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  • 1
    Electronic Resource
    Electronic Resource
    Palo Alto, Calif. : Annual Reviews
    Annual Review of Plant Physiology and Plant Molecular Biology 48 (1997), S. 431-460 
    ISSN: 1040-2519
    Source: Annual Reviews Electronic Back Volume Collection 1932-2001ff
    Topics: Biology
    Notes: Abstract The recent impressive progress in research on gibberellin (GA) biosynthesis has resulted primarily from cloning of genes encoding biosynthetic enzymes and studies with GA-deficient and GA-insensitive mutants. Highlights include the cloning of ent-copalyl diphosphate synthase and ent-kaurene synthase (formally ent-kaurene synthases A and B) and the demonstration that the former is targeted to the plastid; the finding that the Dwarf-3 gene of maize encodes a cytochrome P450, although of unknown function; and the cloning of GA 20-oxidase and 3beta-hydroxylase genes. The availability of cDNA and genomic clones for these enzymes is enabling the mechanisms by which GA concentrations are regulated by environmental and endogenous factors to be studied at the molecular level. For example, it has been shown that transcript levels for GA 20-oxidase and 3beta-hydroxylase are subject to feedback regulation by GA action and, in the case of the GA 20-oxidase, are regulated by light. Also discussed is other new information, particularly from mutants, that has added to our understanding of the biosynthetic pathway, the enzymes, and their regulation and tissue localization.
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 101 (1997), S. 0 
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Gibberellins (GAs) are biosynthesised from the diterpene ent-kaurene by a series of oxidative reactions catalysed by two classes of enzymes. The early steps, involving transformations of highly hydrophobic substrates, are carried out by membrane-associated monooxygenases, probably involving cytochrome P450, whereas the later reactions are catalysed by soluble 2-oxoglutarate-dependent dioxygenases. Some reactions involving substrates, such as GA12 and GA12-aldehyde, that have intermediate polarity are catalysed by enzymes in both classes. The monooxygenases and dioxygenases catalyse the same types of reactions: hydroxylation, desaturation, alcohol and aldehyde oxidation. For both enzyme classes, the oxidant is thought to be an oxyferryl species, depicted as Felv=O, that is derived from molecular oxygen by different mechanisms, the reducing power being supplied by NADPH in the case of cytochrome P450 monooxygenases and by the decarboxylation of 2-oxoglutarate to succinate for the dioxygenases. The recent availability of cDNA clones for several of the dioxygenases and the ability to prepare active enzymes by heterologous expression of cDNAs in Escherichia coli have provided new opportunities for examining the function of these enzymes. They have relatively low substrate specificity and, in many cases, are multifunctional. Consequently, fewer enzymes than expected are required to produce the large number of GA structures encountered in higher plants. In the present review, the major oxygenases of GA biosynthesis are described and their reactions are discussed in an attempt to rationalise this multifunctionality.
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  • 3
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Gibberellin (GA) 20-oxidases are multifunctional enzymes that catalyse reactions at an important branch point in the GA biosynthetic pathway. These enzymes oxidise the C-20 methyl group of a diterpene carboxylic acid precursor (e.g. GA12) to form an alcohol (in our case GA15-open lactone) and an aldehyde (GA24). The aldehyde is either oxidised to a tricarboxylic acid (GA25) or, with loss of carbon-20 and lactonisation, to a C19-GA (GA9). This branching is interesting to study, because C19-GA derivatives function as plant hormones in different tissues, whereas the C20-GA tricarboxylic acids have no known function. We have constructed chimaeric proteins by combining a GA 20-oxidase from immature seeds of Cucurbita maxima L., which produces mainly C-20 carboxylic acids, with a 20-oxidase from Marah macrocarpus immature seeds, which forms predominantly CC19-GAs. The cDNAs encoding these two very similar 20-oxidases were digested with restriction endonucleases Van 911. Bcl 1, and Bsa WI, and six chimaeric sequences were produced by recombination of the DNA fragments. The pCM1 -construct was obtained by exchanging nt 303–809 of the Cucurbita cDNA with the homologous DNA from the March 20-oxidase. In pCM2, pCM3, pCM4, pCM5 and pCM6, nt 810–992, nt 993–end, nt 303–992, nt 810–end, and nt 311–end were exchanged, respectively. All constructs were cloned in a pUC18 vector and functionally expressed in E. coli NM522 cells. GA 20-oxidase activity was detectable in cell-lysates from the transformed E. coli, but the extent and kind of conversion depended on the construct. Highest conversion of GA12was found with pCM1 and pCM3, one-tenth of this conversion was observed with pCM5 and pCM6, and one-hundredth was obtained with the hybrid proteins from pCM2 and pCM4. With pCM2 and pCM4, neither the C19-end product, GA9, nor the C20-end product, GA25-was formed. However, after transformation with constructs pCM1, pCM3, pCM5 or pCM6. GA9accounted for 30, 40, 60 and 90%, respectively, of the end products formed. Thus, the segments originating from M. macrocarpus conferred upon the chimaeric proteins an increasing ability to direct the biosynthetic flow into C19-GAs in this order. Although GA24is the immediate precursor, much less end products were formed by using this substrate.
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  • 4
    Electronic Resource
    Electronic Resource
    [s.l.] : Nature Publishing Group
    Nature biotechnology 21 (2003), S. 873-874 
    ISSN: 1546-1696
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: [Auszug] It can be argued that the introduction into cereals of genes to reduce stem height has saved more lives than any other scientific development. During the past 40 years, the ability to feed a human population, which has doubled from three to six billion, has relied on the development of ...
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  • 5
    ISSN: 1432-2048
    Keywords: Abscisic acid ; Carotenoid ; Gibberellin ; Isoprenoid biosynthesis ; Lycopersicon (isoprenoid levels) ; Sterol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Carotenoids, gibberellins (GAs), sterols, abscisic acid and β-amyrins were analysed in tomato (Lycopersicon esculentum Mill.) pericarp during fruit development and ripening. The contents of these isoprenoids in wild-type (cv. Ailsa Craig) fruit were compared with those in fruit of the carotenoid-deficient R-mutant and a transgenic plant containing antisense RNA to a phytoene synthase gene. In both carotenoid-deficient genotypes, a 14-fold reduction in carotene and twofold decrease in xanthophyll content, compared to the wild type, was found in ripe fruit. Immature green fruit from wild type and R-mutant plants contained similar amounts of the C19-GAs, GA1, and GA20, and their C20 precursor, GA19. Immature fruit from the transgenic plants contained three- to fivefold higher contents of these GAs. In wild-type fruit at the mature green stage the contents of these GAs had decreased to 〈 10% of the levels in immature fruit. A similar decrease in GA19 content occurred in the other genotypes. However, the contents of GA1 and GA20 in fruit from phytoene synthase antisense plants decreased only to 30% between the immature and mature green stages and did not decrease at all in R-mutant fruit. At the breaker and ripe stages, the contents of each GA were much reduced for all genotypes. The amount of abscisic acid was the same in immature fruit from all three genotypes, but, on ripening, the levels of this hormone in antisense and R-mutant fruit were ca. 50% of those in the wild type. Quantitative differences in the amounts of the triterpenoid β-amyrins, total sterols, as well as individual sterols, such as campesterol, stigmasterol and sitosterol, were apparent between all three genotypes during development. Amounts of free sterols of wild type and antisense fruit were greatest during development and decreased during ripening, whereas the opposite was found in the R-mutant. This genotype also possessed less free sterol and more bound sterol in comparison to the other varieties. These data provide experimental evidence to support the concept of an integrated metabolic relationship amongst the isoprenoids.
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  • 6
    ISSN: 1432-2048
    Keywords: Key words: Fruit-set and growth ; Gibberellin (distri-bution in fruit and seed ; identification ; metabolism) ; Pisum (fruit growth ; seed development) ; Seed develop-ment
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. Gibberellins A1 and A3 are the major physiologically active gibberellins (GAs) present in young fruit of pea (Pisum sativum L.). The relative importance of these GAs in controlling fruit growth and their biosynthetic origins were investigated in cv. Alaska. In addition, the non-13-hydroxylated active GAs, GA4 and GA7, were identified for the first time in young seeds harvested 4 d after anthesis, although they are minor components and are not expected to play major physiological roles. The GA1 content is maximal in seeds and pods at 6 d after anthesis, the time of highest growth-rate of the pod (Garcia-Martinez et al. 1991, Planta 184: 53–60), whereas gibberellic acid (GA3), which is present at high levels in seeds 4–8 d after anthesis, has very low abundance in pods. Gibberellins A19, A20 and A29 are most concentrated in seeds at, or shortly after, anthesis and their abundance declines rapidly with development, concomitant with the sharp increase in GA1 and GA3 content. Application of GA1 or GA3 to the leaf subtending an emasculated flower stimulated parthenocarpic fruit development. Measurement of the GA content of the pods at 4 d after anthesis indicated that only 0.002–0.5% of the applied GA was transported to the fruit, depending on dose. There was a linear relationship between GA1 content and pod weight up to about 2 ng · (g FW)−1, whereas no such correlation existed for GA3 content. The concentration of endogenous GA1 in pods from pollinated ovaries is just sufficient to give the maximum growth response. It is concluded that GA1, but not GA3, controls pod growth in pea; GA3 may be involved in early seed development. The distribution of GAs within the seeds at 4 d post anthesis was also investigated. Most of the GA1, GA8, GA19, GA20 and GA29 was present in the testa, whereas GA3 was distributed equally between testa and endosperm and GA4 was localised mainly in the endosperm. Of the GAs analysed, only GA3 and GA20 were detected in the embryo. Metabolism experiments with intact tissues and cell-free fractions indicated compartmentation of GA biosynthesis within the seed. Using 14C-labelled GA12, GA9, 2,3-didehydroGA9 and GA20 as substrates, the testa was shown to contain 13-hydroxylase and 20-oxidase activities, the endosperm, 3β-hydroxylase and 20-oxidase activities. Both tissues also produced 16,17-dihydrodiols. However, GA1 and GA3 were not obtained as products and it is unlikely that they are formed via the early 13-hydroxylation pathway. [14C]gibberellin A12, applied to the inside surface of pods in situ, was metabolised to GA19, GA20, GA29, GA29-catabolite, GA81 and GA97, but GA1 was not detected. Gibberellin A20 was metabolised by this tissue to GA29 and GA29-catabolite.
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  • 7
    ISSN: 1432-2048
    Keywords: Fruit set and growth ; Gibberellin (distribution in fruit, fruit growth, quantitation, transport) ; Parthenocarpy ; Pisum (fruit growth)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In addition to the previously-reported gibberellins: GA1; GA8, GA20 and GA29 (García-Martínez et al., 1987, Planta 170, 130–137), GA3 and GA19 were identified by combined gas chromatography-mass spectrometry in pods and ovules of 4-d-old pollinated pea (Pisum sativum cv. Alaska) ovaries. Pods contained additionally GA17, GA81 (2α-hydroxy GA20) and GA29-catabolite. The concentrations of GA1, GA3, GA8, GA19, GA20 and GA29 were higher in the ovules than in the pod, although, with the exception of GA3, the total content of these GAs in the pod exceeded that in the seeds. About 80% of the GA3 content of the ovary was present in the seeds. The concentrations of GA19 and GA20 in pollinated ovaries remained fairly constant for the first 12 ds after an thesis, after which they increased sharply. In contrast, GA1 and GA3 concentrations were maximal at 7 d and 4–6 d, respectively, after anthesis, at about the time of maximum pod growth rate, and declined thereafter. Emasculated ovaries at anthesis contained GA8, GA19 and GA20 at concentrations comparable with pollinated fruit, but they decreased rapidly. Gibberellins a1 and A3 were present in only trace amounts in emasculated ovaries at any stage. Parthenocarpic fruit, produced by decapitating plants immediately above an emasculated flower, or by treating such flowers with 2,4-dichlorophenoxyacetic acid or GA7, contained GA19 and GA20 at similar concentrations to seeded fruit, but very low amounts of GA1 and GA3 Thus, it appears that the presence of fertilised ovules is necessary for the synthesis of these last two GAs. Mature leaves and leaf diffusates contained GA1, GA8, GA19 and GA20 as determined by combined gas chromatography-mass spectrometry using selected ion monitoring. This provides further evidence that vegetative tissues are a possible alternative source of GAs for fruit-set, particularly in decapitated plants.
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  • 8
    ISSN: 1432-2048
    Keywords: Gibberellin biosynthesis ; Hordeum (gibberellin synthesis) ; Metabolism (cell-free system)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Gibberellin (GA) metabolism from GA12-aldehyde was studied in cell-free systems from 2-d-old germinating embryos of barley. [14C]- or [17-2H2]Gibberellins were used as substrates and all products were identified by combined gas chromatography-mass spectrometry. Stepwise analysis demonstrated the conversion of GA12-aldehyde via the 13-deoxy pathway to GA51 and via the 13-hydroxylation pathway to GA29, GA1 and GA8. In addition, GA3 was formed from GA20 via GA5. We conclude that the embryo is capable of producing gibberellins that can induce α-amylase production in the aleurone layer. There was no evidence for 12β- or 18-hydroxylation and GA4 was neither synthesised nor metabolised by the system. All metabolically obtained GAs, with the exception of GA3, were also found as endogenous components of the cell-free system in spite of ammonium-sulfate precipitation and desalting steps.
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  • 9
    ISSN: 1432-2048
    Keywords: Cucurbita (endosperm) ; Gibberellin biosynthetic enzymes (3β-hydroxylase, 7-oxidase, 20-oxidase) ; 2-Oxoglutarate-dependent dioxygenases
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Three enzymes of the gibberellin (GA) biosynthetic pathway, a 7-oxidase, a 20-oxidase and a 3β-hydroxylase, were partially purified from Cucurbita maxima endosperm by ammonium sulfate precipitation, gel-filtration and anion-exchange chromatography. The enzyme activities, which were assayed by the oxidation of GA12-aldehyde to GA12, of GA12 to GA15 (and GA24) and of GA15 to GA37, respectively, were completely separated from each other. The apparent molecular masses as estimated by gel-filtration high-performance liquid chromatography were 34.5 kDa for the 7-oxidase, 44.5 kDa for the 20-oxidase and 58 kDa for the 3β-hydroxylase. The Michaelis-Menten constants (K m) were 8.6 μM, 0.15μM and 8.7 μM for the respective substrates. All three enzymes had properties typical of 2-oxoglutarate dependent dioxygenases. 2-Oxoglutarate was essential for activity and served as a co-substrate, giving K m values of 6.1 μM, 91 μM and 41 μM with the 7-oxidase, 20-oxidase and 3β-hydroxylase, respectively. Furthermore, 2 oxo[5-14C]glutarate was oxidised stoichiometrically to [14C]succinate when the GA-substrates were oxidised to their respective products, and the 1∶1 ratio was maintained under different oxygen concentrations. Approximately equimolar amounts of 14CO2 were released from 2-oxo[1-14C]glutarate when GA12 was oxidised to GA15/24 by the 20-oxidase. A crude enzyme preparation containing all three enzyme activities (and a 2β-hydroxylase) converted GA12-aldehyde to [18O2]GA4 and [18O5]GA43 under 18O2, showing that all O-atoms incorporated after GA12-aldehyde originate from O2. Accordingly, the reaction rates were near zero under anaerobic conditions, although very low concentrations of O2 sufficed to sustain the reactions. Both Fe2+ and dithiothreitol stimulated the enzyme activities strongly, but if they were added together, catalase was needed to prevent inhibition. The pH dependence showed two opposite trends; the 7-oxidase was most active at pH 6 and below, whereas the other enzymes were maximally active above pH 6.5.
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  • 10
    ISSN: 1432-2048
    Keywords: Cucurbita (endosperm, nucellus) ; Gibberellin metabolism (cell-free system, 12α-hydroxylation, 13-hydroxylation) ; Gibberellin identification ; 2-Oxoglutarate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Gibberellin (GA) biosynthesis in cell-free systems from Cucurbita maxima L. endosperm was reinvestigated using incubation conditions different from those employed in previous work. The metabolism of GA12 yielded GA13, GA43 and 12α-hydroxyGA43 as major products, GA4, GA37, GA39, GA46 and four unidentified compounds as minor products. The intermediates GA15, GA24 and GA25 accumulated at low protein concentrations. The structure of the previously uncharacterised 12α-hydroxyGA43 was inferred from its mass spectrum and by its formation from both GA39 and GA43. Gibberellin A39 and 12α-hydroxyGA43 were formed by a soluble 12α-hydroxylase that had not been detected before. Gibberellin A12-aldehyde was metabolised to essentially the same products as GA12 but with less efficiency. A new 13-hydroxylation pathway was found. Gibberellin A53, formed from GA12 by a microsomal oxidase, was converted by soluble 2-oxoglutarate-dependent oxidases to GA1 GA23, GA28, GA44, and putative 2β-hydroxyGA28. Minor products were GA19, GA20, GA38 and three unidentified GAs. Microsomal 13-hydroxylation (the formation of GA53) was suppressed by the cofactors for 2-oxoglutarate-dependent enzymes. Reinvestigation of the endogenous GAs confirmed the significance of the new metabolic products. In addition to the endogenous GAs reported by Blechschmidt et al. (1984, Phytochemistry 23, 553–558), GA1, GA8, GA25, GA28, GA36, GA48 and 12α-hydroxyGA43 were identified by full-scan capillary gas chromatography-mass spectrometry and Kovats retention indices. Thus both the 12α-hydroxylation and the 13-hydroxylation pathways found in the cell-free system operate also in vivo, giving rise to 12α-hydroxyGA43 and GA1 (or GA8), respectively, as their end products. Evidence for endogenous GA20 and GA24 was also obtained but it was less conclusive due to interference.
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