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1.
H. -D. Knöfel E. Schwarzkopf P. Müller G. Sembdner 《Journal of Plant Growth Regulation》1984,3(1-3):127-140
Starting from the well-known conversion of exogenously applied free gibberellic acid (GA3) to its 3(O)-glucoside by intact immature fruits of runner beans (Phaseolus coccineus L.), a protein fraction has been prepared from this plant material possessing glucosylating activity towards GAs. This glucosyltransferase is located in the pericarp only and utilizes preferably UDP-glucose as a sugar donor. The product formed enzymically from GA3 and UDP-glucose could be identified by derivatization and comparison with the authentic compound to be GA3-3(O)-glucoside. Among 15 native or chemically modified GAs, the enzyme glucosylates only GA3 and to a lower extent GA7 and GA30, indicating a high enzyme specificity with regard to the A ring of gibberellins. The physiological significance of the enzymic GA3-3(O)-glucoside formation inPhaseolus coccineus is not clear, since this glucoside is not known to be endogenous in this plant. The enzyme preparation did not glucosylate substances of phenolic structure, such as hydroquinone, aesculetin, and quercetin. Glucosylation of GA3 was achieved also by enzyme preparations fromVigna sinensis and from cell suspension cultures ofDigitalis purpurea. A number of other plant materials showed no activity.Gibberellins 100. For part 99 see Liebisch et al. 1984a. 相似文献
2.
《Phytochemistry》1986,25(8):1823-1828
A series of chromatographic and derivatization techniques has been developed for the identification of radiolabelled gibberellin (GA) conjugates. The methods are based on reversed-phase HPLC, gel permeation chromatography, anion-exchange chromatography, enzymatic hydrolysis and transesterification of conjugates, and derivatization of free GAs to methoxycoumaryl esters. The procedures have been used to identify GA4-glucosyl ester, GA4-3-O-glucoside, a GA34-O-glucoside and GA8-2-O-glucoside, in addition to GA1 and GA8, as products of [1,2-3H]GA4 metabolism in shoots of light-grown Phaseolus coccineus seedlings. 相似文献
3.
G. Sembdner H. -D. Knöfel Evelin Schwarzkopf H. W. Liebisch 《Biologia Plantarum》1985,27(2-3):231-236
In maturing fruits ofPhaseolus coccineus a soluble glucosyltransferase activity occurs which converts gibberellins into their O-glucosides. The enzyme glucosylates
GA3 and structurally closely related gibberellins (GA7 and GA30) to their 3-O-glucosides by transfer of glucose preferentially from UDP-glucose.
From cell suspension cultures ofLycopersicon peruvianum cytosolic glucosyltransferases were isolated which in the presence of UDP-glucose converted GA7 and GA9 to the corresponding glucosyl esters. In both cases numerous other gibberellins failed to serve as substrates. Thus, the
enzymes are UDP-glucose: gibberellin glucosyltransferases of considerable substrate specificity. 相似文献
4.
Analysis of extracts from 6300 (1.2 grams fresh weight) Phaseolus coccineus suspensors by combined gas chromatography-mass spectrometry has demonstrated the presence of five C19-gibberellins, GA1, GA4, GA5, GA6, GA8, and one C20-GA, GA44. The major GAs present were GA1 and GA8. Data are discussed in relation to previous results obtained in P. coccineus seed as well as in the embryo-suspensor system. 相似文献
5.
J. A. Kuhnle P. H. Moore W. F. Haddon M. M. Fitch 《Journal of Plant Growth Regulation》1983,2(1-4):59-71
Five GAs, GA1, GA3, GA19, GA20, and GA29, were identified in extracts from mature leaf and shoot apical meristem of flowering and non-flowering sugarcane (Saccharum spp. hybrids) by combined GC/MS. The presence of ABA was also confirmed. 相似文献
6.
Paul Gaskin Sarah Jane Gilmour John R. Lenton Jake MacMillan Valerie M. Sponsel 《Journal of Plant Growth Regulation》1983,2(1-4):229-242
Several gibberellins (GAs) and kauranoids were identified in extracts of barley (Hordeum vulgare) by combined capillary gas chromatography-mass spectrometry (GC-MS). A partially purified acidic ethyl acetate extract from 21-day postanthesis developing barley grain (cv. Proctor) contained GA1 (trace), GA4 (trace), GA8 (trace), GA12, GA17, GA20 (tentative) (trace), GA25, GA34, GA48, 18-hydroxy-GA4, 12β-hydroxy-GA9, and 18-hydroxy-GA34 (tentative). A hydrolyzed butanol extract contained GA17, GA20, GA48, and 18-hydroxy-GA34 (tentative). An acidic ethyl acetate extract from 3-day-old germinating barley grain (cv. Maris Otter) contained GA1, GA3 (possibly a contaminant), GA17, GA19, GA20, GA34, GA48, and 18-hydroxy-GA34 (tentative). A hydrolyzed butanol extract contained GA34, GA48, and 18-hydroxy-GA34 (tentative). In germinating grain, levels of all GAs were very low. Two hydroxylated kauranoic acids and a number of other kauranoids were also detected in the above extracts. 1β-Hydroxylated GAs previously found in wheat were not found in barley in this study. 相似文献
7.
Gibberellins in Embryo-Suspensor of Phaseolus coccineus Seeds at the Heart Stage of Embryo Development 总被引:2,自引:1,他引:1
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Gibberellins (GAs) in suspensors and embryos of Phaseolus coccineus seeds at the heart stage of embryo development were analyzed by combined gas chromatography-mass spectrometry (GC-MS). From the suspensor four C19-GAs, GA1, GA4, GA5, GA6, and one C20 GA, GA44, were identified. From the embryo, five C19-GAs GA1, GA4, GA5, GA6, GA60 and two C20 GAs, GA19 and GA44 were identified. The data, in relation to previous results, suggest a dependence of the embryo on the suspensor during early stages of development. 相似文献
8.
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
9.
John Kamsteeg Jan van Brederode Gerrit van Nigtevecht 《Biochemical genetics》1978,16(11-12):1059-1071
An enzyme catalyzing the transfer of the glucosyl moiety of UDP-glucose to the 5-hydroxyl group of cyanidin-3-rhamnosyl-(1→6)-glucoside has been demonstrated in petal extracts of Silene dioica plants. This glucosyltransferase activity was not detectable in green parts of these plants. The enzyme activity is controlled by a single dominant gene M; no glucosyltransferase activity could be demonstrated in petals of m/m plants. The enzyme was purified eightyfold by PVP and Sephadex G50 chromatography. The glucosyltransferase had a pH optimum of 7.4, had a molecular weight of about 55,000, was stimulated by divalent metal ions, and had a “true Km” value of 0.5×10?3 m for UDP-glucose and 3.6×10?3 m for cyanidin-3-rhamnosylglucoside. Pelargonidin-3-rhamnosylglucoside also could serve as acceptor. The enzyme did not catalyze the glucosylation of the 5-hydroxyl group of cyanidin-3-glucoside, although in petals of M/- n/n mutants cyanidin-3,5-diglucoside is present. ADP-glucose could not serve as a glucosyl donor. 相似文献
10.
Twenty known gibberellins (GAs) have been identified by combined capillary gas chromatography-mass spectrometry in extracts from less than 10 g fresh weight of maturing seeds of the cucurbit Sechium edule Sw. The GAs are predominantly 3- and-or 13-hydroxylated. This is the first reported identification of non-conjugated 13-hydroxylated GAs in a cucurbit. Gibberellin A8 and gibberellin A8-catabolite are the major GAs in terms of quantity and are largely accumulated in the testa. The catabolites of 2-hydroxylated GAs are ,-unsaturated ketones which no longer possess of a -lactone. They were hitherto known only in legumes. The presence of GA8-catabolite as a major component of Sechium seeds indicates that the distribution of these GA-catabolites may be more widespread than previously envisaged. The localization of known GAs in maturing seeds of the legume Phaseolus coccineus L. was found to resemble closely that in Sechium. Gibberellin A8, a putative conjugate of GA8 and GA8-catabolite are accumulated in the testa. The localization in the testa of end-products of the GA-biosynthetic pathway, which was first observed in maturing seeds of Pisum sativum, and is now described in Phaseolus and Sechium, may be a general feature of seed development.Abbreviations GAn
gibberellin An
- GC-MS
combined gas chromatography-mass spectrometry 相似文献
11.
[3H]GA20 (1)1, fed toVicia faba seedlings, was converted to [3H]GA20 glucosyl ester (5) and [3H]GA20-13-0-glucoside (6). The GA20 glucosyl ester (5) was identified by HPLC-RC and by GC-MS of GA20-Me formed by transesterification of (5). The [3H]GA20-Me was crystallized to constant specific radioactivity with authentic GA20-Me. On HPLC-RC the GA20-13-0-glucoside (6) was shown to have the same retention time as an authentic sample. Subsequent enzymic hydrolysis gave a product with an HPLC retention time identical to that of authentic GA20 (1). 相似文献
12.
Endogenous pools of presumptive gibberellin (GA) glucosyl conjugates of Phaseolus coccineus were metabolically labelled by feeding of [3H]GA1 to immature fruits. The [3H]GA1 glucoside fraction was isolated and the main constituent tentatively identified by enzymic hydrolysis, ion exchange chromatography and elution volume on HPLC-RC as GA1-3-0--D-glucopyranoside. 相似文献
13.
Piero Picciarelli Alberto Piaggesi Nello Ceccarelli Lorenzo Guglielminetti Amedeo Alpi 《Plant Growth Regulation》1994,14(2):183-185
Endogenous gibberellins (GAs) were extracted from suspensor, embryo and integument of very young seeds of Phaseolus coccineus L. and detected by combined gas chromatography-mass spectrometry (GC-MS). Results show the presence of one C20-GA, GA44 and five C19-GAs in the suspensor: GA1, GA4, GA5, GA6 and GA8, and four C19-GAs in the integument: GA1, GA5, GA6 and GA8. Only traces of GA1 and GA5 were identified in the embryo. A compound structurally related to GAs was identified as tetrahydroxy-Kauranoic acid in suspensor, integument and, only in trace amounts, in the embryo. 相似文献
14.
Summary An extract from 6000 dark-grown Phaseolus coccineus seedlings was purified by countercurrent distribution and G-10 Sephadex followed by gradient elution from a silicic acid partition column with increasing amounts of ethyl actetate in n-hexane. 25 fractions were collected and tested with the barley-aleurone, Tan-ginbozu dwarf-rice, lettuce, cucumber, dwarf-pea, d-1, d-2, d-3 and d-5 maize, oat first-internode, and sugarcane-spindle bioassays. Major gibberellin (GA)-like activity was detected in fractions 4 (500g GA3-equivalents) and 12–13 (270 g GA3-equivalents) with smaller amounts in fractions 6, 8–9, 15–16, 18, 20, 23 and 25. The extracts were also applied to AMO-1618=dwarfed Ph.-coccineus seedlings. Fractions 4, 8 and 12 promoted the growth of both light- and dark-grown seedlings. GA1, GA3, GA4 and GA8 were active in the Phaseolus bioassay but GA8-glucoside was inactive.The biological and chromatographic properties of fractions 4, 8–9 and 12–13 correspond with those of GA4, GA19 and GA1. The identity of GA4 in fraction 4 was conclusively established by combined gas chromatography-mass spectrometry (GC-MS) of the methyl ester and the trimethylsilyl ether of the methyl ester. Gasliquid-chromatography peaks corresponding to these derivatives of GA19 and GA1 were detected on QF-1 and SE-33 columns but their intensities were too weak to permit conclusive identification by GC-MS.Supported by an S.R.C. StudentshipSupported by a NATO Grant.Supported by NRC Grant A-5727. 相似文献
15.
By combined gas chromatography-mass spectrometry the gibberellin present in suspensors of heart-shaped embryos of Phaseolus coccineus has been identified as Gibberellin A1 (GA1). The amount of GA1 in 2000 suspensors (452 mg), as estimated by gas chromatography. was 4g. The presence of GA1 in suspensors of P. coccineus is discussed in relation to our present knowledge of the occurrence of many gibberellins in developing seeds and immature fruits of the same species.Abbreviations FID
flame ionization detector
- GA
gibberellin
- GC
gas chromatography
- MS
mass spectrometry
- PGC
preparative gas chromatography
- Stage A
heart-shaped embryo
- Stage B
cotytedonary embryo
- TMS
trimethylsilyl 相似文献
16.
John Kamsteeg Jan van Brederode Gerrit van Nigtevecht 《Biochemical genetics》1978,16(11-12):1045-1058
An enzyme catalyzing the transfer of the glucosyl moiety of UDP-glucose to the 3-hydroxyl group of cyanidin has been demonstrated in petal extracts of Silene dioica mutants with cyanidin-3-O-glucoside in the petals. This transferase activity was also present in young rosette leaves and calyces of these plants. The highest glucosyltransferase activity was found in petals of opening flowers of young plants. The enzyme was purified ninetyfold by PVP and Sephadex chromatography. The glucosyltransferase had a pH optimum of 7.5, had a “true Km value” of 4.1×10?4 m for UDP-glucose and 0.4×10?4 m for cyanidin chloride, and was not stimulated by divalent metal ions. Both p-chloromercuribenzoate and HgCl2 inhibited the enzyme activity. Pelargonidin chloride and delphinidin chloride at reduced rates also served as substrates. The enzyme did not catalyze the glucosylation of the 3-hydroxyl group of flavonols or the 5-hydroxyl group of anthocyanins. ADP-glucose could not serve as a glucosyl donor. The results of Sephadex G150 chromatography suggest that the glucosyltransferase can exist as dimer of about 125,000 daltons and as active monomers of 60,000 daltons. The genetic control of the glucosyltransferase activity is discussed. 相似文献
17.
A very sensitive and specific bioassay using prohexadione calcium [BX-112, which blocks 2- and 3-hydroxylation of gibberellins (GAs)] with uniconazole (which blocks oxidation of ent-kaurene, ent-kaurenol and ent-kaurenal) in a microdrop assay was developed for several rice (Oryza sativa L.) varieties, including cv. Waito-C, which is already specific to 3-hydroxylated GAs. The sensitivity and specificity of cvs. Waito-C, Tan-ginbozu and Koshihikari to 3-hydroxylated GAs was greatly enhanced by treatment of the seeds with a combination of 40 mM prohexadione calcium and 80 M uniconazole. The minimum detectable doses of 3-hydroxylated GAs (GA1, GA3, GA4 and GA7) in the three cultivars treated with both chemicals were 1 to 10 fmol (i.e. ca. 350 fg to 3.5 pg) per plant. This is equal to 30-fold more sensitive than Waito-C treated with uniconazole alone, and 30 to 1000-fold more sensitive than Waito-C with no growth retardant soak. Minimum detectable doses of 3-nonhydroxylated GAs (GA9, GA19 GA20) and GAs with very low biological activity (GA8 and GA17) were equal to or more than 1000 fmol per plant. This is about equal to the activity in Waito-C treated with uniconazole alone. Application of this assay to an extract from Raphanus sativus was compared with the data by gas chromatography/mass spectrometry (GC/MS), confirming the conclusions reached using authentic test GAs, namely that use of uniconazole plus BX-112 appreciably enhanced the detection sensitivity to fractions shown by GC/MS to contain GA1 and GA4, both 3-hydroxylated GAs.Abbreviations GA
gibberellin
- BX-112
prohexadione calcium 相似文献
18.
cDNA
corresponding to the GA4 gene of
Arabidopsis thaliana L. (Heynh.) was
expressed in Escherichia coli, from which cell lysates
converted [14C]gibberellin (GA)9 and
[14C]GA20 to radiolabeled GA4 and
GA1, respectively, thereby confirming that
GA4 encodes a GA 3β-hydroxylase. GA9 was
the preferred substrate, with a Michaelis value of 1 μm
compared with 15 μm for GA20. Hydroxylation
of these GAs was regiospecific, with no indication of
2β-hydroxylation or 2,3-desaturation. The capacity of the recombinant
enzyme to hydroxylate a range of other GA substrates was investigated.
In general, the preferred substrates contained a polar bridge between
C-4 and C-10, and 13-deoxy GAs were preferred to their 13-hydroxylated
analogs. Therefore, no activity was detected using
GA12-aldehyde, GA12, GA19,
GA25, GA53, or GA44 as the open
lactone (20-hydroxy-GA53), whereas GA15,
GA24, and GA44 were hydroxylated to
GA37, GA36, and GA38, respectively.
The open lactone of GA15 (20-hydroxy-GA12) was
hydroxylated but less efficiently than GA15. In contrast to
the free acid, GA25 19,20-anhydride was 3β-hydroxylated
to give GA13. 2,3-Didehydro-GA9 and
GA5 were converted by recombinant GA4 to the corresponding
epoxides 2,3-oxido-GA9 and GA6.Dwarf mutants with reduced biosynthesis of the GA plant hormones
have been valuable tools in studies of the function of these compounds
(Ross, 1994). In Arabidopsis thaliana, mutations
at six loci (GA1-GA6) that result in reduced GA
biosynthesis have been identified (Koorneef and van der Veen, 1980;
Sponsel et al., 1997), and three of these loci have recently been
cloned. The GA1 locus was isolated by genomic subtraction
(Sun et al., 1992) and shown by heterologous expression in
Escherichia coli to encode the enzyme that cyclizes
geranylgeranyl diphosphate to copalyl diphosphate (Sun and Kamiya,
1994). This enzyme was formerly referred to as ent-kaurene
synthase A but has been renamed copalyl diphosphate synthase
(Hedden and Kamiya, 1997; MacMillan, 1997). The GA5
locus was shown to correspond to one of the GA 20-oxidase genes (Xu et
al., 1995), the products of which catalyze the conversion of
GA12 to GA9 and
GA53 to GA20 (Phillips et
al., 1995; Xu et al., 1995). GA 20-oxidases are
2-oxoglutarate-dependent dioxygenases that are encoded by small
multigene families, members of which are differentially expressed in
plant tissues (Phillips et al., 1995; Garcia-Martinez et al., 1997).The GA4 locus was isolated by T-DNA tagging and, on the
basis of the derived amino acid sequence, was also shown to encode a
dioxygenase (Chiang et al., 1995). Several lines of evidence indicate
that the GA4 gene encodes a GA 3β-hydroxylase. Shoots of a
ga4 mutant, all alleles of which are semidwarf, contained
reduced concentrations of the 3β-hydroxy GAs
GA1, GA4, and
GA8 compared with the Landsberg erecta
wild type, whereas levels of immediate precursors to these GAs were
elevated (Talon et al., 1990). Furthermore, metabolism of
[13C]GA20 to
[13C]GA1 was
substantially less in the mutant than in the wild type (Kobayashi et
al., 1994). In the present paper we confirm by functional expression of
its cDNA in E. coli that GA4 encodes a GA
3β-hydroxylase. In addition, we determine the substrate specificity
of recombinant GA4 using a number of C20- and
C19-GAs and show by kinetic analysis that the enzyme
has a higher affinity for GA9 than for
GA20, which is consistent with the
non-13-hydroxylation pathway predominating in Arabidopsis (Talon et
al., 1990). 相似文献
19.
Gerard W. M. Barendse André Dijkstra Thomas C. Moore 《Journal of Plant Growth Regulation》1983,2(1-4):165-175
The endogenous levels of gibberellins (GAs) determined by a combined HPLC-bioassay procedure and the formation ofent-kaurene, an immediate GA precursor, in cell-free extracts were studied in relation to seed development inPharbitis nil Choisy cv. Violet. Three biologically active GA fractions were obtained, tentatively identified as GA3, GA5/ GA20, and a GA fraction, possibly GA19 and/or GA44, which all increased in activity during early seed development and subsequently declined during maturation of the seeds. The total endogenous GA level reached its maximum at 19 days after anthesis, just before the seeds had attained their maximum fresh weight at about 23 days after anthesis. Similarly, theent-kaurene synthesizing capacity showed a rapid increase during the period of rapid growth of the seeds, followed by a decline during maturation. A direct relationship between the endogenous GA levels and theent-kaurene synthesizing capacity of a particular tissue was indicated. 相似文献