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.