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 2 beta -hydroxylation, a reaction catalyzed by 2-oxoglutarate-dependent dioxygenas es. To isolate a GA 2 beta-hydroxylase cDNA clone we used functional screen ing of a cDNA library from developing cotyledons of runner bean (Phaseolus coccineus L.) with a highly sensitive tritium-release assay for enzyme acti vity, The encoded protein, obtained by heterologous expression in Escherich ia coli, converted GAs to GA(51) (2 beta-hydroxyGA(9)) and GA(51)-catabolit e, the latter produced from GA(51) by further oxidation at C-2. The enzyme thus is multifunctional and is best described as a GA 2 oxidase. The recomb inant enzyme also 2 beta-hydroxylated other C-19-GAs, although only GAs and GA(4) 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 Arabido psis genes were abundant in upper stems, flowers, and siliques, but the thi rd transcript was not detected by Northern analysis. Transcript abundance f or the two most highly expressed genes was lower in apices of the GA-defici ent gal-2 mutant of Arabidopsis than in wild-type plants and increased afte r treatment of the mutant with GA(3). This up-regulation of GA 2-oxidase ge ne expression by GA contrasts GA-induced down-regulation of genes encoding the biosynthetic enzymes GA 20-oxidase and GA 3 beta-hydroxylase. These mec hanisms would serve to maintain the concentrations of biologically active G As in plant tissues.