Vacuoles have been often thought to be storage compartments: excess nitrogen is stored as amino acid in vacuoles. However, vacuolar amino acid concentrations in several plant species are estimated to be lower than cytosolic ones, suggesting active export of amino acids from vacuoles. So far, transporters that are responsible for amino acid efflux at the tonoplast have not been identified in plants.
Yeast Avt3/Avt4 mediate export of neutral amino acids across the vacuole membrane. We found that AtAVT3a , an Arabidopsis homologue of AVT3/AVT4 , was localized at the tonoplast in plant cells. Furthermore, we showed that AtAVT3a could rescue defects of yeast avt3/avt4 mutant in amino acid transport. This is the first report of a plant functional vacuolar amino acid transporter.

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H⁺-pyrophosphatase (H⁺-PPase) is a proton pump coupling with pyrophosphate hydrolysis. Plants have two isoforms of H⁺-PPase, type I and II. The type I enzyme is well known as vacuolar membrane H⁺-PPase. Membrane topology and identification of functional residues of H⁺-PPase has been determined for the enzyme of Streptomyces coelicolor in our laboratory. With the obtained information, we drew the membrane topology models of type I and II H⁺-PPases in plants (Vigna radiate and Arabidopsis thaliana). Type I enzyme consists of 16 transmembrane domains and type II of 17 transmembrane domains. With specific antibodies, we determined the protein amount of the type I and II enzymes and intracellular localization of type II enzyme. The type II enzyme has been demonstrated to be co-localized with markers for Golgi and ER and the protein content was extremely low compared with type I.

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Previously we cloned two Zn/Cd transporter genes (TjZnt1/2) from the Ni-hyperaccumulator Thlaspi japonicum, and reported that these transporters conferred high Ni-tolerance to yeast (Mizuno et al. 2005). The ion-specificity and Ni-tolerance level of TjZNT1 is distinctly different from TjZNT2, even though both transporters exhibit high sequence homology. This suggests that the determinants of ion-specificity and Ni-tolerance of these transporters are same, or related. In this work, we constructed various chimeras with these transporter genes, and heterologously expressed in yeast cells to identify the domain responsible for the ion-specificity and Ni-tolerance. N-terminal (1-7 a.a.) of TjZNT1 was identified as a region needed for high affinity Zn-transport. The truncated TjZNT2 that deleted first 1-36 amino acid residues newly obtained Zn-transport ability, suggesting the region is autoinhibitory domain for Zn-transport. Furthermore, we found that this domain conferred high Ni-tolerance of TjZNT2. The detail functions of the N-terminal domains are currently under investigation.

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To understand the plant responses to ozone, we isolated and characterized an ozone sensitive mutant (ozs1) from Arabidopsis thaliana ecotype Columbia. The mutant plants showed enhanced sensitivity to ozone, desiccation and sulfur dioxide but had normal sensitivity to hydrogen peroxide, low temperature and high light intensity. The OZS1 encodes a transporter-like protein in the tellurite resistance/C₄-dicarboxylate transporter family. Transpiration levels of OZS1, stomatal conductance and stomatal apertures were greater in ozs1 plants than in wild-type. The stomatal apertures of ozs1 plants showed diurnal fluctuations but were always larger than that of the wild-type plants under the same conditions. The stomata of ozs1 and wild-type plants similarly responded to stimuli such as light, abscisic acid and high concentration (1,000 ppm) of carbon dioxide. These results suggest that OZS1 helps to maintain stomata in the closed state rather than being involved in regulation of responses to these stimuli.

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Resveratrol is a phytoalexin against Botrytis cinerea and one of the functional compounds for human health. Resveratrol is a member of terpenoids and has a stilbene chemical backbone. For terpenoid transport, PDR transporters, which belong to ABC transporters, had been reported in Nicotiana plumbaginifolia (NpABC1) and Spirodela polyrrhiza (SpTUR2). Thus we predicted the existence of PDR transporter for resveratrol secretion in grape berry skin. We found an orthologue (VvPDR1) of NpABC1 in grape genome (Genescope) and cDNA of VvPDR1 was cloned with total RNA from grape skin by RT-PCR. Closest homologue to VvPDR1 in Arabidopsis PDRs is AtPDR12, which transports sclareol. Resveratrol accumulation in grape berry skin is increased by ultraviolet (UV) irradiation, thus we treated UV to grape berry and VvPDR1 expression in the skin was determined. Gene expressions of both VvPDR1 and stilbene synthase was induced by UV, suggesting the involvement ofVvPDR1 in resveratrol accumulation in grape skin.

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Sucrose is the compound of translocation of photosynthetically fixed carbons through plant body. Sucrose transporters play a pivotal role for the translocation process. To understand the uptake of sucrose in plant cells we investigated the intercellular localization of tobacco sucrose transporter (NtSUT) homologs. We first made antibodies against NtSUT proteins found in the tobacco EST database. Microsomes were separated by density gradient centrifugation and used to analyze the distribution of these proteins by immunoblotting. Distribution pattern of NtSUT proteins were different from that of known organelle markers. Immunocyochemical analyses indicted that these proteins are primary localized in organelle that showed intracellular puncta. We also expressed NtSUT-GFP fusion proteins in tobacco cells and found that these fusion proteins showed punctate pattern in the cells. The expression of the transporters was higher at log-phase cells than cells reached to stationaly-phase. Sucrose-starvation caused the degradation of these transporters.

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In higher plants, vacuole, which occupies most of a plant cell, plays important roles in a variety of plant growth and development. However, regulatory mechanism of vacuolar development has remained largely unknown. In this study, we focused vacuolar aquaporins which serve in the permeation of water across the vacuolar membranes. We first identified a vacuolar aquaporin from tobacco BY-2 cells (NtTIP1;1) and established transgenic tobacco BY-2 cells that overexpress NtTIP1;1-GFP (BY-TIPG cells). Using model system for synchronous cell elongation in miniprotoplasts prepared from tobacco BY-2 cells, we found that overexpression of NtTIP1:1-GFP accelerated vacuolar regeneration and cell expansion. Moreover in the condition of cell elongation, we also found that the population of divided BY-TIPG cells was twice in the case of wild type BY-2 cells. These results suggest that NtTIP1;1 is involved in accelation of both cell expansion and cell devision via water homeostasis in plant vacuoles.

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It is reported that expression of aquaporin gene is related with cold resistance of rice.
We investigated expression of all aquaporin genes using real-time PCR to know relation with chilling tolerance. The genes of plasma membrane intrinsic proteins (PIP) 1 group were risen expression remarkably when the seedling of chilling tolerant rice varieties was return to normal temperature from low temperature (type V). In the seedling of chilling sensitive rice varieties, the gene expression of PIP1 group was degraded (type L). Aquaporin genes showing V-L pattern is limited to a few genes, and it is thought that gene of PIP1 group have close relation to chilling resistance. Two kind of overexpressed PIP1 group (OsPIP1;1 and OsPIP1;3) rice was made by transformation into chilling sensitive rice variety. PIP1 genes in these rice seedlings showed type V when the seedlings were examined by chilling treatment.

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Membrane protein aquaporin showing high water permeability in various organisms is related to specific disease and physiological function.
Recently analysis of all genome of Koji fungi (Aspergillus oryzae) RIB40 strain was completed, which is important for traditional fermentation/brewing of Japan. Function analysis and establishment of expression system of aquaporin gene seem useful for functional improvement of Aspergillus oryzae. Therefore we show following studies:
1. Genes defined as aquaporin were isolated, and aquaporin expression by Xenopus oocyte was experimented on. These aquaporins showed extremely low water permeability from the coefficient of expansion of oocyte.
2. Using the Aspergillus oryzae mentioned above, establishment of experimental system of foreign aquaporin gene transformation was tried. The condition for expression of foreign gene in the transformed protoplasts is under study now. A result of foreign gene -expression in Aspergillus oryzae will be compared to the oocytes expression system.

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Four full-length cDNAs of plasma membrane intrinsic proteins (PIPs) were systematically isolated, identified and cloned from tulip (Tulipa gesneriana) petals According to the nomenclature of plant aquaporins, these were designated TgPIP1;1, TgPIP1;2, TgPIP2;1 and TgPIP2;2. Of these four homologues, only TgPIP2;2 displayed the significant water channel activity (W_a) in the Xenopus oocytes heterologous expression system. However, in Pichia pastoris expression system, both TgPIP2;1 and TgPIP2;2 showed W_a. The W_a was affected by mercury and inhibitors of protein kinases and protein phosphatases. Site-directed mutagenesis approache and spheroplast bursting assay revealed that Ser-35, Ser-116 and Ser-274 were the putative phosphorylation sites of the TgPIP2;2. Real-time RT-PCR analysis revealed that TgPIP2;2 is ubiquitously expressed in all organs. Collected data suggest that TgPIP2;2 might be modulated by phosphorylation and dephosphorylation for regulating water channel activity, and may play role in transcellular water transport in all tulip organs.

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The transport and gating activities of K⁺ channels are thought to be regulated by posttranslational modification. Several putative phosphorylation target residues exist in the cytosolic region of the Arabidopis K⁺ channel, KAT1. In this study, in vitro kinase assays demonstrated that the C-terminal region of KAT1 acts as a phosphorylation target for an Arabidopsis protein kinase. We have also examined the correlation between KAT1 phosphorylation and channel activity using two-electrode voltage clamp analysis in Xenopus oocytes. In these experiments, KAT1 channel activity was measured both pre- and post-activation of protein kinases in the Xenopus oocytes. Several KAT1 variants have now been generated that contain point mutations at putative phosphorylation target sites. The K⁺transport activities of these variants are also being examined in our Xenopus oocyte system and compared to that of wild-type KAT1.

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Synechococcus elongatus possesses a sulfate permease family transporter (LtnT) having latent nitrate transport activity and whose C-terminal domain inhibits the transport activity. The physiological substrate of this transporter, whose affinity for nitrate is low, remains to be elucidated. To characterize the amino acid residues involved in determination of the affinity for substrates, we aimed to obtain the LtnT transporters with improved affinity for nitrate. A truncated ltnT gene lacking the region encoding the C-terminal domain was amplified by error-prone PCR, the resulting ltnT genes were introduced into a nitrate transport-deficient Synechococcus strain, and mutants that grew on medium containing low concentrations of nitrate were isolated. Characterization of the ltnT genes of the mutants showed that five amino acid residues are involved in determination of the affinity for nitrate of the transporter. Combinations of these mutations synergistically improved the affinity for nitrate of the transporter.

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We find 5 genes for chloride channel in rice and cloned two of them, OsCLC-1 and OsCLC-2. And we obtained the deleted mutant for each gene by insertion of Tos17 and analyzed the phenotypes to clarify the function of each gene. The mutant for OsCLC-2 was sensitive to salt stress than wild type, Nipponbare and recovered the tolerance by the transformation of OsCLC-2 or OsCLC-1 cDNA. However, the deletion of OsCLC-1 gene did not change salt tolerance. Furthermore, the deletion of OsCLC-2 gene caused more requirement of nitrate for optimal growth, but not in the case of OsCLC-1. The requirement was diminished by the transformation of OsCLC-2 or OsCLC-1 cDNA. In the field, similar inhibition of the growth was observed only in the deletion of OsCLC-2. And the yield and size of seeds were also decreased in this deletion.

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We have reported that Arabidopsis transcription factors DREB1s control expression of many stress-responsive genes. In rice, we isolated cDNAs for DREB1 homologues, OsDREB1s. Overexpression of OsDREB1A in rice activated expression of target stress-responsive genes and resulted in improved stress tolerance to drought, high-salinity and cold. Rice has other 9 DREB1/CBF family genes than OsDREB1A. Previously, we have reported that these 10 OsDREB1 genes are expressed in response to different stresses each other and proteins encoded by these genes show different transactivation abilities.
In this study we performed 44k microarray analysis using transgenic rice overexpressing OsDREB1A. Approximately 50 genes were up-regulated in the transgenic rice. Microarray and northern blot analyses of transgenic rice overexpressing other OsDREB1-family showed that other OsDREB1-family genes have different downstream genes from OsDREB1A. These results suggest that there may be different functions between OsDREB1A and other OsDREB1-family genes in rice.

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Plants growth and productivity are affected by various abiotic stresses such as drought, high salinity, and low temperature. When plants are exposed to these stresses, many genes that function in stress tolerance such as rd29 are induced. Previously we have reported a cis-acting element, DRE/CRT involved in drought, high-salt and cold-stress responsive gene expression and cDNAs for DRE/CRT (A/GCCGAC) binding proteins (DREBs) were isolated. One of DREBs, DREB2A was induced by not only drought and salt but also heat stresses. To analyze the regulatory mechanisms of DREB2A induction under stress conditions we analyzed the promoter region of DREB2A. We identified a cis-regulatory region in response to heat stress in the DREB2A promoter. This region contains HSE (GAAnnTTC), allowing us to investigate which HSFs regulate DREB2A expression through the HSE. We found a cis-regulatory region in response to drought stress in the other region of the DREB2A promoter.

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RPK1, a leucine-rich repeat (LRR) receptor kinase in the plasma membrane, is upregulated by ABA in Arabidopsis. Repression of RPK1 expression in Arabidopsis decreased sensitivity to ABA during germination, growth, and stomatal closure. We produced the transgenic plants overexpressing the RPK1 protein and analyzed their phenotypes. The transgenic plants showed growth retardation and increased ABA sensitivity in root growth and stomatal closure. Reactive oxygen species (ROS) act as second messengers in ABA signaling. Oxidative stress responses were altered in the RPK1 knockout and the RPK1 overexpressor. Expression levels of stress- and ROS- responsive genes in rpk1-1 were decreased compared with those in wild-type, whereas these expression levels in the RPK1 overexpressor were higher than those in wild-type. Overexpression of RPK1 improved tolerance to drought stress in Arabidopsis. These results suggest that RPK1 mediates ROS signaling pathway, which regulates ROS production and scavenging during drought stress.

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Receptor-like kinases (RLK) comprise a large gene family and play important roles in growth, development, and hormone and stress responses. Microarray analysis identified a leucine-rich repeat RLK (LRR-RLK), LOW-TEMPERATURE INDUCED RECEPTOR-LIKE KINASE (LIK1), which expression is especially induced during cold stress condition. Expression of LIK1 is induced by cold treatment within 1 hour. The LIK1-GFP protein was localized to the cell surface of root tips, roots vascular bundles, and apical meristem in the transgenic plants expressing the LIK1-GFP fusion protein driven by the LIK1 promoter. The GFP Fluorescence was increased by cold treatment in the root tip. We also analyzed 35S:LIK1 transgenic plants and LIK1 knockout mutants. Sensitivity of the 35S:LIK1 plants and lik1 mutants to freezing temperature wasn't altered but under long-day conditions, 35S:LIK1 plants showed a late-flowering phenotype. The results indicate that LIK1 is induced by cold stress and may regulate flowering and growth under cold stress condition.

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Although the nuclear RNA binding protein FCA and the Mg-chelatase H subunit localized in the chloroplast have recently been reported as receptors for the plant hormone, abscisic acid (ABA), some physiological evidence suggest perception of the ABA signal on the cell surface. By using biotinylated ABA, we visualized and quantitatively characterized the putative ABA perception sites on the cell surface of Vicia faba guard cell protoplasts and barley aleurone protoplasts (Yamazaki et al., 2003; Kitahata et al., 2005). In the present study, in an attempt to identify putative plasma membrane ABA receptors, we applied the phage display technique using the cDNA library from Arabidopsis thaliana to screen for novel biotinlylated ABA-binding proteins. Subcellular localization, ABA sensitivity of the T-DNA insertional mutants in terms of seed germination, root growth and stomatal closure, as well as the ABA binding activity of the putative novel ABA binding proteins will be discussed.

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Arabidopsis ABI1, which encodes a type 2C protein phosphatase (PP2C), negatively regulates many of abscisic acid (ABA) functions in Arabidopsis, such as desiccation tolerance and germination of seeds, and adaptation to stresses such as drought, high salinity and cold. The moss Physcomitrella patens is known to accumulate ABA, and possesses an ABA signaling pathway similar to that of seed plants, however, our knowledge of ABA function in the moss is limited. We have shown that P. patens has two ABI1 homologous genes, PpABI1A and PpABI1B, and that targeted disruption of PpABI1A causes ABA hypersensitive phenotypes. To understand the physiological functions of ABA in P. patens, we analyzed phenotypes of the PpABI1A disruptant. As the result, the PpABI1A disruptant showed enhanced salt tolerance compared to the wild type. This suggests that salt tolerance mediated by ABA signaling was already established when the first drought-tolerant land plants appeared.

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Arabidopsis thaliana ABI1, which encodes a type-2C Ser/Thr protein phosphatase (PP2C), is a negative regulator of ABA-signaling pathway. The abi1-1 is a dominant mutant of ABI1 that carries an amino acid substitution (Gly to Asp) in the catalytic domain, and strongly blocks ABA signaling. Multiple ABI1-interacting proteins have been isolated, but none of these interactions are able to explain the dominant effect of abi1-1 in ABA signaling. Previously we demonstrated that the PP2C-mediated ABA signaling is evolutionarily conserved in the moss Physcomitrella patens. The protonemata of P. patens consist of only two cell types, and ABA response does occur uniformly in protonemal cells. Thus, by exploiting the feature of the protonemata, we started to identify interacting proteins of PpABI1A from P. patens using a yeast-two-hybrid system. We obtained about 250 positive clones from screening of 700,000 clones. We are analyzing these clones, and some of the results will be discussed.

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Protein phosphorylation and dephosphorylation play important roles in signal transduction pathways. We have focused on a PP2C in Arabidopsis thaliana that has a similarity with PP2C in yeast (PTC), and named APC4. It has been reported that PTC negatively regulated stress-responsive signaling pathway. So far, we have confirmed that APC4 has enzymatic properties of PP2C. In order to clarify its function in vivo, we have screened T-DNA tag lines in A. thaliana and isolated two independent lines. In one of these T-DNA insertion lines, we have not obtained any homozygous plants, and the abnormal segregation ratio between wild type and heterozygous mutant plants was observed. Defective APC4 mRNA by T-DNA insertion has been detected in heterozygous plants by northern analysis, suggesting that a mutant protein may act dominant-negatively. Now, we try to analyze the function of APC4 by using over-expressing plants.

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We have screened Arabidopsis mutants for root growth resistance to a putative antiauxin, p-chlorophenoxyisobutyric acid (PCIB), which inhibits auxin action by interfering the upstream auxin signaling events. In addition to two known auxin-related loci, TIR1 and AtCUL1, we have successfully isolated at least three novel antiauxin-resistant mutants (aars). The aar3 mutant was also resistant to 2,4-D as shown by a root growth assay. The AAR3 gene encodes a protein with a domain of unknown function (DUF298). The protein has a putative nuclear localization signal and its nuclear localization was confirmed by a protoplast transient assay. The protein shares homology with the DEFECTIVE IN CULLIN NEDDYLATION-1 protein through the DUF298 domain. However, no significant difference in the auxin responsive markers, DR5:GUS or HS:AXR3NT-GUS, was observed between the wild type and aar3, suggesting that the AAR3 gene regulates 2,4-D sensitivity through a previously uncharacterized mechanism.

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AtPCaP1 was found as a novel-type cation-binding protein in Arabidopsis thaliana. AtPCaP1 is a hydrophilic protein that is rich in glutamate and lysine residues and has no transmembrane domain or enzymatic functional motif. AtPCaP1 is stably associated with the plasma membrane via N-myristoylation. The mRNA level was increased by treatment with copper and a bacterial elicitor peptide flg22. In the present study, we prepared T-DNA inserted mutant of AtPCaP1 (atpcap1) and examined phenotype of the plants grown for 2 to 4 weeks under several stress conditions. The atpcap1 mutant plants became sensitive to deficiency and excess of copper but not of iron. Interestingly, the growth of the mutant plants was markedly suppressed when plants were grown at high density in plastic dishes. Furthermore we observed a significant induction of AtPCaP1 by flg22 in promoter-GUS analysis. Thus, AtPCaP1 may be a key protein involved in response to multiple physiological stresses.

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The site-specific signal of Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] from apices of growing root hairs and pollen tubes promotes cytoskeletal reorganization and membrane trafficking. To uncover how its spatiotemporal pattern is established and which aspect of tip growth it regulates, we identified the Arabidopsis thaliana PIP5K3 gene which encodes a phosphatidylinositol 4-phosphate 5-kinase, a key enzyme producing PtdIns(4,5)P₂, and is expressed preferentially in root hair cells. T-DNA insertion mutations and overexpression of PIP5K3 caused significantly shorter and longer root hairs respectively. A yellow fluorescence protein fusion of PIP5K3, directed by the PIP5K3 promoter, complemented the short root hair phenotype and was found to localize at the plasma membrane and cytoplasmic space of elongating root hair apices. These results provide evidence that PIP5K3 is involved in the localization of PtdIns(4,5)P₂ to the elongating root hair apex and acts as a key regulatory component of the machinery initiating and promoting root hair tip growth.

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In guard cells, ABA evokes production of reactive oxygen species (ROS) and elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt), resulting in inducing stomatal closure. A regulatory A subunit of protein phosphatase 2A, RCN1, functions as a positive regulator upstream of [Ca²⁺]_cyt elevation in the ABA signaling pathway (1). Methyl jasmonate (MeJA) as well as ABA induces stomatal closure. There is the signaling interaction between ABA and MeJA upstream of ROS production (2). Hence we examined the role of RCN1 in the signaling interaction in guard cells using the Arabidopsis rcn1 mutant. Both ABA and MeJA failed to induce stomatal closure in the rcn1 mutant. Furthermore, neither ABA nor MeJA induced ROS production in rcn1 guard cells. These results suggest that RCN1 could function upstream of ROS production in ABA signaling and MeJA signaling in guard cells.

(1) Kwak et al. Plant cell (2002) 2849-2861
(2) Munemasa et al. Plant Physiol. (2007) 1398-1407

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In living organisms, Ca²⁺ functions as an important second messenger and various physiological processes are dependent on alteration of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt). In plants, roles of Ca2+ in root growth, pollen tube elongation, light perception, and regulation of stomatal movements have been well studied. However, the mechanism of Ca²⁺ signaling in plant cells remains unclear compared with animal cells. Here we focused on Ca²⁺ signaling in guard cells where [Ca²⁺]_cyt alters in response to various stimuli including phytohormone abscisic acid and extracellular Ca²⁺ concentration ([Ca²⁺]_o). In order to examine dynamic alteration of [Ca²⁺]_cyt in guard cells, we used Arabidopsis expressing a Ca²⁺ indicator fluorescent protein, yellow cameleon. We also report [Ca²⁺]_cyt oscillations in guard cells of several Arabidopsis mutants defective in stomatal regulation in response to [Ca²⁺]_o.

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In higher plants, metabolism of sugar (C) and nitrogen (N) is regulated each other. Therefore it is suggested the existence in C/N regulator which senses and controls metabolism with relative amount of C and N (C/N balance).
To clarify the regulator, we isolated C/N response mutant ssv1-D (super survival 1-D) in which SSV1 gene was overexpressed. The mutant enables to survive under high-sugar / low-nitrogen conditions while wild-type does not.
The SSV1 gene belongs to Arabidopsis ATL family. Thus functional study on ATL gene family in C/N response was examined. Detailed characterizations of ATL family will be reported.

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MAP kinase transduction pathways are highly conserved in eukaryotes, and are suggested to be concerned in cell proliferation, cell differentiation and stress responses. Plant's MAPKs were thought to be different from those in animals and fungi, although the precise functions in plant were unknown.
Chlamydomonas reinhardtii, a unicellular alga, is most suitable model organism for analysis of MAPK systems in plant because the Chlamydomonas is unicellular and genomic information are available. Recently, genomic sequences were determined in related algae and higher plant species, such as Ostreococcus sp., Volvox carteri, Chlorella sp. and Physcomitrella patens. We have compared the conservation of MAPKs in algae and higher plants and have suggested the function of each MAPK in plants.

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Arabidopsis ht 1 (high leaf temperature 1) is a recessive mutant, which is altered in their ability to control stomatal movements in response to CO₂. The strong allele, ht1-2 exhibits a dramatically impaired CO₂ response but functional reactions to blue light and ABA. The HT 1 gene encodes a protein kinase which is expressed mainly in guard cells. In vitro phosphorylation assays demonstrate that the activity of the HT1 protein carrying the recessive ht 1 mutation is greatly impaired. Recently we isolated a new dominant mutant of HT1, ht1-3D. The ht1-3D is insensitive to CO₂ but responsive to ABA and light. These results suggest that HT1 is a distinct component of CO₂ signalling pathway. We will discuss the effect of ht1-3D mutation on the HT1 kinase activity.

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We recently showed that CERK1 (Chitin Elicitor Receptor Kinase 1), a receptor-like kinase with extracellular LysM domains, is an essential component for chitin elicitor signaling in A. thaliana1). In rice genome, ten receptor-like kinase genes with LysM domains, named as OsLysM-RLKs, were found. Among these, OsLysM-RLK9 showed the highest homology with CERK1 and thus analyzed for its function in chitin elicitor signaling in rice. Knock-down transformants of OsLysM-RLK9 were established to analyze the function of this molecule. Several lines of these transformants showed significantly reduced expression of OsLysM-RLK9 but not of CEBiP (Chitin Elicitor Binding Protein) 2). These lines showed almost no ROS generation as well as phytoalexin biosynthesis in response to chitin oligosaccharide elicitor. These results indicated that OsLysM-RLK9 plays an essential role for chitin elicitor signaling in rice, somehow collaborating with CEBiP.
1)Miya et al., PNAS, in press; 2)Kaku et al., PNAS, 103, 11086 ('06)

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To understand the G-protein related signal transduction system of plant in detail, we tried to obtain various controlling factors influencing on the function of G-protein α subunit in vivo. For this, we designed the efficient vector system, having the TAP tag cassette for isolating the highly purified protein complex and fused with GFP not only to localize the protein but to screen the transformants rapidly with the western blot. Moreover, we used Arabidopsis sgs mutant as a host, not to reject the foreign gene introduction. To test the capability of newly constructed vector, we coexpressed GPA1 with a marker protein, H⁺-ATPase into the Arabidopsis protoplasts. Result showed that the two proteins were observed at the plasma membrane with the fluorescence microscope. After isolation of the GPA1-interacting proteins by this vector system, they were analyzed by MALDI-TOF Mass Spectrometry. Now, we examine the role of individual proteins in the signal network.

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We studied the photoreaction of the BLUF domain of TePixD protein of Thermosynechococcus elongatus. The Tyr8 residue was modified to phenylalanine or alanine residues (Y8F or Y8A, respectively) by the site-directed mutagenesis. At room temperature, illumination induced the red shift of the absorption in the wild type (WT) protein, but not in the Y8F and Y8A mutant proteins as reported. However, the illumination at 80 K accumulated the red shifted forms both in the WT and mutant proteins. The illumination of Y8F protein at 80 K induced the red-shifted form to the extent at 1/2 of that in the WT protein at a 43 times slower rate. Upon the illumination at 150 K, the Y8F proteins did not accumulate the red-shifted forms, but accumulated the flavin anions. It is concluded that the photoconversion occurs even without Tyr8 residue, and that Tyr8 is necessary to enhance the photoconversion efficiency.

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The bending of a plant toward the direction of intense light is called phototropism. This directional growth response is caused by the plant growth regulating substances. In this aspect, Bruinsma-Hasegawa hypothesis (1990) stated that the gradient of growth-inhibiting substances in the illuminated side is a key factor of bending during phototropic curvature. The symbolic growth inhibitors, cis- and trans-raphanusanins have been isolated from radish hypocotyls. These compounds were dramatically accumulated on the side of blue light illumination. To understand the role of raphanusanins in phototropic curvature and the responsible genes for the growth inhibition, DD-PCR was performed between the raphanusanin applied and control hypocotyls. We could trace some candidate genes indicating a close linkage between raphanusanins and phototropic curvature. The detailed roles and functional impacts of these genes for the growth inhibition in response to phototropic stimulation will be presented.

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Stomata in Adiantum capillus-veneris lack blue light-specific response, but open in response to light. We investigated this light response in intact leaves. The spectrum for stomatal opening matched to photosynthesis. The application of red and far-red light induced the synergism of stomatal opening. Stomata in Adiantum lacked the CO₂ sensitivity in dark. Stomatal conductance showed the much higher sensitivity to light when it was applied to the lower leaf surface, where stomata exist, than applied to the upper surface, suggesting that guard cells sensed the light. In isolated epidermis, red light induced stomatal opening and K⁺ accumulation in guard cells, and photosynthetic inhibitor inhibited both of them. Red light-induced stomatal opening was completely inhibited by CsCl, a K⁺ channel blocker. These results indicate that Adiantum stomata lack the sensitivity to CO₂ and that the opening is driven by photosynthesis in guard cell chloroplasts, probably via K⁺ uptake.

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Blue light, perceived by blue light photoreceptor phototropins (phot1 and phot2), induces stomatal opening through the activation of plasma membrane H⁺-ATPase in guard cells. However, little is known about the signaling pathway from phototropins to the H⁺-ATPase. In this study, we isolated the mutants, which show closed stomata phenotype, from EMS-treated Arabidopsis by thermal imaging. First we established the condition for screening to distinguish leaf temperature from phot1 phot2 double mutant and wild type. Then, we performed the screening of mutants under above conditions. We isolated 10 mutants, which show closed stomata phenotype, from 40,000 plants. One of them was rpt2, which acts on the downstream of phot1 in phototropism and stomatal opening, suggesting that this screening is useful for isolation of stomatal aperture mutants in the signal transduction pathway of phototropin. In addition, we isolated open stomata mutants by this screening. We will also report these mutants.

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In non-photosynthetic, yellow or colorless mutant cells of Chlorella kessleri, grown with nitrate as sole nitrogen source, 6-deoxy-D-glucose enhanced the uptake of the glycine, while it did not influence the uptake of nitrate in both growing and resting cells. On the other hand, the addition of glucose inhibited the uptake of nitrate in growing cells. Addition of methionine sulphoximine, a potent inhibitor of glutamine synthetase, to cells incubated with glucose, resulted in intracellular ammonia-accumulation and the uptake of nitrate was also extremely inhibited by extra cellular ammonia. Thus the utilization of exogenous nitrogen compounds such as ammonia, amino acids and nitrate is controlled by ammonia accumulated in inner cells. The role of glucose, ammonia and blue light in the uptake of nitrogenous compounds is discussed.

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In the moss, Physcomitrella patens, phytochrome is involved in branch formation, phototropism, and chloroplast photomovement. At least 4 PHY genes are known in P. patens. In the present study, functions of the phy species were analyzed by overexpression of PHY cDNA fused to YFP. When photoinduction of branch formation was examined, promotion of the response was observed in the overexpressors, being dependent on the phy species expressed. Intracellular localization of phy as detected by YFP fluorescence also showed differences among the phy species. In the light-grown cells, phy located in the cytoplasm and also in the nucleus as speckles. Nuclear-cytoplasmic partitioning showed a difference among the species. Interestingly, nuclear speckles were also seen in the dark-grown cells and their abundance depended on the phy species. These evidences suggest the possible functional difference of each phy species in the photoresponse.

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Red- and far-red-sensing photoreceptors; phytochromes are encoded by small gene families in higher plants. The phytochrome gene family consists of only three members known as PHYA, PHYB and PHYC in rice while it contains five genes in Arabidopsis. Each member of phytochromes has distinct as well as cooperative roles in monitoring and responding to the environmental light signals. In this study, we isolated the promoter region of each phytochrome gene from rice genome and analyzed the distinct expression patterns of each phytochrome. Transgenes composed of each promoter and cDNA were introduced to appropriate phytochrome mutants. Photomorphogenesis of the T1 seedlings indicated that the isolated promoters regulated downstream cDNAs accordingly. We also made promoter-GUS transgenic rice plants and found the different patterns of GUS expression among these promoters. Moreover, we monitored nucleocytoplasmic partitioning of rice phytochromes by using phytochrome-GFP fusion proteins.

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We have demonstrated that cryptochromes mediate blue light dependent photomorphogenetic responses in rice, such as growth inhibition of coleoptile, leaf blade and leaf sheath and declination angles of leaf blades.
To investigate the underlying mechanisms, we analyzed the change of gene expression induced by blue light by using microarray (Agilent rice 44k). Among the blue light induced genes, we found GA2ox genes, which inactivate the active GAs. Individual RT-PCR experiments confirmed that GA2ox genes were induced by blue but not by red light.
Next, we compared endogenous levels of various GAs in dark grown seedlings with those in blue light irradiated ones. GA1, active GA, was decreased by the blue light irradiation. Thus, GA2ox seems to be involved in blue light dependent photomorphogenetic responses in rice.

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We focus on parasitic plants, which lost photosynthetic abilities after had become parasitic in the process of evolution. New insights into the light signal transduction of plants will be obtained by clarifying differences of light signal transduction pathways between parasitic plants and photosynthetic plants. So far we isolated a phytochrome gene in Orobanche minor, which doesn't do photosynthesis at all, and we analyzed functions of the gene. In this study, we focuse on another parasitic plant, Striga hermonthica, which is a closely related species of O. minor but still keeps photosynthetic ability. Relationship between functions of phytochrome and its amino acid sequence will be more clear by comparison of related photosynthetic and non-photosynthetic species. Therefore we isolated phytochrome A and B homologous genes in S. hermonthica. We are verifing photoresponses of S. hermonthica now.

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The photoreceptors for chloroplast photorelocation movement have been known, but the signal transduction pathway remains to be clarified. To investigate the properties of the signal for chloroplast accumulation response, we deduced the speed of signal transfer from light-irradiated site to chloroplasts in the gametophytes of Adiantum capillus-veneris. When dark-adapted cells were irradiated with a microbeam of various light intensities of red or blue light for 1min or continuously, the chloroplasts started to move towards the irradiated area. The speed of signal transfer was calculated from the relationship between the timing of start moving and the distance of chloroplalsts from the microbeam and found to be constant at any light conditions. In prothallial cells, the speed was about 1.0 μm/min and in protonemal cells about 0.8 μm/min towards base and about 2.3 μm/min towards the apex. When chloroplasts stay the longer distance from the microbeam, the faster they could move.

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Chloroplast relocations have been known to be induced in response to light conditions. Chloroplasts move toward weak light and avoid from strong light. Dark-induced relocation, called dark position, has also been known. Although studies on chloroplast relocation by light have been performed since approximately 100 years ago, the studies by other stimuli have been carried out poorly. In this study, we found that low temperature induces chloroplast relocation (termed cold position) in prothalli of the fern Adiantum capillus-veneris. When prothalli were incubated at 25 degrees Celsius under weak light, chloroplasts accumulated along the periclinal cell wall. When the prothalli were subsequently incubated at 4 degrees Celsius under weak light, the chloroplasts relocated to and congregated at the anticlinal cell wall. Cold positioning was not observed in mutant ferns defective of a photoreceptor, phototropin 2. These results indicate that cold positioning is mediated by phototropin 2 under cold condition in fern.

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In epidermal cells of Vallisneria gigantea, hight-intensity blue light (BL) induces the avoidance response of chloroplasts. BL remarkably decreases the resistance of chloroplasts to centrifugal force. This BL-induced dis-anchorage of chloroplasts occurs before the start of directional movement of chloroplasts.
In several plants, BL induces a Ca²⁺ transient. On the other hand, chloroplasts take up or release Ca²⁺, responding to changes in light conditions.
To ascertain a possibility that Ca²⁺ is involved in the induction of avoidance response of chloroplasts, we examined the effects of Ca²⁺-channel blocker (La³⁺), phospholipase C inhibitor (U73122), and photosynthesis inhibitor (DCMU). These drugs suppressed the avoidance response additively, and when all three drugs were present, the response was completely inhibited. The BL-induced dis-anchorage of chloroplasts was substantially suppressed in the presence of La³⁺ and U73122. These results suggest that Ca²⁺ from different stores is involved in the induction of avoidance response of chloroplasts.

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We found the novel Ca²⁺-binding proteins, CCaP1 (cytosolic Ca²⁺-binding protein), CCaP2, and CCaP3. First, we determined their cytoplasmic localization using GFP fusion protein. Second, we examined tissue specificity using promoter-GUS transgenic plants. CCaP1 was predominantly expressed in petioles and CCaP2 and CCaP3 in roots. Next, we analyzed effect of growth conditions on the mRNA level of CCaPs by real-time PCR. The mRNA levels of CCaP1 and CCaP2 were increase about 10-fold of that of the daytime level in a dark period for >20h and decreased to the daytime level by light illumination for <5h. We will report the results from mutant lines of phytochrome and cryptochrome. Expression profiles of CCaPs in seedlings showed the same response. These results suggest the difference mechanisms in stimulation and suppression of transcription. Now we are trying to determine wavelength of light essential for regulation of transcription and quantify the protein level of CCaPs.

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Ultraviolet-B radiation (UV-B: 280-320 nm) has pleiotropic effects on plant development, plant morphology, and plant physiology. To investigate the effects of continuous UV-B irradiation on the inside of cucumber cotyledon, cucumber cotyledon was embedded in Spurr's epoxy resin and was sectioned by ultramicrotome. Observation under the light microscope revealed that longitudinal extension of the cells that compose palisade two layers is inhibited and the shape of the cell walls become waved after continuous UV-B irradiation for 7 days. Longitudinal extension of the cells that compose palisade two layers is more inhibited and palisade two layers were collapsed after continuous UV-B irradiation for 15 days. Also, the surface of the epidermal cells become smooth and epidermal cells were expanded. To clarify the effect of continuous UV-B irradiation on cotyledon morphology and anatomy in cucumber, time course analysis is necessary.

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Cytochrome P450 is a diverse superfamily of hemoproteins found in bacteria, archaea, animals and plants. Especially in plants, the number of this gene family is very large and occupy up to 1 % of the plant genome. Most P450 mainly catalyze a monooxygenase reaction (RH+O₂+NADPH₂⁺→ROH+H₂O+NADP⁺).
We isolated a P450 gene, OsCYP-10, in rice that is grouped into the CYP84A subfamily. In this family, a homologous gene has been reported to be involved in phenylpropanoid biosynthesis. The transgenic plants harboring the OsCYP-10 antisense gene were apparently increased sensitivity to the UV-B irradiation. This result suggests that OsCYP-10 may play some important roles in hydroxylation of the C5 position during phenylpropanoid biosynthesis pathway similery as the other CYP84A members.

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Supplementary UVB radiation inhibited growth, yield and grain development. One of principal causes of such UVB-induced damage is UVB-induced CPD, and increasing CPD photolyase activity can significantly alleviate UVB-caused growth inhibition. How degree of growth inhibition was caused by UVB in natural sunlight? In this work, effects of solar UVB on growth and yield of rice under outdoor conditions were investigated using UV-resistant Koshihikari and the UV-sensitive chromosomal segment substitution line (SL-229) as experimental plant; chromosomal 10 region, on which CPD photolyase gene located, in SL-229 was homozygous for Kasalath allele, whereas all other chromosomal regions were homozygous for Koshihikari alleles. CPD photolyase activity of Kasalath was lower than that of Koshihikari. The grain size and weight of SL-229 under current outdoor conditions were reduced. These results mean that growth and yield of rice grown under current environmental conditions would be inhibited by UVB in natural sunlight.

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Cyclobutane pyrimidine dimer (CPD) photolyase is a crucial factor for determining UVB sensitivity in plants. We previously found that native rice CPD photolyase have two protein of about 54 kDa and 56 kDa. Treatment with protein phosphatase revealed that the 56-kDa native rice CPD photolyase was phosphorylated. Our finding is the first demonstration that the CPD photolyase is phosphorylated. In this study, we investigated the effect of phosphorylation on CPD photolyase activity. First, the purified native rice CPD photolyase was treated with protein phosphatase, and then the activity of dephosphorylated CPD photolyase was compared with the purified native rice CPD photolyase, including phosphorylated CPD photolyase. Next, the phosphorylated CPD photolyase was isolated by phosphate metal affinity chromatography, and then the activity of the phosphorylated CPD photolyase was measured. We discuss the effect of phosphorylation on the CPD photolyase activity.

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Chloroplastic 2-oxoglutarate (2-OG)/malate transporter (OMT) and general dicarboxylate transporter cooperatively function in mediating between carbon and nitrogen metabolic pathways. Additionally, we consider that OMT also participates in malate valve that transports reducing equivalents from stroma to cytoplasm. We found that OMT knockout mutant, omt1-T2, showed growth delay due to reduction of 2-OG transport, GS/GOGAT cycle activity and amino acid synthesis. Here, we compared high-light responsiveness between wild type and omt1-T2 plants. The knockout plant lapsed more prominent photoinhibition in response to high-intensity light. Moreover, activation state of stromal NADP-MDH was increased more rapidly in omt1-T2. These findings suggest that photoinhibition progresses more rapidly in omt1-T2 due to accumulation of more NADPH in stroma compared to wild type.Complemented plants of omt1-T2 showed recovery of growth rate, amino acid contents and high-light susceptibility. Therefore, it is confirmed that OMT gene is responsible for mutant phenotype.

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In higher plants, two types of ascorbate peroxidase are localized in thylakoid membrane (tAPX) and stroma (sAPX) of chloroplasts, and contribute to the regulation of H₂O₂ level. However, these chloroplastic APXs are extremely unstable compared with other APX isoenzymes. Recently, much attention has focused on the function of H₂O₂ as a signaling molecule. To understand the contribution of chloroplastic APXs to the cellular redox regulation in higher plants, we obtained the T-DNA insertion Arabidopsis lines in the tAPX or sAPX genes (KO-tAPX, KO-sAPX). Although KO-tAPX plants showed slight increase in the sensitivity to oxidative stress caused by 25 uM paraquat treatment compared with wild-type and KO-sAPX plants, those changes were no greater than expected. Next, we carried out the transient suppression of tAPX expression by the estrogen inducible RNAi. When the expression of tAPX was transiently suppressed, the transcripts of several oxidative stress-responsive genes were induced.

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