| Teinturier grape cultivars with high anthocyanin concentration,were developed to blend with other grape varieties to improve the color and tannin content of red wines during winemaking,or its wine blended with other wine.Yan73 grape is a vitis vinifera teinturier(redfleshed)grape cultivar.It is vital for the full use of teinturier grape cultivar and the development of red-fleshed grape varieties to investigate the anthocyanin biosynthesis and the molecular mechanisms underlying anthocyanin biosynthesis of Yan73 teinturier grape cultivar.However,relatively little is known about the anthocyanin biosynthesis of Yan73 grape cultivar,and future studies can be grouped as follows:(i)In most red grapes,anthocyanins exclusively or mainly accumulate in the berry skin.However,in teinturier grape cultivars,anthocyanins can accumulate not only in the skin,but also in the flesh and vegetative tissues(leaf,stem and tendril).The molecular mechanisms underlying anthocyanin biosynthesis of Yan73 grape skin have been reported,but it is unclear for Yan73 grape flesh and vegetative tissues;(ii)Yan73 grape skin and flesh has been thought to be able to accumulate pelargonidin-based anthocyanins,but the molecular mechanisms remain unclear.Previously,there was no consensus on whether vitis vinifera grape variety had pelargonidin-based anthocyanin biosynthesis pathway,because it is unclear what is the substrates of DFR protein in grape.In this study,we analyzed the anthocyanin metabolic profiles of the grape skin,flesh and vegetative tissues of Yan73 with metabolome technologies.We screened the differentially expressed structure genes,R2R3-MYB transcriptional activators and repressors that regulate the anthocyanin biosynthesis in Yan73 grape flesh and vegetative tissues using RNAsequencing and q RT-PCR technologies.Then we performed functional analysis for selected R2R3-MYB transcriptional activators and repressors that regulate the anthocyanin biosynthesis in Yan73 grape flesh and vegetative tissues using yeast one-and two-hybrid,dual luciferase and bimolecular fluorescence complementation assays;To further confirm the presence of pelargonidin-type anthocyanins in Yan73 grape skin and flesh,their DFRs were cloned,expressed in Escherichia coli,and purified.An enzyme-activity assay and DFR substrate analysis were performed.The main findings are as follows:1.This study confirmed that the anthocyanin profiles were different among Yan73 grape skin,flesh and vegetative tissues.Yan73 grape skins had higher concentration of delphinidinbased anthocyanins than the corresponding cyanidin-based anthocyanins.Malvidin 3-O-glucoside was the most abundant anthocyanin.While Yan73 grape flesh contained similar levels of delphinidin-based and cyanidin-based anthocyanins,and peonidin 3-O-glucoside was the most abundant anthocyanin.Yan73 grapevine leaf,stem and tendril showed similar anthocyanin profile,with relatively higher percentages of cyanidin-based,acylated and methoxylated anthocyanins than the corresponding delphinidin-based,non-acylated and nonmethoxylated anthocyanins in these Yan73 grapevine vegetative tissues.Furthermore,peonidin-3-O-(trans-6-coumaryl)-glucoside was the most abundant anthocyanin in Yan73 these vegetative tissues.Pelargonidin 3-O-glucoside was detected in the Yan73 skin and flesh.Furthermore,pelargonidin 3-O-(6-acetyl)-glucoside was detected in the Yan73 skin and flesh for the first time.However,no any pelargonidin-based anthocyanins were detected in Yan73 grapevine vegetative tissues.The concentrations of delphinidin-based and cyanidin-based anthocyanins were significantly higher than that of pelargonidin-based anthocyanins.2.This study determined anthocyanin-related structure genes were up-regulated in the flesh of Yan73 grape after veraison and Yan73 grape young vegetative tissues.9811 genes were differentially expressed among the flesh of Yan73 grape before and after veraison.Among the 24 differentially expressed genes related to the flavonoid biosynthesis pathway,16 anthocyanin-related genes exhibited significantly up-regulated expression in the flesh of Yan73 grape after veraison.10323,9324 and 6199 genes were differentially expressed among Yan73 and its male grape leaves,stem and tendril tissues,respectively.Among which,a total of 30,17 and 10 anthocyanin biosynthesis genes exhibited up-regulated expression in Yan73 leaf,stem and tendril,respectively.The expression of anthocyanin-specific Vv UFGT,Vv OMT,Vv3 AT and Vv GST4 genes were all up-regulated in Yan73 grapevine vegetative tissues.3.This study determined the up-regulated R2R3-MYB transcriptional activator and Vv MYBC2-L1 repressor genes in the flesh of Yan73 grape after veraison and Yan73 grape young vegetative tissues.The expression levels of genes encoding Vv MYBA1,Vv MYB4 A,and Vv MYBC2-L1 transcription factors were significantly up-regulated in the flesh of Yan73 grape after veraison.Phylogenetic analysis indicated that Vv MYBA1 is clustered in the anthocyanin related activator clade,whereas Vv MYB4 A and Vv MYBC2-L1 belong to the C2 repressor motif clade.Furthermore,Vv MYBA1 and Vv MYBC2-L1 expression levels are highly correlated with anthocyanin accumulation and the expression of Vv UFGT and Vv LDOX in developing Yan73 berry flesh.Two groups of Vv MYBA transcription activator genes,including Vv MYBA1 on chromosome 2 and Vv MYBA5,Vv MYBA6 and Vv MYBA7 on chromosome 14,were up-regulated expressed in Yan73 grapevine vegetative tissues,with Vv MYBA5 and Vv MYBA6 genes having the highest expression levels.Simultaneously,the expression of a set of R2R3-MYB C2 repressor genes,mainly including Vv MYBC2-L1 and Vv MYBC2-L3,is activated and up-regulated in Yan73 grapevine vegetative tissues.4.This study determined the molecular regulatory mechanisms of anthocyanin biosynthesis in Yan73 grape reproductive and vegetative tissues are different.The Vv MYBA1 transcriptional activators and Vv MYBC2-L1 repressors coordinately regulate the anthocyanin biosynthesis of Yan73 grape flesh.The Vv MYBA1 positively regulates the anthocyanin biosynthesis of Yan73 berry flesh by interacting with Vv WDR1 and activating the expression of Vv CHI3,Vv OMT,and Vv GST4,whereas Vv MYBC2-L1 negatively regulates the anthocyanin biosynthesis of Yan73 berry flesh,possibly by competing with the R2R3-MYB transcriptional activators for Vv MYC1 partners or by repressing Vv OMT and Vv GST4 expression.Two groups of Vv MYBA transcription activators and Vv MYBC2-L1 repressor coordinately regulate the anthocyanin biosynthesis of Yan73 grapevine vegetative tissues.Vv MYBA1 positively regulates the anthocyanin biosynthesis of Yan73 grapevine vegetative tissues by interacting with Vv MYC1 and activating the expression of Vv F3’5’H and Vv UFGT;meanwhile Vv MYBA6 and Vv MYBA7 positively regulates the anthocyanin biosynthesis of Yan73 grapevine vegetative tissues by interacting with Vv MYC1 and activating the expression of Vv UFGT;whereas Vv MYBC2-L1 negatively regulates the anthocyanin biosynthesis of Yan73 berry flesh,possibly by competing with the R2R3-MYB transcriptional activators for Vv MYC1 partners.5.This study confirmed the DFR protein from Yan73 grape skin and flesh could catalyze dihydrokaempferol to produce leucopelargonidin.The similarity levels of the nucleotide and putative amino acid sequences of the cloned DFR genes from Yan73 grape skin and flesh were 100%.Amino acid residue 134 in these DFR was asparagine.The DFR from Yan73 grape skin and flesh was most closely related to DFRs from Malus domestica and Crataegus monogyna.The molecular mass of the purified DFR was 37.6 k Da.The DFR protein from Yan73 grape skin and flesh can reduce dihydrokaempferol to produce leucopelargonidin,although it prefers dihydroquercetin and dihydromyricetin as substrates to produce leucocyanidin and leucodelphinidin,respectively. |
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