The plant hormone, auxin plays a crucial role in many aspects of plant development. Endogenous auxin action was modulated at three steps, auxin metabolism, transport and signaling. Indole 3-acetic acid is major naturally occurring auxin. It has been major issue how this simple molecule elicits profound responses on plant development. Forward and reverse molecular genetics approach using a model plant Arabidopsis have identified fundamental components of auxin signaling and transport, and illustrated the molecular mechanism of auxin action. However, it is still hard to study the physiological role of auxin beyond the model plants. To complement the molecular genetic approach to auxin biology, we have been investigated the chemical probes on auxin signaling and transport. Because chemical probe can potentially overcome the functional redundancy of cognate target proteins and accomplish the regulation of orthologue protein function beyond a species. We have identified auxin signaling inhibitors, yokonolide B and terfestain A by a forward screen of microbial extracts using a transgenic auxin-inducible reporter line. Structure-based drug design approach for auxin analogs identified auxin antagonist specific for TIR1/AFB auxin receptors. We recently developed chemical tools for auxin polar transport, such as caged auxins and competitive auxin transport inhibitors. Our work substantiates the useful chemical tools for plant biology.

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Gibberellic acid (GA) regulates many aspects of plant growth and development, such as promoting germination, stem elongation and flower formation. Hence, GA is used in agriculture as growth-controlling agent, and GA biosynthesis inhibitors are also used as plant growth retardants. Recently chemical biology becomes general, and therefore biologically active chemicals have been recognized as more important tools for analyzing functions in plants. Here we conducted screening with a chemical library to identify GA agonists, anticipating their potency as tools for dissecting mechanisms of plant growth. In our screening we co-applied paclobutrazol, a GA biosynthesis inhibitor, and a chemical from the library in the dark for 6 days, and compared the phenotype with that of plants treated only by paclobutrazol. Finally we found a compound, called 67D, which can recover the phenotypes of Arabidopsis induce by paclobutrazol. Now we are investigating the effects of 67D on plants treated with other plant growth retardants to analyzed the mode of action of 67D. Furthermore we are studying structure-activity relationships of 67D to improve the activity and selectivity.

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The hydroxylation of gibberellin (GA) by GA 2-oxidase (GA2ox), a 2-oxoglutarate dependent dioxygenase, is known to be one of its major inactivation reactions. The regulation of this catabolic step is very important for a fluctuation of endogenous GA in plants, and therefore is a good chemical target to control the gibberellin action. At present, prohexadione is known as a commercially available inhibitor to GA2oxs, however prohexadione has a characteristic that it inhibits other 2-oxoglutarate dependent dioxygenases (2ODD) like GA 3-oxidases. Last year we reported in this meeting that we performed in vitro random screening to obtain novel inhibitors more specific to GA2oxs, and we discovered an inhibitory effect of CBTC to the enzymatic activity of GA2ox2. As a result CBTC promoted both the germination of Arabidopsis seeds and their growth. So in this report, we examined if CBTC inhibits other 2ODD, AtGA2ox1・AtGA2ox6・ATGA2ox8・AtGA3ox1・AtGA3ox4・AtGA20ox3, by using recombinant proteins, and how CBTC and prohexadione inhibit GA2ox2 by binding assay. These results strongly suggest that CBTC specifically inhibits GA2oxs, not GA3oxs, in Arabidopsis. These data well conform with the results obtained in planta assay.

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CYP701A, a gibberellin (GA) biosynthetic P450 enzyme, catalyzes the three step oxidation of ent-kaurene to ent-kaurenoic acid. A selective inhibitor of this enzyme is a promising research tool for chemical genetic studies of GA deficiency. Uniconazole (UNI), paclobutrazol (PAC), inabenfide (IBF) and ancymidol (ANC) have been known as inhibitors of CYP701A. Although their selectivity has little been investigated biochemically, recent researches suggest that UNI, PAC, and ANC are not specific to CYP701A; UNI and PAC act as an inhibitor of CYP707A, a catabolic enzyme of abscisic acid, and ANC inhibits cellulose synthesis. Hence, we launched the development of a novel selective inhibitor of CYP701A. Recently we developed a selective and potent inhibitor of CYP707A, abscinazoles, by screening our library of UNI analogues whose structures are enlarged or conformationally restricted to eliminate structural factors that may cause the low enzyme selectivity. This library may contain a selective inhibitor of CYP701A. We examined inhibitory activities of compounds in the library against recombinant rice CYP701A6 and Arabidopsis CYP707A3 enzymes. Finally we found a conformationally restricted UNI analogue, UFAP2, that strongly inhibited CYP701A6 without exhibiting considerable inhibitory effect on CYP707A3. Here we present the enzyme selectivity, biological activity and water solubility of UFAP2 in comparison with those of other known CYP701A inhibitors.

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The 13-hydroxylation in gibberellin (GA) biosynthesis-the substrate, the enzyme, and the biological significance are still poorly understood. Although recent studies suggest that some CYP714 enzymes catalyze the 13-hydroxylation of ent-kaurenoic acid (KA) or GA_<12>, the biological significance of the 13-hydroxylation remains unclear. The GA receptor, GID1 in Arabidopsis and rice, prefers GA_4 with no hydroxy group at C13 rather than GA_1, the 13-hydroxylated GA. Nevertheless, GA_1 is the major active GA in rice. Why does the 13-hydroxylation exist in GA biosynthesis? Our goal in this study is the development of a selective inhibitor of the 13-hydroxylases in GA biosynthesis as a chemical tool for probing the biological significance of the 13-hydroxylation. We screened inhibitors for Arabidopsis KA 13-hydroxylase, CYP714A, from commercial available azole compounds, and found imazalil (IMZ), which was 30-fold stronger than uniconazole in inhibition of CYP714A. Because IMZ is a fungicide that inhibits fungal CYP51, it is not excluded the possibility that this compound inhibits plant CYP51. Hence, we are constructing the assay system of plant and fungal CYP51 to test the selectivity of IMZ. We are also modifying the structure of IMZ to raise the selectivity.

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S-Uniconazole (UNI), which was developed as an inhibitor of GA biosynthetic enzyme (CYP701A), is a low selective P450 inhibitor that inhibits multiple P450 enzymes including ABA 8'-hydroxylase (CYP707A). Based on our speculation that the low selectivity of S-UNI may be resulted from its small molecule, we developed enlarged UNI analogues to find a selective inhibitor of CYP707A. Finally we found a practical inhibitor of CYP707A, (±)-abscinazole-E2B (Abz-E2B). (±)-Abz-E2B showed strong inhibitory activity (inhibition constant, K_1=36 nM) against CYP707A, which was equivalent to that of S-UNI (K_1=10 nM). The (±)-Abz-E2B-sprayed plants exhibited drought tolerance, stomatal closure, and an increase in the amount of ABA. On the other hand, against CYP701A, Abz-E2B was a poorer inhibitor, and it did not inhibit the growth of rice seedlings, contrary to UNI. In this study, we optically resolved (±)-Abz-E2B and determined the absolute configuration. The K_1 values of S-(-)- and R-(+)-Abz-E2B for CYP707A were 28 nM and 360 nM, respectively. The similar tendency is observed in UNI whose S-enantiomer is more potent than the R-enantiomer. Similarly, in bioassays, Abz-E2B enhanced the effect of ABA in the order of potency: the S-enantiomer, racemic form, and R-enantiomer. Because no side effects were observed by administration of R-enantiomer, the use of the racemic form may be a reasonable alternative option in the field experiments using a large amount of chemicals.

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In the ABA receptor (PYR/PYL/RCAR, PYLs)-ABA complex, ABA intrudes deeply into the ligand pocket of PYLs to be tightly packed by the protein residues. In the solvent-excluded molecular surfaces of some complexes, a narrow tunnel is found in the direction of the C3'-H. This is located on a side of the gate which is closed in accordance with formation of the complex. The crystal structure of PYLs-ABA-protein phosphatases 2C (PP2C) ternary complex shows that this tunnel is covered with PP2C. This indicates that the linear hydrophobic alkyl chain at C3' fits into the tunnel with hydrophobic interactions, and if the chain sticks out over the surface of PYLs, it may interfere with the PYLs-PP2C interaction. Based on this speculation, we designed and synthesized 3'-alkylsulfanilated ABA, ASn, where n is a carbon number of an alkyl chain, for finding an antagonist of PYLs. In seed germination and seedling growth assays using lettuce and Arabidopsis thaliana, ASn acted as an ABA mimic when n<4, whereas an inhibitor of exogenous ABA activity when n>4. AS6 exhibited the antagonistic effect on the PP2C assay and the ABA-responsive transgenic plants (MAPKKK18::GUS) assay, whereas the effect of AS2 was agonistic similarly to ABA. Radish seedlings treated with AS6 wilted more rapidly than the non-treated seedlings. These results suggest that the length of an alkyl chain of ASn modulates an interaction between PYLs and PP2C; ASn acts as the agonist when n<4 and the antagonist when n>4.

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Indole-3-acetic acid (IAA), the most important member of the auxin family, has been reported to play an important role in plant development and growth. TRYPTOPHAN AMINOTRANSFERASE of ARABIDOPSIS 1 (TAA1) is involved in IAA biosynthesis. We reported that L-aminooxy-phenylpropionic acid (AOPP) is an inhibitor of TAA1. However AOPP has side effects, such as inhibition of phenylalanine ammonia-lyase and 1-Aminocyclopropane-l-carboxylic acid synthase (ACS). Here, we developed more stable and specific auxin-biosynthesis inhibitor, KOK1169, using AOPP as a lead compound. We examined the inhibitor activity of KOK1169 for AtACS8. The K, value of KOK1169 is 246 nM, and it was lower than that of AOPP (847 nM). While, the K, value of KOK1169 is higher than that of L-α-(2-Aminoethoxyvinyl)glyeine (AVG), which is a typical inhibitor of the ACS (37 nM). Although KOK1169 showed an inhibitor activity for ACS in enzyme assay, ethylene biosynthesis of Arabidopsis seedling was scarcely affected by KOK1169. Furthermore, the K_i value of KOK1169 for TAA1 is 76 nM, and it was superior to that of KOK1169 for AtACS8. Therefore, we concluded that KOK1169 has the higher specificity for IAA biosynthesis than AOPP. This work was supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN).

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Recently we reported the identification of the auxin-biosynthesis inhibitor, L-aminooxyphenylpropionic acid (AOPP), which blocks L-Tryptophan aminotransferase (PCP (2010) 51 (4): 524-536). We designed and synthesized novel AOPP analogues by modifying functional groups of AOPP and finally identified KOK1169 as more selectivity inhibitor than AOPP by several screening. Thus we examined the effect of KOK1169 treatment on auxin biosynthesis in Arabidopsis and Rice in vivo. The profile of endogenous auxin-biosynthesis related compounds will be reported. This work was supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN).

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Plant hormone indole-3-acetic acid (IAA) has various physiological function, but IAA biosynthetic and signal pathway is still unclear. Also the components mediating the crosstalk between IAA and other plant hormones are little known. As IAA is essential for plant growth, the analysis of IAA biosynthesis knock-out mutants in the main pathway and the selection of double mutant with the knock-out mutant background may have been difficult. There are several pathways in IAA biosynthesis and some of them may function complementarily to each other. Mutants that have defect in such pathway may show no significant phenotype to distinguish mutants from wild type. On the other hand, the use of IAA specific biosynthesis inhibitor may bypass these difficulties and would make it possible to isolate new mutants. We can raise the next point as an overall merit of using IAA biosynthesis inhibitor for isolating IAA mutants. That is, we could isolate double or triple mutant-like mutants by screening mutants with the inhibitor.

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Eucalyptus globulus is one of the major afforestation species, but is known to be difficult for vegetative-prapagation because of its difficulty in root formation. According to the previous research, the endogenous level of indole-3-acetic acid (IAA) in poor-rooting line is three times lower than that of easy-rooting line, which suggests that endogenous IAA level may affect the ability of root formation. Therefore, we screened chemical library of triazole derivatives to find chemicals that is effective in promoting root formation. Consequently, several chemicals were selected as promoters of root formation in E. globulus. One of these chemicals, named MA65, has the ability to increase the number of root and the amount of endogenous level of IAA in Arabidopsis thaliana. On the basis of the chemical structure of MA65 as a lead compound, we started the lead optimization and synthesized 15 triazole derivatives and evaluated their ability to promote root formation and to increase the level of endogenous IAA using A. thaliana. As a result, one of the chemicals synthesized in this study was selected and found to be more effective in promoting root formation in E. globulus than MA65.

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Plants produce plant hormones to regulate plant growth and development in response to biotic or abiotic stresses. The plant hormones interact with each other to control physiological phenomena. Although many researches indicated that plant hormone interactions occur at the level of hormone biosynthetic and signaling pathway, common targets of multiple hormones are little known. In this research, we aimed to develop chemicals that inhibit the common targets in the phytohormone signaling. For this purpose, we use epicotyl section of azuki bean seedling. Its elongation is promoted by exogenously applied brassinosteriod, auxin and gibberellins. We selected the first candidates that show inhibitory effects to the synergistic functions of at least two hormones from a commercially available chemical library. At present, some of these candidates are subjected to the next experiment using Arabidopsis seedling.

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SCF(TIR1/AFB) auxin signaling pathway regulate the auxin-responsive gene expression. In this pathway, auxin is perceived by TIR1/AFB auxin receptors, leading to the degradation of Aux/IAA repressors mediated by SCF(TIR1/AFB) E3 ubiquitin-ligase. Auxin Binding Protein 1 (ABP1) has been believed to be another auxin receptor. However, the physiological function of ABP1 and its down-stream components have been still unknown. Therefore, ABP1 specific ligand would be useful chemical tool to investigate the function and signaling of ABP1. We designed ABP1 ligand according to the crystal structures of ABP1, and assessed the physiological activity of ABP ligand. The ABP1 ligand promoted the hypocotyl elongation and adventitious root formation in Arabidopsis plant, but the ligand did not activate the DRS-GUS expression under the control of SCF(TIR1/AFB) signals. Additionally, axrl-3 and tirl afb2 mutants were sensitive to the ABP1 ligand and the ligand did not induce the interaction between TIR1 and Aux/IAA. On the contrary, the conditional loss of functional mutant on ABP1 was highly resistant to the ABP1 ligand. These evidences suggest that the ligand would specifically activate ABP1 signals.

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Auxin is a master regulators of plant development including apical dominance, gravitropism, and phototropism. The auxin polar transport establish an asymmetric auxin distribution to respond environmental stimulus and consequently modulate the auxin responses of cells. Therefore, the auxin transport system plays a crucial role in plant development. Auxin distribution is regulated by the active transport by auxin influx and efflux carriers/transporters localized in plant cell membrane. Auxin influx transporters, AUX/LAX importers, efflux carrier proteins, PIN and auxin transporters ABCB/PGP are thought to coordinately modulate the endogenous auxin gradient to regulate plant development. Therefore, the dynamic of intracellular auxin distribution are important for the understanding of molecular mechanism for auxin transport system. The radio-labeled IAA and the indirect auxin-responsive reporter marker were utilized to study the auxin distribution and its polar transport. We here demonstrate the new fluorescent auxin analogs that is substrate for auxin transporters, but is unrecognized as auxin by TIR1/AFB auxin receptors. This new fluorescent auxin analogue function as auxin specific for polar auxin transport system, but not for auxin signaling. This fluorescent tools would mimic the cellular IAA distribution and indirectly visualize the dynamic of auxin movement mediated by auxin polar transport system.

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Cytokinins primarily promote cell division to regulate cell growth and differentiation, and also affect apical dominance, axillary bud growth, and leaf senescence. Cytokinin is perceived on the cytokinin receptors CRE1/AHK4 and AHK3 that function as the members of a hybrid sensor histidine kinases and regulates the subset of cytokinin-responsive gene expression. These receptors have a putative ligand binding domain called cyclases/histidine kinases associated sensor extracellular (CHASE) domain and this domain has been thought to be implicated in hormone binding. To identify the cytokinin binding site on CHASE domain of AHK4 receptor, we developed photoaffinity cytokinin probe. The cytokinin probes were designed to show the dual-function on photo-labeling and molecular-tagging for post-labeling. The cytokinin probe has aryl-azide and alkyl-azide for photo-reaction and click-reaction tag with biotin/fluorescent marker, respectively. The cytokinin activity of probe was evaluated by cytokinin-responsive ARR5-GUS reporter line and E. coil bacterial reporter assay expressing AHK4 receptor. The probes were found to be active cytokinin and would be promising tools to dissect cytokinin recognition.

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A series of new triazole derivatives was synthesized and their inhibitory activity against allene oxide synthase (CYP74A), a key enzyme in jasmonic acid biosynthesis, was evaluated. Structure-activity relationship studies revealed that 8-(1-naphthalen-2-y1-2-[1,2,4]triazol-1-yl-ethoxy)octanoic acid methyl ester (4i) and 8-[1-(2,4-dichlorophenyl)-2-[1,2,4]triazol-1-yl-ethoxy] octanoic acid methyl ester (4g) exhibit potent inhibitory activity to allene oxide synthase, with IC_<50> values approximately 0.75±0.3 and 0.84±0.6 μM, respectively.

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P450 are enzymes involved in phytohormone biosynthesis. Several azole type inhibitors have been reported that they targeting the P450 enzymes of gibberellin, brassinosteroid and/or jasmonic acid biosynthesis. It is generally known that azole derivatives exhibit inhibitory activities against P450, apparently due to the intrinsic affinity of the nitrogen electron pair in heterocyclic molecules for the prosthetic heme iron. In this context, we carried out searching for novel phytohormone biosynthesis inhibitors based on the chemical structure of ketoconazole as a molecular scaffold. The biological activities of synthesized compounds were evaluated by determining their inhibitory activity against CYP74A, a key enzyme in jasmonic acid biosynthesis, as well as in assays for brassinosteroid and gibberellin biosynthesis inhibitor using Arabidopsis seedlings grown in the darkness. We found several newly synthesized triazole derivatives exhibit potent inhibitory activity against brassinosteroid biosynthesis.

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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. Furthermore, current trial for chemical screening targeted to brassinosteroid signalling will be presented at this meeting.

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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 BRs 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 candidate 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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Brassinosteroids are essential for plant growth and development. Brz is a specific inhibitor of the brassinosteroid biosynthesis. We had identified 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 bssl (Brz-sensitive-shortl) showed normally etiolated hypocotyl same as wild type without Brz in the dark. However, with Brz, bssl hypocotyl became about 70% length of wild type in the dark. In the weak light, hyocotyl of bssl showed about 50% length of wild type without Brz. These results suggested that bssl hypocotyl is hypersensitive to Brz and the light. In the later growth stage, bssl showed extremely dwarf phenotype by inhibition of axis elongation. An activation tag insertion was identified in non-ORF region on bssl 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 hypoeotyl 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 are a group of plant steroidal hormones that regulate various aspects of plant growth and development. 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-insensitive-long hypocotyls in the dark was applied to Arabidopsis activation-tag lines. We screened Arabidopsis activation-tag lines and isolated bil2 that showed long hypocotyls on the medium containing Brz in the dark. Light-grown bil2 mutants had long petiole phenotype similar to wild-type plants treated with BR or BRIT-OX mutants. These phenotypes suggested that bil2 mutants enhanced the BR signaling. Identification of over-expression the BIL2 candidate gene is progress.

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We had identified several brassinosteroid signaling mutants from EMS-mutation lines, fast neutron-mutation lines and activation-tag lines in Arabidopsis. To identify new brassinosteroid signaling mutants, we used FOX (Full-length cDNA over-expressor gene) hunting system lines, which were overexpressed mutants of a normalized Arabidopsis full-length cDNA library. For these screening, we used Brz that is an inhibitor of brassinosteroid biosynthesis enzyme DWF4. Brz treatment caused to wild type as shorten hypocotyls in the dark. We tried to screen from about 8000 Fox-lines for bil(Brz-insensitive-long hypocotyls) that showed longer hypocotyls than wild type with Brz in the dark, and identified bil7 mutant. At adult stage, bil7 showed taller inflorescence than wild type. It appears that possible bil7 is brassinosteroid signaling factor and promote inflorescence elongation. Further analyses are in progress.

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The gaseous hormone ethylene plays important roles in many physiological and developmental processes in plants. To regulate ethylene signaling, we screened novel chemicals with ethylene mimic activity that induce triple response phenotype of etiolated seedlings. Finally we identified a compound with ethylene mimic activity, named HJ2. Ethylene biosynthetic inhibitor did not suppress HJ2-induced phenotype. On the other hand, ethylene insensitive mutant, ein2, suppressed HJ2-induced phenotype. Moreover, antagonist of ethylene receptor, STS, suppressed HJ2-induced phenotype in dose-dependent manner. These results suggested that HJ2 is an agonist of ethylene receptor. To improve ethylene activity of HJ2, we designed and synthesized HJ2 derivatives. As a result, we developed more effective ethylene agonists. At present, we examine binding of these chemicals to ETR1 protein in detail.

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Plants activate systemic acquired resistance (SAR), a form of long-lasting induced defense, to confer protection against a broad spectrum of pathogen. The induction of SAR requires the accumulation of salicylic acid (SA) and a subset of the pathogenesis-related (PR) genes in both local and systemic tissues. To clarify the SAR signaling pathway using chemical biology technique, we have established a high-throughput system for the easy identification of chemicals affecting SAR. We identified one candidate chemical, 4-phenyl-2-{[3-(trifluoromethyl)anilino]methylidene}cyclohexane-1,3-dione (PAMD), that down-regulates the expression of pathogenesis-related (PR) gene.

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Spermidine (Spd) is a well-known plant polyamine involved in disease resistance responses such as hypersensitive response (HR). Its N-hexanoylated derivative is reported to accumulate in plants progressively in senescing organs. However, regardless of its importance, the effect and action mechanism of Spd remains unclear. Recently we detected N-acylSpd-like compounds in Sp118 that is a disease resistance mutant identified from rice activation-tagging lines, by LC-MSMS. On the basis of this result, we synthesized several acylated Spd derivatives, examined the effect of them on cell death and disease resistance responses to reveal the role of Spd derivatives in plant. As a result, we found that non-anoylated and lauroylated Spds caused cell death in a manner different from that of Spd. Then using rice treated with these Spd derivatives we examined the expression levels of widely-used resistance marker genes, OsPR1b, PBZ1, and WRKY45, performed rice blast resistance assay, and quantified rice phytoalexines. Finally we identified ^4N-(12-hydroxylauroyl)spermidine as the most active plant activator among the acylSpds. To explore the possibility that acylSpds function as natural plant activators and induce disease resistance in rice, we analyzed acylSpds in Sp118 using LC-MSMS MRM mode. As a result, we identified two acylSpds, the content of them was far more than that in WT, respectively. This result could suggest the role of acyl Spds in disease resistance.

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Strigolactones (SLs) were discovered from cotton root exudates as a seed germination stimulant of root parasitic weeds Striga and Orobanche. Since the discovery of SLs, many attempts for the synthesis of SL mimics have been done for the purpose of decreasing the damage by parasitic weeds to crops. Recent studies of highly branched mutants figured out that SLs work as a phytohormone inhibiting shoot branching. We attempted various chemical designs of new SL analogs and finally, we discovered Debranone derivatives as strong inhibitors of the plant shoot branching. Debranones were quite potent for branching inhibition but not for stimulating root parasitic weed germination. Next, we tried structure development of Debranone to increase its activity and selectivity. In this poster, we show the branching inhibition activity in rice and the germination promotion activity in root parasitic seed of recently synthesized Debranone derivatives.

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We screened the chemical library of azole-containing compounds for new strigolactone (SL) biosynthesis inhibitors by estimating the activity of these compounds to reduce the level of 2'-epi-5-deoxystrigol (epi-5DS) that is one of the identified SLs in rice. We found an imidazole-containing compound (1b) as a new compound inhibiting the epi-5DS production. Then we carried out a structure-activity relationship study of lb to discover more potent SL biosynthesis inhibitors and found 6b as the most potent inhibitor of the epi-5DS production among the synthesized compounds, which possesses an extended carbon chain and a triazole moiety instead of an imidazole moiety. Treatment of rice seedlings with 6b at 10 μM reduced SL levels in both roots and root exudates and did not exhibit the growth retardation caused by the inhibition of the gibberellin and/or brassinosteroid biosynthesis. In addition, root exudates of 6b-treated rice seedlings stimulated Striga germination less than those of control plants. These results suggest that 6b is an effective SL biosynthesis inhibitor and has a potential to be applied in the control of root parasitic weeds germination.

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Strigolatones(SLs) were identified from plant root exudates as a germination stimulants for root parasitic weeds which cause severe damage to agriculture in Africa and in the Mediterranean. Arbuscular mycorrhizal (AM) fungi forms symbiosis with many land plants, and SLs also have hyphal branching activity of AM fungi. Recently it was reported that SLs are novel class of phytohormone that inhibit shoot branching, and therefore active studies on the function of SLs in several plants are in progress. However many genes involved in SL biosynthesis or signaling pathway have remained unknown. In the previous work we discovered triazol-type inhibitor of SLs (TIS13). TIS13 reduces endogenous levels of one of major SLs in rice, 2'-epi-5-deoxystrigol, and induce 2nd tiller bud outgrowth. As specific inhibitors are very useful for identifying new genes involved in the function of a biological substance inhibited by the inhibitor, we started to explore SL-related genes by using TIS13 for the screening of new mutants resistant to the function of TIS13. In our mutant screening we used Full-length cDNA over-expressing line (FOX) library, aiming at characterizing genes that cannot be identified from knockout lines because of their functional redundancy. In addition, FOX library easily enables us to identify the genes causing the TIS13 resistance. Here we report the result of the screening of rice FOX library on the basis of the phenotype resistant to TIS13.

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It has been demonstrated that all tested natural and synthetic strigolactones (SLs) with a tricyclic ABC lactone coupled to a methylbutenolide via an enol ether bond showed hyphal branching activity in the AM fungus Gigaspora margarita, and that truncation of the A- and AB-rings in the tricyclic lactone resulted in a drastic reduction in hyphal branching activity. In this study, we synthesized the BC-ring-opening SL analogues and evaluated their biological activity on G. margarita and the root parasitic weed Orobanche minor. Alcoholysis of formyl Meldrum's acid by either benzyl alcohol, o-, m-, p-methylbenzyl alcohol, or cyclohexanemethanol followed by alkylation with racemic 4-bromo-2-methyl-2-buten-4-olide provided trans- and diester BC-ring-opening SL analogues. Among the analogues synthesized, the cyclohexanemethanol-derived diester and trans-analogues showed the most potent activity on G. margarita and O. minor, respectively.

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Arbuscular mycorrhizal fungi (AM fungi) form mutualistic, symbiotic associations with more than 80% of land plants. In one of the first stages of host recognition, the hyphae of AM fungi show extensive branching by recognizing strigolactones (SLs) exuded from host plant roots. Our extensive structure-activity relationship study demonstrated that SLs show potent activity at very low concentrations in a structure-dependent manner, suggesting that the induction of hyphal branching proceeds via a receptor-mediated mechanism. However, the AM fungal SL receptor has not been isolated. Here, we designed and synthesized phenyldiazirine-containing 5-deoxystrigol (5DS) and GR24 probes for photoaffinity labeling of SL receptor in AM fungi. An alkynyl phenyldiazirine photophore was respectively introduced at C-5 of 5DS and at C-7 of GR24. The hyphal branching activities of 5DS photoaffinity probes were much weaker than those of 5DSs (1-10 ng/disc vs 3-30 pg/disc), while the GR24 photoaffinity probes showed comparable activity to GR24 (1 ng/disc vs 1 ng/disc).

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Arbuscular mycorrhizas formed between more than 80% of land plants and arbuscular mycorrhizal (AM) fungi belonging to the Glomeromycota are the most common and widespread symbiosis on our planet. Strigolactones (SLs) exuded from host plant roots act as a host-recognition signal for AM fungi. Very low concentrations of SLs can induce the host recognition response, hyphal branching, in AM fungi. This suggests a highly sensitive perception system for SLs present in AM fungi. However, very little is known about the molecular mechanisms underlying SL perception and induction of hyphal branching in AM fungi. We have recently started to design and synthesize molecular probes for photoaffinity labeling of SL receptor proteins in AM fungi. In this study, we modified ligand SL structure to increase sensitivity and specificity in photoaffinity labeling. 4a-Hydroxy-GR24 synthesized from 1,3-indandione showed comparable activity to GR24 in an AM fungus Gigaspora margarita (100 pg/disc). 8-Desmethylsorgolactone (8-dMS) synthesized from cyclohexanone exhibited strong but slightly weaker activity than 5-deoxystrigol (5DS) (10 pg/disc vs 3 pg/disc). This suggests that the introduction of functionality at C-8 could increase hyphal branching activity. Thus, we are now synthesizing 8-dMS photoaffinity probes that possess a phenyldiazirine photophore at C-8.

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All of the highly complicated and specialized organs of higher plants above ground, the leaves, stems, and flowers, are ultimately generated from a handful of stem cells in shoot apical meristem (SAM). CLV3 is a plant peptide hormone in which the proline residues are post-taranslationally hydroxylated and glycosylated. CLV3 plays a key role in controlling stem cell mass in the SAM of Arabidopsis thaliana. We identified a dodecapeptide (MCLV3) from CLV3-overexpressing Arabidopsis calli; MCLV3 was a smallest functional peptide derived from the CLV3 precursor. Here we designed a series of MCLV3 analogs in which proline residues were substituted with N-substituted glycines (peptoids), and show the structure-activity relationship of the plant peptide hormone. To understand the solution structure of MCLV3, we performed NMR measurements and computer modeling. The NMR analyses indicated trans-conformer is predominant at the three pre-prolyl amide bonds in MCLV3. However, minor signals in the ^1H-NMR spectrum and conformational search suggest the presence of cis-conformers at three amide bonds.

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Stomata play a crucial role in the regulation of gas exchange through the pore between a pair of guard cells. Recently, we have reported a gene named STOMAGEN shows unnaturally increased stomata in the over-expressed lines. The 45-amino-acid cysteine-rich peptide at the C-terminus of the STOMAGEN precursor act as a hormonally active peptide which contains three intermolecular disulfide bonds. STOMAGEN is a member of Epidermal Patterning Factor (EPF) family. EPF1 and EPF2 are also in the family, which lead to the contrary phenotype with decreased stomatal formation when overexpressed in Arabidopsis. However, the essential region of EPF1 and EPF2 is not clear. In this presentation, we produced the bioactive peptides derived from EPF1 and EPF2. We synthesized these peptides using Fmoc chemistry, or obtained recombinant peptides using E. coli, and then refolded these peptides. Folded EPF1 and EPF2 peptides significantly decreased stomatal density in the abaxial leaf epidermis at the concentrations around 10 μM. We analysed the arrangement of the intermolecular disulfide bonds in EPF1 by enzymatic digestion of this peptide, and revealed that the fifth and sixth cysteine residues that are additional, not conserved in the family are bound each other through a disulfide bond. These results suggest that the C-terminal cysteine-rich region of EPF family is sufficient to function as bioactive peptides, and share common backbone fixed by the conserved cysteine residues.

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The phytohormone auxin plays a fundamental role in various aspects of plant growth and development. Indole-3-acetic acid (IAA) has been known as a major naturally occurring auxin in plants, but its biosynthesis has not been fully understood. Previous studies demonstrated that the YUCCA (YUC) family encodes a plant-specific flavin monooxygenase that catalyzes a rate-limiting step in IAA biosynthesis. More recent studies demonstrated that the Tryptophan Aminotransferase of Arabidopsis 1 (TAA1) converts Trp to indole-3-pyruvic acid (IPA). Both the TAA and YUC families play critical roles throughout plant development, but they are proposed to function in two independent pathways. In this study, we provide evidences that the YUC family acts in the IPA-dependent IAA biosynthesis in Arabidopsis. We found that co-overexpression of TAA and YUC genes dramatically increases IAA production and results in hyper-auxin phenotypes, suggesting a synergistic interaction between TAA and YUC genes in IAA biosynthesis. The LC-ESI-MS/MS analysis of IPA levels in TAA and YUC-related mutants indicates that TAA produces IPA from Trp and YUC converts IPA to IAA. We further showed that recombinant YUC proteins catalyze the direct conversion of IPA to IAA in an NADPH-dependent manner. From these results, we conclude that the IPA pathway is the main IAA biosynthesis pathway in Arabidopsis.

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Galls are abnormal plant tissues induced by parasitic insects on various plant parts. It is generally assumed that the insect secretes chemicals at oviposition or ingestion, which induce abnormal cell growth and proliferation leading to gall formation. In this study, we selected a willow (Salix japonica) gall formed by a sawfly of the genus Pontania to investigate the substances involved in gall formation. In the last meeting, we reported that the concentration of indole-3-acetic acid (IAA) in larvae was more than 100 times higher than that of the willow leaves, and that the larvae could synthesize IAA from tryptophan. This time, we analyzed cytokinins (CKs) in willow leaves, gall plant tissue and larval tissue, so as to examine the possible involvment of CKs in gall formation. A major cytokinin in the all analyzed samples was trans-zeatin (t-Z). The levels of t-Z in gall tissue and larval tissue were respectively more than 60〜200 times and 2000 times higher than that of the willow leaves. A correlation between the endogenous level of t-Z both in gall tissue and larvae and the developmental state of galls was observed, which suggests that the t-Z plays a key role in the regulation of gall state. Ttranscript levels of isopentenyltransferase (IPT) genes, key enzymes involved in CK biosynthesis and feed-back regulated by CK signaling, in the gall tissues were about 1/10 of those in the willow leaves, suggesting that the larvae provide gall tissues with t-Z.

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We have reported in the last meeting that a gall-inducing sawfly on a willow tree (Salix japonica) and a gall midge on a mugwort (Artemisia princeps) contained high concentrations of indole-3-acetic acid (IAA), and both showed converting activity of tryptophan (Trp) to IAA. Afterward we found that IAA was contained not only in the gall-inducing insects but also in the non-gall-inducing insects, such as drosophila (Drosophila melanogaster) and silkworm, which (Bombyx mori) also converted Trp to IAA. Here we examined the origin of the converting enzymes and the biosynthetic pathway using silkworms. The regurgitant fluid (RF) of silkworms, which could contain saliva and contents of a foregut and a midgut, showed IAA producing activity. The activity was lost by heat treatment or pronase E treatment, suggesting the enzymatic conversion. In the feeding experiment, indoleacetamide (IAM) and indoleacetaldoxime (IAOx) have been so far detected as metabolites of labeled-Trp. We have concluded that the former was not the intermediate of IAA synthesis based on its non-enzymatic production from Trp and the failure of detecting IAA from JAM feed. The possibility that the symbiotic or contaminated bacteria are responsible for the IAA synthesis was eliminated by preparing sterile silkworm larvae, i.e., their RF showed activity to convert Trp both to IAOx and IAA. On the other hand, the silkworm bait composed of mulberry powder converted Trp to IAA.

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Blue light (BL) induces many adaptive responses in plants, including shoot growth inhibition and phototropism. To clarify the mechanism of growth inhibition in response to unilateral BL illumination, the isolation and identification of various growth inhibitors has been attempted in several plant species. In maize coleoptiles, benzoxazinoids (MBOA and its precursor, DIMBOA), were isolated and identified as candidate inhibitors. Previously, we found in the early stage of phototropism that unilateral BL illumination promotes P-glucosidase (DIMBOA-glucosidase) activity on the illuminated side of maize coleoptiles, releasing DIMBOA and simultaneously producing MBOA. We found that BL induces H_2O_2 accumulation via the up-regulation of DIMBOA and probably of MBOA. We also found that these compounds contribute to their growth inhibitory activity by promoting H_2O_2 synthesis and subsequent lignification (cell-wall stiffness). These results suggest a close association between an oxidative burst and phototropism. In our previous report, the unilateral application of these compounds suppressed the growth of coleoptiles on the treated side and causing them to bend towards the side of application. Furthermore, BL up-regulated the transcriptional level of DIMBOA-glucosidase. Thus, it is concluded that phototropism is triggered by a local gradient, induced at the site of illumination, of BL-induced growth inhibitors interfering with the action of the evenly distributed auxin.

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Gibberellin (GA) are plant hormone that regulates plant growth and environmental responses. We had already reported the method for GA analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Recently, plant hormone researches require tissue/organ specific high sensitive quantification analysis. In this meeting, we will report a derivatization method for high sensitive gibberellin analysis by LC-ESI-MS/MS. In addition to previously reported GA_1, GA_4, GA_8, GA_9, GA_<19>, GA_<20>, GA_<24>, GA_<44> and GA_<53>, kaurenoic acid, GA_<15>, GA_<29>, GA_<34> and GA_<51> were derivatized by 4% of 0.5 M 2-bromoethyltrimethylammonium bromide 70% acetonitrile solution in 75% 1-propanol, 20% water and 1% triethylamine. In case of 20 μl of reaction volume in capped glass capillary, 45 to 85% of GA had been reacted in 2 hours. In case of 100 μl of reaction volume in screw capped glass vial, approximately 30% of GA had been reacted in 3 hours. These results suggest that concentration affects reaction efficiency in this derivatization method. Detected areas of derivatized GA were from similar to 10 times larger than original GA. One product ion was determined in all GA. Acidic fraction of Arabidopsis leaves was prepared by hydrophobic, strong cation exchange and weak anion exchange extraction. As a result of derivatization, more than 25 times larger areas were determined in both GA_1 and GA_4 but higher base lines gave similar S/N compare to original GA analysis method.

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Gibberellins regulate seed germination, shoot elongation and flowering. DELLA proteins are members of the plant-specific GRAS protein family and act as repressors of the GA signaling pathway. DELLA proteins are rapidly degraded in the presence of GA. The degradation mechanism has been uncovered by the discovery of GA receptor and F-box protein. To understand the downstream signaling of DELLA proteins, we have identified GAF1 that interacts with DELLA protein by modified yeast two hybrid screening. GAF1 is a novel transcriptional factor. Arabidopsis plants that overexpress GAF1 are early flowering with larger leaves. In contrast the gaf mutant exhibits a semi-dwarf and late flowering phenotype. BiFC analysis indicated that the interaction of GAF1 and DELLA protein disappeared after GA treatment. Moreover we isolated a WD repeat protein as another GAF1 interaction protein. Our trans-activation assays in yeast and plant cells suggest that DELLA and the WD repeat protein regulates the function of GAF1. These data suggest that GA controls transcriptional activity via alteration of GAF1 complex components. GAF1 also interacts with 5 DELLA proteins and WD repeat protein. The binding of GID1 to the GAF1 complex represses the interaction between GAF and DELLA proteins. We identified each interaction domain of GAF1, and analyze interaction the mechanism of the GAF1 complex using yeast and plant systems.

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In response to water stress, plants increase their endogenous ABA levels and close stomata to limit water loss. It has been shown in Arabidopsis that ABA biosynthetic enzymes are localized in vascular tissues, suggesting that ABA synthesized in vascular tissues is transported to guard cells to close stomata. Two ATP-binding cassette (ABC)-type transporters that mediate ABA export from the inside to the outside of cells (AtABCG25) and ABA uptake from the outside to the inside of cells (AtABCG40), respectively, have been identified. However, the loss-of-function mutants atabcg25 and atabcg40 show milder phenotypes compared to typical ABA-deficient mutants, indicating that the ABA transport system might be highly complex and redundant. We identified a non-ABC-type ABA transporter, AIT1, which mediates cellular ABA uptake. Mutants defective in AIT1 (aitl) were insensitive to exogenously applied ABA during germination, whereas overexpression of AIT1 resulted in ABA-hypersensitivity in the same condition. Interestingly, the inflorescence stems of aitl had a lower surface temperature than those of the wild type because of excess water loss from the stomata. Promoter activities of AIT1 were detected around vascular tissues in inflorescence stems, leaves, and roots. These data suggest that AIT1 functions as an ABA transporter in vivo and plays a role in maintaining the pool size of ABA at the site of ABA biosynthesis.

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Seed dormancy, defined as the failure of viable seed to germinate under favorable conditions, is thought to be an important factor that determines geographical distribution of plants. Increasing lines of evidences have demonstrated that phytohormones, such as abscisic acid (ABA) and gibberellins (GA), play an essential role in seed dormancy and germination. In Arabidopsis, it is widely known that wild-type accessions, which originate from various regions of the world, significantly differ in the degree of seed dormancy. Cvi-0, one of dormant accession, has been reported to show altered endogenous ABA/GA balance in agreement with the reduced germination frequencies. In this study, we analyzed seed germination and phytohormone levels in the 2-months after-ripened seeds of over 100 accessions. Analysis of 6 phytohormones including ABA and GA first revealed that there are significant variations in all horomone levels among these accessions. Interestingly, endogenous levels of ABA, GA and isopentenyladenine (iP) levels after seed imbibition showed negative (ABA) and positive (GA, iP) correlation with germination frequencies among accessions, suggesting the universal role of phytohormone balance in seed dormancy and germination. To identify genetic factors that are associated with these phenotypic variations, we next performed genome-wide association mapping analysis. Several candidate genes that would potentially explain the phenotypic variations have been identified so far.

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Seed coat-imposed dormancy is part of the seed survival strategy of many species. The seed coat (testa) exerts its germination-restrictive action most of the time by being impermeable to water and/or oxygen or by its mechanical resistance to radicle protrusion. These properties have been positively correlated with seed coat color due to phenolic compounds in diverse species. The brown pigments of the seeds are mainly condensed tannins of the procyanidin type, which are end-products of the flavonoid biosynthetic pathway. The seeds of Arabidopsis transparent testa (tt) mutants which have defect in flavonoid metabolism have been reported to reduce dormancy at various levels. We have isolated tt7 allele (tt7-4) as one of the high temperature resistant germination mutants, and found that tt4-1, tt5-1, tt6-1, tt7-1 and fis1, all of which are deficient in quercetin accumulation, were high temperature resistant but tt3-1 and banyuls, which are deficient in proanthocyanidins, were not. We confirmed that quercetin contents in wild type and tt mutant seeds showed good anti-correlation with germination levels at high temperatures. These results suggest that quercetin, quercetin glycosides or its derivatives are responsible for coat-imposed dormancy and thermoinhibition of Arabidopsis seeds. We are now isolating flavonol glycosidase mutants and are planning to see germination phenotypes of the knockout mutant seeds.

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