Study On The 2,3,5,4?-Tetrahydroxystilbene-2-O-?-D-Glucopyranoside(THSG) Biosynthetic Pathway In Fallopia Multiflora

Fallopia multiflora?also known as Polygonum multiflorum,common name Chinese Knotweed?is a popular traditional Chinese medicine and an ingredient of numerous prescriptions.The tetrahydroxystilbene glycoside 2,3,5,4?-tetrahydroxystilbene-2-O-?-D-glucoside?THSG?was first isolated and identified in 1975,the main active component of this plant,and has been the primary indicator of F.multiflora quality in the Chinese Pharmacopoeia.In vitro and in vivo pharmacological studies have demonstrated that F.multiflora extract possesses antioxidant activity.THSG,contributes to the antioxidant activity of F.multiflora and produces anti-inflammatory effects,protects endothelial cells,and inhibits oncogenic enzyme activity.Structurally,THSG has an additional hydroxyl group compared with resveratrol glucoside?polydatin?.Resveratrol isformed viathewell-characterized phenylalanine/polymalonate biosynthetic pathway.The final step in resveratrol biosynthesis involves the condensation of one molecule of p-coumaroyl-CoA and three molecules of malonyl-CoA in a reaction catalyzed by resveratrol synthase.We therefore proposed the hypothetical THSG biosynthetic pathway based on the resveratrol biosynthetic pathway.The suspension cultures of F.multiflora cells were established as the research system and feeding experiments on F.multiflora suspension cultures using ¹³C-labelled precursors were performed to investigate the biosynthetic pathway of THSG.The resveratrol synthase,glycosyltransferase,and hydroxylase activities involved in THSG biosynthesis in various tissue types of wild F.multiflora and callus cultures were characterized.Full-length cDNAs of genes potentially involved in THSG biosynthesis in F.multiflora were isolated based on the existing transcriptome dataset and DGE profile.Bioinformatics analysis was performed and their differential expression patterns in different organs and cell cultures were investigated.The main results of this article are as follows:?1?Our rapidly established 2nd-generation suspension cultures,showed a ability to produce THSG and a stable growth patterns.The flow cytometry profiles indicated that there was no aneuploidy or polyploidy in the cell cultures during the sub-culture process.Thus,there was no change in ploidy level in the F.multiflora cell cultures.The maximum THSG content was 0.0176 mg/g?THSG content/DW?.THSG was not detected during the first 10 days of cultivation.Our results suggest that JA,MeJ,SA,and SN are not appropriate elicitors for inducing THSG production in PM suspension culture systems.?2?First the THSG content was detected by the HPLC-UV system.The results showed that F.multiflora roots and vines contained very high levels of THSG,but THSG was not detected in the leaves or stems?young or old?.THSG was detected in the PM cell cultures at low levels.The THSG contents in the cell cultures?callus,0.022±0.008mg/g DW;suspension cultures,0.014±0.003 mg/g DW?were much lower than those in PM roots and vines?roots,42.751±3.323 mg/g DW;vines,24.125±3.291 mg/g DW?.And then we developed a rapid,convenient,and reliable analytical method to investigate the distributions of PD,resveratrol and THSG in wild plant tissues and cultured F.multiflora cells using a UPLC/Q-TOF-MS system.THSG accumulated to high concentrations within the vines?1504.091±81.672 ug/g FW?and roots?1730.719±37.807ug/g FW?,whereas it accumulated to very low concentrations within the stem tips,stems,leaves and callus.PD was detected in the leaves,vines,roots,and callus at very low concentrations.Resveratrol was not detected in the in wild F.multiflora and cultured cells.?3?¹³C-labelled L-phenylalanine?L-PHE?,sodium pyruvate?SP?,and sodium bicarbonate?SB?were used as putative precursors in the feeding experiment.Labeling of polydatin?PD?and THSG using[¹³C9]L-PHE and[¹³C₁]L-PHE confirmed that the p-coumaric moiety of PD and THSG was derived from PHE.The results of the feeding experiments with[¹³C₁]SB and[2,3-¹³C₂]SP suggested that PD and THSG were derivatives of resveratrol that were synthesized by glycosylation and hydroxylation.?4?The total crude protein extracts?soluble and microsomal?were used for comprehensive and simultaneous analysis of resveratrol synthase,glycosyltransferase,and hydroxylase activities in various tissue types of wild F.multiflora and callus cultures.The activity of each tested enzyme was confirmed in one or more tissue types or cell cultures in vitro.The results of the enzyme activity experiments and the distributions of PD and THSG were used to determine the main site and pathway of THSG biosynthesis in F.multiflora.Experiments were conducted in which the crude enzymes were incubated with substrates of more than one reaction step in the biosynthetic pathway of THSG.The results of the combinatorial biocatalysis experiments indicate that only the hydroxylation reactions in the THSG biosynthetic pathway can proceed following the resveratrol synthesis reaction in a single incubation in vitro.No THSG was produced,which indicated that these three reactions cannot proceed sequentially in vitro in a single incubation.?5?Based on the expression and functional annotation information for the unigene sequences from the F.multiflora transcriptome dataset,we selected contigs annotated as CYP450,UGT,and STS genes,and those potentially involved in secondary metabolite production.15 gene cDNA sequences potentially involved in the THSG biosynthetic pathway were cloned based on a transcriptome dataset.According to bioinformatics analysis,all the genes showed extensive homologies with cytochrome P450?CYPs?,uridine diphosphate-glycosyltransferases?UGT?,and plant-specific type III polyketide synthase?PKS??genes from other plant species,respectively.The expression profiles of the 15 genes in roots,vines,leaves,and callus of F.multiflora were investigated and the relationships between FmSTS,FmCYP,and FmUGT expression levels and THSG contents were analyzed.The results of bioinformatics analysis and relative expression quantification of the 15 gene sequences suggested that four genes?FmSTS4,FmSTS5,FmCYP1 and FmCYP2?were likely to be involved in THSG biosynthesis.