Gibberellin 3-oxidases in developing embryos of the southern wild cucumber, Marah macrocarpus

Abstract

Immature seeds of the southern wild cucumber, Marah macrocarpus, are a rich source of gibberellins (GAs) and were used in some of the earliest experiments on GA biosynthesis. The main biologically active GAs in developing embryos and endosperm of M. macrocarpus are GA(4) and GA(7), which have been shown previously to be formed from GA(9) in separate pathways, GA(4) being formed directly by 3β-hydroxylation, while GA(7) is produced in two steps via 2,3-didehydroGA(9). In order to identify the enzymes responsible for these conversions, three cDNA clones encoding functionally different GA 3-oxidases, MmGA3ox1, -2 and -3, were obtained from young immature M. macrocarpus embryos. Their biochemical functions were determined by expression of the cDNAs in Escherichia coli and incubation of cell lysates with (14)C-labelled substrates. MmGA3ox1 and MmGA3ox3 converted GA(9) to GA(4) as sole product, while MmGA3ox2 produced several products, including GA(4), 2,3-didehydroGA(9), 2,3-epoxyGA(9), GA(20) and GA(5), these last two products requiring 13-hydroxylation of GA(9) and 2,3-didehydroGA(9), respectively. MmGA3ox1 converted 2,3-didehydroGA(9) to GA(7), while MmGA3ox3 converted this substrate to the 2,3-epoxide, and MmGA3ox2 also formed the epoxide, but also GA(5.) Thus, formation of GA(7) requires the sequential activities of MmGA3ox2 and MmGA3ox1, while MmGA3ox3 is not involved in GA(7) production. The enzymes catalysed similar reactions when incubated with 13-hydroxylated GAs, although with reduced efficiencies. The 13-hydroxylase activity of MmGA3ox2 may be responsible for the production of GA(1) and GA(3), which are present at low levels in developing M. macrocarpus seeds.