Hyaluronan is a linear polysaccharide composed of alternating β1,4-glucuronic acid and β1,3-N-acetylglucosamine moieties. Recently, hyaluronan has extended the applications in various fields including cosmetics, medicine, health-care food, etc. Commercially available hyaluronan is isolated from rooster comb or produced with microbial fermentation. We have introduced the chlorella virus hyaluronan synthase (cvHAS) gene into tobacco BY-2 cells or tobacco plants via Agrobacterium-mediated transformation and confirmed the production of hyaluronan in the transgenic plant cells. Production level of hyaluronan in the transgenic BY-2 cells was increased by coexpressing UDP-D-glucose dehydroganase(Ugd) and L-glutamine D-fructose-6-phosphate amidotransferase (GFAT) with cvHAS.

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L-ascorbate content was 2-3 μmol gFW^-1 in the first 20 days after full bloom (DAFB) in peach fruit. The content then decreased to 0.2 μmol gFW^-1 at 92 DAFB and remained till harvest at 112 DAFB. Based on a fluctuation in total amount of L-ascorbate per fruit, four characteristic phases were observed. Feeding of possible precursors suggested L-galactose pathway is dominantly operating. cDNA clones for the last 6 steps were isolated. Northan blot analysis revealed upstream 3 steps (GMPH, GME, GGGT) showed maximum expression at 43 DAFB, whereas downstream 3 steps (GPP, GDH, GLDH) showed two peaks at 21 and 92 DAFB. No clear relationships were observed between biosynthetic gene expression and L-ascorbate content change during fruit development.

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Large amounts of caffeic acid esters accumulate in the vacuole of sweet potato cells in suspension culture. Their precursor, p-coumaroyl-Glc also accumulates in the vacuole. These findings suggest the possibility of vacuole to be a site for their esters biosynthesis. We previously showed that 3-hydroxylation of p-coumaroyl-Glc was catalyzed by polyphenol oxidase (PPO), whereas plant PPO is a chloroplast-localized enzyme, but not vacuole. In the present study, we examined the translocation of plastids into the vacuoles during biosynthesis of caffeic acid esters. 1) Forty-kD PPO occurred from plastid-localized 60-kD PPO by vacuolar protease. 2) Many structures speculated to be cytoplasmic components were observed in the vacuoles by electron microscopy. 3) Many MDC-staining structures are also observed in the cells, mainly in the vacuoles. These observations indicate that PPO-containing plastids are translocated into the vacuole during the biosynthesis of caffeic acid esters in sweet potato cells.

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The viridicaulis1 (vic1) mutant of Lotus japonicus , which was isolated by EMS mutagenesis of seeds, is deficient in anthocyanin and condensed tannins (CTs) accumulation. CTs are flavonoid polymers including catechins, and known for the anti-microbial and anti-herbivorous activities. To clarify the biosynthetic mechanism of CTs, we analyzed the transcript accumulation of biosynthetic genes in the mutant and identified the VIC1 gene by positional cloning. The RT-PCR analysis using the total RNA prepared from the stem suggested that VIC1 is a regulatory factor of the dihydroflavonol 4-reductase genes. The genetic linkage analysis mapped the VIC1 locus near the TM2085 marker, and a gene encoding a putative bHLH protein was found in its flanking clone. The sequence analysis showed that the candidate gene is mutated in the two independent allelic mutants, vic1-1 and vic1-2 . The complementation test of the vic1 mutant with the bHLH gene is in progress.

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We have identified that the feruloyl-CoA ortho-(6'-) hydroxylase of Arabidopsis thaliana (AtF6'H1) catalyzes the key step of scopoletin biosynthesis. AtF6'H1 exhibits the high substrate specificity for feruloyl-CoA. A. thaliana mainly accumulates scopoletin (7-hydroxy-6-methoxycoumarin).
AtF6'H1 homologs are found in the EST database of the plants. Sweet potato (Ipomoea batatas) accumulates both scopoletin and umbelliferone (7-hydroxycoumarin). It is assumed that EST homologs of sweet potato exhibit the different substrate specificity from that of AtF6'H1 and that are involved in biosynthesis of scopoletin and umbelliferone. In order to investigate the substrate specificity of these homologs, we performed cloning and functional analysis of the homologs of sweet potato. The total sequences of the homologs were obtained by RT-PCR, 3'- and 5'-RACE methods. Expression pattern of the homologs were analyzed by RT-PCR.

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Phenylalanine ammonia-lyase (PAL) acts as the key enzyme in regulating phenylpropanoid metabolism. It has been revealed that the expression of DcPAL1 in suspension cultured carrot cells is induced by dilution effect, elicitation and UV-B irradiation and DcMYB1 acted as the transcriptional activator concerning to the induction of DcPAL1 expression by several stresses. Deletion analysis of the DcMYB1 promoter indicated that the promoter region between -138 and -97 were negative regulatory cis-element for the promoter activity. We isolated a transcription factor, DcEIL, that interact with this region using yeast one-hybrid system. The amino acid sequence of DcEIL had the similarity to that of ETHYLENE-INSENSITIVE3 (EIN3) that act in ethylene signal transduction. The level of DcEIL mRNA was unaffected by several stresses, whereas inhibition of proteasome-mediated proteolysis by MG132 treatment led to reduction of DcMYB1 mRNA level, suggesting that the post-transcriptional regulation of DcEIL might involve in the regulation of DcMYB1 expression.

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We established a system in which anthocyanin synthesis of regenerated torenia shoots derived from the leaf discs in vitro was induced on the medium containing high concentration of sucrose. In this system, ABA could also induce anthocyanin synthesis without addition of high concentration of sucrose. The amount of endogenous ABA transiently increased just before the induction of anthocyanin synthesis by high concentration of sucrose. These results suggested that change in the amounts of endogenous ABA might play an important role in the induction of anthocyanin synthesis in the regenerated torenia shoots. In the previous study, we performed cDNA subtraction and obtained the genes up-regulated in the regenerated torenia shoots. In this study, gene expression profiles of 1200 clones obtained by cDNA subtraction were analyzed chronologically by cDNA microarray under the each condition of high concentration of sucrose or addition of endogenous ABA.

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4-Coumarate:CoA ligase (4CL) gene occurs in the form of gene family. Phylogenetic analysis of the gene family distinguishes two classes. Enzymes in the class I are supposed to be related to the synthesis of lignin and those in the class II to the synthesis of flavonoid. However, their substrate specificities and roles under physiological conditions have not been clarified. In Arabidopsis and raspberry, 4CL genes were cloned and Ri4CL3 is likely to participate in flavor and color in the fruit based on its phylogenetic classification and expression patterns. In petunia, many genes related to flower color have been identified, but 4CL genes have not yet. Now I cloned a 4CL cDNA expressing in petunia petal. Transgenic petunia plants suppressing the gene demonstrated decrease of anthocyanin accumulation in petal. Expression experiment using the 4CL promoter revealed that the 4CL gene was specifically expressed in the petal.

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Flavonoids are widely distributed in the plant kingdom, and their functions as antimicrobial agents and UV-B protectants are essential for the normal growth of plants under stressful environments. Flavonoid biosynthesis is thought to proceed in the cytosolic surface of the endoplasmic reticulum, whereas many endproducts of flavonoid are accumulated in vacuoles. In the model plant Arabidopsis, intracellular flavonoid transport mechanisms are investigated for proanthocyanidin pathway, one of the flavonoid subclass being synthesized and accumulated specifically in seed coat. Some factors presumably involved in proanthocyanidin transport/accumulation, such as membrane-bound proanthocyanidin transporter TT12 and putative proanthocyanidin ligandin TT19, have so far been isolated. We describe here the results on metabolic analysis of immature seed extracts from these flavonoid mutants.

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J proteins are functional partners for Hsp70. Yeast has three J proteins in the ER: Scj1p and Jem1p functioning in the ER quality control, and Sec63p functioning in protein translocation across the ER membrane as partners for an Hsp70 in the ER. We found that A. thaliana has orthologs of these yeast ER J proteins. Since expression of AtERdj3A, AtERdj3B and AtP58IPK partially suppressed the growth defects of the yeast jem1Δ scj1Δ mutant, they appear to have functions similar to those of Scj1p and Jem1p. To examine the role in the ER quality control for the Arabidopsis growth, we analyzed T-DNA mutants of these J protein genes. We found that the aterdj3b mutants became sterile when they grew at high temperature. The aterdj3b mutants reduced the number of pollen grains attached to stigma.

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While it is getting a consensus of plant scientists that the endocytic pathway plays very important roles in various plant functions of higher order, the knowledge on the molecular mechanism of endocytosis in plant cells is still very limited. We are attempting to resolve this question by revealing molecular functions of SNAREs with a special interest in the plant-unique machineries. One of such machineries is an R-SNARE, VAMP727, whose homologs are conserved only in seed plants. VAMP727 is characterized by an insertion of 20 amino acids in its longin domain. We have revealed that VAMP727 forms a SANRE complex with VAM3, VTI11, and SYP51 on a subpopulation of PVCs closely associated with vacuolar membrane (Ebine et al., 2008). In this meeting, we will report our recent results on the regulatory mechanism of VAMP727 complex formation.

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SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) regulate specific membrane fusion between transport vesicles or organelles and target membranes in membrane traffic. AtVAM3 and AtPEP12 are Qa-SNAREs, which predominantly localize on the vacuolar membrane and the prevacuolar compartment (PVC), respectively. T-DNA insertion mutants for AtVAM3 (vam3) show pleiotropic phenotypes including semidwarfism and wavy leaves, although a loss-of-function mutant of AtPEP12 (pep12) exhibits no visible abnormality. By genetic analysis, we found that a double mutant for vam3 and pep12 is embryonic lethal. Moreover, promoter swapping analysis revealed that GFP-AtPEP12 driven by AtVAM3 promoter suppresses all phenotypes of vam3. These results suggest that the functions of AtVAM3 and AtPEP12 are redundant and interchangeable. We also report their precise subcellular localization observed by a high-performance confocal laser scanning microscope we developed.

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Among PM Qa-SNARE molecules expressed during root development in Arabidopsis, SYP123 shows root-hair cell specific expression and polarized accumulation at root hair tip region, whereas SYP132 expresses constitutively in all root cell types so far examined. Here we performed the live imaging analysis coupled with the treatment of various inhibitors of membrane trafficking, and examined whether some differences in intracellular dynamics are observed between these PM Qa-SNAREs. We further elucidated the interaction specificity among PM-SNAREs to form complexes using the in vitro complex formation assay and the Split Luciferase complementation assay. From these analyzes, we found the candidate R-SNARE molecules which might be involved in a membrane traffic pathway to tip-region of root hair. In addition, we also present the tissue-selective expression pattern or subcellular dynamics of mRFP-R-SNARE was similar to that of Qa-SNARE. These results indicated the existence of vesicular trafficking mechanism with complicated balance of exocytosis and endocytosis.

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Clathrin vesicle trafficking in plant is mainly composed of three pathways from trans-Golgi network (TGN) to plasma membrane (PM) or to cellular organisms such as vacuoles, and from PM to endosome (ENDO). These pathways are significantly involved in cell growth and development as well as environmental responses. Although plant clathrin vesicle trafficking has similarities to those in animals, its cellular dynamics remains unclear. In this study, we investigated cellular behavior of plant clathrin by using fluorescent protein of clathrin. The clathrin proteins mainly localised at TGN and PM. FM4-64, a fluorescent dye for tracing endocytosis revealed that the clathrin vesicles moved from PM to ENDO marked by Ara7. In addition, RFP-clathrin fusion moved along actin filament visualised by GFP. In conclusion, plant clathrin vesicles localised at TGN, PM, and ENDO, and the vesicles were trafficked by the aid of actin filaments.

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Cells degrade various kinds of cellular components by autophagy under nutrient starvation conditions. However, little is known about how much autophagy contributes to the degradation of each cellular component and whether such degradation is selective or not. In this study, we have examined the change in rRNA contents in tobacco BY-2 cells cultured under sucrose starvation conditions, because we suppose that ribosomes can be good nutrients for starved cells. rRNA was degraded upon sucrose deprivation, and the net degradation was inhibited by the autophagy inhibiter 3-methyladenine by approximately 50%. On the other hand, our morphological observation suggests that 3-methyladenine inhibits autophagy almost completely under the same condition. These results show that the autophagic pathway contributes to the degradation of rRNA and suggest that other pathways exist for the degradation of rRNA. In the presentation, we will report our data, and discuss possible degradation pathways of rRNA.

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Molybdenum (Mo) is a transient element found in the cofactor of several prokaryotic and eukaryotic enzymes. During nodulation, the symbiotic interaction between legumes and soil-living bacteria, atmospheric nitrogen is reduced into ammonia by the nitrogenase, a Mo-containing bacterial enzyme. To investigate the requirements of Mo during nodulation, we set out to identify Mo transporters in L. japonicus. We searched L. japonicus database for homologous sequences of the A. thaliana Mo transporter MOT1 and identified four putative AtMOT1 orthologs (LjMOT1-4). An EMS mutant of L. japonicus, line 4-22, showing a dramatic reduction in its Mo content had previously been isolated. A mutation resulting in a early stop codon in the sequence of LjMOT1 was found in 4-22. Macroscopic nodulation phenotype as well as nitrogenase activity were investigated in this mutant and no significant differences were observed with wild-type plants, suggesting that low amounts of Mo are sufficient to sustain nodulation.

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Compared to yeast and animals, membrane trafficking systems are less understood in plants. Arabidopsis thaliana BOR1 is an efflux-type borate (B) transporter required for xylem loading of B under low B condition. BOR1 accumulates at plasma membrane under B limitation and is degraded via endocytosis under high B condition. BOR1 was the first plasma-membrane transporter protein which was shown to be degraded via endocytosis in plants (Takano et al., 2005). To obtain insights into the molecular mechanisms of this regulation, we performed screening for mutants defective in B-dependent degradation of BOR1. In EMS-treated A. thaliana transgenic line carrying CaMV35S-BOR1-GFP, we selected plants showing high GFP fluorescence under high B conditions. Out of 57,150 M2 plants, we isolated several independent mutants and identified the responsible genes by map-based cloning method. Here, we report isolation and characterization of these mutants and the genes.

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In rice, a boron efflux transporter OsBOR1 mediates efficient boron translocation from root to shoot. We herein generated three independent lines of transgenic rice plants expressing rice BOR1-GFP under the control of the cauliflower mosaic virus 35S RNA promoter. Real-time RT-PCR analysis showed that BOR1-GFP was highly expressed in two out of the three lines and was slightly expressed in the other line. The boron concentration in xylem sap was 1.8-1.9 times higher in two lines over-expressing BOR1-GFP than in non-transgenic plants. The grain yields under the boron deficient condition was 1.7-1.8 times greater in two over-expressing lines than in non-transgenic plants and another transgenic line with slight expression. These results suggest that enhanced expression of the rice boron transporter BOR1 enhanced root-to-shoot translocation of boron, which resulted in improved grain yields under boron deficient condition.

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Observations on the effects to cut flowers were made using bisque fired ceramic balls (patent pending 2002-382862). Tests have indicated the presence of H^- created during a hydrogen (protium) generation process when the ceramic balls are immersed in water. When the balls are placed into tap water, the ORP of the water becomes about -250mV and the pH about 10. This reduction reaction is maintained for more than 90 hours.

Experiments with different numbers of ceramic balls and cut flowers treated with blue dye indicated the concentration of hydrogen ions generated by the ceramic balls influenced plant circulation. Flowers placed in 200ml of plain water, 200ml plus 0.65g of ceramic balls and 200ml of water plus 0.8g of ceramic balls showed significant differences in plant circulation with 0.8g giving the strongest circulation response. Hydrogen's influence on this mechanism is now being examined.

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Expression profiling of aquaporin genes was studied on anthers of 2 varieties of rice plants, Hitomebore and Hidekomochi, Japonica cultivar. Hitomebore is a cool-temperature-tolerant strain that suppresses male sterility in less than 20% of spikelets when it is exposed to cool temperature.
Pollen development is accompanied with drastic change of water content in the anther. We studied expression of 35 aquaporin genes referred to PIPs, TIPs, NIPs and SIPs on whole tissues of anthers during pollen development. 13 genes of them were further studied on the expression levels during the microgametogenesis including pollen mitosis I when the tapetum cells undergo programmed cell death.
Expression of 6 genes including PIPs, TIPs, NIPs and SIPs in both strains decreased approximately tenth after the tetrad stage in the control. Some of these genes, however, did not decrease or increased in the anther of Hidekomochi strain under the cool temperature.

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Although an association between chilling tolerance and aquaporins has been reported, the exact mechanisms involved in this relationship remain unclear. We performed the expression profiles of aquaporin genes using real-time PCR, suggested that PIP1;3 gene is the most closely related to low temperature. Although OsPIP1;3 showed a much lower water permeability than members of the OsPIP2 family, OsPIP1;3 enhanced the water permeability of OsPIP2;2 and OsPIP2;4 when coexpressed with either of these proteins in oocytes. Transgenic rice plants overexpressing OsPIP1;3 showed an enhanced level of chilling tolerance and the ability to maintain high OsPIP1;3 expression levels under low temperature treatment, similar to that of chilling tolerant rice plants. We assume that OsPIP1;3, constitutively overexpressed in the leaf and root of transgenic plants, interacts with members of the OsPIP2 subfamily thereby improving water balance of the plants under low temperatures and resulting in the observed chilling tolerance of the plants.

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The salt stress is one of the representative abiotic stresses that decreases growth and productivity of crops plants. The root water permeability of three barley (Hordeum Vulgare) cultivars that show a difference in the salt sensitivity was measured under salinity stress using a pressure chamber. Water permeability of salt sensitive variety did not show any difference in response to salt stress, in contrast to significant reductions of water permeability found in more salt tolerant varieties. Root water permeability of tolerant cultivars exposed to 100 mM NaCl was found to be extremely down-regulated after 1hr. Interestingly, however, water permeability was partially recovered after 4 hrs, which was followed by a re-down-regulation afterwards. Further measurements using various inhibitors indicated that at least protein phosphorylation plays an essential role in the regulation mechanism. Effects of putative phoshorylation sites on the water transport activity of HvPIP aquaporins are in progress using Xenopus laevis ooctyes.

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Aquaporins facilitate membrane transport of water and other small molecules to support plant growth. We introduced radish aquaporins PIPs into eucalyptus and determined physiological properties of the transformants. Radish PIP2, but not PIP1, has been demonstrated to have water channel activity.
Radish PIP cDNAs linked with 35S promoter were introduced by the Agrobacterium infection method. Plants were generated from the transformed calli and grown in the soil. PIP2 mRNA was detected and PIP2 protein level was remarkably increased in the PIP2 transformants. The PIP2-transgenic lines showed higher values of CO₂ assimilation, water efficiency and branch growth rate. For PIP1-transgenic lines, the protein level of PIP1 was decreased and their growth rate was lower than that of the wild type. These results indicate that PIP1 and PIP2 are essential for normal growth. We also report the relationship of PIP content to physiological properties of wild eucalyptus trees.

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Magnesium deficiency, featuring with etiolated leaves, especially during fruit developmental stage, is a popular symptom of crops. To study this phenomenon, it is very important to understand Mg transportation manner. Therefore, we studied Mg distribution and uptake manner and tried to isolate Mg transporters using a rice plant as a model crop. Under Mg deficient condition, necrosis was observed at old leaves. And Mg concentration in xylem sap was higher than culture solution. These results suggested that there are several types of Mg transporters for Mg uptake and distribution. We isolated 6 candidate transporters, OsMGT1-6, homologues transporters to AtMGT10 which is a Mg transporter in Arabidopsis. Mg transport ability of some OsMGT was confirmed by functional complementation using mutant yeast. The mRNA amount and expression of OsMGT is now under analysis.

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Fortifying food crops with iron (Fe) can combat Fe deficiency, a leading global human nutritional disorder. To generate transgenic rice seeds containing high levels of Fe, an appropriate Fe transporter must be selected and tightly regulated. Nicotianamine, an Fe chelator, is indispensable for Fe translocation within plants. The Fe concentration in rice seeds, especially in the endosperm, was decreased as a result of the knock down or constitutive overexpression of the Fe-nicotianamine transporter OsYSL2, suggesting that tightly regulated OsYSL2 expression is essential for Fe transport to the endosperm. Thus, OsYSL2 expression was driven by the sucrose transporter OsSUT1 promoter. Consequently, we succeeded in producing transgenic rice seeds with a 4.4-fold higher level of Fe in the endosperm.

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We previously reported identification and characterization of Arabidopsis molybdate transporter (MOT1). MOT1 is similar to sulfate transporters. To assess possible involvement of SULTRs in molybdate transport, we measured Mo contents in Arabidopsis 12 sulfate transporter T-DNA mutants grown with 170 nM Mo. SULTR1;2 T-DNA line exhibited lower Mo concentrations both in roots and shoots. Mo concentrations were 37% and 78% of those of wild-type plant in roots and shoots, respectively. To confirm those reductions of Mo concentration were caused by the defect of SULTR1;2, we measured Mo contents in the allelic mutants of sultr1;2 (sel1-8, sel1-9 and sel1-10). The Mo concentrations in roots and shoots of these mutant plants were also reduced to 22% and 56% (sel1-8); 37% and 55% (sel1-9); and 32% and 68% (sel1-10) of those in wild-type plants, respectively. Taken together, SULTR1;2 is involved in Mo uptake.

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Zinc is a trace element essential for all organisms as a cofactor for various enzymes and proteins. However, excess zinc becomes toxic. We study on zinc transporters in plants. In Arabidopsis thaliana, MTP1 and MTP3 function as Zn²⁺/H⁺ antiporter in vacuolar membrane and play a key role in zinc tolerance. In this study, we investigated MTP4. GFP-tag analysis and subcellular fractionation disclosed that MTP4 is localized in the plasma membrane. Yeast complementation assay using a zinc-hypersensitive strain showed the zinc transport activity of MTP4. Promoter GUS assay revealed that MTP4 is expressed in mature pollens and vascular bundles. Analysis using a construct of own promoter driven MTP4-GFP showed that MTP4 exists in pollen tubes. With consideration of structural similarity to MTP1, MTP4 may function as a zinc efflux transporter in the plasma membrane. We also showed the phenotypic properties of the T-DNA inserted mutant lines.

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ZIP transporters are essential for uptake micronutrient ions (Zn, Fe, Mn and Cu), while they also erroneously transport toxic metal ions and cause the accumulation in plant. However, little is known about the structure concerning ion specificity of ZIP. At the last JSPP meeting, we reported that TjZNT2, the ZIP transporter of Thlaspi japonicum (Brassicaceae), deleted N-terminal region (36 a.a.) acquired the ability of Zn transport and suggested the involvement of N-terminal structure in metal ion specificity. We constructed three kinds of TjZNT2 mutants with different N-terminal length and quantified their Zn⁶⁵ transport activity, suggested that the inhibitory effect of Zn transport tend to be proportional to the size of N-terminal. We also found that truncated TjZNT2 lost Mn transport activity and the N-terminal acts not only Zn but also other substrate metal ion. Now, we examine further the function of TjZNT2 in planta.

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We have demonstrated that Cd is detoxified in the Cd hyperaccumulating ecotype of Thlaspi caerulescens, Ganges by sequestering Cd into the vacuoles. However, transporters involved in this process have not been identified. Here, we functionally characterized a P-type ATPase, HMA3, which showed higher expression in Ganges than in Prayon, a non-Cd accumulating ecotype based on a cross-species microarray analysis. HMA3 from Ganges and Prayon shared 99% identity and both of them showed transport activity for Cd, but not for Zn in yeast expression system. TcHMA3 was expressed in both the roots and shoots and the expression level was not altered by high Zn or Cd exposure in both ecotypes. However, the expression was constitutively 4-times higher in Ganges than in Prayon. Transient expression of GFP fusion protein and western-blot analysis showed that TcHMA3 was localized on the tonoplast. These results suggest that TcHMA3 is involved in Cd transport into vacuoles.

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Aluminum (Al) resistance in wheat is primarily controlled by the ALMT1 protein Al-activated malate transporter which was localized on the plasma membrane. Several ALMT homologues were found in Arabidopsis, rice and other plant species, but not in animal and bacteria, indicating that the ALMT family consists of plant-specific transporter proteins. To elucidate the function and structure of wheatALMT1 protein, we have tried to purify the protein using heterologous expression systems as follows. The functional expression of the ALMT1 with a C-terminal 6-histidine tag was confirmed in tobacco cultured cells (BY-2). The ALMT1-His was also expressed in yeast or was synthesized using a wheat germ cell-free protein synthesis system. We will report partial purification of the protein.

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The antiporter of tonoplast defends plants against salt and osmotic stress by maintaining the ion balance between cytoplasm and vacuole. We cloned OsChaC gene having a homology with ChaC which regulates membrane-penetrating antiporter in E. coli as one among several kinds of genes which were induced under submergence, salinity or drought stress in submergence tolerant rice cultivar FR13A. In OsChaC overexpressed tobacco, growth inhibition was reduced and photosynthesis was kept highly under salinity stress compared with wild type. Here, we tried to clarify a function of Arabidopsis ChaC. It was confirmed that three ChaC homologues are present in Arabidopsis. We cloned them separately and examined their expressions under environmental stresses. We generated ChaC overexpressed Arabidopsis and potato, and knockout Arabidopsis and evaluated their stress tolerances. We tried to purify ChaC protein from overexpressed E. coli and isolate antiporter among ChaC -binding proteins using ChaC affinity chromatography.

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The pha1 gene cluster (pha1AB-G) of Sinorhizobium meliloti had been characterized as a necessary component for proper invasion into plant root tissue, and suggested to encode a multi-subunit K⁺/H⁺ antipoter (CPA-3) since mutations in pha1 region rendered S. meliloti cells K⁺ and alkaline sensitive. However, detailed property of Pha1 activity is yet to be determined. Interestingly, most of the CPA-3 transporters are highly selective to Na⁺, unlike Pha1. Here, we report the functional expression of Pha1 in E.coli TO114, which lacks three major cation/H⁺ antiporters, and the measurement of cation/H⁺ antiport activity. We show that the Pha1 system is a K⁺(Na⁺)/H⁺ antiporter. Furthermore, we found that the cation selectivity of Pha1 system is regulated by pH, which might reflect the importance of Pha1 system in S. meliloti to cope with different environmental conditions such as outside or inside of plant cells.

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To investigate the mechanism of mechanosensing in plants, we have isolated MCA1 and MCA2 genes encoding Ca²⁺-permeable mechanosensitive channel candidates from Arabidopsis thaliana (Nakagawa et al. , PNAS 104:3639-3644, 2007). MCA1 and MCA2 have 73 % identity in amino acid sequence and can complement a low Ca²⁺ influx activity of the yeast mid1 mutant, although sequence similarity between MCA1/MCA2 and Mid1 is low. We have reported previously that MCA1 is localized in the plasma membrane in Arabidopsis and yeast. MCA1 and MCA2 have an EF-hand-like motif. Here, we performed further molecular characterization of MCA1/MCA2 using yeast. We found that the EF hand-like motif possessed a weak Ca²⁺ binding activity, as revealed by ⁴⁵Ca²⁺ overlay assays. A fraction of MCA2 proteins expressed from a multicopy plasmid in yeast was found to be localized to the plasma membrane. In addition, we confirmed that MCA1 and MCA2 each formed a tetramer with disulfide bonding.

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The root tip senses the hardness of the soil to decide the direction of root elongation. The Ca²⁺-permeable mechanosensitive channel is postulated to be a key element of touch sensing at the root tip. Recently, we have reported candidates for such channels, named MCA1 and MCA2, both of which share 73% amino acid sequence identity (Nakagawa et al., PNAS 104:3639-3644, 2007). In this report, we have shown that the primary root of mca1 plants cannot enter the lower, harder agar medium from the upper, softer agar medium. Here, we show that the primary root of mca2 plants can enter the lower, harder agar medium, like that of wild-type plants. Interestingly, this phenotypic difference between the two mutant plants may be explained by the results of GUS staining. Namely, MCA1p::GUS was found to be expressed in the root apical meristem, while MCA2p::GUS was not.

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Blue light induces stomatal opening through the activation of the plasma membrane H⁺-ATPase. Recent investigations have demonstrated that blue light activates the H⁺-ATPase via phosphorylation on threonine residue in the C-terminus with subsequent binding of 14-3-3 protein to the phosphorylated C-terminus in guard cells. However, protein kinase and protein phosphatase, which catalyze phosphorylation and dephosphorylation of the H⁺-ATPase, are still unknown. In this study, we analyzed biochemical properties of the H⁺-ATPase phosphorylation. In vitro phosphorylation on threonine residue in the C-terminus of the H⁺-ATPase was detected in microsomal fraction from Vicia guard cells. Furthermore, phosphorylation was also detected in plasma membrane fraction from etiolated seedlings of Arabidopsis. These results suggested that a protein kinase localized in the plasma membrane was a common in plant cells. We will report analysis of in vitro phosphorylation in the purified complex of the H⁺-ATPase from plasma membrane of Arabidopsis.

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Plant plasma membrane H⁺-ATPase creates an electrochemical gradient of H⁺ across the plasma membrane. Recent studies demonstrated that the H⁺-ATPase is activated via phosphorylation on threonine in the C-terminus with subsequent binding of 14-3-3 protein to the phosphorylated C-terminus in plant cells. However, protein kinase and protein phosphatase, which catalyze phosphorylation and dephosphorylation of the H⁺-ATPase, are still unknown. In this study, we analyzed in vitro dephosphorylation of the H⁺-ATPase. Dephosphorylation of the H⁺-ATPase was detected in both microsomal fraction from Vicia guard cells and plasma membrane fraction from etiolated seedlings of Arabidopsis. Furthermore, the dephosphorylations were inhibited in the presence of EDTA, a chelating agent for divalent cations. These results suggest that a divalent cation-dependent protein phosphatase localized in the plasma membrane is involved in dephosphorylation of the H⁺-ATPase. We'll report analysis of in vitro dephosphorylation of the H⁺-ATPase in the purified complex.

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Vacuoles play many important roles in plant cells. In the root meristematic tissue, the presence of mature vacuoles is less evident, but they are always present as small provacuoles. It is known that provacuoles are produced by the trans Golgi network (TGN). When a cell begins to mature, the provacuoles fuse to produce large central vacuoles that are characteristic of most mature plant cells. We tried to prepare ultrathin sections of various plant samples by high-pressure freezing method and immunogold staining. In the root meristematic cells of Arabidopsis spp. and tobacco, vacuolar H⁺-pyrophosphatase (V-PPase) signals were detected in the TGN, tonoplast, and certain ring-like structures. These ring-like structures existed as flat and/or double-membrane structures and contained cellular components. Moreover, the structures fused with the central vacuoles. Therefore, these ring-like structures containing V-PPase may play an important role in vacuole formation and degradation of cellular components.

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MicroRNAs (miRNAs) are small, noncoding RNAs that can play important roles in eukaryotes. To investigate whether miRNAs regulate gene expression in tree species, we analyzed small RNAs expressed at flowering stage of Eucalyptus. Twenty-two bases of the small RNAs were sequenced by MPSS method, and about 130,000 unique sequences were determined. After removing rRNA, tRNA, sn/snoRNA, 30,000 genome sequences including the small RNAs were extracted. A total of 370 candidate miRNAs belonging to 236 miRNA families were identified by prediction of secondary structures of the sequences. Most of the candidate miRNAs are novel and Eucalyptus specific. A total of 404 target genes with not more than 3 mismatches in the miRNA duplexes were predicted for the 65 miRNA families using genome sequence information. The cleavage sites of some target genes were validated by 5'-RACE analysis. These results suggest that miRNAs in Eucalyptus control gene expression in various processes.

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A hydrophilic protein, Arabidopsis thaliana PCaP2 is composed of 168 amino acids and has an N-myristoylation signal at N-terminus. We found that PCaP2 binds Ca and phosphatidylinositol phosphates (PIPs). In this study, we investigated intracellular localization and tissue specificity. When PCaP2::GFP protein was expressed in Arabidopsis cells, green fluorescence was clearly observed in the plasma membrane. But fluorescence of PCaP2^G2A::GFP whose Gly-2 was replaced with Ala was observed in the cytosol. The results indicate that PCaP2 binds to the plasma membrane via myristoylation at Gly-2. Observation of PCaP2promoter::PCaP2:GFP plants confirmed the plasma membrane localization. Promoter-GUS analysis showed the expression in root epidermal cells, root hairs and pollen tubes. PCaP2 is likely to function through the interaction with Ca and PIPs on the plasma membrane of epidermal cells. Since PCaP2 was reported as a microtuble-associated protein (Wang et al. 2007), we will discuss about these facts.

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Innate immunity signaling pathways in plants are regulated by mitogen-activated protein kinase (MAPK) cascades. Arabidopsis MEKK1, a MAPK kinase kinase (MAPKKK) is an important factor of innate immunity signaling in the upstream of MPK4 which has at least two functions, negative regulation of defense-related responses and positive regulation of camalexin biosynthesis upon pathogen challenge. MEKK1 is essential for flg22- and H₂O₂-induced MPK4 activation, however regulatory mechanism of MEKK1 remains to be elucidated.
To identify possible regulator of MEKK1, we performed yeast two-hybrid screening using MEKK1 protein as bait. We found two MEKK1-binding proteins which contain U-box domain. Protein harboring this domain is thought to be function as an ubiquitin E3 ligase. In vitro ubiquitylation assay suggests that these proteins are functional E3 lingases. These results suggest presence of ubiquitin-mediated regulation of MEKK1.

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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 human (PPM1G), and named it APC4. It has been reported that PTC and PPM1G are involved in stress-responsive signaling pathways. 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 an abnormal segregation ratio in F2 generation of heterozygous plants was observed. We have made some transformants such as APC4-over-expressing plants. We are analyzing the function of APC4 by comparing phenotypes between wild-type plants and these transgenic plants in several conditions.

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COP9 signalosome (CSN) is a conserved protein complex that is known to regulate ubiquitin-proteasome mediated protein degradation pathway. CSN is composed of eight subunits designated CSN1 to CSN8 according to their sizes. Each subunit has unique roles and specific functions in the complex. In order to dissect the function of CSN, we focus on analyzing the specific function of CSN1 in the model plant, Arabidopsis.
SAP130 is one of the identified CSN1 binding proteins. SAP130 is a component of the SF3b complex which is involved in mRNA splicing. In Arabidopsis, SAP130 was coded by two genes (AtSAP130a and AtSAP130b) which both map on chromosome 3. The mRNA for both genes were detected in all observed organs. Pull down and immunoprecipitation revealed that AtSAP130 C-terminus directly bound AtCSN1 N-terminus. Here we discuss the possible role of CSN function in post-transcriptional regulation in Arabidopsis.

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COP9 signalosome (CSN) is a nuclear complex regulating signal transduction in plants and animals. To dissect the function of CSN, we focus on the function of CSN1 subunit. The N-terminal region of CSN1 (CSN1N) possessed a transcriptional repression activity in mammals, and was essential for survival in plants. To understand this mechanism, Yeast Two-Hybrid method was utilized to isolate proteins interacting with CSN1N in Arabidopsis. Here, we isolated a transcription factor containing a predicted structural domain composed of three helixes and two loops. In Arabidopsis, transcription factors with this conserved domain form a novel family. This conservation was preserved in plants, but not in mammals, which suggests that these genes may encode proteins with plant-specific functions. In pull-down assays, this transcription factor, and two others from the family, was shown to directly bind CSN1N. Here, we characterize these transcriptional factors, and discuss their role in CSN regulation.

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RECEPTOR-LIKE PROTEIN KINASE1, RPK1, a protein localized to the plasma membrane, functions as a regulator of abscisic acid (ABA) signaling in Arabidopsis. We investigated the effect of RPK1 disruption and overproduction on plant responses to drought stress. The transgenic plants that overexpress RPK1 protein showed increased ABA sensitivity in root growth and stomatal closure and less transpirational water loss, and increased tolerance to drought stress. The microarray analysis of RPK1 transgenic plants revealed the enhanced expression of several stress-responsive genes and H₂O₂-responsive genes. The results suggested that the overproduction of RPK1 enhances ABA and drought stress signaling pathway. Furthermore, RPK1-overexpressing transgenic plants increased tolerance to oxidative stress with ABA-pretreatment. These data suggest that RPK1 overproduction also controls reactive oxygen species (ROS) homeostasis and enhances both water and oxidative stress and results in increased tolerance to these stresses in Arabidopsis.

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Protein phosphorylation plays an important role in ABA-induced stomatal closure, however, little is known about the ABA signaling via protein phosphorylation in biochemical level. Previously, we found that a 61 kDa protein is phosphorylated and bound to a 14-3-3 protein in response to ABA in Vicia guard cells. In this study, we investigated ABA-induced protein phosphorylation using Arabidopsis guard cells. We found that 53 kDa and 43 kDa proteins were bound to a 14-3-3 protein in response to ABA, suggesting that ABA induced the phosphorylation of these proteins. These proteins responded to ABA at 1 μ M within a few minutes as has been found in Vicia 61 kDa protein. Furthermore, we investigated the 14-3-3 protein bindings to these two proteins using some ABA-insensitive mutant plants, including ost1/srk2e, abi1-1 and abi2-1. From these results, we report the location of these proteins in ABA signaling pathways.

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12-oxo-phytodienoic acid (OPDA) is not only an intermediate in jasmonic acid biosynthesis pathway, but also regulates gene expressions as an oxylipin signaling molecule that differ from jasmonic acid. However, the signaling mechanism of OPDA is largely unknown. In this study, to reveal the OPDA signaling mechanism, we analyzed transcriptional responses of OPDA-specific response genes, HsfA2 and DREB2A, in the presence of a protein synthesis inhibitor cycloheximide (CHX) and a HSP90 inhibitor geldanamycin (GDA).
HsfA2 and DREB2A expressions were markedly increased by co-treatment with OPDA and CHX, although these expressions were slightly induced by respective treatment of OPDA or CHX. In addition, these expressions were transiently induced by OPDA and/or GDA, with distinct expression profiles observed in co-treatment of OPDA and CHX. These results suggest that the OPDA-mediated expressions of these genes are suppressed by HSP90. Furthermore, other unknown protein factor(s) may also suppress their sustained expressions.

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We have reported that Arabidopsis transcription factors DREB1s control expression of abiotic stress-responsive genes. Rice has 10 DREB1/CBF family genes and we isolated cDNAs for these DREB1 homologues, OsDREB1s. Overexpression of OsDREB1A in rice activated expression of target stress-responsive genes such as lip9 and improved stress tolerance to drought, high-salinity and cold. Recently, we reported that 10 OsDREB1 genes were expressed in response to different stresses and proteins encoded by these genes showed different transactivation abilities.
In this study, we analyzed expression of the OsDREB1 genes histochemically using transgenic rice containing a GUS gene regulated under promoters of the OsDREB1 genes, respectively. Under cold condition, blue staining was observed at roots of the OsDREB1C-promoter:GUS plants. We examined whether the OsDREB1s could activate the lip9 promoter by transactivation assay using Arabidopsis protoplasts. OsDREB1C, 1E, 1G and 1I could activate the lip9 promoter like OsDREB1A.

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We report two Oryza sative stress-related CCCH-type Zinc Finger proteins genes (OsSCZF1 and OsSCZF2), involved in drought, salt and cold stress responses in rice. The expression of OsSCZF1 was induced by drought and NaCl treatment, whereas OsSCZF2 was induced by drought and cold. Further, the expressions of both genes were induced by methyl jasmonate and salicyclic acid. GFP localization showed that OsSCZF1 was localized to tonoplast and shifted to nucleus upon treatment with abscisic acid, while OsSCZF2 was mainly localized to nucleus. Transgenic plants over-expressing OsSCZF1 gene showed a mimic lesion mutant phenotype. These transgenic plants had an increased expression of pathogenesis related genes, PR1 and PBZ1, compared to control. Transgenic plants over-expressing OsSCZF2 were short in stature and exhibited reduced seed setting. Taken together, these results demonstrate that OsSCZF1 and OsSCZF2 encode functional proteins and may be involved in modulating stress tolerance in rice.

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Jasmonic acid (JA) is a plant hormone that regulates various developmental processes in plants and also acts as a signal molecule in plant defense against herbivores and pathogens. RERJ1 that encodes a bHLH transcription factor was isolated as a JA-responsive gene from rice suspension cells. Previously, we reported that RERJ1 functions as a transcriptional activator. Here, we report the microarray analysis using a rerj1 Tos17 insertion mutant, which was identified in a PCR screen of a Tos17 insertion population.
We carried out microarray analysis using suspension cells of wild-type rice and the rerj1 mutant after 100 μM JA treatment. Hierarchical cluster analysis exhibited 51 candidates that are possibly regulated by RERJ1. Now, we are analyzing RERJ1-dependent expressions of these candidate genes and the binding of RERJ1 to the promoter region of the target genes.

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We studied the transcripts and translation products of the rice Gα in ten alleles of d1, which was a deficient mutant of the α subunit (Gα) gene of heterotrimeric G protein in rice. The transcripts of the Gα gene were analyzed by RT-PCR in all d1 alleles. The Gα proteins were not detected in the plasma membrane fractions by western blot using anti Gα antibody, in the nine d1 alleles except d1-4, in which one amino acid exchange in GTP-binding box (A) was happened. The Gα protein was barely present in d1-4. Gα proteins were not present in d1-8, which one amino acid deletion in the effector binding region 2 was happened. From the results, it is evoked that a proper conformation of Gα is necessary for accumulation of Gα in the plasma membrane.

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