Gibberellin oxidase activities in Bradyrhizobium japonicum bacteroids

Bradyrhizobium japonicum bacteroids isolated from root nodules of soybean (Glycine max.) plants converted the gibberellin (GA) precursor [(14)C1]GA12 into several products identified by combined gas chromatography-mass spectrometry as [(14)C1]GA24, [(14)C1]GA9, [(14)C1]GA15, GA9 17-nor-16-one and unidentified products. The oxidation of GA12, catalyzed by the GA 20-oxidase, was present in symbiotic bacteroids from plants around flowering, but not in bacteroids from plants at either an early vegetative stage or at late growth stages. Expression of cps and ks genes, involved in ent-kaurene biosynthesis, was also demonstrated in bacteroids from soybean plants around flowering. Earlier precursors of the GA pathway, ent-[(14)C1]kaurenoic acid or [(14)C4]GA12-aldehyde, were efficiently utilized by B. japonicum bacteroids to give labelled GA9 plus intermediates partially oxidized at C-20, as well as GA9 17-nor-16-one and an unidentified product. No 3β or 13-hydroxylated [(14)C]GAs were detected in any of the incubations. Moreover the C19-GAs [(14)C1]GA4 or [(14)C1]GA20 were recovered unconverted upon incubation with the bacteroids which supports the absence of GA 3β-hydroxylase activity in B. japonicum. The bacterial 20-oxidase utilized the 13-hydroxylated substrates [(14)C1]GA53, [(14)C1]GA44 or [(14)C1]GA19, although with less efficiency than [(14)C1]GA12 to give [(14)C1]GA20 as final product, while the 3β-hydroxylated substrate [(14)C1]GA14 was converted to [(14)C1]GA4 to a very small extent. Endogenous GA9 and GA24 were identified by GC-MS in methanolic nodule extracts. These results suggest that B. japonicum bacteroids would synthesize GA9 under the symbiotic conditions present in soybean root nodules.