Haplotyped genome mapping and functional characterization of a blueberry anthocyanin acetyltransferase (AAT) controlling the accumulation of acylated anthocyanins

Blueberry has a diversity of anthocyanins that confer its characteristic blue-coloured skin. Whilst most cultivars produce only anthocyanin glycosides, some can add aliphatic or aromatic groups to the sugar moiety to create acylated anthocyanins. Due to their enhanced stability, acylated anthocyanins represent an attractive breeding target in blueberry. In this study, a haplotype-resolved assembly of a previously identified quantitative trait locus on chromosome 2 of 'Hortblue Petite' (Vaccinium corymbosum) was created to identify candidate anthocyanin acyltransferase genes. One full-length gene (VcAAT1a) was selected based on quantitative PCR expression profiling and transient expression in tobacco leaves and in strawberry and blueberry fruit flesh. In all three systems, VcAAT1a was able to produce a range of acylated anthocyanins in planta. Recombinant VcAAT1a protein demonstrated that, while VcAAT1a was able to act on both anthocyanin 3-O-glucosides and 3-O-galactosides, it could only utilize acetyl-CoA as an acyl donor. Protein modelling using AlphaFold suggested that this restricted range in acyl donors may be due to a spatially restricted sub-pocket in the acyl-binding site of VvAAT1. Finally, LUC/REN promoter activation assays revealed that the VcAAT1a promoter was transactivated by the VcMYBPA1 and VcMYBPA2 transcription factors, further expanding our knowledge of anthocyanin regulation in blueberry.