Cystationine gamma-synthase (CGS) of Arabidopsis catalyzes the key step of methionine biosynthesis. Expression of the CGS1 gene is negatively feedback-regulated at the step of mRNA stability in response to S-adenosyl-L-methionine (SAM). CGS1 encodes a fourteen amino acid sequence (MTO1 region) in exon1, which is required for this regulation. When the ribosome is arrested on CGS1 mRNA, the nascent MTO1 peptide is predicted to be located within the ribosomal exit tunnel. We are currently analyzing the relationship between the ribosomal exit tunnel and CGS1 translation arrest.

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Expression of CGS1 is down-regulated at the step of mRNA degradation in response to S-adenosylmethionine. A fourteen amino acid sequence, termed the MTO1 region, encoded within CGS1 exon 1 is responsible for this regulation. Translation arrest was observed prior to mRNA degradation in a cell-free system of wheat germ extract. This arrest occurs at the position where the ribosomal A site resides at the Ser-94 codon. The Trp-93 and Ser-94 residues are located downstream of the MTO1 region and are widely conserved in higher plants. Substitution of Ser-94 with other amino acids confirmed that Ser-94 is important for the efficiency of the translation arrest and for determining the translation arrest position. We next examined the importance the distance between the MTO1 region and the Trp-93/Ser-94 residues. Changes in the translation arrest response suggested that this distance is also important for CGS1 regulation.

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Heat shock stress rapidly inhibits nearly all syntheses of proteins in plant cells. In sharp contrast, mRNAs encoding a small subset of conserved proteins, the heat shock proteins (Hsps), are preferentially translated during heat shock. Here, we used DNA microarray to evaluate the translational regulation (polysome association) of individual mRNA species at the genome-wide scale in non-stressed and heat-stressed Arabidopsis thaliana suspension cell strain T87. Out of 19099 analyzed genes, polysome association of most genes was impaired upon heat shock of 37ºC for 10 min, consistent with the overall inhibition of protein synthesis by heat shock. We also found that some functionally categorized genes show no change in polysome association against the global trend, suggesting the importance of those functions in heat-stressed plant cells.

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MicroRNAs (miRNAs) are small, noncoding RNAs that can play important roles in eukaryotes by mRNA degradation, and translation repression. To investigate whether miRNAs regulate gene expression in woody plants, we had analyzed small RNAs expressed at flowering stage of Eucalyptus, and then isolated about 500 miRNAs. In those miRNAs, there were many new miRNAs that were not identified in Arabidopsis or Populus. We are searching the miRNA target transcripts, and identified some miRNA target genes so far. One of the target genes has homology with Arabidopsis SPL2 gene, and this gene was expressed in all organs in Eucalyptus. Another target gene has sequence conservation with MYB genes, and this gene was also expressed in every organ except young roots. 5'RLM-RACE demonstrated that these two genes were cleaved in the middle of their target sites. These results indicated that miRNAs regulate gene expression of the transcription factors in Eucalyptus.

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The amplification mechanism of siRNAs along the transgene sequence exists in RNAi. The RNA-dependent RNA polymerase synthesizes complementary RNAs by using the transgene mRNA as a template, and the secondary siRNAs are generated from the outside of primary RNAi target. We addressed the transitive RNA silencing of a tobacco endoplasmic reticulum omega-3 fatty acid desaturase gene (NtFAD3). Four independent RNAi vectors which produced primary siRNAs against distinct regions of NtFAD3 were transiently expressed in the leaves of the NtFAD3-overexpressed transgenic plants by agroinfiltration. Irrespective of RNAi vectors used, the secondary NtFAD3 siRNAs were generated only from the 3' downstream region of the transgene but not from the 5' upstream region relative to the annealing site of primary siRNAs. These results suggest that regulatory mechanisms involved in the spreading of RNA silencing differentially act on the 5' upstream and 3' downstream regions of the target sequence, respectively.

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Argonaute (AGO) genes play central roles in RNA silencing. In this study, we analyzed Arabidopsis AGO2 and AGO5. Interestingly, the 5' nucleotide of small RNAs that associated with AGO2 was mainly adenine and that of AGO5 was mainly cytosine. Small RNAs that were abundantly cloned from the AGO2 fraction (miR163-LL and miR390) and from the AGO5 fraction (miR163-UL and miR390*) are derived from the single small RNA duplexes. Furthermore, each strand of the miR163-LL/miR163-UL duplex is selectively sorted to associate with AGO2 or AGO5 in a 5' nucleotide-dependent manner. These results clearly indicate that the mechanism selecting the guide strand is different between AGO proteins even from the identical small RNA duplex. AGO2 localized in the nucleus and in cytoplasm, while AGO5 localized throughout the cytoplasm.

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To evade host defense mechanism, many RNA viruses encode suppressor of RNA silencing. The use of RNA silencing suppressors for the improvement of foreign protein production levels in transgenic plants has been suggested, but the mechanisms involved in the process are not necessarily clear. To evaluate the function of RNA silencing suppressors we tested transient gene expression system by microprojectile bombardment. Bioluminescence reporter genes fused to the Cauliflower mosaic virus (CaMV) 35S promoter were co-introduced into plant cells with silencing suppressors from Cucumber mosaic virus-encoded 2b or Peanut clump virus-encoded P15. Monitoring of luminescence levels by cooled CCD camera after introduction of expression vectors suggested that we could observe the enhanced expression levels of reporter genes by co-expression of silencing suppressors in the transient assay system.

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Heterosis, the superior performance of F₁ hybrids, is commonly evaluated with yields and biomasses, which are directly related to economic value, but genetic factors affecting these values vary for each characteristic evaluated. Meanwhile, recent studies on epigenetic modification of the genome have indicated the importance of chromatin modification in phenotypic variation and its plasticity in F₁ hybrids. To examine the involvement of epigenetic chromatin modification in hybrid growth, we induced changes of chromatin modification in inbreds and their inter-subspecific F₁ hybrids of rice by inhibiting the activity of histone deacetylases (HDACs), and examined the effects on basic growth parameters using large-scale sequential image analysis combined with growth modeling. Changes in chromatin modification led to differential effects on inbreds and their hybrids in the intrinsic rate of single leaf growth. Our results indicate that inbreds depend on HDAC activity to maintain their intrinsic growth rate more than hybrids.

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CACTA transposon in Arabidopsis thaliana is silent in wild type background, but it is mobilized by DNA hypomethylation mutation ddm1 (Decrease in DNA Methylation). Once activated by the ddm1 mutation, CACTA remains mobile even after the introduction into wild type background. Interestingly, the transposition frequency of CACTA increased during self-pollination in the ddm1 background. Here we report that the increase was also detectable even after cross to wild type. In addition, DNA methylation level decreased during the activation process of CACTA. Possible molecular mechanisms of this autoregulatory process will be discussed.

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HYL1/DRB family, which has two double-strand RNA(dsRNA) binding motifs in its N-terminal region, is one of the dsRNA binding protein families in Arabidopsis thaliana. In animal, the homologous genes are associated with RNA silencing/RNAi machinery. Similarly, in A. thaliana, HYL1/DRB1 and DRB4 were reported to be associated with RNA silencing/RNAi. However, the involvement of RNA silencing or the functions of DRB2, DRB3 and DRB5 are not yet to be characterized. Therefore, we performed the functional analysis with transgenic plants over-expressing these DRB genes. These plants showed the leaf morphological abnormality and the change of small RNA accumulation. These results indicate that DRB2, DRB3 and DRB5 are associated with RNA silencing machinery as well as DRB1 and DRB4 are. We are further studying to understand the association between DRBs and RNA silencing and functional diversity of DRB-family proteins.

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Production of reactive oxygen species is induced during many physiological processes. Respiratory burst oxidase homolog (Rboh) genes have been identified in various plants as a major source for reactive oxygen species production. Rbohs possess transmembrane domains, N-terminal regulatory domain containing EF-hand like motifs, and C-terminal catalytic domain containing FAD-, NADPH-binding sites. We have been analyzing the molecular mechanisms for activation of AtrbohD by heterologous expression in HEK293T cells and physicochemical analyses of the regulatory domain. Activation of AtrbohD was mediated by binding of Ca²⁺ to its EF-hand region, and required the Ca²⁺-induced conformational change in the regulatory domain. We further analyzed possible involvement of Ca²⁺-induced dynamic intramolecular conformational change and interaction between the two cytosolic domains in the activation of AtrbohD.

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The 26S proteasome is an ATP-dependent eukaryotic protease responsible for degrading many important cell regulators. The complex is composed of two subparticles, the 20S core protease and the 19S regulatory particle (RP), which binds to either end of the CP. The RP contains a set of six AAA-type ATPase, RPT1-6. In Arabidopsis thaliana, five of six RPT are encoded by two genes, with exception that RPT3 by single gene.
We isolated and characterized T-DNA insertion mutants that affect each RPT genes. Mutants that affects RPT2a have reduced root growth, abnormal phyllotaxy, narrow serrated leaves with increased branching of trichome, and fasciated stems. Some individuals had heavier deficiency in vegetable growth and fertility. The mutants were hyper-sensitive to DNA-crosslinking reagent, Mitomycin C. It is intriguing that this phenotype of rpt2a mutants is similar to that of mutants affects chromatin maintenance.

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Ubiquitination is one of a major protein-modification systems conserved in all eukaryote. The ubiquitinated proteins are mainly degraded by 26S proteasome to be controlled the concentration of the proteins in the cells. Although the ubiquitin-dependent protein degradation is a significant machinery to regulate the various biological processes, less information has been reported in plant as contrast to animal and yeast.
Here, we inform some studies of protein ubiquitination. These studies were supported by a grant in-Aid for scientific researches for plant graduate students from Nara Institute of Science and Technology, supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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COP9 signalosome (CSN) is a conserved nuclear complex essential for survival. CSN regulates proteolysis of nuclear proteins by deconjugating RUB1 from the cullin subunit of CRL-type E3 ligases.
Since the N-terminal of CSN1 subunit harbored transcriptional repression activity, we isolated proteins interacting with this region, and identified SAP130, DdxX15, and CF Im68, which are all involved in mRNA processing.
Although, detailed function of SAP130 is unknown, SAP130 has been identified in the SF3b splicing complex and STAGA/TFTC transcription complexes. Based on our hypothesis that CSN-SAP130 interaction is the key to bridge proteolysis and mRNA processing, we first revealed that SAP130 binds CSN in human and plants. We further showed SAP130 also binds CRL-type E3 ligases, implying novel regulation of CSN.
Currently genetic/reverse-genetic approaches are applied in plants to reveal the CSN-SAP130 function in vivo. Here we will discuss the conservation and specificity of CSN regulation among kingdoms.

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The SUMO gene family in Arabidopsis consists of eight members. The relatively large number of the isoforms comparing to those in other organisms implies the functional specificity of each SUMO gene. In order to test the substrate specify of each SUMO molecule, we initially performed the yeast two-hybrid screening for the SUMO target proteins. Through the screening, CSN5a, a subunit of COP9 signalosome, was isolated as an AtSUMO3 interacting protein. CSN5a was shown to bind exclusively to AtSUMO3. Moreover, the mature form of AtSUMO3 with the mutated C terminus was also shown to interact with CSN5a. Since the CSN5a-AtSUMO3 interaction was reproduced in the co-immunoprecipitation experiment using E.coli, this interaction was proved to be non-covalent. The identification of the SUMO-interacting motif (SIM) in CSN5a and the SIM-interacting position in AtSUMO3 is currently in progress. Furthermore, we are now investigating the CSN5a-AtSUMO3 interaction in planta.

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The SNF1-related protein kinase 2 (SnRK2) family are activated by osmotic stress and/or abscisic acid (ABA), and they contains 10 members in Arabidopsis genome (SRK2A–J). Recently, some SnRK2s were analyzed, for example, SRK2E/OST1 is activated by ABA and positively regulates ABA-dependent stomatal closure, or other two kinases, SRK2D and SRK2I, regulate ABA signaling in seeds and vegetative tissues. Since SnRK2s are activated by both ABA and/or osmotic stress, they are regarded as a hub module in stress signaling. However, the mechanism for SnRK2-mediated signal transduction is still unclear, therefore we have started a project to identify SnRK2-interacting proteins and analyze their roles in signaling networks. We have taken three strategies as follows: 1) Yeast two-hybrid screening, 2) Co-immunoprecipitation and 3) Phospho-proteomic analysis. Although this project on the systematic analysis of SnRK2 regulatory networks is still under way, some topics or progress will be introduced in this session.

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In Arabidopsis, six of NAC transcription factors including RD26 are induced by drought, ABA and high salinity stress. The RD26 transcripts are induced in 30 minutes after the stress treatment and the expression continues to increase for more than 24 hours. However, the protein of RD26 is less accumulated than the transcripts of RD26, which suggests that the level of the RD26 protein may be regulated not only at transcriptional level but also at posttranslational level. Three of RING-type ubiquitin ligases were reported to interact with ANAC, a homolog of RD26 in the yeast two-hybrid system. We found an F box-type ubiquitin ligase that binds to RD26 in yeast. All of these four ubiquitin ligases showed the interaction with RD26 and ANAC by BiFC assay. We report the regulation of the stress-inducible NAC proteins and the functional analyses of these ubiquitin ligases in the ABA-dependent stress-signaling pathway.

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Action of the carboxyl-terminal processing protease (CtpA) is indispensable for appearance of oxygenic photosynthetic competence. As an approach for molecular dissection of this essential protease, we constructed an in vivo assay system for activity of mutagenized CtpA. In this system, spinach CtpA gene was transformed into a Synechocystis heterotrophic mutant deficient in its native ctpA gene. The transformant with WT spinach CtpA regains photoautotrophic growth competence, suggesting that the spinach CtpA is replaceable with the host ctpA in Synechosystis cells. Next, we introduced the mutant CtpA library in which codon for a selected residue was substituted randomly, into the system. Transformants were screened for autotrophy. The surviving cells under the condition certainly have an active CtpA gene and we sequenced the gene to elucidate codon at the residue. Variability of observed codons is likely to be a reciprocal factor to indicate importance of the residue on the CtpA action.

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In higher plants, various Z,E-mixed polyisoprenoids, including dolichol and polyprenol, are biosynthesized showing broad chain length distributions from C₅₀ to C₁₂₀. However, a physiological function of Z,E-mixed polyisoprenoids in higher plants is hardly elucidated. To understand physiological roles of Z,E-mixed polyisoprenoids, we are characterizing Arabidopsis thaliana cis-prenyltransferases (AtCPTs) which catalyze the formation of the basic backbone of Z,E-mixed polyisoprenoids. In this meeting, we report on the enzymatic characterization of AtCPT5.
AtCPT5 fused with a trigger factor as well as a histidine-tag was overexpressed in Escherichia coli and partially purified using Ni²⁺-affinity chromatography. AtCPT5 required Mg²⁺ for its CPT activity, in which optimal concentration was 1 mM. AtCPT5 was activated by the addition of Triton X-100. The optimal allylic substrate was farnesyl pyrophosphate (C₁₅) among various allylic substrates (C₅~C₂₀). Analysis of the product chain length revealed that AtCPT5 had a novel medium-chain CPT activity, producing C₃₅ polyisoprenoid as a major product.

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Higher plants take up sulfate and convert it to adenosine 5'-phosphosulfate (APS). APS can be a substrate of sulfur reduction, or can be phosphorylated by APS kinase to yield 3'-phosphoadenosine 5'-phosphosulfate (PAPS), a donor for sulfation of a range of metabolites. There are four APS kinase genes, AKN1, AKN2, AKN3 and AKN4 in Arabidopsis genome. The isolated recombinant proteins of AKN1, AKN2, AKN3 and AKN4 independently catalyzed an APS kinase reaction. The mRNA levels of AKN2 significantly decreased under sulfur-deficient conditions. Transgenic Arabidopsis plants expressing fusion gene construct of AKN promoter-AKN coding sequence-GFP were generated to analyze organellar-specific and cell type-specific expression of APS kinases. Localization of AKN-GFP fusion proteins suggested that AKN1, AKN2 and AKN4 expressed in plastids whereas AKN3 localized in cytosol. In addition, cell type-specificities of AKN isozymes were different from each other, suggesting that each AKN isozyme plays a specific role in the PAPS synthesis.

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Nudix hydrolases act to hydrolyze various nucleoside diphosphate derivatives. A. thaliana contains 27 genes encoding Nudix hydrolase homologues (AtNUDXs) with a predicted distribution in the cytosol (AtNUDX1~11, 25), mitochondria (AtNUDX12~18), and chloroplasts (AtNUDX19~24, 26, 27). Previously, we have reported that AtNUDX11 specifically hydrolyzes CoA (J. Biol. Chem. 2005 280: 25277-83). The UPF0035 motif for CoA pyrophosphohydrolases was conserved in not only AtNUDX11 but also AtNUDX15 and AtNUDX22. Here we analyzed the molecular characterization of these CoA pyrophosphohydrolases. Recombinant AtNUDX15 hydrolyzed CoA. Furthermore, AtNUDX11 and 15 also showed activities toward CoA derivatives, such as malonyl-CoA and lauroyl-CoA. No activity toward any nucleoside diphosphate derivatives was observed in AtNUDX22. Interestingly, predicted mitochondrial (AtNUDX15) and peroxisomal (AtNUDX15a) isoforms were produced from AtNUDX15 by an alternative splicing event. These results suggest that AtNUDX11 and 15 may directly or indirectly influenced to CoA-relating metabolisms such as TCA cycle and β-oxidation in mitochondria and peroxisome.

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Many living organisms produce small antimicrobial peptides to protect their tissues from infectious microbial agents. Plant defensins are small, highly stable, cysteine-rich antimicrobial proteins that are thought to constitute an important component of plant defense against fungal pathogens. There are a number of such defensins expressed in various plant tissues with differing antifungal activity and spectrum.
We have previously reported the isolation and characterization of broad-spectrum antifungal defensin (BJ-AFP1) from Brassica juncea. Apart from the BJ-AFP1, an increasing number of BJ-AFP-like proteins leads to understand the relation between anti-microbial activity and structure of the BJ-AFP-like proteins. Therefore, we tried to gain a gene coding BJ-AFP-like protein from Brassica juncea.
In this study, we report the cloning of defensin DNAs from total DNA of Brassica juncea and the measurement of anti-microbial assay to several microorganisms.

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Regulation of ribosomal biogenesis strongly depends on the function of GTPases of YawG family members in eukaryotes, which are earmarked by a permutated order of GTPase motifs within the GTP-binding domain. The Nug protein, a member of YawG GTPases, is known to be associated with 60S ribosomal maturation in nucleolus and nucleus. Lack of understanding of plant Nug proteins in spite of their wide distribution in eukaryotes so far led us to characterize a rice nuclear GTPase, OsNug2 as a yeast Nug2 homolog on the molecular level. We showed that the OsNug2 exhibits a circularly permutated GTPase fold and an intrinsic GTPase activity. Complementation analysis using yeast Nug2 temperature-sensitive mutant cells demonstrated that the OsNug2 could replace them. We also provide evidence that OsNug2 localizes to the nucleus mediated by N-terminal OsNug2 domain. From these, we propose that rice nuclear GTPase, OsNug2 might play a role in 60S ribosomal maturation.

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Monoglucosyldiacylglycerol (MGlcDG) is a minor lipid but known as a precursor of monogalactosyldiacylglycerol, a major glycolipid in cyanobacterial membranes. In a strain Synechocystis sp. PCC 6803, sll1377 encodes MGlcDG synthase which catalyzes a synthesis of MGlcDG from UDP-glucose and diacylglycerol. The enzyme contains 'D, DAD, QXXRW' motif conserved in β-glycosyltransferases. In this study, we conducted site-directed mutagenesis and produced four enzymes in which the conserved motif was changed as follows; D147Q, D200A, R329Q, R331A. The enzymes expressed in E. coli showed very low activity of MGlcDG synthesis in comparison with wild-type enzyme. We also report detailed results of their enzymatic properties. In addition, we will show the correlation between the expression level of sll1377 and the accumulation of MGlcDG in cyanobacteria grown under various stresses.

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Plant isoprenoids, produced in cytosol, plastid and mitochondria, are biosynthesized from common C5 isoprene units; isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). Unlike animals, plants synthesize IPP and DMAPP via the cytosolic mevalonate (MVA) and the plastidal 2-C-methyl-D-erythritol-4-phosphate (MEP) pathways. To understand the regulatory mechanisms of these pathways, we isolated and characterized the Arabidopsis mutant lovastatin insensitive 1 (loi1), resistant to lovastatin and clomazon, inhibitors of the MVA and MEP pathway, respectively.
LOI1 encodes a mitochondrial pentatricopeptide repeat (PPR) protein. Most PPR proteins are predicted to take part in the post-transcriptional regulation of organelle gene expression. To identify the target RNA of LOI1, we investigated mitochondrial RNA sequences. RNA editing in some respiration chain related genes were abnormal in loi1. WT treated with some respiration inhibitors show lovastatin and/or clomazon insensitive phenotype. Our investigation suggests that the abnormality of mitochondrial respiration may affect plant isoprenoid biosynthesis.

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Phlorotannins, which are oligomers of phloroglucinol, are secondary polyphenolic metabolites found in brown algae, making up 1-20% of the dry weight. Most of them are located in 'physode' that is a specialized vesicle in brown algae, but its physiological roles are still unknown. It had been shown that the accumulation of phlorotannins was increased by UV-B radiation or methyl jasmonate treatment, implying the involvement of phlorotannins in response mechanism to UV-B radiation or wounding. In this study, we examined the conditions that induced phlorotannin accumulation in sterile cultured gametophyte of Laminaria japonica . Analyses of the fluorescence of phlorotannins, which emits green fluorescence under blue light excitation, revealed that UV-B radiation induced accumulation of phlorotannins presumably in physodes. We determined the optimal conditions that induce phlorotannin accumulation. Now we are trying to identify genes involved in the accumulation of phlorotannins using the suppression subtractive hybridization method.

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Rosmarinic acid (RA), a kind of the polyphenols, attracts attention as a material letting pollinosis and allergy reduce. Our study has previously shown that continuous red light let RA increase. In addition, there is another unknown material showing antioxidant activity influenced by light. We investigated the variation of light condition to identify their contents and relevance to antioxidant activity. As a result, RA increased in order of white, red, and blue, which meant the red involved in both white and red light let much RA increase effectively. The other was increased in order of white, blue, and red, and was mainly influenced by blue. Each antioxidant activity and polyphenol content increased most by white. These results suggest that continuous white light is effective in increasing polyphenols including RA, which lead to the high antioxidant activity. Furthermore, we will present gene expression analysis of RA biosynthesis pathway, especially TAT and CYP98A6.

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Lithospermum erythrorhizon produces rosmarinic acid (RA), a well-known hydroxycinnamic acid ester. In RA biosynthetic pathway, condensation of 4-coumaroyl CoA derived from phenylalanine, with 4-hydroxyphenyllactic acid (HPL) derived from tyrosine, results in formation of 4-coumaroyl-4'-hydroxyphenyllactic acid (CHPL) catalyzed by rosmarinic acid synthase (RAS). We attempted to isolate a cDNA of RAS from L. erythrorhizon.
To isolate cDNA, we screened cDNA library using informatics analysis of EST data. Four cDNA isolated were encoding acyltransferases (LeHCT1~4) with typical characteristics of the BAHD superfamily. We prepared the recombinant enzyme to characterize its catalytic function. LeHCT1 displayed the activity of RAS using 4-coumaroyl- and caffeoyl CoA and HPL as substrates. Shikimic acid was not able to serve as acyl acceptors. In L. erythrorhizon cell cultures, RA production is rapidly stimulated by addition methyl jasmonate (MJ), but RA activity is not induced. LeHCT1 mRNA was not induced in accordance with this pattern.

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Flavonoid 3',5'-hydroxylase (F3'5'H) and flavonoid 3'-hydroxylase (F3'H), cytochrome P450 enzymes, are the key enzymes for biosynthesis of delphinidin and cyanidin, respectively, and belong to CYP75A and CYP75B in the cytochrome P450 classification, respectively. CYP75A and CYP75B diverted before the speciation of seed plants. However, a recent report indicates that the F3'5'H in Asteraceae plants belong to CYP75B and that the F3'5'H gene was derived from the F3'H gene.
Antirrhinum majus produces pelargonidin and cyanidin but not delphinidin, and thus lack violet flower colour while A. kellogii generates delphinidin and violet colour. We isolated two CYP75A and one CYP75B cDNAs from the A. kellogii petal cDNA library. Their over-expression in petunia resulted in elevated amount of delphinidin and cyanidin based anthocyanins, respectively, and flower color alteration. The results indicate that A. kellogii CYP75A and CYP75B encode F3'5'H and F3'H, respectively, and that A. majus lost F3'5'H activity during its evolution.

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We identified anthocyanins in the petals of tartary buckwheat to study physiological roles of them. As materials, we used a tartary buckwheat leading-variety and a mutant-line, which accumulates high amount of anthocyanins. Anthocyanins were identified using HPLC-ESI-MS/MS system. The leading-variety contained Cyanidin-3-Orutinoside (C3r) as a major anthocyanin, and also contained trace amount of Cyanidin-3-O-glucoside (C3g). The mutant-line contained C3r as a major anthocyanin, and also contained C3g, and trace amount of Cyanidin-3-O-galactosyl-rhamnoside. The total anthocyanin content in the mutant-line was 10 times higher than that of the leading-variety. On the other hand, we also measured Cyanidin content in methanol non-extractive proanthocyanidin (ProA). The leading-variety and the mutant lines contained 257 and 8.8 times higher amount of cyanidin moiety in ProA than in anthocyanins. From this finding, Cyanidin moieties may be distributed competitively between anthocyanins and ProA. We will study physiological roles of anthocyanins in tartary buckwheat petals using the mutant-line.

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To identify useful genes from rice, we constructed and analyzed Arabidopsis overexpressing rice full-length cDNAs. Among them, we isolated a transgenic plant (#R10933) with yellow seeds. In aerial part of R10933, the levels of flavonoids including cyanidin, kaempferol, and quercetin derivatives were lower than in wild type. Traits of reduced anthocyanin were genetically dominant. Expression of genes involved in anthocyanin synthesis was not suppressed.
A F-box gene with Kelch repeats was identified in R10933. Introduction of this gene caused repression of anthocyanin level in Arabidopsis and tomato. The F-box protein interacted with chalcone synthase (CHS) protein in yeast two-hybrid system. Transcriptional level of Arabidopsis homologue increased transiently soon after transfer from high-light stress condition to normal condition. These results indicate that the F-box protein plays a role of CHS degradation during rapid suppression of anthocyanin synthesis.

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Flavonoids are secondary metabolites with many important functions in plants, such as acting as pigments and UV-B protectants. Although the mechanisms by which flavonoids are synthesized in the cytosol are considerably elucidated, the mechanisms by which flavonoids are sequestered into the vacuole are poorly understood. We have isolated Arabidopsis mutants with a reduced level of flavonoid pigments, named transparent testa19 (tt19) mutants. The TT19 gene was shown to encode a protein similar to glutathione S-transferase (GST) family. We described here the characterization of flavonoid transport-related factors, such as TT19, in flavonoid accumulating cells of Arabidopsis.

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Prenylated flavonoids have various physiological activities, and have also drawn a large attention as valuable compounds in the pharmaceutical and food industries. Recent studies demonstrated that the prenyl chain is crucial for their activities. In spite of the importance, there has been no report on the identification of flavonoid prenyltransferase genes in plants. Thus, we attempted to isolate flavonoid prenyltransferase cDNA from a leguminous plant, Sophora flavescens, which produces a large amount of prenylated flavonoids.
We screened cDNA library using informatics analysis of EST data and yeast expression system in combination. The isolated cDNA encoded naringenin 8-dimethylallyltransferase (SfN8DT1) that catalyze the transfer of dimethylallyl moiety to the 8-position of naringenin.
To isolate the other flavonoid prenyltransferases from S. flavescens, we applied the homology-based cloning using SfN8DT1 sequence, and we obtained several cDNAs having complete ORF. Their catalytic functions and the molecular evolution on flavonoid prenyltransferases will be discussed.

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Natural rubber (cis-1,4-polyisoprene) ,which is a secondary metabolite of Hevea brasiliensis, is a strategic raw material. However, only little is known about the mechanism of rubber biosynthesis.
Small rubber particle protein (SRPP) and rubber elongation factor (REF) are two major proteins found in latex and are considered to participate in rubber biosynthesis.
In this study, we analyzed the localization of these proteins by employing immuno-histochemical methods with using anti-SRPP and anti-REF antibodies. The results indicated that SRPP and REF were specifically expressed in laticifers, furthermore, both of these proteins were localized on rubber particles. Interestingly, qualitative difference of rubber particle was also suggested in terms of the distribution of these proteins.

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Coleus forskohlii, an Indian herb, has been used as an important folk medicine for heart disease, respiratory diseases, etc. Furthermore, forskolin a secondary metabolite in C.forskohlii has been found to be a potent activator of adenylyl cyclase, that leads to an increase in levels of cAMP. Therefore, forskolin has been used not only as medicine but also as a health supplement. Currently, almost all the forskolin is produced by plant extraction although production of forskolin by in-vitro culture and chemical synthesis has also been pursued. Due to this reason forskolin is very expensive. To make it affordable on a large scale we aim to engineer plants towards higher production of forskolin. Briefly, we use a combination of FOX hunting system, a gain-of-function gene hunting technique in plant and mammalian culture cell for measurement of forskolin. I will touch upon some of the progress made so far.

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Tomato (Solanum lycopersicum) has been an excellent model system for analysis of plant mechanism, which cannot easily be studied in Arabidopsis or rice, such as fruit physiology and secondary metabolites accumulation. To obtain information on the genetic mechanism of fruit ripening, we induced mutations in the tomato cultivar 'Micro-Tom' by irradiation with gamma ray or accelerated heavy ions, and screened for associated phenotypes and tried to identify loss-of-function mutations in some genes that involve in the process. One of the lines we found showed reduced pigmentation from the mature green stage of the fruit, and displayed pink coloration in the fully ripe stage. We've started to monitor the differences of gene expression level by oligonucleotide-based microarray that generated from whole tomato unigene and consists of over 41,000 probe sets.

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In anti-sense and RNA interference transgenic plants of Coffea canephora in which the expression of CaMXMT1 was suppressed, caffeine biosynthesis from [8-¹⁴C]adenine was investigated, together with the overall metabolism of [8^-14C]adenine. Compared with wild type control plants, total purine alkaloid biosynthesis from adenine and conversion of theobromine to caffeine were both reduced in the transgenic plants. As found previously, [8-¹⁴C]adenine was metabolised to salvage products (nucleotides and RNA), to degradation products (ureides and CO₂) and to purine alkaloids (theobromine and caffeine). In the transgenic plants, metabolism of [8-¹⁴C]adenine shifted from purine alkaloid synthesis to purine catabolism or salvage for nucleotides. HPLC analysis revealed a significantly reduced caffeine content in the transgenic plants. A small quantity (less than 20 nmol/g fresh weight) of xanthosine had accumulated in at least one of the transgenic plants.

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The GCAs are new tetraploid interspecific low-caffeine hybrids bred in Madagascar. They are produced from Coffea. eugenioides, C. canephora and C. arabica. Our previous studies indicate that C. eugenioides has the capacity to rapidly degrade caffeine in addition to the low biosynthetic activity. In order to clarify the mechanism of low caffeine accumulation in the GCAs, we examined the overall metabolism of [8-¹⁴C]adenosine in young leaves and immature fruits of the GCAs, C. eugenioides, C. canephora and C. arabica. Although little differences were found in the formation of nucleotides and RNA between four species used, the biosynthetic activity of purine alkaloids was 50-90% reduced in GCA. Instead, increase in the conventional purine catabolic activity initiated from IMP was observed. Catabolism of [8-¹⁴C]caffeine was negligible at least during 18 hours in GCAs. Low caffeine accumulation in the GCA plants seems to be due to lowered caffeine biosynthesis at the N-methyltransferase reactions.

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It has been well characterized that glycerol 3-phosphate (G3P) is exchanged with orthophosphate by the G3P transporter of the inner membrane of Escherichia coli. In contrast, no eukaryotic G3P transporters have been identified while genes encoding membrane proteins homologous to the bacterial G3P transporters are conserved among eukaryotes such as mammals and higher plants. The genome of Arabidopsis thaliana has five genes (AtG3PP1 to AtG3PP5) encoding G3P transporter homologue proteins. The microarray database, GENEVESTIGATOR, shows that the expression of AtG3PP3 and AtG3PP4 are increased by salt, osmotic and cold stresses. To clarify the role of these G3P transporter homologues in higher plants, we are investigating the phenotypes of T-DNA insertional mutants of Arabidopsis for these homologue genes (obtained from the SALK collection). We will report about a homozygous mutant of AtG3PP4 whose initial growth within 5 days after germination is retarded much severer by salt stress than a wild type.

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Boron (B) is an essential trace element for higher plants. B is also toxic to living organisms at high concentrations. It is essential for living organisms to control B transport and distribution to maintain adequate level of B in cells. However, B response and transport mechanisms are not completely understood.
Arabidopsis thaliana BOR1 is an efflux-type 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.
To obtain insights into the mechanisms of this regulation, we screened for mutants defective in this selective degradation of BOR1. We have isolated several independent mutants and at least four loci are found to be involved in the degradation. By analyzing these mutants, we expect to obtain new knowledge about B response mechanism and the endocytosis system in plants.

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Rab5 GTPase is known to regulate not only endosomal fusion but also signaling through endosomes. The Rab GTPases are activated by specific guanine nucleotide exchange factors (GEFs), which accelerate the exchange of GDP for GTP. The Vps9 domain, the catalytic core for the activation of Rab5, is conserved in all Rab5 GEFs. We have already demonstrated that the Arabidopsis VPS9a that we identified can activate all of the three Arabidopsis Rab5 members (ARA6,ARA7 and RHA1). To examine the activation mechanism of Rab5s, we performed mutational analysis of VPS9a based on the crystal structure of the VPS9a/ARA7 complex. We introduced several amino acid changes in the Vps9 domain and revealed that conserved Tyr (225) and Asp (185) are essential for the GEF activity. We also demonstrated that truncated VPS9a shows specific increase in the GEF activity toward ARA6. We are now examining in detail the function of the C-terminal region of VPS9a.

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Endosomes are essential for organisms to percept environmental stimuli and maintain homeostasis. The key molecule that regulates the endosomal function is Rab5, which acts as a "molecular switch". When Rab5 is in the active state, it recruits various molecules, called effectors, to trigger downstream phenomena. Rab5 is conserved in eukaryotic organisms including plants. In addition to the conventional type of Rab5 homologs, land plants possess structurally unique Rab5 homologs. Our functional analysis has shown that these two types of Rab5s are not only functionally differentiated but counteractive. To elucidate the molecular basis of endocytosis in plants, I focused on Ara6, the plant-unique-type Rab5 in Arabidopsis thaliana, and screened for effector molecules. Until now, seven candidates were obtained, three of which have localized to the Ara6-endosomes in protoplasts. These effector candidates are plant specific, suggesting that plants have established a unique molecular machinery to regulate endosomal functions.

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BiP is an Hsp70 in the endoplasmic reticulum (ER) in eukaryotic cells, which functions in various essential processes including protein translocation across the ER membrane and protein folding and quality control in the ER. Arabidopsis thaliana has three BiP genes, BiP1, BiP2 and BiP3. BiP1 and BiP2 are expressed ubiquitously under the normal growth condition, and BiP3 expression is induced only by ER stress. Although single deletion mutants of each BiP gene are viable, genetic analyses have shown that simultaneous deletion of the BiP1 and the BiP2 genes causes female sterility. The bip1 bip2 double mutation did not affect production of fertilization-competent female gametophyte, and fertilization occurred normally. However, embryos derived from the mutant female gametophytes stopped their development at the heart stage. This embryonic lethal phenotype was not rescued by expression of BiP genes from the wild type pollen.

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Myrosin cells are idioblasts specific for Capparales plants, and accumulate large amounts of myrosinases, which hydrolyze glucosinolates to produce toxic compounds for repelling pests. Previously, we showed that myrosin cells were scattered along leaf veins in wild-type leaves, while they were abnormally distributed in atvam3leaves. The mutants developed a network of myrosin cells throughout the leaves: myrosin cells were not only distributed continuously along leaf veins, but were also observed independent of leaf veins. The result indicates that AtVAM3 is involved in development of myrosin cells. AtVAM3 is a SNARE protein belonging to SYP2 family, which is localized to vacuoles and prevacuolar compartments. In this study, we analyzed the involvement of other SYP2 members, AtPEP12 and AtPLP, in development of myrosin cells. We will show the functional differentiation of SYP2 proteins in development of myrosin cells.

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SNARE molecules have a central role in membrane fusion step between a target membrane and a transport vesicle in eukaryotes.
In Arabidopsis genome, about 60 SNARE genes have been identified, and we have already determined the subcellular localizations of almost all SNAREs in Arabidopsis. We found that the greater numbers of SNAREs are localized on the plasma membrane, indicating that SNAREs on the plasma membrane may function in distinct transport pathways to the different subdomains of the plasma membrane. In fact, we already reported in the last meeting that one of the plasma membrane Qa-SNARE molecues, SYP123, is highly concentrated on the tip-region of the root hair.
Here, we performed the split luciferase complementation assay to identify the interacting proteins with plasma membrane Qa-SNAREs and found that SYP132 interacts with SEC1 family proteins, KEU and SEC1B. We also report that molecules which possibly form complexes with SYP123.

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The elongation of pollen tube is a highly coordinated process involving polarized secretion of the cell wall materials and membranes to the tip region and rapid expansion of vacuole. To study the mechanism of pollen tube elongation, we have focused on the SNARE molecules which possibly function in the pollen tube elongation.
We generated transgenic Arabidopsis expressing pollen specific Qa-SNAREs (SYP124, 125 and 131) with GFP-tag under control of their own promoters, and observed the localizations and dynamics of these molecules. SYP125 was highly concentrated on the tip region of the pollen tube. In contrast, SYP124 was concentrated on the proximal region of the tube tip, but not the tube tip region. The localization of SYP131 is uniformly as that of SYP132 in root hair. These results suggest that at least two distinct membrane transport pathways are involved in the pollen tube elongation.

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Graminaceous plants, including rice (Oryza sativa L.), utilize a chelation strategy to acquire Fe from the soil. The roots of these species release low-molecular weight compounds, called mugineic acid family phytosiderophores (MAs), which bind to and solubilize Fe(III) in the rhizosphere. The resulting Fe(III)-MA complexes are reabsorbed by the root cells. Rice produces and secretes deoxymugineic acid (DMA), the first member of the MAs. To examine how to secrete DMA from rice root, the secretion pattern was examined. DMA amounts secreted from rice root have been changed diurnally under iron-deficient condition. Moreover, rER-vesicles were observed in only iron-deficient rice root.
The fusion protein, OsNAS2, the enzyme participated in the DMA synthetic pathway, and sGFP was localized at spots in the cytoplasm of the transgenic rice roots. These vesicles have been suggested to be involved in the DMA production and secretion in rice root.

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Natural rubber obtained from Hevea brasiliensis is an essential industrial material because of its excellent properties (elasticity, cold resistance, abrasion resistance). However, natural rubber biosynthesis has not been well understood. As the basic information to clear the biosynthesis, it is important to analyze the detail structure of laticifers, where natural rubber production and accumulation occur. In previous studies, laticifers have been stained with conventional lipid-staining reagent, Oil red O mainly. But it is difficult to analyze the detail structure using Oil red O and optical microscopes. In this study, we established a structural imaging system and succeeded in visualization of laticifers using spectral confocal laser scanning microscopy (SCLSM). Now, we are trying to profile gene-expression pattern based on difference in structure using laser microdissection and real-time PCR.

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Eucommia ulmoides O. is a native plant in China. E. ulmoides accumulates trans-polyisoprene (TP) in all organs. TP with high-molecular-weight is versatile for rubber industries, and it can change the raw material of substitute for petroleum, because it is composed of the hydrocarbon. Light microscopic observation and FT-IR microspectroscopic analysis have shown that TP is localized in phloem as fibrous material. However, in the conventional method there was not any accurate method of visualizing TP specifically. In this study, TP was labeled by fluorescent probes, and the specific fluorescence bands were separated using spectral confocal laser scanning microscopy. The bioimaging graphics thus obtained were analyzed to understand the localization of TP. Additionally, the detailed observation was carried out on phloem of E.ulmoides using scanning electron microscopy.

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Among the members of NIP subfamily, both NIP5;1 and NIP6;1 are boric acid channels required for normal growth of Arabidopsis under B limitation, but with different roles. NIP5;1 is important for efficient B uptake, while NIP6;1 is required for preferential distribution of B to young leaves. In the present report, we characterized physiology of NIP5;1 and NIP6;1 double T-DNA insertion lines (nip5;1-1 x nip6;1-1and nip5;1-1 x nip6;1-2) . The growth of double T-DNA insertion lines was greater than that of a single NIP5;1 insertion line (nip5;1-1), but lower than that of single NIP6;1 insertion lines (nip6;1-1 and nip6;1-2). The B concentration of nip5;1-1 x nip6;1-1 and nip5;1-1 x nip6;1-2 mutant plants was higher than that of nip5;1-1 mutant plants, but lower than that of nip6;1-1 and nip6;1-2 mutant plants. These findings suggested that NIP6;1 works as an negative growth regulator under the NIP5;1 KO background.

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