2-Phenylethanol (2PE), main aroma component of rose, is biosynthesized from L-phenylalanine by the action of two enzymes, aromatic L-amino acid decarboxylase and phenylacetaldehyde reductase (PAR). The aim of this study is to elucidate biochemical function of PAR in the 2PE synthesis and latters emission process. First we examined enantio-selectivity of the plant PAR and the E. coli-expressed enzyme. We also analyzed the changes in the epidermal structures of rose petals of Rosa 'Yves Piaget' during the unfurling process, followed by the immunohistochemical analysis of PAR using an antibody against PAR protein. From Western blotting analysis, we confirmed the specificity of the antibody. To investigate the enantio-selectivity of PAR, either pro R or S-[4-^2H] labeled NADPH and phenylacetaldehyde (PAld) were used for the enzymatic reactions. 2PE produced after the reaction was analyzed by GC-MS to determine the ratio of [1-^2H]-2PE/2PE. We also investigated the enantio-selectivity of PAR-catalyzed reaction of ketone. Both E. coli-expressed and plant-derived enzyme enantio-selectively utilized pro-S hydrogen at C-4 of NADPH to yield [1-^2H]-2PE from PAld. We found changes in the epidermal layer by light and electron microscopes during the course of the floral development process of R. 'Yves Piaget'.

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A receptor kinase CLV1 and a secreted peptide CLV3, both of which are expressed in adjacent regions in the shoot apical meristem (SAM), are key components of the regulatory network controlling stem cell renewal and differentiation in Arabidopsis. Several lines of evidence suggest that the CLE domain, which is the only region with similarity among CLE family peptides, is the functional domain of CLV3. However, we have reservations about the proposed structure for the CLV3 functional form, mainly because exogenous application of the 12-amino-acid CLE domain peptide does not fully rescue clv3 mutant phenotypes at physiologically relevant concentrations. We thus assume that mature functional form of CLV3 may have undergone as-yet undiscovered posttranslational modifications critical for their function. Here, we show, by nano-LC-MS/MS analysis of apoplastic peptides of Arabidopsis plants overexpressing CLV3, that active mature CLV3 is a 13-amino-acid arabinosylated glycopeptide. We treated clv3-2 mutant seedlings with purified CLV3 glycopeptide and observed that the clv3-2 SAM treated with CLV3 at 30nM were substantially reduced in size comparable to wild-type levels. We also analyzed the binding affinity of CLV3 glycopeptide to CLV1 receptor kinase and confirmed that CLV3 glycopeptide interacted with the CLV1 ectodomain far more strongly than the non-arabinosylated forms. Collectively, we propose that active mature CLV3 is an arabinosylated glycopeptide.

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To date, any concrete evidence for the occurrence of brassinosteroids in ferns, seedless vascular plants, has not been obtained. In this study we analyzed several species of ferns for the presence of brassinosteroids by using GC-MS. As the results, brassinosteroids were identified from all the ferns examined. As biologically-active brassinosteroids, castasterone but not brassinolide was identified. The biosynthetic precursors of castasterone were also identified, being the same as those in seed plants. The endogenous brassinosteroid profiles of the respective ferns will be discussed by comparing with those in Arabidopsis.

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Genetic studies have revealed that brassinosteroids (BRs) are essential plant hormones for growth and development of seed plants. However, little is known about the occurrence and physiological roles of BRs in ferns and moss. The biosynthesis genes of sterols are highly conserved in lower to higher plants. However, the genome of the moss, Physcomitrella patens comprises no genes with high similarity to BR biosynthesis genes of seed plants. In order to know whether P. patens produces BRs, we analyzed endogenous BRs in the protonema using GC-MS. Furthermore, we also analyzed endogenous animal steroid hormones using the same material although their roles and biosynthesis in plants are unknown. As the result, a small amount of castasterone that is a biological active form in seed plants and a series of the precursors such as 6-dexoxocastasterone were identified in P. patens. In addition, animal steroid hormones including progesterone were also identified. These findings may imply that not only BRs but also animal steroid hormones had been utilized as physiologically active steroids before the branching-off from the common ancestor into moss and seed plants.

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Several cytochromes P450 (P450s) are involved in the biosynthesis of phtohormones in higher plants. Genome sequencing projects of Physcomitrella patents and Selaginella moellendorffii have revealed that some orthologous genes to phytohormone-related P450s from the flowering plants may be present in bryophyte and pteridophyte. We think that the comparative analysis of plant P450s conserved across the evolutionary stages can give us a clue to study the evolutionary aspects of phytohormone homeostasis. In this study, we performed functional analysis of putative brassinosteroid-biosynthetic P450s from P. patents and S. moellendorffii. We have found that CYP763A1 and CYP763B1/B2 from P. patents and CYP90E1/E2/E3 and CYP90F1 from S. moellendorffii may be the orthologs of brassinosteroid-biosynthetic P450s from the flowering plants. We have isolated the full-length cDNAs of the P450s except for CYP90F1 by RT-PCR. The recombinant P450 proteins were expressed in E. coli and with a baculovirusinsect cell system in order to analyze their catalytic activities in vitro. Now, we are looking for their proper substrates among the BR-biosynthesis intermediates and try to reveal the enzymatic reactions catalyzed by them.

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Immunomodulation is a means to modulate an organism's function by antibody production to capture either endogenous or exogenous antigens. We screened single chain antibodies (scFvs) against the extracellular domain of BRI1 (BRI1-LRR), which is known as the binding domain of brassinosteroids, from phage display scFv libraries in order to create plants in which brassinosteroid signaling is impaired by immunomodulation. BRI1-LRR was produced as a fusion protein with GST, and was fixed either on immunotubes by adsorption or on glutathione-Sepharose, against which phages expressing different scFvs, J-1 and J-2, were respectively selected. J-1 showed binding activity to GST-BRI1-LRR fusion protein with clearly lower affinity to GST in ELISA, while J-2 showed higher specificity to GST-BRI1-LRR. In Western blot analysis, J-1, but not J-2, could detect GST-BRI1-LRR, suggesting that J-2 recognizes an epitope formed as a three-dimensional structure of BRI1-LRR.

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Brassinosteroids are signal compounds that are involved in the regulation of plant growth and response to environmental stimuli. To analyze endogenous brassinosteroids, gas chromatography-electron impact ionization-mass spectrometry (GC-EI-MS) has been frequently used. An analytical method for castasterone and brassinolide, known as bioactive brassinosteroids, by liquid chromatography-electronspray inonization-tandem mass spectrometry (LC-ESI-MS/MS) has been reported. In this system the detection limit of these compounds were lower than that in common GC-EI-MS systems. We have previously reported a comprehensive analysis method of bioactive hormones such as auxin (indole-3-acetic acid), cytokinins (trans-zeatin and isopentenyladenine), gibberellins, abscisic acid, jasmonates and salicylic acid by LC-ESI-MS/MS. In addition to these compounds, castasterone and brassinolide were analyzed. We first optimized the ionization condition for castasterone and brassinolide using authentic compounds. We found that negative ion mode showed stable intensity of [M-H]- and this molecular ion gave 3 specific product ions that can be used for quantification. By using this optimized condition, a purified sample from 500mg fresh weight of aerial of 4 week old Arabidopsis plants gave 3 specific product ions of castasterone, and the ratio of intensity of these ions were consistent with those of authentic castasterone. In this meeting, we will report the results of other plant materials.

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Brz (brassinazole) was synthesized as the first specific inhibitor of brassinosteroid biosynthesis. Target of Brz220 is the cytochrome P450 enzyme encoded by DWF4. In order to analyze in detail the mechanisms of brassinosteroid signal transduction, we tried to perform the chemical genetics screening by Brz (brassinazole). Brz caused shorten hypocotyl and opening cotyledon to germination of wild type that was called as photomorphogenesis in the dark. We screened for mutants that showed longer hypocotyls than wild type when grown with Brz220 in the dark, and designated bil mutants (Brz-insensitive-long hypocotyl). We identified a semidominant mutant, bil5, from fast neutron-treated lines. Hypocotyl elongation of these plants on Brz medium was at least twice that of the wild type. Adult bil5 plants showed pale green and thin leaves, and thin and shortened inflorescences. By microscope observation, the palisade cell and spongy cell of bil5 mesophyll tissues were smaller and disturbed in comparison with wild type. Number and size of secondary xylem cell of bil5 inflorescences were decreased. The root elongation of bil5 was also inhibited.

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Brassinosteroids (BRs) are perceived by a cell surface receptor kinase BRI1, and then various components are predicted to be involved to transduce the BR signal in the cell. To identify additional components involved in BRs signal, we had analyzed various dwarf and semidwarf mutants from Arabidopsis mutant, which is insensitive to the BRs synthetic inhibitor Brz. We had identified Brz insensitive mutant bill from EMS-mutation lines, and bil5 from fast neutron-mutation lines. We tried to screen new bil mutants from Arabidopsis activation-tag lines and selected bil4 that showed Brz-insensitive phenotype in the dark. Genetic analysis suggested that the candidate genes of bil4 mutation are a novel protein. Transgenic plants overexpressing the bil4 candidte gene display a slender dwarf phenotype and are insensitive to Brz. From genetic and functional analyses to bil4 mutant, we would like to clarify in detail the mechanism of brassinosteroid signaling transduction.

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We had identified brassinosteroid biosynthesis inhibitor Brz insensitive mutant bill from EMS-mutation lines, and bil5 from fast neutron-mutation lines. To identify additional components of the brassinosteroid signaling, trial to screen new mutants as Brz-insesitive-long hypocotyls in the dark was applied to Arabidopsis activation-tag lines. The semidominant mutant bss1 (Brz-sensitive-short1) showed normally etiolated hypocotyl same as wild type without Brz in the dark. However, with Brz, bss1 hypocotyl became about 50% length of wild type in the dark. In the weak light, hyocotyl of bss1 showed about 50% length of wild type without Brz. These results suggested that bss1 hypocotyl is hypersensitive to Brz and the light. In the later growth stage, bss1 showed extremely dwarf phenotype by inhibition of axis elongation. An activation tag insertion was identified in non-ORF region on bss1 genome. Further analysis is in progress.

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Brassinosteroids are plant steroid hormones work for plant growth and development. We had identified brassinosteroid biosynthesis inhibitor Brz insensitive mutant bill (Brz-insensitive-long hypocotyl 1) from EMS-mutation lines, and bil5 from fast-neutron lines. We tried to screen new bil mutants from Arabidopsis activation-tag lines that showed longer hypocotyls than wild type even with Brz in the dark, and selected semidominant mutant bil3. Bil3 showed slender dwarf phenotype related to excessive epinastic leaves and thin inflorescences in the light. We are now establishing transgenic plants that overexpress the candidate genes to identify relation between the genes and bil3 mutants. In the comparison these mutants with mutation genes, we would like to clarify in detail the mechanism of brassinosteroid signaling transduction.

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Brassinosteroids (BRs) are involved in many developmental processes. However, little is known about the BR signal transduction and response network in monocots. To identify novel BR-related genes in rice, we monitored the transcriptomic response of the brassinosteroid deficient1 (brd1) mutant, with a defective BR biosynthetic gene, to brassinolide treatment. Here, we describe a novel BR-induced rice gene BRASSINOSTEROID UPREGULATED1 (BU1, previously called OsBU3), encoding a helix-loop-helix protein. Rice plants overexpressing BU1 (BU1: OX) showed enhanced bending of the lamina joint, increased grain size, and resistance to brassinazole, an inhibitor of BR biosynthesis. In contrast to BU1:OX, RNAi plants designed to repress both BU1 and its homologs displayed erect leaves. This year, we showed that the induction of BU1 by exogenous brassinolide did not require de novo protein synthesis and it was weaker in a BR receptor mutant OsbriI (d61) and a rice G protein alpha subunit (RGA1) mutant d1, compared to the wild type. These results indicate that BU1 protein is a positive regulator of BR response: it controls bending of the lamina joint in rice and it is a novel primary response gene that participates in two BR signaling pathways through OsBRI1 and RGA1. Furthermore, expression analyses showed that BU1 is expressed in several organs including lamina joint, phloem, and epithelial cells in embryos.

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Antheridium of L. japonicum is known to be induced by GA methyl ester. We earlier found that progesterone strongly inhibits GA4-Me induced antheridium formation. This paper reports that IAA and ABA strongly inhibited antheridium formation of L. japonicum. IAA was more biologically active than ABA and progesterorone, 3 times and 14 times, respectively. Protonemal elongation was enhanced by IAA but inhibited by ABA. No biological activity of brassinosteroids, brassinolide and castasterone, was observed in this biological system.

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Strigolactone released from plant root stimulates germination of root parasitic plants and induces hyphal branching of arbuscular mycorrhizal fungi in the rhizosphere. In plant, strigolactone works as a plant hormone inhibiting shoot branching. It was already reported that phosphorus (P) deficiency promoted but P fertilization suppressed strigolactone exudation. In the present study, we demonstrated that auxin increased strigolactone exudation when rice plants were subjected to P fertilization, while cytokinin decreased strigolactone exudation when rice plants faced to P deficiency.

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Orobanche minor seedlings were attached onto red clover (Trifolium pretense) roots grown in rhizotrons. Approximately 30% of the O. minor seedlings formed tubercles on red clover roots 2 weeks after attachment. Root primordia were appeared on the surface of the tubercles, and crown roots were developed 19 days after attachment. A shoot bud differentiated from crown roots 5 weeks after attachment with or without the cotyledon of O. minor. O. minor parasitism induced little morphological changes in red clover roots during the development process from tubercle formation to shoot differentiation. These observations suggest that O. minor produces growth regulators in its own metabolism to control its differentiation. In addition, the crown roots and the shoot bud differentiated without the cotyledon of O. minor, suggesting the developmental process seems to be the process of adventitious root and shoot formation.

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Effects of rhizobial symbiosis on Orobanche aegyptiaca parasitism to Lotus japonicus were studied. Approximately 30% of the parasite attached onto the host root formed tubercles, and root primordia developed from the tubercles. Concurrent inoculation of Mesorhizobium loti doubled the number of tubercles, suggesting some positive effects of rhizobial symbiosis on the parasite invasion. Inoculation of the rhizobium three weeks before attachment of the parasite did not affect the number of tubercles. However, the tubercles turned brown, and root development of the parasite delayed significantly, suggesting the negative effects of the symbiosis on the parasite growth.

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Strigolactones are multifunctional plant molecules that induce the germination of parasitic weed seeds, trigger acteristic hyphal branching of arbuscular mycorhizal fungi, and inhibit the shoot branching in plants. The structural core of strigolactones consists a tricyclic-lactone (the ABC ring) and a butyrolactone group (the D-ring) that are connected via an enol ether bridge. By using structural analogs of strigolactones, the structural requirements for seed germination of parasitic weeds have been thoroughly investigated. On the basis of these structure-activity relationship data, it is conclusion that the bioactiphore of strigolactones resides in the CD part of the molecule. In contrast, the structural requirement for shoot branching in plants has not been fully cleared. For acquisition of the detailed information in plants, we are preparing a library of structural analogs of strigolactones. Our presentation will introduce some analogs with modified the ABC-ring.

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The seeds of root parasitic plants Orobanche spp. and Striga spp. only germinate when they are exposed to stimulant molecules, collectively referred to as strigolactones. In this study, strigolactones and their synthetic analogs with modification at C-4 were evaluated for their potency in inducing seed germination of S. hermonthica, S. gesnerioides, and O. minor. We prepared three synthetic analogs, GR24, OH-GR24 having a hydroxy group at position C-4, and AcO-GR24 having an acetoxy group at C-4, and these compounds were used for the seed germination assay. In the S. hermonthica germination assay, the activity was GR24>OH-GR24 and AcO-GR24. In the O. minor germination assay, the activity was OH-GR24>GR24 and AcO-GR24. These results suggest that the modification at C-4 is an important factor to determine the activity and the specifity of strigolactones for the parasites. In contrast, S. gesnerioides seeds did not respond to any of the synthetic strigolactones while the seeds were responsive to cowpea root exudates that contain orobanchyl acetate as a stimulant. Although it is known that the CD-ring structure of strigolactones is essential for their bioactivity, the results obtained in this study suggested the importance of the A-ring structure for the bioactivity toward S. gesnerioides.

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Strigolactones are host recognition signals for arbuscular mycorrhizal fungi and root plasitic plants, such as Striga and Orobanche. They also inhibit shoot branching in host plants. However, the structural requirements of strigolactones for shoot branching inhibition have not yet been elucidated. Thus, we evaluated the structure-activity relationship of various strigolactone analogues in shoot branching inhibition. 5-deoxystrigol (5DS) is a natural strigolactone in rice. Among 5DS stereoisomers, (+)-5DS and (-)-2'-epi-5DS, whose C2' configuration is R, showed stronger activity than the 2'S-isomers. In rice cv. Shiokari, (-)-2'-epi-5DS was detected as a major endogenous strigolactone. Likewise, (+)-GR24 and (-)-2'-epi-GR24 showed stronger activity among the four stereoisomers of GR24. Therefore, the R configuration at C2' is crucial for the biological activity in shoot branching assays. We also found that 3,6'-dihydro GR24 was inactive, suggesting that the double bond in the enol ether bridge connecting the C and D rings is essential for shoot branching inhibition in rice.

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Shoot branching plays an important role in plant morphogenesis. Previous studies have suggested that the two classical hormones, auxin and cytokinin, play key roles in controlling shoot branching. Recently, it has been reported that strigolactones (SLs) act as a novel hormone in shoot branching, and many efforts to discover these hormone interactions have been made. In this study, we examined the effects of many hormones, and discovered some hormone interactions in shoot branching.

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Strigolactones are plant secondary metabolites that function as host recognition signals for arbuscular mycorrhizal fungi and root parasitic plants, Striga and Orobanche. Recently, it has been reported that strigolactones act as a novel hormone in shoot branching. In this study, we try to design a strigolactone biosynthesis inhibitor. Strigolactones are synthesized from carotenoids, and in a proposed strigolactione biosynthesis pathway, carotenoid cleavage dioxygenase 7 (CCD7) and CCD8 play an important role. 9-cis-Epoxycarotenoid dioxygenase (NCED) is the most characterized CCD in plants, and a couple of NCED inhibitors including abamine and abamineSG were reported from our group. In this context we estimated that some of NCED inhibitors or their derivatives could inhibit strigolactone biosynthesis and we started to screen our chemical libraries targeted to NCED (CCD). In consequence we found that the treatment of abamine reduced the level of 5-deoxystrigol in root exudates from rice plants, while the treatment of abamineSG, a specific and potent inhibitor of NCED, did not reduced the level of 5-deoxystrigol. Now we continue screening strigolactone biosynthesis inhibitors and try to elucidate structure-activity relationship of abamine and its derivatives.

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At the previous meeting, we reported that Physcomitrella patens subsp. patens, a bryophyte which is evolutionary most far from angiosperm, produces strigolactones (SLs). However, we often failed to detect SLs in P. patens grown in liquid culture. In this study, we collected only chloronema and analyzed SLs. The chloronema were obtained from 1-week old cultures of both WT and the CCD8 loss-of-function mutant (Ppccd8), and extracted with ethyl acetate. Characterization of SLs in these extracts was conducted by comparing retention times of germination stimulants on reversed phase HPLC with those of synthetic standards and by using LC-MS/MS. Both WT and Ppccd8 were found to produce SLs including 7-oxoorobanchyl acetate, 7α-hydroxyorobanchyl acetate, and orobanchyl acetate at similar levels. These results indicate that CCD8 homolog in P. patens would be redundant in the chloronema.

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Strigolactones (SLs) function in the rhizosphere as host detection signals for both symbionts arbuscular mycorrhizal (AM) fungi and root parasitic plants, and in the plants they act as a class of plant hormones inhibiting shoot branching. In this study, characterization of SLs produced by rice (Oryza sativa L. cv. Nipponbare) was conducted by comparing retention times of germination stimulants on reversed phase HPLC with those of synthetic standards and by LC-MS/MS. In the roots exudates collected from rice plants grown hydroponically, known SLs including 7-oxoorobanchyl acetate, 2'-epiorobanchol, orobanchol, orobanchyl acetate, and 2'-epi-5-deoxystrigol were detected. An unknown peak was detected in the channel for 7-oxoorobanchol (m/z 383>286) with distinct germination stimulation on Orobanche minor and Striga hermonthica seeds. LC-MS/MS and GC/MS analyses indicated that this is a novel SL. Purification and structural elucidation of the novel SL will be presented.

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Strigolactone are plant secondary metabolites which function as host recognition signals for arbuscular mycorrhizal fungi and root parasitic plants, Striga and Orobanche. Recently, strigolactones have been identified as a novel class of plant hormones inhibiting shoots branching. In this study, characterization of strigolactones in cucumber (Cucumis sativus L.) root exudate was conducted. Cucumber was found to produce at least 9 germination stimulants including orobanchol, orobanchyl acetate, 7-oxoorobanchyl acetate, and 7-oxoorobanchol. In addition to these, two novel strigolactones were isolated and their structures were determined as 7α-hydroxy-orobanchyl acetate and 7β-hydroxy-orobanchyl acetate by 1D and 2D NMR spectroscopy, CD, and ESI- and EI-MS spectrometries.

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Strigolactones (SLs) have been known as germination stimulants of root parasitic plants and hyphal branching factors of symbiotic arbuscular mycorrhizal fungi. Recent studies suggest that they act as a new plant hormone and regulate shoot blanching, however functions of SLs in other stages of development and the regulation of SL-related gene expressions are mostly unknown in Arabidopsis. Analyses of SL-responsive and MAX2 downstream gene candidates provide a clue to broaden our understanding of SL signaling. We previously identified some SL-responsive genes by using real-time RT-PCR and microarray analyses. We also showed MAX2 plays an important role in the expression of those genes and the feedback regulation of MAX3 and MAX4. In this study, we over-expressed SL-responsive genes in Arabidopsis and analyze those mutants phenotype. We also performed yeast two-hybrid screening to identify the target of F-box protein, MAX2. We isolated some proteins that seem to interact with MAX2 Leu rich repeat.

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There is a large cultivar difference in ethylene synthesis during fruit ripening in Japanese pear. Fruit storage potential is closely related to maximum level of ethylene production in Japanese pear. PPACS2 was specifically expressed in cultivars showing moderate ethylene production. We have found that PpACS2 expression can be controlled by regions comprising purine-rich (GAGA repeats) sites that are located near TATA box. PpACS2 promoter of moderate ethylene producers has either (GA)_<14> or (GA)_<10>. On the other hand, that of low ethylene producers has only (GA)_5. Recently, genes have been identified that encode a class of proteins (GABP) that binds GA-rich elements in three plant species. To identify factors that interact with the promoter regions of PpACS2, southwestern screening and 5' and 3' RACE methods following RT-PCR with degenerate primers were performed. We cloned two possible GABPs (PpRTF1 and PpGABP2) and studied their expression during fruit development and ripening. To test whether both genes are able to bind to GA repeats, eletcromobility shift assays (EMSAs) were performed. PpRTF1 protein has shown to bind (GA)_<14>, but PpGABP2 has not. Furthermore, PpRTF1 protein has the ability to bind nucleotides consisting of (GA)_<10>, but no binding activity of nucleotides consisting of (GA)_5. These results suggest that PpRTF1 protein can be involved in the regulation of PpACS2 expression.

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Seed dormancy, defined as the failure of intact seed to germinate in the presence of water and light, is thought to be an important adaptive trait that determines geographical distribution of many plant species. In Arabidopsis and many crop species, seed dormancy is imposed during seed maturation and released by various environmental factors, such as after-ripening (dry storage of matured seed), cold stratification, and nitrate treatment. It is thought that these signals control seed dormancy and germination by regulating sensitivity and metabolism of phytohormones, such as abscisic acid and gibberellins. In this study, we assessed the effect of various environmental factors on seed dormancy, especially embryo dormancy, using hyper-dormant accessions of Arabidopsis. Interestingly, it was found that strong embryonic seed dormancy, which can be released by application of a specific bioactive compound, is imposed under certain conditions. In the next meeting, we will discuss about the role of phytohormone metabolisms in the embryo dormancy induction in these dormant accessions.

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Seed germination is regulated by many environmental signals to complete this critical event exactly. We haven't seen germinated seeds in a fruit. This may be due to seed dormancy, lack of oxygen or available water. But it is assumed that chemical(s) which exist around seeds are also involved. Using extracts of watermelon [Citrullus lanatus (Thunb.) Matsum and Nakai)] flesh, bioassay-guided purification led to three fractions which possess inhibitory activity. Structural analysis following more separation identified six compounds: abscisic acid, 4-hydroxybenzoic acid, indole-3-carboxylic acid, abscisic acid glucose ester, abscisic alcohol glucoside and dehydrodiconiferyl alcohol glucoside. Among these, abscisic acid has very strong activity at a concentration equal to that of watermelon flesh.

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Uniconazole (UNI), which is well known as a plant growth regulator, is a triazole-containing inhibitor of GA biosynthetic enzyme (CYP701A). UNI is a low selective P450 inhibitor that inhibits multiple P450 enzymes including abscisic acid (ABA) 8'-hydroxylase (CYP707A), which is a key enzyme for ABA catabolism. Based on our speculation that the low selectivity of UNI may be resulted from its small molecule, we designed and synthesized enlarged UNI analogues of UNI (UT) that have a 1,2,3-triazolyl alkyl chain at C4 of chlorophenyl group of UNI. Although UT compounds showed the strong inhibitory activity against CYP707A, the biological activity of most of UT compounds, especially those with a long alkyl chain (>C4), was not tested because of their water-insolubility. On the other hand, the introduction of protic functional groups in the alkyl chain diminished the inhibitory activity against CYP707A. Thus we designed and synthesized UT1-E2Ts that has a diethylene glycol chain as a novel, water-soluble UT analogue, and examined its water solubility, inhibitory activity against CYP707A, and biological activity. UT1-E2Ts showed strong inhibitory activity against CYP707A and good water-solubility, equivalent to UNI, whereas it did not inhibit the growth of rice seedlings, contrary to UNI.

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Uniconazole (UNI), which is well known as a triazole-containing inhibitor of GA biosynthetic enzyme (CYP701A), functions as inhibitors of multiple P450 enzymes including ABA 8'-hydroxylase (CYP707A), which is a key enzyme for ABA catabolism. Azole-containing P450 inhibitors bind to a P450 active site by both coordinating to the heme-iron atom via sp2 nitrogen and interacting with surrounding protein residues through the lipophilic region. We speculated that poor selectivity of UNI may be resulted from its small and flexible conformation adjustable for a variety of substrate pockets. Based on this speculation, we designed and synthesized novel UNI analogues with a disubstituted azole ring (DSI). These analogues were expected to have higher selectivity than UNI because the added functional group may have an interaction with the active site to increase a rigidity of the molecule in the active site. DSI-505M, which has an imidazolyl group with a methyl 5-acrylate at C2, strongly inhibited recombinant CYP707A3, whereas it did not inhibit the growth of rice seedlings.

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A plant hormone abscisic acid (ABA) protects plants from water stress, so suppression of its metabolic inactivation could increase resistance of plants against water stress. ABA is metabolized to 8'-hydroxy-ABA by ABA 8'-hydroxylase, which spontaneously isomerizes to phaseic acid (PA). PA is converted to dihydrophaseic acid (DPA) and epi-DPA by PA 4'-reductase. 8'-Hydroxy-ABA and PA still have biological activity although it is low, but DPAs are almost inactive. The gene of ABA 8'-hydroxylase was identified in Arabidopsis, and its inhibitors have been developed. However, research on PA 4'-reductase has not progressed. The objective of this research is identification of PA 4'-reductase gene, and development of its inhibitor. We have already found that the extract of immature garden pea (Pisum sativum) showed the PA 4'-reductase activity. After centrifugation of the extract, the supernatant was purified with an anion exchange resin. The activity was eluted with a buffer of pH 8.0 containing 50mM NaCl. The active fraction was purified on a 2'5'ADP-Sepharose 4B. The activity was eluted by a buffer of pH 8.0 containing 1mM NADP^+. The specific activity was increased to 137-fold. The fraction was purified on Sephadex G-75. The SDS-PAGE analysis of the active fraction showed several bands after silver staining. Further purification of PA 4'-reductase is under progress.

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During seed development, abscisic acid (ABA) is involved in the accumulation of storage compounds, acquisition of desiccation tolerance and induction of dormancy. To understand the regulation of ABA metabolism and the mode of ABA actions, we conducted precise quantification analysis of ABA and its metabolites during Arabidopsis seed development. During middle stage of seed development, ABA was actively synthesized in the tissue derived from maternal origin (seed coat), and ABA synthesized in this tissue appeared to be transported to the embryo. In later stage, ABA accumulated in silique envelopes and to a lesser extent in seeds. ABA accumulated in seeds in this stage was dominantly synthesized in embryos. The levels of ABA metabolites fluctuated according to the changes in ABA levels during seed development. Accumulation of ABA metabolites appeared to be regulated differentially depend on metabolic pathways. Recent studies indicate that hormones interact at the level of both metabolism and signaling. Therefore, it is required to analyze possibly all hormones in order to understand hormonal regulation of physiological processes of plants. We are now conducting precise quantification of gibberelliens, auxin (indole-3-acetic acid), cytokinins, jasmonates and salicylic acid in the context of seed development.

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We have already reported that chlorophyll contents in cucumber plants increased by the application of ABA under low nitrogen conditions. In this study, we investigated the regulation of gene expression for synthesis and degradation of chlorophyll in cucumber plants under low nitrogen conditions by semi-quantitative RT-PCR. The amount of transcripts of genes for chlorophyll biosynthesis, glutamyl-tRNA reductase (GR), Mg chelatase D subunit (Mg-D) and Mg chelatase H subunit (Mg-H), reduced in older cucumber plants grown without ABA. The levels of transcripts of genes for chlorophyll degradation, chlorophyll b reductase (NYC), pheophytinase (PPH), pheophorbide a oxygenase (PAO) and red chlorophyll catabolite reductase (RCCR), increased in older plants without ABA. On the other hand, the levels of transcripts of genes for chlorophyll synthesis were almost same in cucumber plants cultivated for 10 and 20 days in presence of ABA 10-5M. The amounts of transcripts of genes for chlorophyll degradation in cucumber plants with ABA 10-5M were smaller than that in drug free plants. These results suggest that ABA promotes chlorophyll biosynthesis and suppress chlorophyll degradation in cucumber plants under low nitrogen conditions.

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Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is effective against a broad range of pathogen. SAR development in dicotyledonous plants such as tobacco and Arabidopsis is mediated by salicylic acid (SA). Here, using different types of SAR-inducing chemicals, i.e., 1, 2-benzisothiazol-3(2H)-onel, 1-dioxide (BIT) and benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (BTH) that act on upstream and downstream of SA in the SAR signaling pathway, respectively, we show that treatment of abscisic acid (ABA) suppresses the induction of SAR in Arabidopsis. In our previous studies, it was indicated that ABA suppressed PR genes expression and SA accumulation induced by BIT or BTH. Moreover, ABA suppressed PR genes expression in BTH-treated sid2 mutant, defective in SA biosynthesis, suggesting that ABA suppressed both upstream and downstream of SA in SAR signaling pathway. To clarify SAR suppression mechanism of SAR by ABA, we performed microarray analysis.

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RSOsPR10 (Root Specific Oryza sativa Pathogenesis-Related 10) was identified as the root specific protein induced by drought and salt treatment in rice. The RSOsPR10 shows high similarity to PBZ1/OsPR10a, a PR10 protein induced by probenazole. Expression of RSOsPR10 is regulated at the transcriptional level, and stress-dependent RSOsPR10 expression is induced by ethylene and suppressed by salicylic acid. Since RSOsPR10 expression was induced by exogenously applied jasmonic acid (JA), up-regulation of RSOsPR10 is supposed to be mediated through ethylene-JA closs talk. When RSOsPR10 was over-expressed, the transformed rice showed higher tolerance to drought stress than the wild-type. These suggest that RSOsPR10 acts as an important molecule for stress responses. In the study, we conducted promoter analysis to illuminate mechanisms in regulation of RSOsPR10 expression. Transformant expressing GFP under 0.6 or 1.9kb promoter region of RSOsPR10 was prepared and the expression profile of GFP was observed. In the transgenic plants without stress treatment, the GFP signal was detected in their root and shoot. Moreover, level of GFP signal did not change even after salt-treatment. These suggested that regulatory elements for root-specific expression and stress responsibility are located beyond 1.9kb of RSOsPR10 promoter. We are currently investigating the 3 and 4kb promoter region, in which two possible ethylene responsive elements exist.

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In this study, we investigated roles of JA in phytoalexin production using the JA-biosynthetic rice mutant cpm2 or the JA-Ile-biosynthetic rice mutant jar1. First we examined phytoalexin production in CuCl_2-treated mutant leaves. The production of the diterpenoid phytoalexins in cpm2 was comparable to that in a wild-type. On the other hand, the production of sakuranetin, a flavonoid phytoalexin, was suppressed in cpm2, and the suppression was restored by the addition of JA. Next, we examined phytoalexin production in Magnaphorthe oryzae infected leaf sheaths. Upon M. oryzae infection production of both diterpenoid phytoalexins and sakuranetin was severely suppressed in cpm2. These data suggest that, under CuCl_2 treatment, JA is essential for the production of sakuranetin, but not for diterpenoid phytoalexins. On the other hand, upon M. oryzae infection, JA is essential for production of both diterpenoid phytoalexins and sakuranetin. We also analyzed phenotypes of rice jar1 mutant defective in JA-Ile production to extend understanding of roles of jasmonates in phytoalexin production. We examined phytoalexin production by JA or JA-Ile treatment in jar1 leaves. Production of both diterpenoid phytoalexins and sakuranetin by JA treatment was suppressed in jar1. On the other hand, production of these phytoalexins by JA-Ile treatment in jarl was comparable to that in wild type. These data suggest that JA-Ile was essential for JA-induced phytoalexin production in rice.

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WRKY proteins are a large family of plant-specific transcription factors, and they have been suggested to have a regulatory function in the response to pathogen infection and other stresses. OsWRKY53 is a rice WRKY gene identified by microarray analysis as a chitin oligosaccharide elicitor-induced gene. Previously we indicated that OsWRKY53 functions as a transcriptional activator, and overexpression of OsWRKY53 resulted in enhanced resistance to the rice blast fungus Magnaporthe oryzae in rice plants. To identify direct target genes of OsWRKY53, we generated transgenic rice cells which constitutively express an OsWRKY53-3xHA fusion protein, and established an experimental system for chromatin immunoprecipitation (ChIP). Next, we performed ChIP-on-chip analysis using a custom promoter array which covers promoter regions of chitin oligosaccharide elicitor-induced genes in rice. As the result of ChIP-on-chip analysis, we identified several genes as the candidates of the direct target genes of OsWRKY53. These genes included transcription factors and PR protein genes. Moreover, we performed transcriptome analysis using rice cells overexpressing a dominant-nagative OsWRKY53 mutant. As the result, the expression levels of the several candidate genes decreased in the transgenic rice cells treated with elicitor compared with the similarly treated wild-type rice cells. These results strongly suggest that these selected genes are the direct target genes of OsWRKY53.

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Momilactones and phytocassanes are major diterpenoid phytoalexins, which are produced in elicitor-treated rice cells. We recently showed that biosynthetic genes for momilactones (OsCPS4, OsKSL4, CYP99A2, CYP99A3, OsMAS) are clustered on rice chromosome 4, and that those for phytocassanes (OsCPS2, OsKSL7, CYP71Z7) are clustered on rice chromosome 2. Moreover, we reported that an elicitor-inducible bZIP transcription factor, OsTGAP1, is essential for momilactone biosynthesis and regulates the expression of the five genes in the cluster. Here we report OsTGAP1 is involved also in the elicitor-inducible expression of methylerythritol phosphate (MEP) pathway genes (OsDXS3, OsDXR, OsCMS) and phytocassane biosynthetic genes (OsCPS2, OsKSL7). Inductive expression of OsCPS2 and OsKSL7 after elicitation seen in wild-type cells was weakened in ostgap1 mutant, although phytocassane accumulation still occurred. On the other hand, OsTGAP1 over-expressing lines exhibited enhanced expression of OsCPS2 and OsKSL7 and hyper-accumulation of phytocassanes in response to the elicitor. We also found that the elicitor-induced expression of OsDXS3, OsDXR and OsCMS, was significantly suppressed in the ostgap1 mutant and that OsTGAP1 over-expression exhibited enhanced expression of OsDXS3, OsDXR and OsCMS. These results indicate that OsTGAP1 functions as a key regulator of the coordinated transcription of genes involved in the elicitor-induced diterpenoid phytoalexin production in rice.

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Antimicrobial diterpenoid compounds such as momilactones and phytocassanes are a major phytoalexin produced in rice upon pathogen attack. Recently, we reported that the momilactone biosynthetic genes are clustered in rice chromosome 4 and are coexpressed after the elicitation. In this report, we demonstrate that a novel gene cluster for phytocassanes biosynthesis is organized in rice chromosome 2, which consists of two diterpene cyclases genes for phytocassanes (OsCPS2 and OsKSL4) and six uncharacterized cytochrome P450 monooxygenases (P450) genes. Using genetic approaches, we first investigated functions of the CYP71Z7 gene, one of the P450 genes in the middle of the cluster. The knockdown of CYP71Z7 specifically suppressed the elicitor-inducible production of phytocassanes A, B, and D (C-2 oxygenated phytocassanes), whereas phytocassanes C, E, and the metabolic intermediate 1-deoxyphytocassane C (C-2 non-oxygenated phytocassanes) were detected to be more than 10 times accumulated to wild-type plant. These results suggest that the CYP71Z7 probably catalyzes C-2 hydroxylation of possible metabolic substrates phytocassane C and/or 1-deoxyphytocassane C to yield phytocassanes A, B, and D, and that phytocassanes biosynthetic genes are clustered in chromosome 2 like the momilacone biosynthetic gene cluster. We are now beginning to understand functions of remaining P450 genes (CYP76M5, M6, M7, M8) in the phytocassans biosynthetic gene cluster by generating their RNAi lines.

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Rice leaves accumulate tryptophan derived secondary metabolites including serotonin and its hydroxycinnamic acid amides in response to pathogen attack. These compounds have been implicated in the physical defense system against pathogen invasion by deposition in the cell walls. Serotonin is biosynthesized from tryptophan via tryptamine, and tryptophan decarboxylase (TDC) catalyzes the first committed reaction. (S)-α-(Fluoromethyl) tryptophan (S-αFMT) was utilized to investigate the effects of inhibition of TDC on the defense responses of rice leaves. S-αFMT was enatiospecifically synthesized from L-tryptophan. S-αFMT effectively inhibited TDC activity extracted from rice leaves infected by Bipolaris oryzae. The treatment of rice seedlings with S-αFMT suppressed the accumulation of serotonin in a dose-dependent manner in the B. oryzae-inoculated leaves. The lesions formed on the leaves treated with S-αFMT lacked deposition of brown materials, and those leaves were severely damaged in comparison with the leaves that were not treated with S-αFMT. The feeding of tryptamine to S-αFMT-treated seedlings restored the accumulation of tryptophan derived metabolites as well as deposition of brown materials. In addition, the tryptamine feeding reduced the damage caused by fungal infection. Accordingly, the accumulation of tryptophan derived metabolites was suggested to be a part of effective defense responses of rice.

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Phytoalexin are low-molecular-weight compounds produced and accumulated after exposure to microorganism attack, elicitor and ultraviolet, and have been suggested to serve as plant antibiotics. Rice (Oryza sativa) produces 14 diterpenoid phytoalexins, including oryzalexins A-F, phytocassanes A-E, oryzalexin S, and momilactones A and B. We have identified 6 diterpene cyclase genes responsible for biosynthesis of these phytoalexins: OsCPS2, 4, OsKSL4, 7, 8, and 10. In this study, we attempted to produce diterpenoid-phytoalexin-deficient rice plants by knock down of OsCPS2/4, OsKSL4, or OsKSL7 to verify the roles of these phytoalexins on disease resistance in rice. We first found one line of rice plant in which Tos17 was inserted in the 3rd intron of OsCPS4 by PCR screening of Tos17 mutant panel. However, the level of the mature OsCPS4 mRNA in the line slightly lower than that in wild type rice, suggesting that Tos17 in the 3rd intron of OsCPS4 was excluded by splicing. This result showed that this line is not a null mutant for OsCPS4. Next, we prepared transgenic rice, in which each RNAi trigger (approximately 100bp) for OsCPS2/4, OsKSL4, and OsKSL7 was expressed under the control of rice ubiquitin promoter. However, specific knock down of the targeted genes were not observed in the transgenic rice plants. We have made again transgenic rice plants in which approximately 200bp trigger sequences were introduced, and will present the transcript levels in the transgenic plants.

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Hot water (50-60℃) treatment is expected as a new technology to control diseases of greenhouse-grown strawberry. It has been suggested that hot water treatment should induce disease resistance of plants by heat shock reaction. In this paper, we studied the induction of resistance against anthracnose fungus (Colletotrichum gloeosporioides) by hot water treatment in strawberry leaves. Strawberry leaves were dipped in hot water (50℃) for 20s. After one-day incubation, C. gloeosporioides was inoculated. After 7 days, clear disease symptoms appeared in control leaves. However, almost no disease symptoms were observed in hot water-treated leaves. After hot water-treated leaves were injured, 25μL of distilled water was put on the wound. The water was collected after one-day incubation. The inhibitory activity of collected water from hot water-treated leaves against C. gloeosporioides spore germination was higher than that from control leaves.

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For the purpose of estimating heat shock as a protection activator, the aerial portion of seedlings of Fragaria×ananassa Duch. cv. Tochiotome were dipped in water at various temperatures. Salicylic acid accumulation and the expression of defense related genes after the heat shock treatment were examined. The results indicated that treatment at 50℃ was most effective, accompanying the accumulation of salicylic acid and mRNA of defense related genes. These results suggested that the heat shock treatment induced systemic acquired resistance in strawberry plants.

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Systemic acquired resistance (SAR), one of plant defense systems, is induced by pathogen infection through salicylic acid (SA) accumulation. SAR has been well characterized and set of pathogenesis-related (PR) genes has been identified as SAR marker genes. The induction of disease resistance by high temperature treatment has been reported in cucumber, in which SA is likely to take part. To investigate the detailed mechanism of this resistance, effects of high temperature treatment in Arabidopsis were examined. The treatment induced the expression of PR genes, the accumulation of SA, and disease resistance to Pseudomonas syringae pv. tomato DC3000. The High temperature treatment induced the expression the isochorismate synthase 1 (ICS1) gene, encoding an SA biosynthetic enzyme. Analyses using the sid2 (SALICYLIC ACID INDUCTION DEFICIENT2) mutant defective in ICS1 and NahG transgenic plants expressing SA degrading enzyme, indicated that high temperature-induced disease resistance required SA biosynthesis.

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Several iridoid glucosides found in several Oleaceae species such as the olive tree, privet tree and so on. Iridoid glucosides have iridoid group and glucose group. The Olive tree (Olea europaea) is a species of small tree in Oleaceae species, and olive fruit is of major agricultural importance in the word as the source of olive oil. Olive leaves and fruits contain many kinds of iridoid glucosides. However, this compound itself is stable, does not have any of the activities, and is kept in the vacuoles or cytosol of the leaf cell. Several researches reported that iridoid glucoside would be chemical defense compound in Oleaceae species. When the leaves are mechanically damaged by herbivore like olive weevil and cell compartments are broken, enzymatic activity localized in organelles separate from iridoid glucosides starts to activate iridoid glucosides into a very strong protein denaturant. Nevertheless the importance of iridoid glucosides, it has not yet elucidation "HOW" "WHERE", "WHEN" iridoid glucosides biosynthesized in Oleaceae species like olive tree. To investigate the role of iridoid glucosides in chemical defense, we first started to analyze endogenous levels of iridoid glucosides with/without an olive weevil, and isolated a β-D-glucosidase from olive tree. We demonstrate that biochemical function of β-D-glucosidase the chemical analysis of iridoid compounds in chemical defense.

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In recent research for bioactive substances of Arabidopsis thaliana, seven new oxylipins named arabidopsides A〜G were isolated from the aerial parts of this plant. Arabidopsides A〜G are monogalactosyl diacylglycerides or digalactosyl diacylglycerides containing 12-oxophytodienoic acid and/or dinor-oxophytodienoic acid which were known as precursors of jasmonic acid (JA) and have received much attention because they play important roles in regulation of developmental and defense gene expression of plants as JA and methyl jasmonate. On the other hand, it is known that plant volatile compounds like C6 aldehydes, alcohols, and acetates are released from plants in response to mechanical wounding and herbivore attack. In this research, we investigated the effect of volatile compounds as potent inducers for triggering accumulations of arabidopsides A, B, E, and G in mature leaves of A. thaliana. In addition, volatiles-induced arabidopsides were not detected in the coi 1-1 mutant. These results indicate that volatiles-induced arabidopsides are concerned in the mechanism of stress response through COI 1 signaling in A. thaliana.

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Samanea plants close their leaves in the evening, as if to sleep, and open them in the morning according to the circadian rhythm. Potassium β-D-glucopyranosyl-12-hydroxyjasmonate (1) was isolated as leaf-closing factor (LCF) of S. saman. We developed molecular probes consisting of modified LCF1 in order to identify its mode of action. We have already demonstrated that a specific binding protein is involved in the motor cell of S. saman. We synthesized natural-type photoaffinity probe and biologically inactive enantiomer-type probe. We utilized them for photoaffinity labeling of the target protein for LCF1. By using protoplasts of motor cell, we found membrane protein of 38kDa which strictly recognizes the stereochemistry of 1, and it is highly likely that the protein is the target protein for LCF 1. Recently, we observed that LCF shrank motor cell protoplasts prepared from S. saman. And comparing the results of several bioassay using glucosyl jasmonate-type LCF and jasmonic acid, it is also interesting that the mode of action of LCF is completely different with that of jasmonate.

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Jasmonic acid (JA) is a plant hormone that plays important roles for a large number of processes in stress adaptation and development in vascular plants. The functions and signal transduction of JA in vascular plants have been studied extensively however those in non-vascular plants have been overlooked. The UPLC-MS/MS analysis of JA showed the presence of JA in Physcomitrella patens that is a model plant in mosses. The genomic database and protein homology searches predicted that P. patens would possess two allene oxide synthases, PpAOS1 and PpAOS2, evolved in JA biosynthesis. Recombinant PpAOSs were tried to obtain by overexpression in Escherichia coli. Only recombinant PpAOS1 was obtained as a soluble form. PpAOS1 had AOS activity and showed a pH optimum of approximately pH 6.0 using 13-HPOT as a substrate. PpAOS1 had substrate specificity for 13-HPOT, showing that it was 13-AOS. Semi-quantitative RT-PCR of PpAOSs showed that the expression of PpAOS1 was higher than that of PpAOS2 this indicated that PpAOS1 might operate JA biosynthesis in P. patens predominantly. These data suggest that the octadecanoid pathway also exists in P. patens.

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