Plants respond to wounding caused by mechanical stress or chewing of herbivore by induction of a various genes that act on wound healing and protection from subsequent invasion by phytopathogens. We had been reported that tobacco ERF genes were immediate early responsive genes to wounding. In addition, these genes were also induced by a translational inhibitor, cycloheximide (CHX). In this study, we performed macroarray analysis with probe sets for approximately 300 genes including transcription factors (AP2/ERF, Dof and Nac family). Twenty and 12 genes were identified as immediate early wound-responsive genes and CHX-responsive genes, respectively, with a greater than 3-fold change over control. All of overlapped 3 genes were encoding the ERF subfamily. On the other hand, 8 genes that belong to the Nac family were specifically up-regulated by wounding. Moreover, we analyzed in detail the expression pattern of these identified genes by real time PCR.

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TSK-associating protein1 (TSA1), which was identified as a protein that can interact with TONSOKU (TSK)/MGOUN3/BRUSHY1 involved in meristem maintenance, contains N-terminal putative signal peptide, novel glutamic acid-phenylalanine-glutamic acid (EFE) repeats, putative transmembrane domain, and C-terminal TSK-interacting domain. EFE repeats of recombinant TSA1 protein possesses CA2+-binding activity and the fluorescence of TSA1-GFP fusion protein appears as many spots in cytoplasm, suggesting that TSA1 is associated to endoplasmic reticulum (ER) or ER-derived vesicular organelle.
We identified disruptants for TSA1 and TSA1-like, with high sequence similarity to TSA1. However these disruptants or double disruptant did not show any tsk mutant-like morphological defects. The TSA1 and TSA1-like mRNAs were found predominantly in aerial part and roots, respectively. Furthermore expression of both of TSA1 and TSA1-like genes was induced in response to wounding and methyl jasmonate treatment. These results imply that TSA1 and TSA1-like are likely to be associated with wound-inducible organelle ER body.

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The Arabidopsis lesion initiation 3 (len3) mutant develops lesions on leaves without pathogen attack. len3 plants express pathogenesis-related ( PR ) genes and accumulate elevated levels of salicylic acid (SA). To determine the signaling pathway, which is activated in len3 plants, we crossed the len3 plants with nahG, npr1-1, and pad4-1 plants and analyzed the phenotypes of the double mutants. The len3-conferred phenotypes, including cell death and PR expressions were suppressed in len3 nahG plants. The phenotypes of len3 npr1 and len3 pad4 plants were almost the same as those of len3 nahG plants. However, although BTH treatment could restore cell death phenotype in len3 nahG plants, but not in len3 npr1 and len3 pad4 plants. This result might suggest the involvement of the PAD4-dependent signal for the cell death in len3 plants.

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Hypersensitive response (HR), which is a dominant plant response associated with resistance to pathogen attack, leads to rapid local cell death to restrict growth and spread of the pathogen. To clarify mechanisms involved in HR-induced cell death, FOX hunting system was used to isolate gain-of-function mutants. The isolated mutant, which shows brown- or black-colored lesions on leaves, is caused by over-expression of the gene encoding transcription factor that contains an ERF/AP2 domain. Microarray analysis using the mutant revealed that the genes involved in pathogenic resistance such as beta-glucanase, chitinase, defensine and H₂O₂-inducible GST are extremely up-regulated. These results suggest that the cell death phenotype in the mutant is due to activation of HR and biotic stress response. The detailed characterization of the mutant will be reported.

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A collection of 40 ecotypes of Arabidopsis varied in their responses to infection by C. higginsianum. Analyses suggested that the hypersensitive response and reactive oxygen species are important in a defense response. Crosses between the resistant and susceptible ecotypes suggested that the resistance was dominant and was conferred by a single locus, which we named RCH (for Recognition of Colletotrichum higginsianum). We mapped the RCH locus in several resistant ecotypes by using PCR-based microsatellite and amplified polymorphic sequence genetic markers. Our results indicated that the RCH1 locus was linked to the SSLP marker nga8, located at the chromosome 4. In addition, it is shown that Arabidopsis plants have a variety of RCH loci. Furthermore, expression analyses were performed using a microarray. In this meeting, we present expression profiles of regulatory genes in C. higginsianum-inoculation using a microarray.

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tpoxN1 encodes a tobacco peroxidase gene whose wound-induced expression is insensitive to known wound-signal compounds such as jasmonic acid and ethylene. To study the mechanism of the expression, 2-kbp and successive 5^'-deleted tpoxN1 promoters were introduced as GUS fusion genes into tobacco plants. The wound-induced GUS activities indicated that a jA- and ethylene- independent wound-responsive cis-element (JEIW) is present at -239/-200 in the promoter. Gel mobility shift assays suggested that a nuclear factor(s) prepared from wounded tobacco stems binds a 14-bp sequence (-229/-215) in the -239/-200 region in a sequence-specific manner. A mutation in the 14-bp region resulted in a decrease in wound-responsive GUS activity in transgenic plants. The 14-bp sequence contains no known wound-responsive cis-elements. Thus, the characteristic tpoxN1 expression is regulated by a novel 14-bp wound-responsive cis-element, JEIW.

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We previously showed that the promoter of the Ri-plasmid-borne agropine synthase gene (Ri-ags) of Agrobacterium rhizogenes had tissue-specificity to root and callus and wound-responsiveness in leaf and stem in tobacco. We identified a region of the promoter which was necessary for both of these expressional traits. Furthermore, we found binding activity in the nuclear extract of tobacco suspention cells to a 8-base motif inside the region, so we tentatively named the motif the wound responsive element (WRE). In this work, we characterized the WRE-binding activity using the electrophoretic mobility shift assay (EMSA). Comparing the WRE-binding activity in the nuclear extracts from various tissues, we detected a major DNA-protein complex at the same position in all the tissues examined except the hairy root where the mobility was greater. There was no obvious correlation between the WRE-binding activity and the promoter activity in these tissues.

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Calmodulins (CaMs), ubiquitous Ca²⁺ binding proteins in eukaryotes, are consisted of multiple isoforms in plants. It is suggested that plant CaM isoforms may play different roles in the plant cells because (1) these isoforms activate specific target enzymes in vitro respectively (2) they require different Ca²⁺ concentrations to activate enzymes (3) their expression profiles are unique. We have isolated 13 tobacco CaM genes (NtCaM1-13) classified into three isoforms (NtCaM1 / NtCaM3 / NtCaM13). To study their functions and specificity in defense response against pathogens in tobacco, we analyzed their transcriptional activation, and found that transcripts of genes encoding NtCaM1 and NtCaM13 isoforms accumulated during hypersensitive response in Tobacco mosaic virus (TMV)-infected tobacco leaves containing the N resistance gene. Transgenic tobacco with reduced expression of NtCaM1 and NtCaM13 were respectively obtained through an RNAi strategy. Using these plants will report the roles of NtCaM isoforms on defense signals.

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The ATPase associated with various cellular activities (AAA) protein of tobacco is specifically induced during the early phase of the HR, with a role in regulation of the defense system against pathogen. Suppression of NtAAA1 plants exhibited enhanced resistance to pathogen in comparison with WT plants. In order to clarify the molecular mechanism, we performed yeast two hybrid screening and identified a gene encoding an ADP ribosylation factor (ARF), a small GTP binding protein. In animal cells, ARF is known to be directly involved in regulation of vesicular trafficking and activation of phosholipase D. To date, plant ARF was shown to enhance disease resistance to pathogens, to induce salicylic acid (SA) -activated PR genes, and to interact with phospholipase D to increase jasmonic acid (JA) formation. NtAAA1 and NtARF are conceivable to be involved in regulation of PR gene expression by NtAAA1 functioning as a negative regulator for ARF.

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Lipid rafts are relatively resistant to solubilization at low temperature in nonionic detergent. These domains are known to be enriched in sterols and sphingolipids and postulated to be involved in transmembrane signaling. The aim of this study is to analyze the components of lipid raft domains associated with defense responses of rice.
First, we purified lipid raft proteins from rice cultured cells by using Triton-X 100 and sucrose density-gradient ultracentrifugation and we are now identifying the proteins in the raft fraction by mass spectrometry. Also, as our previous studies indicated that OsRac1, a small GTP-binding protein, functions as a key regulator of defense signaling in rice, we analyzed the raft fractions isolated from constitutive active OsRac1 (CA-Rac) and dominant negative-OsRac1 (DN-Rac) expressing rice cells. We investigated whether the raft components among Wild type, CA-Rac and DN-Rac cells were different.

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Generation of superoxide and influx of Ca²⁺ into the cells induced by Magnaporthe grisea hyphal elicitor was observed in the suspension-cultured transgenic cell line of rice (Oryza sativa) expressing aequorin. The patterns of superoxide generation and increase in cytosolic Ca²⁺ concentration were shown to be different from those observed in tobacco cells used as a non-host material.

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Jasmonic acid (JA) plays an important role as a signaling molecule in plant defense responses to wound stress and pathogen infection. Although there are various JA-inducible pathogenesis-related (PR) genes in rice, JA does not enhance the disease resistance against rice blast fungus Magnaporthe grisea. To resolve this discrepancy, we generated and analyzed JA-deficient rice plants (F7Ri) with co-suppression of both two intrinsic plastidial ω-3 fatty acid desaturases, OsFAD7-1 and OsFAD7-2, that supply the JA precursor linolenic acid. Unexpectedly, in F7Ri plants, the disease resistance against M. grisea was enhanced in a non-race-specific manner. Furthermore, the enhanced resistance in F7Ri was not restored by the methyl-jasmonate treatment. These results suggest that JA is not an effective regulator of the fungal resistance. Moreover, this study provides a novel insight that the oxidized derivatives of polyunsatulated fatty acids may be involved in rice-specific fungal resistance.

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We have investigated the function of a rice protein kinase OsPti1a, a homologue of tomato Pti1 isolated as an interactor of the R gene Pto. The OsPti1a functions as a negative regulator of resistance to blast fungus in rice, whereas the Pti1 plays as a positive regulator of bacterial resistance in Solanaceae. Introduction of the Pti1 complements the ospti1a mutant phenotype, suggesting that both proteins similarly act as kinases in signal transduction pathways in defence mechanism, diverged between dicot and monocot. To elucidate the molecular mechanism involving OsPti1a, yeast two hybrid screening was carried out to specify unknown players implicated in defence in rice. Two OsPti1a-interacting partners were isolated; one is a protein kinase showing similarity to the tomato Adi3 and the other is a carboxylesterase similar to the tobacco HSR203J. Their involvement upon the defence and the mechanism of signal transduction via phosphorylation will be discussed.

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Rice blast and bacterial leaf blight disease caused serious damage to the harvest and quality of rice crops. Ribosome-inactivating proteins (RIPs) are antifungal proteins commonly found in the most of plant species. We had found the introduction of rye RIP gene confer the resistance in rice to rice blast disease. In this report, we made modified acetolactate synthase gene (mALS) vector cassettes which contained a rye RIP cDNA under control of a leaf-specific E0082 promoter. We introduced it into a rice cultivar "Dontokoi". T₁ plants obtained from transgenic plants were inoculated with rice blast pathogen (Magnaporthe grisea). Some of transgenic lines showed strong resistance against the pathogen. These transgenic plants also suppressed the development of disease symptoms of bacterial blight pathogen (Xanthomonas campestris pv. oryzae). These results indicated that the introduced RIP genes enhanced the resistance against bacterial as well as fungal pathogens.

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Jasmonic acid (JA) is known to be an important signaling molecule in defense responses against pathogen attack. JA is synthesized in the octadecanoid pathway, which employs several enzymes including allene oxide cyclase (AOC) and 12-oxo-phytodienoic acid (OPDA) reductase (OPR). Recent studies in Arabidopsis suggest that OPDA, an intermediate in the biosynthesis of JA, is also involved in defense responses. However, little is known about physiological roles of OPDA in defense responses in rice. We generated transgenic rice plants in which the expression of the genes encoding AOC and OPR (OsAOC, OsOPR1 and OsOPR3) is suppressed by RNAi method. JA content was undetectable in leaves of the OsAOC-suppressed transformant, and was reduced by approximately 3% in leaves of the OsOPR1-OsOPR3-co-suppressed transformant. We will also report the OPDA content in the OsAOC-suppressed rice.

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Brassinosteroids (BRs) have the ability to protect plants from various environmental stresses, such as drought, salinity, heat stress, and pathogens. Treatment of brassinolide (BL), considered to the most important BRs, induced systemic resistance against a broad range of pathogens in tobacco and rice plant. However, the mechanisms of brassinosteroid-mediated disease resistance (BDR) have not been clarified. In this study, we characterized the mechanism of BDR using Arabidopsis thaliana. BDR was also effective in Arabidopsis and suppressed the growth of the virlent pathogen Pseudomonas syringae pv. tomato DC3000 (Pst). Treatment with BL did not induce the accumulation of mRNA of the systemic acquired resistance-associated PR-1 and the ethylene- and jasmonic acid-dependent PDF1.2 genes. The expression of PR-1 induced by pathogen infection in BDR-induced plants was earlier than in control plants, which is a priming effect of BDR and could be an important mechanism of BDR.

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In paddy field, phytochrome double mutants, phyAphyB, appeared more susceptible to fungi than wild type. In order to investigate the possible roles of phytochromes in defense responses, rice (Oryza sativa L. cv. Nipponbare) seedlings of phyA and phyAphyB mutants were inoculated with the compatible rice blast fungus (Pyricularia oyzae). PhyAphyB showed more serious disease symptoms than phyA. Moreover, the inoculation with fungus induced rapider and higher accumulation of several PR proteins in phyA than in phyAphyB. The crosstalks between phytochromes signaling with Jasmonic Acid (JA) and Salicylic acid (SA) pathway were examined by analyzing expression of defense-related genes in rice seedlings of phyA and phyAphyB treated with exogenous JA or SA.

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Rapid accumulation of reactive oxygen intermediates (e.g., H₂O₂) is a striking early event in the hypersensitive response including programmed cell death. Signaling pathways leading to these responses, however, remain to be dissolved. To elucidate the signaling pathways, eleven rice lesion mimic mutants spl (spl1-11) with spontaneous cell death on their leaves, were investigated using suspension-cultured cell system. Three of the mutants (spl2, spl7, spl11) were found to accumulate higher amount of H₂O₂ than the wild type, when treated with elicitor, indicating that these mutations are involved in accumulation of H₂O₂. Among three mutants, only spl7 mutant accumulated higher amounts of H₂O₂ than the wild type, when treated with calyculin A (CA), an inhibitor of protein phosphatase. Furthermore, spl2 mutant plant exhibited accelerated accumulation of H₂O₂ in response to wounding. We discuss the possible roles of these spl mutations in H₂O₂ generation.

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Higher plants produce reactive oxygen species (ROS) in the defense responses, which acts as a second signal leading to the later responses. We analyzed the interaction between rice and rice blast fungus by monitoring the level of H₂O₂. Spore suspension from different field isolate of the fungus showed different activity to induce ROS accumulation in the media, partially ascribed to the different activities of catalase-like activity in the spore suspension. When spores of an isolate, INA68-137, were washed with water and inoculated on the leaf sheath of a compatible rice cv, Nipponbare, a significant delay in the growth of hyphae was observed in parallel with the increase in the number of cells positive in DAB-staining. These results strongly indicate that the post-spore fraction of INA68-137 includes factors which positively regulate the growth of invading hyphae.

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Elicitor-triggered transient membrane potential changes and Ca²⁺ influx through the plasma membrane are thought to be important during defense signaling in plants. However, the molecular bases for the Ca²⁺ influx and its regulation remain largely unknown. We identified a putative voltage-gated Ca²⁺-permeable channel (OsTPC1) in rice (Plant Cell Physiol. 2005 45: 693-), and developed its overexperssors as well as its retrotransposon-insertional functional knockout mutant (Ostpc1). OsTPC1 has been suggested to determine sensitivity to a proteinaceous elicitor and plays a role as a key regulator of elicitor-induced defense responses, activation of MAPK cascade and hypersensitive cell death. (Plant J. 2005 42: 798-809). Oligomicroarray transcriptome analyses revealed significance of OsTPC1 in regulation of pathogenic elicitor-induced defense gene expression.

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Suspension-cultured cells of Arabidopsis thaliana could be an excellent model system to study physiological/biochemical responses induced by PAMPs and compare with the results obtained from the molecular genetic studies with the use of various Arabidopsis thaliana mutants. We show here that the suspension-cultured Arabidopsis thaliana cells respond to various PAMPs, including bacterial LPSs, flg22 peptide, chitin oligosaccharides, but not to chitosan oligosaccharides and linear laminarioligosaccharides, for the induction of ROS generation. The ROS generation required protein phosphorylation but not protein synthesis. The involvement of PLC/PLD in this process was also indicated. These results were mostly similar to those obtained by suspension-cultured rice cells as well as intact A. thaliana seedlings but partly different. The results indicate the usefulness of the cell culture system for the detailed analysis of PAMPs recognition and responses.

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We have found various double-stranded RNAs (dsRNAs) from many species of crops and wild plants. Some of these dsRNAs are already classified as viruses, but their properties are plasmid-like. The International Committee on Taxonomy of Viruses recently accepted Endornavirus, large (about 14 kbp) plasmid-like dsRNA replions, as a new genus. However, the sequence information of endornaviruses is limited and only three dsRNAs have been sequenced entirely. We have established the partial nucleotide sequences of the RNA-dependent RNA polymerase regions from the large dsRNAs (about 14 kbp) isolated from barley (Hordeum vulgare), kidney bean (Phaseolus vulgaris), melon (Cucumis melo), bottle gourd (Lagenaria siceraria), Malabar spinach (Basella alba), seagrass (Zostera marina), and the fungus Helicobasidium mompa. Phylogenetic analyses of these seven dsRNAs with known endornaviruses and single-stranded RNA (ssRNA) viruses indicate that these dsRNAs are new members of Endornavirus that are widely distributed over plants and fungi.

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Bacterial wilt is caused by a soil borne pathogen, Ralsotonia solanacearum, and damaging the production of major crops such as potato, tomato, and eggplant. The pathogen infection occurs mainly at roots via wounds or secondary roots. In early stages of infection, the pathogen multiply in the vascular element and produce extracellular polysaccharides clogging their vascular system. The pathogenicity and molecular mechanism of the infection of R. solanacearum have been well studied through molecular genetic approaches and the genome sequencing project. On the other hand, only a limited amount of information is reported for plant responses during the infection or development of disease resistance. Here, we report gene expression profiles of Tobacco and Tomato plants during the infection of either an incompatible- or a compatible-strain of R. solanacarum, using a DNA array filter set with 12,158 non-redundant tomato EST sequences.

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We tried to identify a novel gene involved in root nodule formation of Lotus japonicus, a model legume, by activation tagging. We introduced six tandem 35S enhancers to the genome of nod^- castor mutant at random by the hairy root method with Agrobacterium rhizogenes. Among the resulting 4000 transformed lines, 8 plants formed root nodules. The enhancer tag was introduced only one of them, the others probably being somaclonal mutants. TAIR-PCR and inverse PCR analyses revealed the presence of a transcription factor-like gene near the tag. Using the genomic DNA fragment as a probe, we isolated a full-length clone and named the gene TOPAZ (for tag-associated object in putative activation zone). The TOPAZ promoter was fused with GUS gene and introduced into L. japonicus, and then the transformants were inoculated with Mesorhizobium loti. Strong GUS activity was detected at the basis of nodules, suggesting an important role of TOPAZ.

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LjnsRING was identified from L.japonicus as an up-regulated gene in transcriptome analysis during root nodule formation. The LjnsRING encordes a protein containing a RING-finger motif which is potentially involved in the interaction of E3 Ubiquitin-ligase with E2 protein. The LjnsRING homologs were found in arabidopsis, rice and various legume, but no function has been reported. Expression analysis of total RNA from several organs of L.japonicus indicated that LjnsRING was enhanced in nodules of 4-7 days after inoculation with M.loti. To investigate possible roles of LjnsRING in nodulation process, we carried out RNA silencing experiments by hairly root transformation. RNAi suppression of LjnsRING significantly reduced infection frequency as well as nodulation, indicating it is essential for nitrogen-fixing symbiosis. LjnsRING suppressed plants also exhibited impaired growth of roots and shoots, that could not compensated by N-rich medium, suggesting it plays additional roles other than nodulation.

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Nitrogen-fixing symbiosis between legume plants and rhizobia is established on complex interactions between two symbiotic partners. To identify the host legume genes that play crucial roles in such interactions that are responsible in establishment of functional symbiosis, we isolated a Fix^- mutant Ljsym105 from a model legume Lotus japonicus MG-20 regenerated from hypocotyl-derived calli after extensive in vitro culture. The Ljsym105 plants displayed nitrogen deficiency symptoms under symbiotic condition and formed small pale-pink nodules with acetylene reduction activity less than half of the wild type. Microscopic observations revealed that the Ljsym105 nodule infected cells underwent deterioration and/or degradation of the symbiosomes prematurely as well as disintegration of the whole infected cell cytoplasm. Genetic analysis revealed that Ljsym105 was monogenic and recessive, and genetic linkage analysis using DNA markers indicated that Ljsym105 locates on upper portion of chromosome 4 of Lotus japonicus. Map based cloning of Ljsym105 is in progress.

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Laser Microdissection (LMD) allows for the one-step procurement of large homogeneous populations of cells from tissue sections. To date, analyses of global gene expression have generally been conducted on whole organs. Such analyses have the potential to mask genes of interest, that is, those that are specifically expressed in a particular cell type. LMD/microarray analyses will provide a powerful approach to monitor gene expression in some specific cell types of plant mutants.
In this study, we germinated the reduced adh activity (rad) mutant and wild type under submerged conditions. The rad mutant shows that elongation of coleoptiles is repressed under submergence. We used LMD for isolation of coleoptiles of the rad mutant and the wild type, extracted RNA and performed microarray experiments. We identified some genes whose transcript levels were different between the rad mutant and the wild type.

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The mitogen-activated protein kinase (MAPK) cascade is involved in responses to biotic and abiotic stress in plants. In this study, we isolated a new MAPK, NtMPK4, which is a tobacco homolog of Arabidopsis MPK4 (AtMPK4). We found that NtMPK4 was activated by SIPKK, which has been isolated as a SIPK-interacting MAPK kinase. In NtMPK4 activity-suppressed tobacco, wound-induced expression of jasmonic acid (JA)-responsive genes was inhibited. NtMPK4-silenced plants showed enhanced sensitivity to ozone. Inversely, transgenic tobacco plants, in which SIPKK or the constitutively active type SIPKKEE was overexpressed, exhibited greater responsiveness to wounding with enhanced resistance to ozone. We further found that NtMPK4 was preferentially expressed in epidermis, and the enhanced sensitivity to ozone in NtMPK4-silenced plants was caused by an abnormal regulation of stomatal closure in an abscisic acid-independent manner. These results suggest that NtMPK4 is involved in JA signaling and in stomatal movement.

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We developed the system for detection and analysis of mutations occurring on chromosomal DNA in higher plants. Plasmid pML4 carrying Escherichia coli rpsL gene, a target gene for mutagenesis, was inserted into pCGN5138, a plasmid for transformation of plant, and introduced into Arabidopsis thaliana via Agrobacterium. The pML4 was rescued from plant and reconstructed. Rescued pML4 was introduced into streptomycin (Sm)-resistant E. coli to isolate clones carrying mutations in rpsL gene. In this system, Sm-sensitive phenotype is dominant against resistant phenotype. Clones carrying mutated rpsL gene produce colonies on LB agar plate supplemented with Sm. We successfully detected mutational alteration in rpsL sequence from Sm-resistant clones. Typical base change mutations were found in EMS treated Arabidopsis. The assay system developed here is quite useful for detection and analysis of mutations arising on chromosomal DNA in plants and may be valuable to evaluate mutagenicity of environmental mutagens.

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Through the analysis of mutants and gene expression studies, we are beginning to understand proteins involved during acclimation to adverse environmental conditions. In addition, there are many biological signalling events occurring that do not involve a change in protein level, but rather protein activity. It is generally difficult to study such signalling events in plants and there is limited information on upstream signalling events during acclimation responses.

We are developing technology and novel indicators that will allow real-time imaging of such signalling events (calcium changes, phosphorylation events) at high resolution in living tissues as they actually respond to altered environmental conditions. This will allow us to determine the temporal and spatial interactions between specific signalling events. Combined with extensive genetic tools available, we aim to understand which specific signalling events are involved and how they contribute to respective acclimation responses.

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Rad54, one of the Rad52 epistasis group proteins involving in homologous recombination (HR), is a member of the SNF2/SWI2 superfamily of chromatin remodeling factors defined by the presence of conserved helicase motifs. Rad54 is thought to exert its stimulatory effect on the DNA pairing activity of Rad51 by binding to the Rad51 - single-strand DNA nucleoprotein filament. Recent studies with yeast and vertebrates, Rad54 can translocate on DNA, generate super-helical torsion, and furthermore enhance the accessibility to nucleosomal DNA.
We identified a sequence that resembles known RAD54 genes in the Arabidopsis genome, and cloned the corresponding cDNA (AtRAD54). According to analyses of the expression pattern, the protein-protein interaction, and a T-DNA insertion mutant, AtRAD54 is a counterpart gene of RAD54 in Arabidopsis, and is important for HR repair in plants. Currently, we are analyzing AtRAD54 over-expressing Arabidopsis lines to test whether the over-expressed AtRad54 enhances HR.

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We have previously identified multigene family of the molecular chaperone dnaK (dnaK1, 2 , 3) and dnaJ (dnaJ1, 2, 3, 4) in the genome of unicellular cyanobacterium Synechococcus sp. PCC7942. We have found that, among seven genes, DnaK2, K3, J1, J2 and J3 are essential for nomal growth. In order to explore specific functions of these DnaK and DnaJ, we first analysed the expression profiles of each gene under various stress conditions. The results indicated that dnaK2 and dnaJ2 increased remarkably under heat or high-light stress. The results obtained suggested that each dnaK and dnaJ are differently regulated and may have distinct function under a stress conditions. Finally, we are interested in the partnership among multiple DnaK and DnaJ whether there is a specificity between the two structurally related proteins. We observed some specificity between them and the results will be discussed in the viewpoint of chaperone functions.

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We have previously identified four homologues of the molecular chaperone dnaJ (dnaJ1 ~ dnaJ4 ) in the genome of unicellular cyanobacterium Synechococcus sp. PCC7942, with only DnaJ3 protein was localized to thylakoid membrane. Our genetic analysis revealed that syc1092 null mutation suppresses temperature sensitive mutation of dnaJ3 gene. Syc1092 was widely conserved from cyanobacteria to higher plant, but the function is unknown.To investigate Syc1092 function, we carried out yeast two-hybrid screening suggested a specific interaction between Syc1092 and beta subunit of RNA polymerase complex. In addition, Syc1092 overexpressing strain showed temperature sensitive and the heat shock induction of hspA transcripts was compared with that of the wild type strain by using RT-PCR. The results indicated that Syc1092 negatively regulated the hspA induction.

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Although eukaryotic Hsp90 has been well known to play important roles in diverse signal transduction pathways, functions of its prokaryotic counterpart HtpG are not clearly understood. In cyanobacteria Synechococcus sp. PCC7942, we reported previously that HtpG protein increased in response to various stress conditions. In addition, htpG null mutant revealed reduced production of phycocyanin and chlorophyll.
Our systematic analysis of protein-protein interactions by using yeast two-hybrid analysis suggested a specific interaction between HtpG and HemE (uroporphyrinogen decarboxyrase). To analyze the role of HtpG in the HemE activity, the cell lysate of htpG overexpressing strain was prepared and the level of coproporphyrin was compared with that of the wild type strain. The results indicated that HtpG negatively regulated the HemE activity. Furthermore, htpG overexpression inhibited the cell growth, suggesting a detrimental effect of HtpG in cyanobacteria.

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In eukaryotic cells, Hsp90 has been shown to play important roles in regulation of several important cellular processes such as signal transduction pathways, but a role of HtpG, an Hsp90 homologue in bacteria, is not clear yet.
We have demonstrated that HtpG plays roles under various stresses such as heat and oxidative stresses in the cyanobacterium Synechococcus sp. PCC 7942 and the amount of phycocyanin and chlorophyll is reduced in an htpG mutant. We have been searching for in vivo targets for HtpG.
By yeast two hybrid screening analysis, we discovered that the HtpG interacted with uroporphyrinogen decarboxylase (HemE). This enzyme plays an essential role in heme and chlorophyll biosynthesis.
To study the interaction between HemE and HtpG in vitro, recombinant proteins of His-tagged HtpG and HemE were over-expressed in E. coli, and purified. Studies on the interaction between these proteins by pull-down assays or the complex are in progress.

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A rice homolog, OsMLT1 (GenBank AY554051), of the barley low-temperature induced gene blt101 (GenBank Z25537) was identified in the differential clones prepared from cDNA library of rice seedlings treated under drought stress. The OsMLT1 has an ORF of 547 bp that encodes 55 amino acid residues. The deduced amino acid sequences of OsMLT1 showed 54% positional identities with that of barley blt101. Northern analysis with 3-UTR gene-specific probe revealed that OsMLT1 is strongly induced by drought stress. However, Northern analysis on other environmental stress response showed that OsMLT1 is also induced by the moderate low-temperature at 12 degree C, but not by the lower low-temperature at 5 degree C at all. The barley blt101 is known to respond to the lower low-temperature at 5 degree C. Therefore, unknown sensing mechanism may exist that is capable of discriminating degree of low-temperature in regulation of barley blt101 and rice OsMLT1.

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A novel shaggy-like kinase cDNA (TaSK5) was isolated from winter wheat. Northern blots revealed TaSK5 was notably induced by cold and NaCl treatments and lesser by drought and ABA treatments. Complementation experiments utilizing a yeast shaggy-like kinase mutant (Δmck1) revealed that TaSK5 is functional in the yeast to suppress the cold- and salt- sensitive phenotypes of the mutant. Transgenic Arabidopsis plants overexpressing TaSK5 displayed no phenotypic differences from the willd type plants under standard growth conditions. However, transgenic plants significantly improved survival after drought stress compared to the wild type. Interestingly, tolerance against freezing and salt stresses was not altered in the transgenic plants. A microarray analysis revealed that a number of abiotic stress response genes was costitutively induced at non stress conditions.

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Aminoalcoholphosphotransferases (AAPTs) utilize diacylglycerols and CDP-choline/ethanolamine as substrates to synthesize phosphatidylethanolamine (PE) and phosphatidylcholine (PC). We cloned a cDNAs for two AAPTs (TaAAPT1 and TaAAPT2) from a wheat crown. TaAAPT1 and TaAAPT2 complemented the low temperature-sensitive phenotype of the double mutant (Δcpt, Δept) of yeast. Assays with recombinant TaAAPT1 and TaAAPT2 proteins produced in the microsomal fraction of the yeast mutant indicated that TaAAPT1 displayed a greater preference for CDP-ethanolamine than CDP-choline. However, TaAAPT2 mainly utilized CDP-choline. RealTime PCR analysis showed that TaAAPT1 mRNA levels increased in crown tissue during cold treatment, while no increase in TaAAPT2 levels was observed. These results suggest that TaAAPT1 involved in PE biosynthesis is important for cold acclimation of wheat. Fluorescence microscopy images revealed that the TaAAPT1/TaAAPT2::GFP fusion protein are localized in the ER and Golgi apparatus, suggesting that the subcellular sites for the final step of PE/PC synthesis are ER and Golgi apparatus.

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In plants, the most common pathway of trehalose biosynthesis is a two-step process by trehalose-6-phosphate synthase(TPS)and trehalose-6-phosphate phosphatase(TPP). We isolated two major trehalose biosyntheses genes OsTPP1, OsTPP2 from rice and showed that their expression were induced transiently by chilling stress(12°C). Both TPP activity and trehalose levels were transiently increased after chilling stress in accordance with OsTPP1mRNA accumulation. Recombinant OsTPP1 and OsTPP2 proteins were purified and enzymatically characterized. Compared with TPPs from microorganisms, OsTPP1 and OsTPP2 display higher substrate specificity for Tre-6-P and indicated remarkably low K_m value. Futhermore, it was shown these enzymes were unstable against heat. The difference in these enzymatic characteristics suggests the functional differentiation of the trehalose biosynthesis between microorganism and plants. Currently, analysis of a OsTPP1 knock-out mutant and OsTPP1 overexpressors is underway.

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Cold shock domain (CSD) proteins are distributed in bacteria, plant and animal. In E.coli, CSD proteins are highly induced after cold shock and function as a RNA chaperone. The cold-induced wheat WCSP1 protein belong to the CSD protein family. Here we demonstrated that purified recombinant WCSP1 is boiling soluble and binds ss/dsDNA and mRNA. Furthermore, boiled-WCSP1 retained its characteristic nucleic acid binding activity. A WCSP1 deletion mutant, containing only a CSD, lost ssDNA/RNA-binding activity; while a mutant containing the CSD and the first glycine-rich region (GR) displayed the activity. These date indicated that the first GR of WCSP1 is necessary for the binding activity but is not for the heat stability of the protein.

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We have isolated a cDNA clone for wpi6, which encodes a 5.9kDa hydrophobic protein from cold acclimated winter wheat. The predicted protein of wpi6 possesses two membrane-spanning domains, the sequence of which shows high homology with BLT101-family proteins from plants and yeast. Strong induction of wpi6 mRNA was observed in cold-treated root and shoot tissues of both winter and spring wheat cultivars. In contrast to blt101 in barley, wpi6 mRNA was also induced by drought and salinity stresses, and exogenous application of ABA. Expression of wpi6::GFP in an epidermal cell of onion revealed that WPI6 is localized in the plasma membrane. Taken together, these data suggested that the WPI6 might have a protective role in maintaining plasma membrane function during cold acclimation in wheat. We will also present the characterization of the possible transposable element Xumet found in the promoter of blt101 but not of wpi6.

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In response to cold, E. coli accumulates cold shock proteins (CSPs) that function as a RNA chaperone. Plant cold shock domain (CSD) proteins contain a CSD that is highly conserved with bacterial CSPs. A wheat CSD protein (WCSP1) has been characterized and shown to have RNA chaperone activity in our previous studies. Here, we performed functional characterization of one of the four Arabidopsis CSPs (AtCSP3). Nucleic acid binding assays revealed that AtCSP3 binds both ss and dsDNA. AtCSP3 expression was up-regulated in response to cold treatment at 4°C. Promoter-GUS analysis revealed tissue specific expression of AtCSP3 in anthers and shoots and root tips within the seedlings. AtCSP3 knockout mutant was less freezing tolerant than wild -type with or without cold-acclimation, while transgenic plants overexpressing AtCSP3 exhibited higher freezing tolerance than wild-type. These data suggested that AtCSP3 is required for basal and induced freezing tolerance during cold acclimation in Arabidopsis.

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Bromegrass is an extremely cold hardy perrenial grass and its suspension culture provides a unique system where freezing tolerance can be induced by two different stimuli, cold and ABA. The latter process has an optimum temperature at 25C or over. The objective is to characterize each process and find common phenomena. We analyzed gene expression profiles during these two freezing tolerance induction processes (4C vs. ABA at 25C) using rice micro-arrays. We focused on genes expressed after 7 days of treatment. Cold induced many genes involved in protein synthesis, RNA functioning and protein degradation while ABA induced many stress-related genes and genes involved in storage compounds. These processes involve expression of fairly different sets of genes but have some common processes such as induction of anti-oxidating enzymes and cell wall alterations. Plasma membrane ATPase was up-regulated by cold (in agreement of physiological studies) and ABA.

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Many land plants increase freezing tolerance upon exposure to low, non-freezing temperatures, the phenomenon known as cold acclimation. To understand basic mechanisms of plant cold acclimation, we determined the effect of cold acclimation on freezing tolerance, cell structure and compatible solute composition of a green alga (Charophyceae) Klebsormidium flaccidum, which occurs in cold region and is thought to be a phylogenetic ancestor of land plants. Cold acclimation resulted in a significant increase in freezing tolerance, an increase in the size and number of starch grains in chloroplasts, a decrease in the size of vacuoles, and the thickening of cell wall. Osmotic concentration of cells increased after cold acclimation, partly due to the accumulation of soluble sugars (such as sucrose and unidentified compounds) and amino acids (such as glutamine and alanine). These changes are thought to be associated with the increase in freezing tolerance of K. flaccidum during cold acclimation.

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Leaves of African violet (Saintpaulia sp.) are sensitive to decreasing temperature and were damaged when temperature decreased not only from 25 to 10^oC but also from 35 to 20^oC. After temperature decrease, a layer of palisade cells were strongly damaged and died at last, although epidermal cells and spongy cells were not. Decrease of pH in the cytoplasm of palisade cells was determined by fluorescent dye (BCECF-AM) during decreasing temperature, but the fluorescence of chloroplasts was successively decreased after finishing the treatments. These results could suggest the first damage of tonoplast caused by decreasing temperature.

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The aim of this study is to investigate the metabolic regulation of DMSP (dimethylsulfoniopropionate) biosynthesis in Enteromorpha compressa for the environmental adaptation. DMSP is one of the compatible solutes accumulated in many species of marine algae and a few of higher plants.
We fed the radioactive labeled precursor of DMSP (i.e. methionine) to E.compressa in high salinity medium and analyzed the change in DMSP biosynthesis activity from each precursor. The obtained results suggest the biosynthesis of DMSP in salt-treated E.compressa is regulated by the supply of methionine.
If the biosynthesis of methionine is accelerated on response to salt stress, the expression of the salt-responsive genes involved in methionine biosynthesis may be induced. Cystathionine-γ-synthase (CGS) is known as a key enzyme of methionine biosynthesis in plant. So we isolated CGS gene(s) from E.compressa by RACE method and examined the correlation between the CGS expression and salt stress by RT-PCR.

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Some plant-pathogenic microorganisms are known as causes of wilt symptom of plant. The main cause of wilt symptom was described as invasion of vessel elements by microorganisms and blocking water-flow. Phomopsis sclerotioides causes black root rot of cucurbitaceae with wilt symptom. To reveal the mechanism of wilt symptom production by this fungi, xylem sap of inoculated seedlings of cucumber was measured. Xylem sap of seedling indicating wilt symptoms was only 1/10 of non-inoculated plants at 21 days after inoculation. Xylem sap of inoculated seedlings without wilt symptoms was slightly decreased. Furthermore, the plant which grafted one shoot onto two root indicated no wilt symptoms while only one root was infected. But in the root tissue of infected plants, spread of mycelium was detected, but invasion xylem was not detected. This suggest that P. sclerotioides infection cause water flow disturbance in the root tissue by unknown mechanisms.

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Hydrotropism is a response of roots to a moisture gradient. Our previous studies suggest that hydrotropism is interfered with gravitropism, however, this interacting mechanism is hardly uncovered. To approach this problem, we intended to compare the functional tissues responsible for both tropisms. We irradiated either laser beam or heavy-ion beam to root tissues and monitored the changes in tropic responses. Laser beam irradiation to either elongation zone or columella cells resulted in marked decrease in both tropic curvatures. Similar effect was observed when heavy-ion beam was irradiation to elongation zones. Irradiation of either beams to elongation zone resulted in a decrease in root growth. However, the heavy-ion beam irradiation to columella cells affected neither root tropism nor growth. Considering the non-destructive cell inactivation effect of heavy-ion beam, our results suggest that the interacting process between both tropisms, which occurs inside columella cells, do not require de novo gene expression.

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Plants respond and adapt to cold and drought stresses with physiological and developmental changes. These stresses induce some kinds of genes which function in stress tolerance. Overexpression of DREB1A and/or DREB2A in transgenic plants activated the expression of many stress-inducible genes and resulted in improved tolerance to freezing and/or drought. To identify cold- and/or drought-stress-inducible metabolic enzyme genes and downstream genes of DREB1A and/or DREB2A, we performed 22k oligarray analysis. We identified known and new cold- and/or drought-stress-inducible metabolic enzyme genes. Some kinds of metabolic enzyme genes were included in downstream genes of DREB1A and/or DREB2A. To monitor metabolites in the cold and/or drought treated plants and transgenic plants overexpressing DREB1A and/or DREB2A, we assessed metabolites on a large scale by using GC/MS, LC/MS and CE/MS. Some kinds of sugers, amino acids and flavonoides were found to increase in cold and/or drought acclimated plants and transgenic plants.

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DREB2A is a transcriptional factor that functions in drought and high-salinity responsive gene expression. This protein specifically binds to DRE/CRT in the promoter region of stress-inducible genes and seems to require post-translational modification for its activation. DREB2A has a negative regulatory domain in the central region, and deletion of this region makes DREB2A a constitutive active form (DREB2A CA). We generated transgenic Arabidopsis plants overexpressing 35S:DREB2A CA. The microarray analysis of the transgenic plants revealed that many water-stress inducible genes were expressed in the transgenic plants even under non-stressed condition. Induction of heat-shock related genes was also detected by overexpression of DREB2A CA. We have analyzed expression profile of DREB2A under heat-shock condition. The expression of DREB2A was rapidly and transiently induced by heat-shock treatment. This result suggests that DREB2A may be involved in not only drought and high-salinity response but also heat-shock response.

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