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1.
Azospirillum lipoferum strain USA 5b, a gibberellin producing bacterium, was cultured in a nitrogen-free biotin-based chemically-defined medium in the presence of the glucosyl ester or the 13-O-glucoside of [17,17-2H2]-gibberellin A20. The [17,17-2H2]-gibberellin A20 conjugates were added at both the stationary phase of the cultures and at the beginning of the growth curve. Metabolism of the conjugates was examined after 72 h of incubation using capillary gas chromatography-mass spectrometry, with identification by full scan mass spectra. Metabolites identified were [17,17-2H2]-gibberellin A20, [17,17-2H2]-gibberellin A1 and [17,17-2H2]-gibberellin A3. Also, in the Azospirillum cultures fed at the beginning of the growth curve, gibberellin A5 and gibberellin A20 were characterized as endogenous by mass spectrometry/full spectrum. These results support the concept that the growth promotion in plants that is induced by Azospirillum infection may occur by a combination of both gibberellin production and gibberellin-glucoside/glucosyl ester deconjugation by the bacterium.  相似文献   

2.
Azospirillum species are plant growth-promotive bacteria whose beneficial effects have been postulated to be partially due to production of phytohormones, including gibberellins (GAs). In this work, Azospirillum brasilense strain Cd and Azospirillum lipoferum strain USA 5b promoted sheath elongation growth of two single gene GA-deficient dwarf rice (Oryza sativa) mutants, dy and dx, when the inoculated seedlings were supplied with [17,17-2H2]GA20-glucosyl ester or [17,17- 2H2]GA20-glucosyl ether. Results of capillary gas chromatography-mass spectrometry analysis show that this growth was due primarily to release of the aglycone [17,17-2H2]GA20 and its subsequent 3beta-hydroxylation to [17,17-2H2]GA1 by the microorganism for the dy mutant, and by both the rice plant and microorganism for the dx mutant.  相似文献   

3.
The fate of [14C] gibberellin A3 and [3H] gibberellin A1 was examined in senescing fruit of Shamouti orange (Citrus sinensis L. Osbeck) and tomato (Lycopersicon esculentum Mill.). Gibberellin A3 was highly persistent in Citrus peel (t 1/2=18 days) and to a lesser degree in tomato (t 1/2=5.5 days). Ethylene and ethephon caused a slight enhancement of gibberellin A3 metabolism in Citrus and tomato fruit, respectively. Gibberellin A1 was metabolized by Citrus peel at a relatively high rate (t 1/2 < 24 h) and ethylene slightly reduced this rate. It is concluded that the ethylene-induced enhancement of senescence does not involve major effects on the deactivation of applied gibberellins.Abbreviations GA3 gibberellin A3 - GA1 gibberellin A1  相似文献   

4.
The mass production of pure gibberellin A1 (GA1) by shake-culturing Phaeosphaeria sp. L487 was investigated. Its GA1 production was markedly influenced by natural nitrogen sources and NH4NO3. When the fungus was cultured in an 8% glucose-1.5% oatmeal-0.1% NH4NO3-0.5% KH2PO4-0.1% MgSO4 x 7H2O medium for 3 weeks, the amount of GA1 in the culture filtrate was up to ca. 200 microg/ml: the addition of safflower oil to the culture medium two weeks after inoculation prolonged the GA1-production period to produce 300 microg/ml. Further preparation of [U-13C]GA, as a tool for the analysis of a complex of GA1 and its binding protein was attempted by using the fungus. The fungal culture in a [U-13C]glucose-oatmeal medium gave 6 mg of crystalline 13C-enriched GA1. Its 13C-enrichment of ca. 75% and 1J(CC) values were determined by NMR spectrometry.  相似文献   

5.
The relationship between protein synthesis and the incorporation of [3H]gibberellin A1 ([3H]GA1) into a 2,000xg pelletable (2KP) fraction from lettuce (Lactuca sativa L.) hypocotyl sections has been investigated. Concentrations of D-2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide (MDMP) between 10-7 M and 10-4 M caused increasing inhibition of growth, 2KP labelling and incorporation of [14C]leucine into soluble protein. Growth and 2KP radioactivity were highly correlated (r=0.996). Transfer to MDMP early or late in the course of GA response caused reductions in both growth and incorporation into the 2KP fraction. Exposure to the inhibitor had more effect at 4 h than at 20 h. The proportions of alkali-soluble and insoluble radioactivity in the 2KP fraction were also altered by this treatment. The implications of these findings are discussed.Abbreviations GA1 gibberellin A1 - MDMP D-2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide - 2KP a2,000xg pelletable fraction  相似文献   

6.
The steps involved in kaurenolide and fujenoic acids biosynthesis, from ent-kauradienoic acid and ent-6alpha,7alpha-dihydroxykaurenoic acid, respectively, are demonstrated in the gibberellin (GA)-deficient Gibberella fujikuroi mutant SG139, which lacks the entire GA-biosynthesis gene cluster, complemented with the P450-1 gene of GA biosynthesis (SG139-P450-1). ent-[2H]Kauradienoic acid was efficiently converted into 7beta-hydroxy[2H]kaurenolide and 7beta,18-dihydroxy[2H]kaurenolide by the cultures while 7beta-hydroxy[2H]kaurenolide was transformed into 7beta,18-dihydroxy[2H]kaurenolide. The limiting step was found to be hydroxylation at C-18. In addition, SG139-P450-1 transformed ent-6alpha,7alpha-dihydroxy[14C4]kaurenoic acid into [14C4]fujenoic acid and [14C4]fujenoic triacid. Fujenal was also converted into the same products but was demonstrated not to be an intermediate in this sequence. All the above reactions were absent in the mutant SG139 and were suppressed in the wild-type strain ACC917 by disruption of the P450-1 gene. Kaurenolide and fujenoic acids synthesis were associated with the microsomal fraction and showed an absolute requirement for NADPH or NADH, all properties of cytochrome P450 monooxygenases. Only 7beta-hydroxy[14C4]kaurenolide synthesis and not further 18-hydroxylation was detected in the microsomal fraction. The substrates for the P450-1 monooxygenase, ent-kaurenoic acid and [2H]GA12, efficiently inhibited kaurenolide synthesis with I50 values of 3 and 6 microM, respectively. Both substrates also inhibited ent-6alpha,7alpha-dihydroxy[14C4]kaurenoic acid metabolism by SG139-P450-1. Conversely, [14C4]GA14 synthesis from [14C4]GA12-aldehyde was inhibited by ent-[2H]kauradienoic acid and fujenal with I50 values of 10 and 30 microM, respectively. These results demonstrate that kaurenolides and seco-ring B kaurenoids are formed by the P450-1 monooxygenase (GA14 synthase) of G. fujikuroi and are thus side products that probably result from stabilization of radical intermediates involved in GA14 synthesis.  相似文献   

7.
In a carrot (Daucus carota L.) cell line lacking the ability to undergo somatic embryogenasis, and in carrot and anise (Pimpinella anisum L.) cell lines in which embryogenesis could be regulated by presence or absence of 2,4-dichlorophen-oxyacetic acid (2,4-D), in the medium (+2,4-D=no embryogenesis,-2,4-D=embryo differentiation and development), the levels of endogenous gibberellin(s) (GA) were determined by the dwarfrice bioassay, and the metabolism of [3H]GA1 was followed. Embryos harvested after 14 d of subculture in-2,4-D had low levels (0.2–0.3 g g-1 dry weight) of polar GA (e.g. GA1-like), but much (3–22 times) higher levels of less-polar GA (GA4/7-like); GA1, GA4 and GA7 are native to these cultures. Conversely, the undifferentiated cells in a non-embryogenic strain, and proembryos of an embryogenic strain (+2,4-D) showed very high levels of polar GA (2.9–4.4 g g-1), and somewhat reduced levels of less-polar GA. Cultures of anise undergoing somatic embryo development (-2,4-D) metabolized [3H]GA1 very quickly, whereas proembryo cultures of anise (+2,4-D) metabolized [3H]GA1 slowly. The major metabolites of [3H]GA1 in anise were tentatively identified as GA8-glucoside (24%), GA8 (15%), GA1-glucoside (8%) and the 1(10)GA1-counterpart (2%). Thus, high levels of a GA1-like substance and a reduced ability to metabolize GA1 are correlated with the absence of embryo development, while lowered levels of GA1-like substance and a rapid metabolism of GA1 into GA8 and GA-conjugates are correlated with continued embryo development. Exogenous application of GA3 is known to reduce somatic embryogenesis in carrot cultures; GA4 was found to have the same effect in anise cultures. Thus, a role (albeit negative) in somatic embryogenesis for a polar, biologically active GA is implied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellin(s) or gibberellin-like substances - GC-RC gas chromatography-radiochromatogram counting - HPLC high-presare liquid chromatography - Rt retention time - TLC thinlaver chromatography  相似文献   

8.
W. Hartung  I. D. J. Phillips 《Planta》1974,118(4):311-322
Summary Movement of both [3H]GA1 and [14C]GA3 through root segments from P. coccineus seedlings was basipetally polarised. The basipetal/acropetal ratio of radioactivity from [3H]GA1 in agar receiver blocks was 9.2 for apical, elongating segments, and 4.0 for more basal, non-elongating segments. Polarity of gibberellin transport was restricted to the stele, and absent from cortical tissues. Transport of [14C]IAA through root segments to agar receivers was preferentially acropetal, particularly so in the stele. Despite the existence of basipetal polarity of gibberellin transport in the root, [3H]GA1 injected into cotyledons moved into and acropetally along the seedling root.  相似文献   

9.
The properties of the water-soluble metabolites of [3H]gibberellin A1 ([3H]GA1) from lettuce (Lactuca sativa L.) hypocotyls were compared with those of authentic samples of gibberellin (GA) glucosyl esters and ethers. Partitioning against l-butanol at high and low pH was not an efficient method of differentiating between ester and ether conjugates of GA1 or GA3. Extraction into l-butanol at pH 2.5 was, however, useful as a group purification step. Gel-filtration on acrylamide indicated a mean molecular weight of ca. 600 for the polar material and high-voltage electrophoresis separated two compounds (LH 1 and LH 2) with differing charge properties. Both metabolites incorporated 14C from glucose and 3H from GA1. Subsequent enzymatic hydrolysis of LH 1 released material with identical properties to [14C]glucose together with a second uncharacterised component. Feeding with [3H]GA1 methyl ester greatly reduced the formation of LH 1 but not LH 2. The metabolites were provisionally identified as GA1-glucosyl ester (LH 1) and GA1-glucosyl ether (LH 2).Abbreviations GA gibberellin - LH1 GA3-glucosyl ester - LH2 GA1-glucosyl ether - HVE high voltage paper electrophoresis - TLC thin-layer chromatography  相似文献   

10.
The stepwise metabolism of gibberellin A12-aldehyde (GA12-aldehyde) to GA20 is demonstrated from seedling shoots of maize (Zea mays L.). The labeled substrates [13C,3H]GA12-aldehyde, [13C,3H]GA12, [14C4]GA53, [14C4/2H2]GA44, and [14C4/2H2]GA19 were fed individually to dwarf-5 vegetative shoots. Both [13C,3H]GA12-aldehyde and [13C,3H]GA12 were also added individually to normal shoots. The labeled metabolites were identified by full-scan gas chromatography-mass spectrometry and Kovats retention indices. GA12-aldehyde was metabolized to GA53-aldehyde, GA12, GA53, GA44, and GA19; GA12 was metabolized to 2[beta]-hydroxy-GA12, GA53, 2[beta]-hydroxyGA53, GA44, 2[beta]-hydroxyGA44, and GA19; GA53 was metabolized to GA44, GA19, GA20, and GA1; GA44 was metabolized to GA19; and GA19 was metabolized to GA20. These results, together with previously published data from this laboratory, document the most completely defined gibberellin pathway for the vegetative tissues of higher plants.  相似文献   

11.
Winter canola (Brassica napus cv Crystal) is an oilseed crop that requires vernalization (chilling treatment) for the induction of stem elongation and flowering. To investigate the role of gibberellins (GAs) in vernalization-induced events, endogenous GA content and the metabolism of [3H]GAs were examined in 10-week vernalized and nonvernalized plants. Shoot tips were harvested 0, 8, and 18 d postvernalization (DPV), and GAs were purified and quantified using 2H2-internal standards and gas chromatography-selected ion monitoring. Concentrations of GA1, GA3, GA8, GA19, and GA20 were 3.1-, 2.3-, 7.8-, 12.0-, and 24.5-fold higher, respectively, in the vernalized plants at the end of the vernalization treatment (0 DPV) relative to the nonvernalized plants. Thermoregulation apparently occurs prior to GA19 biosynthesis, since vernalization elevated the concentration of all of the monitored GAs. [3H]GA20 or [3H]GA1 was applied to the shoot tips of vernalized and nonvernalized plants, and after 24 h, plants were harvested at 6, 12, and 15 DPV. Following high-performance liquid chromatography analyses, vernalized plants showed increased conversion of [3H]GA20 to a [3H]GA1-like metabolite and reduced conversion of [3H]GA1 or [3H]GA20 to polar 3H-metabolites, putative glucosyl conjugates. These results demonstrate that vernalization influences GA content and GA metabolism, with GAs serving as probable regulatory intermediaries between chilling treatment and subsequent stem growth.  相似文献   

12.
A convenient chemical synthesis of erythro-D-[1-2H1] sphinganine and erythro-D-[1-3H1]sphinganine is described. The approach utilizes a stereospecific starting material (natural sphinganine prepared from bovine brain sphingomyelin) and applies a sequence of selective protection of functional groups yielding 2-acetamido-3-O-benzoyloctadecan-1-ol. Oxidation of the primary alcohol to an aldehyde followed by NaB2H4 or NaB3H4 reduction and hydrolysis of the protective groups yields erythro-D-[1-2H1]sphinganine or erythro-D-[1-3H1]sphinganine. The synthetic intermediates and isotopically labeled sphinganines are characterized by infrared analysis, 1H-nuclear magnetic resonance, optical rotation, and gas-liquid radiochromatographic and mass spectral fragmentation analyses. The [1-2H1] and [1-3H1] derivatives were obtained with overall yields (and isotope enrichments) of 11% (min. 84 mol% 2H1) and 8% (60 mCi/mmol), respectively.  相似文献   

13.
14.
Cell-free systems were prepared from germinating seed and seedlings of Phaseolus coccineus. Gibberellin A4 (GA4)-metabolising activity was detected in vitro using preparations from roots, shoots and cotyledons of germinating seed, but only up to 24 h after imbibition. Cell-free preparations from cotyledons converted [3H]GA4 to GA1, GA34, GA4-glucosyl ester and a putative O-glucoside of GA34, and, in addition converted [3H]GA1 to GA8. Preparations from embryo tissues contained 2-hydroxylase activity, converting [3H]GA4 to GA34 and [3H]GA1 to GA8.The presence of GA-metabolising enzymes was also indicated by in-vivo feeds of [3H]GA4 to epicotyls of intact 4-d-old seedlings, which resulted in the accumulation of GA1, GA8, GA3-3-O-glucoside, GA4-glucosyl ester, GA8-2-O-glucoside and a putative O-glucoside of GA34. Gibberellin A1 was the first metabolite detected, 15 min after application of [3H]GA4, but after 24 h most of the label was associated with GA8-2-O-glucoside. Over 90% of the recovered radioactivity was found in the shoot. Within the shoot, movement was preferentially acropetal, and was not dependent upon metabolism of the applied [3H]GA4.Abbreviations DEAE diethylaminoethyl - GAn gibberellin An - GPC gel permeation chromatography - HPLC-RC high performance liquid chromatography-radio counting - S-1 1000·g supernatant - UDP uridine 5-diphosphate  相似文献   

15.
Certain N-substituted phthalimides (NSPs) have gibberellin (GA)-like activity in a number of GA bioassays. The interaction between representative NSPs and a protein fraction from cucumber (Cucumis sativus L.) hypocotyls that has GA-binding characteristics consistent with those expected of GA receptors was studied. Analysis of in vitro equilibrium saturation data indicated the presence of only one class of high affinity [3H]GA4 binding sites (Kd ~ 30 nanomolar, n = 0.25 picomole per milligram of protein). In the presence of 6 or 60 micromolar 1-[3-chlorophthalimido]-cyclohexanecarboximide (AC-94,377), the Kd for [3H]GA4 increased, whereas the maximum number of saturable [3H]GA4 binding sites did not change significantly. The dissociation of [3H]GA4 from its binding sites was complex and was best described by a bi-exponential equation. AC-94,377 did not affect the rates of [3H]GA4 dissociation from its binding sites. These results implied that AC-94,377 and [3H]GA4 compete for binding to the same sites. A correlation was observed between the activity of over 20 NSPs in the cucumber hypocotyl bioassay and their in vitro affinity for the GA binding sites. Our observations lend further support to the notion that certain GA binding proteins in cucumber cytosol are GA receptors and also provide a molecular explanation for the GA-like in vivo activity of some NSPs.  相似文献   

16.
Phosphatidyl[2-3H]inositol was prepared from Saccharomyces cerevisiae (YSC-2), grown in synthetic medium containing myo[2-3H]inositol. Over 44 microCi (or 81%) of the radiolabeled inositol was taken up by the organism, with 34 microCi incorporated into phosphatidylinositol. Upon purification by silicic acid pressure liquid chromatography (MPLC), a final yield of 24 to 26 microCi of phosphatidyl[2-3H]inositol with a specific radioactivity of 40 X 10(3) dpm/nmole was obtained. The purified phosphatidyl[2-3H]inositol was found to be a suitable for phospholipase C from human platelets.  相似文献   

17.
The native gibberellin A4 (GA4), in radioactive form ([1,2-3H]GA4, 1.06 Ci/mmol), was fed to carrot somatic cell cultures (suspension and immobilized cell systems) and its metabolism over a 48 hr period was investigated. It was found that the [3H]GA4 was metabolized to at least two GAs, [3H]GA1 and [3H]GA8, six GA glucosyl conjugates, [3H]GA1-0(3)-glucoside, [3H]GA1-0(13)-glucoside, [3H]GA1-glucosyl ester, [3H]GA4-glucoside, [3H]GA4-glucosyl ester, a [3H]GA8 glucosyl conjugate(s) and a previously unknown [3H]GA1 glucosyl conjugate ([3H]GA1-0(3,13)-diglucoside-like compound). The GA1-diglucoside-like compound was found only in extracts of cells and was present in significant amounts (33 % of total extractable radioactivity). All other metabolites were present in both cells and medium. For extracts of the medium, no differences between the suspension and immobilized cultures existed in types of [3H]GA4 metabolites although quantitative differences were apparent.  相似文献   

18.
When the metabolism of [13C,3H]gibberellin (GA)20 in Pisum sativum L. was investigated using decapitated plants and stem sections, no evidence was obtained for the recently postulated inhibitor of GA20 3[beta]-hydroxylase (V.A. Smith [1992] Plant Physiol 99: 372-377). Instead, the results are consistent with the hypothesis that the mutation le reduces GA1 production by altering the structure or level of the 3[beta]-hydroxylase.  相似文献   

19.
The multifunctional cytochrome P450 monooxygenases P450-1 and P450-2 from Fusarium fujikuroi catalyze the formation of GA14 and GA4, respectively, in the gibberellin (GA)-biosynthetic pathway. However, the activity of these enzymes is qualitatively and quantitatively different in mutants lacking the NADPH:cytochrome P450 oxidoreductase (CPR) compared to CPR-containing strains. 3beta-Hydroxylation, a major P450-1 activity in wild-type strains, was strongly decreased in the mutants relative to oxidation at C-6 and C-7, while synthesis of C19-GAs as a result of oxidative cleavage of C-20 by P450-2 was almost absent whereas the C-20 alcohol, aldehyde and carboxylic acid derivatives accumulated. Interaction of the monooxygenases with alternative electron transport proteins could account for these different product distributions. In the absence of CPR, P450-1 activities were NADH-dependent, and stimulated by cytochrome b5 or by added FAD. These properties as well as the decreased efficiency of P450-1 and P450-2 in the mutants are consistent with the participation of cytochrome b5:NADH cytochrome b5 reductase as redox partner of the gibberellin monooxygenases in the absence of CPR. We provide evidence, from either incubations of GA12 (C-20 methyl) with cultures of the mutant suspended in [18O]H2O or maintained under an atmosphere of [18O]O2:N2 (20:80), that GA15 (C-20 alcohol) and GA24 (C-20 aldehyde) are formed directly from dioxygen and not from hydrolysis of covalently enzyme-bound intermediates. Thus these partially oxidized GAs correspond to intermediates of the sequential oxidation of C-20 catalyzed by P450-2.  相似文献   

20.
In complementary experiments the metabolism of [1-2H]glucose in H2O and of unlabelled glucose in 2H2O by Zymomonas mobilis was examined. The utilization of [1-2H]glucose by Z. mobilis was monitored by high-resolution 2H NMR. The deuterium-labelling pattern and stereochemistry of the ethanols produced from the metabolism of [1-2H]glucose and unlabelled glucose in 2H2O were determined by a combination of 13C and 1H NMR and selective enzyme action. The labelling patterns were explained in terms of enzyme mechanisms and stereospecificity, and metabolite enolization.  相似文献   

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