Lotus japonicusTriterpenoid Profile and Characterization oftheCYP716A51andLjCYP93E1Genes Involved in TheirBiosynthesis In Planta

Abstract

Lotus japonicusis an important model legume plant in sev-eral fields of research, such as secondary (specialized) me-tabolism and symbiotic nodulation. This plant accumulatestriterpenoids; however, less information regarding its com-position, content and biosynthesis is available comparedwithMedicago truncatulaandGlycine max. In this study,we analyzed the triterpenoid content and composition ofL. japonicus.Lotus japonicusaccumulated C-28-oxidized tri-terpenoids (ursolic, betulinic and oleanolic acids) and soya-sapogenols (soyasapogenol B, A and E) in a tissue-dependentmanner. We identified an oxidosqualene cyclase (OSC) andtwo cytochrome P450 enzymes (P450s) involved in triter-penoid biosynthesis using a yeast heterologous expressionsystem. OSC9 was the first enzyme derived fromL. japonicusthat showeda-amyrin (a precursor of ursolic acid)-produ-cing activity. CYP716A51 showed triterpenoid C-28oxidation activity. LjCYP93E1 convertedb-amyrin into24-hydroxy-b-amyrin, a metabolic intermediate of soyasapo-genols. The involvement of the identified genes in triterpen-oid biosynthesis inL. japonicusplants was evaluated byquantitative real-time PCR analysis. Furthermore, geneloss-of-function analysis ofCYP716A51andLjCYP93E1wasconducted. Thecyp716a51-mutantL. japonicushairy rootsgenerated by the genome-editing technique produced no C-28 oxidized triterpenoids. Likewise, the complete abolitionof soyasapogenols and soyasaponin I was observed in mutantplants harboringLotus retrotransposon 1(LORE1)inLjCYP93E1. These results indicate that the activities ofthese P450 enzymes are essential for triterpenoid biosynthe-sis inL. japonicus. This study increases our understanding oftriterpenoid biosynthesis in leguminous plants and providesinformation that will facilitate further studies of the physio-logical functions of triterpenoids usingL. japonicus.