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Molecular cloning and functional expression of gibberellin 2- oxidases, multifunctional enzymes involved in gibberellin deactivation.

ABSTRACT: A major catabolic pathway for the gibberellins (GAs) is initiated by 2beta-hydroxylation, a reaction catalyzed by 2-oxoglutarate-dependent dioxygenases. To isolate a GA 2beta-hydroxylase cDNA clone we used functional screening of a cDNA library from developing cotyledons of runner bean (Phaseolus coccineus L.) with a highly sensitive tritium-release assay for enzyme activity. The encoded protein, obtained by heterologous expression in Escherichia coli, converted GA9 to GA51 (2beta-hydroxyGA9) and GA51-catabolite, the latter produced from GA51 by further oxidation at C-2. The enzyme thus is multifunctional and is best described as a GA 2-oxidase. The recombinant enzyme also 2beta-hydroxylated other C19-GAs, although only GA9 and GA4 were converted to the corresponding catabolites. Three related cDNAs, corresponding to gene sequences present in Arabidopsis thaliana databases, also encoded functional GA 2-oxidases. Transcripts for two of the Arabidopsis genes were abundant in upper stems, flowers, and siliques, but the third transcript was not detected by Northern analysis. Transcript abundance for the two most highly expressed genes was lower in apices of the GA-deficient ga1-2 mutant of Arabidopsis than in wild-type plants and increased after treatment of the mutant with GA3. This up-regulation of GA 2-oxidase gene expression by GA contrasts GA-induced down-regulation of genes encoding the biosynthetic enzymes GA 20-oxidase and GA 3beta-hydroxylase. These mechanisms would serve to maintain the concentrations of biologically active GAs in plant tissues.

SUBMITTER: Thomas SG

PROVIDER: S-EPMC16395 | biostudies-literature | 1999 Apr

REPOSITORIES: biostudies-literature

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Molecular cloning and functional expression of gibberellin 2- oxidases, multifunctional enzymes involved in gibberellin deactivation.

Thomas S G SG Phillips A L AL Hedden P P

Proceedings of the National Academy of Sciences of the United States of America 19990401 8

A major catabolic pathway for the gibberellins (GAs) is initiated by 2beta-hydroxylation, a reaction catalyzed by 2-oxoglutarate-dependent dioxygenases. To isolate a GA 2beta-hydroxylase cDNA clone we used functional screening of a cDNA library from developing cotyledons of runner bean (Phaseolus coccineus L.) with a highly sensitive tritium-release assay for enzyme activity. The encoded protein, obtained by heterologous expression in Escherichia coli, converted GA9 to GA51 (2beta-hydroxyGA9) an ...[more]

PMID: 10200325

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Project description:Gibberellin (GA), a classical phytohormone, plays significant roles in plant growth and development. It shares the important intermediate diphosphate precursor, GGPP, with the main lipophilic bioactive components, diterpenoid tanshinones in Salvia miltiorrhiza Bunge, one of the most important Traditional Chinese Medicine materials and an emerging model medicinal plant. Analysis of GA metabolism and regulation may help to demonstrate the biological functions of GAs and the crosstalk between GA metabolism and tanshinone biosynthesis in S. miltiorrhiza. However, genes involved in the conversion of ent-kaurene to GAs have not been systematically studied.Through genome-wide prediction and molecular cloning, twenty two candidate gibberellin metabolism pathway genes were systematically identified for the first time. It includes a SmKO, two SmKAOs, six SmGA20oxs, two SmGA3oxs and eleven SmGA2oxs, of which twenty genes are novel. The deduced proteins showed sequence conservation and divergence. Gibberellin metabolism pathway genes exhibited tissue-specific expression patterns and responded differentially to exogenous GA3 treatment, indicating differential regulation of gibberellin metabolism in different tissue types in S. miltiorrhiza. SmKAO1, SmKAO2, SmGA2ox2, and SmGA2ox4-SmGA2ox7 were significantly up-regulated; SmGA20ox2, SmGA3ox1, SmGA2ox1, SmGA2ox8, SmGA2ox10 and SmGA2ox11 were significantly down-regulated; while the responses of many other genes varied among different tissue-types and time-points of GA3 treatment, suggesting the complexity of feedback regulation. Tanshinone biosynthesis-related genes, such as SmCPS1 and SmKSL1, were up-regulated in response to GA3 treatment. Among the 22 identified genes, nine responded to yeast extract and Ag(+)-treatment in S. miltiorrhiza hairy roots. Moreover, tissue-specifically expressed splice variants were identified for SmKO, SmGA20ox3, SmGA2ox3 and SmGA2ox11, of which SmKOv1, SmGA20ox3v and SmGA2ox11v1 were GA3-responsive, suggesting the importance of alternative splicing in regulating GA metabolism.The results show tissue-specifically expressed, feedback-regulated, stress-responsive and alternatively spliced novel genes and reveal multiple layer regulation of GA metabolism and crosstalk between gibberellin metabolism and tanshinone biosynthesis in S. miltiorrhiza.

Dataset Information

Molecular cloning and functional expression of gibberellin 2- oxidases, multifunctional enzymes involved in gibberellin deactivation.

Publications

Molecular cloning and functional expression of gibberellin 2- oxidases, multifunctional enzymes involved in gibberellin deactivation.

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