Overexpression of a Tonoplast Malate Transporter Gene Leads to Enhanced Anthocyanin Biosynthesis in Apple

Anthocyanins, a group of secondary metabolites synthesised in the phenylpropanoid pathway, largely determine the peel colour of fleshy fruits, but it is not known if their synthesis is linked to vacuolar malate accumulation that underlies fruit acidity. Here, we show that when the coding sequence of Ma1 (cMa1), encoding a tonoplast malate transporter for controlling apple fruit acidity, is overexpressed in 'Royal Gala' apple, anthocyanin biosynthesis in the fruit peel is enhanced, corresponding to the downregulation of the expression of MdMYB73, a transcriptional activator for Ma1. RNAi suppression of MdMYB73 expression increases anthocyanin biosynthesis whereas its transient overexpression decreases anthocyanin biosynthesis in apple fruit peel. MdMYB73 binds to all 7 MYB-sites in the promoter of the gene encoding UDP-glucose: flavonoid-3-O-glucosyltransferase (UFGT), the enzyme that catalyses the last step in anthocyanin synthesis, to repress its expression. When MdMYB73 expression is suppressed by RNAi, MdUFGT expression is enhanced, leading to more anthocyanin synthesis, but this effect is blocked by RNAi suppression of MdUFGT expression. In addition, MdMYB73 competes with MdMYB1, a key transcriptional activator of anthocyanin synthesis, by binding to the same MYB-sites in the promoter of MdUFGT. These results indicate that, in addition to being a transcriptional activator for Ma1, MdMYB73 negatively regulates anthocyanin biosynthesis via repressing MdUFGT expression and competing with MdMYB1 for binding to the MdUFGT promoter in apple peel. In cMa1-OE fruits, downregulation of MdMYB73 releases MdUFGT from MdMYB73 repression, which allows more MdMYB1 to bind to the promoter of MdUFGT, leading to enhanced anthocyanin biosynthesis.