Exposure of normal, tall rice(Oryza sativa)seedlings to 5-azacytidine, a powerful inhibitor of DNA methylation in vivo, induced both demethylation of genomic DNA and dwarf plants. Genes that had been affected by teatment were identified by differential screening of a cDNA library, and a gene encoding a small GTP binding protein, rgp1, was subsequently isolated. To determine the physiological role of rgp1, the coding region of rgp1 was introduced into tobacco plants. Transformants showed distinct phenotypic changes; reduced apical dominance and dwarfism. The content of cytokinins was 6-fold higher in transgenic plants that in control wild-type plants. Looking for genes that respond to cytokinins, a protein kinase gene, wpk4 was isolated. In addition to cytokinin treatment, transcripts of wpk4 were increased upon poor nutrients and light. Cytokinin antagonist erased these effects, suggesting that nutrient and light signals were mediated by cytokinins. Our experiments have revealed that DNA methylation controls expression of genes involved in plant growth, and that protein phosphorylation cascade that is regulated by cytokinins is at least a part of signal transduction pathways of plant growth. Further experiments are under progress to determine components that participate in these pathways.

抄録全体を表示

The endogenous levels of ABA, IAA and Gas were analyzed to investigate physiological effects of plant hormones. In tomato fruits, distributions and their changes of phytohormone levels were determined. In citrus, phytohormone levels in fruitlets were determined in parthenocarpic and non-parthenocarpic cultivar. Tha mechanism of fruit drop by GA biosynthesis inhibitor was discussed in relation to hormone levels of mandarin. ABA was repeatedly applied to fruts, suggesting that ABA increases sugar concentration. In asparagus stem had high levels of ABA and IAA in tip region, suggesting that these hormones induce stem growth. Stress-relaxation analysis was applied to examine tissue softening in fruit using a conical probe. The technique was applied to banana. Physical and chemical properties of banana pulp were analyzed during softening. Cellulose contents did not change, but other cell wall components and starch contents decreased with identical pattern, which was similar to those of elasticity and viscosity. However, the partial breakdown of the cell wall preceded breakdown of starch. In cooking banana, the results analyzed suggest that cell wall polysaccharides are the chemical components responsible for the changes in firmness rathar than starch.

抄録全体を表示

Blue-light showed the most promoting effects as compared with green-light or red-light on growth and flowering of shot-day plants such as morning-glory and rice plant. On the other hand, green-light showed the most promoting effects on long-day plants such as spinach, radish and wheat. Blue-light and green-light showed the opposite effects on short-day plants and long-day plants. Under the natural environmental conditions, a ratio of blue-light is high during summer season and low during winter season. On the other hand, a ratio of green-light is high during winter season and low during summer season. The season of flower initiation for long-day plants is during winter and it for short-day plants is during mummer. It is suggested that the seasonal change of the ratio of blue-light and grenn-right have influence on flowering season of long-day plants and short-day plants under the natural conditions.

抄録全体を表示

Acid-induced cell wall loosening was studied by measuring the creep of the cell wall as affected by IAA, BR and cell wall proteins using pumpkin (Cucurbita moschata Duch. Cv. Shirakikuza) hypocotyl segments stored in 50% glycerin at-15 C at least for 2 weeks (glycerinated segments). Effect of BR was also examined using glycerinated internode segments of dwarf(lkb), a BR mutant, and normal (LKB) pea (Pisum sativum L.). Two parameters involved in cell wall loosening, cell wall extensibility (φ) and yield of threshold (Y), were determined. At pH4.5, φ increased and Y decreased, indicating that acid-induced cell wall loosening occurred. IAA (10 uM) applied to segments for 2 hr prior to glycerin storage had no effect on φ and Y when measured at pH4.5. However, at pH6.8, Y decreased while φ was not affected significantly. BR similarly applied for 6 hr had no effect either at PH4.5 or pH6.8. Boiled glycerinated segments showed no acid-induced cell wall loosening. Incubation of boiled segments with proteins extracted form the cell wall by 1 N NaC1 recovered the ability of the segments to show acid-induced wall loosening. A protein fraction precipitated by 250g/L ammonium sulfate was most active. The present result confirms that some cell wall proteins are necessary for acid-induced cell wall loosening. Experiments with lkb/LKB have shown that BR is necessary for acid-induced wall loosening, since lkb did not show acid-induced wall loosening while BR-treated lkb showed the same ability as LKB.

抄録全体を表示

The extensibility of cell walls is conceived to limit the elongation growth of stems and roots. The stress-relaxation method developed by Yamamoto et al. Has been employed for analyzing mechanical properties of stems and coleptiles. Tanimoto recently found the advantage of the creepanalysis method for detecting gibberellin (GA)-enhanced cell-wall extensibility of roots. In this report we used the 6-element model which composed of 3 elastic moduli (E0, E1 and E2) and 3 viscosity coefficients (n0, n1, and n2) and compared the effects of aluminum (A1), acid pH and GA on these parameters in order to understand the extension mechanism of root cell walls in response to environmental pH, metal and hormone treatment. A1(10uM, pH4.5) treatment of wheat roots increased E0 and n0 a little at 3 h, and E0, E1, E2, time-course inhibition of elongation growth. In contrast, acid treatment (pH4.0) of methanol-killed segments of pea roots markedly decreased n0, whereas treatment with A1 in vitro did not affect these parameters examined so far. Whereas the change in n0 was commonly found in GA-induced growth, acid growth and A1-inhibition, the mode of change in other parameters were characteristic for three treatments. It was suggested that A1 decreases cell-wall extensibility via cytoplasmic activity to synthesize and metabolize cell-wall polymers, while the acid effect is brought about by the direct effect on cell-wall polymers. Further investigation is necessary to understand the function of these parameters of root cell walls in acidic soil.

抄録全体を表示

Experiments were undertaken to verify the involvement of ethylene in germination of the hemiparaitic angiosperm Striga hermonthica (Del.) Benth. Germination was inhibited by AVG (aminoethoxyvinyl glycine), an inhibitor of ACC synthase activity, and restored by ACC (1-aminocyclopropane-1-carboxylic asid), the immediate ethylene precursor. Ethylene release from GR24-treated seeds commenced 6 h after treatment, while germination was first visible 12 h later. These results strongly suggest involvement of ethylene is S. hermonthica germination. RNAs, isolated from GR24-treated seeds, were used for PCR-based amplification of DNA fragments encoding the ACC synthase-active site domain. Three DNA fragments, indicating involvement of 3 different ACC synthase genes, were identified.

抄録全体を表示

The restration of thermoinhibition by germinstion-inducers varied in response to the difference of temperatures. The changes of ABA contents could be responsible for the varying effects.

抄録全体を表示

ABA(1)is metabolized to 8'-hydroxy-ABA(2) which isomerizes to phaseic acid(3), and 3 is reduced to dihydrophaseic acid(4). The activity of 3 is lower than 1, and 4 is almost inactive. However, the activity of 2 is unknown since 2 is spontaneously isomerizes to 3 in vitro. In order to estimate the activity of 2 we tested the biological activities of 3'-fluoro-8'-hydroxy-ABA(6), a stable metabolite of 3'-fluoro-ABA(5). Compound 6 was obtained by feeding 5 to suspension culture of rice cells. Compound 6 was stable at pH2-6 although it isomerized rapidly to 3'a-fluorophaseic acid(7)at pH 8-10. Inhibitory activity of 6 was lower than 5, and higher than 7 in assays of lettuce seed germination, rice seedling elongation and stomata opening. The lower activity of 6 could be caused by hydrophilicity of the 8'-hydroxyl group since the C-8' and -9' of 1 seem to interact with a hydrophobic domain of ABA-binding proteins. This result strongly suggests that the activity of 2 is lower than 1. And higher than 3. Therefore, 1would be inactivated stepwise by metabolic enzymes, 8'-hydroxylase, 8'-hydroxy-ABA cyclase, and phaseic acid reductase, in plants.

抄録全体を表示

In the present study, the effects of externally applied IAA and its endogenous level under simulated micro gravity condition were examined using etiolated Alaska pea seedling. Under such condition, the effect of IAA on epicotyl elongation was increased, and its endogenous level in epicotyls was decreased. The clear effect of applied IAA on the epicotyls may be induced relating to lower level of endogenous IAA in buds and hooks.

抄録全体を表示

In citrus growing, rootstocks that regulate scion growth have important roles. But it is not clear how the rootstocks regulate scion vigor. In this study, we selected three citrus rootstock cultivars, flying dragon (Poncirus trifoliata var. monstrosa) as a dwarfing rootstocks and swingle citrumelo (P.trifoliata X Citrus paradisi) as a vigorous with trfoliate orang (P.trifoliata) as a control. Tender buds from new shoot of Lemon 'Eureka' were cleft grafted on etiolated rootstocd seedlings. Eighteen months after grfting, dry matter of each part of young trees was measured. The order of top weight was the best on swingle citrumelo and worst on flying dragon. But the difference among rootstocks was not detected in the underground part. Endogenous indole acetic acid (IAA) and abscisic acid (ABA) in the new shoot was measured. IAA levels on swingle citrumelo was the highest and lowest on flying dragon. There were no distinct differences in ABA levels.

抄録全体を表示

Higher molecular weight (M.W.2, 500) promoting activity for greening of etiolated cotyledon of cucumber was detected in squash xylem sap, and subjected to column chromatographies. It is suggested that xylem sap contains new peptidy substance which promotes greening

抄録全体を表示

The metabolism of C1 compounds in higher plants is an attractive research target. As a first step, we investigated the effect of formate on the growth of rice plant.Growth stimulation of the aerial part and increase of fresh weight were observed by the (Formate Dehydrogenase)specific activity was increased. These findings suggest the existence of formate metabolism in rice plant. We obtained FDH homologue sequence based on potato FDH sequence by RT-PCR method. The cDNA contains 1230 bp open reading frame. The deduced amino acid sequence shows high homology to other plant FDHs, so far reported.

抄録全体を表示

In cell suspension cultures of lily, exogenously applied teasterone was metabolized to teasterone-3-O-B-D-glucopyranoside. Structure of the glucoside was determined by comparison with authentic one using FAB-MS and 1H-NMR spectra. This is the first teasterone glucoside and the biological activity of it is comparable to that of teasterone by the rice-lamina inclination test.

抄録全体を表示

In this study, we examined the volatile compound-inducing acivity of OPC homologous series. The upper 20cm of a 40cm high tobacco stem was cut, and the lower 2 leaves were removed. The remaining stem was placed in a 50ml Falcon tube which was filled with a 10-3M solution of each of the OPC homologous series. 16 hr after treatment, the stem was put in a plastic desiccator (10L) and flushed with 120L purified air (300-350ml/min.)The volatile compound-containing air was led through a glass trap containing Tenax TA. The Tenax was extracted with distilled diethyl ether, and the extract was concentrated under a nitrogen stream. The major component, (E)-β-ocimene, was identified by the retention time of GC and MS spectrum pattern. Quantative analysis was done using readily available (Z)-β-ocimene as a standard compound. Table 1 shows the (E)-β-ocimene amounts of tobacco treated with each of the OPC homologous series. 3-Oxa and 2-F homologous series did not induce ocimene formation. On the other hand, some of the OPC homologous series, especially those with an even number carbon chain, induced ocimene formation. Despite having an odd number carbon chain, OPC-7 induced a large amount of (E)-β-ocimene These facts indicated that OPC homologous series might be oxidized to jasmonic acid (epi-jasmonic acid) by β-oxidation, and jasmonic acid is the active form of these OPC analogs. Further investigation is needed to examine the high activity of OPC-7.

抄録全体を表示

It has been suggested that gravitropic curvature in plant stem and root is induced by a lateral gradient of endogenous IAA. However, very little information is available about involvemint of auxin in gravitropic plant stem. In this respect, the hypocotyl elongation of the lower and upper sides, and endogenous IAA distribution in sunflower (Helianthus annuus L.) hypocotyls during gravitropic responses were described. The distribution of endogenous IAA in the lower and upper sides was analyzed using physicochemical assay, and it was indicalted that no significant difference in IAA distribution has observed between the upper and lower halves of gravi-stimulated sunflower hypocotyls.

抄録全体を表示

9-Hydoroxy-10, 12-octadecadienoic acid was isolated form indian mustard (Brassicajuncea) shoots as a photo-induced growth inhibitor, which must explain the difference of cotyledon length between the plants grown under illuminated and dark conditions.

抄録全体を表示

Light-induced growth inhibitors were isolated from light-grown radish seedlings, and named raphanusanins. Recently natural raphanusanins were determined to be isomers of 3-(methylthio)methylene-2-pyrrolidinethione. The raphanusanins are produced from 4-methylthio-3-butenyl glucosinolate(MTBG)via 4-methylthio-3-butenyl isothiocyanate(MTBI). The contents of raphanusanins and MTBI in hypocotyls increased by uni-and bilateral illumination, whereas the content of MTBG decreased. The biological activities of raphanusanins and their precursors are studied by means of the radish hypocotyl growth test and the cress root growth test.The raphanusanins had strong inhibitory activities in both bioassays, whereas MTBI shwed inhibitory activity at concentration greater than 3mM. MTBG showd no inhibitory activity. These data suggest that MTBI is released from MTBG by light-promoted thioglucosidase and spontaneously converted into bioactive raphanusanins.

抄録全体を表示

We examined the thickening of tuberous roots in sweet potato with treatments of four auxin and one anti-auxin. In this field test, we understood the tuberous roots excessive with auxin treatment.Secondly we investigated the auxin of tuberous roots in sweet potato and could ascertain the material is 4-Chloroindole-3-acetic acid.

抄録全体を表示

The beautiful Cholodny-Went theory that had held for decades, based on the lateral translocation, from light to shaded side, of auxin within the tip of seedlings, is it true? To solve the question, a direct examination and repetition of Boysen-Jensen's classical experiment and the experiment on the migration of radiolabeled auxin performed by Pickard and Thimann were performed. Even if oat coleoptile tips were divided with mica perpendicularly to light, the coleoptiles showed the same bending as the coleoptiles divided parallel with mica. When a unilateral blue light dose corresponding to the first-or second-positive curvature was administered, the radioactivity recovered in the basal agar blocks was equal in the lighted and shaded sides both in the curvatures. All these data indicate that any phototropic stimulation does not induce uneven distribution of endogenous or exogenously applied IAA in the lighted and shaded sides, suggesting that phototropic curvature is not explained by the Cholodny-Went theory.

抄録全体を表示

Efficiently utilizing the wastes form food industry is an important way of protecting the environment and seeking the possibilities of their applications in agriculture. Conversion of the strained lees of carrot into plant growth substances was demonstrated. The cell wall polysaccharides extracted from the carrot lees were hydrolyzed and separated by HPLC. Some organic acid and sugar showed promotive activities for the hypocotyl growth of cockscomb seedlings. Their isolation and identification are in progress.

抄録全体を表示

It has been well known that vegetation under the Japanese red pine trees was very sparse. This phenomenon was contributed to allelopathic substance related from fallen leaves, bark or pinecones. Some allelochemicals released to the phenomenon have already been isolated from the extracts of fallen leaves or bark. In pre-observation of the Japanese red pine forest, we found that the fewer weeds in the ground with many pinecones grow than that in the ground without pinecones. In this study, we investigated the allelopathic substance released from pinecones, since to our knowledge allelopathy of pinecones has not yet been studied. The exudates of the pinecones showed high biological activity. When some species of germinating seeds were cultured with pinecones, the growth of the plants was inhibited. These results suggested that some substances released from the pinecones play some roles as allelochemical(s) in Japanese red pine trees. The isolation and the identification of the substances are under study.

抄録全体を表示

In order to clarify biosynthetic intermediates of fungal diterpenoid fusicoccins as an H^+-ATPase activator as well as a potent plant growth stimulant, we investigated fusicoccin-related metabolites in detail from the fungus Fusicoccum amygdali F6. A new derivative of fusicoccin J (FC-J) was isolated along with FC-J and 16-O-demethyl 3-epi-FC-J (FC-1) from a polar neutral fraction of the Et OAcextract, and was identified as 16-O-demethyl FC-J (FC-2) by NMR spectrometry. Isolation of FC-1 and FC-2 as 3-epimers of fucicoccane skeleton has much interest for us from biosynthetic and bioactive points of view.

抄録全体を表示

Fusicoccanes including fusicoccins (FCs), cotylenins (CNs), ophiobolines, and their synthetic analogs were examined for their effects on seed germination of important root parasitic weeds, witch-weed [Striga hermonthica (Del.) Benth] and clover broomrape [Orobanche minor Smith]. Since FCs and CNs carrying different sugar moieties showed similar levels of stimulative activity and, in addition, aglycones were more active than the corresponding parent compounds, aglycones appeared to contain the essential structure for stimulative activity. Furthermore, 9-deoxy-19-hydroxycotylenol was the most active among the compounds tested, indicating that the substituent on the 9-position is not needed for the acitivity. By contrast, the presence of hydroxy group either on the 3- or 12- position seemed to be essential for the activity.

抄録全体を表示

Occurrence and properties of hydroxamic acids glucosyltransferase and hydroxamic acid glucosides glucosidase in wheat seedlings were investigated to elucidate their roles during juvenile stage of growth. The fashion of the appearance and disappearance of these enzyme activities were concurrent with that of hydroxamic acids. Partialy purified glucosyltransferase was more specific to DIMBOA than to DIBOA, and did not convert their precursors, HBOA and HMBOA, to their respective glucodsides.The glucosidase was suggested to be a oligomer of about 60 kD polypeptides. It showed higher specificity to DIMBOA-G than to othar hydroxamic acid glucosides. These results indicate that the occurrence of hydroxamic acids is for defense at juvenile stage, and this is regulated by these specific enzymes.

抄録全体を表示

We found that a glucosyltransferase activity occurred concurrently with the specific occurrence of DIMBOA-G in maize during juvenile stage of growth. The partial purification revealed that there are two isozymes, GT1 and GT2, and GT1 was puridied 74-fold, and GT2 38-fold. GT2 catalyzed the glucosylation of both DIMBOA and DIBOA, while GT1 only DIMBOA.

抄録全体を表示

(I) We have prepared the methyl ester of 9, 11-didehydrogibberellin A_24 and shown it to be the biosynthetic precursor of the potent antheridiogen, gibberellin A_73 methyl ester (GA_73-Me), in protharia of the ferns Lygodium flexuosum and Lygodium circinnatum. (II) The structure of a new gibberellin-like antheridiogen from the fern Lygodium circinnatum has been confermed as the methyl ester of 12 α-hydroxy-GA_73 (GA_117) by synthesis of an authentic sample from the 17-nor-16one derived from GA_73. Comparative bioassays of the synthetic compound as an antheridium-inducing substance in Lygodium showed that it is highly potent, with activity in the picomolar range, and that it is considerably more active than the 12β-epimer(GA_96).

抄録全体を表示

Cultured broths of actinomycetes isolated from plants and rhizopheres were examined for plant growth-regulating activities in 3 bioassays. Results reveal that 8 strains produce the inhibitors of lettuce seeds germination or rice seeds growth, or the cytokinin-like compounds.

抄録全体を表示

Pythium red blight is one of important diseas of bent grass, and is caused by infection of Pythium aphanidermatum. IAA, linoleic acid and P-hydroxybenzaldehyde were isolated from the metabolite of Pythium aphanidermatum. IAA and linoleicacid completely inhibited tea pollen tube gowth at a concentration of 100mg/l. IAA inhibited the hypocotyl elongation and the root growth of lettuce seedlings by 13% and 8% at a concentration of 100mg/l. However, IAA, linoleicacid and p-hydroxybenzaldehyde showed no inhibitory activity against the growth of rice seedlings at the sama concentration. IAA completely inhibited the growth of bent grass at a concentration of 1000mg/l.On the other hand, linoleic acid and p-hydroxybenzaldehyde did not show any remarkable inhibitory activity against the gowth of bent grass at the same concentration.

抄録全体を表示

A sterile dark mycelial fungus (SD fungus), which has been isolated from wheat roots cultured in its replanted soil for over 25 years, was found to be a new genus close to the genus Geomyces and Blumeria in Eymycota on the basis of 18S rRNA sequence analysis. As a metabolic function of the SD fungus, cerevisterol and Δ^9^(11)-cerevisterol were isolated from the fermentation mycelia and further the presence of related steroids were confirmed.

抄録全体を表示

Oat phytoalexins, avenanthramides, were induced by the treatment with penta-N-acetylchitopentaose. The induction was proceeded by an increase in enzyme activities of phenylalanine ammonia lyase(PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate: CoA ligase(4CL). The dependency on elicitor concentraton of induced enzyme activities was well correlated with that of avenanthramide induction. Among tested oligo-N-acetylchitooligosaccharides, tetra-and pentasaccharides strongly induced these enzyme activities as well as avenanthramide production.These findings indicate that PAL, C4H and 4CL activities are involved in biosymthesis of avenanthramides and that accumulation of avenanthramides is the result from activation of de novo synthesis.

抄録全体を表示

Tyramine feruloyltransferase (TFT) catalyzes the condensation reaction to feruloyltyramine from tyramine and feruloyl-CoA. Wounding strongly induced this enzyme activity in maize leaves. TFT activity started to increase 3 hr after wounding and reached a maximum by 36 hr, being 11 times higher than the activity in control leaves. For characterization, TFT from wounded leaves was partially purified by (NH_4)_2SO_4 precipitation and two steps of anion exchage chromatography on DEAE-Sepharose and Mono Q. The best substrate was tyramine among tested amines, although phenethylamine, tryptamine and dopamine served as acyl acceptors. On the other hand, for acyl donar, feuloyl-CoA was most rapidly converted to a respective amide by TFT among hydroxycinnamoy-CoA esters.

抄録全体を表示

PSK-α is a plant peptidal growth factor isolated from conditioned medium of Asparagus mesophyl cell culture. PSK-α is a modified pentapeptide whose tyrosine residues are sulfated. The apply of PSK-α to Asparagus mesophyll cell culture is known to result in activation of cell division at low cell density. PSK-α was found in vaious plant cell culture systems not only in monocotyledonous but also in dicotyledonous plants. These observations indicate that PSK-α is an indispensable factor in cell division of higher plants. In this study, we examined the effect of PSK-α on cell division cycle using synchronous culture of carrot non-embryogenic cells(NC). Synchronized cell division of NC was established by aphidicolin treatment. In this system, the first peak of cell division was observed 13-14 hours after elimination of aphidicolin, even though NC was precultured in MS medium with or without 2, 4-D (1mg/l). When PSK-α(1x10^-7M) was treated before or after aphidicolin treatment, mitotic index (MI) increased in both cases. However, the time after aphidicolin elimination showing maximum MI was the same in both cases and equivalent to that in negative control (no PSK-α treatment). These results may indicate that PSK-α functions in G1 or G0 phase but not in G2 or M phase and that PSK-α acts very rapid after the application.

抄録全体を表示

It is well known that proliferation of plant cells in culture is promoted by addition of conditioned medium(CM) to a fresh medium. Recently, a peptide plant growth factor, phytosulfokine-α(PSK-α), has been isolated from CM of asparaqus mesophyl cell cultures. However, there is no information on the physiological roles of PSKs. To clarify effects of PSKs on plant morphogenesis, we examined it using carrot cell cultures. When PSK-α was added to the induction medium of carrot somatic embryogenesis, the number of somatic embryos formed increased depending on the concentration of PSK-α, and the cell numbers also increased throughout the culture period by addition of PSK-α. 【1-4】PSK-α and 【1-3】PSK-α showed similar effects. On the other hand, addition of Tyr-SO_3H and 【2-5】PSK-α, negative control chemicals, showed no effect on somatic embryogenesis nor cell proliferation. When embryogenic cells(EC) were cultured in the medium containing 2, 4-D and PSKs, proliferation of EC was stimulated depending on the concentration of PSK-α, 【1-4】PSK-α and 【1-3】PSK-α. When non-embryogenic cells (NC), Which lost the ability to form somatic embryo, was used, cell proliferation in the medium containing 2, 4-D(1mg/L) was not affected by PSK-α, 【1-4】PSK-α, 【1-3】PSK-α and negative contol chemicals, Tyr-SO_3H and 【2-5】PSK-α but those in the medium containing low concentrations of 2, 4-D stimulated by addition of PSK-α. While in 2, 4-D deleted medium, cell proliferation of NC was not stimulated by PSK-α. These results suggest that PSKs stimulates carrot somatic embryogesis though promotion of cell division and PSKs may support the fuction of auxin, 2, 4-D.

抄録全体を表示

Both tracheary element(TE) differentiation and proliferation of the mesophyll cells Zinnia elegans are suppressed in low cell density cultures with indication of cell-to-cell interaction involvement. We have investigated the effects of a sulfated pentapeptide, phytosulfokine-α(PSK-α), orginally isolated as a growth factor from asparagus suspension culture, on differentiation and proliferation of Zinnia mesophyll cells cultured at low density. When cells were incubated at an initial density of 2.5x10^4 cells/ml in TE inductive medium containing NAA(0.1mg/l) and (6-BA(1.0mg/l) in the presence of various concentrations of PSK-α, differentiation was strongly stimulated in dose-dependent fashion;more than 30% of the living cells were differentiated into TEs by the 4th day of culture in the presence of 10 nM of the factor . PSK-α also promoted proliferation of Zinnia cells in medium specified for cell division containing NAA (0.1mg/l) and 6-BA (0.1mg/l), at concentrations of 3.2nM and above. ELISA and mass spectrometry further confirmed that cultured Zinnia mesophyll cells produce nanomolar levels of PSKs under these conditions. This is the first study to demonstrate that a peptide factor riggers both cell differentiation and proliferation in plant cells.

抄録全体を表示

Endogenous brassinosteroids in the of baley (Hordeum vulgare L., cv. Masakadomugl and Kantohardaka No.77) and wheat (Triticum aestlvum L., cv Bandowase) were studied. In every sample, shoots at their maximum tillening stage collected at the end of March were homogenlzed and extracted in methanol. After the purification, their active fractions on bioassay were analyzed by full scan GC/MS. Catasterone and 6-deoxocastasterone were identified from all extracts. Brassinolide was identified from the extract of Masakadomugl. In the extract of Bandowase, brassinolide was identified by GC/SIM analysis. This repot first demonstrated the occurrence of brassinosterolds in the shoots of barley and wheat.

抄録全体を表示

Brassinosteroid deficient mutants, being known only in Arabidopsis, tomato and pea, are playing important roles to clarify the biological significances of brassinosteroids in plants. In order to investigate the function of brassinosteroids in other plants, a novel brassinosteroid biosynthesis inhibitor (Brz) was treated to cress, rice, cucumber. Brz induced photomorphorogical changes in the dark, and dwarfisim and early development of leaves in the light. These morphological changes of the plants should be due to interferring with biosynthesis of brassinosteroids.

抄録全体を表示

Some of cytochrome P450 (P450) inhibitors are triazole analogs. Because plant P450s are also affected by triazole analogs, we designed and synthesized triazole analogs as biosynthetic inhibitors of BRs (Brassinosteroids). Effect of chemicals synthesized in this study on BRs biosynthesis was examined using Arabidopsis seedling test and rice lamina inclination test. One of analogs, MA 91 showed inhibitory effect on Arabidopsis seedling at 1mM, which were recovered by BR treatmen. Study on structure-activity relationship of synthetic compounds indicated that biological activities of these compounds varied significantly according to the difference of functional group of benzene ring and a substituent at C2 where hydroxyl group attached. The data suggested that MA 91 should block' step(s) in the biosynthetic pathway of BRs. We named Ma 91 as Brz.

抄録全体を表示

In the investigation of brassinosteroids (BRs), the isotopically labeled BRs are powerful tools to elucidate the biosynthesis, metabolism, mode of action at the molecular level. Since nowadays some natural BRs are commercially available. we planned a general approach to labeled BRs from the parent BRs, which included three basic steps, C-25 h droxylation, dehydration to introduce a Δ^<25>^<(26)>-double bond and catalytic deuteriogenation or tritiation or the double bond. By following this strategy, [25, 26, 27-^2H_n] brassinolide (BL) was synthesized from BL with 5 steps, where the catalytic deuteriogenation of Δ^<25>^<(26)>-BL using 5% palladium-on-charcoal resulted in abundant incorporation of deuterium atoms to give [25, 26, 27-^2H_7] BL with 60% isotopic purity. In addition, during this work, we found that BL easily epimerized at C-5 by treatment with sodium methoxide in refluxing methanol to give 5-epi-BL. The prepared 5-epi-BL had neitheragonistic nor antagonistic effect on the rice lamina inclination test, providing the first experimental proof that A/B trans fused ring junction of BRs is an essential structural factor not only for the higt BR activity, but also for interacting with the binding site of the BR receptor.

抄録全体を表示

Two novel Arabidopsis dwarf mutants, dwf5 and dwf7, were originally identified in a screen of 14000 T-DNA transformed lines of Arabidopsis. To determine the defective step in brassinosteroid biosynthetic pathway, we have analyzed endogenous sterols and BRs in these mutants. In addition, feeding studies with ^<13>C-MVA and compactin were performed. Quantitative analysis revealed that endogenous levels of 24-methylenecholesterol and its downstream compounds in biosynthetic pathway of brassinolide were greatly reduced in dwf5 and dwf7, suggesting that each of dwf5 and dwf7 is blocked in a step before 24-methylenecholesterol. Further biochemical characterization was performed with ^<13>C-labeled mevalonic acid (MVA) and compactin, a MVA biosynthetic inhibitor. ^<13>C_5-Episterol, ^<13>C_5-24-methylenecholesterol and ^<13>C_5-campesterol were detected as metabolites of ^<13>C-MVA in wild type. In dwf7, ^<13>C_5-24-methylenecholesterol and ^<13>C_5-campesterol was not detected, whereas ^<13>C_5-episterol accumulated 4-fold as compared to wild type. In addition, an uncommon sterol, ^<13>C_5-7-dehydrocampestanol, greatly accumulated. In addition to ^<13>C_5-episterol and ^<13>C_5-7-dehydrocampestanol, ^<13>C_5-7-dehydrocampesterol was detected in dwf5. These results indicate that dwf5 and dwf7 are brassinosteroid deficient mutants, and defective steps in dwf5 and dwf7 are Δ^7-sterol-C7-reduction and Δ^7-sterol-C5-desaturation, respectively.

抄録全体を表示

Gibberellin 3β-hydroxylase gene (Pv3βOH-3) was cloned from immature seeds of Phaseolus vulgaris. Full-lemgth cDNA was expressed in E. coli resulting a recombinant protein that catalysed both 3β-hydroxylation and epoxydation of GA_<20>, GA_9, GA_<19>, GA_<29>, and GA_<44> were also 3β-hydroxylated by this enzyme.

抄録全体を表示

We have previously reported that over-expression of a tobacco homeobox gene, NTH15, (Nicotiana tabacum homeobox 15) in transgenic tobacco led abnormal morphologies with drastic decrease in the content of active gibberellin, GA_1. RNA gel blot analysis and supplementary experiments revealed that the over-expression of NTH15 specitically inhibits the activity of GA 20-oxidase. In this study, we generate a glucocorticoid-inducible system to elucidate the kinetics underlying between the over-production of homeodomain protein and the inhibition of gibberellin biosynthetic pathway. The expression of GA 20-oxidase gene was down-regulated until 6 hours after induction of the NTH15 function. The content of GA_<20> also decreased after 6 hours from the induction and was not detected after 12 hours. While, the expression of 3β-hydroxylase gene has doubled until 24 hours after the induction. Because the content of GA_1 has also been decreased at this time, up-regulation of the 3β-hydroxylase gene is probably due to the feed-back regulation of the gibberellin biosynthetic pathway. These results clearly suggest that the overproduction of homeodomain protein immediately down-regulates the expression of GA 20-oxidase gene and inhibits the biosynthesis of GA_1 in transgenic plants.

抄録全体を表示

A sigle gene dwarf mutant of maize, D8 is insensitive to exogeneously applied GA, despite its high level of endogeneous GA. This suggeste that D8 has defects either in receptor(s) or in signal transduction pathways. In order to isolate genes that are affected in the D8 mutant, we have screened and compared mRNA population between wild-type and D8 shoots by differential display. Subsequently, one cDNA clone, of which mRNA level was lower in D8 than in wild-type plants, was isolated and designated as gar2. The gar2 cDNA sequence contained a 900 base open reading frame capable of encoding a polypeptide of 177 amino acid residues. DNA blot analysis indicated two to three copies for gar2 in maize genome. RNA blot analysis showed that mRNA of gar2 was increased in response to exogeneously applied GA in maize GA-deficient mutant, d5, and that gar2 mRNA was abundant in leaf sheath containing meristematic tissues in wild-type plants.

抄録全体を表示

Young developing seeds of several dicotyledonous plants contained a large amount of gibberellins (Gas). Their physiological roles, however, remain unknown. Their localization and fluctuation may give informations about their roles in seed development. We focused on the localization of GAs, especially so-called "active GAs" in developing seeds of Pharbits nil with an immunohistochemical technique. To avoid the drift of GAs during their fixation, we adopted the combination of lyophilization and fixation using gaseous diisopropylcarbodiimide. Seeds clipped at various developing stages were subjected to the fixation process, embedded in paraffin, sliced into sections of 10 micrometer thick, and stained with an antiserum specific for 7-substituted GA_1. The staining was visualized with the streptavidin-biotin peroxidase system. The specific staining due to active GAs was observed in outer-integument and immature cotyledons of seeds. At higher magnification, many starch grains were observed in outer-integument. GAs were localized at an antipole of the hilum of the starch grains. The relationship between the transition of this localization of GAs and the metabolism of the starch grains will be discussed.

抄録全体を表示

The perception of the specific molecule is the first step in the signal transduction of plant hormones leading to the regulation of physiological phenomena observed in the life cycle of higher plants. The induction of α-amylase in cereal grains and promotion of shoot elongation of higher plants are typical physiological events induced by gibberellin (GA). The signal transduction pathway of GA has been studied mainly in the α-amylase system which showed the localization of GA-receptor on plasma membrane. However, little is known about the GA perception in shoot elongation systems. We successfully detected in cytosolic fractions from etiolated azuki bean seedlings the protein capable of binding active GAs selectively, and partially purified this GA-binding protein (GBP). The apparent Kd value for GA_4, one of the active Gas, was 6 X 10-^<10> (M) by Scatchard plots analyses. The molecular size of the GBP was estimated to be 25 kDa by HPLC with a gel filtration column. We assume this GBP to be one of the receptor candidates and are further purifying it with various chromatographic methods.

抄録全体を表示