Growth and flowering regulation of horticultural plants such as fruit trees, vegetables and ornamental plants is one of the most important subjects for the management of agricultural production. Gibberellin (GA) is known as a big family in plant hormones which participate in many aspects of plant growth and flowering. In this study, GA biosynthesis and metabolic pathways were identified in many horticultural plants and then, possibility of growth and flowering regulation was investigated by changing GA biosynthesis and metabolism.

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Plants in semi-arid regions often have unique biochemical mechanisms to tolerate environmental stresses such as temperature extremes, draught, strong light, and salinity. In some cases, secondary metabolites have been shown to play an important role in such tolerance mechanisms. Phloroglucinol derivatives, grandinol and homograndinol, found in Eucalyptus grandis are inhibitors of germination and also of photosynthetic electron transport. Chemical modifications of these compounds afforded extremely potent photosystem II (PSII) inhibitors. These PSII inhibitors, however, are not herbicidal because they do not inhibit turnover of D1 protein. These phloroglucinol derivatives exhibit abscisic acid (ABA)-like, antiinflammatory, and anti-tumor-promotion activities. Seeds of root parasitic plants, Striga and Orobanche, require exogenous stimulants to germinate. Orobanchol and alectrol, germination stimulants for Orobanche minor, have been isolated and characterized from root exudates of its host, red clover. The production of these stimulants by red clover roots is promoted by nitrate but is inhibited by ammonium and phosphate. Amounts of orobanchol in the red clover root exudates were quantified by LC/MS/MS, and actively growing roots were found to be major parts of stimulant production.

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There has been limited information how BR biosynthesis is regulated and where BRs are synthesized and used. To address this issue, we isolated two BR-6-oxidase genes (BR6ox) from Arabidopsis and analyzed their gene expression with distribution of endogenous BRs. The results suggested that BRs are synthesized in all of the organs tested, but most actively synthesized in young actively developing organs. To further gain insight into the site of BR biosynthesis, transgenic Arabidopsis carrying reporter, BR6ox-GUS gene were produced. Using these lines, the site of biosynthesis and action is now under investigation at the cellular level. Although recent studies of biosynthesis and sensitivity mutants have provided insights into the functions of BRs, the mode of action of BRs is poorly understood. Using microarray analysis, we identified BR-regulated genes. The list of BR-regulated genes includes transcription factor genes, auxin-related genes, P450 genes, and genes implicated in cell expansion and cell wall organization. These results provide a comprehensive view of the actions of BRs, using BR-regulated genes as molecular markers. Despite numerous physiological studies addressing the interactions between BRs and auxin, little is known about the underlying molecular mechanisms. Using auxin-reporter gene system and auxin-related mutants, signaling interaction between auxin and BR have been investigated.

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[in Japanese]

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We have found that etiolated pea (Pisum sativum L. cv. Alaska) seedlings grown under true and simulated microgravity conditions on a 3-D clinostat showed automorphosis and automorphosis-like growth and development, respectively. We have also suggested an important role of auxin polar transport for automorohosis in etiolated pea seedlings since auxin polar transport activity of etiolated pea seedlings were substantially inhibited under true and simulated microgravity conditions. This suggestion was confirmed by the fact that automorphosis was phenocopied by the application of auxin polar transport inhibitors. In Arabidopsis auxin polar transport has considered to be regulate by efflux and influx carrier proteins located in plasma membranes, AtPINs and AtAUX1,respectively. In order to know how gravistimuli affect auxin polar transport in etiolated pea seedlings at molecular levels, strenuous efforts has been done, resulting in successful identification of PsPIN2 and PsAUX1 whose products are considered to locate in plasma membrane and to be involved in auxin transport. Significantly high levels in homology were found on deduced amino acid sequences among PsPIN2,PsPIN1 and AtPINs, and also found between PsAUX1 and AtAUX1. The expression of PsPIN1 and PsAUX1 were suppressed under simulated microgravity conditions. On the other hand, the expression of PsPIN2 was affected little under simulated microgravity conditions. These results suggest that PsPIN1 and PsAUX1 are genes encoding putative efflux and influx carrier proteins for auxin polar transport, respectively although the function of PsPIN2 has not been clear yet. Based on the results described above, a possible role of PsPINs and PsAUX1 for auxin polar transport in etiolated pea seedlings will be discussed.

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In order to identify the gibberellin (GA_3)-regulated protein factors to regulate the mechanical properties of cell walls, we analyzed cell-wall proteins by SDS-PAGE extracted from pea (Pisum sativum L. cv. Alaska) roots, which had been treated with or without GA_3 and/or Ancymidol. GA_3-affected protein bands were separated, N-terminal amino acids were sequenced, and their homologies were surveyed on data bases. Eight protein bands were sequenced among 11 bands separated. Homologous proteins found on data base were as follows : GA_3-enhanced bands : pectin methylesterase (Pisum sativum, Vigna radiata), peroxidase (Spinacia oleracea, Glicine max, Arabidopsis thaliana) GA_3-suppressed bands : chitinase class III; (Vigna angularis, Nicotiana tabacum, Oriza sativa), cystein proteinase (Pisum sativum, Arabidopsis thaliana), peroxidase (Spinacia oleracea, Medicago truncatula). GA_3-enhanced methylesterase and peroxidase were suggested to participate in cell wall modification for GA-induced growth. GA_3 was suggested to regulate the amount of these apoplastic proteins. Further identification of unsequenced protein bands is in progress.

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We have isolated two cDNAs, OsCyc1 and OsCyc2,encoding diterpene cyclases that are involved in phytoalexin biosynthesis from rice (Oryza sativa L. cv. Nipponbare) leaves. Oscyc1 and OsCyc2 encode syn-copalyl diphosphate (CDP) synthase and ent-CDP synthase, respectively. In this report, we isolated a cDNA, OsCPS, encoding ent-CDP synthase responsible for the biosynthesis of gibberellins (GAs) in rice. It has been shown that a loss-of-function mutation in the OsCPS gene results in a GA-deficient phenotype. Using a bacterial expression system, it was suggested that the translated product of OsCPS could convert geranylgeranyl diphosphate into ent-CDP. Transcript levels of OsCPS did not change in rice leaves after ultra-violet (UV) irradiation, whereas those of OsCyc1 and OsCyc2 drastically increased in response to UV treatment. These results suggest that two ent-CDP synthases, one for the biosynthesis of GAs and the other for phytoalexins, are encoded by two distinct genes in rice.

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We have reported the localization of active gibberellins (GAs) and GA-inducible α-amylase (PnAmy1) around the starch grains in the integuments of immature seeds of Pharbitis nil cv. Violet. We also proposed that the GAs play an important role for the seed development of P. nil through the induction of α-amylase and degradation of starch temporarily accumulated in integuments. To examine whether the similar process proceeds in the seed development of other dicotyledonous plants, we analyzed immature seeds of Arabidopsis thaliana, because further experiments by transformation can be applied for this plant. We harvested green siliques at different maturities from wild type plant (Col.), and prepared their paraffin-embedded sections. The KI/I_2 staining of the embedded sections showed the positive blue stains in seed coat where some granules were observed at the same time. In accordance with the maturation of siliques, both of the positive stains and the granules harmoniously disappeared. The expressions of GA 20-ox genes and a-amylase genes were analyzed by using RT-PCR technique. All specific primers required for the analysis were designed based on the sequence information from online database. In situ hybridization analyses are proceeding for some clones that are expressed at comparatively high levels.

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Seed dormancy and germination are controlled by multiple environmental and endogenous factors. In Arabidopsis, both gibberellin (GA) and cold treatment (imbibition at 4℃ in the dark) promote seed germination. We have previously shown that the GA biosynthesis gene, AtGA3ox1,encoding a GA3-oxidase, is upregulated in response to cold treatment. Consistent with the increase in AtGA3ox1 expression, endogenous GA_4 content and the level of GA-responsive transcripts were elevated following cold treatment. To predict how cold treatment stimulates germination through GA action, GA-dependent gene expression during cold treatment was monitored using DNA microarray analysis. We have previously analyzed gene expression profiles during (A) wild-type seed germination at 22℃ in the light, and (B) germination of GA-deficient ga1-3 mutant seeds upon exogenous GA treatment (22℃, light). These microarray data indicated that the majority of GA-responsive genes during cold treatment were categorized into a gene group of which expression is up-or down-regulated at early time points after imbibition of wild-type seeds (A). We also found that 25% of GA-regulated genes during cold treatment have previously been classified as GA-responsive genes in ga1-3 seeds (B). The rest of GA-regulated genes during cold treatment appear to be unique to the low temperature environment. In addition, we noted that only a few genes responded to 4℃ commonly at seed germination and seedling stages, based on microarray data reported by other groups. These results suggest that both GA biosynthesis and response are modulated under different environmental conditions to control seed germination, and that the response to low temperature is altered at different developmental stages.

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Germination of lettuce (Lactuca sativa L. cv. Grand Rapids) seeds is regulated by phytochrome, which is mediated by gibberellin (GA). We have shown that expression levels of LsGA3ox1 increased and those of LsGA2ox2 decreased after red-light irradiation, resulting in an increase in the levels of physiologically active GA, GA_1,during germination of lettuce seeds. It has been reported that application of abscisic acid (ABA) suppresses germination of the lettuce seeds treated with red light or GA. In this study, to investigate the mechanism for the suppression of germination by application of ABA, we analyzed expression patterns of genes encoding GA biosynthesis and deactivation enzymes in imbibed seeds irradiated with far-red light or red light. Quantitative RT-PCR analysis showed that transcript levels of LsGA3ox1 decreased, whereas those of LsGA2ox2 increased, by ABA treatment. Transcript levels of other GA-related genes that we examined were not altered by ABA under both light conditions. These results suggested that application of ABA affected GA biosynthesis and catabolism genes, of which expression is regulated by phytochrome. Such changes in transcript levels might result in the suppression of the increase in GA_1 content even in red-light-treated seeds.

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Arabinogalactan-proteins (AGPs) are proteoglycans rich in hydroxyproline. Glycosylphosphatidylinositol (GPI)-anchoring is another characteristics of AGPs, suggesting their possible involvement in signal transduction. We have shown that plasma membrane-AGPs are essential to GA signal transduction in barley aleurone layers. AGPs extracted from aleurone layers are separated into two major fractions by RP-HPLC (FrA of a smaller tR and FrB of a larger tR). FrB was specifically detected in aleurone layers. Characterization of AGPs present in aleurone layers in molecular level is important to understand their roles in GA signaling. We examined AGPs in rice brans because of their availability in larger scale than barley, and of another advantage of the accessibility to rice database. We purified AGPs in rice brans by using β-Yariv reagent that binds AGPs selectively. Rice brans showed two major AGPs fractions corresponding to FrA and FrB on RP-HPLC as barley did. Peptide sequencing of FrA showed two different N-terminal sequences. One of them corresponded to two different ORFs encoding a same mature AG-peptide backbone. The other was assigned to a classical AGP-like protein. AGPs in membrane fraction of rice brans were also studied. Membrane extract gave a β-Yariv-reactive fraction (FrC) of a larger tR than FrB on RP-HPLC. Treated with PI-PLC, the β-Yalive reactive peak in FrC shifted to a smaller tR identical to that of FrA. This result indicates that FrC contains GPI-anchored AGPs and at least part of AGPs in FrA are derived from those in FrC by cleavage of GPI-anchor. Identification of AGPs in FrB and functional analysis of aleurone AGPs have yet to be done to show their involvement in GA action.

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Previous cDNA microarray analysis data from the xylem tissue of gibberellin (GA) over-producing transgenic hybrid aspen (Populus tremula×P. tremuloides) with increased secondary growth, has shown pectate lyase (Pel) and arabinogalactan peptide (AGp) encoding genes to be highly up-regulated when compared to WT. These genes have been demonstrated to be highly expressed in the cell division and cell expansion zones of WT developing xylem. These results suggest that Pel and AGp are important in the early stages of secondary growth and that the expression might be affected by GAs. We have cloned putative full-length cDNA clones of both PttPell and PttAGp1 from hybrid aspen. The PttPell deduced amino acid sequence exhibited high similarity to several plant Pel, and the PttAGp1 deduced amino acid sequence showed high homology to Arabidopsis AGp's AtAGP12,AtAGP13 and AtAGP14,which have very short GPI-anchored protein backbones. To evaluate the role of GAs in inducing the transcription of PttPell and PttAGp1,the expression patterns of respective genes in (GA)_4 infiltrated hybrid aspen plants compared to non-treated plants have been examined. The results showed that the expression level of both PttPell and PttAGp1 have been up-regulated by GA treatment. To further investigate the role of PttPell and PttAGp1,the genes were independently over-expressed under the CaMV 35S promoter in Arabidopsis thaliana. Finally, experiments are underway to analyse the enzyme activity of PttPell using recombinant proteins, and to determine the protein backbone amino acid sequence of PttAGp1 isolated from hybrid aspen by using LC/MS.

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In dicotyledonous plants including Arabidopsis, brassinosteroids deficiency induces morphological features of light-grown plants in the dark, i. e. short hypocotyls, expanded cotyledons, and true leaves, in a dose-dependent manner. While how brassinsteroid deficiency does affect the growth of monocotyledonous plants in the dark is still not clear. In order to investigate the effect brassinosteroid deficiency in monocotyledonous plants, a brassinosteroid biosynthesis inhibitor was applied to rice in the dark and the morphological change was intensively observed and the expression of genes induced under light condition was examined.

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Brassinazole is a potent brassinosteroid (BR) biosynthesis inhibitor and is shown to be useful to elucidate the function of BR. We have modified the chemical structure of brassinazole to improve selectivity. Development of the derivatives that have rigid conformation could lead to creation to the drugs without side effects. Therefore, introduction of double bond to the chemical structure of brassinazole was conducted. Moreover, these compounds have been synthesized using the rational approach of Topliss' decision tree. Generally, this approch allows us to obtain the most active derivatives in a few steps. A variety of substituents introduced at phenyl ring have investigated in this series, together with the determination of the effects of double bond of brassinazole is isomerization (E and Z geometry). These compounds were tested against cress stem elongation. As a result, inhibition of BR biosynthesis by these new compounds appears to be linked to combination of E geometry and 4'-chrorine at phenyl ring. The new lead for BR biosynthesis inhibitory activity has been predicted and will be used in further structure-activity relationship studies on brassinazole.

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We had already identified new brassinosteroid-related photomorphogenesis mutant bill from EMS-mutation lines, and bil5 from fast neuron-mutation lines. Trial to screen new mutants as Brz-insensitive-long hypocotyl in the dark was applied to activation-tagging-mutation lines. Furthermore, we also tried to screen mutants brassinosteroid-regulated chloroplast development and leaf formation. In the alignment of these mutants and mutation genes, we would like to clarify the mechanism of brassinosteroid signaling.

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In order to analyze the mechanism of brassinosteroid signal transduction in detail, we screened for mutants that showed resistance to brassinosteroid biosynthesis inhibitor Brz220 effects. In the Brz 3μM medium, that wild-type plant showed a quite short hypocotyl, but the mutant bill showed a hypocotyl length as long as the wild type grown on the unsupplemented medium. The phenotype of bil5 was specially related with chloroplast development and vascular tissue formation. Relation of Brassinosteroid and bill to chloroplast development was also analyzed in detail. In the alignment of these mutants and mutation genes, we would like to clarify the mechanism of brassinosteroid signaling.

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In higher plants, isoprenoids are synthesized via mevalonate pathway (MVA pathway) and non-mevalonate pathway. 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) is a key enzyme of MVA pathway and is encoded by two genes, HMG1 and HMG2,in Arabidoposis. To understand the role of HMGR on plant development, we analyze hmg1 and hmg2 mutants. hmg1 mutant showed dwarf, male sterile and early senescence phenotypes. The microscopic analysis and the gene expression analyses revealed that dwarf phenotype of hmg1 is due to the inhibition of cell elongation. Sterility in hmg1 is due to a loss of the ability of germination in pollen. It was found that the lipids supplied from tapetum cells are important for the development of pollen. HMG1 is expressed in tapetum cells and organelles rich in lipids are decreased in hmg1 tapetum cells. Therefore, it was suggested that hmg1 pollen can not germinate because of decreased lipids supplied from tapetum cells. SAG12,the senescence marker gene, is expressed earlier in hmg1 than in a wild type (WT). Exogenously applied trans-cytokinins, hormones known to inhibit senescence, inhibited the early induction of SAG12 expression in hmg1,however, levels of trans-cytokinins were not lower in hmg1. WT plants treated with squalene synthase inhibitor mimicked hmg1 phenotypes but WT plants treated with brassinosteroids synthase inhibitor did not show similar phenotype with hmg1. Chemical complementation also showed triterpenoids downstream of squalene is contribute to the pleiotropic phenotype of hmg1. The sterol levels in hmg1 mutants were lower than in the WT. These findings suggest that HMG1 is a critical role in triterpene biosynthesis, and that sterols and/or pentacyclic isoprenoids contribute to cell elongation, senescence and fertility.

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Brassinosterois (BRs) play important roles throughout plant growth and development. Despite the importance of clarifying the mechanism of BR-related growth regulation in cereal crops, BR-related cereal mutants have been identified only in rice. We previously found that semi-dwarf barley accessions carrying the "uzu" gene, called "uzu" barley in Japan, are non-responding for brassinolide (BL). We then performed chemical and molecular analyses to clarify the mechanisms of uzu dwarfism using isogenic line pairs of uzu gene. The response of the uzu line to BL was significantly lower than that of its corresponding normal line. Measurement of BRs showed that the uzu line accumulates BRs, similar to known BR-insensitive mutants. The marker synteny of rice and barley chromosomes suggests that the uzu gene may be homologous to rice D61,a rice homolog of Arabidopsis BR-insensitive 1 (BRI1), encoding a BR-receptor protein. A barley homolog of BRI1,HvBRI1,was isolated by using degenerate primers. A comparison of HvBRI1 sequences in uzu and normal barley varieties showed that the uzu phenotype is correlated with a single nucleotide substitution. This substitution results in an amino acid change at a highly conserved residue in the kinase domain of the BR-receptor protein. These results may indicate that uzu dwarfism is caused by the missense mutation in HvBRI1. The uzu gene is being introduced into all hull-less barley cultivars in Japan as an effective dwarf gene for practical use, and this is the first report about an agronomically important mutation related to BRs.

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Glycosylation is known to be a possible metabolic pathway of plant hormones such as gibberellin and auxin. Several glycosides of BRs were isolated from plants by metabolic studies, however, isolation of endogenous glycosides was limited to glucosides of 25-methyl-and 2-epi-25-methyldolichosterone, and teasterone, and their functions remain unclear. We have planned to establish the glycosides library of BR, and utilize them for identification of endogenous glycosides and investigation of metabolic mechanisms. Castasterone 2-, 3-, 22- and 23-O-monoglucoside were synthesized and their Arabidopsis det2 hypocotyl elongation activities were examined.

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Three 7-oxalactone brassinosteroids (BRs), 7-oxateasterone (7-OXTE), 7-oxatyphasterol (7-OXTY) and brassinolide (BL), were identified from leaves of Distylium recemosum and pollens of Lilium longiflorum. 7-OXTE was a novel BR from plants. Biological activities of 7-oxalactone BRs were examined. The activity of 7-OXTE and 7-OXTY using rice lamina joint inclination test showed lower than that of castasterone and BL. In contrast, hypocotyl elongation activity of 7-OXTY using Chinese cabbage seedlings, which was treated with brassinosteroid biosynthetic inhibitor propiconazole, showed higher than that of castasterone. These results suggested that a novel BR biosynthetic pathway, early lactonization pathway, from 7-OXTE to BL via 7-OXTY may operate in higher plants. To elucidate the biosynthetic origin of 7-OXTE, it is important to identify a deduced precursor of 7-OYTE, 3β-hydroxy-7-oxa-campest-6-one. We succeeded a synthesis of a model compound 3β-hydroxy-7-oxa-stigmast-6-one from stigmasterol. We are synthesizing also a stable isotope-labeled 3β-hydroxy-7-oxa-campest-6-one and exploring intermediates of 7-oxalactone BRs.

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We investigated CYP72B1,an Arabidopsis cytochrome P450,to determine the biochemical and physiological functions of this enzyme. Using a yeast functional assay, we have demonstrated that CYP72B1 is a steroid C-26 hydroxylase that converts brassinolide (BL) to 26-hydroxybrassinolide (26-OHBL) and castasterone (CS) to 26-hydroxycastasterone (26-OHCS). We tested the ability of an Arabidopsis CYP72B1-null mutant, the wild type, and a CYP72B1 over-expressor to metabolize BL or CS. Reduced levels of 26-OHBL and 26-OHCS were detected in the null mutant, and increased levels were detected in the over-expressor, demonstrating that 26-hydroxylation of BRs is an endogenous biochemical function of CYP72B1. Bioassays with BL and 26-OHBL have provided evidence that 26-hydroxylation is an inactivation step. We also showed that CYP72B1-mediated brassinosteroid inactivation provides positive modulation of photomorphogenesis.

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Brassinosteroid and Jasmonic acid are newly approved plant hormones and play various physiological roles in plant growth and development. Effects of their exogenous application on the flowers and pods of soybean were examined. Soybean (cv. Tamasudare-No.2) was sowed in plastic nursery box, and one-week old seedlings were transplanted in the field to cultivate until harvesting. Both hormones were applied at 10 days before flowering or at the beginning of flowering. The field experiment was carried out twice in 2002 and 2003. The numbers of pods were decreased by Brassinosteroid application. especially applied at the beginning of flowering, due to the decrease of the rate of podding. Jasmonic acid application increased the number of pods with the increase of the number of flowers, though the number of seeds in a pod was reduced null or one in many pods.

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Brassinosteroids (BRs) are steroidal phytohormones that are essential for many processes in plant growth and development, such as cell expansion, vascular differentiation, and responses to stress. The effects of BRs on cell division are unclear, as attested by contradictory published results. To determine the effect of BRs on cell division, we used the tobacco BY-2 cell line, which is a widely-used model system in plant cell biology. We found that brassinolide (BL) promoted cell division only during the early phase of the culture and in the absence of auxin (2,4-D). This promotion of cell division was confirmed by RNA gel blot analyses using cell-cycle-related gene probes. At later stages in the culturing periods of BL-supplied and 2,4-D-supplied BY-2 cells, differences in cell multiplication and cell-cycle-related gene expression were observed. To determine whether suppressed organellar DNA replication limited this promotion of cell division during the early culture phase, we examined this replication and found that BL treatment had no effect on activating organellar (plastid-and mitochondrial-) DNA synthesis. As preferential organellar DNA synthesis, which is activated by 2,4-D, is necessary during successive cell divisions in BY-2 cells, our data suggest that the mechanism of the promotion of cell division by BL treatment is distinct from that regulated by the balance of auxin and cytokinin.

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We analyzed the endogenous brassinosteroids (BRs) in the callus derived from root of Arabidopsis thaliana to examine whether BRs are involved in the cell division of plant callus. From the callus, 6-deoxocastasterone, 6-deoxotyphasterol, and 6-deoxo-3-dehydroteasterone, 6-deoxoteasterone, and 6-deoxocathasterone were identified by GC-SIM. These BRs are biosynthetic intermediates deemed biologically inactive. The fact that the callus did not produce castasterone (CS), which is contained in Arabidopsis seedling and thought to be biologically active, indicates that BRs are not required for callus growth. Conversely, it seems that differentiation requires the synthesis of CS. The absence of CS in the callus also suggests that C-6 oxidase is not working in callus.

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Germination of lettuce (Lactuca sativa L. cv. Grand Rapids) seeds is regulated by phytochrome. Endogenous levels of physiologically active gibberellin (GA), (GA)_1,increased and those of abscisic acid (ABA) decreased after red-light irradiation during germination of lettuce seeds. These changes of the two phytohormone levels might be important for induction of germination by light. In this research, we isolated cDNAs encoding 9-cis-epoxycarotenoid dioxygenases (NCEDs), an ABA biosynthetic enzyme, and analyzed these expression patterns during germination by quantitative RT-PCR in order to investigate the mechanism on decrease of endogenous ABA levels. Four cDNAs, LsNCED1,2,3,4 were isolated from the extracts of lettuce seeds and seedlings. The expression levels of LsNCED2,4 decreased by red-light irradiation, which might be involved in the decline of the ABA levels.

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Two pathways, the carotenoid pathway and the direct pathway, have been proposed for biosynthesis of abscisic acid (ABA) in fungi. This controversy has not come to a conclusion. We have proposed that an ABA-producing fungus, Botrytis cinerea, biosynthesizes ABA by a new direct pathway in which C-1 of FDP is reduced to give 2E-allofarnesene (1), following its conversion to 2Z-α-ionylideneethane (4), and 4 is converted to ABA by oxidation with molecular oxygen. However, conversions of the intermediates to ABA and isomerization step at C-2 have not been proved. Isomerization at C-2 seems to occur at 1 or 2E-α-ionylideneethane (3), suggesting that the pathway has two possible routes via 1→2Z-allofanesene (2)→4 or via 1→3→4 (Figure). We investigated the new direct pathway in B. cinerea by a feeding experiment with [2-^<13>C]-1-4. A feeding experiment was performed in another ABA-producing fungus, Cercospora cruenta also. A feeding experiment with [2-^<13>C]-1-4 in B. cinerea showed that the fungus converted these compounds to ABA with incorporation ratios of 0.7,0.2,2.6 and 17.1%, respectively. The incorporation ratio of 2 was lower than that of 1. However, the lower incorporation of 3 than that of 4,and the ratios of 1 and 2 (4 : 3), and of 3 and 4 (1 : 9) in the mycelia strongly suggested that isomerization at C-2 occurs at 1,not at 3. The low incorporations of 1 and 2 could be due to their volatility and instability. The stereochemistry at C-1' of ABA biosynthesized from (±)-[2-^<13>C]-4 was (S)-configuration (98% ee), showing that (R)-4 was selectively converted to ABA. These findings prefer the route via FDP→1→2→(R)-4→ABA (Figure). 2Z-γ-Ionylideneethane was converted to ABA in C. cruenta, showing that the new direct pathway is working also in this fungus.

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Abamine (abscisic acid biosynthesis inhibitor with amine moiety) is abscisic acid (ABA) biosynthesis inhibitor targeting the oxidative cleavage reaction of 9-cis-epoxycarotenoids, which is catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). To examine the effects of abamine during developmental stage, we compared the phenotype of abamine-treated Arabidopsis or cress seedlings grown in the dark with those of non-treated plants. In abamine-treated seedlings, hook formation was inhibited. It has been reported that this inhibition of hook formation is observed in ABA deficient mutant, aba2-2. These results suggest that the inhibition of hook formation induced by abamine should be due to ABA deficiency.

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With the increase of conditioning periods (7,14,21,28 days) and the decrease of osmotic potentials (0,-10,-20 bars), the germination rate of O. ramosa seeds decreased progressively. Similar results were found in O. aegyptiaca seeds, but O. minor seeds were more tolerant to stress. ABA contents in Orobanche seeds decreased remarkably after conditioned in water and -10 and -20 bars. After that, ABA levels kept relatively low until conditioned for 37 days. No clear difference in ABA levels was observed among various stress treatments.

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Abscisic acid (ABA) controls many aspects of physiological processes including seed dormancy and drought tolerance. These hormonal action of ABA depends on the endogenous level of ABA which are controlled by the precise balance between biosynthesis and catabolism. Although much is known about its biosynthesis, ABA catabolism is poorly understood. ABA is mainly catabolized into phaseic acid through 8'-hydroxylation. The enzyme ABA 8'-hydroxylase was shown to be a member of cytochrome P450 (P450), however, its gene had not been identified. Based on the phylogenetic analysis, DNA microarray analysis, and comparison with the rice genome, the candidate genes for this enzyme were narrowed down from among 272 Arabidopsis P450 genes. Functional expression of these genes revealed that members of CYP707A family, CYP707A1 through CYP707A4,code for the target ABA 8'-hydroxylase. Expression analysis of these genes showed that CYP707A2 is responsible for rapid decrease in ABA level during seed imbibition. During drought stress, all four genes were upregulated, and upon rehydration, a significant increase in mRNA level was observed which correlated well with the change in the level of ABA.

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Catabolic inactivation of (+)-abscisic acid (ABA) proceeds via hydroxylation at the 8' position to form the unstable intermediate, 8'-hydroxy ABA (8'-OH ABA), which subsequently cyclizes spontaneously to form (-)-phaseic acid (PA). ABA 8'-hydroxylase has been known as P450,but not isolated. Arabidopsis genome contains 246 cytochrome P450 (CYP or P450) genes, which are classified in two main clades : A-type and non-A-type. Several P450 genes involved in brassinosteroids and gibberellins biosynthesis are clustered in the 85 clan of non-A-type. CYP707A family is also in the 85 clan, but its function has not been identified yet. In this study, we have characterized biochemical properties of CYP707A. RT-PCR analysis of expression profiles in response to the phytohormone treatment revealed that expressions of CYP707A genes significantly increased in response to ABA. In addition, expressions of CYP707A genes also induced by various stress conditions such as high salinity, osmotic and drought stresses. These results suggest the involvement of CYP707A in ABA catabolism. To confirm the hypothesis, CYP707As were expressed in insect cells. (+)-ABA was added to the suspension-cultured cells. An EtOAc extract was analyzed by HPLC. PA and 8'-OH ABA were detected. For biochemical characterization, the microsomal fractions were prepared from the insect cells expressing CYP707A3. The recombinant CYP707A3 catalyzed the hydroxylation of ABA to form PA in the presence of NADPH, but not NADH. The recombinant CYP707A3 was solubilized and partially purified by column chromatography. Type I spectra were detected by adding (+)-ABA to the purified CYP707A3 in a dose-dependent manner. Thus, these results demonstrate that CYP707A genes encode ABA 8'-hydoxylase.

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Abscisic acid (ABA) is a phytohormone, which controls numerous aspects of physiological processes including seed dormancy and germination. ABA response is a consequence of ABA biosynthesis, catabolism, transport and signaling. To dissect the mode of ABA action genetically, we have taken advantage of an ABA analog, (-)-R-ABA, which weakly inhibits Arabidopsis germination. Previously we reported isolation and characterization of (-)-R-ABA-insensitive mutants. Here we report isolation of (+)-S-ABA-insensitive mutants and comparison of this mutant population with the mutants obtained from (-)-R-ABA screen. Genetic analyses of (+)-S-ABA-insensitive mutants revealed three known loci that are common with (-)-R-ABA-insensitive loci and one new locus that is not found in (-)-R-insensitive mutants. This finding is consistent with the notion that these chemicals discriminate redundant ABA response pathways. Next, to see the site of discrimination of these chemicals, recently cloned ABA 8'-hydroxylase, CYP707A, was tested in vitro whether this enzyme is able to use these chemicals as the substrate. The result showed that (+)-S-ABA, but not (-)-R-ABA, was converted into the oxidation product in heterologous yeast expression system. This indicates that the ABA 8'-hydroxylation, a predominant ABA in Arabidopsis seed, acts as one of the sites of discrimination of these chemicals. This gene family will be a powerful molecular probe to genetically dissect the redundant ABA response pathways.

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Probenazole (PBZ) and its active metabolite, 1,2-benzisothiazole-3(2H)-one 1,1-dioxide (BIT), induce PR genes expression and resistance against a broad range of pathogens in tobacco and Arabidopsis, by activating upstream of salicylic acid (SA) in SAR (systemic acquired resistance) signaling pathway. To investigate influence of environmental stress on SAR induction, we analyzed effects of abscisic acid (ABA) on SAR induction by BIT. In Arabidopsis, BIT treatment reduced the growth of a virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 in leaves. However, ABA treatment reduced the BIT-induced disease resistance. ABA treatment suppressed BIT-induced PR genes expression and SA accumulation, indicating that ABA suppressed the induction of SAR. BTH (benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester), a SAR inducer, activates downstream of SA in SAR signaling pathway. ABA treatment also suppressed BTH-induced SAR in Arabidopsis. These results suggested that ABA downregulates SAR signaling pathway at upstream and downstream of SA accumulation.

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A plant hormone, ethylene functions in various aspects of the plant life cycle. Ethylene regulates many defensive gene expressions via GCC box, cis-acting element. ERFs (Ethylene-Responsive Factors) are transcriptional factors that bind to GCC box with a highly conserved DNA-binding domain, ERF domain. Whereas all four ERFs isolated from tobacco are induced by ethylene and exogenously applied cycloheximide (CHX), these ERFs have different activities ; e.g. ERF1,ERF2,and ERF4 are activators and ERF3 is a repressor. It is still unclear how these ERFs interact with each other in the complicated hormone signal pathway and how many genes are under the regulation of ERFs. In this study, we first tried to establish the transformants which have suppressed expression of each ERF. For this purpose, we used selective suppression by RNAi. Specific double stranded RNA expression vectors for each ERF, i.e., ERF2ir, ERF3ir, ERF4ir were constructed and introduced into tobacco (Nicotiana tabacum, cv. Samson NN). After selection on kanamycin medium, 8 ERF4ir transformants, 7 ERF3ir transformants, and 2 ERF2ir transformants were obtained. Whereas these transformants have been under investigation, we report here the result of ERF4ir transformants. ERF4ir tobacco showed normal appearance as non-transformant, whereas the mRNA of ERF4 was hardly detected even after CHX treatment. This result suggests the RNAi method is effective to suppress the expression of ERF4. When the expression of other ERFs, ERF1/2/3,and basic PR5 (osmotin) were analyzed after CHX treatment, ERF4ir transformants showed similar induction as control plant, indicating that ERF4 was not the main regulator for the expression of ERF1/2/3 and osmotin. Characterizations of other ERFir transformants are on going.

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Jasmonic acid and the related compounds (JAS) are now considered to be the leading candidates for a new phytohormone due to their diverse physiological activities and ubiquitous distribution throughout the plant kingdom. In 1984,we focused our attention on the potential of JAS as agrochemicals and started the investigation aimed for searching a high-performance JAS analog and exploring its potential uses in fields. Since 1996,the well-screened analog, PDJ (n-propyl dihydrojasmonate, ISO name : prohydrojasmon), had been jointly developed by Riken, Zeon Corporation and Japan Science & Technology Corporation, and last April, "Jasumomeito Ekizai (commercial name)" containing PDJ as the active ingredient was registered as a plant growth regulator (PGR) in Japan. This is the first registration of JAS analogs in the world. Last July, this agrochemical was launched by Meiji Seika Kaisha, Ltd. as an accelerator for color development of apple fruits (Malus domestica cv. Tsugaru). This paper presents the outline of our history of the development, and the performance and safety of PDJ. Some PGR performances of PDJ expected in agriculture in the future are also presented.

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In suspension-cultured rice (Oryza sativa L. cv. Nipponbare) cells, transcription of the 12-oxo-phytodienoic acid reductase gene OsOPR1 was shown to be jasmonic acid (JA)-inducible and the increase in OsOPR1 mRNA levels by JA appears not to require de novo synthesis of proteins. A genomic DNA fragment was isolated containing 5' upstream sequences of OsOPR1,and transient gene expression assays using the firefly luciferase reporter gene were performed to identify a promoter region involved in JA-responsive expression. Analysis of 5' promoter deletions indicated that sequences between -880 and -860 conferred JA-responsive expression. This 20-bp fragment contains the motif TGACG, which has been previously identified as a binding site for bZIP transactivation factors. Site-directed mutagenesis on the TGACG motif abolished JA-responsive expression, clearly identifying this motif as a JA-responsive element.

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Jasmonic acid is known to be biosynthesized via three times of β-oxidation of 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid (OPC-8 : 0). However, the final product of the β-oxidation must be jasmonoyl-CoA, thioester of jasmonic acid and coenzyme A. Therefore, the final step of the biosynthesis of jasmonic acid should be the hydrolysis of jasmonoyl-CoA, the reaction of which is catalyzed by jasmonoyl-CoA thioesterase. In this study, we tried to detect jasmonoyl-CoA thioesterase activity in rice leaves.

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Relative growth rate of plant cells in vitro is considerably affected by the initial cell densities. This troublesome effect has been interfered with the establishment of an efficient plant cell culture system, especially in cases where only a small number of surviving cells are expected such as genetically transformed cells under the antibiotic selection. To promote the growth of these cells, some workers have used "nurse culture systems" such as a feeder layer, in which the feeder cells produce growth factor(s) inducing the division of target cells. Now the active principle involved in the density effect was identified as PSK, a small sulfated peptide easily prepared by chemical synthesis. Here we report that PSK significantly improves the efficiency of genetic transformation of carrot hypocotyl cells by supporting the growth of transformed cells. Chemical nursing using PSK might present a powerful tool for basic research in plant biology and biotechnological applications.

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Plants produce common isoprenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP), through the methylerythritol phosphate (MEP) pathway in plastids and the mevalonate (MVA) pathway in the cytosol. To assess which pathway contributes to supplying DMAPP to cytokinin (CK) biosynthesis, metabolites from each isoprenoid pathway were selectively labeled with ^<13>C in Arabidopsis seedlings. Efficient ^<13>C-labeling was achieved by blocking the endogenous pathway genetically or chemically during the feed of a ^<13>C-labeled precursor specific to the MEP or MVA pathways. Liquid chromatographymass spectrometry (LC-MS) analysis demonstrated that the prenyl group of trans-zeatin is mainly produced through the MEP pathway in Arabidopsis seedlings. In contrast, the LC-MS data showed that a large fraction of the prenyl group of cis-zeatin derivatives in this system is provided by the MVA pathway. When expressed as fusion proteins with green fluorescent protein (GFP) in Arabidopsis cells, four isopentenyl transferases (IPTs) that utilize AMP/ADP/ATP as substrates were found in plastids in agreement with the idea that the MEP pathway primarily provides DMAPP to the biosynthesis of trans-zeatin. On the other hand, two (putative) GFP-IPTs with tRNA : DMAPP transferase activity were detected in the cytosol and mitochondoria. Because the prenylated adenine moiety of tRNA is usually cis-zeatin-type, but not trans-zeatin-type, a major route for the formation cis-zeatin in Arabidopsis seedlings might involve the transfer of DMAPP from the MVA pathway to tRNA in the cytosol. Distinct origins of DMAPP for the biosynthesis of cis- and trans-zeatin suggest that plants are able to modulate the level of these CK species independently.

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Gibberellins (GAs) and brassinosteroids (BRs) play important roles in plant growth and development including stem elongation. GA and BR signal transductions have been well characterized, but little is known about the control mechanisms of gene expression that induce diverse physiological phenomena. We investigated genes controlled by the interaction between GA and BR in rice seedlings by cDNA microarray, concluding that thionin genes were most striking GA/BR-responsive genes. Although rice thionin genes are constitutively expressed in coleoptile, they do not confer resistance to Burkholderia, a major seed-transmitted phytopathogenic bacterium. However, the contribution of rice thionins to resistance to other bacteria is largely unknown. We analyzed the detail expression patterns of rice thionins by Northern-blot analysis and RT-PCR. These analyses suggested that rice thionin mRNA level in coleoptile rapidly decreased in 3 days after germination, and that was suppressed by GA/BR treatment. Furthermore, BR functioned more effectively than GA, and simultaneous treatment showed synergistic effect. Under dark condition, rice thionin gene expression in coleoptile also decreased gradually, but it was kept at higher level than under light condition. These results indicate that the thionin gene expression in rice coleoptile can be a novel monitoring system to study function of plant hormones. This could contribute to elucidate the regulatory gene expression mechanism in response to GA/BR interaction, the enhanced resistance to pathogens by BR, and the relationship between photomorphogenesis and plant hormones.

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Heterotrimeric G-protein-mediated signal transduction is a highly conserved signaling mechanism in animal and yeast. In this pathway, extracellular signals are transduced to intracellular effectors through 7-transmembrane receptors (G-protein coupled receptor : GPCR) and heterotrimeric G-protein consisting of three subunits; α, β, and γ. Up to now each subunit has been identified in some species of plants, suggesting that plants have similar signaling mechanisms to those of animal. Because these subunits, however, have not been proved to compose heterotrimer and no GPCR have been identified in plants, plant G-proteins may constitute their own signaling mechanism. AtGPA1,an arabidopsis Gα, has been reported to involved in pleiotropic signaling such as plant hormone functions, cell division cycle, light signaling, pathogen signalsing and so on, but its biochemical function has yet to be clarified. In this study, we performed a yeast two-hybrid screening to identify interacting partners. We used the constitutively active form of AtGPA1 (AtGPA1^<Q222L>; with a Q222L amino acid substitution) in addition to the wild-type form AtGPA1^<WT> as baits. QL21,one of the clones selected by interaction with AtGPA1^<Q222L>, encoded a protein kinase and showed interaction with AtGPA1^<Q222L>, but not with AtGPA1^<WT> in yeast two hybrid system. Their in vitro interaction was confirmed by immunoprecipitation using recombinant AtGPA1 and QL21 expressed in E. coli. QL21 transcript was detected in all the following parts examined; flowers, siliques, upper stems, lower stems, leaves, rosette leaves, and roots. This expression pattern was similar to AtGPA1,suggesting their cooperative biochemical function.

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We screened Arabidopsis plants transformed with pSKI015 and obtained a mutant, 90-2. In 90-2,T-DNA was inserted in a receptor-like kinase (90-2-RLK) gene. Green fluorescence protein (GFP) fused to the 90-2-RLK protein was strongly detected in root apices, lateral root primordia, while weakly in procambium and pericycle cells. In root tissues, expression pattern of 90-2-RLK pro-GUS was identical to that of 90-2-RLK pro-90-2-RLK : : GFP. In aerial parts, GUS activity was localized in stipules and floral organ abscission zones. In leaves and stems, GUS activity was not detected but induced by wounding, salt and osmotic stress. To identify components of 90-2-RLK mediated signal transduction pathway in plants, we performed yeast two-hybrid screening of Arabidopsis cDNA library using the kinase domain of 90-2-RLK as a bait. 20 clones were obtained as truly positive clones that activate His3 and lacZ reporter genes reproducibly. Sequence analysis revealed that seven encoded a protein with unknown function, three encoded a protein containing tetratricopeptide repeat (TPR) domain, and two encoded a vacuolar sorting protein 35 homolog. The former two proteins showed interaction in yeast two-hybrid system even with the enzymatically inactive form of the kinase domain of 90-2-RLK as a bait. This result suggests that these proteins are not phosphorylation substrates but interacting partners of 90-2-RLK.

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We have been studying one of the receptor-like kinase (RLK) genes, 90-2-RLK, in which T-DNA was inserted in a mutant line 90-2. The phenotype of 90-2 does not show apparent difference from that of wild type, suggesting that other RLKs might possess redundant function with 90-2-RLK. Then this study is focused on RKL1,which shows highest homology with 90-2-RLK. As rkl1 knock-out lines did not show any distinguishable phenotype from WT, we generated 90-2/rkl1 double knock-out plants. These plants did not show distinguishable phenotypes from WT either at least under normal growth condition. RKL1 mRNA can be detected in all tissues tested by Northern analysis, and showed strong expression in inflorescence and roots. In RKL1-pro-GUS transgenic lines, GUS activity was detected in floral organ abscission zones, carpel apex, and guard cells of anthers, sepals, and stems. In leaves, GUS was expressed in vascular bundle and hydathode. Furthermore, GUS staining was observed in cutting site of stems and petioles. These results suggest that RKL1 is induced by environmental stress such as water loss or wounding. Some positive clones were identified by a yeast two-hybrid screen using the RKL1 kinase domain as a bait. Two of these clones were identical to those obtained in yeast two-hybrid using the 90-2-RLK kinase domain as a bait. This result suggests that 90-2-RLK and RKL1 have common biochemical functions.

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Cytosolic free Ca^<2+> rise in various plant signal transduction pathways are postulated to be mediated by changes in membrane potential followed by activation of plasma membrane voltage-gated Ca^<2+> channels. However, molecular bases for the plasma membrane Ca^<2+> influx and their regulation in plant growth, development and stress responses remain largely unknown. We have identified a putative voltage-gated Ca^<2+> channel in rice. It was ubiquitously expressed in calli, mature leaves, shoots and roots. Overexpression of the gene restored growth rate of a yeast Ca^<2+>-requiring mutant cch1. To elucidate its physiological roles, we generated transgenic rice plants overexpressing its mRNA. Suspension-cultured overexpressing cells showed higher growth rate under Ca^<2+> limitation but the growth of overexpressing plants was remarkably slower under normal condition. A retrotransposon (Tos17) insertional mutant of the gene was isolated. Growth of the knockout cultured cells was less sensitive to extracellular [Ca^<2+>]. Possible physiological roles of the voltage-gated Ca^<2+> channel in growth regulation and stress signaling will be discussed.

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Organ regeneration as well as cell proliferation in plant are mainly controlled by auxin and cytokinin. We established about 1,000 activation tagging lines of tobacco, and isolated three mutants (regeneration-less 1-3 ; rel1-3) which showed abnormal regeneration on hormone-free medium. rel1-3 did not regenerate root and rel1 could not grow, whereas they grew vigorously on the shoot-inducing medium which contained (10)^<-7>M NAA and (10)^<-5>M BA. More detailed analysis of the growth on the media with different concentration of NAA and BA showed that rel1 growth was dependent on the cytokinin. rel2 also showed no root formation in any combination of NAA and BA. To get more information about the molecular mechanism of mutants, we examined the gene expression profiles in shoot culture using hormone-responsive genes as "molecular maker". rel1 showed increased expression of ethylene responsive gene (OLP). Whereas rel2 showed enhanced expression of OLP, and decreased expression of gibberellin responsive gene (Ntc12), and D-type cyclin (Nicota ; CycD3 ; 2). rel3 showed increased expression of auxin responsive gene (Nt-iaa4.3), and ethylene responsive genes (OLP and ERF3), and also showed decreased expression of cytokinin responsive gene (cig2), jasmonic acid responsive gene (JIGT), and D-type cyclin. To isolate the target gene in mutants, we determined the numbers of inserted T-DNA by Southern blot analysis. rel1,rel2,and rel3 had 2,2,1 T-DNA insertion. Flanking region of T-DNA was isolated by TAIL-PCR method, and one of T-DNA in rel1 was found to be inserted adjacent of receptor-like kinase gene, Ntrek1. RT-PCR analysis indicated the increased expression of Ntrek1 in rel1. The physiolosical role of Ntrek1 is now examined using overexpression of Ntrek1 in transgenic tobacco.

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Plant resist invading microbial pathogens by mounting rapid defense responses. For instance, tobacco plants carrying the N gene show a hypersensitive response (HR) against tobacco mosaic virus (TMV). This response can act only when the plant has a resistance gene and the pathogen has an avirulence gene. In the case of tobacco and TMV, N is a resistance gene product and the 50 kDa TMV helicase domain (p50) is an avirulence gene product. However, it is not yet known how the N factor recognizes p50. We investigated the interaction of the N protein and p50 by the yeast Two-hybrid system. When both the N protein and p50 are present, β-galactosidase activity is about 10 times higher as compared to the presence of only one of the two. Next, the specificity of the interaction was investigated by the growth test using a histidine synthase inhibitor (3-AT). Our results shown that, only when both the N factor and p50,but not one of the two alone, are present growth was possible. Several strains of TMV have been reported not to induce the HR in tobacco. One of these strains has an amino acid exchange in the hericase domain. A possible explanation of the no-HR phenotype would be a disturbed interaction with the N protein. For this reason we checked this mutated p50 in the yeast Two-hybrid assay. In contrast to the WT protein, the mutated p50 resulted in a very lowβ-galactosidase activity. Moreover, it did not grow in the 3-AT containing culture medium. The results suggest that p50 has combined with N factor directly

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Brassinazole (Brz) is a specific inhibitor for brassinosteroid biosynthesis. The inhibitor is useful to examine functions of brassinosteroid. Our previous experiments demonstrated that Brz treatment to seedlings enhanced greening when they were transferred from dark to light. In this study, we tried to isolate mutants showing different greening phenotypes from T-DNA activation tag lines of Arabidopsis. Four thousands of T-DNA activation tag lines were screened under the Brz treated condition, and a hayamidori (ham) mutant was isolated. When seedlings were transferred from dark to light, the mutant showed fast greening phenotype compared with a wild type. After 7 hours-transfer to light, chlorophyll content of the mutant was twice higher than that of a wild type. ham mutant also showed a dwarf, reduced apical dominancy, and male sterile phenotypes. THese phenotypes were maintained only in heterozygote plant, which suggested that the mutant is dominant and that ham is caused by the activation of some genes by the enhancers on the inserted T-DNA region. To identify the insertion position of the T-DNA in the mutant, plasmid rescue was performed. As a result, the T-DNA was inserted in the 6th exon of a gene with unknown function on the chromosome 4. Expression of 8 genes near the inserted gene was examined by RT-PCR and found out that 3 genes including a putative Myb-type of transacting factor were activated in the mutant. We are now establishing transgenic plants that overproduce candidate genes to identify the genes responsible for ham mutation.

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WRKY proteins are a large family of transcription factors that specifically bind to the DNA sequence motif TTGAC (C/T) (W-box). Many WRKY proteins have been suggested to have a regulatory function in the response to pathogen infection and other stresses. In suspension-cultured rice cells, diterpenoid phytoalexins are produced in response to exogenously applied elicitors such as chitin oligomers and cerebrosides from the pathogen Magnaporthe grisea. By means of microarray analysis of 8987 randomly selected rice expressed sequence tags (EST), we isolated two rice WRKY genes, OsSPF1 and OsABF2,that were induced after treatment with a chitin elicitor, N-acetylchitooctaose, in suspension-cultured rice cells. OsSPF1 and OsABF2 cDNAs encoding the full-length ORF were isolated by RACE-PCR with the primers designed on the basis of EST sequences. OsSPF1 belongs to groupI WRKY proteins (two WRKY domains) and OsABF2 belongs to group II WRKY proteins (one WRKY domain that contains the Cys_2His_2 zinc-finger-like motif). Transcripts og both genes in suspension-cultured rice cells began to accumulate 15 min after addition of the elicitor and reached a maximum level after 30 min, and then decreased gradually.

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