Inactivation Pathway of Diterpenoid Regulator in the Moss Physcomitrium patens

The endogenous levels of plant hormones, including gibberellins (GAs), are strictly regulated and maintained during growth and development in seed plants. The regulation of endogenous levels of bioactive GAs is mediated by the mechanisms of their biosynthesis and inactivation. The moss Physcomitrium patens harbors a partial GA biosynthetic pathway from geranylgeranyl diphosphate to ent-kaurenoic acid (KA). Recently, we have identified ent-3β-hydroxy kaurenoic acid (3OH-KA) as a biologically active metabolite of KA to control the protonemal cell differentiation. In addition, ent-2α-hydroxy kaurenoic acid catalyzed by KA 2-oxidase (KA2ox) was also identified as inactive product. Although the activation and inactivation pathways from KA have been identified, the inactivation pathway of 3OH-KA remains to be elucidated. Considering the GA inactivation mechanism of flowering plants, in which GA2ox hydroxylates the C-2 position of GAs as part of the biosynthetic pathway, it was presumed that 3OH-KA was converted to 2,3-dihydroxy KA by PpKA2ox; however, this work shows that PpKA2ox undergoes hydroxylation at the C-16 position to synthesize a new compound ent-3β,16β-dihydroxy kaurenoic acid (3,16diOH-KA) from 3OH-KA. The protonemal cell differentiation activity of 3,16diOH-KA was low, and 3,16diOH-KA was detected in wild-type strains. These results indicate that 3,16diOH-KA was the major inactivating metabolite of 3OH-KA.