The grapevine R2R3-MYB transcription factor VvMYBF1 regulates flavonol synthesis in developing grape berries

Abstract

Flavonols are important ultraviolet light protectants in many plants and contribute substantially to the quality and health-promoting effects of fruits and derived plant products. To study the regulation of flavonol synthesis in fruit, we isolated and characterized the grapevine (Vitis vinifera 'Shiraz') R2R3-MYB transcription factor VvMYBF1. Transient reporter assays established VvMYBF1 to be a specific activator of flavonol synthase1 (VvFLS1) and several other promoters of grapevine and Arabidopsis (Arabidopsis thaliana) genes involved in flavonol synthesis. Expression of VvMYBF1 in the Arabidopsis mutant myb12 resulted in complementation of its flavonol-deficient phenotype and confirmed the function of VvMYBF1 as a transcriptional regulator of flavonol synthesis. Transcript analysis of VvMYBF1 throughout grape berry development revealed its expression during flowering and in skins of ripening berries, which correlates with the accumulation of flavonols and expression of VvFLS1. In addition to its developmental regulation, VvMYBF1 expression was light inducible, implicating VvMYBF1 in the control of VvFLS1 transcription. Sequence analysis of VvMYBF1 and VvFLS1 indicated conserved putative light regulatory units in promoters of both genes from different cultivars. By analysis of the VvMYBF1 amino acid sequence, we identified the previously described SG7 domain and an additional sequence motif conserved in several plant MYB factors. The described motifs have been used to identify MYB transcription factors from other plant species putatively involved in the regulation of flavonol biosynthesis. To our knowledge, this is the first functional characterization of a light-inducible MYB transcription factor controlling flavonol synthesis in fruit.

Figure 1.

Simplified representation of the flavonoid biosynthetic pathway of grapevine leading to the three major classes of end products: flavonols, PAs, and anthocyanins. Note that the activity of FLS is required for the synthesis of flavonols, the activity of UFGT is required for the synthesis of anthocyanins, while the activities of ANR and LAR are specifically required for the synthesis of the precursors catechin and epicatechin of PA formation. ANR, Anthocyanidin reductase; 4CL, 4-coumaroyl-CoA synthase; C4H, cinnamate-4-hydroxylase; CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; F3H, flavanone-3β-hydroxylase; F3′H, flavonoid-3′-hydroxylase; F3′5′H, flavonoid-3′,5′-hydroxylase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase; LDOX, leucoanthocyanidin dioxygenase; OMT, O-methyltransferase; PAL, Phe ammonia lyase; RT, rhamnosyl transferase; UFGT, UDP-Glc:flavonoid-3-O-glucosyltransferase; UGT, UDP-glycosyltransferase. Note that for simplification, flavonoids catalyzed by the enzymes DFR, LDOX, LAR, and ANR have been omitted; therefore, only the final products anthocyanins and PAs are shown.

Figure 2.

A, Alignment of R2R3-MYB-type transcriptional regulators of flavonol synthesis from Arabidopsis and grapevine and presumptive flavonol regulators from other plant species and the maize phlobaphene regulator ZmP. The position of the R2R3-type MYB domain is indicated below the alignment, while the SG7 and SG7-2 domains are highlighted by black boxes. Amino acids identical in all sequences are shaded black, and amino acids found in more than eight or nine sequences are highlighted in light gray and dark gray, respectively. B, Phylogenetic tree showing selected plant MYB transcription factors from the GenBank or EMBL database. GenBank accession numbers of MYB factors are listed in “Materials and Methods.” Functions of most of the proteins are given in boldface. The scale bar represents 0.1 substitutions per site, and the numbers next to the nodes are bootstrap values from 1,000 replicates.

Figure 3.

Expression of VvMYBF1 during grape berry development. A, Early development. B, Skin. C, Seeds. Transcript levels of VvMYBF1 were determined by qPCR using gene-specific primers and corrected to VvUbiquitin1 (TC32075) gene expression. Data points are given as weeks from onset of ripening (veraison; labeled with the arrow), with expression values as means of three replicate PCRs and error bars indicating se. Note that from 8 weeks before veraison (−8), berry skin has been separated from seeds. Flowering occurred 8 weeks before veraison and is labeled with the star.

Figure 4.

Light induces expression of VvMYBF1 and VvFLS1 and subsequent flavonol synthesis in Chardonnay cells. Transcript levels were determined by qPCR and are shown relative to VvUbiquitin1 (TC32075) expression. Each data point represents the mean of three replicate PCRs with error bars indicating se. A, VvMYBF1 expression. B, VvFLS1 transcript levels in Chardonnay cell samples exposed to light. C, Accumulation of total flavonols in Chardonnay cell samples exposed to light (gray bars) and kept in the dark (black bars) determined by HPLC analysis. D, Positions of putative cis-regulatory elements in the promoters of VvMYBF1 and VvFLS1 from Shiraz, Pinot Noir, and Chardonnay relative to the transcriptional start codon. The putative transcriptional start sites are referred to as positions +1, while the translational start codons are marked by the bent arrows. Numbers above the boxes indicate the distance from the transcriptional start. ACS, ACGT-containing sequence similar to ACE in the Arabidopsis CHS promoter (accession no. S000355); MRS, MYB recognition sequence similar to MRE in the Arabidopsis CHS promoter (S000356); RRS, R response sequence similar to RRE in the Arabidopsis CHS promoter (S000407); IBOXCORE element (S000199), LREBOXIPCCHS1 consensus sequence (S000302), MYBCORE (S000176).

Figure 5.

VvMYBF1 specifically activates grapevine and Arabidopsis promoters of flavonoid pathway genes involved in flavonol synthesis in a transient reporter assay system. A to F, The MYB- and bHLH-type transcription factors and promoters used for transient expression in Chardonnay grape cell cultures by particle bombardment. Each transfection contained the renilla luciferase plasmid pRluc for normalization. Columns in A to E represent fold induction of the corresponding promoter plus MYB factor to the respective control (without MYB factor). Each column represents the mean value of at least six independent experiments with error bars indicating se. For F, basal activities of grapevine promoters are relative to renilla luciferase activity. G, A transient Arabidopsis At7 protoplast reporter gene assay system was used to analyze the activation potential of VvMYBF1 on promoters of Arabidopsis flavonoid biosynthesis enzymes. Each column represents the mean of six independent cotransfections with error bars indicating se. ANR, Anthocyanidin reductase; CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; FLS, flavonol synthase; LDOX, leucoanthocyanidin dioxygenase; UFGT, UDP-Glc:flavonoid-3-O-glucosyltransferase.

Figure 6.

Flavonol accumulation in Arabidopsis seedlings. A to D, Seedlings were stained with the flavonol-specific dye DPBA. Flavonol staining in Arabidopsis seedlings was visualized using epifluorescence microscopy. Images of representative seedlings of Col-0 (A), Col-0 expressing VvMYBF1 (Col-0/proAtMYB12∷VvMYBF1; B), myb12 mutants (C), and one representative complemented line expressing VvMYBF1 (myb12/proAtMYB12∷VvMYBF1; D) are shown. Bars = 0.1 cm. E, Comparison of the relative flavonol content of methanolic extracts of Arabidopsis seedlings by HPLC analysis. The results are from one representative experiment that was repeated with similar relative values. FW, Fresh weight.