Molecular cloning and functional expression of gibberellin 2- oxidases, multifunctional enzymes involved in gibberellin deactivation.
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.
Thomas
S G
SG
Institute of Arable Crops Research (IACR)-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS41 9AF, United Kingdom.
Phillips
A L
AL
Hedden
P
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D001711:Biotransformation; D003001:Cloning, Molecular; D017931:DNA Primers; D007887:Fabaceae / Q000201:enzymology* / Q000235:genetics; D005875:Gibberellins / Q000737:chemistry / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010946:Plants, Medicinal; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D005875:Gibberellins; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C089210:gibberellin, 2-oxoglutarate-oxygen oxidoreductase (20-hydroxylating, oxidizing)