Monogalactosyldiacylglycerol(MGDG) is a main component of the thylakoid membrane. In Arabidopsis thaliana, there are two types of MGDG synthases, type A and B. Under phosphate-limited growth conditions, amounts of phospholipids in extraplastidic membranes are decreased, whereas galactolipids produced by type B MGDG synthases substitute for phospholipids outside plastids. Type A MGDG synthase, MGD1, contributes to synthesis of the bulk of MGDG under normal growth conditions, but functions of MGD1 under stress conditions remained unknown.
There are microarray data sets indicating increases of MGD1 gene expression in response to high salinity, drought, and wounding stresses. Thus, we analyzed in detail the MGD1 expression levels after exogenous abscisic acid(ABA) treatment and under those three stresses by quantitative RT-PCR. MGD1 expression levels were increased after salt and wounding stresses, but unchanged during ABA treatment and drought stress. In this presentation, we will discuss a role of MGD1 in the stress response mechanisms.

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The Arabidopsis transcriptional factor DREB2A plays important roles in response to water and heat stresses. The DREB2A protein is degraded under non-stressed condition by the ubiquitin-proteasome system, but under the water- and heat-stress conditions, the DREB2A protein accumulates in plant cells and induces the expression of downstream genes. The stability of DREB2A is regulated by the "negative regulatory domain (NRD)" composed of 30 amino acids, and the DREB2A protein lacking of NRD shows the constitutive stability and activity. However, details about the post-translational regulation of DREB2A have mostly yet to be revealed.
In this study, we identified a member of the NF-YC family as a novel interacting protein with DREB2A using yeast two-hybrid screening. Domain analysis revealed that the N-terminal region of DREB2A is sufficient for the protein-protein interaction. The interaction was confirmed in protoplasts prepared from Arabidopsis mesophyll cells by a bimolecular fluorescence complementation (BiFC) system. Now, we are analyzing the expression patterns of DREB2A downstream genes using over-expressing and knockout lines of the NF-YC gene.

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The ATP-binding cassette (ABC) transporters form one of the largest known protein families, and are widespread in bacteria, archaea, and eukaryotes encoded in the largest set of paralogous genes. In Synechocystis sp. PCC6803 genome, over 50 ABC transporter related genes have been found from the genome sequences. The substrates of many other ABC transporters are still unknown. To identify ABC transporters involved in acid tolerance, deletion mutants of other substrate unknown ABC transporter genes were screened for acid stress sensitivities in low pH medium. It has been found that the mutant of slr1045 was more sensitive to acid stress condition than wild type cells. The abundance of expression of slr1045 analyzed under low pH conditions by quantitative real time RT-PCR. Expression of slr1045 in acid stress showed twenty fold higher than that of in the BG-11 medium(pH8.0). When we have examined sensitivities to various stresses (Osmotic, Salt, Pi deficient), the mutant also displayed increased resistant to osmotic stress. Metal contents of wild type cells and the mutant cells were determined using ICP-MS to identity the candidate of the substrate of Slr1045.

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Although the degradation pathway of purine bases constitutes one of the fundamentals of plant nitrogen metabolism, the physiological function of this catabolism remains unclear. Previously, we unmasked that RNAi-mediated knockdown of xanthine dehydrogenase (XDH), the first enzyme in purine catabolism, not only impairs normal growth and development, but also causes the hypersensitivity to drought stress in Arabidopsis [1,2]. Recently, we found that transient accumulation of osomoprotectant proline, a typical example of plant stress response, was diminished in XDH-knockdown plants exposed to drought shock. Coincident with this observation, real-time PCR analysis revealed that stress-induced gene expression of proline metabolism was somehow altered in these plants. The results suggest that there exists the possibility of physiological connection between purine degradation and proline accumulation, which might influence the acclimatization process to stress in Arabidopsis.
[1] Nakagawa et al. (2007) Plant Cell Physiol. 48: 1484-1495
[2] Watanabe et al. (2010) FEBS Lett. 584: 1181-1186.

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Salinity is one of major factors in limiting crop production in the world, and improving plant salt tolerance is very crucial and urgent. Silicon (Si) is the second most abundant element on the surface of the earth, and many studies have shown its beneficial effect for plant growth and development and as well as stress tolerance. To test the function of Si in salt stress, we applied Si on sorghum under hydroponic condition. We found that Si could maintain both shoot and root growth under salt stress significantly. To further explore the mechanism of Si-induced salt tolerance, we investigated the effect of Si on polyamines (PAs) and ACC (1-amino- cyclopropane-1-carboxylate) accumulation, and also the expression of SbSAMDC (S- adenosylmethionine decarboxylase) gene. With Si, PAs level was increased, especially putrescence and spermidine which increased 2-4 times, while ACC content was decreased under salt. Moreover, the expression of SbSAMDC was up-regulated by Si. It suggests that Si-induced improvement of salt tolerance is related with the regulation of PAs and ethylene synthesis, and PAs which are essential for plant growth and survival may play an important role in this process.

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Alkali grass (Puccinellia tenuiflora) is an expected prominent plant to achieve crop production and phytoremediation in saline-alkaline environment because of its high tolerance to saline-alkali soil. However, little is known on its behavior during initial growth process under high pH conditions. In this work, we compared germination rates and initial growth under different pH conditions. The germination rates of alkali grass at pH 5.5, 8 and 10 were 6.7%, 10% and 20.7%, respectively. It was striking that the highest germination rate was obtained when the seeds were sawn at the highest pH condition. To observe initial growth, the length of seedlings transplanted and grown on media prepared at pH 5.5,8 and 10 were measured. The fasted growth was recorded on the medium of pH 10. Since acidification of the media by alkali grass was expected, we traced the pH of the media for 15 days. Alkali grass decreased the pH of the media to 5.73 during the first 5 days. After the fifth day, the pH increased slowly to 6.95. The results suggested that alkali grass expresses a high adaptation ability to alkali soil from germination, and remediates its rhizospare.

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Buckwheat is not only highly tolerant to Al, but also accumulates Al at high concentration in the shoots. Physiological studies have shown that buckwheat secretes oxalate in response to Al and sequesters Al into the vacuoles in the leaves. Many transporters are proposed to be involved in these processes, but none of them has been identified so far. To identify these transporters, we established an EST library of buckwheat roots. Sequence analysis with next generation sequencer and short-gun analysis generated 100,000 reading contigs and as well as the gene expression level. In the present study, we focus on FeIREG2, which showed higher expression in transporter candidate genes. The full length of this gene shows 60% similarity with Arabidopsis IREG2, which is a tonoplast-localized efflux transporter of Fe, Co and Ni. FeIREG2 was expressed in the buckwheat roots and the expression was greatly induced by Al. Transient assay using FeIREG2-GFP fusion showed that this protein is localized to the tonoplast. Based on these evidences, we propose that FeIREG2 might be involved in the sequestration of Al into vacuoles in buckwheat. Further characterization of this gene is being undertaken.

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Here, we report identification of selenium resistance gene by QTL analysis using Arabidopsis RILs obtained from Col-0 (selenite resistance) and Ws-2 (selenite sensitive). Our detailed QTL analysis indicates that selenium resistance gene locates between G3883-1.4 and 1H1L-1.4 markers on chromosome 4. Although 199 genes exist at this region, we could not find any candidate gene by gene annotation. Therefore, we narrow down selenium resistance gene from EST database. That is we selected genes that expression is only identified in Col-0. By using this criterion, we found four genes. PCR analysis showed that a genomic deletion, containing At4G19780 and At4G19790 genes, was found only in Ws-2. Furthermore, replacement of promoter region 650bp upstream of At4G19770 gene was also identified in Ws-2. Thus, we carried out gene expression analysis of these genes. The At4G19780 expressed only in Col-0 shoots, while we could not detect all other gene expression in both accessions.

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Most terrestrial higher plants usually retard their growth and sometimes die under salt stress. Since soil salinity becomes a cause of low agricultural production in many places, it is crucial to analyze a mechanism of salt tolerant responses of plans.
In the present study, we aimed to elucidate the salt tolerant mechanism of the mangrove plant (Bruguiera sexangula) , which can grow under high salt conditions at the cellular and molecular levels.
Using suspension-cultured cells of Bruguiera sexangula, we observed changes in the vacuolar structure and measured intracellular ion concentrations under salt treatment. The vacuolar volume increased, while ion concentrations decreased after long treatments of high salt. To clarify the molecular mechanism of ion transport, genes of the plasma membrane Na⁺/H⁺ antipoter (BsSOS1) and the vacuolar Na⁺/H⁺ antipoters (BsNHX2, BsNHX14) were closed and their expression levels under different salt concentrations were measured with the quantitative real-time PCR.

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Plants have strategies to adapt phosphorus (P) deficiency and aluminum (Al) stress by secreting some low molecular weight metabolites from roots. Organic acid secretion from plant roots into the rhizosphere plays an important role in insoluble P mobilization and Al detoxification. However, there was few study on comprehensive analysis about root exudates involved in each stress and combination of them. Therefore, in this study, we investigated profiles of Arabidopsis root exudates under stressed conditions with the combination of P and Al treatments.
Arabidopsis (Col-0) was cultivated under P sufficient (+P) and deficient (-P) conditions for 19 days on 1/2 MS media. Root exudates were collected in water 0 or 10 µM Al for 3h, and used for the comprehensive analysis of low-molecular weight organic compounds by GC/MS. Many amino acids and sugars were increased by both -P and Al stress. Among organic acids, malic, fumaric and succinic acids were increased by both -P and Al stress. On the other hand, citric and lactic acids increased by only -P. These results suggested that the profiles of root exudates were different but related between -P and Al stress.

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We have reported that a subunit of 19S proteasome, RPT5a, is required for high-boron (B) tolerance in roots of A. thaliana. 19S is a subcomplex of 26S proteasome regulating many cellular processes via active proteolysis. 19S mediates recognition and unfolding of targets. We further established that 19S subunits RPN2a, RPN8a and RPT2a were also required for B tolerance, but RPN2b, RPN8b, RPT2b and RPT5b were not. Considering specific requirement for high-B tolerance among 19S subunits, it is possible that subunit-specific substrates degraded by proteasome in response to high-B are present. In this study, we attempted to screen that kind of substrate using rpt5a mutant. Proteomics of affinity-purified poly-ubiquitinated proteins using iTRAQ and LC-MS/MS found that 28 proteins were induced and were highly accumulated in rpt5a-6 than in the wild type by high-B treatment. 17 of 28 proteins have at least 1 motif associated with the degradation by proteasome, suggesting that our method is effective for substrates screening. We also isolated at least 2 high-B insensitive revertants of rpt5a-6 which are expected to be defective in high-B dependent substrates recognized through RPT5a.

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Phosphorus in soil is often present in the forms of which plants are not easily accessible, such as organic phosphate and sparingly soluble inorganic phosphate. Plants exposed to low P alter the root architecture and mobilize these phosphates by secreting acid phosphatases and organic acids.
Arabidopsis thaliana has a worldwide distribution. Each accession should have adapted to each habitat with different low P responses and strategies to acquire phosphorus. However, low P responses of accessions grown on soil are not well known. In this study, P acquisition ability of each accession grown on soil was compared. In addition, accessions were grown on +P and -P medium to characterize their strategies against low P. Accessions were grown on soil under 3 different P conditions and shoot dry mass and shoot phosphate contents were measured. Each accession exhibited different characteristics. The shoot growth and P content increased under more P sufficient condition in most of the accessions but some exhibited a reduction in both growth and P content under the 300 mg P/kg-soil condition. In order to better characterize each low P response, 32 accessions were categorized into groups.

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Synechocystis sp. strain PCC6803 can grow under the wide-ranging environmental stress conditions. Over the course of our study on membrane transport system involved in adaptive response to salinity stress, we found that Synechocystis produced biofilm (exopollysaccharide) at the high salt environment. With increase of amount of biofilm formed around cells, growth rate of the cells decreased. It is known that polyamines play an important role in the cell division and protection against salt stress. To examine the relationship between polyamine content and the cell growth rate, we measured polyamins content. It is found that salinity stress deceased the content of spermidine. Therefore we generated the mutants at putative arginine decarboxylase which is one of the key enzymes in polyamine biosynthesis process in order to measure the amount of biofilm by environmental changes. The mutant exhibited a high rate of biofilm compared with the wild type. To further analyze the regulatory mechanism on the biofilm formation, we are in progress on gene screening of salt stress sensor proteins.

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Wood is the largest sustainable biomass on the land. Thus it has been expected to increase the yield of woody biomass by adding environmental stress tolerance with transgenic technology. However, there are only limited reports on production of transgenic trees with the environmental stress tolerance and on the strategy to evaluate their abilities for stress tolerance. Here, we generated and analyzed transgenic poplar (Populus tremula x tremuloides) overexpressing Arabidopsis GolS2 and SRK2C genes which are known as salt- and drought-stress tolerance genes. First, five independent lines each of transgenic plants in which the Arabidopsis genes were highly expressed were selected by using RT-PCR. Second, we examined the method of propagation of transgenic plants. And then, we investigated conditions of salt stress effective for evaluation: (1) the rooting rate after transfer of shoot explants onto solid medium containing 30 mM NaCl and (2) the observation of growth of soil-grown plants in 100 - 150 mM NaCl condition. In addition, we would like to report on the results of ongoing microarray analysis of the transgenic poplar plants.

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Magnesium (Mg) is an essential element for plants and required for a number of enzyme reactions in plants. We obtained six lines of Arabidopsis mutants from EMS mutagenized M2 population with altered Mg response to understand response mechanism of plant against Mg. They showed very short roots in high-Mg condition (14~20 mM) compared with the normal Mg condition. The phenotypes of mutants was indistinguishable between the media containing MgCl₂ and MgSO₄, suggesting that the phenotypes of mutants depend on Mg concentration not anions. These lines also showed a very similar phenotype in the low-Calcium condition (0.15~0.2 mM). Mg and Ca concentration in above ground tissue of mutants were measured. Concentration of Mg and Ca are lower than WT. We mapped the cause gene of this phenotype. In the result, both 2 lines have mutation on chromosome No.1. In the future, we will carry out gene mapping in detail together with physiological and molecular biological studies.

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Selenium (Se) is an essential micronutrient of many organisms including a human but over intake of Se through foods is toxic. Se-pollution in soils leads accumulation of excess Se in foods, causing toxicity. One of most effective strategies to remove Se from Se-contaminated soil is phytoremediation. In this study, we examined Se-tolerance, Se-accumulation, and expression of Se metabolism-related genes in sulfate transporter SULTR1;2 knockout mutant, sultr1;2, which is tolerant to selenate.
The sultr1;2 was more tolerance to selenate than WS. When these plants were treated with selenate, the amount of total Se in sultr1;2 was the same as that of WS and the amount of organic Se in sultr1;2 was higher than that of WS. Moreover, the expression of Se metabolism-related genes in sultr1;2 were higher than that in WS. These results suggest that sultr1;2 obtains Se-tolerance due to release of Se into the atmosphere via conversion from toxic selenate into non-toxic dimethyl selenide not due to reduction of Se uptake.

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Salsola komarovii Iljin is an amaranthaceous C4 halophyte (NADP-ME type) that grows on coastal sand ground in Japan. Glycine betaine is a compatible solute, which functions in adjusting osmotic balance and protecting enzymes, and synthesized in response to salt stress in S. komarovii. In this study, to examine the physiological role of glycine betaine in the salt tolerance of S. komarovii, we have investigated the tissue localization of glycine betaine synthetic enzymes in the plants grown under control and saline conditions.
The last step of glycine betaine biosynthesis is catalyzed by betaine aldehyde dehydrogenase (BADH). Western blot analysis of BADH protein showed that the expression level of BADH protein was increased by salt treatment in the leaves. In the immunolocalization experiment of BADH protein in the leaves, the signal of BADH protein was detected in vascular tissues under control condition, and in vascular and bundle sheath tissues under a saline condition. These results indicate that production and/or accumulation of glycine betaine in vasucular and bundle sheath tissues may be important for the salt tolerance of S. komarovii.

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We performed comprehensive gene expression profiling of rice root using Laser Capture Microdissection and microarray approach. Root samples were obtained from japonica rice grown in nutrient solution for 10 days. The crown roots were divided into eight sections from the root tip to the basal zone corresponding to various stages of root development. Two individual zones were further divided into three sections corresponding to distinct root tissues. RNAs were extracted from each sample and used for hybridization using the rice microarray (Agilent). Clustering analysis showed preferential gene expression in each stage and tissue. Many DNA replication and translation related genes that maybe involved in cell division and differentiation were identified in the cell division zone. Transporter genes associated with transport and uptake of water and nutrients were observed in the root hair zone. Moreover, highly expressed genes were identified in tissues involved in the formation of root hair and lateral root. A comprehensive gene expression profile of root will provide valuable clue in understanding the gene regulatory networks of root development and morphogenesis in rice.

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The anther tapetum, which lies the most inner layer of anther locule, is believed to play important roles in pollen development and fertility, but the function of tapetum remains unclear. In addition, tapetum is known to undergo programmed cell death (PCD) and is degraded during pollen maturation, although it is unclear what triggers PCD.
To analyze the functions of the tapetum, we produced transcriptomes of Arabidopsis anthers using laser capture microdissection (LCM) strategy. The anther tapetum, microspores and anther wall cells from Arabidopsis buds were separately isolated using LCM, and then comparative cDNA microarray were performed. Some examples of genes which show tapetum-specific expression will be shown.

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Protein prenylation is one of the post-translational lipid modifications, which affects protein-membrane interactions, protein-protein interactions, and activities of proteins. Protein prenyltransferases (PPTases) catalyze the transfer of a prenyl moiety derived from farnesyl diphosphate or geranylgeranyl diphosphate to the cysteine residue in recognition motifs at the C-terminus of target proteins. In higher plants, some PPTase mutants show phenotypes including hypersensitivity to ABA and NAA, enlarged meristems, and increase of floral organs, indicating important roles in stress responses and developmental regulations. Despite the importance of this modification, the identification of most putative target proteins is solely based on their C-terminal motifs identified to be prenylated in studies of mammalian PPTases. Therefore, not much is known about actual target proteins and the significance of prenylation in protein function regulation in higher plants.
As a first step to comprehensively identify prenylated proteins, we report the results from a high-throughput proteomics study of prenylated proteins from Arabidopsis suspension culture cells, labeled with azide-substrates.

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Well-coordinated flower development is crucial for successful fertility and prosperity of their species. Lipid is an emerging multifunctional component which plays pivotal roles in various aspects of plant development. However, the involvement of lipids in flower development is yet to be characterized comprehensively. By exploiting the novel system to synchronize flower development in Arabidopsis, a global stage-specific profiling of polar glycerolipids and phosphoinositides was carried out together with transcriptomic analyses on relevant lipid biosynthetic genes. The result showed that each lipid class showed dynamic changes, and gene expression pattern was clearly categorized into several groups. On the basis of global analyses, functional link between glycerolipids and flower development will be presented.

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In recent years, metabolomics become powerful for identification of novel gene functions by integrating a transcriptome approach. In the present study, we chose silent phenotypes among 50 lines of Arabidopsis EMS mutants that have metabolic changes confirmed by targeted analyses. Silent phenotypes have mutations in their genes but they show little change in their appearances; however, these mutants may show metabolomic changes. Morphological observations for EMS mutants and GC-TOF/MS-based metabolite profiling enabled us to perform silent phenotype selection. The Arabidopsis wild-type (Col-0) and the 50 mutant lines were grown on MS medium. Of these, 64 percent of the mutants were chosen as silent phenotypes because of their morphology. The selected lines were extracted and then analyzed using GC-TOF/MS. Multivariate analysis revealed that 19 percent of the silent phenotypes showed metabolomic changes. Among these silent phenotypes, we selected the mutant called pad2-1, which has reported as the glutathione-deficient mutant. Now, the detailed metabolite profiling of the mutant samples under the oxidization stress condition are conducted using three MS-based metabolomics pipelines.

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Much amount of chemical fertilizers and pesticides have been widely used to improve crop production. However, consumers have been interested in safety and security of food. Organic food is encouraged to be produced, especially in the aspect of environmental protection. Organic foods are believed to be superior than conventionally cultivated food in taste, nutrition and flavor, moreover for human health. There is no scientific data about the difference in metabolites between organic and conventional food. Mini tomatoes were cultivated in either organic or chemical fertilizers with or without pesticides. Fruits were harvested and soluble extracts were analyzed by proton NMR. The spectrum were analyzed by principle component analysis (PCA). Proton NMR is very useful for metabolite fingerprinting or profiling, because it can detect metabolites without isolation procedures. GC and HPLC are necessary to isolate metabolites before detection. PCA shows that there are some difference between organic and conventional cultivars. Loading values shows that sugars seem to contribute these differences. Two dimensional 13C-HSQC are required to identify contributed metabolites.

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As the world population continues to grow geometrically, great pressure is being placed on arable land, water and biological resources to provide an adequate supply of food while maintaining the integrity of our ecosystem. However, it is hard to expand the lands suitable for plant raising on the earth. Instead, a number of closed-type plant factories have been constructed to provide steady production of vegetables. To improve food quality by irradiating different LED light wavelength, we investigated changes of visual phenotypes and metabolite profiles of sunny lettuce grown under different LED light conditions. Radiation of red LED light wavelength drastically altered visual phenotypes of sunny lettuce, while samples grown under other light conditions did not change phenotypes in morphology. Multivariate statistical analysis using metabolite profiles of these samples demonstrated that the groups grown under red LED wavelength showed good separation from the others. We investigated significantly changed metabolites by red LED irradiation, there were significant increase in the revels of sugars and phospholipids, while decrease in the levels of amino acids and organic acids.

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The world population is expected to increase up to nine billion by the year 2050. To secure foods for maintenance of such a large population, it is required to increase yield of crops and vegetables per cultivated acreage. However, it is hard to expand the lands suitable for plant raising on the earth. Instead, a number of closed-type plant factories have been constructed to provide steady production of vegetables. Particularly, controlling light intensity and wavelength can affect plant growth and yield. Therefore, we chose LED light to grow tomato plants because it is easy to change both light conditions by this light. When we grew tomato plants (cv. Reiyo) under the red LED light condition with two different light intensities (weak and strong), we could obtain bigger tomato fruits under the strong light condition than that under the weak light condition. Next, we conducted systems biology approach by integration of metabolite and transcript profiling of leaf and fruit samples harvested from the two light conditions. We applied next generation sequencing as well as microarray analysis for comprehensive transcript profiling. The up-to-date results will be presented.

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We have collected Japanese beech trees (Fagus crenata) superior in growth and stem straightness in Tohoku area by graft multiplication. In this process, difficulties in providing enough count of rootstocks every year raised from its specific characteristic called masting (fruit yield oscillate each year, and abundant harvest year comes only once 5 to 7 years). Most of the unpreserved lines are localized so apart to access in winter due to deep snow. In addition, branches provided for grafting must be collected after defoliation, its available period is limited to mid November before snowfall. This problem also awaken technical difficulty in preserving branches for a long term, at least 4 months till spring. Heating rootstocks in greenhouse can shorten its period but it also spends costs to appoint this method as standard.
To overcome the problems, we studied tissue culture of winter buds available from September. 1/2 WPM medium with IBA and BAP was suitable for multiplicity of bud formation and subcultured every month by cutting on fresh medium and maintained for more than three years. Higher concentration of IBA induced fine roots and succeeded in growing to plantlet.

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Shoot primordia of Micro-Tom, a model cultivar of tomato (Solanum lycopersicum L.), were induced from apical domes of shoot tips in liquid MS medium supplemented with 20 or 30 g/l sucrose, 0, 0.02, 0.2, 2.0, or 4.0 mg/l alpha-naphthaleneacetic acid (NAA), and 0, 0.02, 0.2, 2.0, or 4.0 mg/l 6-benzylaminopurine (BAP) at pH 5.8 on a rotary shaker at 2 rpm under 10,000 lux illuminated continuously. Multiplication of these shoot primordia was most effective in a liquid MS medium supplemented with 20 g/l sucrose, 0.2 mg/l NAA, and 2.0 mg/l BAP. The shoot regeneration from the shoot primordia was observed in static culture on MS medium supplemented with 30 g/l sucrose, 3.0 g/l Gelrite, and 1.0 mg/l zeatin under illumination at 4,500 lux for 16 h and dark for 8h. Their roots were formed from the regenerated shoots in static culture on 1/2 MS medium supplemented with 15 g/l sucrose, 3.0 g/l Gelrite under illumination at 4,500 lux for 16 h and dark for 8 h.

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To the global environmental issues, application of transgenic plants, such as enhanced abiotic-stress tolerant plants, would be a technological solution. We are developing the transgenic Eucalyptus globulus plants containing the choline oxidase (codA) gene derived from a salt-tolerant bacterium, Arthrobacter globiformis, and have confirmed their salt-tolerant trait in the contained and semi-contained experimental conditions. Since March 2008, we started the non-contained trial cultivation in the isolated field, and performed long term follow up of growth and environmental biosafety assessment of risk to precinct vegetation and the microbial community. For precinct vegetation, two kinds of allelopathic bioassay, e.g. sandwich method and Sukikomi method, are performed. For the microbial community, total number, varieties and activity of microbe were assayed by the plate cultivation method and the soil enzymatic activity measurement. These assays are performed every three months and will perform for five years. Then, we will show data from past two years and discuss impacts of cultivation of the transgenic Eucalyptus plants to the effect on the diversity of the ecosystem.

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Mutant plants are useful tools, not only as parents of new cultivars, but also as materials for clarifying physiological mechanisms. Tomato (Solanum lycopersicum) has been an excellent model system for analysis of plant mechanism, which cannot easily be studied in Arabidopsis or rice. To obtain information on these mechanisms, we induced mutations in the tomato cultivar 'Micro-Tom' by irradiation with accelerated heavy ions. It is reported that irradiation with accelerated heavy ions is more effective per dose in creating mutations in plants than irradiation with other sources and is suggested that mutagenesis by accelerated heavy ions could be uniquely used for both forward and reverse genetics in plants.
In attempt to find the conditions for high efficient mutagenesis, we start to investigate the dose-, the ion species- and the linear energy transfer (LET)-dependence of survival rate and the appearance of mutants.
This work carried out under the NIVTS Priority Research Program and Strategic Promotion Program for Basic Nuclear Research by Japanese Ministry of Education, Culture, Sports, Science and Technology.

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Plants have evolved unique systems for plant body formation. Plant vascular development is a paradigm of plant body formation. This process includes the regulation of stem cell fates, differentiation of various types of vascular cells, and cell-cell communication between vascular cells. To understand vascular development, we have established a few culture systems including Zinnia xylogenic cell culture in which mesophyll cells differentiate into tracheary elements (TEs) and an Arabidopsis xylogenic culture. Using these cultures and microarray systems, we have revealed gene expression profiles associated with vascular development. Based on these analyses, we discovered two novel master regulators of TE differentiation, VND6 and VND7. To understand cell-cell communication among vascular cells to form a well-organized tissue, we searched extracellular factors that affect vascular development, using the Zinnia culture. As a result, we discovered novel factors; TE differentiation-promoting and inhibiting factors, xylogen and TDIF, respectively. The analysis of in vivo function of these novel factors in Arabidopsis casted a new light on understanding of vascular development.

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The shoot apical meristem (SAM) is the fundamental structure that is located at the growing tip and gives rise to all aerial parts of plant tissues and organs, such as leaves, stems and flowers. In Arabidopsis thaliana, the CLAVATA3 (CLV3) pathway regulates the stem cell pool in the SAM, in which a small peptide ligand derived from CLV3 is perceived by two major receptor complexes, CLV1 and CLV2-CORYNE (CRN)/SUPPRESSOR OF LLP1 2 (SOL2), to restrict WUSCHEL (WUS) expression. We identified that the SOL2 function as a CLV3 peptide perception, and the SOL2 function as a heterodimer with CLV2. We further used the functional, synthetic CLV3 peptide (MCLV3) to isolate CLV3-insensitive mutants and revealed that a receptor-like kinase, RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2), is the third receptor of CLV3 signal. We also identified that the CLV3 ligand induces CLV1 phosphorylation and MAPK activation. Here I will introduce about our recent work and review about CLV3 system on meristem maintenance system in Arabidopsis.

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Host legumes control root nodule numbers by sensing external and internal cues. A major external cue is soil nitrate, whereas a feedback regulatory system in which earlier formed nodules suppress further nodulation through shoot-root communication is an important internal cue. The latter is known as autoregulation of nodulation (AON), and is believed to consist of two long-distance signals: a root-derived signal that is generated in infected roots and transmitted to the shoot; and a shoot-derived signal that systemically inhibits nodulation. In Lotus japonicus, the leucine-rich repeat receptor-like kinase, HAR1 mediates AON and nitrate inhibition of nodulation, and is hypothesized to recognize the root-derived signal. Here we identify L. japonicus CLE-RS1 and LjCLE-RS2 as strong candidates for the root-derived signal. Overexpression of LjCLE-RS1 and -RS2 in roots inhibits nodulation systemically and in a HAR1-dependent manner. Moreover, LjCLE-RS2 expression is strongly up-regulated in roots by nitrate addition. Based on these findings, we propose a simple model for AON and nitrate inhibition of nodulation mediated by LjCLE-RS1, -RS2 peptides and the HAR1 receptor-like kinase.

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In the UK, information has been collected about the harmful element Cd in soils and crops. This is useful for the targeting of control measures, and as input data into legislation on Cd in soil and foodstuffs.
Most rocks and soils contain low concentrations of Cd, apart from those developed on black shales and those associated with mineralised deposits. Anthropogenic sources of Cd can be significant, including the use of phosphatic fertilisers, and atmospheric deposition. Until recently, deposition of Cd from the atmosphere was estimated to be 3 g /ha/yr. Other important local sources of cadmium include the application of sewage sludge, and metalliferous mining and smelting of zinc and sulphide ores.
The effect of these exposures, and interactions with Zn on the Cd concentration in wheat will be discussed and how to decrease Cd concentrations, thus enabling crops to be grown that conform with the Food Regulations. Models for crop uptake and phytoremediation have been constructed. Other mitigation measures, including adding materials to soil which bind Cd to remediate strongly polluted land and the feasibility of phytoremediation will be discussed.

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Human beings are normally exposed to only a very small amount of environmental cadmium (Cd) in food,leading to gradual Cd accumulation in their bodies,especially in the liver and kidneys, with aging due to the long biological half-life of Cd. The sentinel sign of adverse effect is renal tubular dysfunction, which is characterized by low-molecular-weight proteinuria, and can occur in concert with anemia or bone mineral loss if the exposure level is high, known as Itai-itai disease; it is found in a heavily Cd-polluted area. The accumulation of Cd in the kidneys to some extent without apparent toxic effect is because of the formation of metallothionein, a metal-protein complex. However, when the levels of Cd exceed the critical concentration, the release of free Cd from the complex causes dysfunction of proximal tubular reabsorption of low molecular weight proteins. In general, current exposure levels of Cd among Japanese is sufficiently lower than current provisional tolerable monthly intake (PTMI). But there exist concerns on adverse health effects especially among farmers in Cd-high farmland, because there are small margin of exposure.

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Cadmium (Cd) causes Itai-itai disease and rice is a major source of Cd intake. Therefore, reducing Cd accumulation in rice grain is an important issue for human health. We have identified three QTLs responsible for Cd accumulation in rice by using natural variation in rice Cd accumulation. Among them, one QTL gene on the chromosome 7 has been cloned and functionally characterized (Ueno et al., 2010). The protein encoded by this gene OsHMA3 belongs to P-type ATPase and is localized to the tonoplast of all root cells. There were no differences in the expression level and tissue- and cellular localization of this protein between a high and a low Cd-accumulating cultivar. However, the proteins from the low Cd-accumulating cultivar showed transport activity for Cd, whereas that from high Cd-accumulating cultivar did not. When the functional OsHMA3 was over-expressed in rice, the Cd concentration was remarkably reduced in the grain of rice even if grown on Cd-contaminated soil. These results indicate that functional OsHMA3 reduces translocation of Cd from the roots to the shoots by sequestering Cd taken up into the root vacuoles. Ueno et al., PNAS, 107, 16500-16505(2010).

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Cadmium (Cd) in rice grains is a major source of human Cd intake. We studied the role of OsLCT1 in Cd accumulation in grains. OsLCT1, is predicted to encode the only rice homolog of the low-affinity cation transporter 1 (TaLCT1) of wheat. TaLCT1 transports cations, including Cd in yeast. The cDNA of OsLCT1 was cloned from Nipponbare and was deduced to encode 511 amino acids with 11 transmembrane domains. When expressed in yeast, OsLCT1 reduced Cd accumulation, suggesting that OsLCT1 is a Cd transporter. OsLCT1-GFP was localized primarily to plasma membrane. We generated RNAi-mediated OsLCT1 knockdown rice plants and found that Cd concentration in seeds of transgenic plants is reduced by 30-50%. Interestingly, Cd concentration in xylem sap and leaf blades was not altered, suggesting possible involvement of OsLCT1 in Cd transport into grains.

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Previously we reported that ferrous Fe transporters in rice, OsIRT1 and OsIRT2, take up Cd (Nakanishi et al., 2006). Furthermore, overexpression of OsIRT1 increased Cd accumulation in rice (Lee and An, 2009). We investigated the role of OsNRAMP1, an Fe transporter, in Cd uptake and transport in rice.
OsNRAMP1::GFP fusion proteins localised to the plasma membrane in onion epidermal cells. The growth of yeast expressing OsNRAMP1 was impaired in the presence of Cd compared to yeast transformed with empty vector. Moreover, the Cd content of the OsNRAMP1-expressing yeast exceeded that of the vector control. Overexpression of OsNRAMP1 in rice increased Cd accumulation in the leaves.
The expression of OsNRAMP1 in the roots was higher in a high Cd-accumulating cultivar (Habataki) compared to a low Cd-accumulating cultivar (Sasanishiki) irrespective of the presence of Cd, and the amino acid sequence of OsNRAMP1 showed 100% identity between Sasanishiki and Habataki. These results suggest that OsNRAMP1 participates in cellular Cd uptake and Cd transport within rice, and the higher expression of OsNRAMP1 in the roots could lead to the increase of Cd accumulation in the shoots.

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Photosynthetic proteins for antenna, photosystems and electron transport mostly occur as protein complex or supercomplex in the thylakoid membrane to support coordinated flow of energy and reducing equivalents. The organization of these complexes can be probed by various techniques and the blue-native polyacrylamide gel electrophoresis (BN-PAGE) is one of the powerful tools of high resolution. We will review the general principle and behavior of the photosystem II (PSII), photosystem I (PSI), and light-harvesting antenna complexes in BN-PAGE. The PSII core complex of cyanobacteria and plants can be separated as a monomer and a dimer and their ratio often depends on the condition of solubilization: stronger treatment with detergent tends to yield higher dimer-to-monomer ratio. By contrast, organization of PSI is diversified in evolution. Generally, a trimer and a monomer are popular in cyanobacteria, while a monomeric PSI-LHCI supercomplex is predominant in green plants and chlorophyll c-containing algae. However, we recently demonstrated "tetrameric" organization of PSI in heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 and a glaucocystophyte Cyanophora paradoxa.

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In higher plants, the chloroplast NDH complex interacts with PSI to form the unique NDH-PSI supercomplex. Although it is well known that NDH mediates PSI cyclic and chlororespiratory electron transport, the nature of its electron input module is a long-standing mystery. To reveal the structure of NDH, we separated the NDH-PSI supercomplex by using BN-PAGE and analyzed it by LC/MS/MS. In addition to the NDH and PSI subunits, many proteins with unknown function were identified. Lhca5 and Lhca6 are required for the NDH-PSI formation. We also identified many proteins which are essential for the accumulation of NDH. Furthermore, we discovered three assembly intermediates of one NDH subcomplex (Subcomplex A) present in the chloroplast stroma. These complexes were immunoaffinity purified and analyzed by LC/MS/MS. On the basis of the results from proteomic and genetic analyses, we propose a working model of the NDH subcomplex A assembly.

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Biological processes are not only performed by the cooperation of individual proteins, but also performed by various protein complexes. Notably, the chloroplast is the place where photosynthesis occurs and also the place where many other metabolic pathways are inter-connected via the metabolic products. Therefore, it is an intriguing challenge to detect the uncharacterized protein complexes in the chloroplast for understanding the regulation of photosynthesis and the metabolic flows. However, conventional approaches for comprehensive detection of the protein complexes are often time-consuming, labor-intensive process.

Here, I have developed the rapid method by combination of BN-PAGE, LC-MS/MS, and bioinformatics. In the preliminary experiment using Arabidopsis thylakoid membranes, we successfully detected the known protein complexes including the core complexes and light-harvesting complexes of photosystems I and II, cytochrome b₆/f, NAD(P)H dehydrogenase complex, and ATP synthase. I would like to talk about the present status and future prospects of this development for investigating the protein-protein interaction networks.

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Many recent high throughput proteomic investigations have focused either on the whole chloroplast or on independent suplastidial fractions. However, these previous studies raised the question of the accurate localization of lots of proteins that were identified in different suplastidial compartments. We recently went a step further into the knowledge of the accurate localization of these proteins within the chloroplast. To achieve this goal, we first obtained highly pure subfractions of envelope, stroma and thylakoids, and evaluated their cross-contaminations using biochemical methods. We then performed a comprehensive analysis of the chloroplast proteome starting from the whole chloroplast and its three main compartments. Then, we assessed the partitioning of each identified protein in the three above-cited compartments using a semi-quantitative proteomic approach (Ferro et al., MCP 2010; AT_Chloro database: http://www.grenoble.prabi.fr/at_chloro/).). An in depth investigation of the proteins identified within the purified envelope fraction allowed new insights over this subplastidial compartment to be revealed (Joyard et al., Mol Plant 2009 & Prog Lipid Res 2010).

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RNA-directed DNA methylation (RdDM) represents one of the most extensively studied RNA silencing pathways. RdDM contributes substantially to epigenetic regulation of the genome in plants. RdDM in plants involves a specific process in which 24 nt 'heterochromatic' small interfering RNAs (siRNAs) produced by the RNA silencing pathway induce de novo methylation of cytosines in all sequence contexts (CpG, CpHpG and CpHpH, where H is A, T or C) based on RNA-DNA sequence complementarity.
In plants, the RdDM pathway is thought to be required for establishing and/or maintaining the silened status of transposons or repeats in euchromatic regions, which may be too small to be packaged into constitutive heterochromatin.
Presented in this talk is the current model for the molecular mechanism of RNA-directed DNA methylation.

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Transcriptionally silent genes are associated with heterochromatic epigenetic modifications such as DNA methylation and histone H3 Lysine9 methylation. Although removal of such modifications is essential for gene de-repression, the molecular mechanisms underlying the removal of the heterochromatic modifications are still poorly understood. Using model plant Arabidopsis, we are studying regulation mechanisms of heterochromatic modifications, and trying to identify novel factors involved in this process. By taking a forward genetic approach, we demonstrated that a jumonji-domain protein IBM1 is required for negative regulation of DNA methylation and histone H3 Lysine9 methylation at transcribed genic regions. ibm1 plants exhibit various developmental abnormalities, suggesting that the H3K9 demethylation pathway is essential for regulation of gene activities during development. I will discuss the pathway and factors regulating heterochromatic modifications in genic regions.

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Recent studies suggest that the DNA methylation status of a number of genes is dynamically regulated by methylation and demethylation. In Arabidopsis, active DNA demethylation is mediated by the ROS1 (repressor of silencing 1) subfamily of 5-methylcytosine DNA glycosylases through a base excision repair pathway. These demethylases have critical roles in erasing DNA methylation and preventing TGS of target genes. However, it is not known how the demethylases are targeted to specific sequences. Here we report the identification of ROS3, an essential regulator of DNA demethylation that contains an RNA recognition motif. Analysis of ros3 mutants and ros1 ros3 double mutants suggests that ROS3 acts in the same genetic pathway as ROS1 to prevent DNA hypermethylation and TGS. Gel mobility shift assays and analysis of ROS3 immunoprecipitate from plant extracts shows that ROS3 binds to small RNAs in vitro and in vivo. Immunostaining shows that ROS3 and ROS1 proteins co-localize in discrete foci dispersed throughout the nucleus. These results demonstrate a critical role for ROS3 in preventing DNA hypermethylation and suggest that DNA demethylation by ROS1 may be guided by RNAs bound to ROS3.

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Arabidopsis mom1 mutation releases TGS (Transcriptional Gene Silencing) of hypermethylated genes residing in heterochromatin without major changes of their DNA methylation¹. This suggests that TGS mediated by MOM1 acts downstream or independently of DNA methylation. Recent studies revealed that at some loci MOM1 acts downstream of the RNA-directed DNA methylation^2,3. MOM1 protein contains conserved plant-specific motif, named CMM2 (Conserved MOM Motif 2), which is necessary for MOM1 TGS activity⁴. The molecular function of CMM2 is still unknown. Here we present structural and functional analyses of CMM2, which revealed that CMM2 needs to form multimers to perform its TGS function. In addition, we performed a forward genetic screen for mom1 suppressors. So far 12 suppressor mutants have been isolated and the genomes of some of them were re-sequenced to identify the causative mutations. Current status of the suppressors characterization will be presented.

1. Nishimura and Paszkowski, (2006) BBA-Gene struct. Expr. 1769, 393-8.
2. Yokthongwattana et al., (2010) EMBO J 29, 340-51.
3. Numa et al., (2010) EMBO J 29, 352-62.
4. Caikovski et al., (2008). PLoS Genet 4, e1000165.

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Epigenetic regulations, including histone modifications, are crucial for the maintenance of cell states. Reprogramming from differentiated cells to pluripotent stem cells is a cell state change. In this study, the state change from a leaf cell to a protonemal apical cell of Physcomitrella was used as a model system for reprogramming. Epigenome studies for histone modifications were conducted by chromatin immunoprecipitation followed by sequencing (ChIP-seq) using a next-generation sequencer. Among several types of histone modification, the trimethylation of Lys4 (K4) and Lys27 (K27) of histone 3 (H3) were selected for ChIP-seq analysis representing active and inactive chromatin marks, respectively. Nucleosome occupancy was also surveyed with the pan-histone 3 antibody. So far, a set of ChIP-seq data for leafy gametophores (differentiated sample), protoplast derived from the protonema-apical cells (stem cell sample) and dissected gametophores (reprogramming samples) have been obtained. Integrated analysis of ChIP-seq and gene expression will produce useful information for epigenetics-based regulation of the reprogramming process.

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The acetylation and methylation status on N-terminal regions of histones are dynamically changed and corresponded to gene regulation in eukaryotes. The quantitative and qualitative alterations of these histone modifications contribute to the regulation of structural changes in chromatin, and are associated with the status of gene activity. In plants, histone modification plays an important role in the regulation of gene expression in the process of development and vernalization. It is well known that gene expression is dynamically regulated genome-wide in response to environmental stresses. However, the detailed mechanisms of chromatin regulation in environmental stress responses have not well understood yet. We have studied the alterations of histone modifications under dehydration and rehydration conditions, and the function of histone modifying enzymes under abiotic stresses in Arabidopsis. Here, we will discuss the chromatin regulation mechanism involving histone modifications in environmental stress responses.

References
1) Kim et al., Plant & Cell Physiology (2008) 49, 1580-1588.
2) Kim et al., Plant,Cell & Environment (2010) 33, 604-611. Review.

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Plant cell walls are highly sophisticated fiber composite structures composed mostly of polysaccharides and glycoproteins. In the majority of seed plant species, the dominant structural feature of the primary cell wall is the cellulose/xyloglucan networks which is embedded in an amorphous matrix of pectins and glycoproteins. However, various kinds of cell walls have evolved to fulfill a wide range of biological roles during the diversification of land plants. For example, the primary cell walls from commelinoid monocotyledons, which include cereals, have relatively little xyloglucan, and the predominant glycan that cross-links the cellulose microfibrils is instead (1,3)(1,4)-beta-D-glucan (MLG) and glucuronoarabinoxylan (GAX). Moreover, the cell wall contains less pectin and higher amounts of phenylpropanoids.
Today, cereals represent potential feedstocks for biofuel production. In rice, MLGs, GAXs and xyloglucans are considered to be essential roles in not only regulation of plant growth that directly affect biomass production but also accessibility of cellulase to the cellulose microfibrils. For this reason, we specifically aimed at elucidating roles of these polysaccharides.

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Of all the renewable resources, wood or lignocellulosic biomass is the most abundant. In addition, it is carbon-neutral and its use does not compete with that of food. For these reasons, increasing attention is being paid to the sustainable production and use of lignocellulosics. However, lignocellulosics are structural materials that confer mechanical strength to plant bodies, and therefore, they are inherently recalcitrant to biochemical conversion. To overcome this recalcitrance, there has been substantial research and development of metabolic engineering to reduce lignin content to levels that are effective for enzymatic saccharification. As well as enzymatic saccharification coupled with fermentation, there are a number of other biomass conversion methods. Hence, it is now important to metabolically engineer lignocellulosics for optimal conversion using these other methods. During the last 20 years, significant advances have been made in understanding lignin biosynthesis, and it is now possible to control the structures and contents of lignins in various plant species. In this seminar, the current status and future prospects of lignin metabolic engineering will be discussed.

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Due to shortage of fossil fuel and response to global warming, biofuel production from plant materials has become one of the most pressing challenges of our times. At present biofuel is produced from cereals and sugar. But these are competitive with food and international cereal price increased dramatically. To dissolve these problems biofuel production from agricultural residues has been paid attention.
Plant cell walls are the most abundant and renewable organic compounds on Earth. Cell walls have been used for wood, pulp, fiber, and industrial materials. Plant cell walls consist of cellulose, hemicellulose and lignin. In grasses a major hemicellulose is arabinoxylan, which cross-links cellulose and lignin to make up of tough structure. Due to its strong structure, it is hard to hydrolyze them with cellulase. Therefore, pretreatments using acid or alkali are required. But these treatments are costly and environmental loaded. Production of genetically modified crop is one of the challenging issues. We will introduce structure, biosynthesis, and genetic modification of arabinoxylan.

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