Running head : VvMYBA regulates an anthocyanin acyltransferase

61 Anthocyanins are flavonoid compounds responsible for red/purple colours in the leaves, fruit and flowers 62 of many plant species. They are produced through a multistep pathway which is controlled by MYB 63 transcription factors. VvMYBA1 and VvMYBA2 activate anthocyanin biosynthesis in grapevine (Vitis 64 vinifera) and are non-functional in white grapevine cultivars. In this study, transgenic grapevines with 65 altered VvMYBA gene expression were developed, and transcript analysis was carried out on berries using 66 a microarray technique. The results showed that VvMYBA is a positive regulator of the later stages of 67 anthocyanin synthesis, modification and transport in Shiraz. One up-regulated gene ANTHOCYANIN 368 O-GLUCOSIDE-6”-O-ACYLTRANSFERASE (Vv3AT) encodes a BAHD acyltransferase protein, 69 belonging to a clade separate from most anthocyanin acyltransferases. Functional studies (in planta and in 70 vitro) show that Vv3AT has a broad anthocyanin substrate specificity and can also utilise both aliphatic 71 and aromatic acyl donors, a novel activity for this enzyme family found in nature. In V. vinifera cv. Pinot 72 Noir, a red-berried grapevine mutant lacking acylated anthocyanins, Vv3AT contains a nonsense mutation 73 encoding a truncated protein that lacks two motifs required for BAHD protein activity. Promoter 74 activation assays confirm that Vv3AT transcription is activated by VvMYBA1, which adds to the current 75 understanding of the regulation of the BAHD gene family. The flexibility of Vv3AT to use both classes 76 of acyl donors will be useful in the engineering of anthocyanins in planta or in vitro. 77