Mass spectrometric and enzymatic evidence confirm the existence of anthocyanidin 3,5-O-diglucosides in cabernet sauvignon (Vitis vinifera L.) grape berries.

It has been widely accepted that anthocyanidin 3,5-O-diglucosides do not exist in Vitis vinifera L. Cabernet Sauvignon (CS) berries. However, our anthocyanin analyses using HPLC-ESI-MS/MS detected the existence of a low level of anthocyanidin 3,5-O-diglucosides in the Cabernet Sauvignon grape berries grown in China. The authenticity of these samples was confirmed with microsatellite markers. The existence of anthocyanidin 3,5-O-diglucoside was further verified by the enzymatic evidence for the first time. Four putative 5-O-glucosyltransferase (5GT) genes were isolated from the Cabernet Sauvignon berries. The enzymatic analysis showed that a recombinant protein (designated as Vv5GT3) glucosylated the 3-O- and 5-O-positions of anthocyanidins and flavonols. A phylogenetic analysis revealed that this bifunctional enzyme belongs to the 5GT subfamily of UDP-glycosyltransferases. This finding brought a new understanding of the anthocyanins' profile and their biosynthesis in V. vinifera and would be helpful for further investigations of the mechanism of accumulation of anthocyanidin diglucosides in Cabernet Sauvignon berries in China's wine-producing regions.