Enzymatic basis for stepwise C‐glycosylation in the formation of flavonoid di‐C‐glycosides in sacred lotus (Nelumbo nucifera Gaertn.)

Lotus plumule, the embryo of sacred lotus (Nelumbo nucifera) seed, contains high accumulation of secondary metabolites including flavonoids and possesses important pharmaceutical value. Flavonoid C-glycosides, which accumulate exclusively in lotus plumule, attract considerable attention due to their unique chemical structures and special bioactivities in recent decades. Besides mono-C-glycosides, lotus plumule also accumulates various kinds of di-C-glycosides by unclear mechanisms. In this study, we identified two C-glycosyltransferases (CGTs) genes by mining sacred lotus genome data and provided in vitro and in planta evidences that these two enzymes (NnCGT1 and NnCGT2, also designated as UGT708N1 and UGT708N2, respectively) exhibited CGT activity. Recombinant UGT708N1 and UGT708N2 can C-glycosylate 2-hydroxyflavanones and 2-hydroxynaringenin C-glucoside, forming flavone mono-C-glycosides and di-C-glycosides after dehydration, respectively. In addition, the above reactions were successfully catalyzed by cell-free extracts from tobacco leaves transiently expressing NnCGT1 or NnCGT2. Finally, enzyme assays using cell-free extracts of lotus plumule suggested that flavone di-C-glycosides (vicenin-1, vicenin-3, schaftoside and isoschaftoside) were biosynthesized through sequentially C-glucosylating and C-arabinosylating/C-xylosylating 2-hydroxynaringenin. Taken together, our results provide novel insights into the biosynthesis of flavonoid di-C-glycosides by proposing a new flavone C-glycosides biosynthetic pathway of N. nucifera and identifying a novel UGT (UGT708N2) that specifically catalyzes the second glycosylation, C-arabinosylating and C-xylosylating 2-hydroxynaringenin C-glucoside.