Gibberellin 2-oxidases from seedlings of adzuki bean (Vigna angularis) show high gibberellin-binding activity in the presence of 2-oxoglutarate and Co 2+

Abstract

Five full-length cDNA encoding gibberellin 2-oxidases, VaGA2oxA1, VaGA2oxA2, VaGA2oxB1, VaGA2oxB2, and VaGA2oxB3, were cloned from etiolated adzuki bean (Vigna angularis cv. Dainagon) seedlings, and their enzymatic characteristics were examined using recombinant enzymes fused with glutathione S-transferase (GST). Recombinant VaGA2oxA1 (rVaGA2oxA1) and rVaGA2oxA2 showed 2β-hydroxylation activity by converting GA1, GA4, GA9, GA20, GA4-methyl ester, and 16,17-dihydro-GA4 to the corresponding 2β-hydroxylated gibberellins, which were identified by GC/ MS. rVaGA2oxB1, rVaGA2oxB2, and rVaGA2oxB3 showed similar activity by converting [3H 4]-16,17-dihydro-GA4 to a metabolite showing an R f value of 16,17-dihydro-GA34. RNA-blot analysis showed that VaGA2oxA1 and VaGA2oxA2 were the major ones expressed in etiolated hypocotyls. The addition of Co2+ instead of Fe2+ to the assay medium apparently reduced the enzymatic activity, but increased the binding of [3H4]-16,17-dihydro-GA4 to rVaGA2oxA1, indicating the possibility that VaGA2oxs can be detected as gibberellin-binding proteins under certain conditions.