Accumulation Of Free And Glycosidically-bound Terpenes And Its Transcriptional Regulation In Wine Grapes

Terpenes in grape berries are major contributors to the floral/fruity odors of wine,and particularly are responsible for the varietal flavor of aromatic wines.Terpenes are present in both free and glycosidically bound forms in grapes.Bound-form terpenes are potential contributors to wine aroma odors because they can be converted into free volatile compounds through acidic and enzymatic hydrolysis during wine making.The ratio of free and bound-form terpenes varies with grape varieties,berry development and regions.Recently,a series of genes required for the synthesis of monoterpenes and their glycosidically-ally bound forms were biochemically characterized in grape berries.However,the genes that dominate the differential accumulation of terpenes in different grape varieties or grapes from different regions are not identified yet,and the transcriptional regulation of these genes remains also unclear.This project aimed to study the synthesis of free and glycosidically-bound terpenes and its transcriptional regulation in wine grapes,using aromatic grape varieties with high terpene concentration as experimental materials,and using modern molecular biology and analytical chemistry methods.The results were shown as follows:The comparison of the free and glycosidically bound terpene profiles was conducted amongst three aromatic varieties:'Gewurztraminer','Tchervine Muscat'and 'Muscat blanc a Petit grain'.The results showed that geraniol and cis/trans-rose oxides were the main compounds contributing to varietal aroma of 'Gewurztraminer',while linalool and geraniol were the main volatile contributors to aroma of'Tchervine Muscat'and 'Muscat blanc a Petit grain'.The expression patterns of terpene synthase and monoterpene glucosyltransferase were also examined.Two linalool synthase genes VvPNLinNerl and VvPNLinNer2 had high transcript abundance in ripening berries,suggesting that they are mainly responsible for linalool synthesis during berry development.The gene expression of VvPNGer coding for geraniol synthase exhibited a weak correclation with the accumulation of free geraniol in grape berries.Both the expression of VvGT7 and VvGT14 closely correlated with the accumulation of glycosidically bound monoterpenes in grape berries.Using the integrative analysis of transcripts and metabolites to propose key genes for differential terpene accumulation across two regions(Changli county,Hebei province and Gaotai county,Gansu province)with significantly distinct climates,we demonstrated that mature grape berries from CL region contained significantly higher concentrations of free and glycosidically bound terpenes than berries from GT.Transcriptome analysis revealed that some key genes involved in terpene synthesis were up-regulated in the CL region.Particularly in the MEP pathway of terpene biosynthesis,the expression of VvHDR(1-hydroxy-2-methyl-2-butenyl 4-diphosphate reductase)paralleled with the accumulation of terpenes,which suggests that this enzyme plays a key role in controlling the flow of isopentenyl diphosphate(IPP)into the terpene synthetic pathway in Muscat grapes.A weak correlation existed between the transcript abundances of terpenoid synthase genes(VvTPSs)and the concentrations of relevant metabolites,which implies that monoterpene synthesis could be regulated at the post-transcriptional level.The glycosidically bound monoterpenes accumulated differentially along with maturation in both regions,which closely correlated with the expression of a monoterpene glucosyltransferase gene(VvGT14).We thus infer that VvGT14 should dominate the production of glycosidically bound terpenes in the late stages of berry development.Other genes were also found to be related to the differential accumulation of terpenes and monoterpene glycosides in the grapes between regions.Transcription factors that could regulate terpene synthesis were predicted through gene co-expression network analysis.Additionally,the genes involved in abscisic acid(ABA)and ethylene signal responses were expressed at high levels earlier in GT grapes than in CL grapes,which could explain the shorter mature duration for GT grapes.To further understand the regulation of VvGT14 at a transcriptional level,we analyzed the VvGT14 gene promoter activity and the DNA methylation level in VvGT14 gene promoter region of grapes from Changli and Gaotai region at E-L35 stage.The results showed that the VvGT14 promoter can be activated by weak light and ABA induction,while high tempreture could reduce the activity of the promoter.The VvGT14 gene promoter region showed a low methylation level.The two CpG sites on this promoter displayed different methylation levels in the grapes from the two regions.These two sites were located in the cis-element area,suggesting that they may affect the combination of the transcription factors with the promoter.Based on these findings,it is inferred that transcriptional regulation of VvGT14 gene may be involved in both response of cis-elements on this gene promoter to external stimuli and the interaction of transcription factors with promoter elements.