Identification of candidate UDP-glycosyltransferases involved in protopanaxadiol-type ginsenoside biosynthesis in Panax ginseng

Abstract

Ginsenosides are dammarane-type or triterpenoidal saponins that contribute to the various pharmacological activities of the medicinal herb Panax ginseng. The putative biosynthetic pathway for ginsenoside biosynthesis is known in P. ginseng, as are some of the transcripts and enzyme-encoding genes. However, few genes related to the UDP-glycosyltransferases (UGTs), enzymes that mediate glycosylation processes in final saponin biosynthesis, have been identified. Here, we generated three replicated Illumina RNA-Seq datasets from the adventitious roots of P. ginseng cultivar Cheongsun (CS) after 0, 12, 24, and 48 h of treatment with methyl jasmonate (MeJA). Using the same CS cultivar, metabolomic data were also generated at 0 h and every 12-24 h thereafter until 120 h of MeJA treatment. Differential gene expression, phylogenetic analysis, and metabolic profiling were used to identify candidate UGTs. Eleven candidate UGTs likely to be involved in ginsenoside glycosylation were identified. Eight of these were considered novel UGTs, newly identified in this study, and three were matched to previously characterized UGTs in P. ginseng. Phylogenetic analysis further asserted their association with ginsenoside biosynthesis. Additionally, metabolomic analysis revealed that the newly identified UGTs might be involved in the elongation of glycosyl chains of ginsenosides, especially of protopanaxadiol (PPD)-type ginsenosides.

Figure 1

Differentially expressed UDP-glycosyltransferase (UGT) genes in P. ginseng. Using the edgeR Bioconductor package based on normalized fragments per kilobase per million (FPKM) read values of three biological replicates of each adventitious root sample, 11 UGT genes were identified as being differentially expressed between control (CTR: 0 h) and methyl jasmonate (MeJA)-treated samples (12, 24, and 48 h MeJA treatment). The heatmap showing hierarchical clustering FPKM was generated using the heatmap.2 function provided by the R-package gplots.

Figure 2

Phylogenetic analysis between candidate UGTs in P. ginseng and other plant UGTs including SiUGT94D1 (Sesamum indicum); CaUGT3 (Catharanthus roseus); AtUGT91A1, AtUGT91B1, AtUGT91C1, AtUGT79B1, AtUGT79B5, AtUGT79B2, AtUGT79B8, AtUGT73D1, AtUGT73C7, AtUGT73C2, AtUGT74F2, AtUGT74F1, AtUGT74B1, AtUGT74E1, AtUGT74D1 (A. thaliana); GmUGT73P2, GmUGT91H4, GmUGT73F2, GmUGT73F4 (Glycine max); MtUGT73F3, MtUGT73K1, MtUGT71G1 (Medicago truncatula); BvUGT73C10, BvUGT73C11, BvUGT73C12, BvUGT73C13 (Barbarea vulgaris); PgUGT71A27, PgUGT74A1 (P. ginseng); SvUGT74M1 (Saponaria vacaria); ZmUGT74A1 (Zea mays); BpUGT94B1 (Bellis perennis). The red circle indicates newly identified UGTs in this study and blue boxes represent previously identified UGTs of P. ginseng. Pink boxes show the UGTs involved in the triterpene pathway in other plant species. The transparent blue big rectangular boxes group these UGTs into different sub families including UGT74, UGT71, UGT73, UGT94 and UGT91.

Figure 3

Relative expression UDP-glycosyltransferase genes differentially expressed between control (0 h) and methyl jasmonate-treated (after 12, 24 and 48 h) adventitious roots analysed by RT-quantitative polymerase chain reaction (RT-qPCR) assay. Results were normalized to a validated control gene, PgActin.

Figure 4

Liquid chromatography–mass spectrometry base peak ion chromatograms of adventitious roots of P. ginseng collected after 0–120 h treatment with methyl jasmonate. Numbers on chromatographic peaks annotate the ginsenosides identified and listed in Supplementary Table 1.

Figure 5

Principal component analysis (PCA) score plot (A) and loading plot (B) of liquid chromatography–mass spectrometry ginsenoside profiles of the adventitious roots if P. ginseng collected at different time points following treatment with methyl jasmonate. Numbers in the PCA loading plot annotate the compounds identified and listed in Supplementary Table 1.

Figure 6

Relative abundances (fold changes) of the major groups of ginsenosides in adventitious roots of Panax ginseng treated with methyl jasmonate and analysed by liquid chromatography–mass spectrometry. Error bars represent standard deviations from the mean of three replicates. PPD, protopanaxadiol; PPT, protopanaxatriol.

Figure 7

Proposed pathway of protopanaxadiol (PPD)-type ginsenoside biosynthesis in adventitious roots of Panax ginseng. Dotted arrows represent glycosylation steps for which the mediating UDP-glycosyltransferases (UGTs) are as yet unknown. Blue and red lines show two possible pathways for the addition of two glucose residues at the C3-OH position (see text). Glc, β-d-glucopyranosyl; Ara(p), α-l-arabinopyranosyl; Ara(f), α-l-arabinofuranosy; Xyl, β-d-xylopyranosyl.