We are establishing large population of novel lines independently overexpresing each transcription factor for functional genomics in Arabidopsis. We have employed glucocorticoid receptor (GR)-mediated functional induction system to avoid lethality caused by overexpression of some transcription factors. We have isolated a mutant that shows long hypocotyls phenotype by DEX application under long day condition. The long hypocotyls phenotype was observed under blue, red and far-red light conditions, but not under dark conditions. The gene encodes zinc finger type transcription factor, and transcriptional repression domain was predicted near C-terminal. These results suggest that the novel transcription factor function as negative regulator in light signal transduction of this mutant. Presently, we are producing knockdown mutants of this gene, because T-DNA insertion mutant of this gene does not exist.

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The moss Physcomitrella patens is a model plant in which various genetic resources including the genome sequence have been well established. To clarify molecular action of plant totipotency, we developed molecular tools for reverse genetic analyses in P. patens. We searched P. patens genome for targeting sites of transgenes. For gain-of-function analyses, we constructed inducible expression systems, one using heat-shock promoter (HSP) and another one induced by estrogen application. Although gene knock-out (KO) in P. patens has been useful for loss-of-function analyses, it is important to avoid lethality of gene KO because the moss is haploid in most of its life cycle. Thus, we developed a conditional KO system by using Cre-loxP system, in which Cre recombinase is expressed under the control of HSP facilitating excision of the target gene flanked by loxP sites. We will discuss results and applicability of our reverse genetic tools.

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As a part of National Bio-Resource Project Tomato (NBRP tomato, MEXT, Japan), Kazusa DNA Research Institute has been developing DNA-level resources. The major focus of our effort is to sequence the full-length cDNA clones derived from dwarf cultivar Micro-Tom. In the year 2008, the full-length cDNA sequencing-project is accelerated by the support of National Bio-Resource Project Genome Program. In April 2009, we will release full-length sequences of more than 13,000 cDNA clones. Information including the similarity to Arabidopsis and rice genes, the prediction of the function of encoded proteins, and the similarity to tomato genome sequences will be provided as well in the database KaFTom (http://www.pgb.kazusa.or.jp/kaftom/). The second focus of our effort is to collect tomato promoter clones. Since the number of tomato promoters with experimentally confirmed expression patterns is limited, a collection of promoters with various tissue-specificity will be usefull for tomato functional genomics.

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It has reported that intake of isoflavonoids is effective for osteoporosis prevention in postmenopausal women. Kudzu belongs to legume, same as soybean, and its vine also includes much isoflavonoids.
When natural resources are used as industrial raw materials, quality control (QC) in its supply process is important. Aiming to use kudzu vine as a raw material of isoflavonoid supplement, prediction model of total amount of isoflavonoids in kudzu vine as a QC technique was constructed with Fourier transform near-infrared (FT-NIR) spectroscopy.
Freeze-dried powder samples of kudzu vines, which were harvested from 2006 to 2008, were measured by FT-NIR, and prediction model was constructed using the amount of total isoflavonoids in each kudzu vine and the FT-NIR spectra. The prediction model was validated using other test samples and its predicted values were close to measured values.
This study has been funded and supported by Japan Science and Technology Agency (JST).

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Identification of target protein has been an integral but rate-limiting part in chemical genetics studies. Using Arabidopsis full-length cDNA over-expression (FOX) lines and chemicals that inhibit the MEP pathway as model bleaching compounds, we developed a novel target protein screening system. With this system, we effectively identified chemical-resistant plants that over-express a target protein in the MEP pathway from the FOX plants. Moreover, chemical-resistant plants that over-express a novel UDP-glucosyltransferase, which presumably inactivates the MEP pathway inhibitor in Arabidopsis, have been identified. These results suggested that the FOX screening system can be used for target protein identification in chemical genetics studies.

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Triterpenoid saponins are suger-modified triterpene derivatives that have various functions, anti-cancer, anti-oxidant, and anti-microbial. Cereals and grasses are generally deficient in this secondary metabolite with the exception of oats. Our final goal is that the anti-microbial saponin is available for rice. We reported already that 1) triterpenoid saponins and the intermediates were not detected in rice young seedling by LC/MS and GC/MS analyses, 2) we found twelve Oxidosqualene cyclase homologues in rice genome using knowledge in oats, but it was likely that no one was involved in the triterpeneoid biosynthesis pathway. Here, we report that we found twelve OsCYP51 homologues in rice genome that are in next step of the pathway, and it is likely that one is for rice steroids biosynthesis pathway, and another is for rice triterpeneoids biosynthesis pathway, by genome structure and gene expression analyses.

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Many conifer species synthesize copious amounts of oleoresin, composed mainly of monoterpenes and diterpene resin acids in addition to smaller amounts of sesquiterpenes. A diverse array of hundreds of chemical compounds which are initially produced by terpene synthases (TPSs) and further modified by cytochrome P450 monooxygenases (P450), is important in conifer defence against herbivores and pathogens. Our recent gene discovery and phylogenetic cluster analysis for spruce and pine P450s revealed that the CYP720 subfamily consists of over 30 P450s. Here, we report new results from the biochemical characterization of Sitka spruce CYP720B4. To investigate the biochemical functions, we expressed CYP720B4 and the Sitka spruce NADPH-P450 reductase in E.coli, respectively. In vitro assay results show that this P450 catalyze three oxidation steps at the C-18 position of abietadiene to form abietic acid, and also oxidize other diterpenoid olefins (resins) to the corresponding resin acids.

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Latex of a succulent shrub Euphorbia tirucalli includes flamable metabolites sterols and triterpenoids, and so can be processed into biofuel. We have isolated a gene for squalene synthase (EtSS), which produces the first lipophilic intermediate in the sterol-biosynthesis pathway. The 411 residues of EtSS amino-acid sequence exhibits high homology with those from other dicot plants, in contrast to low amino-acid identity to those from monocot plants. In situ hybridization analysis indicated that EtSS expression is dominant in cambia within bundle sheathes. N-terminal 380-residues of EtSS was overexpressed as a fusion protein in E. coli and this protein was subjected to in vitro enzyme reaction and GC-MS analysis to detect a product squalene. Overexpression of entire region of EtSS in E. tirucalli callus resulted in upregulation of phytosterol accumulation. This work was partly performed as one of the technology development projects of the "Green Biotechnology Program" in NEDO.

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Multiple copies of flavonoid biosynthetic genes with different patterns of expression in several tissue and induction following environmental stimuli were identified in Legume. We are trying to analyze regulatory networks controling flavonoid biosynthesis in Legume. Sequence homology searches in model legume Lotus japonicus revealed gene duplication in the transcription factors regulating proanthocyanidin biosynthesis. Three copies of a homologue of Arabidopsis TRANSPARENT TESTA2 (TT2), which is an MYB transcription factor, were present in the Lotus japonicus genome. Transient assay suggest that three LjTT2s are different in protein-protein interaction property. To understand the protein interaction of LjTT2s, we isolated genes encoding bHLH factors and a WDR protein in Lotus japonicus. We also made chimeric constructs exchanging R2R3-MYB domain and C-terminal region of each LjTT2 and mutated constructs that possess amino acid substitution in R2 domain. Analysis using these constructs and isolated bHLH/WDR factors are in progress.

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Leguminous plants have many paralogous genes encoding enzymes involved in the flavonoid biosynthetic pathway. In a model legume Lotus japonicus genome, five dihydroflavonol 4-reductase (DFR) genes encoding the first committed enzyme of the anthocyanin and proanthocyanidin pathway are found to form a cluster. Expression analysis revealed that all the genes were expressed with different organ specificities, suggesting five DFR genes are regulated independently. To clarify the transcriptional regulation of DFR we cloned a homologue of Arabidopsis TT2 and PAP, which are MYB transcription factors that control transcription of DFR gene. Transient assay showed that the DFR2 promoter were activated by LjTT2a, LjTT2b and LjPAP. While LjTT2a and LjTT2b activated ANS and ANR promoter which are pivotal enzymes of the proanthocyanidin pathway, LjPAP didn't activate ANR. These result suggest that proanchocyanidin and anthocyanin biosyntheses are regulated respectively by different transcription factors.

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We have recently reported that sugar concentration (Brix %) in tomato fruit was enhanced by root-chilling cultivation (Fujimura et al. 2008). In this study, we analyzed the concentration of starch and sugars and the gene expression of the major enzymes for starch and sugar metabolisms during tomato fruit development, to help define the mechanism. Root chilling did not affect the pattern in which starch accumulated in early expansion stage and disappeared before ripening while fructose and glucose increased with the fruit development. Root chilling, however, enhanced the accumulation levels of starch, fructose and glucose. Root chilling also enhanced the expression of genes for starch synthesis (AGPase large subunit and SuSy), but not for starch degradation: The expression of SPS gene was not affected, and that of an apoplastic invertase (LIN5) gene was inhibited, by root chilling. These results suggested that root chilling activated the starch synthesis during expansion stage.

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Recent studies indicate that raffinose-related oligosaccharides are synthesized in response to various stresses, salt stress and drought in higher plants and that the oligosaccharides play important roles in protection of proteins and biomembranes as osmolytes and antioxidants. The raffinose-related oligosaccharides are synthesized by galactinol synthase (GolS) with UDP-galactose and myo-inositol. In Arabidopsis, a GolS gene is expressed in seed and multiple GolSs are differentially regulated in plant under drought, salt stress and chilling. We isolated a full length cDNA of galactinol synthase (SlGolS2) in tomato (Solanum lycopersicum L. cv. Micro-Tom), based on partial sequences of GolS homologs in MiBASE (Kazusa DNA Institute). SlGolS2 was transiently induced in response to salt, heat and chilling stresses. Involvement of phytohormones and oxidative stress on SlGolS2 expression and characterization of a recombinant SlGolS2 will be discussed.

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Myo-inositol is required for maintenance of the essential components of cells such as cell wall polysaccharides and membrane phospholipids. Myo-inositol monophosphatase (IMP) is known as the enzyme that catalyzes the last step of myo-inositol synthesis from glucose 6-phosphate to myo-inositol. Myo-inositol is also produced by the dephosphorylation of other inositol phosphate, inositol triphosphate, which is produced in the recycling pathway of inositol. Some upstream or related genes of these two pathways have been known, but detailed analysis of the relationship with IMP genes has not been carried out. In this work, we identified three members of IMP-like genes in Arabidopsis genome database with significant amino acid sequence similarity to the known human IMP genes. Here, we report the expression pattern of these AtIMP-like and related genes in various organs by the analysis with real-time PCR. Our results suggest possible functions of AtIMP-like genes in plant development.

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Lipid metabolism in flower is distinct from that in leaves and potentially involved in many aspects of flower development or reproductive processes. In Arabidopsis, however, lipid metabolism in flowers is largely unknown mainly because flowers are too tiny to be sampled according to the developmental stages. Here, we established Arabidopsis Super Plant, which shows synchronized flower development, as a tool for flower lipidomics analysis. By introducing inducible APETALA1 transgene into ap1-1cal-5, whose flower development is arrested, flower development was synchronized till the anthesis. Using this system, we conducted lipidomics analysis on membrane lipids in different developmental stages. The results revealed that lipid contents were dynamically changed during flower development. These results suggest that lipid metabolism is actively regulated by induction of distinct set of gene during flower development, and that Super Plant is a powerful tool to investigate flower lipid metabolism in Arabidopsis.

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Bax Inhibitor-1 (BI-1) is a widely conserved cell death supressor. Arabidopsis BI-1 (AtBI-1) is an ER membrane protein which has 7 transmembrane domains, and suppresses the cell death induced by oxidative stress in plants. However, the precise molecular mechanism has not been clear.
Recently, it has been demonstrated that AtBI-1 interacts with 2-hydroxylase (AtFAH) via cytochrome b₅ (AtCb5) (Nagano et al., Plant Journal, in press). Moreover, an analysis using Saccharomysis cerevisiae suggests that fatty acid elongase (ScELO2, ScELO3) are related to cell death supression mediated by AtBI-1. ScELOs are condensing enzymes, which catalyze the first step of fatty acid elongation, and essential for production of very long chain fatty acid. Although higher plants have ELO homologs, their functions are not clear. Here, we show the functions of Arabidopsis ELO homologs and the relationship between ELO and AtBI-1.

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Sphingolipids are a large class of lipids ubiquitously present in eukaryotic cell membranes and essential for various cellular reactions such as signal transduction, protein transport and programmed cell death. The structural diversity of sphingolipids derives from more than 300 distinct head groups as well as various modifications on hydrocarbon chains of the hydrophobic ceramide moiety. One of the main modifications of ceramide structure is 2-hydroxylation of the fatty acids. The 2-hydroxylation of the fatty acids is catalyzed by fatty acid hydroxylase (FAH). Two Arabidopsis FAHs (AtFAH1 and AtFAH2) are localized in endoplasmic reticulum (ER) membrane. In addition, AtFAHs were activated by ER-localized cytochrome b5 through their direct interaction. Here we show the functions of AtFAHs and the roles of 2-hydroxy fatty acids in Arabidopsis.

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Phosphatidylserine (PS) represents 10% of total lipids from flowers in A. thaliana, where PS biosynthesis is regulated by the base-exchange-type PS synthase gene AtPSS1 (At1g15110). To understand the role of PS in reproductive development, we previously showed that the null alleles pss1-1 (T-DNA insertion) and pss1-2 (EMS-tilling) mostly showed embryonic lethality and that heterozygotic mutants partly showed cell death of pollen microspores. To extend our understanding of the role of PS in pollen development, we herein isolated homozygotes for pss1-1 and pss1-2mutants. Homozygous pss1-1 and pss1-2 plants were raised to show dwarfism and produce infertile flowers. Pollen grains were found on the stigma, but no fertile embryo was found within the ovary. Pollen from homozygotic mutants partly showed leakage of cytoplasmic materials through the cell wall, suggestive of damaged cell walls due to malfunction of tapete cells.

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Cyanobacterial thylakoid membranes contained the four glycerolipids, monogalactosyldiacylglycerol, digalactocyldiacylglycerol, sulfoquinovosyldiacylglycerol and phosphatidylglycerol. In addition to these glycerolipids we found that filamentous cyanobacteria (such as Anabaena sp. PCC7120, A. variabilis and Spirulina )contain a fifth glycerolipid. Structural analysis by NMR revealed that this lipid is three galactose residues linked to the glycerol back bone with methylation at the third galactose, diacyl -O-(O-methyl-α-Galactopyranosyl-(1,6)-α-Galactopyranosyl-(1,6)-β-galactopyranosyl)-glycerol. We will report to identify the genes for the enzymes involved in this glycolipid synthesis.

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Green photosynthetic bacteria have a unique light-harvesting antenna system called chlorosome. The envelope of a chlorosome consists of monolayer of glyco/phospholipids containing proteins. These glyceroglycolipids were reported to be monogalactosyldiacylglycerol (MGDG) and rhamnosylgalactosyldiacylglycerol (RGDG), but not characterized at the molecular level, especially for their fatty acids. In this study, we elucidated the molecular structures and compositions of MGDG and RGDG extracted from Chlorobium (Chl.) tepidum using combination of liquid-chromatography with electrospray-ionization mass spectrometry and evaporative-light-scattering detection. Major two fatty acids in MGDG and RGDG were determined to be cyclopropane acid (C17:1) and saturated acid (C16:0). Their covalently linked positions with glycerol moieties were determined by selective hydrolysis of the esters using a lipase. Moreover, we investigated changes in the glycolipid composition of the cells of Chl. tepidum grown at different temperatures, indicating that the composition drastically changed with lowering the culturing temperature.

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Cyclobutane pyrimidine dimer (CPD) is a major type of UV-induced DNA damage. CPD photolyase is a crucial factor for determining the sensitivity of rice to UVB. We previously reported that the native rice CPD photolyase is phosphorylated. To investigate the effect of phosphorylation on CPD photolyase activity, we separated phosphorylated and unphosphorylated CPD photolyase from the native rice CPD photolyase, and then we measured the activity of each CPD photolyase. As a result, there was no difference in the activity between phosphorylated and unphosphorylated CPD photolyase. Furthermore, we found that CPD photolyase localizes to chloroplasts, mitochondria and nuclei and efficiently repairs UVB induced CPDs in all three organelles in rice plants. We prepared organelle-enriched fractions from rice plants, and detected CPD photolyase phosphorylated status. Mitochondria demonstrated a high proportion of non-phosphorylated CPD photolyase, whereas nuclei and chloroplasts had a relatively high proportion of the phosphorylated form.

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Our previous study using mutant and transgenic of tobacco and Arabidopsis cell suspension cultures suggested that reactive oxygen species (ROS), calcium and salicylic acid signaling involved UVC-induced cell death. In UVC-induced salicylic acid signaling, SID2 and NPR1 did not play key role as cell death inducers as cells with sid2, npr1 and NPR over expression showed. UVC-induced PR1 gene expression is generally known. Same date was shown by our researches using Arabidopsis and tobacco cell suspension cultures. It was suggested UVC-induced PR1 gene expression was depended on ROS, calcium and NPR1. It was known that NPR1, TGA1 and WRKY70 were important gene in salicylic acid signaling. Those gene was induced by UVC irradiation in Arabidopsis cell suspension was shown. We analyzed UVC-induced NPR1, TGA1 and WRKY70 gene expression.

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The coordinated high-light response of genes encoding subunits of photosystem I (PSI genes) is achieved by the AT-rich region located just upstream of the core promoter in Synechocystis sp. PCC 6803. The upstream element enhances the basal promoter activity under low-light conditions, whereas this positive regulation is lost immediately after the shift to high-light conditions. We found that a high light regulatory 1 (HLR1) sequence exists in the upstream element of every PSI gene and a response regulator RpaB binds to the HLR1 sequence. In the case of the psaAB genes encoding reaction center subunits, there are two light-responsive promoters and three additional HLR1 sequences, suggesting the complicated mechanism of transcriptional regulation. In this study, we examine the involvement of the HLR1 sequence to the regulation of two promoters in the psaAB genes using reporter constructs with or without mutation in the HLR1 sequences.

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In cyanobacteria, small transcriptional regulators that consist solely of a helix-turn-helix motif of LuxR type are highly conserved. These regulators possess three cysteine residues in their C terminal region. In Synechocystis sp. PCC 6803, a small LuxR-type regulator, PedR, regulates the expression of several high-light responsive genes when the activity of photosynthetic electron transport is low. Amino acid substitution in PedR revealed that Cys80 is essential for dimerization. But amino acid residues involved in redox sensing were not identified by this experiment. In order to identify regulatory factors interacting with PedR, pull down analysis was performed. We detected the interaction of thioredoxin with PedR and confirmed that purified thioredoxin reduced PedR effectively. This suggests that PedR is inactivated when reducing equivalents were supplied by thioredoxin in vivo. We are now trying to identify cysteine residues interacting with thioredoxin.

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Elongation factor G (EF-G) is a key protein responsible for translational elongation. We have recently found that EF-G is a primary target, by reactive oxygen species (ROS), of inhibition of the translational machinery in Synechocystis sp. PCC 6803. In the present study, we found that inactivation of EF-G by ROS was attributed to oxidation of two specific cysteine residues and formation of a disulfide bond. The disulfide bond in the oxidized EF-G was reduced by thioredoxin. Substitution of the target cysteine residues by serine in EF-G resulted in the increased tolerance to ROS of EF-G as well as of the translation system in vitro. These observations suggest that the activity of translation might be regulated by the redox state of EF-G, which is determined by a redox signal that is emitted by photosynthetic electron transport and mediated by thioredoxin.

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Thioredoxin is a small ubiquitous protein that regulates a number of phenomena through formation or dissociation of a disulphide bridge in the target enzyme. Cytosolic thioredoxin has been reported to regulate several membrane proteins that involve in the self-incompatibility and in the innate immunity of plant so far. However, a membrane protein involved in the redox cascade is still poorly understood.
To uncover a whole picture of membrane proteins involved in the redox cascade, we screened target proteins of thioredoxin in plasma membranes prepared from the suspension culture of Arabidopsis thaliana. The resulting proteins were identified by MALDI-TOF/TOF MS/MS. A list of potential targets for thioredoxin contained some signaling molecules. We prepared some recombinant protein of the molecules and analyzed their redox regulation in vitro and in vivo. The result allows us to presume a molecular mechanism for redox regulation of the membrane signaling molecule in plant cells.

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We reported that the activity of Arabidopsis protein tyrosine phosphatase (AtPTP1) is regulated by redox status of the non-catalytic cysteine 175 and a substitutional mutant C175S AtPTP1 was not inhibited by GSSG (the JSPPJ Annual Meeting, 2008). To elucidate whether C175 is redox-regulated in vivo, we investigated seed germination, a redox-regulated phenomenon, in transgenic plants over-expressing wild-type or C175S mutant AtPTP1. Seed germination of these plants was little affected by GSSG alone. The suppression of seed germination by ABA was enhanced with increasing AtPTP1 expression, and the suppressive effect was strongest when C175S was expressed. The suppression was reduced by GSSG, and the reducing effect was strongest in C175S plants. These suggest that AtPTP1 promotes the ABA-dependent suppression of seed germination and that the abolishment of the promotion is caused by direct inhibition pathway via C175 and suppressive pathway downstream of AtPTP1.

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The unsaturated fatty acyl group linolenic acid of lipids (LA) suppresses flowering in Arabidopsis thaliana. Compared to 22°C, a low temperature (15°C) increases LA and delays flowering in wild-type plants (Col). Since the delay of flowering by low temperatures was reduced by triple mutations in FAD3, FAD7 and FAD8 genes encoding LA- synthesizing enzymes, it is considered to be attributed to LA. The early-flowering phenotype of 35S-AP1 plants was suppressed by low temperatures or a cross with 35S-FAD3 plants, LA suppresses the flowering determination by AP1. The delay of flowering by low temperature was reduced by certain mutations in AP1 gene. To elucidate the LA-controlled domain of AP1, we introduced several truncated AP1 constructs into wild-type plants and characterized flowering phenotypes.

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Genes involved in defense systems against various types of stress particularly prone to alternative splicing in higher plants. We demonstrated that Arabidopsis splicing factors, atSR30 and atSR45a, are predicted to participate in regulation of the alternative splicing under high-light stress (Plant Cell Physiol. 48: 1036-1049, 2007). Here, we characterized T-DNA insertion mutants of atSR30 (KO-sr30) and atSR45a (KO-sr45a), and their double knockout mutants (WKO-sr30/sr45a). The transcript levels of atSR45a and atSR30 increased in KO-sr30 and KO-sr45a, respectively, under high-light stress. WKO-sr30/sr45a showed a dwarf phenotype, although KO-sr30 and KO-sr45a showed no difference in the phenotypes compared with wild-type plants. The transcript levels of various heat shock proteins (HSP), including HSP70, HSP17.4 and HSP101, decreased in WKO-sr30/sr45a. These results suggest that atSR30 and atSR45a coordinately function in the regulation of alternative splicing for genes involved in the induction of HSPs expression.

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We reported that an Arabidopsis ADP-ribose/NADH pyrophosphohydrolase, AtNUDX7, functions in regulation of the poly (ADP-ribosyl)ation (PAR) reaction via hydrolyzation of ADP-ribose/NADH, and plants overexpressing AtNUDX7 show increased tolerance to oxidative stress. The PAR reaction is a post-translational modification for responding early to DNA damage caused by oxidative stress in mammalian. Here we studied the effect of the overexpression or depletion of AtNUDX7 on expression of genes encoding factors involved in the DNA repair factors (AtXRCC1, AtRAD51, AtDMC1, AtXRCC2, AtXRCC3, and AtMND1) by quantitative RT-PCR. The expression levels of AtXRCC1 and 2 paralleled that of AtNUDX7 under normal conditions and oxidative stress. Whereas, an inverse correlation was observed between the levels of AtRAD51, AtXRCC3, AtDMC1, and AtMND1 and AtNUDX7. These findings indicated that AtNUDX7 confers enhanced tolerance of oxidative stress on Arabidopsis plants, resulting from regulation of the defense mechanisms against DNA damage via modulation of the PAR reaction.

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In higher plants, phosphomannose isomerase (PMI) catalyzes the reversible isomerization of D-fructose 6-phosphate and D-mannose 6-phosphate as the first step of GDP-mannose and ascorbate (AsA) biosynthesis. In this study, we analyzed molecular properties of Arabidopsis PMI isoenzymes (PMI1 and PMI2). The induction of PMI1 expression and an increase in the AsA level were observed in leaves under continuous light, while the induction of PMI2 expression and a decrease in the AsA level were observed under long-term darkness. Moreover, a reduction of PMI1 expression through RNA interference resulted in a substantial decrease in the total AsA content of leaves of knockdown-PMI1 plants, while the complete inhibition of PMI2 expression did not affect the total AsA levels. Therefore, PMI1 is essential for biosynthesis of AsA, while PMI2 is involved in the utilization of Mannose-derived carbohydrates as an energy source in leaves under sugar-starved conditions (J. Biol. Chem. 283: 28842-28851, 2008).

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Ozone induces the synthesis of phytohormones, including ethylene and salicylic acid (SA), and these phytohormones act as signal molecules that enhance cell death. However some studies have shown that these phytohormones can decrease the magnitude of ozone-induced cell death. Here, we studied the defensive roles of ethylene and SA against ozone. Unlike wild-type plants, Arabidopsis mutants deficient in ethylene-signaling (ein2) or SA-biosynthesis (sid2) exhibited visible leaf injury, indicating that these phytohormones can reduce ozone damage. Induction of glutathione synthetase (GSH2) expression and the enzymatic activity in ein2 and sid2 were lower than those in Col-0. Increases of glutathione occurred in Col-0 was also suppressed in ozone-exposed ein2 and sid2 mutants. Moreover, ozone-induced leaf damage observed in these mutants was mitigated by artificial complementation of glutathione. Our results suggest that ethylene and SA protect against ozone-induced leaf injury by increasing de novo biosynthesis of glutathione.

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Acylamino Acid-Releasing Enzyme (AARE) is protease conserved widely among microorganism, mammal, and plant. AARE catalyzes the N-terminal hydrolysis of N^α-acylpeptide, which releases N^α-acylated amino acids. It was found in vitro that AARE degraded oxidized and glycated protein. We analyzed plant AARE function in the degradation of oxidized protein using AARE silencing Arabidopsis thaliana.
By oxidative stress treatment, AARE knockdown Arabidopsis increased ion leakage in comparison with WT. Accumulation amounts of oxidized proteins were lower in the knockdown plants than wild ones. When oxidized proteins were separated by 2D electrophoresis and were visualized, the pattern was different between WT and AARE knockdown. Therefore, these results suggests the AARE involvement in oxidized protein degradation in plants.
Cellular localization of AARE was analyzed by fluorescence microscope. By MS analysis, it was confirmed that fructose-1,6-bisphosphate aldolase, dehydroascorbate reductas eand etc. were oxidized in AARE knockdown more than in WT.

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We reported previously that plants grown in the air containing nitrogen dioxides (NOx) at background levels were almost two times greater in the growth rate and biomass yield than those grown in the air without NOx¹⁾. We designated this plant vitalization by NOx. In this study, we investigated genes involved in the regulation of this effect on Arabidopsis thaliana. The DNA microarray analysis showed that more than 130 genes increased their expression by more than two times in response to fumigation with atmospheric NOx. T-DNA insertion mutants were then analyzed for the vitalization by NOx. It was found that lines in which these genes were tagged by T-DNA insertion lost this effect, showing that genes collected by the microarray analysis are involved in the regulation of the vitalization by NOx.
1) M. Takahashi et al. (2005) New Phytologist 168: 149-154.

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The powdery mildew is an obligate biotrophic fungus that infects various crops and vegetables, thus causing economical damages. Powdery mildew infection is highly race-specific, and its infection and growth are limited to the plant surface. Plant defense response against pathogens is known to be multidimensional. Pathogens have to overcome (1) physical defense by cell wall, (2) defense induced by pathogen itself at entering, and (3) defense caused by pathogen and plant protein interaction to successfully establish infection. Recent research has mostly focused on the defense response at powdery mildew entering, and much of defense mechanism remains to be unknown. To obtain a clear view of how Arabidopsis responds against powdery mildew Golovinomyces orontii, we have performed detailed morphological analyses on early infection process. We found that powdery mildew has a tissue preference, and that the accumulation of wax crystal was one of factors affecting the preference.

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PEN2 was isolated as a gene involved in nonhost resistance of Arabidopsis thaliana against powdery mildews, and it also functions in nonhost resistance against Colletotrichum species. In this study, we identified lic1 mutants (lesion induced by nonadapted Colletotrichum) that failed to maintain nonhost resistance against Colletotrichum. Nonadapted Colletotrichum did not elevate the efficiency of plant invasion in the lic1 mutant, suggesting that lesion development in the lic1 mutant is not due to fungal invasion. Map-based cloning revealed that LIC1 is allelic to NSL1 encoding a protein with a MACPF domain. The lic1 pad4 and lic1 eds16 double mutants reduced lesion formation compared with the lic1 mutant, indicating the involvement of salicylic acid signaling in the lic1 phenotype. Surprisingly, the pen2 mutation also partially suppressed lesion development in the lic1 mutant, suggesting a link between PEN2-mediated antimicrobial response and the MACPF protein in cell death regulation.

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PEN2 is required for nonhost resistance of Arabidopsis thaliana against powdery mildews, and it also functions in resistance against Colletotrichum species. In this study, we report that EDR1 is involved in nonhost resistance of Arabidopsis against Colletotrichum. It has been reported that the edr1 mutant increased resistance against adapted powdery mildew via enhanced activation of defense responses mediated by salicylic acid (SA). We show that nonadapted Colletotrichum increases the efficiency of plant invasion and formed lesions in the edr1 mutant. The edr1 pad4 mutant is still defective in nonhost resistance, suggesting that reduced resistance in the edr1 mutant is not due to activation of SA signaling. To assess the relation between PEN2 and EDR1, we generated the edr1 pen2 mutant. Importantly, the double mutant exhibits more severe defects in resistance against nonadapted Colletotrichum in comparison with each single mutant, indicating that EDR1 regulates nonhost resistance independently of PEN2.

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One of the most serious and widespread diseases of rice is blast caused by the fungus Magnaporthe grisea. An understanding of the mechanisms underlying pathogenesis and host response is critical to future progress in rice blast pathology. Many studies on the resistance responses have been made on rice. However, non-host resistance against M. grisea is not well known. So, we investigated the molecular basis of non-host resistance in Arabidopsis against the rice blast pathogen, M. grisea. Conidia of M. grisea germinated and formed appressoria over the Arabidopsis leaf surface. However, they could not penetrate epidermal cells. Next, we assayed the response of Arabidopsis mutants to this fungus and identified a mutant with defective penetration resistance. In this mutant, M. grisea could penetrate epidermal cells. However, most of the penetrated cells underwent hypersensitive-response-like cell death. This result suggests that non-host resistance to rice blast is composed of penetration and post-penetration resistance.

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The DEAR1(DREB and EAR motif protein 1), which encodes the protein containing DREB domain and EAR motif, is likely to function as a transcriptional repressor to the DRE-binding protein. The DEAR1 overexpressor showed constitutive cell death on rosette-leaves and accelerated leaf senescence. The overexpressor also stimulated expression of PDF1.2 gene which is controlled ERF (ethylene-responsive element binding factor) genes.
DNA microarray on DEAR1 overexpressor and expression analysis showed that ERF9, which contains the EAR motif and have DRE region in the promoter, is the target of DEAR1. It is hypothesized that the ERF9 usually represses expression of target genes including PDF1.2, and that release the repression by the DEAR1 in pathogen infection.
The ERF9 gene under the DEAR1 control will be discussed.

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Protein degradation by ubiquitin / 26S proteasome system (UPS) is involved in various aspects of cellular activity. Selection of target protein in the UPS is catalized by ubiquitin ligase (E3).
In higher plants, metabolism of sugar (C) and nitrogen (N) is competitively regulated. To clarify the C/N regulator, we isolated novel C/N regulatory E3 ligase SSV1. The SSV1 gene belongs to ATL family. Although some ATLs have been reported to be involved in plant immunity, the detailed mechanisms are still unknown. To investigate the relationship between ATL family and pathogen resistance, we examined pathogen resistance using Pseudomonas syringae pv. tomato DC3000 . Plants overexpressing SSV1 and ATL6 which is most closely related gene to the SSV1 in ATL family showed increase in pathogen resistance, whereas double mutant.
Relationships between C/N balance regulation and plant immunity will be discussed in terms of ATL family.

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Defense response of plants against pathogenic microorganisms includes changes in metabolism, such as the production of key signaling compounds, salicylic acid (SA) and jasmonic acid (JA). The mechanisms controlling SA- and JA production in response to the pathogen attack have been studied extensively. On the other hand, there have been few studies describing a comprehensive profile of metabolites associated with pathogenesis. Here, we report comprehensive metabolite profiles of transgenic Arabidopsis lines that have different levels of resistance to viral infection using metabolome approach.
Resistant lines without provoke hypersensitive response (HR), so called extreme resistance (ER), were strongly restricted metabolite changes at the early stage of viral infection whereas susceptible Col-0 plants and resistant lines with HR were not. We would like to discuss a mechanism of ER type resistance in terms of changes in metabolite profiles.

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SGT1-HSP90 complex is essential for disease resistance in plants triggered by resistance (R) proteins and is also necessary for innate immunity in animals induced by R protein homologues, Nod family proteins. Here, we present the structural and functional characterization of the plant SGT1-HSP90 complex. X-ray crystal structural analysis, NMR-based interaction surface mapping and mutational analyses reveal that (1) the CHORD-II domain of RAR1 and the N-terminal domain of HSP90 interact with opposite sides of the CS domain of SGT1, and (2) SGT1 binds to different surface of HSP90 than those to which other co-chaperones, P23 and AHA1 bind. This analysis allowed us to obtain a compensatory mutant pair between both partners that is able to restore virus resistance in vivo through Rx (Resistance to potato virus X) immune sensor stabilization. These results directly show the critical role of SGT1-HSP90 interaction in innate immunity in plants.

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There are many reports that virus infection leads to plant death. Clover yellow vein virus (ClYVV) infects and kills legume crops such as broad bean and pea. We previously screened the host factors involved in ClYVV virulence using broad bean and isolated the functionally unknown gene, Vfk158. Vfk158 is a glycine-rich protein (29.3%) and has nine XYPPX repeats in the N-terminal region. In this study, we used Potato virus X-Nicotiana benthamiana pathosystem. N. benthamiana has two Vfk158 homolog candidates (Nbk158-1 and -2). To examine whether Nbk158 is involved in PVX virulence, we suppressed or ectopically expressed Nbk158 using PVX vector. The suppression of Nbk158 enhanced the symptoms and over-expression of Nbk158-1 attenuated. Tissue print showed that Nbk158-1 over-expression suppressed PVX spread. Furthermore, Agrobacterium-mediated transient assay showed that Nbk158-1 over-expression suppressed PVX accumulation. These results suggest that Nbk158 has a role in resistance against PVX in N. benthamiana.

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Rice has been infected with many kinds of viruses transmitted by the vector insects. Once a virus disease occurs, great deal of loss of grain production will be assumed. For long time, extensive studies on rice virus have been carried out and many kinds of knowledge on the virus structures, genome sequences and functions of encoded proteins have been accumulated. However, the information about how the virus knocks over the host system, how host resists against virus propagation are very poor. In this study we have tried to understand the battle between virus and host in the molecular level by performing the transcriptome analysis through rice 44K microarray system. Transcriptome analyses and data mining results from RDV, RSV, RTSV or RTBV infected rice plants susceptible or resistant, will be presented and discussed.

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In N gene-containing tobacco (Nicotiana tabacum) cultivars, infection with Tobacco mosaic virus (TMV) results in the rapid death of infected cells, resulting in the localization of the virus by necrotic lesions. Although the mitogen-activated protein kinases WIPK and SIPK have been implicated in TMV resistance by regulating hypersentitive response (HR) cell death, evidence linking them directly to disease resistance has been insufficient. WIPK and SIPK were silenced in a TMV-resistant tobacco cultivar using RNA interference to define their roles in TMV resistance. Viral multiplication was reduced in WIPK/SIPK-silenced plants. Inoculation of lower leaves resulted in movement of TMV through the plant and necrosis in uninoculated upper leaves. These results suggest that WIPK and SIPK function cooperatively to enhance multiplication of TMV, but inhibit systemic viral movement, probably by regulating HR cell death.

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Rapid production of nitric oxide (NO) and reactive oxygen species (ROS), called NO burst and oxidative burst, respectively, is characterized during resistance to biotic and abiotic stresses. Our previous study showed that MAPK cascades regulate NO and oxidative bursts induced by INF1, which is produced by the oomycete pathogen Phytophthora infestans (Asai et al. 2008 Plant Cell). We have screened candidates related to cell death, termed CELL DEATH ASSOCIATED (CDA) genes, in randomly genes-silenced plants by VIGS. Silencing CDA1 compromised HR-like cell death induced by INF1 and MEK2^DD, a constitutively active mutant of MEK2, in N. benthamiana. CDA1 encodes the gene for riboflavin synthesis. Riboflavin converts to FAD and FMN known as coenzyme of NADPH oxidase, NO synthase and nitrate reductase, indicating that CDA1 might be responsible for NO and oxidative bursts. Here, we will show effects of CDA1 silencing on production of NO and ROS and on disease resistance.

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Possible involvement of autophagy in immune responses and programmed cell death both in animals and plants have recently been discussed extensively. However, little is known on the dynamics and physiological significance of autophagy in plant innate immunity. Cryptogein, a proteinaceous elicitor from an oomycete, induces a series of cell cycle-dependent defense responses and hypersensitive cell death synchronously in tobacco BY-2 cells (Kadota et al. 2004), which provides a suitable model system to characterize dynamic intracellular cell biological events during innate immunity (Higaki et al. 2007; 2008). We established a BY-2 cell line in which dynamics of autophagosomes can be visualized with Atg8-YFP. We will report the effects of cryptogein on the dynamics of autophagy and possible involvement of autophagy in the regulation of cryptogein-induced defense responses and programmed cell death.

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Plants, which lack the immune cells, have evolved their own defense strategy against invading pathogens. We reported that jasmonic acid, one of plant stress hormones, induced ER body formation in leaves of Arabidopsis thaliana. ER body is an ER-related organelle that accumulates PYK10, a beta-glucosidase with an ER-retention signal. To clarify the function of ER bodies and their component of PYK10 in plant defense system, we compared wild-type with two mutants (nai1, lack of ER body mutant, and pyk10 ) on the growth of bacteria and the expression of PYK10 gene after infection of Pseudomonas syringae pv. tomato (Pst) DC3000(avrRpm1). After infection of Pst DC3000(avrRpm1), the number of bacteria increased in nai1 more than that in wild-type. The PYK10 gene expression also increased after 6 hour-post-inoculation with Pst DC3000(avrRpm1). These results suggest that PYK10 is involved in plant defense system of Arabidopsis thaliana.

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Plants are known to have a type of programmed cell death (PCD) to defend against invading pathogens. We show that following a bacterial attack, the proteasome β1 subunit (PBA1) mediates fusion of the vacuolar and cell membranes. This process results in the discharge of vacuolar proteins to the outside of the plasma membrane. This then leads to PCD for the efficient disease resistance. To our knowledge, this is the first report of fusion of the vacuolar and cell membranes in plants. PCD may be induced by the release of vacuolar hydrolytic enzymes as a result of the PBA1-dependent membrane fusion. Fusion of the membranes may also release vacuolar defense proteins to kill bacteria on the outside of the cells.

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Higher plants protect themselves against pathogens through their immune systems. It has been suggested that membrane trafficking plays an important role in some of these immune systems. However, the molecular mechanism is not known. Previously, we developed an efficient and powerful method to isolate mutants deficient in vacuolar sorting (1) and designated these mutants as green fluorescent seed (gfs) mutants. To identify vacuolar sorting factors responsible for disease resistance, we infected the leaves of 64 gfs lines with Pseudomanas syringae pv. tomato (Pst) DC3000 (avrRpm1). We succeeded in isolating two gfs mutants that exhibited a lower resistance against the bacteria and designated them gfs11 and gfs12. Our results suggest that vacuolar sorting is involved in the disease resistance in plants.
(1) Fuji et al., Plant Cell, 19,597-609 (2007).

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A nuclear Gibberellin (GA) receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1), has a primary structure similar to that of the hormone-sensitive lipases (HSLs). Here we analyze the crystal structure of rice GID1 (OsGID1) bound with GA₄ and GA₃ at 1.9 Å resolution. The overall structure of both complexes shows an alpha/beta-hydrolase fold similar to that of HSLs. The GA-binding pocket corresponds to the substrate-binding site of HSLs. On the basis of the OsGID1 structure, we mutagenized important residues for GA binding. The replacement of Ile 133 with Leu or Val, residues corresponding to those of the lycophyte GID1s, caused an increase in the binding affinity for GA₃₄, a 2β-hydroxylated GA₄. These observations indicate that GID1 originated from HSL and has been further modified to have higher affinity and more strict selectivity for bioactive GAs by adapting the amino acids involved in GA binding in the course of plant evolution.

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