Onset of such heterochronic gene expression is detected as early as before the heart stage. These data suggest that postgerminative gene programs are actively repressed during embryogenesis through the function of the LEC/FUS3 factors. Base on these analysis as well as trascripotome data of various source, we will discuss about seed dormancy and germination viewed from switching of gene expression programs.

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The completion of the human genome project opened the new era where various biological phenomena are elucidated with massive molecular data. Although many things have been elucidated with the massive data, the data were obtained by averaging ensembles of cells which may mask important information on a real life system. These days, many people want to analyze single-cells because heterogeneity in cell pools sometimes plays important roles in a life system. However, the analysis methods for single-cells are limited now. The next generation DNA sequencer can be used for that, however, only a part of mRNA in a single-cell can be analyzed.
We have developed a new accurate quantitative analysis method for mRNA in single-cells. It uses q-PCR coupled with a single-cell cDNA library for analyzing multiple gene expression levels in single-cells. As its ability is very high, it can analyze mRNA as small as several copies in a single-cell. We are making a tool to easily carry out the accurate multiple gene expression analysis with the method. The technology and its applications will be presented in the symposium.

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Recently, MALDI (Matrix Assisted Laser Desorption/Ionization) imaging mass spectrometry has been a powerful tool to map spatial distribution of molecules on the surface of materials. In sample preparation in MALDI imaging mass spectrometry, in principle, sample is not purified prior to the analysis, so MALDI process generates mixture of various different molecular ions. To distinguish these ions to identify unknown molecular species exist on the sample surface, ultra-high resolution and ultra-high mass accuracy is required. To achieve high spacial resolution of MALDI imaging mass spectrometry, the LASER spot size should be smaller enough. However, smaller LASER spot size also decreases the ablated material amount, causing worse overall sensitivity. To overcome such problems, we developed new microscope MALDI ion source and built-in to the commercial Q-FTICR-MS. The combination of matrix compounds which absorb UV (Ultra-Violet) light and short pulsed UV-LASER is difficult to be applied simply to plant tissue slices, in some fundamental issues. We describe our approach towards identifying and locating unknown molecular species in plat tissue slices.

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In the next generation of structural studies of biological samples, coherent X-ray diffraction microscopy (CXDM) technique will provide a novel tool to visualize non-crystalline biological particles, which are, in principle, unable to be crystallized. For the diffraction patterns recorded using coherent and highly brilliant X-rays, the phase-retrieval algorithm reconstructs the electron density distribution within the samples directly. Toward the utilization of this technique in biological field, we are developing an X-ray diffraction apparatus for micro to sub-micrometer biological samples at cryogenic temperatures. In the talk, I would like to introduce the theoretical background of the image reconstruction from the diffraction patterns and the details of the diffraction apparatus.

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When an intense femtosecond laser pulse is focused into biological tissue or cells through an objective lens, local disruption and/or explosion is induced at the laser focal point with an effective multiphoton absorption. Especially, since the time duration of the femtosecond laser pulse is extremely shorter than the time scale to convert from the light energy to the heat energy, the disruption and explosion are initiated in the time scale before the heat conversion. Namely, the local disruption and explosion with less heating are realized by the femtosecond laser irradiation. Previously, we have developed manipulation and stimulation techniques for single animal cells by utilizing these phenomena. Here I present our new trial to apply these techniques to plant cells or tissue. We have succeeded in function inhabitation of a plant by drilling a part of the tissue and single cell dissection and isolation by scanning the laser on the tissue. Furthermore, we are proposing techniques to stimulate a plant cell mechanically by the local explosion and to introduce outside molecules into the cell.

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Temporal induction of gene expression is an important technique to examine cell lineage, gene function and intercellular communication in vivo. In many species, several gene induction systems were reported already, such as steroid hormone, ethanol, and heat shock. Recently, we have developed a new method of gene induction in a single cell of living organisms using an infrared laser-evoked gene operator (IR-LEGO) system. We have chose heat shock system for temporal induction, because almost all species such as animals and plants have the system. And we have employed infrared laser for heating cell, since the light can heat water molecules effectively and does not induce photochemical reaction which produces radicals. We reported the application of IR-LEGO to some species including Arabidopsis. In transgenic plant line for HSP 18.2 promoter : GUS, we successfully induced gene expression in a single cell of a seedling. Furthermore, we have succeeded constitutive gene expression in single-cell using cre/loxP recombination system. IR-LEGO has the potential to be a useful tool in extensive research fields for cell marking or targeted gene expression in the plant.

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Plant cells exhibit highly dynamic protein and membrane trafficking pathways. Many key protein and membrane sorting events occur in very small compartments or even in subdomains within an organelle, which makes their visualization by light microscopy very challenging. To analyze the endosomal sorting of plasma membrane proteins for degradation and the vacuolar transport of storage proteins in seeds, we perform dual-axis electron tomography of high-pressure frozen/freeze substituted plant material. We obtain 3D reconstructions of large areas of the cells containing organelles of interest and subject them to segmentation, model calculation, and quantitative analysis. Electron tomography in combination with structural pattern recognition of known macromolecular complexes and/or immunogold labeling allow for the correlation between structure and biochemical composition. Several examples on how these imaging approaches have been applied to the understanding of plant trafficking pathways will be discussed.

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Recently it is reported that negative regulars play an important role on the regulation of gene expression in plants. In addition, we lately showed that, in the transgenic plants that express the chimeric repressor, the expression of group of genes, which are involved in the phenotype induced by the chimeric repressor, are upregulated, such as shown in TCPs. These suggest that plant genome contains a number of negative regulators and those factors control various biological function and maintenance of homeostasis via the regulation of the gene expression antagonistically with positive regulators. Negative regulation includes transcriptional suppression by a repressor, posttranscriptional regulation by miRNAs, active protein degradation and chromation modification. However, the mechanisms of regulation of the negative regulators or their function has not been fully characterized. In this session, we introduce negative regulation in leaf and floral stem cell development, floral patterning, response to salt stress, gibberellin signaling and Circadian Rhythm. We discuss functional role of the negative regulators involved in the regulation of these activities.

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Plants exploit numerous strategies to adapt to changes in their environment. The development of plants with enhanced tolerance to abiotic environmental stress, such as drought, salinity and heat stress should enhance crop yield. Regulators of transcription are important in the acclimation of plants to environmental stresses, and several have been shown to confer tolerance to abiotic stress. Because some loss-of-function mutants have elevated tolerance to abiotic stresses, we postulated that application of CRES-T (Chimeric REpressor gene Silencing Technology) might allow the generation of factors that can improve tolerance to abiotic stress. We show here that five independent chimeric repressors derived from MYB, NAC, GARP, C2H2ZnF and ERF transcription factors confer tolerance to salt or osmotic stress in Arabidopsis. We also show that chimeric repressors derived from rice homologs of two of these factors confer salt tolerance on rice plants. Furthermore, negative regulator(s) that is involved in abiotic stress tolerance will be discussed.

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Coordination of the maintenance of the undifferentiated fate in the shoot meristem and the promotion of cellular differentiation in organs is essential for the development of plant shoots. CINCINNATA-like (CIN-like) TCP transcription factors are involved in this coordination via the negative regulation of CUP-SHAPED COTYLEDON (CUC) genes, which regulate the formation of shoot meristems and the specification of organ boundaries. However, the mechanism of this negative regulation is poorly understood. We show here that TCP3, a model of CIN-like TCPs of Arabidopsis, directly activates the expression of genes for miR164, ASYMMETRIC LEAVES1 (AS1), IAA3/SHY2, and SAUR proteins. Gain of function of these genes resulted in the fusion of cotyledons and the defect of shoot meristems, whereas their loss of function induced ectopic CUC expression in leaves. Our results indicate that miR164, AS1, IAA3/SHY2, and SAUR partially but cooperatively suppress the CUC expression. Since CIN-like TCP genes act dose-dependently in the leaf differentiation, we propose that CIN-like TCPs have important roles to generate different leaf forms, without having any lethal effects on shoots.

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In 2000, plant specific transcriptional repressors were first isolated, and next year, the repression domain named EAR-motif, was identified. Using the EAR-motif, the CRES-T system, which is useful tool to induce the knock out phenotype of each transcription factors, was developed. By contrast, biological functions of transcriptional repressors have not been fully analyzed. We present here a functional role of WUSCHEL (WUS) as a representative of transcriptional repressors.
WUS is known as a regulator of stem cell populations in shoot meristems. All activities of WUS, including maintenance of meristems and induction of AG, were eliminated when the WUS-box was mutated (WUSm1), indicating that the WUS-box is essential for WUS function. The fusion of exogenous repression domain (SRDX) complemented the defective activity regarding the induction of stem cell identity in WUSm1. By contrast, fusion of an exogenous activation domain (VP16) to WUSm1 induced flowers similar to those induced by the ectopic expression of AG. These results demonstrate that WUS is a bifunctional transcription factor, which acts mainly as a repressor and as an activator in the case of induction of AG.

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Floral stem cell activity is terminated in flower development. The negative feedback loop of the stem cell determinant WUSCHEL (WUS) and the floral homeotic protein AGAMOUS (AG) plays a major role for the meristem determinacy control in Arabidopsis. AG is directly induced by WUS in the center of floral primordia. WUS and AG are co-expressed for about two days, and then AG represses WUS expression to terminate the stem cell activity. We recently showed that KNUCKLES (KNU), a C2H2-type zinc finger protein is a direct target of AG, mediating the repression of WUS. We show that the KNU locus contains the repressive histone mark H3Lys27 tri-methylation (H3K27me3) and that the AG-dependent removal of the mark happens in a cell-cycle dependent manner. We further show that AG binding sites contain the landing site for a Polycomb Group (PcG) protein necessary for the maintenance of H3K27me3. These results indicate that the competition of AG with the PcG leads to dilution of H3K27me3 during cell division. I will present our model that the negative feedback loop is regulated in a proper developmental timing through histone modification.

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Circadian (about one-day) rhythm is endogeneously generated and self-sustained system that makes organisms to adjust to daily changes in environment. Genetic approaches have identified clock-associated genes in Arabidopsis. However, little knowledge about biochemical properties of clock-associated proteins has prevented us to understand the clock system in molecular level.
In this talk, we will present the molecular function of PSEUDO-RESPONSE REGULATOR 9, 7, and 5 proteins as transcriptional repressors in the clock. Using a glucocorticoid-induced PRR5-GR construct, we found that PRR5 directly down-regulates CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) expression. PRR9, PRR7, and PRR5 suppressed CCA1 and LHY promoter activities, and gave transcriptional repressor activity to a heterologous DNA binding protein. Furthermore, PRR9, PRR7, and PRR5 associated with the CCA1 and LHY promoters in vivo from early daytime to midnight, coinciding with the time these genes were repressed. In turn, CCA1 and LHY activate PRR9 and PRR7 expression in the morning. We propose that the negative feedback system with time lag of several hours constitutes circadian rhythm.

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Gibberellins regulate seed germination, shoot elongation and flowering. DELLA proteins are members of the plant-specific GRAS protein family and act as repressors of the GA signaling pathway. DELLA proteins are rapidly degraded in the presence of GA. The degradation mechanism has been uncovered by the discovery of GA receptor and F-box protein. To understand the downstream signaling of DELLA proteins, we have identified GAF1 that interacts with DELLA protein by modified yeast two hybrid screening. GAF1 is a novel transcriptional factor. Arabidopsis plants that overexpress GAF1 are early flowering with larger leaves. In contrast the gaf mutant exhibits a semi-dwarf and late flowering phenotype. BiFC analysis indicated that the interaction of GAF1 and DELLA protein disappeared after GA treatment. Moreover we isolated a WD repeat protein as another GAF1 interaction protein. Our trans-activation assays in yeast and plant cells suggest that DELLA and the WD repeat protein regulates the function of GAF1. These data suggest that GA controls transcriptional activity via alteration of GAF1 complex components. Based on this model, we are searching for the target genes of GAF1.

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Flowers consisting primarily of four basic organ types are the most remarkable feature that characterizes the angiosperms. Floral organ patterning in Arabidopsis requires activation of floral homeotic genes by the floral meristem identity gene, LEAFY (LFY). Here we discuss how precise activation of expression of class B and C homeotic genes in floral meristems is regulated by three flowering time genes, SHORT VEGETATIVE PHASE (SVP), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and AGAMOUS-LIKE 24 (AGL24), through direct control of a LFY co-regulator, SEPALLATA3 (SEP3). Orchestrated repression of SEP3 by SVP, AGL24 and SOC1 is mediated by recruiting two interacting chromatin regulators, TERMINAL FLOWER 2/LIKE HETEROCHROMATIN PROTEIN 1 and SAP18, a member of SIN3 histone deacetylase complex. Our finding of coordinated regulation of SEP3 by flowering time genes reveals a novel genetic pathway that prevents pre-mature differentiation of floral meristems and determines the appropriate timing of floral organ patterning.

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Two modes of electron flow exist in the chloroplast-linear electron flow (LEF) from water to NADPH via PSII and PSI in series and cyclic electron flow (CEF) around PSI. Although CEF is essential for satisfying the varying demand for ATP, the exact molecule(s) and operational site were remained elusive. In the unicellular green alga Chlamydomonas reinhardtii, the electron flow shifts from LEF to CEF upon preferential excitation of PSII, which is brought about by an energy balancing mechanism between PSII and PSI (state transitions). We isolated a protein supercomplex composed of PSI with LHCI, LHCII, Cyt bf, FNR, and the integral membrane protein PGRL1 from the cells under PSII-favoring conditions. Spectroscopic analyses indicated that upon illumination, reducing equivalents from downstream of PSI were transferred to Cyt bf, while oxidized PSI was re-reduced by reducing equivalents from Cyt bf, indicating that this supercomplex is engaged in CEF. Thus, formation and dissociation of the PSI-LHCI-LHCII-FNR-Cyt bf-PGRL1 supercomplex not only controlled the energy balance of the two photosystems, but also switched the mode of photosynthetic electron flow.

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It is estimated that diatoms perform photosynthesis corresponding about 20% of the global annual primary production. Therefore, diatoms contribute greatly to the global carbon cycle and hence to the regulation of the global climate. Toward the precise understanding of light-acclimation mechanisms in diatoms, we investigated the composition changes in their photosynthetic systems and photosynthetic pigments under different growth irradiances using a centric marine diatom, Chaetoceros gracilis, and a pennate marine diatom, Phaeodactylum tricornutum.
The molar ratio of accessory pigments to chlorophyll a stayed almost constant under a wide range of irradiance indicating their large antenna size. The relative amount of xanthophyll cycle pigments increased under high light, which would facilitate the effective protection mechanisms of photosystems under high light. The ratio of photosystem I to photosystem II, when grown in high-light, increased in C. gracilis but decreased in P. tricornutum. Divergent strategies among diatoms against the environmental light condition will be discussed.

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Aquatic photosynthetic organisms have acquired unique CO₂-concentrating mechanism (CCM) to maintain photosynthesis in aquatic environments, in which CO₂ diffuses slower than in the air. Although CCM is assumed to be composed by many low-CO₂ inducible gene products, cell membrane-bound inorganic carbon transporters have not been described in green algae. We have identified a plasma membrane-bound inorganic transporter LCI1 by characterizing a transgenic Chlamydomonas cells (Ohnishi, Plant Cell 2010). In addition, one of the low-CO₂ inducible genes, LCIB/LCIC, was shown to be essential to CCM under air conditions (Yamano, PCP 2010). I would like to discuss about possibilities to support photosynthesis by over expression the CCM components in eukaryotic algae. In addition, changing of the metabolic pathway and to enhance the productivities in eukaryotic cells.

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Photosynthetic organisms have a wide variation. The primary symbionts such as chlorophytes and rhodophytes are organisms that cyanobacteria got into non-photosynthetic eukaryotes. The second symbionts such as heterokontphytes, haptophytes and dinohytes are that the primary symbionts got into non-photosynthetic eukaryotes. The second symbiotic algae are defined as the second symbiotic eukaryotes driving oxygen evolving photosynthesis. In this paper, I will introduce wide variation of carbon metabolism of the secondary symbiotic algae including a novel pathway of beta-carboxylation in the chloroplast of haptophytes. The second symbiotic algae are known as one of sources of biofuel and limestone production in ancient ara. Present species of those algae are producing a huge bloom in the ocean and transporting CO2 into the oceanic sediments and therefore their carbon fixation and metabolism play key function in the global environment. The important role and their potential of such algae in the global environmental issues also will be discussed.

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Microalgae have been considered as a source of high-lipid material for the production of biofuels. Our research group has launched the selection of marine microalgae with high neutral lipid content from microalgae culture collection. Among of them, a marine pennate diatom, Fistulifera sp. strain JPCC DA0580, was selected as the highest triglyceride producer (The lipid content: 60 wt%). Here, I describe whole genome analysis to clarify the mechanism for high triglyceride productivity in strain JPCC DA0580. The genome size of strain JPCC DA0580 was approximately 50 Mbp, which was much larger than other diatoms sequenced i.e. marine centric diatom, Thalassiosira pseudonana (32.4 Mbp) and marine pennate diatom, Phaeodactylum tricornutum (27.4 Mbp). Comparative genome analysis of three species (JPCC DA 0580, T. pseudonana and P. tricornutum) was performed. Furthermore, analysis of mRNA expression under different culture conditions was performed to identify the functional genes on genomic DNA.

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The characteristic feature of plants is represented by their photosynthetic function and their huge chemical diversity. The so-called plant secondary products (recently often referred as 'specialized compounds') estimated over 200,000 compounds existing are used as medicines, functional ingredients of foods, flavors and industrial materials, which are all indispensable in our lives. Phytochemical genomics elucidating the genomics basis for this chemical diversity is recently recognized as one of major topics of plant science. This study has been initiated and developed with Arabidopsis, and now it is extending to crops and medicinal plants by taking the advantages of technology advancement of next-generation sequencing and cutting-edge metabolomics analysis. Overview for phytochemical genomics will be given as an introduction of the symposium.

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Plant genomes contain numerous genes originating from duplication events. Therefore, it is of interest to address how duplicate genes contributed to metabolic diversities in plants. We showed that recently duplicated genes contribute to a diversity of secondary metabolites in lineage-specific fashion. However, we also showed that these duplicate genes contribute to genetic robustness with preserving the same function. The diversity and redundancy in duplicate genes can be the cause for the hardness to identify genes related to each secondary metabolite. To solve the complexity, we propose a new approach to identify genes related to secondary metabolites by the diversities of secondary metabolites within a species. Using the genome association approach of both genome data and transcriptome data among 20 accessions of A. thaliana, we can infer genes related to each of about 500 known and unknown secondary metabolites in A. thaliana. Finally, I introduce some examples to identify known enzymatic genes of a secondary metabolite by this approach.

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For the last decade genomics has operated by a "white paper" process whereby genomes have been prioritized for sequencing one-by-one by arguing their importance to science or industry. That made sense when genomes cost a fortune to do, but it also meant that (by definition) the next genome was always less interesting than the previous. We are now able to sequence thousands of genomes but we have to answer the critics who rightly ask "why". 1KP has taken a different approach by posing questions of importance to science or industry that might be answerable by sequencing multiple species, none of which need qualify as a species of interest in its own right. We started with transcriptomes instead of genomes only because of the lower costs, but the idea is extensible to genomes and not restricted to plants. Along the way we addressed practical problems: developing light sensitive molecules for optogenetic studies of mammalian brains and understanding the mechanism of resistance to chemotherapy treatment of cancer. In the process we finally begin to explore the vast biodiversity that to date has barely been touched by genomics.

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In a metabolomics research, assignment of measured spectra to a specific metabolite is one of the most fundamental process. The assignment is usually made by taking a match with known compounds and therefore, it is necessary to scan the spectra against whole previously studied compounds. This means the survey of whole natural products reported in the literatures which is an extremely tedious and daunting process. In order to make this process feasible, we have developed a metabolite database that contains species-metabolite relations called KNApSAcK, which currently contains 102,005 species-metabolite relations involving 50,054 metabolites. In the present study, we review the current status of KNApSAcK database and its application to metabolomics and introduce the multifaceted retrieval system, KNApSAcK Family, which consists of seven parts for the purpose of retrieving metabolites from several different aspects. "Pocket" includes search system for species related to human living such as edible plant in Japan ("Lunch Box") , traditional Japanese medicine ("KAMPO"). "KNApSAcK from around the world" includes medicinal and edible plants utilized in each country.

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Medicinal plants produce a wealth of pharmaceutical compounds such as digitoxin and vincristine. Unfortunately, the specialized biochemical pathways leading to such compounds remain poorly understood and progress in elucidating and manipulating these taxonomically-restricted metabolic pathways has been correspondingly slow. Development of "omics"-level resources for medicinal plants has been limited, which means that research in these species has not benefited to the same extent as has research in model plants and agronomic crop species. With the combined use of state-of-the-art sequencing technologies, metabolomics capabilities, and bioinformatics, we are developing an unrestricted, public resource to address this growing gap in our knowledge base of species-specific plant metabolism. This resource is designed to accelerate the identification and functional analysis of genes involved in natural product biosynthesis. Progress towards the discovery of the pathways and genes responsible for biosynthesis of key pharmaceuticals and their diversification will be discussed.

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Triterpenoids, with increasing evidence of their health beneficial properties, is a diverse group of secondary metabolites. Legumes biosynthesize various triterpenoids, including soyasaponins and glycyrrhizin. Glycyrrhizin is a triterpenoid glycoside (saponin) derived from licorice that is used as a natural sweetener and as a crude drug. Most legume triterpenoid saponins are derived from the common triterpene skeleton, β-amyrin. Cytochrome P450 monooxygenases (P450s) play critical roles in site-specific oxidations of triterpene skeleton; thereby create structural variation of triterpenoid aglycones. P450s exist as large gene families in plants, making it difficult to predict potential involvement of specific P450s in triterpenoid biosynthesis. In this study, the analysis of licorice ESTs and Medicago truncatula gene co-expression data led to the identification of several β-amyrin-oxidizing enzymes, including CYP88D6 (β-amyrin 11-oxidase) as the initial P450 in glycyrrhizin biosynthesis. Our observations indicate that in legumes, the P450 enzymes involved in triterpenoid biosynthesis are recruited in at least four very distant CYP families.

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The evolution of enzyme function is the driving force behind the metabolic diversity of plant natural product biosynthesis. Synthetic biology involves the reduction of biological systems to their basic components and the engineering of new processes through the novel assembly of these parts. The PhytoMetaSyn Project (www.phytometasyn.ca) is a consortium of Canadian researchers whose goals are to establish a functional genomics pipeline to characterize plant natural product biosynthetic genes and to establish a process engineering technology to deploy these genes in yeast. We are using massively parallel DNA sequencing to probe the expressed genomes of plants producing bioactive metabolites. Robust bioinformatics and targeted metabolite profiling allows the selection of candidate genes. The project will deliver (1) a public repository of transcriptome libraries from 75 non-model plants; (2) engineered yeast strains producing plant metabolites; (3) a catalogue of biocatalysts for plug-and-play synthetic biology; (4) an efficient functional genomics strategy for identifying unknown plant biosynthetic genes; and (5) an analysis of associated regulatory, ethical, and economic issues.

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Autophagy is a catabolic mechanism activated in response to nutrient deprivation in eukaryotic cells. In this process, a double-membrane vesicle, termed an autophagosome, non-selectively sequesters cytoplasmic materials and fuses to the lysosome/vacuole to deliver the cargo for degradation. The generated pool of nutrients can be then used by the cells to survive starvation periods. Recently, it has been reported that autophagy also contributes to many higher-order cellular functions such as selective removal of infected bacteria and aberrant proteins that may cause pathogenic amyloid formation, and immunity.
In the budding yeast Saccharomyces cerevisiae, autophagy not only plays a role in adaptation to nutrient starvation but also contributes to a transport of specific vacuolar hydrolases via the cytoplasm to vacuole targeting pathway and selective degradation of mitochondria and peroxisomes. In this presentation, I will give an overview of mechanisms of selective autophagy in yeast. Similarities between the yeast systems and mechanisms of clearance of aberrant proteins and bacteria in mammalian cells will be also discussed.

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Autophagy is an evolutionarily conserved intracellular process for the vacuolar degradation of cytoplasmic components. We developed autophagy monitoring systems in planta and revealed that plants in which ATG (autophagy-related) genes are disrupted (atg mutants) are defective in autophagy. Phenotypic analysis of the atg mutants showed that autophagy defects in higher plants result in early senescence and excessive immunity-related programmed cell death (PCD), irrespective of nutrient conditions, but the mechanisms by which cell death occurred in the absence of autophagy were unclear.
We have now identified a conserved requirement for salicylic acid (SA) signaling in these processes in atg mutants and found that SA signaling can induce autophagy. In this symposium, we will present our latest data concerning the physiological role of autophagy during senescence and the innate immune response in plants, and speculate about possible mechanisms for the negative regulation of cell death by autophagy.

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Cryptogein, a protein from a pathogenic oomycete, rapidly triggers cytosolic Ca²⁺ rise and production of reactive oxygen species (ROS), followed by synchronous induction of defense-related gene expression, cell cycle arrest and PCD in tobacco BY-2 cells. To analyze the possible involvement of autophagy in the regulation of plant immunity, we have established a transgenic BY-2 cell line in which dynamics of autophagosomes can be visualized with various fluorescent proteins fused with the tobacco homolog of Atg8, and analyzed the effects of cryptogein and cell cycle phases on the dynamics of autophagosomes. We also tested effects of various autophagy modulators on cryptogein-triggered initial signaling events including Ca²⁺ mobilization and ROS production, as well as gene expression and PCD. We will discuss novel regulatory mechanisms of plant immune responses involving autophagy, and possible potentiation of plant immunity by modulating autophagy.

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Because plants cannot move from the habitat, the supply of nutrients can vary by environmental changes. Several responses to nutrient limitation were observed in plants. Among them, autophagy is the degradation system that is induced upon starvation. We developed previously a method to quantify the autophagic degradation using a red-fluorescent reporter protein. This system was recently improved using color-convertible fluorescent protein to distinguish the synthesis and degradation of the reporter protein. Using this system we investigated the effect of phosphite, which is a non-metabolizable analog of phosphate, on the induction of starvation dependent autophagy. We observed that the phosphite supresses both the autophagy induction and decrease in protein synthesis specifically under phosphate limitation condition. Independently, we observed that a sucrose transporter, which is localized to the trans-Golgi network, specifically degraded upon sugar starvation possibly by autophagy. In this symposium, we present these recent findings and discuss the pathways for the induction of intracellular degradation system and various nutritional deficiencies.

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For autotrophic plants, recycling of nutrients and energy is especially important to survive under severe environments. Much of the total leaf nitrogen is allocated to chloroplasts, especially to Rubisco. During senescence and times of stress such as starvation, chloroplast proteins are rapidly degraded and their nitrogen and carbon are recycled.
While it is considered that bulk degradation of proteins and nutrient recycling in plants occurs mainly by autophagy, its actual role is still unclear. We have studied a role of autophagy on the degradation of chloroplast proteins since Rubisco-containing bodies (RCBs) were found in senescent leaves. RCBs were mobilized to the vacuole for the degradation by an ATG -gene dependent macroautophagy. Following RCBs, shrunk chloroplasts were also transported into the vacuole by autophagy when leaf senescence was promoted. RCB production was promoted by deficiency of leaf carbon but not nitrogen, thus the regulation was distinct from non-RCB-type autophagy. In this presentation, we discuss how autophagy contributes to the degradation of chloroplasts and their proteins.

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We have been investigating the physiological roles of autophagy using cultured tobacco cells, Arabidopsis roots, and the protonemata of Physcomitrella. When tobacco cells cultured in a nutrient-sufficient medium are transferred to sucrose-deprived medium, the net degradation of cellular proteins and rRNA occurs, and these components decreased to approximately half the initial values in 2 days. The degradation of protein and rRNA are inhibited by the autophagy inhibitor 3-methyladenine, suggesting that cells degrade their own constituents by autophagy for recycling and obtaining energy under nutrient starvation conditions. Root tips excised from Arabidopsis seedlings grow in a culture medium. Their growth depends on sucrose in the culture medium. In atg5 mutants, in which the macroautophagic pathway is impaired, the growth of root tips is retarded. This suggests that autophagy contributes to cell growth. When the protonemata of Physcomitrella are placed in the dark, senescence accompanying the degradation of chlorophyll and Rubisco is induced. It proceeds faster in atg5 mutants than in the wild type. This suggests that senescence is negatively regulated by autophagy.

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Plants and animals face microbial attacks as a hazard of everyday life, and have evolved innate immunity systems to defend against these threats. The initial step of the immunity signaling pathway is recognition of intra- or extracellular pathogen-derived molecules. Quite remarkably, both plants and animals utilize proteins with similar structures for this purpose. Externally oriented transmembrane-type proteins containing leucine-rich repeat (LRR) domains detect extracellular molecules, whereas cytoplasmic sensors possess nucleotide-binding (NB) and LRR domains. To understand how plants utilize these proteins and how pathogens sabotage the immunity system, we initiated structural analyses of both host immunity-related proteins and pathogen-derived proteins. Recent findings on the structure-based functional analysis of these proteins will be discussed.

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In plants, there are three major classes of immune receptors. The largest class encodes intracellular NB-LRR type R proteins. The two other classes of immune receptors are transmembrane receptor-like kinases (RLKs) and receptor-like proteins (RLPs). Unlike RLKs, RLPs do not have an intracellular kinase domain. We have obtained a series of mutants such as snc2-1D, bir1-1 and mkk1 mkk2 that constitutively activate resistance responses mediated by different classes of immune receptors. snc2-1D contains a gain-of-function mutation in a RLP. bir1-1 activates cell death and immunity mediated by the RLK SOBIR1, whereas mkk1 mkk2 displayed phenotypes similar as bir1-1. We have performed suppressor screens in these mutant backgrounds to identify new regulatory components of plant defense. Recent progress on studies of the suppressor mutants of snc2-1D, bir1-1 and mkk1 mkk2 will be presented.

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Besides roles of cytokinins as a plant growth promoting hormone on specification of embryonic cells, maintenance of meristemetic cells, and vasculature development, cytokinins have been emerged as a major player in plant-microbe interactions during nodule organogenesis and pathogenesis. Microbe-originated cytokinins confer abnormal hypersensitivity of cytokinins to plants augmenting the sink activity of infected region. However, recent discovery shed light on a distinct role of cytokinins in plant immune responses. Plant-borne cytokinins systemically induce resistance against pathogen infection, orchestrated by endogenous cytokinin and salicylic acid signaling. I will discuss how plant- and pathogen-derived cytokinins inversely affect plant defense response and, furthermore, present molecular mechanisms underlying plant-derived cytokinin action in the plant immunity.

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Plants can detect invading pathogens through the recognition of so-called microbe-associated molecular patterns (MAMPs) and initiate various defense responses. It has been believed that the MAMP-triggered defense responses play a major role in the basal resistance against various potential pathogens. On the other hand, it has been becoming clear that some pathogenic/symbiotic microorganisms inhibit or escape from such a plant defense machinery for successful infection.
Chitin, a major component of fungal cell walls, is a representative fungal MAMP and known to trigger defense responses in a wide range of plant species. We recently identified two LysM receptors, CEBiP and CERK1/OsCERK1, involved in the perception of chitin elicitor in rice and Arabidopsis1-3) and are analyzing the signaling mechanism by these receptors. In this symposium, present situation of such studies as well as the pathogenic/symbiotic interactions relating to chitin signaling will be discussed.
1)Kaku et al., PNAS, 103, 11086 (2006); 2)Miya et al., ibid, 104, 19613 (2007); 3)Shimizu et al., Plant J., 64, 204 (2010).

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We have been studying function of a small GTPase OsRac1 and its associated proteins during immune responses in rice. We have identified a number of proteins which directly or indirectly interact with OsRac1. They include two types of immune receptors, chaperones and co-chaperones, signaling proteins, and downstream enzymes for immune responses. A protein complex containing OsRac1 and its associated proteins is called Defensome. We found that proteins involved in PTI and ETI defense pathways are found in Defensome, suggesting that it is a major protein complex involved in rice innate immunity in general. In this talk results on early signaling events in PTI and biochemical analysis of Defensome including its dynamics after signal perception will be presented.

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Auxin regulates a bewildering array of processes during plant growth and development. This complexity is belied by the apparent simplicity of the auxin-signaling pathway. Auxin regulates transcription via the TIR1/AFB-Aux/IAA-ARF pathway. The hormone directly promotes Aux/IAA degradation through the action of SCFTIR1/AFB thus permitting ARF-dependent transcription. In the case of the TIR1/AFB proteins, recent results indicate that different members of the family have distinct activities both with respect to auxin binding and Aux/IAA interaction. We are currently exploring the possibility that these differences contribute to the complexity of auxin response. We are also studying the evolutionary history of auxin signaling. Our results indicate that the mechanism of auxin signaling is conserved in land plants.

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Auxin is an essential regulator for almost every aspect of plant growth and development. Our research focuses on the molecular mechanisms of how auxin regulates plant growth and development, particularly organogenesis. Genetic screens for mutants with altered responses to exogenous auxin have led to major breakthroughs in auxin biology. We approached the key questions in auxin biology from the other end of the auxin spectrum by analyzing partially auxin deficient mutants. We have demonstrated that the YUC family of flavin monooxygeanses catalyzes a rate-limiting step in auxin biosynthesis. We have shown that YUC-mediated auxin biosynthesis is essential for all major developmental events. We conducted large-scale genetic screens for mutants that fail to make flowers in the auxin biosynthetic yuc mutant backgrounds, but not in a wild type background. Analyses of the mutants have led to the discovery of a major new pathway through which auxin regulates plant organogenesis. We further showed that this new pathway for auxin-regulated organogenesis is analogous to the pathway used in blue-light mediated phototropic bending and gravity-mediated directional root growth.

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Auxins are signaling molecules that have critical roles in intercellular communication in plants. Indole-3-acetic acid (IAA) is the main auxin regulating plant growth and development, yet its biosynthetic pathway remains unclear. We have been dissecting the IAA biosynthetic pathway by LC-MS/MS analysis of proposed IAA precursors. We recently found that Arabidopsis has a unique IAA biosynthetic pathway branching from a crucifer-specific secondary metabolism. The YUCCA and indole-3-pyruvate pathways are proposed as common IAA biosynthetic routes in plants. Phenylacetic acid (PAA) has been known as a naturally occurring auxin, but not much is know about physiological role of PAA in plants. To better understand auxin-mediated regulation of plant growth and development, we dissected the biosynthetic and metabolic pathways for PAA. By LC-MS/MS analysis of PAA and its possible metabolites in IAA biosynthesis-related mutants, we found that YUCCA genes contribute to PAA biosynthesis in Arabidopsis. Moreover, we demonstrated that GH3 genes encoding IAA-amido synthases are implicated in PAA metabolism. Possible biosynthetic and metabolic pathways for PAA in plants will be discussed.

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Cellular dedifferentiation, in which adult somatic cells regress to less differentiated states and regain cellular proliferative competence, occurs widely in multicellular organisms. One of the most profound examples of cell dedifferentiation is provoked by wounding. It is well discovered that the ratio of two plant hormones, auxin and cytokinin, is critical to induce dedifferentiation and subsequent redifferentiation of plant explants but molecular mechanisms underlying this control are poorly understood in plants. We discovered a WOUND- INDUCED DEDIFFERENTIATION 1 (WIND1) participates in the regulation of cell dedifferentiation in Arabidopsis. WIND1 is rapidly induced at the wound site and it promotes cell dedifferentiation and subsequent cell proliferation to form mass of cells termed callus. We further demonstrate that ectopic overexpression of WIND1 is sufficient to establish and maintain the dedifferentiated status of somatic cells without exogenous phytohormones. We discuss the possible function of WIND1 for the regulation of dedifferentiation in relation to auxin and cytokinin responses.

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