Lotus plumule, the embryo of the seed of the sacred lotus (Nelumbo nucifera), contains a high accumulation of secondary metabolites including flavonoids and possesses important pharmaceutical value. Flavonoid C-glycosides, which accumulate exclusively in lotus plumule, have attracted considerable attention in recent decades due to their unique chemical structure and special bioactivities. As well as mono-C-glycosides, lotus plumule also accumulates various kinds of di-C-glycosides by mechanisms which are as yet unclear. In this study we identified two C-glycosyltransferase (CGT) genes by mining sacred lotus genome data and provide in vitro and in planta evidence that these two enzymes (NnCGT1 and NnCGT2, also designated as UGT708N1 and UGT708N2, respectively) exhibit 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, respectively, after dehydration. In addition, the above reactions were successfully catalysed 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) are 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 biosynthetic pathway for flavone C-glycosides in N. nucifera and identifying a novel uridine diphosphate-glycosyltransferase (UGT708N2) that specifically catalyses the second glycsosylation, C-arabinosylating and C-xylosylating 2-hydroxynaringenin C-glucoside.
