CYP701A8: A Rice ent-Kaurene Oxidase Paralog Diverted to More Specialized Diterpenoid Metabolism    

Abstract

All higher plants contain an ent-kaurene oxidase (KO), as such a cytochrome P450 (CYP) 701 family member is required for gibberellin (GA) phytohormone biosynthesis. While gene expansion and functional diversification of GA-biosynthesis-derived diterpene synthases into more specialized metabolism has been demonstrated, no functionally divergent KO/CYP701 homologs have been previously identified. Rice (Oryza sativa) contains five CYP701A subfamily members in its genome, despite the fact that only one (OsKO2/CYP701A6) is required for GA biosynthesis. Here we demonstrate that one of the other rice CYP701A subfamily members, OsKOL4/CYP701A8, does not catalyze the prototypical conversion of the ent-kaurene C4a-methyl to a carboxylic acid, but instead carries out hydroxylation at the nearby C3a position in a number of related diterpenes. In particular, under conditions where OsKO2 catalyzes the expected conversion of ent-kaurene to ent-kaurenoic acid required for GA biosynthesis, OsKOL4 instead efficiently reacts with ent-sandaracopimaradiene and ent-cassadiene to produce the corresponding C3a-hydroxylated diterpenoids. These compounds are expected intermediates in biosynthesis of the oryzalexin and phytocassane families of rice antifungal phytoalexins, respectively, and can be detected in rice plants under the appropriate conditions. Thus, it appears that OsKOL4 plays a role in the more specialized diterpenoid metabolism of rice, and our results provide evidence for divergence of a KO/CYP701 family member from GA biosynthesis. This further expands the range of enzymes recruited from the ancestral GA primary pathway to the more complex and specialized labdane-related diterpenoid metabolic network found in rice.

Plant P450s as versatile drivers for evolution of species-specific chemical diversity

“…With the exception of the moss Physcomitrella, which lacks GA phytohormones, the product of CYP701A is further oxidized in higher land plants by a second P450 to GA 12 . In the genome of rice (O. sativa), five CYP701A subfamily members are found, while only one, CYP701A6, fulfils the vital function of ent-kaurene oxidation en route to GA phytohormones [44]. Additionally, the five P450s are organized in one genomic cluster of 115 kb on chromosome 6.…”

Discovery, Biosynthesis and Stress-Related Accumulation of Dolabradiene-Derived Defenses in Maize

“…The ensuing diTPS products often undergo further structural modifications through the activity of P450 enzymes, as exemplified in rice by the P450-catalyzed formation of several bioactive diterpenoid metabolites (Swaminathan et al, 2009;Wang et al, 2011Wang et al, , 2012aWang et al, , 2012b. Comparatively, less was known about the complexity of maize diterpenoid biosynthesis.…”

Probing the promiscuity of ent -kaurene oxidases via combinatorial biosynthesis

“…The difference between the activity exhibited by AtKO and OsKO2 may reflect their distinct evolutionary contexts. In particular, although Arabidopsis seems to be representative of the Brassicaceae plant family, which does not produce any more specialized LRDs, only GA, and contains just one CYP701 family member, rice is representative of the Poaceae plant family, where it has been indicated that more specialized LRDs arose early, with widespread retention (20), and rice contains a number of CYP701A subfamily members with distinct function (9)(10)(11). This presumably exerted differing selective pressures on these genes, which was examined by molecular phylogenetic analysis of all characterized members of the CYP701 family ( Fig.…”