Two glycoside hydrolase family 1 proteins mediate glycosylated modification at the 5-position of anthocyanin in grape hyacinth

Glycosylation modification of anthocyanins is important as a preceding step to acylation modification. Cyanidin-3-O-(p-coumaroyl)glucoside-5-O-malonylglucoside (Cy3pCG5MaG) is one of the major anthocyanin substances in blue-flowered grape hyacinth, but its 5-position glycosylation is unknown. Here, we identified two glycoside hydrolase family 1 genes, MaAGGT1 and MaAGGT5, which use acyl-glucose as a donor and are involved in the glycosylation modification of anthocyanins in grape hyacinth. MaAGGT1 and MaAGGT5 are localized in vacuoles and primarily expressed in the flowers, coinciding roughly with the accumulation of total anthocyanins and Cy3pCG5MaG. In vitro enzyme activity assays of recombinant proteins showed that MaAGGT1 is substrate-specific for Cy3G and Pt3G, while MaAGGT5 is substrate-specific for Mv3G. Suppressing the expression of MaAGGT1 or MaAGGT5 significantly inhibits the accumulation of total anthocyanins in blue-flowered grape hyacinth, but only MaAGGT1 affects the accumulation of Cy3pCG5MaG. Additionally, the anthocyanin activation factor MaMybA can bind to the promoters of MaAGGT1 and MaAGGT5, positively regulating their transcription, while MaAN2 binds only to the promoter of MaAGGT5, significantly enhancing its expression. In summary, our results provide evidence that two glycoside hydrolase family 1 proteins mediate the glycosylation modification at the 5-position of anthocyanins in grape hyacinth, with MaAGGT1 playing a key catalytic role in the formation of Cy3pCG5MaG.