Plant nitrate transporters (NRTs) are classified into NRT1 and NRT2. NRT2 transporters play major roles in high-affinity nitrate uptake. Plants usually have several NRT2 molecular species, some of which require NAR2 for its expression and function. Physcomitrella patens has eight NRT2 species and three NAR2 species. Among the eight NRT2 species, NRT2;1, NRT2;2 and NRT2;4 are more closely related to one another than to NRT2;3 and have higher affinity for nitrate than NRT2;3, whereas no such functional differentiation is known for NAR2. Functional relationships between the NRT2 and NAR2 molecular species is also unknown. To analyze the function of each NAR2 species, we constructed single and double mutants of NAR2;1 and NAR2;2 . Analysis of the expression of NRT2;3 protein in the mutants using anti-NRT2;3 antibody showed that the NRT2;3 protein was stably expressed in a single mutant of NAR2;1 and NAR2;2. By contrast, the protein was not detectable in a double mutant of NAR2;1 and NAR2;2, indicating that NAR2;3 cannot support expression of the NRT2;3 protein. We are currently trying to further clarify the function of NAR2;1 and NAR2;2 and to construct a NAR2;3 knockout mutant.

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Bet v 1 family proteins of the Fagales order are the major pollen allergens. Fourteen genes of the Bet v 1 family were randomly recombined in vitro by means of DNA shuffling. A resultant low allergenic Tree Pollen Chimera 7 (TPC7) has high immunogenicity against multiple Fagales pollen allergy in vitro and in vivo. Towards development of rice seed-based edible vaccine against multiple Fagales pollen allergy, we generated transgenic rice that accumulate TPC7 in the endosperm. TPC7 high accumulating lines were screened by immunoblot using anti-TPC7 antibody. TPC7 in high accumulating lines was detectable as two visible bands on CBB-stained SDS-PAGE gel. After glycosidase reaction, two bands became a single band, indicating that rice seed-derived TPC7 was glycosylated. In rice endosperm cells, seed storage protein prolamins are deposited in the ER-derived protein body-I (PB-I), and glutelins and globulin are deposited in the storage vacuole PB-II. Since TPC7 was deposited into the novel organelle other than PB-I or PB-II, we termed this as TPC7 body. We will report biochemical properties of TPC7 protein in the transgenic rice and the character of TPC7 body.

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In rice seed, alcohol soluble seed storage proteins, prolamins are classified into Cys-rich (CysR) and Cys-poor (CysP) molecules containing and not containing cysteine residue, respectively. The former is composed of CysR10, CysR14, CysR16 molecules and the latter is composed of CysP13 molecule. These prolamins are accumulated within the endoplasmic reticulum as protein body type I (PB-I). CysR10 is accumulated in the center of PB-I. To elucidate the role of CysR10 in PB-I formation, we produced the RNAi transformant to silence the gene which encodes the CysR10. In the CysR10-RNAi transformant, the malformation of PB-I and the reduction in the level of CysP13 were detected. On the other hand, esp3 mutant which reduces almost all CysR prolamins, showed the hypertrophy and deformation of PB-I. These results suggested that the existence of CysR10 is important for the orderly arrangement of the other prolamins in PB-I. Additionally it is inferred from the structural difference of PB-I between CysR10-RNAi transformant and esp3 that the malformation of PB-I in CysR10-RNAi transformant is induced by the presence of the CysR14 and CysR16.

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Cyanidium caldarium is a unicellular primitive red alga which grows in acidic environments (pH 1-3), but cannot survive above pH 7. In our previous study, we found that a large amount of only one protein was secreted from C. caldarium when the alga was cultured at pH 6. This protein showed relative high homology with lysyl oxidase (LOX), which found in animal cells but not in plant cells. Therefore ,we named it lysyl oxidase like protein (LOL). In this study, we examined pH dependence of the secreted amount of LOL protein, and found that a slight amount of LOL was secreted even at acidic pH. The similar gene of LOL protein was found in another acidophilic primitive red alga. Thus, we examined the secretion of LOL protein from C. melorae cells, but the protein was not secreted. There is no information about the localization and function of LOL protein. Thus, we prepared an antibody against LOL protein to elucidate the localization of LOL protein, and found that a large amount of LOL protein was also presented in intracellular fractions. We will discuss the localization and function of LOL protein.

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In higher plants, metabolism of sugar (C) and nitrogen (N) is mutually regulated. To clarify the C/N regulator, we isolated novel C/N regulatory ubiquitin ligase ATL31 (Sato et al. Plant J, 60: 852, 2009). Plants overexpressing ATL31 showed insensitivity to C/N stress conditions, whereas the knockout mutants did the hypersensitivity.
To investigate the detailed functions of ATL31, we performed identification analysis of the ATL31-interacting proteins. Thus, 14-3-3s were found as the candidate for substrates of ubiquitination by ATL31. From further exploring ATL31-interacting proteins with cross-linker (DSP), we also identified the membrane traffic-related proteins.
Relationships between C/N balance regulation and membrane traffic will be discussed in terms of functions of ATL31.

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Intracellular protein trafficking affects a wide range of developmental processes. However, little is known about its role in resistance to environmental stresses. To better understand the role of protein trafficking in cold stressed induced root growth and development, we investigated the response of the available Arabidopsis trafficking mutants to cold stress, and also screened new mutants. Both the SNARE mutant vam3, and retromer mutant snx1 showed a delayed recovery to gravity after cold stress, while the knockout mutant of RabA4C showed a faster recovery. For root growth recovery, vam3 and snx1 showed a delayed response compared with wild-type. Collectively, these results suggest that intracellular protein trafficking regulates the plant growth and development under cold stress. Among the screened mutants, ems41-16 showed delayed recovery to root growth and gravity response after cold stress, while ems38-1 showed an opposite phenotype, faster recovery of root growth and gravity response. Identification of these mutated genes will enable us to better understand the role of protein trafficking in cold stress induced inhibition of growth and development.

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Among transport vesicle responsible for protein trafficking in eukaryotic cells, COPII coated vesicles mediate anterograde transport from endoplasmic reticulum (ER) to Golgi apparatus. The COPII coat complex is compose of small GTPase, Sar1 and 2 types of protein complexes (Sec23/Sec24 complex and Sec13/31 complex). Sec23/Sec24 contributes to recognition and sorting of specific cargo, and Sec13/Sec31 involve in concentration of cargo and curvature of ER membrane. We found that Sec31 homologue (ATSEC31A) affect development of guard cells and pollen in Arabidopsis thaliana. From these results, we started the study of another component of COPII, Sec23, in A. thaliana. Previously, we found there are 7 types of Sec23 homologue (ATSEC23) in Arabidopsis genome and determined the sites of gene expression and subcellular localization of their products. We also analyzed function of each ATSEC23 using knockout plants. Here, we report involvement of ATSEC23 in development of A.thaliana base on result of functional analysis.

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Molybdenum (Mo) is an essential trace element which is an electron donor and/or acceptor in Mo requiring enzymes. We previously isolated MOT1, the first molybdate transporter in eukaryotes, from Arabidopsis thaliana.
In this study, we found a low Mo accumulation Lotus japonica mutant by screening EMS-mutagenized seeds. Based on the genome analysis, Lotus has four genes that are closely related to MOT1. By sequencing theses putative Mo transporters in mutant, we found that there is a single nucleotide substitution (G876 to A876) in ST53 (LjMOT1) gene, a MOT1 like gene. This substitution changes amino acid Met to stop codon. To confirm the low Mo phenotype is due to the mutation in LjMOT1, the mutant was transformed with wild type LjMOT1 genomic fragment including promoter. Several independent transgenic plants were generated and the Mo concentrations in leaves of the transgenic lines were about 4 times higher than mutant. The mutant does not exhibit defect in nodulation and transcript accumulation is not much affected by nodulation. These data demonstrated that LjMOT1 is a major Mo transporter of Lotus for taking up Mo from soil, but not involved in the symbiosis.

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We analyzed gene expression of 9 candidates of Mg transporters in Oryza Sativa, OsMRS2, homologous transporters to AtMRS2 which are Mg transporter family in Arabidopsis.
OsMRS2 gene expression of rice plant was investigated using semi-quantitative reverse transcription-PCR and real-time PCR. As a result, most of the OsMRS2 members were expressed in whole tissue of the plant. In addition, the expression level in shoot was higher than the level in root during the vegetative stage. Then, the expression of OsMRS2 gene in root and up-ground part of the seedling under Mg deficiency was examined during 8 days of development. It was found that the expression levels of all 8 genes (one of the genes was not detected) were not induced by Mg deficient treatment during 8 days. On the other hand, gene expression level in sheath of developing leaf was increased up to about 30 times higher while the growth.
To detect histochemical localization of the OsMRS2, GUS reporter gene construct comprising the upstream region of OsMRS2 was introduced into a rice plant. The stain was observed in the sheath part of the transgenic plant and further analysis is now underway.

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Plants have various strategies to adapt to available phosphate amount from environment. One of the most important mechanisms is regulation of transporters. Plants change the kind and expression manner of transporter genes within a root. Therefore, seems to contain the different function in each part of the root for phosphate absorption. To understand the mechanisms we studied the real time absorption site and movement of phosphate in roots using radioactive tracer as well as a real time imaging system we developed. We supplied phosphate, ³²P(PO₄^3-), to the different part of the root, main root and side root of Arabidopsis thaliana. The amount of phosphate transferred from the main root to up ground part was about 2 times higher than that from the secondary root. When phosphate was supplied to the leaves, it was moved down to the roots within one hour. The result suggested that phosphate is moving in two orientations in a plant, up and down.

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Starch is an important form of carbon reserve in plants whereas in animals it is the glycogen. While the mechanism of initiating glycogen synthesis is well characterized, initiating starch synthesis in plants remained largely inconclusive. Since starch is synthesized in the chloroplast, we investigated the subcellular localization of the rice glycogenin glucosyltransferase (OsGGT) which was localized to the plasmamembrane. Although transgenic rice plants overexpressing OsGGT showed 1.3 folds higher starch contents compared to wild type (WT) plants, repressing OsGGT expression did not result in complete depletion of the starch pool. After two days of complete submergence, OsGGT overexpressing plants maintained 61% of its original starch contents prior to submergence compared to only 34% in WT plants, whereas after four days the level of the starch was similar in both plants. Our results suggest that OsGGT might be actively involved in protecting starch from the rapid depletion under submergence conditions.

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The molecular evolution of SOD exhibits a characteristic distribution of its isozymes and isoforms among organisms in response to oxidative stress. However, the divergence point of chloroplastic (chl) and cytosolic (cyt) CuZn-SOD isozymes is uncertain. We have previously cloned the chl SOD gene from the green alga Spirogyra, and also found two chl SOD isoform genes in the moss Pogonatum and the fern Equisetum. Since recent genome data revealed the presence of chl SOD gene in the prasinophyte alga Ostreococcus, and the occurrence of two chl isoform genes in Physcomitrella, we analyzed the divergence point of chl and cyt SOD and the phylogenetic relationship of chl SOD isoforms in moss and fern. Ostreococcus chl SOD gene showed a high homology with that of Spirogyra, although their exon-intron structures were completely different. The phylogenetic analysis showed a sister-group relationship between chl and cyt SOD genes, suggesting the divergence of both genes from an ancestor before the appearance of streptophyte algae from chlorophytes. Furthermore, it was shown that chl SOD isoform genes in moss and fern were derived from gene duplication after separation of each species.

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The mechanochemical coupling in myosin motor have mainly been studied by using muscle myosin. However, recent studies show that various non-muscle myosin with quite different mechanochemical properties will provide useful information about the mechanism of myosin motion.
Arabidopsis thaliana, a model plant, has 13 class XI myosins. We studied enzyme properties of one of them, myosin XI-I, because this myosin is expressed in considerable amount in Arabidopsis cells but is poorly characterized. Myosin XI-I showed relatively low actin-activated ATPase activity (Vmax = 3 Pi/head/sec) and low sliding velocity (0.02μm/sec using motor domain). However, its affinity for actin was the highest among all myosins so far measured (Kapp = 0.5 μM).
Mutation study revealed that this high affinity for actin is partly due to loop 4.

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The prokaryotic Hsp90, HtpG, has structural properties that are similar to its eukaryotic homologue. However, its function is among the most enigmatic of the major molecular chaperones. Eukaryotes require a functional cytoplasmic Hsp90 for viability under all conditions. In contrast, HtpG is dispensable under normal growth conditions and even under heat stress in heterotrophic bacteria. Thus, function of HtpG and its cellular substrates, if any, remained to be clarified. We showed an indispensable role of HtpG for survival of the cyanobacterium Synechococcus elongatus PCC 7942 under heat and other stresses. Recently, we found that a polypeptide component of phycobilisome and uroporphyrinogen decarboxylase are in vivo protein substrates for HtpG in the cyanobacterium. We further identified the middle domain as the major chaperone site. Unlike eukaryotic Hsp90, no evidence for the interaction of HtpG with other (co)chaperones has been shown. However, we obtained evidence that HtpG collaborates with the DnaK/DnaJ/GrpE system. I will present these our recent studies which may shed light on the function of this neglected chaperone.

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There are two kinds of translocating sugars in the Rosaceae; sorbitol and sucrose. It is important to understand the mechanism of sugar sensing and signaling in the Rosaceae because sorbitol and sucrose ratio synthesized in source leaves affects fruit quality and vegetative growth. Sorbitol-6-phosphate dehydrogenase (S6DPH) and sucrose-phosphate synthase (SPS) are key enzymes of sorbitol and sucrose synthesis, respectively. In this study, effects of sugars on their gene expression were investigated. Leaf-petiole cuttings of loquat were treated with sugar solution and an analysis of the transcript levels was performed using qRT-PCR. The S6PDH transcript levels were decreased by sorbitol, but increased by sucrose. Simultaneous treatments of sorbitol and sucrose revealed that that was not result from osmotic effects. Glucose and fructose decreased the level. The SPS transcript levels were decreased by any sugars tested. These results suggest the presence of the mechanisms to make sorbitol and sucrose ratio high in source organs of the Rosaceae and that sucrose could be associated with the mechanism as a signal molecule.

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Generally, major constituents of cellular membrane lipids are phospholipids, whereas those of chloroplast membrane lipids in plants are glyceroglycolipids such as monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). When higher plants are exposed to phosphate (Pi) starvation, they decrease a proportion of phospholipids and instead increase the DGDG content in extraplastidic membranes. In Arabidopsis, MGDG synthase (MGD) 2 and MGD3 are highly upregulated by Pi starvation and contribute to the accumulation of DGDG by synthesizing MGDG, a precursor of DGDG. To analyze the regulatory mechanisms of MGD2 expression, in this study, we introduced various truncated promoter sequences of MGD2 fused to its initial 3 codons and β-glucuronidase (GUS) gene into Arabidopsis. Analyses of these transgenic plants revealed two important regions for the MGD2 expression; one is involved in the suppression of MGD2 expression under Pi-sufficient conditions while the other is required for its upregulation under Pi-deficient conditions.

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Cardiolipin (CL) is widely distributed in various prokaryotes and mitochondria of eukaryotes as a membrane phospholipid. We identified the Arabidopsis gene CLS for a CL synthase as the first gene from the multicellular eukaryotic organisms and isolated mutants in which the CLS gene is disrupted by T-DNA insertion. In the mutants, development of embryos was delayed and the mutant cells had mitochondria with abnormal morphology. In vivo labelling experiments of seedlings with [³³P] Pi revealed that the incorporation of radioactivity into CL in the total phospholipids was higher in the wild-type plants than in the mutant plants. Fatty-acid composition and molecular species of CL from the wild-type and mutants of Arabidopsis and other plants were also analyzed by GC and MALDI-TOF-MS. Based on the obtained results roles of CL in plants will be discussed.

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Sphingolipid metabolites, sphingoid long-chain base (LCB) 1-phosphates (LCB-1Ps), are involved in ABA signaling pathways. The LCB-1Ps synthesized by LCB kinase are dephosphorylated by LCB-1P phosphatase or degraded by LCB-1P lyase. In the present study, we show that the At3g58490 gene encodes AtSPP1, a functional LCB-1P phosphatase involved in ABA-mediated stomatal responses. Transient expression of green fluorescent protein fusion in suspension-cultured Arabidopsis cells showed that AtSPP1 localizes on the endoplasmic reticulum. We isolated and characterized Arabidopsis spp1 mutants, in which the AtSPP1 gene is knocked out by T-DNA insertion. After drought stress, the rate of decrease in fresh weight was significantly slower in spp1 mutants than in the wild-type plants. A stomatal aperture bioassay showed that stomatal closure occurs in responses to ABA in spp1 mutants, suggesting that AtSPP1 is involved in guard-cell signaling. spp1 mutants also showed decreased sensitivity to ABA with respect to primary root growth but not to seed germination. The response to fumonisin B1 did not differ between wild-type and spp1 mutant.

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Cyclobutane pyrimidine dimer (CPD) photolyase, which is widely distributed amongst species, ranging from bacteria to plants, is a crucial factor for determining UVB sensitivity in plants. We previously found that the native CPD photolyase of rice was phosphorylated in vivo. There is no report that CPD photolyase is phosphorylated in organism other than rice. First, we investigated the phosphorylation state on CPD photolyase in poaceous species, such as wheat, barley, and maize, by the technique of protein phosphatase treatment or two-dimentional gel electrophoresis. As a result, native wheat CPD photolyase was also phosphorylated, while barley or maize was not.
It is important to identify the phosphorylation site of CPD photolyase, in order to understand the phosphorylated CPD photolyase function in vivo. We estimate the putative phosphorylation site by comparing the amino acid sequence of phosphorylated and nonphosphorylated CPD photolyase, and then tried to identify the phosphorylation site of rice CPD photolyase using site-directed mutagenesis method and insect cell-free translation system. As a result, we identify the phosphorylation site of native rice CPD photolyase.

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Plants use sunlight as energy for photosynthesis; however, plant DNA is exposed to the harmful effects of ultraviolet-B (UVB) radiation in the process.UVB radiation damages nuclear, chloroplast, and mitochondrial DNA by the formation of cyclobutane pyrimidine dimers (CPDs), which are principal cause of UVB-induced growth inhibition in plants. Repair of CPDs is therefore essential for plant survival while exposed to UVB-containing sunlight. Nuclear repair of the UVB-induced CPDs by CPD photolyase is well known, but repair processes in plant chloroplasts and mitochondria are not understood.
Here, we report the photoreactivation of CPDs in chloroplast and mitochondrial DNA in rice. Biochemical and subcellular localization analyses using rice strains with different levels of CPD photolyase activity and transgenic rice strains showed that full-length CPD photolyase is encoded by a single gene, not a splice variant, and is expressed and targeted not only to nuclei but also to chloroplasts and mitochondria. The results indicate that rice may have evolved a CPD photolyase that functions in all three organelles that contain DNA to protect cells from the harmful effects of UVB radiation.

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Ultraviolet-B (UVB) radiation induces photodamage in DNA, including the formation of cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photodimers [(6-4) photoproducts]. Two major repair mechanisms for such DNA damage are excision repair and photoreactivation. In photoreactivation, the enzyme photolyase mediates the repair of damaged DNA by binding to CPDs or (6-4) photoproducts in a lesion-specific manner. In plants, the CPD photolyase is a crucial factor for determining UVB sensitivity. In rice, however, the relative contribution of (6-4) photolyase to UVB resistance is unclear. We measured the activity of (6-4) photolyase and the UVB sensitivity of three rice cultivars. There was no correlation between (6-4) photolyase activity and UVB sensitivity. Furthermore, we investigated the UVB sensitivity of (6-4) photolyase-deficient mutant rice. No difference was found between wild-type rice and the mutant. It was reported that the Arabidopsis mutant defective in (6-4) photolyase was more sensitive to UVB than the wild-type. On the other hand, our results suggested that the activity of (6-4) photolyase was not significantly affected the UVB sensitivity of rice.

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We will report our IPA gene expression analysis results on the effect of metal hydride ion water on plants. At previous meetings we made presentations regarding the ion binding properties of metal hydrides, such as CaH₂ and the ability to control plant growth and promote life extension, blooming and so on. We have shown the phenomenon whereby cut flowers treated in ionized hydrogen water, which was made from ion binding metal hydride, continued growing with increased height as well as the continual blooming of new flowers. The ion binding property of metal hydride has the potential to control ion channels such as calcium. This time we conducted gene expression analysis using a DNA array method to study plant reactions in metal hydride treated water. Results indicate up-regulation of peroxidase 40, CYP71B36 and GRP16. However, mRNA such as thioredoxin-dependent peroxidase 2, vitamin C permease family protein, and domains linked to heavy metal containing proteins, showed down-regulation.

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Following our Part 1 analytical report on plant gene expression influences by metal hydride water, we will describe the phenomena of plant growth promotion, life extension and blooming control. Cut flowers, which absorbed this water, exhibited mysterious phenomena. The cut flowers grew, becoming higher, developed new flowers and had their condition maintained. Some produced only leaves, and others produced only flowers. Some suddenly withered and died. Based on the fact that among metal hydrides, the ion binding property of metal hydrides such as CaH₂ is capable of creating hydrogen in a negative ion condition, we can infer that this negative hydrogen ion is responsible for the observed phenomena on plants.

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A Nipponbare Tos17 mutant XC20 showed decreased field resistance against bacterial blight disease, and a putatively responsible gene xc20, was identified by the Tos17 tagging method. The field resistance of the XC20 was recovered after the reintroduction of the native the xc20 gene. The other five Nipponbare mutants in which the Tos17 was inserted in their xc20 gene showed the decreased field resistance as XC20. We concluded that xc20 is one of the responsible genes for field resistance against bacterial blight.
The amino acid sequences deduced from the xc20 gene had a similarity to the sequence of SAUR family protein. The SAUR proteins are induced by auxin accumulation after the infection of pathogens and considered to act as one of the negative regulators for auxin synthesis. Therefore, the xc20 gene might be involved a field resistance mechanism against bacterial blight disease by suppressing the auxin accumulation.
The XC20 has distinctive yellow-green leaves. However, because the other five mutants mentioned above have wild type leaves and showed decreased resistance, the change of leaf color in XC20 might not affects its field resistance against bacterial blight.

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Momilactones and phytocassanes are major phytoalexins in rice. We showed that biosynthetic genes for momilactones and phytocassanes are respectively clustered on rice chromosome 4 and chromosome 2, and that an elicitor-inducible bZIP transcription factor, OsTGAP1, is involved in the regulation of the expression of biosynthetic genes for diterpenoid phytoalexins. Here we performed microarray analysis using OsTGAP1 over-expressing rice cells. As a result, 1352 genes were up-regulated more than 2-fold in OsTGAP1 over-expressing cells compared to wild-type cells without elicitor treatment. Moreover, 1539 and 1267 genes were up-regulated 6 h and 24 h after elicitor treatment, respectively. We performed hierarchical clustering of gene expression of these up-regulated genes in OsTGAP1 over-expressing cells. We focused on one clade that contains biosynthetic genes for diterpenoid phytoalexins. There were PR10 genes and chitinase genes in this clade, and these genes were respectively clusterd. Now, we are performing the expression analysis of these genes, and searches for the OsTGAP1-binding sites in the promoter region of genes up-regulated in over-expressing cells by ChIP analysis.

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When plants are attacked by pathogenic microorganisms, they respond with a variety of defense reactions including production of PR proteins or phytoalexins. We identified OsWRKY53 by microarray analysis as a chitin oligosaccharide elicitor-induced gene, and indicated that OsWRKY53 functions as a transcriptional activator, and that overexpression of OsWRKY53 resulted in enhanced resistance to the rice blast fungus Magnaporthe grisea in rice plants. It has been suggested that MAPK-mediated phosphorylation of homologs of OsWRKY53 in other plant species results in increase in transactivation acitivity. Using a transient reporter gene assays in rice cells, we found that a phospho-mimic OsWRKY53 (S/D), a constitutively active form, has higher transactivation activity than the native OsWRKY53 and an alanine-substituted OsWRKY53 (S/A). Now, we are trying to determine whether OsMKK4-OsMPK6 cascades are involved in phosphorylation of recombinant OsWRKY53 by in vitro kinase assay. We are also trying microarray analysis to compare downstream responses between OsWRKY53-overexpressing lines and phospho-mimic OsWRKY53-overexpressing lines.

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Rapid recognition and signal transduction of mechanical wounding are necessary early events leading to stress resistance in plants. Accelerated propagation of a wound response operates through various signaling molecules, including Ca²⁺, protein phosphorylation, and ROS. However, the roles of these molecules in early steps of wounding signaling are largely unknown. Here we report a novel function of Arabidopsis mitogen-activated protein kinase 8 (MPK8), which connects protein phosphorylation, Ca²⁺ and ROS in the wound-signaling pathway. MPK8 is activated through mechanical wounding, and this activation requires direct binding of CaMs in a Ca²⁺-dependent manner. MPK8 is also phosphorylated and activated by MKK3 in the prototypic kinase cascade, and full activation of MPK8 needs both CaMs and MKK3 in planta. Loss-of- and gain-of-function analysis revealed that the MPK8 pathway negatively regulates ROS accumulation through controlling expression of the Respiratory burst oxidase homolog D gene. These findings suggest that two major activation modes in eukaryotes, Ca²⁺/CaMs and the MAP kinase phosphorylation cascade, converge at MPK8 to maintain an essential part of ROS homeostasis.

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We have identified RSOsPR10 as the root specific protein induced by drought and salt treatments in rice. Expression of RSOsPR10 was up-regulated by JA and ACC, while SA strongly suppressed its expression. We conducted analyses for putative transcription factors involving RSOsPR10 expression. The results revealed the involvement of OsERF1, an ET/JA-responsive factor, in RSOsPR10 induction, indicating that there is the the ET/JA signaling pathway. OsTGAP1, encoding an elicitor-inducible bZIP transcription factor, was also induced synchronously with RSOsPR10 by salt and JA. These results suggested that RSOsPR10 expression is regulated by the complex interaction of multiple transcription factors. Because GCC-box and TGACG-motifs (as-1-like elements, TGACG (T/G)) were found around 2- to 3-kb RSOsPR10 promoter region, we are currently investigating the cis-elements for RSOsPR10 expression using the rice plants transformed with promoter (2-, 3-, and 4-kb)::GUS constructs. In addition, the transient expression system using rice culture cells by introducing promoter::LUC constructs is under establishing.

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Plants have evolved a unique hormonally regulated self-protection system to defend themselves from various stresses, such as pathogen attacks and environmental stress. In plants, salicylic acid (SA), jasmonic acid (JA) and abscisic acid (ABA) play important roles in adaptation to biotic and abiotic stresses. In addition, recent studies have shown that the mutually antagonistic interactions between stress-related hormones in complex networks of various signaling pathways. For instance, treatment with ABA suppresses the induction of systemic acquired resistance (SAR) by inhibiting the pathway both upstream and downstream of SA accumulation. Conversely, the activation of SAR suppresses the expression of ABA biosynthesis-related and ABA-responsive genes. These results show that antagonistic crosstalk occurs at multiple steps between the SA-mediated signaling of SAR induction and the ABA-mediated signaling of environmental stress responses. Here, we will discuss the complex plant hormone-networks by analyzing expression of plant hormone related-genes in hormone mutant of Arabidopsis.

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Systemic acquired resistance (SAR), one of plant defense systems, is induced by pathogen infection through salicylic acid (SA) accumulation. SAR has been practically utilized in Japan rice fields by exploiting the plant activators capable of induction of SAR, however, the effect of SAR is broken by pathogens. Benzisothiazole (BIT) is a plant activator that induces SAR in rice, tobacco and arabidopsis. In Arabidopsis, environmental stress responses via abscisic acid (ABA) suppresses SAR induction by BIT. Because of the economical importance of the stable induction of SAR, we have investigated the effects of environmental stresses on SAR using rice.
BIT induced the resistance against rice blast fungus, however, these were suppressed by environmental stress pre-treatment. ABM is ABA biosynthesis inhibitor that suppresses effect of environmental stress. ABA treatment reduced the BIT-induced disease resistance. These data indicate that environmental stress response suppresses SAR development on rice.

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The two-component systems (TCS) are a mode of widely conserved signal transduction in plants. The best-characterized representative of plant TCS is the mechanism underlying cytokinin responses,in which His-kinases function as the hormone receptor. Although these ideas came from extensive studies on Arabidopsis thaliana, certain plant species belonging to legume are currently of much interest, because it was found that the formation of beneficial nodules is regulated somehow through the CK receptor His-kinase, named LHK1.We are currently characterizing the Lotus japonicus TCS from the genome-wide viewpoint. For instance, we now know that (i) L. japonicus has three CK receptor His-kinases (LHK1/2/3), as does A. thaliana. (ii) In addition to type-A RRs, LHK1 and one of B-type RR, named LjRRb2 are specifically induced by CK in roots. Taking these together, we are examining roles of L. japonicus TCS factors in nodulation through reverse genetics by adopting the method of transformed hairy-root formation. We will discuss about these results in the context of current knowledge about the genetic networks underlying the nodule formation.

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The symbiotic nitrogen fixation enables legume plants to grow even in the nitrogen-limiting soil. However, developing too many nodules inhibits the host plant growth because the nitrogen fixation is a high energy-consuming process. Thus the number of nodules is tightly restricted by the host plant. One of the negative regulations requires the receptor kinase HAR1 which functions in shoots, indicating that the long distance signal-mediated regulation might exist. The aim of this research is to understand the detailed molecular mechanism using the hypernodulating mutant of a model legume Lotus japonicus named too much love (tml.) Previous studies have shown that TML functions in roots, and TML and HAR1 function in the same genetic pathway. Because tml is a large deletion mutant, it would be very challenging to define the TML gene. Recent reports have shown that "Transcript-Based Gene Cloning" is effective for mutants with abolished expression of their responsible genes. Following this method, we narrow down the candidates in the deleted region. Introducing the genomic region of one candidate tends to rescue the tml mutant phenotype, suggesting this gene is a strong candidate for TML.

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Host legumes control nodule number by autoregulation of nodulation (AON), in which the presence of root nodules inhibits further root nodule formation. AON consist of at least two long-distance signals, i. e. a root-derived infection signal and a shoot-derived signal that inhibits nodulation. In Lotus japonicus, LjCLE-RS1 has been identified one of the strong candidates as the infection signal. However, it is still not clear how the nodulation is repressed in the roots. Abscisic acid (ABA) inhibits nodulation and antisense of LjGlu1 (ABA responsible β-1,3-glucanase) enhances nodulation and nitrogen fixation. We focused on the expression level of LjGlu1 under activating of AON. Split-root system was employed to investigate an involvement of LjGlu1 on AON in L. japonicus. The expression level of LjGlu1 was also analyzed in the transgenic hairy roots expressing LjCLE-RS1 constitutively.

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Lipopolysaccharide (LPS) of rhizobia is involved in establishing symbiosis between rhizobia and their host legumes. However, LPS signaling system is still not clear in plant. In mammals, LPS binding protein (LBP) forms a complex with LPS and activates innate immune system. On the genome of Lotus japonicus, four genes, LjLBP1, 2, 3, and 4, were identified as candidates for plant LBP.
Expression level of LjLBPs was analyzed with or without microsymbiont Mesorhizobium loti. The expression of LjLBP3/4 was strongly repressed in nodulated plants 28 days after inoculation compared with that of plants supplied with 0.5 mM NH₄Cl. Whereas expression of LjLBP1 and LjLBP2 was stable at low level. LjLBP3/4 was up-ragulated transiently at 4h after inoculation of M.loti, Ralsonia solanacearum and Pseudomonas syringae pv. pisi. Furthermore, RNAi of LjLBPs resulted in the failure of symbiosis. These results indicate that the expression of each LBP is regulated in different manner, and LPS signaling via LBP of the host plant will be essential for establishing symbiosis especially after the microsymbionts invasion.

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Monodehydroascorbate reductase (MDHAR) is a key enzyme of the ascorbate-glutathione cycle that maintains reduced pools of ascorbic acid (AsA) and serves as an important antioxidative enzyme. cDNA encoding MDHAR was isolated from acerola (Malpighia glabra), a plant that accumulates very large amount of AsA. MDHAR transcript and enzyme activity were significantly up-regulated in acerola leaves under cold and salt stress conditions, indicating that expression of MDHAR gene is transcriptionally regulated under these stresses. Acerola MDHAR cDNA was then introduced into tobacco plants using an Agrobacterium-mediated gene delivery system. Transgenic tobacco plants accumulated higher amount of AsA and showed higher MDHAR activity than the untransformed control plants. Lipid peroxidation and chlorophyll degradation were restrained in the transgenic plants under salt stress conditions compared to untransformed control plants. These results indicate that overexpressing of acerola MDHAR provided higher tolerance to salt stress.

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We have demonstrated that an Arabidopsis homologue (atSR30) of mammalian ASF/SF2, a serine/arginine rich splicing factor, interacts with the factors for the initial definition of 5' and 3' splice sites and the other SR proteins and its expression is markedly induced by high-light stress. These findings suggest that atSR30 is involved in the regulation of stress responsive-alternative splicing (AS).
Here, we identified genes of which AS efficiencies are regulated by atSR30, by a whole genome tiling array technology. The expression levels of genome regions within 230 genes in the knockout atSR30 (KO-sr30) plants were significantly altered compared with the wild-type plants under high-light stress. Many genes encoded factors involved in signal transduction, protein turnover, and transcription. By semi-quantitative RT-PCR analysis, 22 genes exhibited changes in the transcript levels and/or AS patterns under high-light stress. The sequencing analysis of the AS variants indicated that atSR30 functions as a regulator of the exon skipping- and alternative 5',3' splice sites-types of the AS event.

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Two structurally unrelated protochlorophyllide reductases, light-dependent reductase (LPOR) and dark-operative reductase (DPOR), coexist in chlorophyll biosynthesis in cyanobacteria. DPOR is a nitrogenase-like enzyme that is rapidly inactivated by exposure to oxygen. To understand how the oxygen-sensitive enzyme, DPOR operates in oxygenic photosynthetic cells, we isolated a mutant lacking LPOR in Synechocystis sp. PCC 6803. While the mutant was sensitive to high light, it grew under low light conditions, suggesting the presence of some protection system for DPOR against oxygen. Some candidate genes involved potentially in this system were artificially overexpressed at a neutral site (slr2030-slr2031) in the LPOR-lacking mutant. Unexpectedly, the light-sensitivity was relieved in a negative control transformant with an empty vector as well as in the other transformants. Genome analysis revealed that the parental strain retains the intact slr2031 coding sequence while "Kazusa" strain, whose genome has been fully sequenced, has a 154-bp deletion in slr2031. Thus, a deletion of slr2031 caused by the plasmid insertion presumably resulted in the tolerance to high light intensity.

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Ascorbte peroxidase (APX) catalyzes the scavenging of hydrogen peroxide and plays an important role for reactive oxygen scavenging in chloroplasts. However, chloroplastic APX is highly sensitive to inactivation by hydrogen peroxide and is inactivated under severe stress conditions. Therefore the enzyme is thought to be one of the primary targets of photooxidative damages. In this study, we investigated the role of chloroplastic APX in stress tolerance by overexpression of stromal APX (sAPX). We produced transplastomic tobacco plants which overexpress sAPX from tobacco under the control of psbA promoter. The transplastomic tobacco showed a 27-32 fold increase in total APX activity compared with WT. However, the chlorophyll content and Fv/Fm of the transplastomic lines (APX7 and APX11-1) were lower than WT under non-stressed condition. In addition, the treatment of APX7 with low temperature stress (15 degree C for 4 weeks) caused a significant bleaching and decreases in Fv/Fm, ΦII, total APX activity and sAPX protein level. These results indicate that the sAPX overexpressing lines unexpectedly suffer from photooxidative damages caused by low temperature stress.

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Peroxiredoxins are ubiquitous thiol peroxidases which catalyze the reduction of hydroperoxide. Among them, 2-Cys peroxiredoxin has been proposed to play a key role in the regulation of H₂O₂ level. We have described that an NADPH thioredoxin reductase C (NTRC) is a reductant for a 2-Cys peroxiredoxin which is called BAS1 in a thermophilic cyanobacterium Thermosynechococcus elongatesBP-1. In this work, we discovered that NTRC has a chaperone activity in addition to the peroxiredoxin reductase activity. Although the thermal aggregation of citrate synthase was not reduced by the addition of BAS1, it was efficiently prevented by the addition of NTRC in dose dependent manner, indicating that NTRC protects citrate synthase against thermal aggregation. In the absence of NADPH, NTRC and BAS1 formed aggregates which showed no chaperone activity. The aggregates were dissociated by the addition of NADPH, resulting in recovery of the chaperone activity of NTRC. The molecular mechanisms of the reversible formation of the aggregates of NTRC and BAS1 will be discussed.

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The dune grass Leymus mollis (Triticeae; Poaceae) is wild relative of wheat (Triticum aestivum) and grows mainly along sea coasts and in inland dry areas. L. mollis is highly tolerant to salt and drought stresses, diseases and adaptive to harsh environmental conditions. The genetic bases controlling its physiological tolerance to drought and salt stresses remain largely unexplored. We used Subtractive Subtraction Hybridization to identify candidate genes for drought tolerance from L. mollis. New unknown gene was identified as highly expressed in response to drought stress, up-regulated in response salt stress and jasmonic and gibberellic acid treatments. Using the corresponding homologous gene from Arabidopsis, we found that this gene is localized in the chloroplast. This gene may be a candidate to generate drought tolerant plants by overexpressing it. Furthermore, it represent good DNA marker for selecting drought tolerant wheat since L. mollis genes can be introduced into wheat by cross hybridization.

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We successfully preserved cultured moss cells in liquid nitrogen by desiccation methods. In this study, we cryopreserved cultured rice cells by desiccation methods.
Subcultured rice cells could not survive desiccation, then the cells were precultured in MS medium containing 0.4 M sucrose to enhance the desiccation tolerance. As desiccated moss (Pogonatum inflexum) cells can be preserved for 20 weeks at 4 deg. C and 8 weeks at 16 deg. C, precultured and desiccated rice cells were also preserved at other temperatures. Precultured and desiccated rice cells died after preservation for only 1 day at 26 deg. C, in spite that 60% of the cells survived for 1 week preservation at 4 deg. C. Precultured and desiccated rice cells did not survive higher temperature treatment, though 60% of Pogonatum cells survives 80 deg. C treatment for 1 hour.

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Under abiotic stress conditions such as drought and high salinity, abscisic acid (ABA) is accumulated in plant cells and induces many genes that function in the stress tolerance. In the promoter regions of such ABA-inducible genes, the cis-elements, ABRE (ABA-responsive element), are conserved. AREB/ABF proteins were isolated as ABRE-binding proteins by using yeast one-hybrid method. Among nine AREB/ABF genes in Arabidopsis, AREB1, AREB2, and ABF3 are induced by dehydration, NaCl, and ABA treatments in vegetative tissues. We have demonstrated that AREB1, AREB2, and ABF3 are master transcription factors that regulate ABRE-dependent ABA signaling involved in drought stress tolerance by using the areb1 areb2 abf3 triple mutant. In addition to the three AREB/ABFs, ABF1 is also in the same clade in the phylogenetic tree. Here, we will report a role of ABF1 in ABA signaling based on the results of the comparative analyses using the areb1 areb2 abf3 abf1 quadruple mutant and the areb1 areb2 abf3 triple mutant.

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We had already selected the desiccation-responsible genes from a terrestrial cyanobacterium, Anabaena sp. PCC7120 and some of the genes were disrupted. The phenotype of the gene-disruptants indicated low viability under desiccation in N₂-fixing condition. These results may suggest that desiccation-tolerant genes contain nitrogen fixation relating genes and express to stabilize intra-and outer-cellular condition under desiccation. We were also shown that the no-inducible photosynthesis gene, psb28, was related to desiccation tolerant. Psb28 protein associates with photosystem II but function of Psb28 is not enough to understand.
Desiccation tolerant N₂-fixing cyanobacterium, Nostoc commune is related to the Anabaena. The Nostoc has ability to use scientific research for desiccation tolerance system, food and soil for plantation. These abilities expect to improve devastating soil to nutrient-rich soil including space agriculture. To confirm ability of the Nostoc(axenically) soil, the Nostoc was used plantation as nutrient containing plate. The result of difference plant growth between N₂-deficient plate and cyanobacterial mat is now in progress.

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The population of the earth is increasing rapidly, setting food security one of the major issues in the world. Drought, flooding, unpredictable epidemics, soil erosion and pollutants further threaten sustainable agriculture. We have shown in Arabidopsis that cytokinins (CKs) regulate plant adaptations to various abiotic stresses. In this study, we performed a genome-wide identification of CK metabolic genes encoding isopentenyl transferases and CK oxidases in soybean, and analyzed their expression profiles under normal and dehydration conditions. We found 14 and 17 genes encoding GmIPTs and GmCKXs, respectively, with high homology to its Arabidopsis counterparts. The promoter regions of these genes were found to contain several abiotic-stress inducible cis-elements. Next, we performed expression analysis of these two gene families and found that six GmIPT and six GmCKX genes were induced upon dehydration; three GmIPT and five GmCKX genes were down-regulated by dehydration. Taken together, our data indicated a substantial component of the CK metabolism is involved in the adaptation to dehydration stress in soybean.

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We describe an Oryza sativa stress-related CCCH-type Zinc Finger protein gene (OsSCZF1) involved in stress responses in rice. The expression of OsSCZF1 was induced by drought and high salinity. OsSCZF1 gene expression was also induced by abscisic acid, methyl jasmonate and salicylic acid. GFP localization analyses showed that OsSCZF1 is cytoplasmic and also localized in cytoplasmic foci. Histochemical GUS activity in P_SCZF1-GUS transgenic rice plants was observed in young leaf, panicle and anthers. Transgenic rice plants over-expressing OsSCZF1 constitutively exhibited poor seed germination, a lesion mimic phenotype upon maturity and low reproductive yields. These transgenic rice plants showed improved tolerance to high-salt stress and delay in leaf senescence and also exhibited increased tolerance to rice blast and RSV virus diseases. A number of biotic and abiotic stress related genes were regulated in OsSCZF1 over-expressing rice plants. These results demonstrate that OsSCZF1 encodes a functional protein involved in modulating stress tolerance in rice.

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Potassium plays important roles for plant growth and development, affecting nutrition, membrane potential, enzyme function and the homeostasis of many other ions. Recent studies have reported the important roles of K+ transporters and their functions in plants under abiotic stresses. However, several of putative transporter proteins still remained unclear how they function. To understand function of K+ transporters that play a major role in drought stress, we focused on a putative K+/H+ antitransporter AtKEA5, which showed the increased expression under drought stress. AtKEA family belongs to CPA (Cation Proton Antiporters) family, and consists of six members (AtKEA1-6). AtKEA5 exhibited higher homology to AtKEA4 and AtKEA6 than the other members. Among AtKEA family members, the expression levels of AtKEA2 and AtKEA5 were increased under abiotic stress, and particularly expression of AtKEA5 was highly induced in the root under drought stress. GFP-AtKEA5 protein was localized to the plasma membrane and endomembrane. Phenotypic analysis of the knockout mutants and transgenic plants of AtKEAs, and the functional analysis of AtKEA2, AtKEA4 and AtKEA6 are also being characterized.

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DREB2A is an Arabidopsis transcriptional factor that binds to the DRE/CRT motif and activates the expression of target genes in response to drought and heat stresses. Although DREB2A is a stress-inducible gene, accumulation of its transcript is not sufficient for induction of downstream genes and post-translational activation of the DREB2A protein is also necessary. The DREB2A protein was supposed to be modified by ubiquitination and subjected to 26S proteasome-mediated proteolysis. Removal of a negative regulatory domain from the DREB2A protein transforms it into a constitutively stable and active form. However, it is not clear whether stabilization of DREB2A is sufficient for its activation or further activation is required.
To address this question, we treated transgenic Arabidopsis plants constitutively expressing DREB2A with the proteasome inhibiter MG132 under non-stressful condition. Although the treatment induced accumulation of the DREB2A protein, the expression of downstream genes was not induced. Therefore, transcriptional activation of target genes by DREB2A seems to require stress-specific activation of the DREB2A protein in addition to its stabilization.

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Abiotic stresses induce various physiological changes, including growth inhibition, that result in stress tolerance in plants. ZPT2-related proteins with two Cys2/His2-type zinc-finger motifs and an ERF-associated amphiphilic repression (EAR) motif are thought to function as transcriptional repressor under various stress conditions. We analyzed the function of the two Arabidopsis ZPT2-related genes that were induced by osmotic stress and ABA: AZF1 and AZF2. The nuclear localization of these two proteins was constantly observed in the roots, and the accumulation of AZF2 was clearly detected in the nuclei of leaf cells under stress conditions. The overexpression of AZF1 and AZF2 caused severe damage to plant growth and viability. Transcriptome analyses of the transgenic plants demonstrated that AZF1 and AZF2 repressed various genes that were downregulated by osmotic stress and ABA treatment. Moreover, many auxin-related genes were commonly downregulated in the plants, and most of them were small auxin-up RNAs (SAURs). These results indicate that AZF1 and AZF2 function as transcriptional repressors that are involved in the inhibition of plant growth under abiotic stress conditions.

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Sll1558 in Synechocystis sp. PCC 6803 is identified as D-mannose-1-phosphate guanylyltransferase that catalyzes the transfer of D-mannose-1-phosphate to GDP-mannose involved in cell wall biosynthesis. DNA microarray and real-time RT-PCR analysis revealed that relative expression levels of sll1558 were increased by acid stress. Mutants defective in this gene and sll1496, which is a paralog of the gene, were constructed. The deletion mutants of sll1558 were more sensitive to acid stress compared with wild type cells. Compositions of capsular polysaccharide and lipopolysaccharide were compared with wild type and the deletion mutants by silver staining after the SDS-PAGE. It showed different patterns of the bands between the mutant of sll1558 and wild type. It was found that cyt1, mutants of homolog of sll1558 in Arabidopsis thaliana, strongly retarded root growth under acid condition. These results suggest that Sll1558 contribute to cell wall biosynthesis to adapt acid conditions.

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The AP2/ERF-type transcription factor DREB2A specifically interacts with the DRE/CRT cis-acting element and controls the expression of many abiotic stress-inducible genes in Arabidopsis. DREB2A has a negative regulatory domain (NRD) and transgenic Arabidopsis overexpressing DREB2A deleted of NRD shows improved abiotic stress tolerance. Soybean is a globally important crop and genetically modified varieties are largely produced. To develop abiotic-stress tolerant transgenics, we are analyzing functions of DREB2-type transcription factors in soybean.
Previously, we identified GmDREB2A;2 as one of eighteen DREB2-type transcription factors in soybean. GmDREB2A;2 was strongly induced by drought and heat stresses and exhibited high transactivation activity. Overexpression of GmDREB2A;2 in Arabidopsis enhanced expression of genes related to heat-stress response and improved heat stress tolerance. We will report existence of an NRD-like sequence in GmDREB2A;2 and enhanced transactivation activity of GmDREB2A;2 deleted of this sequence in soybean stem protoplasts. At present we are analyzing phenotypes of transgenic Arabidopsis overexpressing GmDREB2A deleted of the NRD-like sequence.

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