The Metabolism of Gibberellin A20 to Gibberellin A1 by Tall and Dwarf Mutants of Oryza sativa and Arabidopsis thaliana

Kobayashi, Masatomo; Gaskin, Paul; Spray, Clive R.; Phinney, Bernard O.; MacMillan, Jake

doi:10.1104/pp.106.4.1367

Cited by 31 publications

(13 citation statements)

References 9 publications

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“…13 C]GA 20 to [ 13 C]GA 1 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 3␤-hydroxylase.…”

supporting

confidence: 77%

Function and Substrate Specificity of the Gibberellin 3β-Hydroxylase Encoded by the Arabidopsis GA4Gene1

et al. 1998

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cDNA corresponding to the GA4 gene of Arabidopsis thaliana L. (Heynh.) was expressed in Escherichia coli, from which cell lysates converted [14 C]gibberellin (GA) 9 and [ 14 C]GA 20 to radiolabeled GA 4 and GA 1 , respectively, thereby confirming that GA4 encodes a GA 3␤-hydroxylase. GA 9 was the preferred substrate, with a Michaelis value of 1 M compared with 15 M for GA 20 . Hydroxylation of these GAs was regiospecific, with no indication of 2␤-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 GA 12 -aldehyde, GA 12 , GA 19 , GA 25 , GA 53 , or GA 44 as the open lactone (20-hydroxy-GA 53 ), whereas GA 15 , GA 24 , and GA 44 were hydroxylated to GA 37 , GA 36 , and GA 38 , respectively. The open lactone of GA 15 (20-hydroxy-GA 12 ) was hydroxylated but less efficiently than GA 15 . In contrast to the free acid, GA 25 19,20-anhydride was 3␤-hydroxylated to give GA 13 . 2,3-Didehydro-GA 9 and GA 5 were converted by recombinant GA4 to the corresponding epoxides 2,3-oxido-GA 9 and GA 6 .

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supporting

confidence: 77%

Function and Substrate Specificity of the Gibberellin 3β-Hydroxylase Encoded by the Arabidopsis GA4Gene1

et al. 1998

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“…There are dwarf mutants in plants other than maize that block the conversion of GA20 to GA1, e.g., the le mutant of pea (13), the ga4 mutant of arabidopsis (14,24), and the dy mutant 19-20 9 Typical identification data are shown in Table 3.…”

Section: Discussionmentioning

confidence: 99%

“…(14,25). For each mutant, the position of the block was determined by bioassay and metabolic studies.…”

Section: Discussionmentioning

confidence: 99%

“…For each mutant, the position of the block was determined by bioassay and metabolic studies. However, it is unlikely that the three genes, Le, GA4, and Dy, have the same function as the Dl gene of maize, since normal seedlings of pea (13), arabidopsis (14), and rice (14) do not metabolize GA20 to GA5, and GA5 is metabolized to GA3 in normal and le seedlings of pea (26). In addition, no evidence has been published for the presence of endogenous GA5 and GA3 in seedlings of pea, arabidopsis, and rice.…”

Section: Discussionmentioning

confidence: 99%

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The dwarf-1 (dt) Mutant of Zea mays blocks three steps in the gibberellin-biosynthetic pathway.

Spray

Kobayashi²,

Suzuki³

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

111

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In plants, gibberellin (GA)-responding mutants have been used as tools to identify the genes that control specific steps in the GA-biosynthetic pathway. They have also been used to determine which native GAs are active per se, i.e., further metabolism is not necessary for bioactivity. We present metabolic evidence that the Dl gene of maize (Zea mays L.) controls the three biosynthetic steps: GA20 to GA1, GA20 to GA5, and GA5 to GA3. We also present evidence that three gibberellins, GA1, GA5, and GA3, have per se activity in stimulating shoot elongation in maize. The metabolic evidence comes from the injection of [17-13C,3H]GA20 and [17-13C,3H] GA5 into seedlings of dl and controls (normal and d5), followed by isolation and identification of the 13C-labeled metabolites by full-scan GC-MS and Kovats retention index.For the controls, GA20 was metabolized to GA1, GA3, and GA5; GA5 was metabolized to GA3. For the dl mutant, GA20 was not metabolized to GA1, GA3, or to GA5, and GA5 was not metabolized to GA3. The bioassay evidence is based on dosage response curves using dl seedlings for assay. GA1, GA3, and GA5 had similar bioactivities, and they were 10-times more active than GA20.linear and there is no evidence for a metabolic grid with the other pathways, as has been shown for cell-free preparations obtained from seeds of bean, cucumber, and pea (for review, see ref.3).The biological significance of the metabolic studies in maize comes from the use of GA mutants that exhibit a dwarf phenotype, yet respond by normal growth to applied GAs. Thus, the relative responses of the mutants to specific GAs together with information on the role of the genes in controlling specific steps in the pathway have led to the conclusion that only a limited number of GAs in the pathway are active per se, i.e., they do not require further metabolism to be bioactive (for reviews, see refs. 10-12).The purpose of the present study was to examine the metabolic steps, GA20 to GA5, GA5 to GA3 and GA20 to GA1 in relation to the dl mutation (blockage after GA20). To this end, [17-13C,3H]GA2o and [17-13C,3H]GAs were fed to dl seedlings and to controls (normal and d5 seedlings) and the metabolites from the feeds were analyzed by full-scan GC-MS. In addition, the bioactivities of GA20, GA1, GA5, and GA3 were determined using dl seedlings for assay.The gibberellins (GAs) are tetracarbocyclic diterpenes that occur naturally in higher plants (1). There is continued interest in the biosynthetic origin of the GAs since some of them are known to act as native regulators controlling a range of growth responses, including seed germination, floral development, and shoot elongation (for reviews, see refs. 2 and 3).All GAs are biosynthesized from trans-geranylgeranyl diphosphate (GGDP) via ent-copalyl diphosphate (CDP) and the tetracyclic hydrocarbon, ent-kaurene. ent-Kaurene is sequentially oxidized to ent-7a-hydroxykaurenoic acid, which is then rearranged to GA12-aldehyde and oxidized to GA12. At least three pathways diverge from GA12-aldehyde and ...

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“…Seedlings of this mutant are deficient in GA 1 and accumulate its immediate precursor, GA 20 (14). The metabolism of GA 20 to GA 1 is lower in d18-dy plants than in normal ones (15). It has been proposed that the D18 gene encodes a GA 3␤-hydroxylase (15).…”

mentioning

confidence: 99%

Cloning and functional analysis of two gibberellin 3β-hydroxylase genes that are differently expressed during the growth of rice

Itoh

Ueguchi-Tanaka

Shibata

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

270

208

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We have cloned two gibberellin (GA) 3␤-hydroxylase genes, OsGA3ox1 and OsGA3ox2, from rice by screening a genomic library with a DNA fragment obtained by PCR using degenerate primers. We have used full-scan GC-MS and Kovats retention indices to show function for the two encoded recombinant fusion proteins. Both proteins show 3␤-hydroxylase activity for the steps GA20 to GA 1, GA5 to GA3, GA44 to GA38, and GA9 to GA4. In addition, indirect evidence suggests that the OsGA3ox1 protein also has 2,3-desaturase activity, which catalyzes the steps GA9 to 2,3-dehydro-GA9 and GA20 to GA5 (2,3-dehydro GA20), and 2␤-hydroxylase activity, which catalyzes the steps GA 1 to GA8 and GA4 to GA34. Molecular and linkage analysis maps the OsGA3ox1 gene to the distal end of the short arm of chromosome 5; the OsGA3ox2 gene maps to the distal end of the short arm of chromosome 1 that corresponds to the D18 locus. The association of the OsGA3ox2 gene with the d18 locus is confirmed by sequence and complementation analysis of three d18 alleles. Complementation of the d18-AD allele with the OxGA3ox2 gene results in transgenic plants with a normal phenotype. Although both genes show transient expression, the highest level for OsGA3ox1 is from unopened flower. The highest level for OsGA3ox2 is from elongating leaves.

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The Metabolism of Gibberellin A20 to Gibberellin A1 by Tall and Dwarf Mutants of Oryza sativa and Arabidopsis thaliana

Cited by 31 publications

References 9 publications

Function and Substrate Specificity of the Gibberellin 3β-Hydroxylase Encoded by the Arabidopsis GA4Gene1

Function and Substrate Specificity of the Gibberellin 3β-Hydroxylase Encoded by the Arabidopsis GA4Gene1

The dwarf-1 (dt) Mutant of Zea mays blocks three steps in the gibberellin-biosynthetic pathway.

Cloning and functional analysis of two gibberellin 3β-hydroxylase genes that are differently expressed during the growth of rice

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