Plant hormone auxin (indole-3-acetic acid (IAA)) and abscisic acid (ABA) have important roles in almost all plant life including various environmental responses. Here, I report molecular studies of IAA and ABA biosynthesis, showing the importance of their biosynthetic sites. We first revealed that maize coleoptile tip is the vigorous biosynthesis site of IAA, and using this tissue we demonstrated the in vitro system of IAA synthesis from tryptophan. Aldehyde oxidase is a component of the in vitro system and expression of AAO1, a member of Arabidopsis aldehyde oxidase family, is significantly higher in auxin over producing mutant, surl, than in wild type. During course of our study of Arabidopsis Aldehyde oxidase gene family, we found AAO3 has an activity to produce ABA from abscisic aldehyde. It was clearly indicated that AAO3 acts an important role for drought induced ABA accumulation in Arabidopsis leaves and that the vascular systems might be the main site of the ABA synthesis.

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Brassinosteroids (BRs) are essential plant growth hormones. In order to obtain compelling evidence that BRs are prerequisite hormones, we aimed to find out dwarf mutants. As the result, we discovered dwarf pea mutants lka and lkb as BR-insensitive and BR-deficient mutants, respectively. The dwarf phenotype of the lkb mutant was rescued by exogenous application of BRs. GC-MS analysis showed that dwarfism of the lkb mutant is due to BR deficiency caused by blocked synthesis of plant sterol, campesterol. The lka mutant was insensitive to exogenous BRs, but it accumulated castasterone to a great extent. The responsible gene was determined to be a BRI1 homolog encoding a BR receptor. Overproduction of castasterone in the lka mutant was ascribed to the depletion of a feedback control mechanism due to the impaired BR receptor. In this work, a new cytochrome P450 was also discovered that catalyzes the synthesis of brassinolide. A null mutation in the cytochrome P450 CYP85A1 gene of tomato (d^x) causes severe dwarfism due to BR deficiency, but the d^x mutant still produces normal fruits. I found that d^x fruits contain brassinolide at high level and that a new CYP85A gene, CYP85A3, is preferentially expressed in tomato fruits. Tomato CYP85A3 expressed in yeast catalyzed the Baeyer-Villiger oxidation to produce brassinolide from 6-deoxocastasterone and castasterone. In summary, the present work demonstrated that BRs are essential plant growth hormones and provided much information on the roles and functions of the biosynthesis and signaling genes of BRs.

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Abscisic acid (ABA) is an essential mediator of plant responses to environmental stresses. The initial perception and metabolic inactivation of ABA by target cells remain obscure at the molecular level. We have designed and synthesized ABA analogues for probing the mechanism of ABA reception and metabolic inactivation. (1) The biological activities of four fused-type bicyclic ABA suggested that the active conformation of ABA is close to a half-chair with the side-chain axial. (2) The di-and tri-fluorination, methoxylation, or unsaturation of the 8'-methyl group of ABA resulted in the long-lasting bioactivity. (3) The 3'-fluorination of ABA stabilized the 8'-hydroxylated metabolite for isolation. The tautomeric isomerization of 8'-hydroxy-ABA to phaseic acid is significantly affected by pH. (4) The lead compound for the future design and development of an ideal ABA 8'-hydroxylase inhibitor was generated by investigation of the structural requirements for ABA 8'-hydroxylase inhibition and for ABA activity.

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Gibberellins (GAs) are phytohormones that are essential for many developmental processes in plants. Recent molecular biological and genetical studies have identified several positive and negative regulators of GA signalling pathways in higher plants. Among them, the DELLA protein has been considered one of the most important factors, which functions as a negative regulator of GA signalling; its degradation through the ubiquitin/proteasome pathway is a key event in the regulation of GA-stimulated processes. Recently, we isolated a G__-A i__-nsensitive d__-warf mutant of rice, gidl. The GID1 encodes an unknown protein with similarity to the hormone sensitive lipases (HSL) and the GID1-GFP signal was observed preferentially in nuclei. Recombinant GST-GID1 had a high affinity only for bioactive GAs, while mutated GST-GID1 corresponding three gidl alleles, had no GA-binding affinity. The K_d value for GA_4 was estimated to be around 10^<-7>M, enough to account for the GA dependency on shoot elongation. Moreover, GID1 bound to SLR1, a rice DELLA protein, in a GA dependent manner in yeast cells. GID1 overexpression resulted in a GA-hypersensitive phenotype. Taken together, these results indicate that GID1 is a soluble receptor mediating GA signaling in rice.

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The plant hormone auxin (indole-3-acetic acid (IAA)) plays an important role in physiological responses such as gravitropism and phototropism. We reported that in maize coleoptiles the main site of IAA biosynthesis is 0-2mm tip region and the produced IAA moves constantly to the lower part. It has been reported that red light irradiation to the coleoptiles caused the reduction of the IAA level in the tip region and therefore the diffusible IAA from the tip to the lower parts was also decreased. In the present work, to investigate the mechanism(s) for the IAA reduction by red light irradiation we examined free and conjugated IAA levels, and rate of IAA transport. IAA was extracted by an improved method using Micro-Smash (TOMY) and determined with GC-SIM-MS. The results revealed that the red light did not cause any changes in the amount of conjugated IAA and the rate of polar transport, indicating that red light suppresses the rate of IAA biosynthesis in the coleoptile tip.

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The plant hormone auxin (indole-3-acetic acid (IAA)) plays important roles for response to external factors such as light and gravity. We reported that IAA was biosynthesized mainly at 0-2mm tip region and that the synthesized IAA was continuously transported to basal part in maize coleoptiles. It has been well investigated that IAA distribution rapidly changes at upper and lower side of the coleoptile within 20 minutes after gravi-stimulation. Here, we examined not only free IAA level in the tissues but also diffusible IAA level from upper or lower tip region after gravi-stimulation. The results revealed that flow direction mainly changed at 0-3mm tip region, not at 4-8mm region, indicating that apical zone was a main site for gravi-response, where IAA was transported to lower parts causing asymmetric IAA distribution throughout the coleoptile.

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Plant response to gravity is an important adaptive strategy to survive their environments. We have investigated the mechanism of the gravity response using cucumber seedlings. When cucumber seeds germinate in a horizontal position, a protuberance called a peg forms on the lower side of the transition zone between the root and the hypocotyl. The peg helps the cotyledons and the plumule to pull themselves out from the seed coat. In space-flight experiments, we found that cucumber seedlings grown in microgravity developed a peg on each side of the transition zone. This result suggests that gravity response results in the suppression of peg formation on the upper side of transition zone on Earth. It has been shown that auxin induces peg formation. When cucumber seedlings were treated with inhibitors of auxin efflux carrier, a peg was formed not only on the lower side but also on the upper side of the transition zone, suggesting that activation of auxin efflux carriers is required for the suppression of peg formation. To identify auxin efflux carriers involved in suppression of peg formation by graviresponse, we isolated 6 cucumber cDNAs of PIN auxin efflux carrier genes and investigated their mRNA accumulation by in situ hybridization. Our results showed that mRNAs of CsPIN1 and CsPIN6 accumulate in several tissues including endodermis postulated to be gravisensing cells, suggesting that CsPIN1 and CsPIN6 could play a role in the gravity-response of the transition zone.

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Relationships between morphogenesis and auxin polar transport in etiolated pea (Pisum sativum L. cv. Alaska) seedlings were studied, especially in molecular levels. Activities of auxin polar transport in proximal side of epicotyls to cotyledons in etiolated pea seedlings were much higher than those in distal one. Accumulation of PsPIN1 mRNA was observed in proximal side of epicotyls to cotyledons, it showing a good correlation with activities of auxin polar transport. Exogenously applied TIBA (2, 3, 5-triiodobenzoic acid) induced automorphosis-like growth and development of etiolated pea seedlings which were found in microgravity conditions in space. In this case, expression of PsIAA4/5, one of auxin-inducible genes in pea seedlings, was extremely suppressed in proximal and distal sides of epicotyls to cotyledons. These results suggest that the activity of auxin polar transport and intercellular auxin level are essential factors to regulate morphogenesis of etiolated pea seedlings. Further studies on other PsPIN genes and MDR genes, which affect auxin polar transport in higher plants, will also be required.

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Root systems are responsible for acquisition of all the water and nutrients from the soil. Therefore, it is important for plants to develop root systems for avoiding stressful environments and establishing their stand. Thus, roots have been shown to recognize many environmental signals that regulate root system architecture; namely, gravity, light, touch, and moisture gradients. Hydrotropism is the response of roots to a moisture gradient and thought to play an impotant role in avoiding water stress. However, only gravitropic response has been extensively studied among these responses, for gravitropism generally dominates other tropisms. In fact, gravitropic interference with hydrotropism has been shown in several plant species. At the same time, hydrotropism and gravitropism have been proved to share common features. But, how different stimuli lead to a common response remains unclear. To gain new insights on the problem, we searched the molecules that affect either or both gravitropism and hydrotropism. We treated Arabidopsis seedlings with various inhibitors and examined their effects on gravitropism and hydrotropism in roots. The results showed that inhibitors of polar auxin transport inhibited only gravitropism but not hydrotropism, while Ca^<2+> chelator was inhibitory to both tropisms. These results suggest that Ca^<2+> is essential for both gravitropism and hydrotropism, whereas polar auxin transport is necessary only for gravitropic response.

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Although it has been considered to date that a large amount of endogenous indole-3-acetic acid (IAA) in plant is present as conjugated forms, the identified compounds account for only a minor part of the estimated total IAA. In this study, we searched for novel IAA-conjugates and its oxidative catabolites, in order to better understand the IAA metabolism in Arabidopsis. The extract of Arabidopsis (Col-0) seedlings was partially purified using solid-phase extraction cartridge. The presence of IAA and 2-oxoindole-3-acetic acid (OxIAA) metabolites was monitored by LC-ESI-MS/MS systems operated in multiple reaction monitoring (MRM) mode. By comparing the LC behavior and MS/MS data with synthetic standards, two new oxidative conjugates, OxIAA-Phe and OxIAA-Glc were identified. Furthermore, the presence of the conjugates of hydroxy IAA with Asp, Glu, Phe and Tyr was suggested. In order to determine their substitution position of hydroxy group on the indole ring, a series of amino acid conjugates of 4-OH-IAA, 5-OH-IAA, 6-OH-IAA and 7-OH-IAA were respectively synthesized. These synthetic standards were compared with natural products in terms of LC behavior and MS/MS data for identification.

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Effects of tryptophan over-production on the indole-3-acetic acid homeostasis in rice were investigated using the transformants expressing a feed-back insensitive form of anthranilate synthase a subunit gene (OASA1D). The over-expression of OASA1D transgene caused a drastic accumulation of Trp in two-week-old rice plant in a tissue-dependent manner. The Trp level in the youngest leaf of the transformant (HW5) reached ca 3000 nmol/gFW, while the increase in other leaves was more moderate. Stem and root also contained high amount of Trp. The level of IAA was increased by the OASA1D expression with the distribution pattern in the plant similar to that of Trp. The youngest leaf accumulated the highest amount of IAA, which was 20 times as much as that in untransformed rice plant (Nipponbare). No anomaly was observed in the morphology of the transformant. These results suggests that the IAA biosynthesis in OASA1D rice was activated as a result of drastic increase in the Trp level without affecting its growth.

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Mechanism of indole-3-acetic acid (IAA) to induce stem growth in tulips (Tulipa gesneriana L. cv. Apeldoorn) was studied. The growth of stem with or without cooled bulbs was very small when lanolin was applied to the cut surface of the top internode of shoot, of which all leaves and flower bud were removed. IAA (0.1%, w/w) applied as a lanolin paste instead of flower bud extremely promoted the growth of stem with or without cooled bulbs. More conspicuous effect of IAA was observed in the stem with cooled bulbs. The fresh weight of stem segments treated with IAA was much higher than that of untreated ones although the total dry weight of stem segments was almost similar. Dry weight of the first internode decreased, whereas that of the upper internodes increased, and IAA substantially enhanced these changes in dry weight, showing that dry materials are essential factors for IAA-induced growth of the stem. These results suggest that IAA enhances translocation of solutes to stem intenode elongating actively from other part of shoots and/or bulbs, in which solutes act as a driving force in osmoregulation. Further studies of IAA on osmoregulation and solute translocation as well as mechanical properties of cell walls will be required.

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In the previous study, we found that carotenoid biosynthesis inhibitors such as fluridone promoted the germination and emergence of Monochoria vaginalis var. plantaginea and several weeds. In this study, effects of four carotenoid biosynthesis inhibitors, fluridone, norflurazon, diflufenican and benzobicyclon hydolysane, on dormancy of M. vaginalis seeds were examined. Germination rate of the M. vaginalis seeds treated with fluridone and norflurazon under submerged condition in the dark was higher than that of control. It is suggested that fluridone and norflurazon inhibited the secondary dormancy of the M. vaginalis seeds induced under submerged condition in the dark.

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Effects of fertilizers on germination and emergence of Monochoria vaginalis var. plantaginea seeds under low temperature and submerged conditions were examined in pots and glass vials. In both of the pots and the glass vials, among the fertilizers tested, (NH_2)_2CO, (NH_4)_2SO_4, NH_4NO_3 and NH_4Cl promoted the emergence of the M. vaginalis seedlings. These fertilizers promoted the germination of the M. vaginalis seeds stimulated by addition of rice seeds into the glass vials at 27℃ in the dark. These results indicate that nitrogenous fertilizers promote germination of M. vaginalis seeds.

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Among mineral nutrients, reduced supply of phosphorus significantly enhanced production and/or exudation of the germination stimulant orobanchol for Orobanche minor by red clover plants grown hydroponically. Therefore, response of orobanchol exudation to phosphorus availability was examined in detail. Orobanchol exudation greatly increased 48 hours after transferring to low P condition (8μM P). The plants exuded more orobanchol when grown in media with lower P levels. When the plants were continuously grown at 8μM P, orobanchol exudation continued to increase further with incubation period. By contrast, when the plants grown at 8μM P were transferred to 80 or 160μM P, orobanchol exudation decreased within the first 24 hours. These data suggest that red clover plants sense and respond quickly to changes of phosphorus availability in soil and regulate biosynthesis and/or exudation of strigolactones.

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Orobanche minor is one of obligate root parasites of dicotyledonous plants such as red clover (RC). We examined effects of defense-response-inducers, salicylic acid (SA), benzo [1, 2, 3] thiadiazole-7-carbothioic acid S-methyl ester (BTH) and methyl jasmonate (MJ), on resistance of red clover against O. minor infestation. Treatments of RC root with SA and BTH had little effect on germination of the O. minor seeds. By treatment with MJ, the germination rate of O. minor seeds located around the RC roots near the base was reduced, while it tended to increase in those near the ends of the roots. It was presumed that reduction of germination stimulants and/or production of germination inhibitors occurred in the roots directly treated with MJ, and production of germination stimulants was enhanced in the roots developed after treatment. Treatments with SA and BTH inhibited most of the penetration of haustoria into the host vascular bundle and prevented the success of parasitism. By contrast, MJ treatment increased the chance of infestation and facilitated the formation of the tubercles. These results suggest that SA and BTH rendered the RC roots resistant to penetration of haustoria and MJ rendered them susceptible.

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When Lemna paucicostata 441 subjected to drought, heat or osmotic stress were immersed in water, factors which show strong flower-inducing activity after incubation with Norepinephrine (NE) appeared in the water (Takimoto et al., 1994). Yokoyama et al., (2000) isolated the essential component of factors, clarified its chemical structure as 9-hydroxy-10-oxo-12(z), 15(z)-octade-cadienoic acid (KODA). Yamaguchi et al., (2001) detected FN1, which showed a strong flower-inducing activity, in the mixture of KODA and NE after incubation, and the structure of FN1 was determined. On the other hand, we suggested that the induction of systemic acquired resistance in rice and tobacco and the floral induction of Lemna plant have some common processes. Thus, in the present work, we examined the possibility that KODA, NE or FN can induce systemic acquired resistance. Systemic acquired resistance is accompanied with pathogen-related (PR) gene expression. Therefore we first examined the expression of PR-1 gene induced by KODA, NE or FN in tobacco. KODA induced PR-1 gene expression but NE or FN could not induce the expression of the gene. KODA also induced the other PR genes such as Acidic glucanase (PR-2), Basic glucanase (PR-2), Class I basic chitinase (PR-3), Class II acidic chitinase (PR-3), Acidic chitinase (PR-4), Class III acidic chitinase (PR-8) and Class III basic chitinase (PR-8). The present results suggest the possibility that KODA induces systemic acquired resistance in tobacco.

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For the purpose of estimating heat shock as a protection activator, the aerial portion of the seedlings at the three-leaf stage of tomato cv. 'Natsunokoma' were dipped in water at various temperatures. Challenge inoculation test with Botrytis cinerea were performed onto the cut of the first and second true leaves 24h after the heat shock treatment. Although the infiltrated area spread beyond the region of inoculation part in the leaves of non-treated control plants, the less disease symptoms were observed in the leaves dipped into hotter water. To clarify the resistance mechanisms, salicylic acid accumulation and the expression of PR genes after the heat shock treatment were examined. The results indicated that treatment at 45℃ was most effective, accompanying the salicylic acid accumulation and PR-1 gene mRNA. These results suggested that the heat shock treatment induced systemic acquired resistance in tomato plants.

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12-Oxophtrodienoic acid reductase (OPR) is one of jasmonic acid (JA) biosynthetic enzymes. Although in Arabidopsis thaliana and Lycopersicon esculentum there exist a few OPR isozymes, only AtOPR3 and LeOPR3 have been shown to be involved in JA biosynthesis in A. thaliana and L. esculentum, respectively. Biological functions of AtOPR1, AtOPR2, and LeOPR1 remains to be clarified. We found that more than 10 OPR genes exist in rice using a rice (O. sativa L. cv. Nipponbare) genome database recently opened to the public. One of these OPRs, tentatively named OPR8, has the peroxisomal targeting signal, as in the cases of AtOPR3 and LeOPR3, and fusions of OsOPR8 with Green Fluorescent Protein were detected exclusively in peroxisomes of onion epidermal cells. OsOPR8 expressed in a functional form in E. coli exhibited AtOPR3-like activity that reduces effectively naturally occurring (+)-cis-OPDA. It was also shown that both of the OsOPR8 mRNA and endogenous JA levels in rice leaves began to increase within 1/4 h after the wounding and drought treatments. These results strongly suggest that OsOPR8 is the OPR responsible for JA biosynthesis in rice. In addition, we are now investigating whether the male-sterility in opr3 (a T-DNA-tagged, male-sterile mutant of Arabidopsis) is alleviated by transformation of the mutant with an OsOPR8 cDNA.

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Salicylic acid (SA) is well known as an important regulator of plant resistance to pathogens. Ozone, a major photochemical oxidant, induces SA biosynthesis and leaf injury. SA participates in both the formation of leaf injury and the defense response when ozone was fumigated. In pathogen-infected plants, SA is synthesized via two pathways involving phenylalanine or isochorismate. Biosynthesis of SA in ozone-fumigated plants had not been well defined, so we examined it in tobacco and Arabidopsis. Salicylic acid accumulated in tobacco exposed to 0.2ppm ozone for 6h. At the same time, phenylalanine ammonia-lyase (PAL) activity and its mRNA level were increased remarkably. When we applied ^<14>C-labeled benzoic acid to ozone-exposed tobacco leaves, it was effectively metabolized to SA. However, isochorismate synthase (ICS) activity and its mRNA level were not increased. These results suggest that ozone-exposed tobacco synthesized SA from phenylalanine. Salicylic acid levels also increased in ozone-exposed Arabidopsis, but not in sid2 (salicylic acid induction-deficient 2) mutants, in which ICSI is defective. Furthermore, ICS activity and the mRNA level of ICSI increased dramatically in wild-type Arabidopsis after the start of ozone exposure. These results suggest that ozone-exposed Arabidopsis synthesizes SA from isochorismate. Therefore, our results imply that the main pathway of ozone-induced SA biosynthesis differs between tobacco and Arabidopsis.

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Stresses are one of the environmental factors which induce flowering as photoperiod and temperature are. Although Pharbitis nil is a short-day plant, it flowers under long-days responding to stresses such as low temperature, poor nutrition and high irradiance. We found that metabolic pathway producing benzoic acid is involved in the stress-responding flowering in P. nil, and suggested that salicylic acid may be the compound which actually induces flowering. Here, we examined the involvement of salicylic acid. When P. nil, Violet was cultivated in tap water for 20 days, flowering was induced under long-days. This poor nutrient stress-responding flowering was inhibited if aminooxyacetic acid (AOA) was added to tap water. This inhibition by AOA was reversed if t-cinnamic acid or benzoic acid was added together with AOA. These results are consistent with the previous results in the same experiments with low temperature. It suggests that the same metabolism is involved in the stress-responding flowering irrespective of the kind of stress factors. Further, the inhibition by AOA was reversed also by salicylic acid. Then, flower-inducing activity of salicylic acid was assayed in the culture with nutrient solution under long-days, a non-stress conditions. Salicylic acid did not induce flowering. These results together indicate that stresses may induce not only the synthesis of salicylic acid but also some other factors which are necessary for the flowering.

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ent-Kaurene is a tetracyclic hydrocarbon precursor for gibberellins (GAs) in plants and fungi. In plants, GGDP is converted to the Arabidopsis-kaurene by two separate cyclases, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). In contrast, fungal ent-kaurene synthase (GfCPS/KS) converts by bifunctional activities with both CPS and KS. Previously we reported the following results; 1) When co-cultivated with the GfCPS/KS transgenic lines in a closed environment, the dwarf phenotype of ga1 and ga2 mutants was able to fully complement. 2) Co-cultivation of the ga3 mutant, which accumulate ent-kaurene, or of a filter paper containing ent-kaurene also rescued the dwarf phenotype of the mutants. 3) ent-Kaurene was identified from the headspace of the lines and ga3 mutant by GC-MS analysis. These results suggest that ent-kaurene might be metabolized into bioactive GAs in Arabidopsis when supplied as a volatile. To address whether structural differences in the diterpene hydrocarbon affect the emission into the headspace, we generated transgenic Arabidopsis plants overexpressiong a novel fungal diterpene cyclase. The hydrocarbon produced by the enzyme was detected in the headspace of the transgenic lines by GC-MS analysis. This result indicates that Arabidopsis plant is able to release heterologous diterpene to the air. We are currently investigating the effect of the heterologous diterpene on the emission and perception of ent-kaurene by Arabidopsis plants.

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In rice (Oryza sativa) genome sequences, there are 3 and 7 genes encoding type-B and type-A diterpene cyclases, respectively. OsCPS1 (type B) and OsKS1 (type A) are responsible for biosynthesis of gibberellins. OsCPS/OsCyc2, OsCPS4/OsCyc1 (type B), OsKS4, OsDTC1/OsKS7, OsDTC2/OsKS8, OsKS10 (type A) are for diterpene phytoalexins, such as momilactones, oryzalexins and phytocassanes. Transcript levels of the 6 genes dramatically increased after ultra-violet (UV) irradiation. On the other hand, Arabidopsis genome sequences contain only two diterpene cyclase genes, which are responsible for gibberellin biosynthesis, AtCPS (type B) and AtKS (type A). In this study, we attempt to isolate and characterization of cDNAs encoding diterpene cyclase from wheat (Triticum aestivum cv. Norin 61-gou) in order to address the question whether multi gene family encoding diterpene cyclase is specific to rice. Two and 4 fragments of cDNAs encoding type-B and type-A diterpene cyclases, respectively, were amplified by RT-PCR using primers designed based on the EST data base sequences. Expression levels of these genes did not increase after UV irradiation. And no peaks of diterpene cyclic hydrocarbons were detected by full-scan GC/MS in the extract from leaves of wheat, which were irradiated by UV. We plant to carry out in vitro enzyme assay using heterologusly expressed recombinant proteins from the wheat cyclase genes.

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We have isolated and characterized cDNAs, OsCyc1, OsCyc2, OsKS4, OsKS7, OsKS8, OsKS10, encoding syn-CDP synthase, ent-CDP synthase, 9β-pimara-7, 15-diene synthase, ent-cassa-12, 15-diene synthase, stemar-13-ene synthase, and ent-sandaracopimaradiene synthase, responsible for rice (Oryza sativa) diterpene phytoalexin biosynthesis. In rice genome sequences, we found other translatable diterpene cyclase genes; OsCPS1, OsKS1, OsKS5, and OsKS6. It has already been suggested that OsCPS1 and OsKS1 are responsible for gibberellin biosynthesis. In this study, we show that recombinant proteins expressed in E.coli from OsKS5 and OsKS6 converted ent-CDP into ent-pimara-8(14), 15-diene and ent-kaur-15-ene like substance, respectively. These results suggest that the two genes are responisible for biosynthesis of neither gibberellins nor phytoalexines. In addition, we attempted to investigate the subcelluar localization of diterpen cyclases, responsible for biosynthesis of gibberellins or phytoalexins, using GFP system. Our results suggest that OsCyc2, OsKS1, OsKS8, OsKS10 shoud localize in plastids, like gibberellin biosyntheis ditrpene cyclases in other plants. Studies on the subcelluar localization of OsCyc1, OsCPS1, OsKS4, and OsKS7 are now progressing.

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In suspension-cultured rice cells, four types of diterpenoid phytoalexins, momilactones A, B, oryzalexin S, (-)-phytocassans A-E and oryzalexins A-F, are produced in response to treatment with the chitin elicitor N-acetylchitooctaose. Until now, we isolated and identified six diterpene cyclase genes involved in biosynthesis of diterpenoid phytoalexins (OsCyc1, 2, OsDTC1, 2 and OsKS4, 10). We report identification of cis-acting elements responsible for the elicitor-inducible expression of two diterpene cyclase genes, OsDTC2 and OsKS4, in rice cultured cells. OsDTC2 and OsKS4 encode stemar-13-ene synthase and 9βH-pimara-7, 15-diene synthase that are involved in biosynthesis of oryzalexin S and momilactones A, B, respectively. To identify cis-acting elements responsible for chitin elicitor-inducible expression of OsDTC2, the OsDTC2 promoter was analyzed using dual luciferase assay in suspension-cultured rice cells. Deletion and mutation analysis revealed that six W-box motifs between -1634 and -1549 from the translational start site are responsible for the elicitor-inducible expression of OsDTC2. Using the similar methodology, the OsKS4 promoter was analyzed. The results suggest that a TGACGT motif between -1224 and -991 is responsible for the elicitor-inducible expression of OsKS4. The above results suggest that WRKY and bZIP transcriptional factors are involved in elicitor-inducible expression of OsDTC2 and OsKS4, respectively.

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Diterpenoid phytoalexins such as momilactones and (-)-phytocassanes are produced in suspension-cultured rice cells treated with a chitin elicitor and in the rice plant treated with UV irradiation. The diterpenoid phytoalexins are biosynthesized from geranylgeranyl diphosphate via ent- or syn-copalyl diphosphate (CDP) and diterpene hydrocarbons. Most of biosynthetic steps from the diterpene hydrocarbons into respective diterpenoid phytoalexins are considered to be catalyzed by P450 enzymes. However, functional analysis of the P450 enzymes involved diterpenoid phytoalexins has not been reported. We found that there are six P450 genes, ent-CDP synthase gene (OsCyc2), and ent-cassa-12, 15-diene synthase gene (OsDTC1) in a 215 kb region on chromosome 2, and that there are two P450 genes, syn-CDP synthase gene (OsCyc1), and 9βH-pimara-7, 15-diene synthase gene (OsKS4) in a 155 kb region on chromosome 4. Expression of the above eight P450 genes on chromosomes 2 and 4 were induced in suspension-cultured rice cells treated with a chitin elicitor and/or in the rice plants irradiated UV light. RNAi of the two P450 on chromosome 4 resulted in suppression of production of momilactones, suggesting that these two P450 genes in addition to OsCyc1 and OsKS4 are involved in momilactone biosynthesis. We also detected a chitin elicitor-inducible dehydrogenase gene in the 155 kb gene cluster on chromosome 4 that might encode momilactone A synthase.

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WRKY proteins are a large family of plant-specific transcription factors that specifically bind to the W-box elements. Many WRKY proteins have been suggested to have regulatory functions in the response to pathogen infection and other stresses. In suspension-cultured rice cells, PR proteins and phytoalexins are produced in response to exogenously applied elicitors from the pathogen Magnaporthe grisea. By means of microarray analysis, we isolated the cDNAs encoding two chitin elicitor-induced WRKY genes, OsWRKY53 and OsWRKY71, from suspension-cultured rice cells. Expression of these genes were induced by a sphingolipid elicitors as well as a chitin elicitor in suspension-cultured rice cells, and were induced in rice plants infected with M. grisea. By means of gel mobility-shift assay, we confirmed that these WRKY proteins could specifically bind to the W-box motifs. The survey of target genes of OsWRKY53 and OsWRKY71 was performed by microarray analysis using OsWRKY53 or OsWRKY71-overexpressing mutants. As the results of microarray analysis, several defense-related genes such as PR protein genes and WRKY genes were upregulated, and the presence of W-boxes was observed in the 5' upstream regions of the upregulated genes. Moreover, the expression of these defense related genes were induced by a chitin elicitor in suspension-cultured rice cells. These results strongly suggest that OsWRKY53 and OsWRKY71 are involved in elicitor-induced defense responses in rice.

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Glucosylated plant hormones occur in many plant species, and they are supposed to be catabolized forms to maintain the hormones at suitable levels in tissues, or the forms for translocation. However their physiological roles remain unclear. We cloned cDNAs encoding glucosyltransferases (GTases) that catalyze glucosylation of phytohormones from developing seeds of morning glory (Ipomoea nil) to have information on the metabolic dynamics of phytohormones in seed development. After degenerate-PCRs and RACEs using primers for the conserved region in many orthologous GTase genes and using cDNAs prepared from immature seeds at various maturities (3-33 day-after-flowering) of morning glory, we obtained nine full-length cDNAs, prepared GST-fused recombinant proteins using an E. coli expressional system, and studied their enzymatic activities. The recombinants were incubated with gibberellins (GAs), zeatin, indole-3-acetic acid (IAA), abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA) in the presence of ^<14>C-UDP-glucose as a sugar donor, and the radioactive metabolites were visualized on imaging plates after TLC. A recombinant, designated InGTase1, showed a GTase activity by converting ABA, IAA, JA, and SA to polar metabolites.

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A putative O-methyltransferase cDNA was cloned from UV-irradiated rice leaves based on the amino acid sequence reported as that of naringenin 7-O-methyltransferase, which is involved in the biosynthesis of rice phytoalexin, sakuranetin [Plant Sci., 155, 213-221 (2000)]. However, the recombinant protein (approximately 41 kDa) expressed in Escherichia coli showed no naringenin 7-O-methyltransferase (NOMT) activity but showed caffeic acid 3-O-methyltransferase (COMT) activity. Thus the gene was designated as caffeic acid 3-O-methyltransferase gene (OsCOMT1). The result of the tblastn search of the GenBank rice genome DNA database indicated that the rice plant contained only one copy of the COMT gene. And it was found to be located at 27.1 cM on chromosome 8 by searching the rice EST map. The result of the quantitative RT-PCR showed that OsCOMT1 was expressed in all tissues tested and was not affected by CuCl_2, jasmonic acid and UV treatments. The optimal pH was found to be 7.5 in Tris-HCl buffer. The apparent K_m values for caffeic acid and S-adenosyl-L-methionine were 69 and 51μM, respectively. The enzyme also methylated flavonoids which have adjacent two hydroxyl groups in the B ring. The COMT activity of crude protein extract was detected at almost a constant level in different plant growth stages and different leaf positions; only a slight increase was detected with increasing age. No induction effect was found by CuCl_2, jasmonic acid and UV treatments.

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Glycyrrhizin, a sweet triterpene saponin of Glycyrrhiza uralensis, exhibits various pharmaceutical effects. To produce a better quality of pharmacologically active G. uralensis plant by metabolic engineering approach, it is necessary to understand the regulatory mechanism of glycyrrhizin biosynthesis. For this purpose, we are investigating intermediates and candidates for biosynthetic genes for glycyrrhizin biosynthesis. Since glycyrrhizin should be biosynthesized via β-amyrin by a series of oxidation and glycosylation, several oxygenated and/or glycosylated β-amyrin derivatives are examined as the putative biosynthetic intermediates. The stolon of G. uralensis was analyzed by GC-MS and LC-MS using authentic samples prepared by organic synthesis. All the putative biosynthetic intermediates are detected, strongly supporting the proposed pathway. We genetated over 12,000 expressed sequence tags (ESTs) derived from stolon. Database search revealed that 27, 12 and 40 EST clones showed significant similarities to known P450, dioxygenase, and glucosyltransferase or glycosyltransferase sequences, respectively.

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Coumarins are often found as plant secondary metabolites in the plant kingdom. Their exact roles in plants are unclear. However, they are thought to play some roles in plant defense due to the induction of their biosynthesis following various stresses as well as their antimicrobial activities. We detected the coumarins, scopoletin and its glucoside, scopolin, in Arabidopsis thaliana roots. Details of coumarin biosynthesis pathway in plants are also unclear, although the coumarins are known to be biosynthesized via phenylpropanoid pathway. We quantified the coumarins in the T-DNA insertion mutants of p-coumaroylshikimate/quinate 3'-hydroxylase (C3'H) and caffeoyl CoA O-methyltransferase (CCoAOMT) of A. thaliana to collect information on the coumarin biosynthesis. Analysis of the coumarins in the T-DNA insertion mutants was performed by HPLC and LC-ESI/MS/MS. Mutation of C3'H and CCoAOMT1 resulted in change of the profiles of the coumarins. The level of skimmin, a glucoside of umbelliferone, rose to be detected in the c3'h roots. The levels of scopoletin and scopolin in the c3'h roots and in the ccoaomt1 roots were 〜5% and 〜15% of those in the wild type, respectively. These results suggest that scopoletin and scopolin are biosynthesized via C3'H and CCoAOMT in A. thaliana.

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All terpenoids are derived from dimethylallyl diphosphate (DMAPP) and/or isopentenyl diphosphate (IPP) in organisms. Isotopelabeled DMAPP and IPP will be powerful tools for screening and biosynthetic studies of terpenoid compounds. Here, we report the enzymatic preparation of the isoprenoid compound, farnesyl diphosphate (FDP). [U-^<13>C_6] mevalonic acid (MVA) was prepared by incubation of [U-^<13>C_6] glucose with an yeast Saccharomycopsis fibuligera. To synthesize FDP from MVA, five enzymes are involved; mevalonate kinase (MVK), phosphomevalonate kinase (PMVK), diphosphomevalonate decarboxylase (DPMVDC), IPP isomerase (IPPI) and farnesyl diphosphate synthase (FPS). cDNAs encoding these enzymes were cloned depending on the information of genome database of Neurospora crassa. Each open reading frame of these cDNAs was ligated into pQE30 vector to produce the recombinant proteins in E. coli. Recombinant proteins of MVK, DPMVDC, IPPI and FPS were produced as soluble proteins and recovered from E. coli cells. Since recombinant PMVK was produced as the inclusion body, the ORF was ligated into pGEX vector to produce it as the glutathion-S-transferase fusion protein. Enzymatic preparation of FDP, DMAPP and IPP are in progress.

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Some specific kinds of plants are known as hyperaccumulators of heavy metals. It was reported that Chinese brake fern (Pteris vittata L.) contained large amounts of arsenic (As : ca 22,000μg/g dry weight) when grown in contaminated soils. In the present study, Microbeam Synchrotron-Radiation X-ray Fluorescence (micro-SR-XRF) analysis was applied to the fern of in order to reveal the distributions of elements in the plant's tissues and cells. The chemical speciation of As at the cellular level was also carried out by XAFS analysis utilizing the microbeam. The results showed that the fern a took up As (V) from the cultivation medium and accumulated arsenic inside their cells in the As (III) form.

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Pyrroloquinoline quinone (PQQ), an element discovered as a novel cofactor in bacterial dehydrogenase, has been identified as an important player in mammals and now recognized as a new vitamin. PQQ is reported to be in common foods, such as meat, fruits, vegetables, and crops with HPLC or GC/MS analyses, but the quantitative analysis of PQQ has been difficult because it readily forms hydrate and/or adducts with nucleophilic reagents such as amino acids. Here, we report a method for quantifying various forms of PQQ and its derivatives in plants. This methods will be an important tool for solving a question whether PQQ is synthesized in plants or not, and how plants synthesize PQQ. Multiple reaction monitoring (MRM) of LC/MSMS was used for that purpose because this system has an advantage over the traditional LC or GC/MS in that MRM has a shortened analysis time and can eliminate the need for a derivatization step. We used Arabidopsis thaliana grown in germ-free environments on MS agar plates as our sample. The results demonstrated that PQQ and its various derivatives exist in Arabidopsis. To date, it has long been thought that only bacteria biosynthesize PQQ, but above result could suggest that higher plants can synthesize PQQ. Highly homologous genes with bacterial PQQ metabolic enzyme genes had not been identified in Arabidopsis genome yet, but a new analytical method for PQQ described above will allow us to uncover PQQ biosynthesis and function in plants.

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ALA promotes the growth of plants and their yield increase. Although it is assumed that concentration of endogenous ALA is very low, there are few reports studied in detail. Therefore we investigated the concentration and distribution of ALA in plants. ALA was found in all plants determined in this study. ALA was found at high concentration in the central parts of the fluits. Furthermore, the concentration was much higher in young fluits than matured fluits.

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Opening and closing of stomata are controlled by various conditions. The substance controlling stomatal closing is elucidated to be an abscisic acid. However, the substance controlling the stomatal opening is not completely clear. The aim of our study is to search for the endogenous substance that triggers stomatal opening in higher plants. We searched the active compound in several plants extracts using bioassay with epidermal strips of Commelina communis. Bioassay-guided fractionation of a 50% MeOH extract of Ipomoea tricolor enabled the isolation of two active compounds, cis-12-oxophytodienoic acid (OPDA) and sn-1-O-(cis-12-oxophytodienoyl)-sn-3-O-(β-galactopyranosyl)glyceride. These compounds enhanced stomatal opening of Commelina communis in darkness at micromolar concentrations. These results suggest that these compounds play a crucial role in stomatal opening. OPDA is a known precursor of jasmonic acid. Now, we examine the stomatal opening activity of other jasmonates.

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In the course of our screening search for plant growth regulators among fungal metabolites, we found that the presence of root growth promoters of lettuce seedlings in the filtrate of Penicillium citrinum. Two new compounds possessing a coumarin skelton were isolated and named as 304f88m and 304f26. Each structure was supposed to be a dimer by the physical and chemical evidence. The effects of these compounds were examined by lettuce and rice seedlings. As the result, these compounds accelerated the root growth of both seedlings at the concentration of 300mg/l.

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In general, the way of allelochemicals are released from plants to the environment is as follows : 1) exudation from roots, 2) leaching from plants by rain, 3) decomposition of residues. It is reported that the aqueous leachate of wheat straw and the extract of decomposed wheat straw inhibit the growth of several plant species. Some plant growth inhibitory phenolic acids were detected in the soil incubated with wheat straw, i.e. decomposed wheat straw. Since these studies were concentrated on the extracts of decomposed wheat straw, the information of the plant growth inhibitory substance(s) leached from the wheat straw is limited. In this study, we focused on the allelopathic potential of aqueous leachate of wheat straw and isolated and identified the plant growth inhibitory compound(s) from aqueous leachate of wheat straw. When lettuce, cress, rice and wheat were incubated with leachate of wheat straw, seedling growth, especially the root growth of lettuce and cress were inhibited. The inhibitory activity was stronger as the content of leachate of wheat straw was larger. These results suggested that allelochemical(s) inhibiting the root growth of lettuce and cress are leached from the wheat straw into the water. Two potent compounds were isolated from the leachate of wheat straw and identified as Syringoylglycerol 9-O-β-D-glu-copyranoside and L-tryptophan by spectral analyses.

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【Object】 Molisch (1937) coined the term "allelopathy" to refer biochemical interactions between plants including microorganisms. Since then, various chemical agents involved in allelopathy have been isolated and identified from different adult plants. However, information on allelopathy during seed germination is limited. We have hitherto reported on the stimulatory allelopathy during the seed germination stage. As the stimulatory allelochemicals, lepidimoide was isolated and identified from the exudates of cress seeds, and arctigenin and arctigenic acid from those of burdock seeds, respectively. In the present study, we report on the isolation and identification of stimulatory allelochemicals exuded from germinating oat seeds. 【Methods】 We got the exudates of oat seeds by sowing them in water. The culture solution was purified by Sep-Pak cartridge chromatography and HPLC. Biological activities were tested using the bioassays of cockscomb and lettuce hypocotyls growth. The isolated stimulatory allelochemicals were identified by NMR and MS. 【Results】 The exudates of oat seeds were separated into 0, 20, 40, 60, 80 and 100% methanol in water by C_<18> Sep-Pak. The 0 and 20% methanol fractions showed the strong growth-promoting activity. The 20% fraction was subjected to HPLC (ODS). A peak (Rt. 10-12.5min) that shows growth-promoting activity were isolated and its identification is in progress.

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Ailanthus altissima has been known to be allelopathic plant since no plant can grow under the tree. Heisey et al (1990) showed that crude extracts of root and stem bark of A. altissima exhibited strong herbicidal activity. We confirmed plant growth inhibitors against Lactuca sativa in the aqueous extract of A. altissima bark. The compound was isolated and identified to be ailanthone known as quassinoid or allelopathic substance. Here, we report plant growth activities of ailanthone against Lactuca sativa, Pariucam crus-galli, Raphanus sativa L. and Daucus cavota L. in detail.

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We reported that clomazone and ketoclomazone inhibited 1-deoxyxylulose-5-phosphate synthase (DXS) in 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway and induced chlorosis to plants. As MEP pathway is indispensable for the formation of isoprenoids not only in plants but also in several bacteria, inhibitors of MEP pathway in plants can be inhibitors of MEP pathway in bacteria and affect the growth of bacteria. That is, such inhibitors could be useful as medical drugs. For example, the target site of fosmidomycin, an antimaralial drug, is the second step of MEP pathway. In this context we started the chemical modification of keto-clomazone and found that some of the derivatives inhibited DXS from E.coli. They also induced chlorosis to plants and this was recovered by the coapplication of DX with inhibitors, suggesting that the target site of these chemicals should be DXS. The relationships between DXS inhibition and chlorosis will be discussed.

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Effect of 5-aminolevulinic acid on the efficiency of dry-matter production in cabbage pot-seedlings was investigated. The rates of Po, CGR, RGR and NAR in cabbage seedlings were increased by 5-ALA treatment. This result suggest that the 5-ALA treatment was very useful for the good pot-cultivation of cabbage seedlings.

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The changes of flower shape and pigmentation were observed in petunia corollas treated with 0.5mM glyphosate. The corollas treated with glyphosate increased free amino acid content to 136% of control. Free amino acid profiles in petunia corollas revealed that glyphosate had no significant effect on aromatic amino acid levels but increased level of proline. Soluble protein content in glyphosate-treated corollas did not cause any significant changes. The contents of soluble phenolics, lignin, IAA in the corollas were not significantly affected by glyphosate treatment. In contrast, glyphosate reduced the nitrate content of petunia corolla to 45% of control. And glyphosate reduced the RNA content of petunia corollas to 63% of control. However, the DNA content in glyphosate-treated corollas was similar to that of control. Low concentrations of glyphosate did not show any phytotoxic effects on the whole plants and any remarkable changes on aromatic amino acid metabolism and protein synthesis. However, glyphosate reduced RNA content of petunia corollas and changed flower symmetry from actinomorphic type to zygomorphic type. The results of nonprotein nitrogen metabolism in glyphosate-treated petunia corollas suggested that low concentration of glyphosate might effected on the regulation of the corolla growth and the flower symmetry through the reduction of RNA biosynthesis.

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