Gene transcript accumulation, tissue and subcellular localization of anthocyanidin synthase (ANS) in developing grape berries

Anthocyanidin synthase (ANS; EC 1.14.11.19) is one of the key enzymes in the biosynthesis of anthocyanins and proanthocyanidins, which contribute to color and mouthfeel of grape berry and wine. Previous studies concerning the ANS gene in reproductive organs like grape berries mainly focused on gene expression and regulation at transcriptional level. Little was reported about translational controlling and cellular localization. In the present study, the full ANS cDNA with 1068bp was cloned from grape berries (Vitis vinifera L. Cabernet Sauvignon) via PCR, the recombinant protein was obtained by prokaryotic expression and a polyclonal antibody against grape berry ANS was prepared. On this basis, the tissue and subcellular localization of ANS during grape berry development were investigated by using the techniques of immunohistochemical, immuno-electron microscopy and confocal microscopy. The results showed that ANS distributed in both exocarp and mesocarp cells of grape berries and the changes of ANS signals correspond well to the temporal accumulation patterns of ANS gene transcript and ANS protein during grape berry development. Furthermore a trio cytoplasmic/plastid/nuclear subcellular localization of ANS was observed. This work will provide new insight for the regulation of different branch pathways leading to diverse flavonoid compounds in grape berry.