Transcription Factor VviMYB86 Oppositely Regulates Proanthocyanidin and Anthocyanin Biosynthesis in Grape Berries

Abstract

Proanthocyanidins (PAs) and anthocyanins are two vital groups of flavonoid compounds for grape berries and red wines. Several transcription factors (TFs) have been identified to be involved in regulating PA and anthocyanin biosynthesis in grape berries. However, research on TFs with different regulatory mechanisms for these two biosynthesis branches in grapes remains limited. In this study, we identified an R2R3-MYB TF, VviMYB86, whose spatiotemporal gene expression pattern in grape berries coincided well with PA accumulation but contrasted with anthocyanin synthesis. Both in vivo and in vitro experiments verified that VviMYB86 positively regulated PA biosynthesis, primarily by upregulating the expression of the two leucoanthocyanidin reductase (LAR) genes in the Arabidopsis protoplast system, as well as in VviMYB86-overexpressing grape callus cultured under 24 h of darkness. Moreover, VviMYB86 was observed to repress the anthocyanin biosynthesis branch in grapes by downregulating the transcript levels of VviANS and VviUFGT. Overall, VviMYB86 is indicated to have a broad effect on flavonoid synthesis in grape berries. The results of this study will help elucidate the regulatory mechanism governing the expression of the two LAR genes in grape berries and provide new insights into the regulation of PA and anthocyanin biosynthesis in grape berries.

Keywords: R2R3-MYB; flavonoid; grape (Vitis vinifera L.); leucoanthocyanidin reductase; light; regulation.

FIGURE 1

The deduced peptide sequences of VviMYB86 and related MYB’s. (A) Polypeptide alignment analysis of the full-length amino acid sequences of VviMYB86 and other PA-related R2R3 MYB transcription factors (TFs) as well as TFs with high homology. Conserved residues were highlighted in black, and partial conservation was indicated in gray. The R2 and R3 MYB DNA binding domains were indicated above the alignment. The alpha helices of R2 and R3 repeats were indicated with red lines. Motif 1 [DNEI(A/S/G)N(D/A/N)V] and C1-Motif (lsrGIDPxT/NHR) were indicated by underlining with red line. (B) Phylogenetic tree of MYB TFs related to VviMYB86. The phylogenetic tree was constructed using Neighbor-Joining method of MEGA 7.0 software. The scale bar represents the number of substitutions per site. The putative regulatory functions of most of the proteins in the control of flavonoid biosynthesis are indicated. GenBank accession numbers are as follows (in parentheses): AtTT2 (Q9FJA2), AtMYB12 (NP_182268), AtMYB111 (NP_199744), AtMYB60 (AAC83617), AtPAP1 (AAG42001), AtPAP2 (NP_176813), VviMYB5a (AAS68190), VviMYB5b (AAX51291), VviMYBPA1 (CAJ90831), VviMYBPA2 (ACK56131), VviMYBPAR (XP_003633091), VviMYBF1 (ACT88298), VviMYBA1 (BAD18977), VviMYBA2 (BAD18978), VviMYBC2-L1 (ABW34393), VviMYBC2-L2 (ACX50288), DkMYB4 (BAI49721), FaMYB1 (AAK84064), GhMYB38 (AAK19618), GhMYB10 (ABV53918), and MdMYB6 (AAZ20429).

FIGURE 2

Subcellular localization of VviMYB86 and its transcription activation. (A) GFP activity in onion epidermal cells transiently expressing a VviMYB86-GFP construct. Bar: 100 μm. GFP, green fluorescence channel; Marker, blue fluorescence channel; DIC, bright light channel; Merge, the GFP and DAPi overlap. Arrows were the sites of GFP and DAPi overlap. (B) Transcription activation by VviMYB86 in yeast. The pGBKT7-VviMYBPAR plasmid was employed as a positive control. The pGBKT7-VviMYBC2L1 and pGBKT7 plasmids were employed as the negative controls. SD/-Trp, SD medium lacking tryptophan; SD/-Trp –His, SD medium lacking both tryptophan and histidine; SD/-Trp –His -Ade, SD medium lacking tryptophan, histidine, and adenine.

FIGURE 3

Spatiotemporal expression profile of VviMYB86 and some PA-related and anthocyanin-related genes in grape skins and seeds. The gradient arrows indicated the time-point of véraison. Gene expression was determined by real-time quantitative PCR and normalized with the expression of VviUbiquitin1. Each sample was individually assayed in triplicate. Error bars indicated the standard error of the mean.

FIGURE 4

The influence of VviMYB86 on the promoters of structural and regulatory genes in PA and anthocyanin synthesis. (A) The characteristics of cis-elements for binding of MYB, MYC and other proteins in structural and regulatory gene promoters of PA and anthocyanin synthesis. (B) Effects of VviMYB86 on the activities of gene promoters of PA and anthocyanin synthesis as determined by dual luciferase assay. Control indicated the activity of the promoter transfected with the empty vector (pCAMBIA 1301). Normalized luciferase activity was calculated as the ratio between firefly and Renilla reniformis luciferase activities. Each column represents means ± SD from four biological replicates.

FIGURE 5

VviMYB86 positively regulated proanthocyanidin (PA) synthesis in grape callus. (A) Identification of transgenic callus. The top was hygromycin gene expression detected by PCR and analyzed by gel electrophoresis. The below was the expression level of VviMYB86 in wild type (WT) callus and different transgenic lines (L1-L6). The value above the column referred to the transcripts of VviMYB86 in transgenic and WT calluses cultured under dark conditions. (B) The photos of WT callus and two independent VviMYB86 transgenic lines (L3 and L5). The left was WT callus and transgenic lines in their natural growth states cultured in darkness. The right was WT callus and transgenic lines after 4-Dimethylaminocinnamaldehyde (DMACA) staining. (C) The soluble and insoluble PA content in the WT callus and transgenic lines. FW, fresh weight. Data was expressed as means ± SD of three replicates. Asterisks indicated significant differences relative to the control by one-way ANOVA test (^∗∗∗p < 0.001). (D) The relative expression of flavonoid pathway related structural genes. After several successive rounds of subculture, stable transgenic callus lines were established on selectable medium. Callus grown for 25 days was collected for each assay. CHS, chalcone synthase; CHI, chalcone isomerase; DFR, dihydroflavonol-4-reductase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; ANS, anthocyanidin synthase; UFGT; UDP-glucose: flavonoid-3-O-glucosyltransferase. (E) The relative expression of known flavonoid regulators. Data was expressed as means ± SD of three replicates. Asterisks indicated significant differences relative to the control by one-way ANOVA test (^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001).

FIGURE 6

VviMYB86 negatively regulated anthocyanin synthesis in grape callus. WT-Light, wild type callus cultured under light conditions; L3-Light, transgenic line 3 cultured under light conditions; L5-Light, transgenic line 5 cultured under light conditions. (A) The cis-acting elements present in VviMYB86 promoter predicted by PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). (B) The nature status of WT callus and transgenic line 3 and line 5 under light conditions. (C) The expression levels of VviMYB86 in WT and transgenic calluses. Control: wild-type callus and transgenic calluses that cultured under dark conditions. Light: wild-type callus and transgenic calluses that cultured under light conditions. The value above the column referred to the transcripts of VviMYB86 in transgenic and WT calluses. (D) Anthocyanin contents in the WT callus and transgenic lines cultured under light conditions. The anthocyanin contents in callus samples measured by using the pH-differential method. FW, fresh weight. (E) Expressions of corresponding genes of anthocyanin biosynthesis pathway in the WT callus and transgenic lines cultured under light conditions. ANS, anthocyanidin synthase; UFGT, UDP-glucose: flavonoid-3-O-glucosyltransferase. Data was expressed as means ± SD of three replicates. Asterisks indicated significant differences relative to the control by one-way ANOVA test (^∗p < 0.05; ^∗∗∗p < 0.001).