Leguminous plants are unique in their ability to form mutually beneficial root-microbe associations with rhizobial bacteria and arbuscular mycorrhizal (AM) fungi. Recent genetic studies in model legumes have revealed the existence of common symbiosis pathway (CSP) that is essential for both root nodule (RN) and AM symbioses. Among the host plant genes located in CSP, calcium calmodulin-dependent protein kinase (CCaMK) is thought to be a putative decoder for microsymbionts-induced Ca2+ signals and play a central role in rhizobial/mycorrhizal infections and nodule organogenesis. However, it remains unknown how CCaMK is activated in response to symbiotic partners. To elucidate the activation mechanisms of CCaMK and involvement of its functional domains in the activation processes, we carried out a detailed complementation analysis of Lotus ccamk mutant with various truncated or amino-acid substituted CCaMKs. Based on our study, we propose models for CCaMK activation during RN or AM symbioses.

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In root nodule symbiosis, coordinated regulation between rhizobial infection and nodule organogenesis is essential for development of fully effective nodules in which rhizobia reside and fix atmospheric nitrogen. Recent studies with non-nodulating mutants of Lotus japonicus have led to isolation of a number of symbiotic genes. However, involvement of these genes in infection thread formation in epidermis and/or nodule organogenesis in cortex remains obscure. To dissect symbiotic cellular responses occurred in different cell layers, epidermis/cortex specific expression systems which make it possible to express the symbiotic gene only in epidermis or cortex, are required. In Arabidopsis thaliana, the promoter of ExpansinA7 gene (AtEXPA7) is known to contain root hair-specific cis elements (RHEs), which confer root hair-specific expression to AtEXPA7. Using a promoter region of AtEXPA7 homologs in Lotus, we developed an epidermis specific expression system, epi-system, in Lotus roots. Based on the results of complementation tests of symbiotic mutants with causative genes expressed by epi-system, we will propose a model for regulating mechanism of epidermal infection process in Lotus.

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In recent years, a number of legume genes involved in the root nodule symbiosis has been identified in the model legumes, Lotus japonicus and Medicago truncatula. Among them, the distinct set of genes has been characterized as common sym, because they are also essential for another mutual interaction, the arbuscular mycorrhiza symbiosis.
Common sym are functionally conserved in non-nodulating plants, such as rice, and are essential for the arbuscular mycorrhiza symbiosis in rice. Furthermore, it has been revealed that common sym from rice could fully complement symbiosis defects in the corresponding Lotus japonicus mutants for both the root nodule symbiosis and the arbuscular mycorrhiza symbiosis.
In this presentation, we will report that the functional conservation analyses of legume-rhizobium symbiosis genes from rice in Lotus japonicus.

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The infection thread is a tubular structure formed in the root hair infected by rhizobia, which reach the cortex through the IT and differentiate into nitrogen-fixing root nodule bacteroids. alb1 mutants develop short and aberrant ITs. We have reported that ALB1 encodes an atypical receptor kinase with leucine-rich repeats in its extracellular domain at the 2009 annual meeting. ALB1 kinase domain (KD) contains substitution of amino acid residues at positions important for a protein kinase activity. We demonstrated that ALB1 KD, purified form E. coli cells, did not have the kinase activity in vitro. A kinase-dead version of ALB1, in which an amino acid residue of the ATP binding site was mutated, suppressed the alb1 phenotype as well as the wild type ALB1 did also. However, a KD-deleted version of ALB1 failed to suppress the phenotype, suggesting that the KD is important for the ALB1 function, although the kinase activity is not required for. Furthermore, we found that ALB1-GFP fusion protein localized to the IT plasma membrane in L. japonicus. These results suggest that ALB1 positively regulates IT development at the IT membrane without the kinase activity.

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Lipopolysaccharide (LPS) is a structural component of the outer membrane of Gram-negative bacteria and LPS of pathogen induces the plant resistant. LPS is also involved in the establishment of symbiosis between legumes and rhizobia. Thus, the elucidation of the LPS recognition system is essential for understanding the plant-bacteria interaction. However, LPS recognition system is not clear in plants. In animal, LPS receptor and LPS binding protein (LBP) have been identified. Referring LPS recognition systems of animal, we tried to clone LBP gene of Lotus japonicus. As the result of BLAST research in the genome library of L. japonicus, four high regions showed high similarity with human LBP. We referred to these genes as LjLBP1, 2, 3 and 4 respectively. N-terminal domain of LjLBP1 and LjLBP2 showed the LPS binding activity. Symbiosis was aborted in the nodules formed on RNAi hairy roots of LjLBP. These results suggest that LjLBP is necessary for the normal symbiosis.

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The actinobacterium Frankia alni is able to form nodules on the roots of a large spectrum of actinorhizal plants, where it converts dinitrogen to ammonia in exchange for plant photosynthates. In the present study, transcriptional analyses were performed on nitrogen-replete free-living cells and on Alnus glutinosa nodule bacteria, using whole genome microarrays. Distribution of nodule-induced genes on the genome was mostly over regions with high synteny between three Frankia genomes, while nodule-repressed genes were spread around the genome. Genes related to symbiosis, such as nif, hup2, suf and shc were highly induced. The expression of genes involved in ammonium assimilation and transport was strongly modified suggesting that bacterial ammonium assimilation was limited. Genes involved in transcriptional regulation, signaling processes, protein secretion, surface popolysaccharide biosynthesis that may play a role in symbiosis were also identified. The symbiotic transcriptome was highly similar among phylogenetically distant host plants. Finally, comparison with rhizobia transcriptome suggested that Frankia is metabolically more active in symbiosis than rhizobia.

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Growth and movement of Ralstonia solanacearum harboring the phage-modified plasmid were monitored in tomato seedlings. The plasmid contains a gene for green fluorescent protein (GFP) and is stably maintained in R. solanacearum cells without selection pressure. Bacteria harboring the plasmid can be tracked in planta by visualizing GFP fluorescence. For real-time monitoring of bacteria in planta, tomato seedlings were grown on agar medium and bacterial suspension was applied to the root apex. The bacterial invasion process was monitored by fluorescent microscopy. In susceptible tomato cultivars, strong GFP fluorescence was observed in hypocotyls and lateral roots as well as the taproot. In resistant cultivars, however, GFP fluorescence was rarely observed on lateral roots. Our results show that this monitoring system can be used to assess bacterial pathogenicity. GFP labeled filamentous phage (φRSS1) was infected for labeling R. solanacearum cells. Labeled cells by using phage showed similar behaviors in tomato plant. Interestingly, infection of R. solanacearum cells containing φRSS1 caused formation of organs similar to a lateral root.

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Culture filtrate of Penicillium simplicissimum GP17-2 that is the plant growth promoting fungi induces systemic resistance (ISR) to Arabidopsis thaliana. We performed microarray analysis of the gene expression that responded to culture filtrate of GP17-2. Microarray data of stress response, pest response, phytohormone response, nutrient response and others. of A. thaliana are publicly available. We performed comparative analysis of expression pattern in pathogen response, phytohormone response, hydrogen peroxide response, wound response and culture filtrate of GP17-2 response. The response to GP17-2 showed the closest relationship with abscisic acid response. Plant stomata can play an active role in limiting bacterial invasion as part of the plant innate immune system and signal transduction pathway through abscisic acid involve in stomatal closure. A. thaliana inoculated with bacterial pathogen after soaking the root in culture filtrate of GP17-2 was significantly induced stomatal closure. This result suggest that stomatal closure involved in ISR by culture filtrate of GP17-2.

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Transcriptome analyses of rice genes infected by eight rice viruses (RDV, RRSV, RBSDV, RSV, RGSV, RTYV, RTSV and RTBV) were done. Severe disease signs of infection were observed after RDV and RSV, while in the cases of RTYV and RTSV disease signs were not observed. Numbers of the up- and down-regulated genes were well correlated with the intensity of disease sign. Host genes were classified into the metabolic pathways, transcription, translation, protein processing processes, family of transcription factors, biosynthesis of phytohormones and responsive genes to phytohormones. According to these classifications, what happened in infected cells were analyzed. As for cell-cycle related genes, CycB genes were down-regulated in many virus infections. As for transcription factors, AP2-EREBP, Tify, WRKY families were actively changed after virus infection and they were further classified into some subfamilies by the up- and down-changes. Gene expression profiles of RTSV- and RTBV susceptible (Taichung Native 1) and resistant hosts (TW16: NIL originated from Utri Merah) will also be reported.

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Plants defend themselves against pathogens by a sophisticated innate immune system. The primary immune response is induced by Microbe-associated molecular patterns (MAMPs). MAMPs receptors recognize the presence of pathogens and turn on an appropriate basal defense response. Since stomata are one of the main entry pathways of pathogenic invaders, plants have developed an active system to close stomata quickly in response to pathogen attack. However, its molecular mechanism is largely unknown. Previously, we demonstrated that elicitors could induce a transient increase in stromal Ca2+ concentration within a few minutes. This implies that chloroplasts could quickly recognize the extracellular pathogens via Ca2+ signaling. CAS is a plant-specific Ca2+-binding protein that is localized in chloroplast thylakoid membranes. In this study, we found that CAS is essential for flg22-induced stomatal closure in Arabidopsis. Furthermore, we revealed that CAS is also involved in the flg22-induced expression of pathogen-related genes (PR-1). These results imply that chloroplast protein CAS is crucial for plant innate immune response, such as MAMP-induced stomatal closure.

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Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in eukaryotes. In plants, the MAPK cascade plays a crucial role in various biotic and abiotic stress responses, hormone responses, cell division, and developmental processes. In Arabidopsis genome, there are 20 genes encoding possible MAPKs, and these MAPKs can be divided into at least four groups (A-D). Group D has 8 members, and forms the largest subgroup. Group D MAPKs contain TDY motifs, which is generally phosphorylated by MAPKKs, instead of TEY motifs in their T-loop and larger C-terminal regions relative to other groups. We have focused our work on MPK8, one of the plant specific group D MAPK. MPK8 was shown to function as a member of MAPK in yeast mpk1 mutant based on complementation analysis. We also found that calmodulins (CaMs) interact with MPK8 by using yeast two-hybrid screening. The BiFC analysis in Arabidopsis protoplast revealed that MPK8 interacts with CaMs not only in yeast but also in plant cells. We have shown that CaM can activate MPK8. These results suggest a novel activation mechanism of MAPK by CaM. We will discuss the activation mechanism of MPK8.

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Calcium-dependent protein kinases (CPKs) have been predicted to mediate the signaling following Ca2+ influx after insect herbivory. To investigate the roles CPKs play in a herbivore response-signaling pathway, we screened the characteristics of Arabidopsis CPK mutants damaged by Spodoptera littoralis. Following insect attack, the cpk3 and cpk13 mutants showed lower transcript levels of plant defensin gene PDF1.2 compared to wild-type plants. In vitro kinase assays of the CPK proteins with a suite of substrates demonstrated that the protein phosphorylates transcription factors (including HsfB2a). In vivo agroinflitaration assays showed that CPK-derived phosphorylation of HsfB2a promotes PDF1.2 transcriptional activation in defense response. Furthermore, both CPKs are involved in the transcript level of heat shock protein (HSP) genes, which resulted in impairment of basal thermotolerance in cpk3 and cpk13 mutants. However, HsfB2a played a role in HSP transcripts as suppressor. These results reveal an intricate array of CPK-associated signal transduction networks that are in part common, but by distinct mechanisms, between plant-insect interactions and heat-shock signal transduction.

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In N gene-containing tobacco (Nicotiana tabacum) cultivars, infection with Tobacco mosaic virus (TMV) results in the rapid death of infected cells, resulting in the localization of the virus and formation of necrotic lesions. The tobacco mitogen-activated protein kinases WIPK and SIPK are activated by TMV infection. In a TMV-resistant tobacco cultivar, silencing of WIPK and SIPK resulted in suppression of TMV multiplication and increased accumulation of salicylic acid (SA). In a TMV-susceptible tobacco cultivar, TMV accumulation was not affected by silencing of WIPK and SIPK, suggesting that N-mediated signaling is required for suppression of TMV multiplication in WIPK/SIPK-silenced plants. When SA accumulation was prevented by expression of SA-degrading salicylate hydroxylase (NahG), suppression of viral multiplication in WIPK/SIPK-silenced plants was partially compromised. These results suggest that N-mediated activation of WIPK and SIPK enhances TMV multiplication through suppression of SA accumulation.

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Plants are exposed to many types of abiotic or biotic stresses. Many researchers have analyzed the mechanism of these stress responses using Arabidopsis plants. At the present day, creation of plants having tolerance for several abiotic or biotic stresses has reported. On the other hand, insect damage is very serious problem that decrease the crop yields. However, the mechanism of plant response to feeding damage has not been well understood. We analyzed the interaction between Arabidopsis and western flower thrips (Frankliniella occidentalis) and serpentine leafminer (Liriomyza trifolii), which is one of the most serious insect pests. The emergence worldwide of insecticide resistance among these insect pests makes them difficult to control The feeding mode of thrips is piercing and sucking, in contrast leafminers is mining. We focused on the function of the immunity-related plant hormones jasmonate (JA), ethylene (ET), and salicylic acid (SA) on plant tolerance to thrips and leafminers with different feeding mode.

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Herbivore-infested plants are known to emit a specific blend of volatiles. Such a blend is specific in plant and herbivore species. It is reported that these volatiles can induce the defensive responses in plants: an intact plant that is exposed to these volatiles from neighboring infested plants becomes more defensive against herbivores than an unexposed conspecific plant. In this study, we observed induction of defensive response in intact tomato plants against common cutworm (Spodoptera litura) when the intact plants were exposed to the volatiles emitted from tomato plants infested by the caterpillars. We showed that the performance of common cutworm on the exposed intact tomato plants were lower than that on plants exposed to intact plant volatiles (control). We focused on the accumulation of a putative glycoside compound in the exposed plants over the control plants by metabolomic analysis. From these results, we show the intact tomato plants neighboring to herbivore-damaged plants increased their defense against herbivores by changing their metabolism after volatile perception. Now we try to identify the structure and physiological function of this compound.

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Colletotrichum higginsianum is a fungal pathogen that infects a wide variety of cruciferous plants causing important crop losses. We have used map-based cloning and natural variation analysis of 19 Arabidopsis ecotypes to identify a dominant resistance locus against C. higginsianum. By analyzing natural variations, we found that alleles of RRS1 from susceptible ecotypes contain SNPs that may affect the encoded protein. Furthermore, two susceptible mutants, rrs1-1 and rrs1-2, were identified by screening a T-DNA-tagged mutant library for the loss of resistance to C. higginsianum. The screening identified an additional susceptible mutant (rps4-21) which has a 5 bp deletion in the neighboring gene, RPS4-Ws, a well-characterized R gene that provides resistance to Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 (Pst-avrRps4). The rps4-21/rrs1-1 double mutant exhibited similar susceptibility levels to C. higginsianum as the single mutants. We also found that both RRS1 and RPS4 are required for resistance to Ralstonia solanacearum and Pst-avrRps4. Thus RPS4-Ws and RRS1-Ws function as a dual resistance gene system that prevents infection by three distinct pathogens.

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AP2/ERF-type transcription factors DREBs specifically interact with the DRE/CRT cis-acting element and control the expression of many abiotic stress-inducible genes in Arabidopsis. Soybean is a globally important crop and genetically modified varieties are largely produced. To develop abiotic stress tolerant transgenics we analyzed function of DREB-type transcription factors in soybean.
To identify soybean DREB-type transcription factors containing an AP2/ERF domain, we performed soybean microarray analysis. We identified six DREB-like genes which were strongly induced by cold, drought, and salt stresses. Two of six DREB-like proteins exhibited transactivation activity in the Arabidopsis mesophyll protoplasts. We named them GmDREB1A and GmDREB2A. These proteins accumulated in nuclei and exhibited high transactivation activity in the soybean stem protoplasts. Overexpression of GmDREB2A in Arabidopsis plants resulted in upregulation of some genes related to heat shock and consequently these transgenic plants showed improved heat stress tolerance. These results indicate that GmDREB2A functions as a transcriptional activator in the heat-stress-responsive gene expression.

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SNF1-related protein kinases 2 (SnRK2s) are activated by ABA- and/or osmotic stress and are involved in stress signaling pathways in plants. Ten members of Arabidopsis SnRK2 are classified into three subclasses. Recently, three subclass III SnRK2s including SRK2E/OST1/SnRK2.6 were shown to play crucial roles in ABA signal transduction pathways. However, the functions of other two subclasses of SnRK2s are still unclear. In this study, we studied in planta functions of the subclass II SnRK2s (SRK2C/SnRK2.8 and SRK2F/SnRK2.9) in Arabidopsis. To analyze their functions, we established the srk2cf double mutant. Microarray analysis showed that the many ABA-inducible genes were downregulated in srk2cf under drought stress conditions. However, those genes showed normal response to ABA even in srk2cf, suggesting that the subclass II SnRK2s mediate drought stress signaling significantly, but that their roles are only supportive in ABA-dependent pathway. We could not observe significant phenotypes of srk2cf. We will discuss the role of the subclass II SnRK2s in Arabidopsis.

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In plants, under drought stress conditions, abscisic acid (ABA) levels increase, resulting in regulating the expression of many genes that function in the stress tolerance. In the presence of ABA, PYR/PYL/RCAR family START proteins that function as ABA receptors recognize and bind to group-A protein phosphatases 2C (PP2Cs). Then the subclass III of SNF1-related protein kinases (SnRK2s; SRK2D/SnRK2.2, 4SRK2E/SnRK2.6, and SRK2I/SnRK2.3) are released from the PP2C-dependent negative regulation, which allows SnRK2s to phosphorylate downstream transcription factors such as AREB/ABFs (AREB1/ABF2, AREB2/ABF4, and ABF3) and activate ABA-responsive gene expression or other ABA-related responses. In both srk2d srk2e srk2i and areb1 areb2 abf3 triple knockout mutants, the expression of the ABA-responsive genes such as group-A PP2Cs was drastically and globally impaired in comparison to the WT plants. Among the PP2Cs, three PP2C genes strongly induced in the vegetative tissues after exogenous ABA treatment were named HAI1, HAI2 and HAI3 (for highly ABA-induced PP2C genes). Here, we report the functional analysis of these HAI genes involved in drought stress response in Arabidopsis thaliana.

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ABA is a phytohormone that plays an essential role in dehydration stress responses, such as stomata closure, gene expression and stress tolerance. Many dehydration-inducible genes are regulated by ABA. In Arabidopsis ABA biosynthetic pathway, NCED3 (9-cis-epoxycarotenoid dioxygenase) was required for dehydration-inducible ABA accumulation that is essential for dehydration tolerance. In the present study, using transcriptome data of NCED3 knockout mutant (nc3-2) and wild type plants, we analyzed co-expressions of stress-inducible genes. We found 120 co-expression modules and focused on some modules in which stress-inducible genes were co-expressed. Co-expression analysis showed that the global gene-to-gene correlations occurred among ABA-regulated genes and JA-regulated genes. In ABA-regulated genes module, two cis-acting elements, ABRE and G-box, were significantly observed in their promoter regions. In JA-regulated genes module, many unknown motifs existed significantly in addition to ABRE and G-box. Co-expression analyses revealed new molecular mechanisms of dynamic gene networks in response to dehydration stress. We will also report network analysis of stress-repressed genes.

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Wild watermelon is a desert plant showing the strong tolerance to drought stress. This plant is unique in that development of its root system is significantly promoted in response to the onset of drought, which offers adaptive advantage for absorbing water from deep soil layer under water deficit conditions. However, molecular mechanism responsible for this physiological response has been largely uncharacterized. To gain information on the genetic factors involved in this process, change in the root transcriptome under drought was compared between wild and domesticated watermelon plants by microarray analysis. Out of 8,069 genes analyzed in this study, expression levels of 79 genes were found to be up-regulated by drought in the root of wild watermelon but unchanged in domesticated watermelon. These differentially-expressed genes included zinc-finger-type and myb-type transcriptional factors, raising the possibility that these genes may function as key regulators for promoting root growth in wild watermelon under drought. We are currently analyzing the function of these genes using transgenic approach in hairy root system for wild and domesticated watermelon plants.

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Citrulline is a compatible solute in wild watermelon, a drought-tolerant plant native to the Kalahari Desert, Africa. Citrulline is an intermediary amino acid in the arginine biosynthetic pathway, and has a potent activity for scavenging hydroxyl radicals. In wild watermelon, accumulation of citrulline in the leaves is triggered by the onset of drought stress in the presence of high light. Previous results showed that the activity of N-acetylglutamate kinase (AGK), a key enzyme in the citrulline biosynthetic pathway, was significantly up-regulated in the leaves under the stress. In this study, a cDNA for this enzyme, designated CLAGK, and a cDNA for the putative regulator of AGK, CLP_II, were isolated from wild watermelon. Deduced amino acid sequences of both proteins had putative chloroplast transit peptides at their N-terminus, and showed 67-79% deduced amino acid sequence similarity with those of corresponding genes reported from Arabidopsis. Moreover, western blot analysis showed that abundance of CLP_II was markedly up-regulated in the leaves under drought. We are currently performing kinetic analysis of CLAGK and CLP_II using their recombinant proteins.

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Although rice produces little glycine betaine, it has two betaine aldehyde dehydrogenase (BADH) gene homologs (OsBADH1 and OsBADH2). We found that OsBADH1 catalyzes the oxidation of acetaldehyde efficiently, while the activity of OsBADH2 is extremely low. The accumulation of OsBADH1 mRNA decreases following submergence treatment, but quickly recovers after re-aeration. We confirmed that OsBADH1 localizes in peroxisomes. In the present study, a possible physiological function of OsBADH1 in the oxidation of acetaldehyde produced by catalase in rice plant peroxisomes is discussed.

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QTL analysis using chromosome segment substitution lines (CSSLs) derived from Sasanishiki(SA)/Habataki(HA) has carried out to identify the locus participate in ozone-induced yield decrease in rice. The 39 lines of CSSL and their parental cultivars were grown under ozone-exposure (77 ppb) or ambient air (32 ppb) conditions. Ozone-induced visible leaf injury was detected in SA, and photosynthetic rate, stomatal conductance, transpiration rate were not different. Total grain yield was decreased in HA by ozone. Measurement of compositional units of grain yield showed that culm length, first branch number, filling rate and 1000 seed weight were decreased by ozone in HA, suggesting, these involve in ozone-derived yield decrease in HA. QTL analysis of CSSLs showed a loci located at chromosome 6 involve in the increase of first branch number in the ambient air condition but not in plants grown in ozone-exposed condition, suggesting, yield decrease in HA by ozone might be result in decrease of first branch number derived from the change of activity of genes located at chromosome 6. This work is supported by the Global Environment Research Fund (Ba-086) of the Ministry of the Environment.

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Oxidative signaling via H₂O₂ is essential for plant response to environmental stress. Two types of ascorbate peroxidase are localized in thylakoid membrane (tAPX) and stroma (sAPX) of chloroplasts in higher plants. In order to clarify involvement of chloroplastic APX in the regulation of oxidative signaling via H₂O₂, we characterized single mutants lacking either tAPX or sAPX. Under high-light, H₂O₂ accumulated at higher levels in both mutants than the wild-type plants. However, the absence of sAPX or tAPX suppressed drastically the expression of H₂O₂-responsive genes under high-light. These findings suggest that H₂O₂ derived from chloroplasts negatively regulates H₂O₂-responsive genes under high-light. When the expression of tAPX was transiently suppressed by an estrogen inducible RNAi, the levels of oxidative protein increased in chloroplasts, and the expression of genes involved in several hormonal or stressful response was changed.

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We isolated a high-light (HL) inducible NAC transcription factor named ANAC078. To identify the genes targeted by ANAC078, we conducted a transcriptome analysis of the transgenic Arabidopsis plants overexpressing ANAC078 (Ox-ANAC078) and wild-type plants using a DNA microarray. In Ox-ANAC078, the transcription of 166 genes was up-regulated compared with the levels in wild-type plants under HL. These genes included some for transcription factors regulating the expression of genes related to the biosynthesis of flavonoids. Interestingly, the transcript levels of some genes related to flavonoid biosynthesis and the levels of anthocyanins were significantly increased in the Ox-ANAC078 plants and reduced in knockout ANAC078 plants (KO-ANAC078) compared with the wild-type plants under HL stress. The present findings suggest that ANAC078 protein is associated with the induction of genes related to flavonoid biosynthesis, leading to the accumulation of anthocyanins, in response to HL stress (Morishita et al., 2009).

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Heat shock transcription factor A2 (HsfA2) plays an important role in regulating induction of defenses against different types of oxidative stress (Plant J. 2006). Here, we identified a cis-element and regulatory factor involved in the induction of HsfA2 expression in response to oxidative stress. The transient reporter assay using luciferase reporter constructs with different fragments of HsfA2 promoter showed that the deletion of the sequence from -191 bp to -108 bp markedly reduced the luciferase activity under high-light. This promoter region contained HSE elements, suggesting that some Hsfs mediate the induction of HsfA2 expression. Next, to identify the Hsfs regulating the expression of HsfA2, we took advantage of the chimeric repressor silencing technology (CRES-T). We found that ectopic expression of the chimeric HsfA1d or HsfA1e repressor significantly suppressed the induction of HsfA2 expression in response to high-light. We are currently investigating that transcriptionally regulation of HsfA2 via HSE by HsfA1d/A1e and the effect of loss-of-function and overexpression of HsfA1d and/or HsfA1e on the phenotype in Arabidopsis under oxidative stress.

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We reported previously that plants grown in the air containing nitrogen dioxide (NO₂) at the environmental standard concentration were almost two times greater in growth rate and biomass yield than those grown in the air without NO₂ (designated plant vitalization)¹⁾. Based on the DNA microarray analysis, we obtained about 30 key candidate genes responsible for the plant vitalization in Arabidopsis thaliana. In this study, we investigated the responses of T-DNA insertion mutants defective in these genes to NO₂. One-week-old seedlings of mutants were cultivated for 3 weeks in the air containing or not containing 50 pbb NO₂, after which the biomass of plants were determined. We found that a line (designated vita1) lacked the response to NO₂. We made 35S:VITA1 and amiR-VITA1 constructs, produced transformant plants bearing each one of the constructs, and are currently analyzing them.
1) M. Takahashi et al. (2005) New Phytologist 168: 149-154.

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Cold acclimation (CA) is associated with changes in lipid and protein compositions of the plasma membrane (PM), which is critical for the increase in freezing tolerance of plant cells. Recently, we demonstrated that CA dramatically changed composition of sphingolipid- and sterol-enriched PM microdomains, in which various functional protein complexes are localized to function efficiently (Minami et al., 2009). Proteomic and subsequent western blot analyses revealed that dynamin-related protein 1E (DRP1E), which is one of 16 Arabidopsis DRP family proteins, increased in PM microdomains after CA. DRP1E expression also increased after 12 hours of CA. With DRP1E::GFP-overexpressed Arabidopsis plants, DRP1E was demonstrated to be localized in/near the PM and the GFP signals came from the proximity of the PM non-uniformly. The capacity of CA was much less in the drp1e T-DNA insertion mutants than wild type. Because DRPs are thought to be associated with membrane trafficking and membrane fission processes, our results suggest that DRP1E contributes to freezing tolerance by helping reconstruction of the PM architecture through a vesicle-trafficking pathway in Arabidopsis during CA.

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Overwintering plants are capable of exhibiting high levels of freezing tolerance, which is acquired through cold acclimation. In contrast, the acquired freezing tolerance is rapidly reduced in deacclimation and plants resume growth after sensing warm temperature. It is important to clarify the mechanism of deacclimation for understanding plant growth and development. However the detailed mechanism of deacclimation is not fully understood.
In order to understand the molecular mechanism of deacclimation, we focused on the RNA masking system, which is an RNA regulation mechanism in translational step. RNA masking regulates protein expression by repressing translation and the translation is started by environmental changes or developmental signals. To identify the target mRNAs of RNA masking, we performed comparative analysis between transcriptome and proteome. According to these analyses, we identified several candidates of target mRNAs whose proteins were specifically increased in deacclimation. These target mRNAs encoded enzymes involved in primary metabolism providing energy to resume plant growth. The initial response of plants in deacclimation will be discussed.

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Potamogeton malaianus (MAL) shows phenotypic plasticity and grows under both submerged and terrestrial conditions. In contrast, its allied species P. perfoliatus (PER) can survive only in water. From cultivation experiments, we found a significant difference in thermotolerance between them, especially PER lacked the ability to acclimate to high temperature.
Heat shock transcription factor HsfA2, the most strongly induced by heat stress among Hsf family, is required for basal and acquired thermotolerance in Arabidopsis. We attempted to isolate this gene from MAL and PER, and obtained two genes, namely HsfA2a and HsfA2b. By heat treatment, HsfA2a was strongly expressed in both plants, while HsfA2b was induced in MAL but not in PER. The lack of PER-HsfA2b expression may be due to mutations in heat shock element (HSE) and a long insertion in the promoter region. The RT-PCR analysis of HsfA2 and Hsp during long-term acclimation revealed that Potamogeton HsfA2s may be differentiated functionally: HsfA2a is involved in basal thermotolerance and HsfA2b is essential for acquired thermotolerance.

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Plants are directly and strongly affected by abiotic stresses that relate to water, temperature, light and nutrients. Plant reproductive development is more sensitive to abiotic stresses than vegetative growth. Especially high temperatures cause male sterility due to abortion of anther development in several plant species. We have studied high temperature injury to male reproductive development using barley and Arabidopsis. These previous data indicate that anther tissue-specific auxin reduction is a cause in high temperature injury, which leads to the abortion of pollen development and the short filaments. It has been well known that importance of cross talk and interaction between phytohormones. In this study, we focused the ethylene signaling in developing anther cells and its response to high temperature stress. The ethylene insensitive mutant showed tolerance to increasing temperatures and suppressed the reduction of filaments. Promoter analysis implied some of ACS genes and ERF1 gene constitutively expressed in anther layer cells. These results suggest that high temperature injury to anther development occurs through ethylene signaling.

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In certain cyanobacteria, green-light irradiation induces accumulation of a green-absorbing pigment, phycoerythrin, whereas red-light irradiation induces accumulation of a red-absorbing pigment, phycocyanin, in the photosynthetic light-harvesting phycobilisome. In this study, we show that genes of a cyanobacteriochrome CcaS and cognate response regulator CcaR are essential for green light induced PE accumulation in Nostoc punctiforme ATCC 29133 that performs group II chromatic adaptation. In addition, we analyzed the chromophore-binding domain of another cyanobacteriochrome RcaE that is suggested to regulate group III chromatic adaptation in Fremyella diplosiphon. We will discuss about molecular mechanism and evolution of CcaS and RcaE with two different types of complementary chromatic adaptation.

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Cyanobacteria harbor many GAF-containing photoreceptors that bind a linear tetrapyrrole as a chromophore (cyanobacteriochrome). We have reported that the cyanobacteriochrome TePixJ of Thermosynechococcus elongatus: (1) TePixJ exhibits reversible photoconversion between blue and green-absorbing forms, (2) TePixJ covalently binds phycoviolobilin (PVB), which is isomerized from phycocyanobilin (PCB) by TePixJ itself. Here, we performed site-directed mutagenesis of some conserved amino acid residues and expressed the mutant proteins in Escherichia coli producing PCB. E497A mutant showed photoconversion and isomerization, which are comparable to the wild type but a small amount of novel photoinactive species were generated during photoconversion. D492A mutant did not isomerize PCB to PVB and PCB-bound protein showed photoconversion much less efficiently than the wild-type protein. H523A mutant has mostly PVB but showed unusual photoconversion from blue to orange/red-absorbing form. These suggest that hydrogen bonds interacting with the chromophore are critical for the photoconversion of TePixJ and the isomerization from PCB to PVB.

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Cyanobacteria harbor many photoreceptors (cyanobacteriochromes) that bind a linear tetrapyrroles and respond to a wide range of lights. In this study, we characterized putative photoreceptor Tlr0924 of Thermosynechococcus elongatus. Tlr0924 has the chromophore-binding GAF domain and GGDEF domain that synthesizes cyclic dimeric guanosine monophosphate (c-di-GMP) as a second messenger. It was expected that the activity of Tlr0924 is regulated by light. We prepared full length of Tlr0924 protein, which was expressed in E. coli and Synechocystis. Purified proteins bound a tetrapyrrole chromophore and showed reversible photoconversion between blue light-absorbing form and green light-absorbing form. We measured the diguanylate cyclase activity as accumulation of the by-product pyrophosphate. Detection of c-di-GMP by mass spectrometry is now in progress. In addition, photoregulation of the diguanylate cyclase activity is now being measured. We are also trying to identify the phenotype of the tlr0924 disruptant under various light conditions.

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AnPixJ is a novel cyanobacteriochrome of a cyanobacterium Anabaena (Nostoc) sp. PCC 7120. Upon the irradiation of red- light and green-light, it shows a reversible photoconversion between a green-absorbing (λmax=543 nm) and red-absorbing (λmax¬=648 nm) forms (1) . The crystallographic structure in the state of Pg is not understood though that in the state of Pr is understood (2) . His-tagged chromophore-binding GAF domain of AnPixJ protein (AnPixJ-GAF) with a molecular weight of about 20 kDa was purified by using Ni affinity column. Then, we illuminated the protein at 77 K and followed worming up to examine the reaction process. We succeeded to trap 4 intermediate states in the photoconversion process. The absorption spectra of these 4 states confirmed the ones measured by the time-resolved spectroscopy at room temperature. We discuss the photoconversion mechanism of this protein.

(1) Narikawa J.Mol.Biol. (2008) 380, 844-855
(2) Narikawa Acta Cryst. (2009). F65, 159-162

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We are establishing novel lines for functional genomics in Arabidopsis, in which each transcription factor ectopically is expressed. We have employed glucocorticoid receptor (GR)-mediated induction system to avoid lethality that may be caused by overexpression of transcription factors. We have isolated a mutant that has long hypocotyl phenotype and small cotyledons by application of dexamethasone (DEX) under long day condition. Interestingly, this long hypocotyl phenotype was observed on medium with low concentration but not high concentration of DEX. Oppositely, the phenotype of small cotyledons was dependent on the concentration of DEX. These phenotypes were observed under blue, red and far-red light conditions, but not under dark conditions. These results indicate that this mutant exhibits light dependent dwarfism. The gene encodes zinc finger type transcription factor, and the result of in vivo assay showed that this transcription factor is transcriptional repressor. We will discuss the functions of this transcription factor in light signal transductions.

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Expression of PtCA1, a chloroplastic carbonic anhydrase in the marine diatom Phaeodactylum tricornutum, is regulated by ambient CO₂ concentration and light at the transcriptional level, that is down regulated under high CO₂ or dark conditions. In previous studies, 3 critical CO₂/cAMP-response elements (CCRE1-3) were identified at the promoter region of ptca1 (Pptca1) and it was reveled that these elements are under control of cAMP as second messenger. However, light-response mechanisms of Pptca1 in not clarified yet. In this study, light-response elements in Pptca1 were determined. GUS reporter assay using one base replaced constructs of CCRE2 and 3 and their adjacent sequences, it was shown that light-response element exactly corresponded to CCRE2 and 3. We tested the light-response element of CCRE1 and found that CCRE1 is also responsible for light-response of the Pptca1. These results indicated that light-response elements are identical to CO₂-response elements, CCRE1-3, in Pptca1. Further analysis of light-response of Pptca1 using dibutylyl cAMP (dbcAMP), an analogue of cAMP, demonstrated that light signal is also transduced by cAMP as a second messenger.

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To better understand the mechanism of phytochrome-mediated signaling, we used a basal land plant, the liverwort Marchantia polymorpha L.. Phytochrome in M. polymorpha, Mpphy, is encoded by a single-copied gene, MpPHY. Mpphy exhibits red/far-red photoreversibility in vitro, which is lost in Mpphy with a single amino-acid substitution, MpphyY241H. Mpphy has been shown to promote cell division after spore germination, development to thallus, and thallus regeneration. In this study, chromosomes were visualized by tdTomato fused with histone H2B, which revealed that meristem formation and cell division are red-light dependent. Comparison of nuclear phase between transgenic plants carrying MpphyY241H and WT implied that Mpphy could facilitate transition from G2 phase to M phase. Mpphy was largely localized in the nucleus after red-light irradiation, while MpphyY241H was detected in the nucleus as well as in the cytoplasm regardless of light condition. Silencing of MpPHY by RNAi caused transition into reproductive phase without far-red light. These results indicate that Mpphy is functional in the nucleus, and also that Mpphy regulates morphogenesis directly or indirectly by cell division.

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Phytochrome has been shown to be the major photoreceptor involved in photomorphogenetic and physiological responses in plants, and there are three rice genes for them. Our previous study showed that one of the genes encoding ethylene biosynthetic enzymes, ACO1, was highly expressed in phytochrome triple mutants compared to the wild-type (WT). To examine the role of ACO1 in internode elongation at the heading stage, ACO1-deficient (aco1) and overexpressing (ACO1-OX) mutants were characterized and used for study. The first internode of ACO1-OX mutants showed no significant difference in length compared to that of WT plants, but lower elongated internodes were longer in ACO1-OX mutants than in WT plants. On the other hand, the first internode of aco1 mutants was shorter than that of WT plants, whereas the lower elongated internodes were similar in length. We further examined expression of ethylene biosynthesis/signaling genes and found that some of them were upregulated in aco1 mutants. From these results, we discuss about the molecular mechanism of internode elongation mediated by ACO1.

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Contrary to the established notion that the apical hook of dark-grown dicotyledonous seedlings opens in response to light, we have previously discovered in tomato that the apical hook curvature is exaggerated by light through low- and very low-fluence responses of phytochrome. The novel phenomenon is not limited to tomato, but found also in some other species. This led us to seek the significance of the phenomenon in the field. Here we present the results of field simulation experiments and time-lapse movies, and a hereby-deduced role of the hook exaggeration that it may contribute to the release of the seed coat; i.e., seedlings germinated in soil respond to first photons coming through soil gaps by hook exaggeration which delays the emergence of the seedlings above soil surface. The delay provides the time for the yet small, immature cotyledons to develop and become ready to unroll themselves and eject the seed coat. It also allows the seed coat to stay longer in humid conditions needed for the release of seed coat. Furthermore, we found that the hook exaggeration requires the seed coat and/or endosperm, which was assumed to supply some essential factor to the hook part.

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Phytochromes (phys), which absorb red (R) and far-red (FR) light, are expressed in almost all the organs throughout the life cycle of plants. Hence, the light stimuli perceived in different parts of a plant should be properly integrated to trigger the ultimate response at the whole plant level. To fully understand this phenomenon, we treated Arabidopsis seedlings with micro-beam LED light. The seedlings were grown under day/night cycles and treated with a micro-beam FR pulse at the end of the day to induce hypocotyl elongation, which is one of the typical shade avoidance responses. Consequently, we demonstrated that phys in cotyledons almost exclusively regulated the hypocotyl elongation, indicating the inter-organ signaling from cotyledons to the stem. In addition, we found that Pr formation during the first 4 hr of the dark period efficiently promoted the elongation, whereas the actual elongation took place in a much later phase of the dark period. The RT-PCR analysis is now in progress to search for key genes underlying the above inter-organ communication.

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Light is the most important environmental factor that controls the leaf shape and structure. The leaf blade has a wide and flat lamina structure to absorb light efficiently. Leaf flattening is enhanced in response to blue light, whereas the downward curing of leaves is observed under red light. Phototropin mediate the above leaf flattening response to blue light. By contrast, little is known about the involvement of phytochrome in the regulation of leaf flatness.
In this study, we described the activity of PHYB to suppress the leaf flattening. We found that the flatness of the rosette leaves was enhanced by end-of-day far-red light treatment or phyB deficient mutation. To examine the interaction of PHYB with Phototropin, we made the phyBphot1phot2 triple mutant. The leaf blade of the triple mutant was as flat as that of the phyB leaf, although intense downward curling leaves were observed in the phot1phot2 leaf. Hence, PHYB appears to act downstream of PHOT1/PHOT2 in the leaf-flatness regulation. On the basis of these results, we suggest that antagonistic interaction between PHYB and PHOT1/PHOT2 is involved in the regulation of leaf flatness.

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Regulatory mechanisms of stress responses in rice largely remain unclear. Using rice microarray, we identified many abiotic stress-responsive genes. Among them, a gene for a bHLH transcription factor down-regulated by drought stress has been studied. The bHLH protein showed a high sequence homology with Arabidopsis PIF, driving us to name the transcription factor OsPIF1. Overexpression of OsPIF1 in transgenic rice plants promoted internode elongation. In contrast, dominant loss-of-function rice mutants with a chimeric repressor resulted in short length of the internode sections. These data suggest that OsPIF1 functions as an important regulatory factor of plant height in response to drought stress. Recently, we analyzed down-stream genes of OsPIF1 by oligoarray system using node sections of OsPIF1 transgenic rice plants. Those putative down-stream genes of OsPIF1 were down-regulated by drought stress. In a transient experiment using rice protoplasts prepared from shoots, the transcription of a GUS reporter gene driven by the promoter containing E-box elements was activated by OsPIF1.

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Arabidopsis thaliana changes its architecture dependent on environmental conditions, among which light and temperature are prominent cue. Light regulates the plant growth in the quantity- and/or quality-dependent manner. Moderate high temperature promotes the cell elongation and low temperature suppresses it. Recently, Phytochrome Interacting Factor4 (PIF4), which is a positive cell elongation factor, has been identified as an integrator for the light- and temperature-signaling.
Here, we report destabilizations of PIF4 protein under blue light or low temperature (16 degrees) conditions. Blue light induced the PIF4 degradation mediated through the 26S proteasome pathway in a quantity-dependent manner, which required the Active Phytochrome Binding (APB) domain of PIF4 as like as red light does. On the other hand, ΔN-PIF4, which lacked the APB domain, was also degraded under low temperature-condition through the 26S proteasome pathway. These data suggest that the low temperature-induced degradation mechanism is distinguishable from the mechanism of light-induced degradation.

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BLUF (sensor of Blue Light Using FAD) protein PixD controls phototactic movement of cells. The X-ray crystal structural analysis of the SyPixD of synechocystis sp. PCC6803 at 1.8 A resolution showed a high-ordered decameric structure formed of a pair of pentameric SyPixD rings. Photoexcitation of a FAD chromophore in PixD induces decomposition of the complex into dimmers. It is proposed that the light-induced decomposition regulates the optical response in cells. PELDOR (Pulsed ELectron elctron DOuble Resonance) was applied to the SyPixD multimers to determine the relative arrangement of each SyPixD monomer in the complex. A Pake's doublet type EPR signal was detected at 10 K after preillumination at 150 K for 30 min and indicated a magnetic dipole interaction between a neutral flabosemiquinone radical FADH and a neutral tyrosine radical Y8. PELDOR envelope signal induced by the selective excitation was modulated with the dipolar coupling frequency depending on the arrangements of radical pairs. We will discuss the distance and relative orientation between proteins in a SyPixD multimer.

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In Arabidopsis, two phototropins, phot1 and phot2, function as photoreceptors for hypocotyl phototropism, chloroplast movement, and stomatal opening. Rice (Oryza sativa) has two PHOT1 gene homologs (OsPHOT1a and OsPHOT1b) and one PHOT2 gene homolog. We investigated phototropism of a rice phot1a mutant that has a Tos17 insertion in OsPHOT1a. Seedlings of this mutant showed normal coleoptile and root phototropisms. We therefore produced RNA interference transgenic lines that have substantially reduced OsPHOT1a/OsPHOT1b expressions and also isolated a phot1a/phot1b double mutant from the phot1a mutant mutagenized by γ ray. These transgenic and mutant lines showed no detectable seedling phototropisms to continuous blue light stimulation (0.01-100 μmol m^-2 s^-1). The phot1b single mutant isolated from the phot1a/phot1b double mutant showed near wild-type levels of phototropisms. These results demonstrated that phot1a and phot1b redundantly mediate phototropisms of coleoptiles and primary roots and are nearly the sole photoreceptors of these phototropisms. It was further found that phot1 functions as a major photoreceptor for the photonastic movement observed in the second leaf.

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Earlier studies of phototropic fluence-response curves with maize coleoptiles identified first and second pulse-induced phototropisms and time-dependent phototropism. In this study, we established a phototropism assay system that allowed quantitative determination of hypocotyl phototropism and investigated the molecular basis of multiphasic fluence-response relationships by using phototropic mutants of Arabidopsis. With this assay system in which red light-adapted seedlings were used and phototropic stimulation was directed parallel to the symmetrical plane of the seedling shoot, the first pulse-induced and time-dependent phototropisms were resolved and, in addition, a weak second pulse-induced phototropism was identified in high fluences. It was uncovered that all these responses are mediated by phot1 with no apparent contribution from phot2. It was also uncovered that NPH3 limits all the responses whereas RPT2 is involved specifically in time-dependent phototropism. Further analysis using continuous blue light stimulation indicated that phot2 can cause a weak phototropism at high fluence rates when phot1 is absent, but it does not mediate phototropism in wild-type hypocotyls.

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Phototropins, which are members of AGCVIII kinase family, are plant specific blue-light photoreceptors having photosensory LOV domains in the N-terminus. The Arabidopsis genome contains 23 AGCVIII kinases exhibiting different expression patterns and functions. Since the kinase domains are well conserved among them, there is a possibility that AGCVIII kinases other than phototropin transduce the signal by using the pathway similar to phototropin. To investigate this possibility, we produced transgenic plants that over-expressed the kinase domains of representive AGCVIII kinases, and compared them with respect to physiological activities. In particular, we produced transgenic plants expressing the GFP-fused kinase domains of PHOT1, PHOT2, AGC1-3, KIPK, PID and OXI1 under the control of the heat-inducible promoter. The resulted plants were subjected to the physiological analysis. Consequently, distinct AGCVIII kinases interfered with the endogenous phot2 actions in the chloroplast positioning and phototropic responses. These results suggest that AGCVIII kinases have physiological acitivities similar to the PHOT2 kinase. Currently, we are analyzing stomatal opening in those plants.

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Plants possess a blue-light receptor, phototropin, which mediates phototropism, chloroplast relocation, stomatal opening and leaf flattening. Phototropin is a light-regulated kinase whose authentic substrate has remained unknown. The "flippase-kinase" (Fpk) in Saccharomyces cerevisiae (budding yeast), which shows a high similarity to phototropin, phosphorylates and activates "phospholipid translocase" (flippase). Flippase in turn affects cell polarity, membrane transport, and actin remodeling through modifying the tansbilayer lipid asymmetry of cellular membrane.
We hypothesized that phototropin also acts as a flippase-kinase in plants. As the first step, we demonstrated that phototropin complemented multiple phenotypes in the fpk-deficient mutant strains of budding yeast. Hence, phototropin could regulate the flippase activity at least in budding yeast. We then asked whether flippase was involved in the phototropin-mediated responses in plants. Among 12 members of the flippase family (ALA1 - 12), we found that the ala3 mutant of Arabidopsis was defective in stomatal opening induced by blue light. Thus, phototropin may regulate ALA3 directly to trigger stomatal opening.

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