The molecular mechanisms of transdifferentiation from mature somatic cells to stem cells are largely unexplored in both plants and animals. The moss Physcomitrella patens has a remarkable capacity for such transdifferentiation.
In normal development of the moss, a spore germinates to produce a protonemal apical cell, a type of stem cells. Subsequently, the apical cell changes its nature and raises a leafy shoot, gametophore. Meanwhile, in dissected leaves, cells facing to cutting surface are transdifferentiated into protonemal apical cells. We established an experimental system in which this transdifferentiation can be synchronously induced at high frequency. We further investigated the involvement of phytohormones in this process. Exogenous auxin or abscisic acid at an excess concentration inhibited the transdifferentiation, while cytokinin, gibberellin, or brassinosteroid did not. The suppression of the transdifferentiation by antiauxin was specifically restored by exogenous auxin. Thus, intrinsic auxin accumulation with proper concentration is necessary for this process.

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We have been working on a comparative study of abscisic acid (ABA) signaling and the function between the moss Physcomitrella patens and higher plants. Although P. patens possesses an ABA signaling pathway similar to that of higher plants, our knowledge of ABA function in the moss is limited. In this study, we generated transgenic P. patens lines that overexpress Arabidopsis CYP707A3 gene encoding an ABA 8'-hydroxylase. Because ABA 8'-hydroxylase catalyzes the first step of ABA catabolic pathway, the moss overexpressors (CYP707 OEs) were expected to accumulate less ABA. We found that the CYP707 OEs resulted in the appearance of macrochloroplasts under stress conditions. The macrochloroplast phenotype of CYP707 OEs was relieved by exogenously applied ABA, and application of an ABA biosynthesis inhibitor Abamine SG to wild type caused the macrochloroplasts phenotype. These results suggest that ABA is involved in the chloroplast division under environmental stress.

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Abscisic acid (ABA) is involved in plant development, and mediates responses to environmental stress. The Arabidopsis thaliana abi1-1 is a dominant mutant allele of ABI1, which encodes a type-2C protein phosphatase (PP2C) and functions as a negative regulator of ABA-signaling. We demonstrated that transient expression of abi1-1 also resulted in the repression of ABA-signaling in Physcomitrella patens, suggesting that PP2Cs are involved in ABA-signaling in the moss. The moss abi1-1 overexpressor (abi1-1 OE) lines were less sensitive to ABA and osmotic stress. The gametophores of abi1-1 OEs were bigger compared to wild type, and sporophyte development was abnormal in abi1-1 OEs. Furthermore, an ABI1 homolog from P. patens (PpABI1) strongly repressed ABA-signaling in P. patens. These results suggest that PP2C-regulated ABA-signaling is involved not only in stress tolerance but also in development in P. patens, and that PpABI1 functions as a negative regulator of ABA-signaling in P. patens.

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ABI1 gene, which encodes a type 2C protein phosphatase (PP2C), functions as a negative regulator of the ABA-signaling in Arabidopsis. We have indicated that the moss Physcomitrella patens also conserves the PP2C-mediated ABA-signaling pathway, and have identified an ABI1 homologous gene, PpABI1 from P. patens. From the transient assay of P. patens, PpABI1 was suggested to be a negative regulator of the ABA-signaling. Here we analyzed the in vivo function of PpABI1 by gene targeting. ABA application is known to increase the freezing tolerance of P. patens protonemata in a dose-dependent manner. Protonemata of PpABI1 disruptant (PpABI1 KO) showed 2 times higher freezing tolerance than the wild type at 0.1µM ABA concentration. PpABI1 KO was more sensitive to exogenously applied ABA, as judged by the redifferentiation of protonemal cells into brood cells. These results clearly show that PpABI1 functions as a negative regulator of ABA-signaling in P. patens.

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A plant-specific transcription factor ABI3/VP1 is expressed mainly in seeds, and plays an important role in seed maturation and dormancy. We have identified three copies of ABI3/VP1 homolog (designated as PpABI3A, PpABI3B and PpABI3C) from the moss Physcomitrella patens, which forms not seeds but spores. Although PpABI3A was partially able to complement the function of ABI3/VP1 in higher plants, the in vivo function of the PpABI3 gene in P. patens was yet to be elucidated. Here we report that PpABI3A, PpABI3B and PpABI3C were expressed though the life cycle of P. patens, in contrast to ABI3/VP1. The overexpressors of PpABI3A were more sensitive to ABA, and the triple knockout of PpABI3 genes was less sensitive to ABA, as judged by the protonemata growth on media containing ABA. These data strongly suggest that PpABI3 functions as a positive regulator of the ABA-signaling in vivo and regulates the protonemal growth.

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Methyltransferases (MT) are involved in a variety of metabolic pathways in plants. We found two Arabidopsis MTs that are able catalyze methylation of gibberellins (GAs) and named them GAMT1 and GAMT2. Ectopic expression of GAMT1 or GAMT2 in Arabidopsis resulted in dwarfism with reduced levels of bioactive GAs. GAMT1 and GAMT2 are both expressed mainly in siliques during Arabidopsis development. Siliques of the plants homozygous for null alleles at the GAMT1 and GAMT2 loci accumulated high levels of various GAs, with siliques of the double mutant showing particularly high levels of GA₁. Seeds of the gamt1 gamt2 double mutant showed reduced inhibition of germination, compared with wild type, in the presence of a GA biosynthesis inhibitor. These results suggest that GAMTs play a role in GA deactivation in Arabidopsis seeds.

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Rice eui mutants show drastic elongation of the final internode at the heading stage. We have reported that the EUI gene encodes a cytochrome P450, CYP714D1, and function as a novel gibberellin (GA) deactivation enzyme that epoxidizes the C16(17) double bond of GAs. In Arabidopsis, CYP714A1 is the closest gene to the CYP714D1 in sequence. To clarify the possible function of CYP714A1 in the GA metabolism pathway, enzymatic activity of recombinant CYP714A1 proteins were analyzed in a yeast expression system. We found that GA₁₂ was metabolized into an unknown compound, which was distinct from 16α,17-epoxy GA₁₂, the product of CYP714D1 protein. In comparison with GA₁₂, this product did not induce growth promotion when applied to the GA-deficient ga1-3 mutant. Furthermore, transgenic Arabidopsis plants overexpressing CYP714A1 showed severe dwarfism like the ga1-3 mutant. These results indicate that CYP714A1 functions as a GA deactivation enzyme in Arabidopsis.

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Gibberellin (GAs) affects various aspects of plant development. Thus both biosynthesis and signal transduction of GA must be regulated precisely. GAs controls biosynthesis of themselves by GA-negative feedback through regulating the expression of GA biosynthetic and metabolic genes. For maintaining the amount of endogenous GAs, GA-negative feedback is indicated to play a central role. Among genes regulated by GA-feedback, GA3ox might be most important because it catalyzes the final step to generate active GAs. We have found only AtGA3ox1 is under GA-negative feedback among GA3oxes in Arabidopsis. We also localized cis element, designated GNFEI, necessary for the feedback, and isolated trans factor, designated AGF1, binds to GNFEI. To investigate the function of AGF1, we generated AGF1 constitutive expressor. Up-regulation of the expression of AtGA3ox1 under GA-negative feedback was observed in AGF1 constitutive expressor. We will report about further analysis of AGF1 function.

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Recently, we isolated a GA insensitive dwarf mutant of rice, gid1. GID1 encodes a soluble GA receptor that shares sequence similarity with a hormone sensitive lipase (HSL). The GID1-GA complex directly interacts with SLR1, a DELLA repressor protein associated with GA signaling. In this study, we performed the domain analysis of GID1 protein using 94 GID1 proteins, which were independently mutated in the manner of alanine scanning. We analyzed both in vitro GA binding activity and SLR1 interacting activity of each mutated GID1 protein.
We also examined the interaction between GID1 and SLR1 protein in vivo. GID1-GFP was over-expressed in rice callus and the callus was treated with or without GA₄. SLR1 was co-immnoprecipitated with GID1-GFP protein only from GA₄-treated callus, which indicated that GID1 also interacts with SLR1 in vivo in GA dependent manner.

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Although some types of ferns and mosses produce endogenous gibberellins (GAs), it has been unknown whether they possess the GID1-dependent GA signal transduction pathway as seen in seed plants. We analyzed the GA signaling pathway in a fern (Selaginella moellendorffii) and in a moss (Physcomitrella patens) to explore when plants came to utilize GA as a plant hormone. In rice, the GA receptor (OsGID1) and DELLA protein (SLR1) interact in a GA dependent manner resulting in degradation of SLR1 by OsGID2. Then, degradation of SLR1 induces some GA actions.
Interaction between GID1 and DELLA of P. patens was not found by Y2H assay. In contrast, S. moellendorffi contains SmGID1s (SmGID1a and SmGID1b), SmDELLA1 and SmGID2 involved in GA perception and signaling. SmGID1a has similar function of OsGID1, whereas SmGID1b shows some different characters from OsGID1, suggesting an existence of a fern specific GA receptor.

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Growth of horizontally-grown Arabidopsis thaliana seedlings was almost half (0.57-fold) as compared to that of vertically-grown ones. Comparison of the root growth analysis ethylene mutants has showed that ethylene may play an important role in this process. The simplest explanation of growth inhibition is that mechanical and gravitropic stimulations affect continuously upon the horizontal root tips during the elongation, resulting in greater ethylene production. However, no significant difference was found in ethylene production between horizontal and vertical. In addition, the estimation of ethylene responsive genes by real-time PCR showed marked enhancement of the expression in horizontal roots; 5.2-fold for Ethylene Responsive Factor, ERF and 2.7-fold for basic chitinase, BACH, respectively. Relationship between the mechanical stimulation and the ethylene responsive genes will also be discussed briefly.
Plant physiologists, who often test biological activities of chemicals on filter paper, should pay much attention on potential blind spots of the common methodology.

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Our previous work suggests that leaf gibberellin (GA) is required for tissue-reunion of cortex in the cucumber and tomato cut hypocotyls, but molecular mechanisms are still unclear. In this study, morphological and gene expression analyses during tissue-reunion were performed using the Arabidopsis cut flowering stem. The lowest internode of stem was cut transversely and reunion process was analyzed. Cell division was initially observed in 3 days and tissue-reunion almost completed in14 days. In contrast to the case of cucumber, normal reunion was observed in ga3ox1/ga3ox2 or Paclobutrazol treated-plants. The microarray analysis during reunion process was performed. A number of genes involved in cell division, cell wall-modification, phyohormone-synthesis and signal transduction were determined to be up-regulated. The ACC synthase gene was significantly up-regulated at 1 day and ethylene signaling-deficient mutant showed lower efficiency in the tissue-reunion. The gene expression analysis of XTH showed that cell wall-modification occurred during reunion process.

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MPN+ domain proteins play a pivotal role in the demodification of ubiquitin and ubiquitin-like proteins. Among the three identified MPN+ domain proteins, RPN11 (deubiquitylation, 26S proteasome subunit) and CSN5 (deneddylation, COP9 signalosome subunit) are well characterized. AMSH, the third and obviously the last member of the MPN+ family, still remains to be characterized. In mammals, AMSH was reported to have deubiquitylating activity towards K63-linked ubiquitin chains, and therefore thought to be involved in the regulation of endocytosis.
In our study, we aim to elucidate the functional mechanism of the 3 AMSH proteins in Arabidopsis thaliana, taking advantage of available mutant lines. We found that all three AMSH proteins possess MPN+ domain dependent deubiquitylating activity in vitro, and AMSH3 was found to be essential for plant growth. Together with localization and biochemical studies, we would like to discuss the function of AMSH in higher plants.

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Ubiquitination of proteins is one of post-translational modification in eukaryote and is catalyzed by the three components, E1, E2 and E3. Although main function of the ubiquitination is known to lead to protein degradation, more recent studies showed that ubiquitination might function as a regulator of signal transduction and gene expression. Genomic sequence of model plant Arabidopsis thaliana revealed more than 500 genes encoding the components of ubiquitination. Thus plant might have a highly complicated ubiquitination network. In this study, by combining the wheat cell-free expression system and high-throughput detection system, AlphaScreen, we aimed to construct the comprehensive method for analysis of the ubiquitination network. Twenty-four of 32 E2 genes from RAFL were expressed as soluble proteins, and twenty of them were markedly detected the ubiquitination from rabbit E1. Now we are challenging to investigate the ubiquitination of proteins involved in signal transduction by using this method.

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Sumoylation is a post-translational modification which alters protein function, activity and subcellular localization. Covalent SUMO conjugation requires an E1/E2/E3 enzyme cascade similar to the ubiquitin-conjugating cascade. In most cases, the target proteins interact with both SUMO and E2 enzyme. Although sumoylation is known to be involved in various cellular processes, few SUMO-targeted proteins are reported in plants. In order to identify SUMO-targeted proteins, we performed the yeast two-hybrid screening using E2 enzyme, AtSCE1a, which is encoded by the only one gene in Arabidopsis. Three candidate proteins were isolated through the screening, including At2g20310, which is a plant-specific protein of unknown function. At2g20310 and its paralogous protein, At4g28690, interact with AtSUMO1, 2, and 3, respectively. Furthermore, sumoylation of both proteins were confirmed by in vitro and in vivo symoylation assays. At2g20310-GFP fusion protein is localized at the nucleus in root tissue. The functional analysis of these genes is currently in progress.

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The COP9 signalosome (CSN), a nuclear protein complex essential for plant and animal development, regulates proteasome-mediated proteolysis. To characterize a novel intravital mechanism of CSN, CSN1 binding proteins were isolated and analyzed in the model plant Arabidopsis.
SAP130, one of the identified CSN1 binding proteins, 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. The expression level of AtSAP130b was found to be significantly higher than that of AtSAP130a. Pull down experiments, in vitro, revealed that AtSAP130 directly bound AtCSN1 at the N terminal portion of the protein. Here we discuss the possible role of CSN function in post-transcriptional regulation in Arabidopsis.

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Arabidopsis bZIP transcription factor AtbZIP10 directly interacts with programmed cell death (PCD) regulator LSD1 in cytosol and its function is also involved in the regulation of PCD (1). The interaction with exportin, AtXPO1, would be required for the nuclear export of AtbZIP10 (1). To identify unknown proteins involved in the regulation of AtbZIP10 subcellular localization, we performed Yeast-Two-Hybrid screening and have focused on the microtubule-plus-end-binding protein AtEB1(end-binding 1) from plural positive clones identified by screening. We found that AtbZIP10 interacts with all Arabidopsis EB1(EB1a,b,c) and that the leucine zipper-like region at C-terminal domain of AtEB1s specifically binds to C-terminal domain of AtbZIP10 which does not contain bZIP domain. The function of AtEB1 is not clear yet, but the interaction between AtbZIP10 and AtEB1 suggested that it participates in the control of AtbZIP10 localization in cytosol. (1) Kaminaka et al., EMBO J. (2006)

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We analyzed the structure and composition of starch and the effects of the other SS isozymes derived from SSIIIa-deficiency in rice endosperm. Aactivities of isoamylase, pullulanase, branching enzyme IIa, BEIIb and phosphorylase in the mutants showed no obvious differences with those of the wild type. In constant, the amount of SSI protein was increased by 1.3-1.7-folds and it resulted in the increase of degree of polymerization (DP) 10-15 chains of the mutant amylopectin. Moreover, GBSSI protein was also increased by 1.4-1.7-folds and it resulted in the increase of amylose content and extra-long chains (DP≥500) of amylopectin. These changes in the structure and composition of starch of the mutants were caused directly by the reduction of long chains (DP≥33) of amylopectin by the SSIIIa-deficiency itself, and indirectly the enhancement of the activities of SSI and GBSSI. The combined effects brought about the dramatic reduction of pasting viscosity of the endosperm starch.

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To elucidate the effect of high temperature on grain filling metabolism, rice developing caryopsis was exposed to high temperature during milky stage. Comprehensive gene screening by 22K microarray and differential hybridization, and following expression analysis by semi-quantitative RT-PCR revealed that several starch synthesis-related genes such as GBSSI and BEIIb were down-regulated by high temperature whereas those for starch-consuming α-amylases and HSPs were up-regulated. Biochemical analyses of starch showed that the high temperature-ripened grains consisted of decreased content of amylose and long chain-enriched amylopectin, which might be attributed to repressed expression of GBSSI and BEIIb, respectively. SDS-PAGE and immunoblot analysis of storage proteins revealed decreased accumulation of 13-kD prolamin, which is consistent with diminished expression of prolamin genes under elevated temperature. Ripening under high temperature resulted occurrence of grains with chalky appearance and decreased weight. The possible metabolic pathways affected by high temperature and their relevance to grain chalkiness are discussed.

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Fumonisin B1 (FB1) elicits programmed cell death (PCD) in plants, probably through competitive inhibition of sphingosine-N-acyltransferase. Because this inhibition may result in the accumulation of free sphingoid bases in cells, it is important to characterize the enzymes that control the intracellular levels of sphingoid bases. To assess the functional consequence of the loss of function of an Arabidopsis gene encoding sphingosine-1-phosphate lyase, AtSPL1, we characterized two independent Arabidopsis mutants (atspl1-1 and atspl1-2) with T-DNA insertions in the AtSPL1 gene. These mutants were more sensitive to FB1 than wild-type plants, suggesting that the cell levels of sphingolipid catabolites may be affected to cell death signaling. Sphingolipid synthesis is initiated by serine palmitoyltransferase (SPT) that catalyzes the condensation of serine with palmitoyl-CoA to form 3-ketosphinganine. Here, we report the expression of the SPTLC2 genes in the protoplasts from suspension-cultured tobacco BY-2 cells suspended in the solution containing FB1.

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In all seed plants, the assembly of galactolipids in the thylakoid membrane involves the endoplasmic reticulum (ER), where the glycerolipid precursor phosphatidic acid is synthesized. We have isolated mutants of Arabidopsis thaliana that are disrupted in the biosynthesis of galactolipids from ER-derived precursors. These mutants rely on the de novo assembly of galactolipids from newly synthesized fatty acids in the plastid. The mutants accumulate triacylglycerols and oligogalactolipids, trigalactosyldiacylglycerol, in the leaves leading to their trigalactosyldiacylglycerol (tgd) designation. One of the mutant allele proteins, TGD2 is similar to a substrate binding protein of multipartite ABC transporters as found in bacteria. It is localized in the inner chloroplast envelope membrane and specifically binds phosphatidic acid. Based on this evidence, we hypothesize that the TGD2 is involved in transport of phosphatidic acid from the outer envelope membrane to the inner envelope membrane, thereby providing the ER-derived DAG precursor for galactolipid biosynthesis.

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Plants produce a lot of hydrophobic metabolites including phospholipids, glycolipids. These compounds generally classified into lipids are diverse with their chemical structures and biological functions. To comprehend the chemical profile and biosynthetic network of various plant lipids, we have started the analysis of lipid extract using LC-MS.
Lipid fractions of Arabidopsis thaliana were separated and analyzed using hydrophilic interaction chromatography (HILIC) coupled to Shimadzu ion-trap (IT)-TOF mass spectrometer. Separation was archived by using the mixture of acetonitrile, methanol, and water containing ammonium formate as HPLC solvent. The peaks attributable for typical membrane lipids (phospholipids and glycolipids) and glycosylated terpenoids were determined by chromatographic behavior and their accurate mass. During the optimization of analytical conditions, lipids extracted from different tissue and mutant plants were also analyzed. We would like to report some of the data.

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We previously found that sulfoquinovosyl diacylglycerol (SQDG), the major sulfolipid in the chloroplasts, was degraded under the sulfur-starved condition in Chlamydomonas reinhardtii. We here report the novel role of SQDG as sulfur-source for acclimation to sulfur-starvation. After [³⁵S]-labeled cells of C. reinhardtii were shifted to the sulfur-starved condition, the radioactivity of SQDG, which had occupied 11% of total cellular-sulfur, was decreased to 2% in 6h. On the other hand, sulfur released from SQDG was incorporated into proteins. Since an SQDG-lacking mutant of C. reinhardtii, as compared with the wild type, showed defects in synthesis of chlorophyll-protein complexes, induction of sulfate transport activity, and growth during sulfur-starvation, the re-distribution of sulfur from SQDG proved critical for acclimation to sulfur-starvation.

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The lipid composition of photosynthetic membranes is highly conserved in organisms performing oxygenic photosynthesis among higher plants and cyanobacteria. However the biosynthetic pathways of the major glycolipid monogalactosyldiacylglycerol (MGDG) are not conserved. Higher plants synthesize MGDG from diacylglycerol (DAG) and UDP-galactose while cyanobacteria produce monoglucosyldiacylglycerol (MGlcDG) from DAG and UDP-glucose followed by the epimerization of the sugar for generating MGDG.
We found that Chlamydomonas, evolutionally positioned between higher plants and cyanobacteria, has genes homologous to those for MGDG synthase and MGlcDG synthase, and confirmed that the two genes were transcriptionally expressed in the cell. To determine the biosynthetic pathway of MGDG in Chlamydomonas, we analyzed glycosyltransferase activities using UDP-galactose or UDP-glucose as a sugar donor after subfractionation of the cell. The result showed that the plant-type MGDG synthesis occurs in the membrane fraction. We will also show the results of the cloning of Chlamyomonas MGDG synthase and the biochemical properties.

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Rosmarinic acid (RA) is a well-known constituent of members of Lamiaceae and Boraginaceae and exhibits various pharmacological activities such as antiviral, antimicrobial and anti-inflammatory. RA is an ester of caffeic acid and 3,4-dihydroxyphenylalanine, which is formed from the amino acids L-phenylalanine and L-tyrosine.
We have worked on the environmental control methods for increasing the anitioxidants of Lamiaceae. In our experiments, RA was the main antioxidants of sweet basil and irradiation of blue and/or UV-B light at night had effects in the increasing of RA. We have isolated sweet basil genes involved in the biosynthesis of RA (phenylalanine ammmonia-lyase, tyrosine aminotransferase, hydroxyphenyl pyruvate reductase, etc.) and are currently analyzing expression of these genes.

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Quinolizidine alkaloids, referred to as lupine alkaloids, occur primarily in the genus Lupinus. Lupinus angustifolius L. cultivated as a crop has a sweet cultivar cv. Uniharvest and a bitter cultivar cv. Fest. In this study, we profiled the cDNA fragments specifically expressed in cv. Fest by PCR-select cDNA subtraction technique. Among 71 bitter-specific cDNA fragments, several fragments showed homology with ornithine decarboxylase and amine oxidase that might be involved in alkaloid biosynthesis. Using the sequence information of these fragments, the full length cDNAs (LaDC , LaAO) were obtained by 5'- and 3'- RACE techniques. The full length cDNA of LaDC encodes 439 amino acids showing 70% identity with ornithine decarboxylase from Glycine max. The full length cDNA of LaAO encodes 774 amino acid showing 79% identity with peroxisomal-copper-containing amine oxidase from G. max. LaAO possesses peroxisome targeting signal 1 (PTS1) in its carboxy terminal end.

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It is an important problem that why do the two pigment anthocyanin and betacyanin never occur together in the same plant. DOPA 4,5-dioxygenase (DOD) is an important enzyme to synthesize betalain pigment in higher plant. However, it is known that DOD homologs exist in the genome of betalain non-producing plants. To analyze the difference between the DOD of betalain producing plant and betalain non-producing plants contribute to elucidate the problem. We cloned DOD homolog cDNAs from arabidopsis, torenia and carnation. Each recombinant protein extracted from Escherichia coli harboring these cDNA were added to a reaction mixture containing L-DOPA. When recombinant tereina DOD was added to the reaction mixture, the mixture color turned to yellow and and a reaction product in the mixture was betalamic acid confirmed by HPLC.

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Coumarins are often found as secondary metabolites in various plant species. Hydroxylation of cinnamates at the ortho-position is a key step to form the coumarin structure, and however ortho-hydroxylase still remains to be characterized. We previously reported the high accumulation of scopolin, a β-glucoside of scopoletin, in the roots of Arabidopsis. We have also found that scopoletin biosynthesis strongly depends on CCoAMT1, which catalyzes methylation of the 3'-hydroxy group of caffeoyl CoA. We therefore selected several oxidase genes, which show co-expression with CCoAOMT1, as candidates of ortho-hydroxylase, and their T-DNA insertion mutants were obtained from SALK institute. One of the mutants exhibited severe reduction in scopolin/scopoletin levels in the roots. The recombinant protein expressed in E. coli exhibited ortho-hydroxylase activity. Biochemical characterization of the recombinant protein is in progress.

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Flavonoids are one of the well-studied secondary metabolites with over 6,000 known structures. The huge diversity is derived from modifications such as glycosylation, acylation and methylation. The structures of nineteen flavonoids in Arabidopsis suggest that at least nine family 1-type glycosyltransferases (UGT) are involved in flavonoid modification pathway. However, only four UGT genes were identified. In Arabidopsis, UGTs are encoded by 107 genes. Their huge diversity makes it difficult to estimate their physiological functions distinctively.
By coexpression analysis of public microarray datasets, we found a UGT gene, UGT1, which is highly correlated with flavonoid biosynthetic genes. The deduced amino acid sequence and flavonoid target analysis of the mutants suggest that UGT1 encodes flavonoid 7-O-rhamnosyltransferase (7RhaT). We identified that UGT1 functions as flavonol 7RhaT by UGT1 recombinant protein and a complementation test of the T-DNA insertion mutants. Substrate specificity of UGT1 and putative flavonol glycosylation pathway are also discussed.

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The functional involvement of cyclic GMP (cGMP) in phytochrome-dependent producton of anthocyanin has been reported, whereas little is known about molecular mechanisms controlling gene expression by cGMP. Northern analyses using photomixotrophic soybean cell-culture (SB-P) revealed that gene expression of several flavonoid-biosynthetic enzymes was enhanced by cGMP. All cGMP-induced genes that we tested were also stimulated by nitric oxide (NO) and illuminaton of white light. Furthermore, we found that cGMP, NO, and white light could increase anthocyanin in SB-P cells suggesting that white light-dependent production of anthocyanin is mediated by cGMP and/or NO. On the other hand, promoter analyses of chalcone reductase gene indicated that the upper region of the promoter (-991 to -576 bp) was responsible for the induction by cGMP. We are now investigating the relation between the cGMP and/or NO signaling and phytochrome signal-transduction for gene regulation of flavonoid biosynthetic enzymes by pharmacological analysis.

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Prenylated flavonoids have various physiological activities, and have also drawn a large attention as valuable compounds in the medical and food industry. Recent studies showed 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 a cDNA of flavonoid prenyltransferase from a leguminous plant, S. flavescens.
To isolate cDNA, we screened cDNA library using informatics analysis of EST data and yeast expression system in combination. The cDNA isolated was encoding naringenin 8-dimethylallyltransferase (SfN8DT) that catalyzes the substitution of dimethylallyl moiety with an aromatic proton. In S. flavescens plants, prenylated flavonoids are detectable only in the root bark. In accordance with this accumulation pattern, SfN8DT mRNA was detectable only in root bark, suggesting that prenylated flavonoids were biosynthesized specifically at root bark, where they are accumulated without translocation.

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Family 1 glycosyltransferases (UGTs) attract considerable interest as suitable candidates of biocatalysts for metabolic engineering from the biotechnology community. Current investigation on 3D-structures of UGTs revealed the role of the conserved amino acid residues in the PSPG-box that constitute the donor-sugar binding pockets. However, the roles of less conserved amino acids in this motif that may decide the characteristics unique to particular enzymes such as substrate recognition and catalytic potential have been less understood. We have recently cloned a cDNA encoding a novel glucosyltransferase, UDP-glucose: curcumin glucosyltransferase (CaUGT2), from Catharanthus roseus cultured cells. In this study, we report that a single amino acid in the PSPG-box plays important role in the catalytic activity of CaUGT2.

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(+)-Sesamin, a furofuran class lignan, is widespread in vascular plants and represented by Sesamum spp. (+)-Sesamin has been of rapidly growing interest because of its beneficial biological effects in mammals such as protection against ethanol- and carbon tetrachloride-induced liver damage. At the later phase of seed development, a S. indicum P450, CYP81Q1, catalyzes (+)-sesamin biosynthesis from (+)-pinoresinol via (+)-piperitol by forming two methylenedioxy bridges. (+)-Sesamin is catalyzed into soluble (+)-sesaminol glucosides by hydroxylation and glucosylation, however, biosynthetic mechanisms and physiological roles of lignan glucosides remain to be clarified. Here we show that Sesamum UGT71A subfamily glycosyltransferases catalyze the glucosylation of (+)-pinoresinol as well as (+)-sesaminol. S. indicum UGT71A9 gene is highly expressed during seed development. Moreover, two UGT71A homologs of S. alatum UGT71A8 and S. radiatum UGT71A10 also possess lignan glucosylating activities, demonstrating that UGT71A glucosyltransferases are structurally and functionally conserved among Sesamum spp.

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While the wild-type Japanese morning glory (Ipomoea nil) exhibits blue flowers with red stems, due to anthocyanin pigmentation, and produces dark-brown seeds, the white-flower mutants c-1 and ca display dark-brown seeds with red stems and ivory seeds with green stems, respectively. We have characterized the tissue-specific expression of three R2R3-MYB genes, three bHLH genes, and two WDR genes in I. nil. Subsequently, we have shown that the c-1 and ca mutations are frameshift mutations caused by a 2-bp deletion and 7-bp insertions in the genes for the R2R3-MYB and WDR transcriptional regulators designated as InMYB1 and InWDR1, respectively. In addition to the defects in flower, stem, and seed pigmentations, we also found that the ca mutants show reduced trichome formation in seeds, which is a novel epidermal traits associated with the WDR transcriptional factor.

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The plant epidermal cell traits including anthocyanin and proanthocyanidin pigmentation, leaf trichome and root hair formation are regulated by proteins containing R2R3-MYB domains, bHLH domains, and conserved WD40 repeats. Spontaneous ivs mutants of Ipomoea purpurea displaying pale-pigmented flowers, and ivory seeds are caused by insertions of the transposons into the bHLH2 gene. In these mutants, the expression of the anthocyanin biosynthesis genes were partially reduced in their flower buds whereas only the DFR and ANS transcripts were abolished in their seed coats. The wild-type dark-brown seeds accumulated proanthocyanidin and phytomelanin, and these pigments were reduced in the ivs mutant. In addition, the trichomes in the ivs seed coats were smaller in size and fewer in number than those in the wild type. These results indicated that bHLH2 controls not only anthocyanin and proanthocyanidin biosynthesis but also phytomelanin pigmentation and seed trichome formation.

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Multiple copies of flavonoid biosynthetic genes with different patterns of expression of tissue or cells and induction following environmental stimuli were identified in Legume. We try to analyze regulatory networks controling flavonoid biosynthesis in Legume. Sequence homology searches in Lotus japonicus genome were performed to gain the information about transcription factors involved in flavonoid biosynthesis. Three genes encoding MYB-like proteins that have high similarity to TT2, the R2R3-MYB transcription factor regulating proanthocyanidin biosynthesis were found. RT-PCR revealed that three MYB-like genes, designed LjMYB-1, 2, 3 display diverse patterns of expression. Transient expression experiments using BANYULS (BAN) promoter-reporter gene construct were conducted in planta. BAN promoter was activated by heterologously expressed LjMYB-1 and LjMYB-3, suggesting that LjMYB-1, -3 may function as TT2 orthologue. Further analysis on the transcriptional complexes including LjMYB proteins is in progress.

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In the former annual meeting held on 2005, we introduced the metabolome pipeline by using GC-TOF/MS. Here we show the application of plant metabolic profiling by using our developed pipeline.
The aerial parts and roots of wild-type (Col-0) and well-studied mutants (mto1-1 and tt4) were harvested, and subsequently the obtained samples were extracted, derivatized and then analyzed by using our GC-TOF/MS-based metabolic profiling pipeline. Finally, we could acquire 163 annotated peaks (96 peaks as known metabolites) in aerial parts and 166 annotated peaks (97 peaks as known metabolites) in roots, respectively. The behaviors of metabolites detected could be represented in combination with statistical analysis and correlation analysis. The pattern of metabolite accumulation observed in aerial parts of mto1-1 was largely consistent with the findings presented in the previous publications. For tt4, the unique metabolite-metabolite relationships were observed from the result of metabolite-metabolite correlation analysis when compared with those of Col-0.

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