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 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.

Identification of flavonoids and chlorogenic acids in elm fruits from the genus Ulmus and their antioxidant activity.

Elm fruits were once an important food source in the years of famine. Research on the functional compounds in elm fruits was almost unavailable. In this study, we established an efficient high-performance liquid chromatography method for the simultaneous separation of eight chlorogenic acids and 28 flavonoids in elm fruits for the first time. Total flavonoid contents ranged from 286 mg/100 g (Ulmus laciniata) to 1228 mg/100 g (U. pumila). High concentrations of rutin, quercetin 3-O-glucoside, and kaempferol derivatives were present in U. laevis, U. castaneifolia, and U. pumila, respectively. Furthermore, the fruit extracts of U. americana, U. castaneifolia, U. davidiana, and U. pumila showed higher antioxidant activity. These results suggest that fruits of these species can be used as bioresources for the extraction of the corresponding functional compounds. This work provides informative data and can be an important reference for future research on elm fruits as a renewed food resource.

Fatty acid desaturase 3 (PsFAD3) from Paeonia suffruticosa reveals high α-linolenic acid accumulation.

α-linolenic acid (ALA) deficiency and a skewed ω6: ω3 fatty acid ratio in the diet are thought to be a major cause for the high incidence of cardiovascular, inflammatory, and autoimmune diseases. Recent years, tree peony (Paeonia suffruticosa Andr.) with the high proportion of ALA (more than 45% in seed oil) is widely concerned. However, the underlying accumulation mechanism of the ALA in tree peony seeds remains unknown. In this study, comparative transcriptome analysis was performed between two cultivars ('Saiguifei' and 'Jingshenhuanfa') with different ALA contents. The analysis of the metabolic enzymes associated with ALA biosynthesis and temporal accumulation patterns of unsaturated fatty acids demonstrated the importance of microsomal ω-3 fatty acid desaturase 3 (FAD3). Moreover, PsFAD3 gene was identified from tree peony seeds, which was located in endoplasmic reticulum and the expression levels of PsFAD3 were consistent with ALA accumulation patterns in seeds. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated PsFAD3 protein could catalyze ALA synthesis. These results indicated that PsFAD3 was involved in the synthesis of ALA in seeds and could be exploited by the genetic breeding of new cultivars with high ALA content in tree peony as well as other potential crops.

Identification of microRNAs and long non-coding RNAs involved in fatty acid biosynthesis in tree peony seeds.

MicroRNAs (miRNAs) and long noncoding RNAs (lncRNAs) act as important molecular regulators in a wide range of biological processes during plant development and seed formation, including oil production. Tree peony seeds contain >90% unsaturated fatty acids (UFAs) and high proportions of α-linolenic acid (ALA, > 40%). To dissect the non-coding RNAs (ncRNAs) pathway involved in fatty acids synthesis in tree peony seeds, we construct six small RNA libraries and six transcriptome libraries from developing seeds of two cultivars (J and S) containing different content of fatty acid compositions. After deep sequencing the RNA libraries, the ncRNA expression profiles of tree peony seeds in two cultivars were systematically and comparatively analyzed. A total of 318 known and 153 new miRNAs and 22,430 lncRNAs were identified, among which 106 conserved and 9 novel miRNAs and 2785 lncRNAs were differentially expressed between the two cultivars. In addition, potential target genes of the microRNA and lncRNAs were also predicted and annotated. Among them, 9 miRNAs and 39 lncRNAs were predicted to target lipid related genes. Results showed that all of miR414, miR156b, miR2673b, miR7826, novel-m0027-5p, TR24651|c0_g1, TR24544|c0_g15, and TR27305|c0_g1 were up-regulated and expressed at a higher level in high-ALA cultivar J when compared to low-ALA cultivar S, suggesting that these ncRNAs and target genes are possibly involved in different fatty acid synthesis and lipid metabolism through post-transcriptional regulation. These results provide a better understanding of the roles of ncRNAs during fatty acid biosynthesis and metabolism in tree peony seeds.