In the last decade, evoiutional development of molecular biology and moiecular genetics has brought about noticeable advance in research on plant hormoncs at a molecular level: there has been a rapid about noticeable advance in research on plant hormoncs at a molecular level: there has been a rapid these investigations, the functional probes of plant hormones have been playing an important role, including i) compound libraries consisting of biosynthetic intermediates and metabolites, ii) isotopically labeled compounds, iii) hybrid molecules carrying proper functional molecules on plant hormones, iv) antagonists of biosynthetic enzymes, and v) agonists and antagonists of hormones. In this work, expecting the specified function to each probe, we aimed to assemble various kinds of molecular probes of jasmonates, gibberellins and brassinosteroids, based on fine and precision synthetic chemistry. For this purpose, we developed the new strategies and reactions for refining the synthetic routes to the targeted molecules and elaborated the structures of the targeted molecules by molecular design in order to introduce the new functions and distinguished features to the hormones. For molecular design, the data on the structure-activity relationships of each hormone were accumulated by bioassay of most of synthetic compounds, which was essential especially for designing the molecular probes iii)-v) mentioned above. In the present lecture, the outline will be discussed by some selected topics focusing on assembling the probes along with their achievements in plant science.
The metabolism of C1 compounds in higher plants is an attractive research target. Plant growth test under various C1 compounds showed that formate stimulates the growth of young rice plant. The feeding experiment with ^<13>C-HCOOH and ^<13>C-CH_3OH revealed that these two compounds were readily incorporated from the root and converted into CO_2 by the stepwise oxidation pathway. Application of formate to the root part of the rice plant induced the increase of the activity of formate dehydrogenase (FDH) responsible for the conversion reaction of formate into CO_2 and also the accumulation of mRNA of FDH in the root part. These results indicated that formate could enhance plant growth under the CO_2-limited condition. Strong sunlight is known to be harmful to photosynthesis under CO_2-depleted conditions. This phenomenon is understood as photoinhibition. Application of potassium formate or methanol before the photoinhibitory treatment could lower the damage of rice photosynthesis from photoinhibition. This fact indicated that the substantial concentration of antioxidants capable of protecting PS II from photooxidative damage is not always endorsed and it is strongly suggested that formate is involved in endogenous radical scavenging mechanisms or formate (and/or its metabolite) suppresses ROS generation in the vicinity of the damage site of PS II. The other non-natural C1 compounds are also tested using rice as an experimental plant. Rice plant growth test revealed that methylamine treatment inhibited the aerial part but stimulated the growth of root part. The gene homologue of cytosolic phosphoglycerate kinase was isolated as the gene, which was stimulated by methylamine treatment, by differential display RT-PCR method. Further Northern blot analysis of the genes related to glycolytic pathway indicated that glycolysis stimulated by methyl amine treatment. A series of result mentioned above indicates that exogenous C1 compounds should be activated specific genes. That is interesting as a result suggesting the existence of specific signal-transducing pathway for exogenous C1 compounds.
An isolation method to obtain various kinds of actinomycetes in plant leaves has been devised. All isolates were classified into genus based on their taxonomical characteristics. They included not only the genus Streptomyces but also the non-Streptomyces (rare actinomycetes) genera. In particular, strains belonging to the genus Microbispora represented more than 50% of the total share belonging to the non-Streptomyces genera. Three strains of isolates produced new herbicidal substances: Herbicidin H by "Streptomyees saganonensis" SANK63997, γ-Glutamyimethionine sulfoxmine by Microbispora Sp. SANK 62597 and Substances A-79197-2 & -3 by Dacty1osporangium aurantieum SANK 61299.
B-ring modified brassinolides (BLs): iso-BL [(22R,23R,24S)-2α,3α,22,23-tetrahydroxy-24-methyl-6-oxa-B-homo-5α-cholestan-7-one], 6a-deoxo-iso-BL, 6-aza-iso-BL, N-methyl-6-aza-iso-BL, 6-deoxo-BL and seco-BL diol, were assayed by rice lamina inclination (RLI) test and by rescue test of hypocotyl elongation using a brassinosteroid (BR) deficient mutant, Arabidopsis det2, which is tentatively termed as det2 hypocotyl elongation (DHE) test. Most of the compounds exhibited DHE activities higher than those on RLI, and iso-BL and 6-deoxo-BL showed DHE activities comparable to that of BL. These results, along with those of 6a-carbaBL analogs previously reported, show that the recongnition of BR molecules at the receptor involved in RLI is more strict than at the receptor involved in DHE at the B-ring region, and that each receptor perceives BR molecules in different structure manner at least at the B-ring region. In addition, an intriguing activity of 5-epi-6a-carba-B-homoBL was observed. This compound unambiguously inhibited DHE at high concentrations, but markedly enhanced DHE activity of BL by co-application. This result indicates that 5-epi-6a-carba-B-homoBL functions as an efficient accelerator of DHE activity of BL. The same effect was observed in RLI as well.
Arabidopsis dwf4 was shown to be defective in ateroid 220α-hydroxylation step that is the putative rate-limiting step in brassinosteroid (BR) biosynthesis. To better understand the role of DWF4 in BR biosynthesis, transgenic plants ectopically overexpressing DWF4 (AOD4) were generated, and the phenotypes were characterized. The hypocotyl length of both light- and dark-grown AOD4 seedlings was increased dramatically as compared to wild type. The total number of branches and siliques increased in AOD4 plants, leading to a significant increase in the number of seeds produced. Analysis of endogenous BR levels in dwf4, Ws-2, and AOD4 revealed that dwf4 accumulated the precursors of 22α-hydroxylation steps, whereas overexpression of DWF4 resulted in increased levels of downstream compounds relative to Ws-2, indicative of facilitative metabolic flow through the step. These results suggest that it will be possible to control plant growth by engineering DWF4 transcription in plants.
Brassinosteroid (BR) am now recognized as a major hormone controlling plant development and growth. Recently several cytochrome P450 genes have been isolated from the mutants deficient in BR (Arabidopsis cpd, dwf4, bas1 and tomato dwarf). The physiological function of these P450s has been proposed from analysis of the corresponding routants in chemical levels. But enzymatic characters of these P450s are not yet clear. Recently, Arabidopsis BAS1 (CYP72B1) has been isolated and suggested to catalyze the C26-hydroxylation of brassinolide. To investigate regulation of BR metabolism in tomato, we have obtained P450 cDNA belonging to CYP72 family from tomato. The tomato cDNA tomato CYP72B, showed 60% identity to BAS 1. in this study, we produced recombinant proteins, which was over-expression of tomato CYP72B in the hetelogous expression system of baculovirus-insect cell. The tomato CYP72B over-expressed protein has typical character of P450 enzyme. Secondary, we cultivated a recombinant tobacco transformed by tomato CYP72B. The transgenic tobacco had typical brassinosteroids deftdent phenotype.
Brassinozole is a specific brassinosteroid (BR) biosynthesis inhibitor and is shown to be useful to elucidate the function of BRs. In the course of Structure-activity relationship study on brassinazole, we found more specific and potent BR biosynthesis inhibitors Brz2001 and Brz220, each of which consist of 4 isomers. Generally, azole derivatives showed different activity in response to their stereoisomers. Therefore we separated Brz2001 and Brz220 into 4 isomers on silica gel HPLC and chiral column, respectively, and tested their ability to inhibit BR biosynthesis. The biological activity of these compounds was evaluated by cress stem elongation test. The activity of their compounds clearly depend on their stereostructure. In care of Brz220, the absolute structure of each stereoisomer was determined by the combination of chemical synthesis and NMR analysis.
The levels of endogenous brassinosteroids (BRs) in rice shoots grown under white lightt were higher than those grown in the dark. Especially the levels of 6-deoxocastasterone, typhasterol and castasterone were increased markedly. The BR levels were not elevated by red light and far-red light irradiation as compared with the dark condition. However, blue light increased the BR levels as much as white light did. The contents of 6-deoxocathasterone and 6- deoxoteasterone were not altered under the light conditions used. It is concluded that blue light enhances the synthesis of BRs in the latter half of the biosynthetic pathway, thereby elevating the BR levels. In rice roots, no effect of light on the BR synthesis was observed, suggesting that roots may have no light-sensing mechanism that controls the level of BRs.
Light has been suggested to be a negative regulator of brassinosteroid biosynthesis, but there have been no direct evidences. We analyzed light regulation of brassinoseroid biosynthesis both from biochemical and molecular biological approach. We isolated brassinosteroid-6-oxidase II gene (BR6oxII) from Arabidopsis. Total RNAs were extracted from Arabidopsis seedlings grown in light or darkness for 5 to 7 days. They were then subjected to real-time monitoring RT-PCR. Transcript levels of BR biosynthetic genes were organ specific.Some of them were up-regulated upon transfer from darkness to light. We conclude, together with endogenous BR levels determined by GC-SIM, that BR biosynthesis is activated during photomorphogenesis.
Brassinazole (Brz) is a specific inhibitor of brassinosteroid biosynthesis. The dark-grown Brz-treated Arabidopsis and non-Brz-treated der2, which is a brassinosteroid biosynthesis-deficient Arabidopsis mutant, show characteristics of de-etiolated plants, i.e., short hypocotyls, expanded cotyledons and development of true leaves, when grown in darkness. In this context, brassinosteroids seem to function in the suppression of photomorphogenesis in the dark. In general, the application of cytokinin reduces hypocotyls length and induces development of true leaves in the dark. Here, we examined that brassinosteroid-deficiency and cytokinin-excess exhibit whether common effects or different effects in photomorphogenesis in the dark. In development of true leaves in the dark, Brz induced expanding of leaves mainly and a little promote of proliferation of shoot apex. In contrast, Arabidopsis treated by benzyladenine (BA) exhibited many, slender and wind true leaves. Treatment of Brz plus BA induced many true leaves which is alike to normal leaves in light. Both of brassinosteroid-deficiency and cytokinin-excess may be necessary to shape normal leaves. The Brz--treated and BA-treated plants exhibited fast greening after transferring from dark to light. Then, we examined content of protochlorophylide (Pchlide) and protochlorophyll (Pchl) of Brz-treated and BA-treated Arabidopsis. The results showed that Brz induces accumulation of high contents of Pchl. However, BA induced accumulation of Pchlide rather than Pchl. Thus, brassinosteroid-deficiency and cytokinin-excess exhibited different effects in photomorphogenesis in the dark.
Recent studies of the brassinosteroid-deficient mutants have revealed that brassinosteroid plays essential roles to control plant growth and development, such as cell elongation, division and photomorphogenesis. However, its regulatory mechanisms and signal transduction pathway are still under unclear. We used DNA microarray (Gene Chip, Affymetfix) to analyze BR-regulated genes comprehensively. BL inhibited the expression of the gene for phytochrome-.interacting factor 3, indicating that BRs modulate the light signaling pathway rather than function as signaling molecules downstream of light. After a lag period, BL also increased the expression of early auxin-inducible genes, suggesting that BR signaling overlaps with the auxin signaling pathway to controlcellular processes.
The dwarf mutant lka of garden pea (Pisum sativum L.) has reduced sensitivity to brassinosteroids and increased levels of endogenous brassinosteroids, suggesting that Lka dwarfs are defective in the perception or signal transduction pathway of brassinosteroids. We show that the Lka gene is a pea ortholog of the Arabidopsis BRII gene encoding a leucine-rich repeat kinase which functions as a brassinosteroid receptor. Low-stringency screening of a pea cDNA library with the Bill cDNA probe produced a full-length BRIIhomolog (termed PsBRII). The predicted amino acid sequence of PsBRI1 shows 78% similarity (68% identity) with that of the BRII gene. Sequence analysis indicated that the PsBRII gene in lka plants has a single base substitution resulting in the missense of highly conserved aspartic acid into asparagine just before brassinosteroid-binding region. Furthermore, co-segregation between the RFLP and the tall/dwarf phenotype strongly implied that the mutation results in the dwarf phenotype of the mutant lka plant. Thus, the PsBRII gene functions as a brassinosteroid receptor kinase in pea and can be mapped to the Lka locus. We also report the northern analyses of the PsBRII gene.
We found that light seems to enhance the conversion of 6-deoxocastasterone to castasterone in rice seedlings. So we cloned the rice DWARF gene which is involved in the conversion from 6-deoxocastasterone to castasterone, and compared the effect of light quality on its expression by the semi-quantitative RT-PCR. Total RNAs were extracted from whole shoots and whole roots of rice grown in dark for 1 week, and then under different light conditions (white, blue, red, far red, and dark) for 24 hours. However, different light conditions caused no significant alteration in the expression of the rice DWARF gene. We are now attempting to analyze younger, rapid-growing regions of the rice seedlings.
The ice plant (Mesembryanthemum crystallinum L.) is a succulent plant and a model to study stress response because it can be switched from C3 3 photosynthesis to Crassulacean acid metabolism (CAM) by development or salinity. Although roles of several plant hormones for its CAM induction had been tested, the mechanism was not able to elucidate clearly. To determine whether the plant hormone, brassinosteroid (BR), is responsible for this mechanism, we investigated a role of BR in the ice plant. BR activity in extracts of twelve-week-old plants was determined by rice lamina-joint inclinatkm assay. We found two distinct fractions containing BR activities after HPLC. Castasterone (CS) was identified in one of these fractions by GC-MS. Another active fraction was eluted at the same retention time of authentic brassinolide (BL), and the identification is in progress. We compared BR compositions between C3 and CAM plants. BL activity was observed in the CAM plant but not in the C3 plant, while CS activity was found in both plants. These results may lead a hypothesis that BL play a role in CAM induction of the ice plant.
Not a few plant growth regulators including plant hormones participate in the regulation of plant growth, and some of them interact each other. Gibberellins (GAs) are well known to promote shoot elongation and recently brassinolide (BL) were also shown to play an important role in shoot elongation of dicotyledonous plants. To see whether BL is involved in the regulation of shoot elongation of rice, we tried to identify the genes responsive to GA and/or BL by cDNA microarray, and obtained some genes which show interesting expression patterns. As a result of northern-blot analysis, four genes showed the same expression patterns as cDNA microarray. One of them was a putative farnesylated protein. Others are in progress to obtain the details of their expression profiles by northern-blot analysis.
Several cytochromes P450 are involved in gibberellin (GA) biosynthesis. We have isolated. two cDNAs from tomato, to88B1 and to707A5, specifically expressed in ovary. The cDNAs showed high homology to CYP88A (ent-kaurenoic acid oxidase in GA biosynthesis) in phylogenetic analysis, suggesting that both P450s may be in GA biosynthesiS. Because of only 40% identity to CYP88A, to88B1 may have a different function from CYP88A. Furthermore, the Thr residue conserved in most P450s, which plays an important role to stabilize the ferrous P450-O_2 intermediate, is substituted for Pro in to88B 1. GA biosynthetic pathway has a side branch that lead ent-kaurenoic acid to kaurenolides by C6-desaturation, and to88B1 may be involved in the pathway, together with to707A5. To confirm this, the P450s were expressed in insect cells and the measurement of their activities is in progress.
Cucumber hypocotyls were cut in half transversely, and morphological and histochemical analyses of the process of tissue-reunion in the cortex were performed. Cell division in the cortex commenced 3 days after cutting, and the cortex was nearly fully united within 7 days. DAPI- staining and BrdU-labeling experiments indicate that nuclear DNA synthesis occurred during this process. A specific accumulation of pectic substances was observed in the cell wall of attached cells in the reunion region of the cortex. Cell division in the tissue-reunion process in the cortex was strongly inhibited by removal of the cotyledon, and this inhibition was reversed by the application of gibberellin (10^<-4> M GA_3), but not auxin (10^<-4> MIAA), to the tip of the cotyledon-excised plant. Furthermore, cell division in the cortex was inhibited by treatment of the cotyledon with 10^<-4>M uniconazole-P, and this inhibition was also reversed by simultaneous application of GA. In contrast, removing the shoot apex did not inhibit tissue-reunion, and normal cortex tissue-reunion was observed when 10^<-4>M TIBA was applied to the upper hypocotyl above the position of the cut. Furthermore, similar phenomena were observed in tomato seedlings, and cell division in the tissue-reunion process did not occur in cut hypocotyls of a tomato GA-deficient mutant (gib-1). These results suggest novel functions for GA in cell division during the process of tissue-reunion in the cortex, as well as involvement of the cotyledon in the production of GA.
Gibberellin responsive gene 092 was isolated from cucumber hypocotyls by means increased in hypocotyls of cucumber by GA treatment. The sequence data suggested that the clone encoded an arabinogalactan-protein. The ful1-length cDNA of 092 was introduced into tobacco plants under the control of 35S promoter The AGP was extracted and purified from the transgenic tobacco by reversed-phase-high performance liquid chromatography (RP-HPLC) and gel filtration chromatography (GFC) and analyzed Using β-glucosyl Yariv reagent which binds selectively with AGPs. The transgenic plants gave aβ-Yariv-reactive peak in addition to those present in wild type plants. β-Yariv regent inhibited hypocotyl elongation promoted by GA and IAA treannent in intact cucumber seedling and hypocotyl segments of azuki bean. Together with results using anti-AGP antibodies, the product of 092 was suggested to be an AGP. The transgenic tobacco plants showed earlier flowering than wide ty'pe plants.
Fiuridone and norfiurazon, inhibitors of carotenoid biosynthesis, did not induce shoot elongation of semi-dwarf rice, Tan-ginbozu, but significantly promoted shoot growth in the presence of GA_3. Levels of ABA in the seeds decreased to less than 20% that of dry seeds within 48 hrs after imbibition, and approximately 60% of the reduction was due to leaching to incubation media. These inhibitors of carotenoid biosynthesis seem to increase the GA sensitivity in rice plants.
Gibberellin, a class of plant hormones,induces transcription and secretion of α-amylases during seed germination of crop plants. Arabinogalactan-proteins (AGPs) are plant proteoglycans that have been implicated in plant growth and development. The possible involvement of AGPs in the action of gibberellin (GA) was examined by applying β-glucosyl Yariv reagent (β-Glc)_3Y, a synthetic phenyl glycoside that interacts selectively with AGPs, to barley aleurone protoplasts. Induction of α-amylase was clearly inhibited by (β-Glc)_3Y. Transfection analysis, using an α-amylase promoter-GUS fusion gene in the protoplasts, indicated that the transcriptional activation of the α-amylase promoter was inhfbited specifically by (β-Glc)_3Y. An aleurone-specific AGP was detected by reversed-phase HPLC and supported the idea that an AGP may play an important role in aleurone-specific events.
PCIB (p-chlorophenoxyisobutyric acid), known as antiauxin, is believed to inhibit the auxin action by competing with auxin in the binding site of the auxin receptor, although the detailed mechanism of inhibition is unknown. We have investigated the effects of PCIB on Arabidopsis root system. PCIB acted as auxin antagonist on auxin-induced gene expression; it inhibited root elongation independently to auxin. Futhermore, We have successfully isolated a PCIB resistant mutant from ion beam-irradiated seed population.
Under true microgravity conditions in space as well as simulated ones on a 3-dimensional clinostat, epicotyls of etiolated pea seedlings have been demonstrated to be almost straight but the most to be oriented toward the direction far from their cotyledons with about 45 degrees from the vertical direction, being designated automorphosis. Kinetic studies revealed that in both seedlings grown under 1g and simulated microgravity conditions on the clinostat, epicotyls bent at their basal region or near cotyledonary node toward the direction far from the cotyledons with about 45 degrees within 36 hrs after watering. Thereafter epicoWls grew keeping this orientation under simulated microgravity conditions on the clinostat, whereas those grown on 1g conditions changed the growth direction to antigravity direction as negative gravitropic response. Treatment with inhibitors of auxin polar transport such as 2,3,5-triiodobenzoic acid (TIBA),N-(1-naphtyl)phthalamic acid (NPA) and 9-hydroxyfiuorene- 9-carboxylic acid (HFCA) induced automorphosis-like epicotyl bending, but that with p-chlorophenoxyisobutyric acid (PCIB), an inhibitor of auxin action, did not. These results suggest that auxin polar transport activity seems to be important for negative gravitropic response of epicotyls of etiolated pea seedlings on 1g conditions.
To analyze genes involved in auxin signal transduction, we screened Arabidopsis plants transformed with pSKI015, a binary vector for activation tagging. From approximately 9000 Arabidopsis transformants, we obtained a mutant, 90-2, which showed severer epinasty and root growth defect than WT on the MS-solid medium plate containing 0.05ppm 2,4-D.Southern blotting analysis showed that more than two T-DNAs were integrated in 90-2. Backcross experiment revealed that the mutation of 90-2 was recessive and the T-DNAs insertion occurred at one position. By the plasmid rescue and sequencing, one of the T-DNA was shown to be inserted in the intron of a receptor-like kinase (RLK) gene on the 3rd chromosome. Northern hybridization analysis showed this RLK gene was not transcribed in 90-2. We are studying whether the phenotypes of 90-2 are due to the disruption of this RLK gene.
In cultured Bright Yellow-2 tobacco (Nicotiana tabacum) cells, the depletion of 2,4- dichlorophenoxyacetic acid (2,4-D) in the culture medium induces amyloplast development. This differentiation also includes a decrease in cell multiplication, and an increase in cell size. These changes were primarily triggered by the depletion of 2,4-D, and accelerated by the addition of benzyladenine. Thus, this simple in vitro amyloplast-inducing system provides an opportunity to analyze and compare heterotrophic cells in meristematic and starch-storing phases. In order to analyze the mechanisms controlling starch-storing cell differentiation, we started to isolate genes specifically expressed during amyloplast differentiation. Using differential display method, we cloned six cDNA fragments whose transcripts accumulate at high levels during starch-storing cell differentiation. Three clones had homology to known genes;i.e. clone 26, 30, and 60 had significant identities with cellulose synthase, glutamate dehydrogenase, and malate dehydrogenase, respectively. Other three clones (clone 10, 22, and 25) had no significant identities with well-characterized genes, while sequence similarity search using BLAST algorithm suggested that they are homologous to Arabidopsis genes of unknown functions. Analyses on these genes are now in progress.
Absicsic acid (ABA), an apocarotenoid synthesized by oxidative cleavage of epoxy-carotenoids, is involved in developmental progresses such as seed maturation and dormancy and the response to various environmental stresses in higher plants. The use of carotenoid biosynthesis inhibitors, such as norflurazon and fluridone, has been shown to be useful major tools in studies on the determination of physiological fimctions of endogenous ABA and regulation of the pathway in plants. They give the serious damage to plants due to their ability to reduce the amount of carotenoid in plants. Therefor, the regulation of biosynthetic pathway downstream of zeaxanthin by inhibitors has been reported to be important for the regulation of ABA levels without any side effect on plants. For this reason, we have investigated the discovery of specific ABA biosynthesis inhibitors for the purpose of regulation of endogenous ABA biosynthesis.
The C_15 pathway via the direct cyclization of farnesyi diphosphate has been proposed for the abscisic acid (ABA) biosynthetic pathway in fungi. However, feeding experiments with ^<18>O_2 have suggested that a fungus biosynthesizes ABA via oxidative cleavage of carotenoids as well as higher plants, although 9Z-carotenoids that would be substrates for the oxidative cleavage have not been found. We examined whether two ABA-producing fungi, Cercospora cruenta and Botrytis cinerea, produce carotenoids including 9Z-isomers. C.cruenta was cultured on a liquid medium with cotton cap or silicon cap. ABA in the medium, and carotenoids in the mycelia were analyzed by HPLC equipped with a photodiode array detector. The maximum level of ABA was 3.6 mg/L. in the culture with cotton cap, and 0.17 mg/L in the culture with silicon cap. all-E-β-Carotene (4.0 mg) was detected in the mycelia cultured with cotton cap, while 15Z-phytoene (0.7 mg) and unidentified carotenoid (0.2 mg) were found in addition to all-E-β-carotene (0.6 mg) in the mycelia cultured with silicon cap,. The cultural condition of silicon cap is more anaerobic than that of cotton cap. Suppression of oxidation under the anaerobic condition may cause accumulation of metabolic intermediates of carotenoids and result in decrease in ABA production. B. cinerea also produced a small amount of a carotenoid-like compound
Abscisic acid (ABA) is a phytohormone which regulates many aspects of plant growth and development, including seed development and dormancy, and tolerance of a variety of environmental stresses. ABA is synthesized via C_<40> carotenoid. The carotenoid is cleaved to C_<15> xanthoxin in plastids. Followed by two successive reactions in cytosol, xanthoxin is thought to be converted to ABA via abscisic aldehyde. Although the Arabidopsis ABA deficient mutant aba2 has been considered to be impaired in the conversion of xanthoxin to abscisic aidehyde, isolation of ABA2 gene and characterization of the gene product were not reported. Map-based cloning of ABA2(GIN1/SIS4/ISI4) gene revealed that the gene encoded a 30-kDa protein consist of 286 amino acids. Homology search data showed that the protein was a member of short chain alcohol dehydrogenase family. Northern analysis showed that ABA2 mRNA was highly expressed in roots and stems. In rosette leaves, ABA2 mRNA was much abundant in petiols than blades. These results suggest that ABA2 mRNA is mainly expressed in vascular tissues. ABA2 protein was functionally expressed in a heterologous yeast expression system, and the result represented that the protein had activity to convert xanthoxin to produce a product having the same retention time as abscisic aidehyde. The product was determined to be abscisic aldehyde by GC-MS. The reaction proceeded in NAD dependent manner. These results indicate that ABA2 is a novel short chain alcohol dehydrogenase that catalyzes the conversion of xanthoxin to form abscisic aidehyde. Analysis of further enzymatic properties such as substrate specificity for several compounds having similar structure to xanthoxin and reaction kinetics is now in progress.
Abscisic acid (ABA) is a dormancy factor and stress messenger in plants. The initial perception of ABA by target cells remains obscure at the molecular level, and no ABA-binding protein has yet been isolated. We synthesized 3'-azido-ABA as a potential photoaffinity reagent for ABA-binding proteins. This compound was stable in organic and aqueous solutions in the dark, but was decomposed by UV irradiation. Its biological activity was equivalent to that of ABA, suggesting that it may be an effective photoaffinity reagent.
Abscisic acid (ABA) plays prominent roles on plant growth and development. To understand the mechanism how ABA is perceived and its signal is transduced, we screened Arabidopsis mutants, which were able to germinate in the presence of (-)-ABA, an unnatural stereoisomer of (+)-ABA. The concentration of (-)-ABA, which inhibits wild-type germination, was 3-fold higher than that of (+)-ABA. We screened 360,000 M2 seeds, isolated 40 mutants, and characterized 28 mutants. Twenty-two mutants were able to germinate on both 10 micro M (-)-ABA as well as 3 micro M (+)- ABA. These mutations fell into 3 complementation groups; abi3, abi4, and abi5. In contrast, other 6 mutants showed germinability on 10 micro M (-)-ABA, but they failed to germinate on 3 micro M (+)-ABA. These mutations fell into 3 complementation groups; abi3, chol, and cho2. The chol and cho2 loci were mapped onto bottom of chromosome 5 and chromosome 4, respectively. All mutants exhibits less sensitivity to both (+)-ABA and (-)-ABA, but the degree of reduced sensitivity to (+)-ABA or (-)-ABA were different among the mutants. The abi4, chol, and cho2 mutants showed more reduced sensitivity to (-)-ABA rather than (+)-ABA. In contrast, the abi5 mutants showed more reduced sensitivity to (+)-ABA compared to (-)-ABA. The overlapping ABA response pathways in seeds that are distinguished by the stereoisomers will be discussed.
Inhibitory activities of abscisic acid and its analogues on the open pollination of barley (Hordeurn vulgare L.) were studied. Activities of all the analogues as well as abscisic acid were higher than that of MJA. Activities of (+) 8'(9')-alkyl(oxy)-ABAs were higher than that of (+)-8'-fluorinated-ABAs. Activities of (-)-8'-alkyl(oxy)-ABAs were lower than those of each (+)-derivatives, however, those of (-)-8'-fluorinated and 9'-methoxy-ABAs were similar with those of each (+)-derivatives. Based on these resutls, structure requirement for higher activity are discussed. In the spraying experiment of ABA on a field condition, slight inhibition of open pollination were observed.
The inhibition of seed germination at supraoptimal temperature is called thermoinhibition. The thermoinhibition at 28℃ of lettuce seed germination was rescued with fiuridone, an ABA mhibitor. At 33℃, an application of exogenous GA with fluridone was needed for inducing the germination. This application decreased ABAcontents of the seeds to the levels of those at 18℃, an optimal temperature for germination. In addition, the elevated temperatures made the seeds more sensitive to ABA. In contrast to ABA, no significant difference was observed between GA_1 levels at 18℃ and 33℃. These results indicate that ABA, but not GA, plays the crucial role in regulation of lettuce seed germination at supraoptimal temperatures in the dark.
Jasmonic acid (JA,1) is known to work as a cellular signaling compound concerning plant's self-defensive mechanisms, stress responses and secondary metabolites production and so on. On the other hand, exogenously applied JA effectively inhibits seed germination and plant growth. JA analogues with benzyl group were synthesized and found to possess herbicidal activity. An analogue (2) controlled barnyard grass at concentrations at 100 to 300 ppm. The activity was influenced by the particular aromatic substitution pattern.
Jasmonic acid (JA) plays important roles in production of secondary metabolites including phytoalexins in suspension-cultured cells of rice, soybean, and many other plantS. As the first step of our study to isolate JA-binding proteins from the rice and soybean cells, we have prepared chemical probes that are useful for isolation and functional analysis of JA-binding proteins. In this studY, JA-amino acid conjugates (JA-Gly, (+)-JA-(S)-lle, (-)-JA-(S)--lle), JA-FITC conjugate, and JA-biotin conjugates were prepared by the active ester method, and their biological activities were determined by micro-drop method using rice seedlings. JA- amino acid conjugates showed almost the same growth inhibitory activities as JA. JA-FITC conjugate and JA-biotin conjugates were almost inactive. The effects of the JA analogues on production of phytoalexins in rice plants and on induction of phenylalanine ammonia-lyase (PAL) in suspension-cultured soybean cells will also be reported.
Jasmonic acid (JA) is a plant hormone which regulates various developmental processes in plants, and it acts as an signal molecule in plant defense against herbivores and pathogens. We reported JA is a key signal transducer in the elicitor (N-acethylchitoheptaose)-induced phytoalexin production in suspension-cultured rice cells. To obtain a clue to unravel the signal transduction pathways of JA, we attempted cloning and chractarization of cDNAs for JA-responsive genes by differential screening of a cDNA library constructed using poly(A)^+mRNA from suspension-cultured rice cells treated with JA for 30 min, resulting in isolation of a cDNA clone tentatively named cRERJ1 that responded to exogenous JA by increase in the mRNA level. Northern blot analysis indicated that RERJ1 was early responsive to JA, the maximum level of RERJ1 mRNA being observed 30 min after the start of JA treatment. Based on the sequence analysis, RERJ1 is deduced to encode a transcriptional regulator having a basic region/helix-loop-helix motif. According to analysis of phenotypes of transgenic rice plants overexpressing sense- or antisense-RERJ1, it was strongly suggested that RERJ1 plays key roles in various jasmonic acid-mediated, biological phenomena such as inhibition of vegetative growth and induction of necrosis in the leaves. Investigetion of effects of JA on phytoalexin production in the transgenic rice plants is now under way.
The effects of jasmonic acid (JA) on secondary metabolism in barley were investigated. Reversed-phase HPLC analysis revealed that the amount of a compound increased in excised barley leaves treated with JA. The compound was purified, and identified to be 6‴-feruloylsaponarin by spectroscopic analyses and alkaline hydrolysis. A related compound, 6‴-sinapoylsaponarin, was also found to accumulate in excised leaves independently of JA treatment. The accumulation of these 6‴-hydroxycinnamoylsaponarins was accompanied by a decrease of saponarin content. [8,9-^<13>C]p-Coumaric acid and [2,3,4,5,6-^2H]L-phenylalanine were effectively incorporated into the hydroxycinnamoyl moleties in 6‴-hydroxycinnamoylsaponarins, while the incorporation of the labeled precursors into saponarin part was small. These findings indicated that the hydroxycinnamoyl moieties of 6‴-hydroxycinnamoylsaponarins are de novo synthesized from phenylalanine via the phenylpropanoid pathway, and that the saponarin part is mainly provided by the constitutive saponarin pool.
S-Adenosyl-L-methionine:DIMBOA-Glc (2-(2,4-dihydroxy-7-methoxy-l,4-benzoxazin-3-onc)-β-D-glucopyranose) 4-O-methyltransferase was purified from JA (jasmonic acid)-treated wheat leaves. This methyltransferase catalyzed the methylation of the hydroxyl group at position 4 of DIMBOA-Glc with S-Adenosyl-L-methionine as the methyl donor to form HDMBOA-Glc (2-(2-hydroxy-4,7-dimethoxy-l,4-benzoxazin-3-one)-β-D-glucopyranose). The activity reached a maximum 9 hours after treatment with JA, and then decreased gradually. The optimum pH and temperature was 7.5 and 35℃, respectively. The activity increased by the application of 1 mM EDTA (ethylenediamine tetra acetate), while heavy metal ions (1 mM) inhibited the activity completely.
Occurrence of N-acetylhexosaminidase was found in young maize seedlings. Most of activity was localized in the embryo before germination. After germination, the strongest activity was detected in scutellum, while the activity was hardly detectable in roots and shoots. The high activity in scutellum was sustained until 80 hours after germination. When N-acetylchitobiose [(GIcNAc)_2] was used as substrate, this enzyme showed a pH optimum at pH 4.5 and a temperature optimum at 55℃. The native molecular mass and isoelectric point (pI) were determined as 44 kDa and 6.45, respectively. Apparent K_m value for (GlcNAc)_2was estimated to be 0.1.24 mM, while the value for p-nitrophenyl-N-acetylglucoside was 0.400 mM.
Theobroxide is an epoxy cyclohexene compound isolated from the fungus Lasiodiplodia theobromae and induces potato microtuber formation in vitro as low as 5x10^<-6>M (Nakamori et al., 1994). When sprayed on potato (Solanum tuberosum L.) and morning glory (Pharbitis nil) plants, which require short days to induce tubers and flower buds, respectively, these plants kept in non-inducing conditions (long days) produced corresponded tubers and flower buds (Yoshihara et al., 2000). However, the action mechanism of theobroxide is still not clear. The examination of the hypothesis that theobroxide may play an important role in the accumulation of endogenous jasmonoids was carried out in this study. In vitro bioassay using cultures of single-node segment of potato stems was performed with the supplement of theobroxide in the medium at different concentrations, the endogenous jasmonic acid (JA), tuberonic acid (TA, 12-hydroxy jasmonic acid) and its glucoside (TAG) in segments, stolons and microtubers were respectively quantitative analyzed. As concern as morning glory, plant seedlings were sprayed with theobroxide solution at a two-day interval, the endogenous JA and IA in the leaves were analyzed. An increase in the content of endogenous JA correlated with the concentration of theobroxide was observed in both segments and microtubers. Endogenous TA was only detected in segments, and the content increased with the concentration of theobroxide. The increase in the content of endogenous TAG caused by the treatment of theobroxide was also observed in microtubers. However, this trend was not found in stolons and segments. It was also found that spraying theobroxide on morning glory plants for 2-3 weeks under short and long days respectively increased 1.6 times and 2.3 times of JA in leaves. The theobroxide spray under short days caused 1.5 times increase in TA level in all treatments.
Herbivore attacks induce leaves to emit a specific blend of volatiles. It is thought that accumulation of jasmonic acid (JA) is elicited in higher plants in response to herbivore attack, which leads to emit the volatiles to attract natural enemy of herbivores. We found that lima bean.leaves, in response to the infestation with herbivorous mites, not only accumulate JA but also emit ethylene. Ethylene is thought as one of the signaling mediators, as like as JA, for wound response in plants. Thus, we studied the synergistic effects of exogenous 1-aminocyclopropane-t-carboxylic acid (ACC: a precursor of ethylene) and JA on production of induced volatiles by excised lima bean leaves. Application of ACC alone to leaves induced trace amounts of volatiles. However, ACC positively affected three JA-induced volatiles, (E)-and (Z)-β-ocimene, and (Z)-3- hexenyl acetate, when lima bean leaves were treated with JA and ACC. The ethylene inhibitor, silver thiosulphate, inhibited the production of these compounds. The results suggest synergic effects of JA and ethylene on inducible volatile production by lima bean leaves. Furthermore, lima bean leaves treated with JA plus ACC became more attractive to predatory, mites, phytoseiulus persimilis, than those treated with JA alone. We therefore conclude that ethylene is involved in one of the signal mediators for indirect defense systems.
Infestation by spider mites (Tetranychus urticae) induces lima bean leaves to emit a blend of volatiles that attracts the predatory mites. This blend of volatiles also induces plants downwind from the infested plants to become more attractive to the predatory mites and, thus, less susceptible to spider mites. Here we show that exposure to T. urticae-induced volatiles, as well as T. urticae infestation and artificial wounding, activates the transcription of the genes involved in the biosynthesis of ethylene [S-adenosylmethionine (SAM) synthetase and 1-aminocyclopropane-l-carboxylic acid oxidase] and a gene involved in the biosynthesis of polyamines from SAM (SAM decarboxylase) in lima bean leaves. Moreover, exposure of leaves to any one of the 7 major chemical components of T. urticae-induced volatiles also induces expression of these genes. Furthermore, we found that, when lima beans were exposed to Z urticae-induced volatiles, they emitted ethylene. Lima bean plants infested by T. urticae and artificially wounded leaves also emitted ethylene. Endogenous polyamine levels were not increased in the exposed leaves or the infested leaves, suggesting that polyamine production from SAM was only slightly promoted at the metabolic levels present in the leaves. We found that jasmonate (JA), but no salicylate (SA), accumulated in leaves exposed to T. urticae-induced volatiles, and that both JA and SA accumulated in leaves infested by T. urticae. These findings, as well as results of pharmacological analyses, suggest that, in leaves exposed to T. urticae-induced volatiles, ethylene biosynthesis might be regulated by pathways involving JA and the ethylene positive feedback loop.
Sex expression of cucumber plants is affected by the day length and a plant hormone, ethylene, Short-day conditions or the treatment with ethylene induce the femaleness whereas long-day conditions or the treatments with inhibitors of ethylene biosynthesis induce the maleness. In this study, we examined the correlation between the effects of day length and ethylene on sex expression of monoecious cucumber plants. The application of ethephone, an ethylene releasing agent, to the shoot apices of cucumber plants, when the leaf blade of the fourth leaf was approximately 2-cm long (defined as the 4-leaf stage), induced femaleness under the long-day (16h) condition. Also, the application of aminoethoxyvinyl glycine (AVG), an inhibitor of ethylene biosynthesis, to the shoot apices of cucumber plants at the 4-leaf stage induced maleness under the short-day (8h) condition. Ethephone treatment did not induce femalehess significantly under the short-day condition. Also, the cucumber plants grown under the short-day condition produced more ethylene than those grown under the long-day condition at the 4-leaf stage. Ethylene production from the shoot apices of cucumber plants and the expression of CS-ACS2 and CS-ERS showed circadian rhythm with a peak in the middle of the photoperiod. These results indicate that the femaleness of the cucumber flowers under the short-day condition mediate ethylene production. CS-ACS2 and CS-ERS genes may contribute to this ethylene signaling cascade in the femaleness of cucumber plants.
We detected endogenous propylene as a peculiarity of the genus Erigeron plants during the analysis of physiological properties of ethylene among plant species and their cultivars. The unknown peak with about 5.8 min. retention time in our GC system in Erigeron plants was identified as propylene by GC-MS. Propylene was determined in the leaf (and callus) of E.philadelficus, E.annuus , E.canadensis, Conyza sumatrensis(E.fioribundus). We could not find it in other species including some Compositae plants. Propylene production fluctuated by light/dark condition and it was remarkably promoted in light. This behavior is very different from that of ethylene which is produced in the darkness much more than in light.