Rice OsMac1 gene contained a 5'untranslational region (5'UTR) consisting of an approximately 500 bases upstream of the major ORF. This region contained three upstream ORFs (uORFs), and CU rich region. We found three forms of 5'UTR, named as UTRa, UTRb, and UTRc, which were derived from alternative splicing on the junction between Exon1 and Exon2. UTRa and UTRb was 54 and 38 base shorter than UTRc, respectively. These sequences were expected to be involved in control of translation of the major ORF. A reporter gene, in which the GUS gene was connected downstream the UTRc, was expressed with a high translational efficiency, but GUS genes connected with UTRa and UTRb resulted in a low translational efficiency as shown in those without 5'UTR. Deletion of the CU rich region resulted in decrease of the translation of the major ORF. In contrast, the GUS gene attached with modified 5'UTRs, in which the initiation codons of uORFs were defected, showed no significant changes on the GUS activities. These results suggest that the 5'UTR possesses two kinds of the enhance sequences, the CU rich region and the 38 bp region, affecting the efficiency of the translation of the major ORF.

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Cystathionine γ-synthase (CGS), which catalyzes the key step of methionine biosynthesis, is encoded by CGS1 gene in Arabidopsis. Expression of CGS1 is feedback-regulated at the step of mRNA degradation in response to S-adenosyl-L-methionine (AdoMet). A 14-amino-acid sequence, termed the MTO1 sequence, located near the N-terminal region of CGS itself is important for this regulation by acting in cis. This CGS1 mRNA degradation is preceded by temporal translation arrest that occurs at Ser-94 located immediately downstream of the MTO1 sequence. This implies that the MTO1 peptide exists in the exit tunnel of the arrested ribosome.
In an attempt to gain insights into the molecular function of MTO1 sequence in the exit tunnel, we explored the possibility that AdoMet induces a structural alteration of the CGS1 nascent peptide covering the MTO1 sequence. We examined this possibility by pegylation assay using an in vitro translation system. The results indicated that AdoMet induces a compact structure formation in CGS1 nascent peptide in an MTO1 sequence-dependent manner. Correlation between nascent peptide compaction and translation arrest will be discussed.

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Upstream open reading frames (uORF) are small ORFs often found in 5' leader region of eukaryotic genes. The presence of uORF can negatively affect translational efficiency of the downstream main ORF. Although the effects of most uORFs are independent of their encoded peptide sequences, several uORFs have been shown to control translation of the main ORF in an amino acid sequence-dependent manner. In order to further identify such uORF-encoded regulatory peptides from Arabidopsis, we searched for uORFs whose amino acid sequences are conserved among eudicots by using a comparative genomic approach. We found 14 novel conserved uORFs, and tested whether these uORFs affect expression of the downstream ORF in a uORF peptide sequence-dependent manner. From this screen, we identified 5 novel genes whose expression is controlled by a uORF-encoded peptide. Some of these genes contain multiple uORFs in their 5'UTR. Mutational analyses revealed an involvement of the multiple uORFs in the translational regulation. It is also suggested that a uORF located upstream of the conserved uORF have a role to modulate translational efficiency of the conserved uORF.

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In clonal plants producing vegetative offspring by growing vegetative organs, a genetic individual (genet) is constructed by multiple independent plants (ramets), widely distributing in its population. In such a plant species, ramets of the same genotype are exposed to the heterogeneous environment. Therefore, epigenetic variations as well as genetic variations may play important roles to adapt to the fine scale habitat conditions.
In this study, we explored the extent and patterns of DNA methylation in a wild population of a clonal plant Cardamine leucantha, by using the technique of MSAP (methylation-sensitive amplified polymorphism) that makes used of the differential sensitivity of a pair of isoschizomers to cytosine methylation. As a result, the methylation patterns were different between genets even if they were related and/or neighboring genets. Within a single genet, the similarities of methylation depended on the spatial distance between ramet pairs. The correlations between the vegetative coverage reflecting light capture and methylation loci varied from positive to negative, which suggested that such epigenetic variations were beyond wide sites in the genome.

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fwa is a late flowering epi-mutant in Arabidopsis thaliana. FWA is stably silenced in A. thaliana but in related Arabidopsis species, FWA expression and DNA methylation levels vary in vegetative tissue. In this study, we show that variation in FWA expression in field isolates having identical DNA sequences is associated with changes in DNA methylation and may change over time. Vegetative FWA expression is correlated with decreased methylation at non-CG sites in the region upstream of the transcription start site in species related to A. thaliana and we conclude that methylation of this region is critical for FWA silencing in these species. In A. thaliana, FWA expression is affected by methylation in regions both upstream and downstream of the transcription start site. Ectopic thaliana FWA expression causes a late flowering phenotype, but over-expression of lyrata FWA does not. In A. thaliana, stable silencing of FWA to prevent late flowering may have evolved through the selection of large tandem repeats and spread of the critical methylated region to include these repeats.

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SAP130 is a component of the SF3b mRNA splicing complex and the STAGA/TFTC transcription complexes. Although SAP130 has been identified to bind CSN through its interaction with the N-terminal half of CSN1, the molecular function of SAP130, in vivo, has remained unclear.
In Arabidopsis, SAP130 is encoded by two genes, AtSAP130a and AtSAP130b, which both map on chromosome 3. The transcripts for both genes were detected in all observed organs. The promoter activity of AtSAP130a and AtSAP130b was detected in similar tissues displaying identical patterns at a specific stage in the developing anther. These data suggest redundant roles of the two genes in vivo. Furthermore, detailed histological analyses revealed that AtSAP130 RNAi mutants show a pollen developmental defect at the microspore stage to the bicellular stage transition, presumably contributing to the reduction of the final seed number. These results revealed that AtSAP130 function is essential for proper pollen development in Arabidopsis.

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COP9 signalosome (CSN) is a nuclear complex regulating signal transduction in plants and animals. To dissect the function of CSN, we focused on the function of CSN1 subunit. The N-terminal region of CSN1 (CSN1N) possessed a transcriptional repression activity in mammals, and was essential for survival in plants. To understand this mechanism, we isolated proteins interacting with CSN1N in Arabidopsis, and identified a transcription factor belonging to a plant-specific trihelix family. Through a series of pull-down assays, this transcription factor and the two homologous were shown to directly bind CSN1N in a specific manner. We characterized T-DNA-inserted mutants, RNAi plants, and 35S promoter driven overexpressing plants. Two independent lines with increased mRNA accumulation exhibited slightly shorter hypocotyl compared to wild-type plants. Here, we report the promoter activity, the gene expression pattern of this transcription factor and the phenotype of its mutants. We will further characterize this transcriptional factor and discuss its role in CSN-mediated signal regulation.

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Autophagy is a degradation system induced by nutrient limitation. We have developed a quantitative monitoring system of autophagic degradation in transformed tobacco BY-2 cells, which express a fusion protein of cytochrome b5 (Cyt b5) and a photo-convertible fluorescent protein (Kikume). Using this system we found that tobacco cell does not stop protein synthesis immediately after phosphate limitation. Under phosphate starvation proteins synthesized both before and after starvation were degraded by autophagy.
To compare the mechanism of the induction of autophagy under phosphate, sugar and nitrogen limitation conditions, we conducted experiments including phosphite under starvation conditions. We found that phosphite in phosphate starvation is effective in delaying the induction of autophagy. This result was also shown by analysis of the stability and degradation of Atg8, which is a marker protein of autophagosomes. Interestingly, we could not find any effect of phosphite on nitrogen- and sucrose-starvation conditions. Thus the signal transduction pathways of nutrient-responsive autophagy induction varied on the type of nutrients.

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Dynamic modification of Ser/Thr redidue with O-linked β-N-acetylglucosamine is known to be very common in nucleocytoplasmic proteins. O-GlcNAcylation has been well studied in mammalian, and more than 500 proteins have been identified to be O-GlcNAcylated. In Arabidopsis, it has two O-GlcNAc transferase (OGT), SPINDRY (SPY) and SECRET AGENT (SEC). And the double mutant of these two has shown to be lethal at embryo stage, which likely indicates that they should essentially function in overlapped and uniquely in plants. In spite of those, OGT targets remain to be found yet in plants. So in this study, we will show the occurrence of O-GlcNAcylation and characterization of the apparent O-GlcNAcylated targets at various development stages in Arabidopsis.

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Triterpenoid saponins are sugar-modified triterpene derivatives that have various functions, anti-cancer, anti-oxidant, and anti-microbial. Cereals and grasses are generally deficient in this secondary metabolite except for oats. Our final goal is that the anti-microbial saponin is available for the rice. We reported already that 1) we found twelve Oxidosqualene cyclase homologues in rice genome using knowledge in oats, but it was likely that no one was involved in the triterpenoid biosynthesis pathway, 2) we also found twelve CYP51 sterol demethylase homologues in rice genome. It was likely that one is for rice steroids biosynthesis pathway, and another is for rice triterpeneoids biosynthesis pathway. Here, we report that 1) we made several transgenic rice lines introduced Avena strigosa beta-Amyrin Synthase 1 gene, 2) analyzed the triterpenoids and their intermediates extracted from the transgenic lines by GC/MS, 3) and demonstrated the accumulation of only beta-Amyrin in the transgenic rice without the other intermediates and/or the triterpenoids.

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Caffeine and theobromine are purine alkaloids that are present in high concentrations in plants of some species of Camellia. The main caffeine biosynthetic pathway involves in three S-Adenosyl-_L-methionine (SAM)-dependent methylation steps. SAM-dependent N-methyltransferases play an important role in the regulation of caffeine biosynthesis. Caffeine synthase catalyzes 1-N and 3-N-methylation reaction of mono- or di-methylxanthines, on the other hand, theobromine synthase catalyzes 3-N-methylation reaction of 7-methylxanthine. Recently we reported occurrence of theobromine synthase genes in purine alkaloid-free species of Camellia plants. Caffeine synthase and theobromine synthase are members of motif B' methyltransferase family which have motif B' and YFFF region.
In this study, we isolated 20 motif B' methyltransferase genes from 12 species of Theaceae. Recombinant enzymes from Gordonia acuminata and Eurya japonica showed theobromine synthase activity and salicylic acid carboxylmethyltransferase activity, respectively. The molecular evolution of these methyltransferases were discussed.

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Japanese morning glory (Ipomoea nil) shows various flower colors except black and yellow. In the Edo era, dark yellow flowers were drawn, but these mutants were extinct. Carotenoid is one of the yellow pigments in flowers. For example, an Ipomoea sp., distant relative of Ipomoea nil bears dark yellow flowers with carotenoid. Expression of genes in carotenoid biosynthetic pathway after the isopentenyl pyrophosphate isomerase (IPI) in a white flower of Ipomoea nil, is lower compared to the yellow flower of Ipomoea sp. (Yamamizo et al., 2009). Then, using genetic transformation, we introduced genes in carotenoid biosynthetic pathway to Ipomoea nil to make flower yellow. We first examined the flavanone 3-hydroxylase (F3H) promoter derived from chrysanthemum, and found pF3H::GUS shows petal specific expression in Ipomoea nil. Next, we transformed genes of two carotenoid biosynthetic enzymes, namely, genranylgeranyl pyrophosphate synthase (GGPS) and phytoene synthase (PSY) under F3H promoter. Results of HPLC analysis showed transgenic flowers contained higher level of β-carotene compared to non-transformant.

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S-oxygenation of S-allyl-L-cysteine (SAC) is the last step of alliin biosynthesis. The degradation of alliin produces various organosulfur compounds, which show antimicrobial and anticancer properties. In Arabidopsis thaliana, the S-oxygenation of glucosinolates is catalyzed by flavin-containing monooxygenases (FMOs). Under the assumption that SAC is S-oxygenated by FMOs, we cloned the FMO-like gene (AsFMO1) from garlic by using the primers designed from FMO-like EST sequences of onion. We conducted RT-PCR and 5'/3'-RACE using RNA extracted from a garlic as a template. The ORF of AsFMO1 encodes 457 amino acids containing FAD-binding motif and NADP-binding motif, which are essential for FMO activity. AsFMO1 showed 51-55% identities with FMOs, which catalyze S-oxygenation of glucosinolates in A.thaliana. The functional analysis of AsFMO1 recombinant protein expressed in Escherichia coli is in progress.

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Glucosinolates (GSLs) are sulfur containing glycosides, characteristic secondary metabolites to Brassica family. GSLs are reported to function as defense chemicals against harmful insects or pathogens. GSLs are considered as a potential storage source of sulfur, because they are accumulated in seeds and their decomposition is promoted under sulfur deficiency.
In the case of Arabidopsis thaliana, it has been reported that GSLs content in seeds is the highest among the tissues and there is a difference in chemical composition of GSLs between seeds and leaves. However, the detail of GSLs content and profile during the developmental stage is not clear.
In this study, we measured the changes of 24 types of GSLs during the developmental stage of siliques, until 10 days after flowering. The total content of GSLs in siliques was not increased until 5 days after flowering and was increased from 6 days to 10 days after flowering. During this period, 4-Methylsulfinylbutyl GSL was decreased, whereas 4-Methylthiobutyl GSL and 4-Benzoyloxybutyl GSL were increased. We also analyzed aop3, one of the mutants with different GSLs profile compared to that of wild type, in the same way.

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Botryococcus braunii is a green colonial fresh water microalga that produces large amounts of hydrocarbons. This alga is classified into three races (A, B, and L), depending on the type of hydrocarbons synthesized. The B race produces triterpenoid hydrocarbons, referred to as botryococcenes, whereas the A race accumulates alkadienes and alkatrienes derived from fatty acids. The L race produces a tetraterpene hydrocarbon called lycopadiene. We reported the botryococcenes accumulated by B70 strain (race B) were derived from the MEP pathway in the 51st annual meeting. In this study, we isolated two genes (BbDXS1, BbDXS2) encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which catalyzed the first step of the MEP pathway. Two genes showed features similar to known DXS genes from other plants, including the presence of an N-terminal targeting sequence and a conserved thiamine pyrophosphate binding domain typical of transketolase-like sequences. The characterization of two DXSs was identified by the analysis of the recombinant enzymes. Further studies are now in progress to examine expression profiles of two DXS genes.

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Cyanobacteria possess a CO₂-concentrating mechanism including some transporters that take part in this mechanism. One of these is the sbtA encoding Na⁺ dependent HCO₃^- transporter. It have also been reported that the PxcA deficient mutant was inhibited CO₂ uptake under acidic conditions.
To examine the introduction of the inorganic carbon transportation mechanism of the cyanobacteria into plants, we attempted to generate transgenic plants in which pxcA or sbtA was overexpressed. The cyanobacteria gene expression binary vectors were introduced into A.thaliana via Agrobacterium-mediated gene transfer. First generation lines were obtained by drug resistance screening. These have not visibly shown morphological abnormality. In the next generation, we examined mRNA expression level by RT-PCR and finally got some transgenic plants overexpressing each transporter gene of cyanobacteria. However, the expected protein could be detected by western blotting in the only PxcA overexpressed transgenic plants. Analysis of the effect of salt or pH conditions on growth in early period of self-pollinated transgenic plants is in progress.

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Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are play an important role in human health, prevention of heart and circulatory disease and brain development in infancy. At present, fish oil is used as a major commercial source of EPA and DHA, but its purification involves many problems. Therefore, searching for alternative sources of EPA and DHA is important and necessary. Marine microalgae can be considered as a good alternative source of EPA and DHA. In this study, we screened and identified the marine microalga KU01 with high EPA and DHA contents, and studied the optimal cultivation condition providing maximum EPA and DHA yields. The growth rate of the marine microalga KU01 was the highest at 25 degrees C under a constant light intensity at 70 μE/m²s at an air flow rate of 150 mL/min in medium contained 0.5 mM CH₄N₂O (pH 7.0). In contrast, there were no significant changes in the content of EPA and DHA. The maximum EPA and DHA yields achieved in flasks were 6.1 and 1.3 mg/L, respectively.

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Oxidative signaling via reactive oxygen species (ROS) is essential for plant response to abiotic and botic stresses. To clarify molecular mechanism of oxidative signaling derived from chloroplasts, we have created the system for transient suppression of thylakoid membrane-bound ascorbate peroxidase in Arabidopsis plants. Microarray analysis using this system revealed that ROS derived from chloroplasts affect the expression of approximately 800 genes, including genes involved in the oxidative signaling. Here we isolated and characterized paraquat-induced photooxidative stress sensitive (pss) and insensitive(psi) mutants from knockout lines lacking each ROS-responsive gene.
Among 241 lines, several psi and pss mutants were isolated and characterized. Some PSI and PSS genes encoded transcription factors and protein kinases, which are responsive to pathogens. The transcript levels of almost PSSs and PSIs were changed in response to high light or treatment with paraquat. Now, we are analyzing sensitivity of the mutants to treatment with salicylic acid or elicitor to clarify the function of each PSI and PSS gene.

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Chloroplasts are one of the major sources of reactive oxygen species (ROS), and therefore are also sources of oxidative signaling. Recently we have created the system for chemical-inducible suppression of thylakoid membrane-bound ascorbate peroxidase (tAPX) in Arabidopsis plants in order to clarify physiological function of oxidative signaling via ROS derived from chloroplasts. Microarray analysis revealed that ROS derived from chloroplasts affect the expression of a large number of genes, some of which are involved in plant response to chilling and pathogens. Here we analyzed the effect of tAPX suppression on plant sensitivity to stresses.
The suppression of tAPX expression decreased expression of the genes encoding key regulators of chilling signaling, and thus increased plant sensitivity to chilling stress, while it decreased the transcript levels of genes involved in pathogen response without increasing salicylate level. These findings suggest that the oxidative signaling via ROS derived from chloroplasts is involved in response to abiotic and biotic stresses. Now, we are analyzing the effect of tAPX suppression on plant sensitivity to elicitors and pathogen.

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In response to cold, E. coli accumulates cold shock proteins (CSPs) that function as RNA chaperones. Plant cold shock domain (CSD) proteins contain a CSD that is highly conserved with bacterial CSPs. Wheat and Arabidopsis CSD proteins have been characterized and shown to have an RNA chaperone activity in our previous studies. Here, we performed functional characterization of AtCSP2, one of the four Arabidopsis CSPs (AtCSP1-4). AtCSP2 overexpression plants showed delayed germination, smaller plant size, late flowering and were less freezing tolerant than wild-type after cold acclimation. A T-DNA mutant with decreased AtCSP2 expression exhibited no perceptible phenotype on plant growth and freezing tolerance. The mutant was crossed with a knock-out mutant of AtCSP4 which shows the highest homology to AtCSP2, and the resulting double mutant showed early flowering and increased freezing tolerance after cold acclimation. Real-time PCR analysis of the double mutant revealed up-regulation of CBF transcription factors and their downstream genes. These data suggested that AtCSP2 regulates induced freezing tolerance during cold acclimation in Arabidopsis.

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Stomata open in response to blue light. Blue light is absorbed by phototropin as a receptor and activates the plasma membrane H+ pump in stomatal guard cells. The activated pump creates a driving force to uptake K+ through the inward-rectifying K+ channels. However, entire signaling components for the stomatal opening are largely unknown. In order to identify the components, we screened and isolated Arabidopsis mutants impairing light-induced stomatal opening using infrared thermography. In this study, we characterized a mutant named as 24-H9 that indicated the high leaf temperature under the light condition. Stomata of 24-H9 did not open by light in both intact leaves and epidermis, but the mutant guard cells normally showed blue light-induced activation of the H+ pump. Furthermore, expressions of the K+ channel KAT1 and AKT1 decreased 65% in guard cells of 24-H9. We performed map-based cloning and found that 24-H9 gene was the DWARF5 gene, which encodes one of the biosynthetic enzymes for phytohormone brassinosteroids. The results suggest that brassinosteroids may regulate the blue light-induced stomatal opening via upregulation of the K+ channels in guard cells.

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A novel protein PCaP1 (plasma membrane associated cation-binding protein-1) was found in Arabidopsis thaliana. PCaP1 is localized in the plasma membrane via N-myristoylation and binds Ca²⁺, Cu²⁺, PtdInsPs, and CaM/Ca²⁺ (Nagasaki et al. 2008a, b). A loss-of-function mutant pcap1 becomes sensitive to excessive Cu²⁺ and pathogens. In this study, we investigated intracellular localization and dynamics of PCaP1 by expressing a PCaP1-GFP construct. Fluorescence of PCaP1-GFP was clearly detected on the plasma membrane at even intensity in almost all tissues. Under stress conditions, such as excess Cu²⁺ and an elicitor, the fluorescence intensity of PCaP1-GFP increased. In guard cells, PCaP1-GFP was detected in outside half part of the plasma membrane. When we measured stomatal apertures of the wild type and pcap1 leaves, the stomatal apertures of pcap1 were higher than that of wild type plants. Thus, we compared stomatal conductance and growth of them under dark and high CO₂ concentrations. From these results, we estimate that PCaP1 is involved in signal transduction through interaction with PtdInsPs and CaM/Ca²⁺ in various physiological phenomena including stomatal closing.

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Production of reactive oxygen species (ROS) by NADPH oxidases has been shown to play crucial roles in plant signaling including regulation of biotic and abiotic defense responses, programmed cell death and root hair development. By heterologous expression in HEK293T cells, we showed that a respiratory burst oxidase homolog (rboh) proteins, AtrbohC and D, possess ROS-producing NADPH oxidase activity that is synergistically activated by binding of ^Ca2+ and phosphorylation. Ten boh genes, AtrbohA-J are present in Arabidopsis. Though genetic studies suggest functional diversity of each Atrboh, their molecular characterization still remain limited. We are attempting to perform comprehensive comparative analyses of the 10 isozymes, AtrbohA–J. ROS-producing activity and its regulation of AtrbohA-J heterologously expressed in HEK293T cells, as well as expression patterns of each isozyme will be discussed.

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Receptor-like kinases (RLKs) comprise a large gene family in Arabidopsis genome and play important roles. We analyzed a receptor-like cytosolic kinase gene, ARCK1, whose expression was induced under water stress. ARCK1 belongs to a cysteine-rich repeat (CRR)-RLK subfamily, although ARCK1 has only a cytosolic kinase domain. Expression of the ARCK1 gene was elevated by water stress and abscisic acid (ABA) treatment mainly in leaves. The microscopic analysis of transgenic plants expressing sGFP-ARCK1 revealed that the ARCK1 protein was localized on the cytosol. We also analyzed phenotypes of ARCK1 knockout mutants, arck1-1 and arck1-2. The arck1 mutants showed higher sensitivity in the inhibition of cotyledon greening by ABA compared to the wild-type plants. In Arabidopsis genome we found an ARCK1 homologous gene (ARCL1) whose expression is also induced by water stress in leaves. ARCL1 is a CRR-RLK protein and the ARCL1-GFP fusion protein was observed on the plasma membrane. Using the yeast two-hybrid system and BiFC analysis in plant cells, we detected interaction between ARCK1 and ARCL1. Currently we are analyzing a double mutant of arcl1 and arck1 concerning the ABA responsiveness.

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Cold tolerant plants acquire freezing tolerance in response to exposure to non-freezing temperature. In higher plants, cold stress can cause increased accumulation of ABA, which could affect the signaling and regulatory pathways that direct the low temperature responses. In contrast, the role of ABA in cold acclimation process is not clearly understood in bryophytes. Protonema cells of the moss P.patens acquire freezing tolerance in response to cold and ABA treatment. In this study, we examined effects of low temperature treatment on freezing tolerance of the mutant and transgenic lines of P.patens. Low temperature treatment for seven days of wild type plants increased the freezing tolerance significantly but the treatment had little effect on that of ABA-insensitive lines. Protein and sugar analyses indicated that cold treatment induces accumulation of specific LEA-like proteins and soluble sugars in wild type, whereas the treatment increased only soluble sugars but not LEA-like proteins in the ABA-insensitive lines. These results indicate that low temperature affects ABA signaling in P.patens,leading accumulation of LEA-like proteins, which are required for freezing tolerance.

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Although a histidine kinase Hik33 in the cyanobacterium Synechocystis sp. PCC 6803 responds to various stress conditions and regulates the expression of several genes, it is not clear how it perceives the stimuli. In general, histidine kinase consists of two modules, a signal-input domain (SID) in the N-terminal region and a signal-transmitter domain in the C-terminal region. In order to analyze function of SID of Hik33 in vivo, we developed a technique to express a chimeric histidine kinase containing a SID from Hik33 and a signal-transmitter domain from a phosphate-deficient responding histidine kinase, SphS, in Synechocystis cells. The deletion of the PAS domain in the chimeric histidine kinase increased the activity of alkaline phosphatase whose expression was regulated via the chimeric histidine kinase. Then we substituted each 14 conserved amino acids in the PAS domain. Two of them decreased the kinase activity of the chimeric histidine kinase. These results indicate that the PAS domain of Hik33 is important for the fine-tuning of the kinase activity.

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In rice, plant activator such as benzothiadiazole (BTH) acts on the salicylic acid signaling pathway and thereby transcriptionally upregulates a transcription factor WRKY45 leading to defense induction. Overexpression of WRKY45 (WRKY45-ox) conferred rice with strong resistance against fungal blast (M. grisea) and bacterial leaf-blight (X. oryzae) diseases. To investigate the mechanism underlying the disease resistance conferred by WRKY45, we performed microscopic and microarray analysis in M. grisea-inoculated WRKY45-ox and BTH-treated rice plants.
Upon inoculation of M. grisea to WRKY45-ox rice, strong inhibition of the invasion of fungal penetration peg was observed. Hypersensitive-response-like cell death was also observed where rice cells permitted hyphal invasion. Microarray revealed that the genes for biosynthesis of diterpenoid phytoalexin, momilactone A, upregulated after M. grisea infection in WRKY45-ox rice. The genes were not upregulated in BTH-treated rice plants, but upregulated after M. grisea infection to them. The upregulation was not observed in WRKY45-knockdown rice plants. These results indicate that WRKY45 primes defense genes in BTH-treated rice.

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To improve immune responses to the chitin elicitor (CE) in rice, we have constructed the chimeric receptor CRXA, which consists of a receptor for CE, CEBiP, and the intracellular kinase domain of XA21 that confers resistance to rice bacterial blight. Rice plants expressing CRXA increase cell death after treatment with CE and show more resistance to rice blast disease (Kishimoto et al., 2010). To test whether the combination of CEBiP and other types of receptor-like protein kinases (RLKs) works as a chimeric receptor, we constructed CRPi, which encodes ectodomain of CEBiP and the intracellular region of a homologue of R protein for rice blast identified in rice cultivar Nipponbare. Rice cultured cells expressing CRPi showed the increased level of oxidative burst after treatment with CE, but the level of cell death was not as much as in CRXA-expressing cells. Rice plants expressing CRPi showed more resistance to the blast fungus in leaf sheaths and blades. These results suggest that the fusion protein of CEBiP and the intracellular region of RLKs other than XA21 also works as a functional receptor leading to the enhancement of defense responses and resistance to rice blast.

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Rice transcription factor WRKY45 plays a central role in chemical-induced resistance through salicylic-acid pathway. Transgenic rice plants overexpressing WRKY45 driven by maize ubiquitin promoter exhibited strong multi-diseases resistance to blast and leaf-blight diseases. However, they showed growth retardation and yield decrease, which hampers their practical application. To optimize WRKY45 expression in regard to disease resistance and agricultural traits, we generated several rice lines overexpressing WRKY45 driven by various promoters.
We isolated 2-kb upstream sequences of 22 genes with various expression characteristics, fused them upstream of WRKY45 cDNA, and introduced the constructs into rice. Among transformants, those that express WRKY45 under the control of OsUbi1, EF1α, OsUbi7 (P_OsUbi7) promoters exhibited enhanced resistance to both blast and leaf-blight diseases, as well as improved agricultural traits. Especially, P_OsUbi7:WRKY45 lines exhibited agricultural traits roughly comparable to those of non-transgenic line in a greenhouse whose conditions follow external ones. These results make them promising candidates for practical application.

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Pseudomonas cichorii causes bacterial rot in lettuce, and apoptotic programmed cell death is closely associated with the disease development. To elucidate the molecular mechanisms of lettuce-P.cichorii interaction, we have been isolated and analyzed P. cichorii-responsive genes (PcRG) from lettuce plants. In this report, we focused on PcRG4-5-2 gene, of which expression was strongly induced in lettuce leaves inoculated with P. cichorii. Deduced amino acid sequence of PcRG4-5-2 contained Plant AT-rich sequence and Zn-binding protein (PLATZ) motif. Then, we designated PcRG4-5-2 as Lactuca sativa PLATZ (LsPLATZ). Expression of LsPLATZgene was not induced by inoculation with Pectobacterium cartovora subsp. cartovora, P. syringae pv. syringae and P. syringae pv. phaseolicola. The expression was scarcely affected by the treatment with salicylic acid, jasmonic acid, and hydrogen peroxide. These results suggested thatLsPLATZ gene may regulate novel intracellular signaling pathway(s) and have important roles in development of bacterial rot disease in lettuce.

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AAL-toxin is a pathogenicity factor of Alternaria alternata f. sp. lycopersici. AAL-toxin is sphinganine-analog and disturbs ceramide biosynthesis, resulting in the inducing programmed cell death (PCD) in host plants. To investigate candidate genes involved in AAL-toxin-induced cell death, we employed virus-induced gene silencing (VIGS) and Nicotiana umbratica that is sensitive to the AAL-toxin. We have showed that ethylene signaling pathway plays a pivotal role in AAL-toxin-induced cell death and identified NuERF4, which is AP2/ERF transcription factor, as a mediator for AAL-toxin-induced cell death. To further investigate role of ethylene signaling in PCD, we used Arabidopsis thaliana and fumonisin B1 (FB1) that is structural analog of AAL-toxin. To find A. thaliana ERF4 homolog (AEH1), multiple alignment analysis was performed using amino acid sequences of the AP2/ERF domain. In aeh1 mutant, FB1-induced cell death was suppressed. Furthermore, we produced AEH1-overexpressed plants and identified genes up- or down-regulated by AEH1-overexpression by microarray analysis.

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Plants have the ability to recognize microbe-associated molecular patterns (MAMPs) from wide range of potential pathogenic microbes and trigger multiple defense responses. We so far identified two LysM receptor-like kinases in Arabidopsis and rice, CERK1 and OsCERK1, respectively and showed that both of them play a critical role in chitin elicitor signal transduction in each plant species(1). In addition, it was reported that OsCERK1 interact with Hsp90 and Hop/Sti1, which form a complex with various factors and contribute for the transportation from ER to plasma membrane and downstream signaling. We surveyed for the candidate proteins that interact with cytoplasmic domains of these receptors and participate in the downstream signaling, by using yeast two-hybrid analysis with kinase-active or inactive construct as a bait protein. As a result, we obtained several candidates such as E3 ubiquitin ligases, cytoplasmic protein kinases, and proteins containing forkhead-associated domains. Presently, we are analyzing the function of these candidates in chitin signaling by using corresponding knockout mutants.
(1)PNAS, 104, 19613; Plant J., 64, 204.

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Chitin is a typical MAMP derived from fungal cell walls and has an ability to induce immune responses in plants and animals. We recently identified CEBiP (rice) and CERK1/OsCERK1 (Arabidopsis/rice) as critical components for chitin signaling in these plants. They are a GPI-anchored protein and a receptor-like kinase (RLK), respectively. To understand whether Arabidopsis requires the presence of CEBiP-like molecule(s) for chitin signaling, we characterized CEBiP homologues in Arabidopsis. Chitin binding activity of CEBiP homologs were analyzed by using tobacco BY-2 expression system and affinity labeling with biotinylated-chitooctamers. One of these homologues (AtCEBiP1) expressed in BY-2 cells showed a high-affinity binding for chitin oligosaccharides. The binding characteristics of AtCEBiP1 was very similar to rice CEBiP. However, the knock-out mutant of AtCEBiP1 showed chitin-induced ROS generation similar to wild type Arabidopsis. These results indicated that AtCEBiP1 is biochemically functional as a chitin binding protein but does not significantly contributes to signaling, in other words, AtCERK1 may be sufficient for chitin perception and signaling in Arabidopsis.

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In recent years, it has been emerging that the defense systems based on the recognition of microbe-associated molecular patterns (MAMPs) in plants and animals show various similarity. We previously reported that bacterial LPS act as a potent elicitor to induce defense responses in rice cells(1). We also found that simultaneous treatment of LPS and chitin amplify defense responses synergistically.
Although LPS is detected through the recognition of Lipid A in animal innate immunity, it is not clear which part of LPS is recognized by plants. To confine the structure required for the biological activities of LPS in rice, we used LPS from Pseudomonas aeruginosa and tried to dissect the structure required for defense induction. Examination of the Lipid A and polysaccharide obtained by mild acid hydrolysis of LPS showed that the Lipid A induced defense responses in rice cells but the remaining portion, polysaccharide fraction, did not. These results showed that rice cells recognize LPS through Lipid A, indicating that the recognition mechanism of LPS is evolutionary conserved between animals and plants.
1)Desaki et al.,Plant Cell Physiol.,47,1530(2006)

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Pb1 (Panicle Blast1) is a panicle blast resistance gene derived from indica rice "Modan". The Pb1 resistance is durable and of broad spectrum for fungal races. We previously reported that Pb1 encodes a CC-NB-LRR-type R protein and the protein interacts with WRKY45, which plays a role in induced resistance via the salicylic pathway. To test the WRKY45 dependence of Pb1-mediated blast resistance, we knocked down WRKY45 in a Pb1 cultivar (Pb1-line/WRKY45-kd) and Pb1-ox Nipponbare (Pb1-ox/WRKY45-kd). In both the cases, Pb1-mediated blast resistance was largely compromised by WRKY45 knockdown, suggesting that Pb1 resistance is dependent on WRKY45. Addition of nuclear export sequence to Pb1 largely abolished the resistance upon its overexpression, indicating that nuclear localization is necessary for Pb1 function. Recently, WRKY45 was found to be regulated by ubiquitin proteasome system (UPS). We found that Pb1 protected WRKY45 from UPS via specific protein-protein interaction. Blast infection induced higher levels of WRKY45 accumulation in Pb1-ox rice than in control rice. These results suggest that Pb1 resistance is mediated by protection of WRKY45 from its proteasome degradation.

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Hypersensitive response (HR) cell death that is one of the plant immune responses is induced by the effector protein secreted into plant cells through the Hrp secretion system (Type III secretion system, T3SS). Acidovorax avenae N1141 avirulent strain induces plant immune responses in rice. To clarify the induction mechanism of HR cell death in Oryza sativa (rice), we analyzed genome sequence of N1141 strain and found 30 kbp hrp gene cluster encoding T3SS. Since the deletion mutant of T3SS in N1141 strain (N1141Δhrc) did not induce HR cell death, effectors secreted through T3SS would cause HR cell death in rice. Therefore we next attempted to identify the effectors using proteomics. Several proteins were specifically accumulated in N1141Δhrc strain when accumulated proteins in N1141 and N1141Δhrc strains were compared. Among the identified protein, disruption mutation in Ahp1 gene resulted in no HR cell death in cultured rice cells. Further transformation of Ahp1 in rice cells caused induction of HR cell death, suggesting that Ahp1 is a candidate of effector involved in induction of HR cell death.

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Lipopolysaccharide (LPS), which is a primary constituent of the outer membrane of gram-negative bacteria, is a pathogen-associated molecular pattern. Although LPS causes defense-related responses in plants, none of the molecules consisting of the LPS recognition mechanism have been identified. We focused on human LPS-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI), which can bind to LPS directly and regulate the physiological responses to LPS. Arabidopsis has two LBP/BPI-related genes (AtLBPs). We investigated their function on LPS-induced plant defense responses.
We demonstrated the direct binding between recombinant proteins of AtLBPs and LPS. After LPS treatment, AtLBP2 mutants showed the earlier initiation of pathogenesis-related 1 gene expression than wild-type. Furthermore, AtLBP2 mutants showed callose deposition with a lower concentration of LPS. Thus, AtLBP2 mutants exhibited higher sensitivity to LPS than wild-type, suggesting that AtLBP2 play an important role in negative regulation of LPS-induced signal transduction.

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To clarify cell death processes involved in plant immunity response, we have isolated and characterized Arabidopsis mutant nsl2( necrotic spotted lesion 2; also reported as the cad1) showed hypersensitive response seen in the lesion mimic mutants. In addition, the nsl2 mutant activated PR gene (pathogen related gene) and SA and JA pathway. Inoculation of nsl2 with virulence pathogen showed restriction of bacterial growth (Plant Cell Physiol. 2005, 46: 902-912). To understand NSL2 functions, we carried out yeast two-hybrid screening. We succeeded in isolation of NSL2 interacting-proteins which may relate to plant immunity. Further characterization will be reported.

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Many gram-negative bacteria that infect plants directly inject lots of effector proteins into host cells by using a protein secretion system, a so-called type III secretion (TTSS) system. The TTSS effector proteins are considered to be the primary virulence factors and strongly contribute to cause disease on the host plants. Therefore, it is likely that these effectors block the important steps in plant immune response. XopP is one of effector proteins secreted into rice cells through the TTSS of Xanthomonas oryzae pv. oryzae (Xoo : Furutani et al ., MPMI 22(1): 96-106 2009). To understand the inhibitory mechanism of host immune response by XopP, we have generated transgenic rice plants expressing XopP . The transgenic plants showed an enhanced susceptibility to the TTSS-deficient hrpX mutant of Xoo , indicating that XopP had the abilities to inhibit PAMPs-triggered immunity (PTI) inside plant cells. Interestingly, PR gene expression induced by the chitin elicitor was blocked in suspension-cultured rice cell expressing XopP . Furthermore, we found that XopP interacts with ARM repeating protein containing U-box domain. The role of target factor of XopP in PTI will be discussed.

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It is estimated that nitric oxide is produced and has functions during defense responses against pathogens in plant cells and has a function in signal transduction. S-nirtosylated is one of the post-translational protein modification which is caused by nitric oxide and regulates the protein functions. The identification of S-nirtosylated proteins during plant defense responses is important for elucidation of signal transduction mechanisms mediated by nitric oxide. In this research, we detected S-nirtosylated proteins in GSNO treated potato (Solanum tuberosum) leaf and tuber extract by using biotin switch method. S-nirtosylated proteins were purified and analyzed using nano liquid chromatography in combination with mass spectrometry. We identified proteins from tuber and leaves that represent candidates for S-nitrosylation, including many redox-related proteins.

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Chitin is a major MAMP (Microbe-Associated Molecular Pattern) for various fungi and its fragments, chitin oligosaccharides, are known to induce various defense responses in plant cells. Our recent study showed that two types of receptor, CEBiP¹⁾ and OsCERK1²⁾, play an important role for chitin elicitor signaling in rice. Yeast two-hybrid analyses suggested that CEBiP and OsCERK1 have an ability to form the receptor complex(es) as a hetero- or homo-oligomer. However, Blue Native PAGE and chemical-cross linking experiments indicated that these two molecules are separated from each other, or very weakly associated, in the plasma membrane. To investigate the possibility of the CEBiP-OsCERK1 receptor complex formation in rice cells, we applied immunoprecipitation analysis using the membrane preparation from the rice myc-tagged OsCERK1 cells and found both receptors are directly formed the receptor complex under the chitin elicitor treatment. Recently, we also found that CEBiP is a GPI-anchor typed receptor and is presence in lipid microdomain of rice plasma membrane. Further analyses are in progress.
1) Kaku et al, PNAS, 103, 11086 ('06)
2) Shimizu et al, Plant J., 64, 204 ('10).

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The oomycete pathogen, Phytophthora infestans, is the causal agent of potato late blight, which is one of the most destructive and economically important plant diseases. We previously showed that mature N. benthamiana is resistant to P. infestans. Screening with virus-induced gene silencing (VIGS) identified genes for calreticulin 3 (NbCRT3), a molecular chaperone for glycoproteins in ER, and N-acetyl glucosamine phosphotransferase (NbGPT), an enzyme for N-glycosylation, as essential genes for the resistance of N. benthamiana against P. infestans. In NbCRT- and NbGPT-silenced plants, production of reactive oxygen species (ROS) induced by the treatment of INF1 elicitor, a secretory protein derived from P. infestans, was significantly reduced. Similarly, silencing of UDP-glucose:glycoprotein glucosyltransferase (UGGT), another component of the ER quality control of glycoprotein folding, compromised ROS production and hypersensitive cell death in response to INF1 treatment. These results suggested that N. benthamiana have N-glycosylated extracellular receptor, which undergoes ERQC during their folding and maturation, for the recognition of elicitor from P. infestans.

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Mature N. benthamiana shows strong resistance to potato blight pathogen Phytophthora infestans. Previously, we reported that ethylene signaling mediated by EIN2 is regulating the expression of genes for phytoalexin biosynthesis, NbEAS and NbEAH, which is essential for resistance of N. benthamiana to P. infestans. By screening using virus-induced gene silencing (VIGS), we isolated genes for a PDR-type ABC transporter, NbPDR1, as a required gene for disease resistance. Given PDR1 from N. plumbaginifolia is previously reported as an exporter of a terpenoid sclareol, the role of NbPDR1 was expected to be a transporter of terpenoid phytoalexin capsidiol to the site of pathogen infection. However, NbPDR1-silenced plant showed significantly reduced accumulation of capsidiol induced by treatment of INF1, an elicitor protein derived from P. infestans. In contrast, INF1-induced production of reactive oxygen species was not affected by the silencing of NbPDR1. These results support the conclusion that PDR1 is involved in phytoalexin production in defense of N. benthamiana to P. infestans, possibly intracellular transport of capsidiol precursor in the process of phytoalexin production.

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Most studies of plant-pathogen interactions have focused on establishment of plant immunity. On the other hands, little is known about host factors required for pathogen fitness and modified by the invading microorganism to avoid plant immunity.
To elucidate the molecular mechanisms of disease susceptibility in plants, we identified NbPAP (Nicotiana benthamiana phosphatidic acid phosphatase), which is specifically expressed in Nicotiana plants by inoculation with pathogenic strain of Ralstonia solanacearum.
Phosphatidic aicd, which is substrate for PAP, functions as an important signaling molecule involved in diverse physiological processes. To elucidate the role of PAP in plant-pathogen interactions, we created NbPAP-silenced plants using virus-induced gene silencing. Silencing of NbPAP reduced PAP activity leading to accumulation of PA. In the silenced plants, growth of R. solanacearum and the appearance of wilt symptom decreased. These results indicate that PAP negatively regulates plant immunity via accumulation of PA.

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GTH4, cultured cells of interspecific F₁ hybrid of N. gossei and N. tabacum, both reproductively isolated, dies at 26 C transferred from 37 C in cultures. We have shown that cell death at 26 C was controlled by quantitative balance between reactive oxygen species (ROS) and nitrogen monoxide (NO), especially presence of both NO and H₂O₂ were critical to initiation of cell death. Involvement of MAPK signalling in ROS generation was also verified. However, little is known about how NO generate in cell under impermissible temperature. We present here effects of inhibitors on NO generation. L-NAME, inhibitors of animal nitrogen monoxide synthetase (NOS), suppressed cell death but had no effect on extracellular NO amount. Neither NO amount nor cell death was affected by AG, inhibitor of inducible NOS. Tungstic acid, inhibitor of nitrate reductase, did not affect NO generation, but effectively reduce rate of cell death. Analysis of these inhibitors on intracellular NO amount is in progress. We further analyzed whether ROS and NO in plants act as same way as in cultured cells, and found they act as death signal in plants under quantitative balancing way.

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Spiraea thunbergii (Rosaceae) allelochemicals, cis-cinnamonyl acid (cis-CA), had high growth-inhibitory activity against several plants. To elucidate the mechanism of growth-inhibitory activity of cis-CA, gene expression of Arabidopsis thaliana was analyzed by using DNA microarrays. After 6-h exposure, the expression of 75 genes was significantly up-regulated, and that of 116 genes was significantly down-regulated. The up-regulated genes mainly consisted of pathogenesis-related genes, and the down-regulated genes consisted of jasmonic acid/ethylene-responsive genes, superoxide dismutase genes encoding defensive enzymes against reactive oxygen species, the genes expressed specific to cell walls essential for cell-enlargement process, and a transcription factor up-regulated in response to environmental stresses. Based on these results, cis-CA acted mainly in two mechanisms: an influence on plant defense systems against environmental stresses and deregulation of cell wall-associated genes involved in cell elongations.

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Sulfoquinovosyl diacylglycerol is one of lipids that construct chloroplast membranes. We recently reported that a green alga, Chlamydomonas reinhardtii, when starved for sulfur (S), degrades sulfoquinovosyl diacylglycerol to ensure an intracellular S-source for protein synthesis [1]. In the course of this study, it was newly found that green algae accumulate triacylglycerol (TG) under S-starved conditions. C. reinhardtii contained TG and free fatty acids as neutral lipids at minor amounts under normal growth conditions, then increasing the TG content specifically 24-96 h after onset of S-starvation. A similarly specific increase in the TG content was observed also with another green alga, Chlorella kesslerli 11h. In contrast, neither TG nor free fatty acids accumulated in C. reinhardtii when starved for phosphorus. These results indicated that accumulation of TG is a response that is not general to nutrition-deficient stresses, but specific to S-starvation at least. Green algae are known to accumulate TG under nitrogen-starved conditions. Accumulation of TG may be related to a decrease in protein synthesis.
[1] K. Sugimoto, M. Tsuzuki and N. Sato: New Phytol. 185, 676 (2010).

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We have been analyzing the role of actin filaments on chloroplast surface in the chloroplast photorelocation movement, using Marchantia polymorpha thallus cells expressing tdTomato-talin. In the previous experiments using cytoskeletal inhibitors, actin filaments were shown to play a crucial role in the chloroplast movement. On the chloroplast surface, short actin filaments (cp-actin filaments) were seen and they showed continuous change in their organization. When reorganization of cp-actin filaments during accumulation and avoidance responses induced by microbeam irradiation with low and high fluence-rate was examined, biased relocalization of cp-actin filaments on the leading half of chloroplast was revealed both in avoidance and accumulation movement. The speed of chloroplast movement correlated with the degree of biased localization of cp-actin filaments. Blue light irradiation on whole cells also induced reorganization of cp-actin filaments similar to that under microbeam irradiation. The evidences indicate the significance of biased configuration of cp-actin filaments in the mechanism of chloroplast relocation.

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Contrary to the established notion that the apical hook of dark-grown dicotyledonous seedlings opens in response to light, we found in tomato that the apical hook curvature is exaggerated by light through phytochrome actions. Based on some simulation experiments for germination in the field and anatomical observations of the seed part of germinating seedlings, we have proposed that light-induced hook exaggeration (LIHE) plays an important role in survival by securing the seed coat release in the field. Interestingly, for the LIHE to occur, the presence of the seed coat and/or endosperm is essential. Once the seed coat carrying the yet unconsumed endosperm has been released, no LIHE occurs any more. Furthermore, LIHE is not limited to tomato, but also found in several other plant species, cotyledons of which are enclosed with unconsumed endosperm in the seed coat. Evidence such as these has led to the test of the ability of endosperm to induce LIHE and we found that the water extract from tomato endosperm applied to the tomato cotyledons free from seed coat and endosperm causes hook exaggeration under red light. Endosperm seems to provide some factor essential for LIHE.

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In Arabidopsis, UV and blue light stimulate expression of anthocyanin biosynthesis gene. In the strawberry Fragaria ananassa, UV light increases the red coloration in receptacle, whereas the effect of blue light or other wavelength is not reported yet. In this study, we examined the effect of irradiation on the red coloration of strawberry (cv. Sachinoka) by using various wavelength of LED. 24 h after irradiation of LED and incubated for 36 h in darkness, we measured anthocyanin content. As a result, 465 nm of blue LED irradiated fruits increased 3-fold amount of anthocyanin than green LED (525 nm) or red LED (625 nm) irradiation.
In addition, we examined functional analysis of blue light photoreceptor genes, phototropin (phot) and cryptochrome (cry), by agrobacterium-mediated RNA interference (RNAi). We observed the reduction of red color in the epidermis and the internal region of receptacle in the RNAi fruits compared with control fruits. We found that anthocyanin content in the RNAi fruits was lower than controls (control 100%, phot 56%, cry 45%).
These results indicate that both photoreceptors may be involved in anthocyanin biosynthesis pathway in the strawberry fruit.

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