Protein sequence alignment of VvMYB5b and other R2R3-MYB transcription factors from various plant species. Identical residues are shown in black, conserved residues in dark gray, and similar residues in light gray. The lines above the alignment locate the different domains and motifs within the proteins. The DNA binding domain corresponds to the R2R3-MYB repeats. The ID domain, involved in the interaction with bHLH proteins, and the C1 and C3 motifs are in bold and italics. GenBank accession numbers are as follows (in parentheses): VvMYB5b (AAX51291), VvMYB5a (AAS68190), BNLGHi233 (AAK19611), PH4 (AAY51377), AtMYB5 (AAC49311), OsMYB4 (BAA23340), VvMYBPA1 (CAJ90831), and VvMYBA1 (BAD18977).

VvMYB5b expression in grapevine tissues. A, VvMYB5b expression in developing Cabernet Sauvignon berries. Southern blots of semiquantitative RT-PCR products were blotted onto a nylon membrane and hybridized with the radiolabeled VvMYB5b 3′-UTR probe. Elongation factor EF1-γ was used as a quantitative control. B, VvMYB5b expression in vegetative tissues of Cabernet Sauvignon vines. Southern blots of semiquantitative RT-PCR products were blotted onto a nylon membrane and hybridized with the radiolabeled VvMYB5b 3′-UTR probe. Elongation factor EF1-γ was used as a quantitative control. C to E, Expression of VvMYB5b in tissues of developing berries from the Shiraz cultivar. Numbers correspond to weeks relative to the onset of ripening (veraison). VvMYB5b expression was analyzed in whole berries from 10 to 6 weeks before veraison and specifically in seed and skin tissues from 6 weeks before up to 8 weeks after veraison. Data are means ± ses of three real-time PCR experiments.

Phenotypic analysis of transgenic tobacco flowers overexpressing VvMYB5b. Flowers of transgenic plants (B and C) showed an increased pigmentation in petal and stamen epidermal cells compared to control flowers (A). DMACA staining of petals (E) and stamen (G) cells from transgenic flowers shows condensed tannin accumulation when compared to control lines (D and F). H and I, Delay in pollen release observed in anthers at developmental stages 10, 11, and 12 (from left to right, according to Koltunow et al. [1990]) from VvMYB5b sense lines (I) compared to control plant anthers (H). J and K, Cross sections of anthers at stage 10 from control (J) and VvMYB5b-overexpressing tobacco plants (K). Boxed areas are magnified in L and M, respectively. Arrows indicate endothecial cells developing complete (L) or incomplete (M) lignified fibers. Bars = 500 μm (J and K) and 75 μm (L and M).

Summary of the possible implication of R2R3-MYB transcription factors in the regulatory mechanisms of the flavonoid pathway during grape berry development. The roles of VvMYBPA1, VvMYB5a, VvMYB5b, VvMYBA1, and VvMYBA2 were assigned according to gene expression levels during berry development together with functional characterization data presented in this article for VvMYB5b and VvMYB5a, in Bogs et al. (2007) for VvMYBPA1, in Deluc et al. (2006) for VvMYB5a, and in Walker et al. (2007) for the VvMYBA genes. Gene expression levels throughout berry development were determined by quantitative RT-PCR and are presented in Figure 3 (VvMYB5b) and in Supplemental Figure S1 for the other genes. The same RNA samples extracted from Shiraz berries collected at various developmental stages (see “Materials and Methods”) were used to analyze the expression level of each gene. According to these data, VvMYBPA1, VvMYB5a, and VvMYB5b appear particularly involved in the regulation of PA synthesis before veraison in skin and seed tissues. At veraison, PA synthesis is complete and anthocyanin synthesis begins in skin cells where UFGT gene expression is specifically regulated by VvMYBA1 and VvMYBA2, whereas genes encoding enzymes of the general flavonoid pathway required for anthocyanidin synthesis appear to be regulated by VvMYB5b. Abbreviations are as follows: PAL, Phe ammonia lyase; C4H, cinnamate 4 hydroxylase; 4CL, 4 coumarate CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3 hydroxylase; F3′5′H, flavonoid 3′5′ hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; and UFGT, UDP-Glc:flavonoid 3-O-glucosyltransferase.

Phylogenetic relationships between VvMYB5b and R2R3-MYB transcription factors from grape and other plant species. Phylogenetic and evolutionary analyses were performed using the neighbor-joining method by the MEGA version 4 program (Kumar et al., 2004). The scale bar represents the number of substitutions per site and the numbers next to the nodes are bootstrap values from 1,000 replicates. The putative regulatory functions of the different R2R3-MYB proteins in the control of secondary metabolite biosynthesis or other biological processes are indicated. GenBank accession numbers are as follows (in parentheses): VvMYBA1 (BAD18977), VvMYBA2 (BAD18978), VvMYBA3 (BAD18979), LeANT1 (AAQ55181), TT2 (CAC40021), AtMYB75 (AAG42001), PH4 (AAY51377), AmRosea1 (ABB83826), AmVenosa (ABB83828), MdMYB10 (ABB84753), ZmMYBPL (AAB67720), ZmMYBC1 (AAA33482), VvMYBPA1 (CAJ90831), PmMBF1 (AAA82943), OSMYB4 (BAA23340), AtMYB5 (AAC49311), BNLGHi233 (AAK19611), VvMYB5b (AAX51291), VvMYB5a (AAS68190), FaMYB1 (AAK84064), AtMYB7 (AAA98762), HOS10 (AAF20989), HvMYB5 (CAA50221), TFMYB6 (AAS19480), GhMYB1 (AAN28270), LeTHM27 (CAA64614), TaMYB1 (AAT37167), and ZmMYB38 (BAD34380).

Activation of flavonoid gene promoters by VvMYB5b and VvMYB5a. Control indicates the activity of the respective promoter in the absence of a MYB factor. Each transfection contained the 35S:EGL3 construct encoding the bHLH protein EGL3 (GenBank accession no. NM20235) from Arabidopsis and as internal control the Renilla luciferase plasmid pRLuc. The normalized luciferase activity was calculated as the ratio between the firefly and the Renilla luciferase activity. Each column represents the mean value of three independent experiments with error bars indicating ses. Abbreviations are as follows: VvUFGT, V. vinifera UDP-Glc:flavonoid 3-O-glucosyltransferase; VvANR, V. vinifera anthocyanidin reductase; AtBAN, A. thaliana Banyuls; VvLAR1, V. vinifera leucoanthocyanidin reductase 1; VvCHI, V. vinifera chalcone isomerase; VvF3′5′H, V. vinifera flavonoid 3′5′ hydroxylase; and VvANS, V. vinifera anthocyanidin synthase.

Analysis of flavonoid gene expression in flowers of transgenic tobacco overexpressing VvMYB5b. A, Transcripts for four flavonoid biosynthetic genes were detected by semiquantitative RT-PCR in petals and stamens from three VvMYB5b independent lines (1–3) and compared to control lines (Ct). VvMYB5b indicates the transgene expression level. UBI was used as a quantitative control. Abbreviations are as follows: CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3 hydroxylase; and F3′5′H, flavonoid 3′5′ hydroxylase. B, Northern-blot analysis of anthocyanidin synthase (ANS) gene expression in petals and stamens from control (Ct) and VvMYB5b lines. Total RNA was extracted from tissues harvested at the opening of flowers. Experiments were repeated with three VvMYB5b and control lines with similar results.