Screening and verification of late embryogenesis abundant protein interacting with anthocyanidin reductase in grape berries
Anthocyanidin reductase (ANR, EC 18.104.22.168) catalyzes the conversion of anthocyanidins to 2R, 3R-flavan-3-ols in Vitis vinifera grapes. These are basic structural units of proanthocyanidins (PAs). The regulation of PA biosynthesis at protein level is not yet clear. Here, we find a VvANR interaction partner called late embryogenesis abundant protein (LEA), which can interact with VvANR in the yeast two-hybrid (Y2H) system. We verified the interaction between VvANR and Vv-LEA by Y2H and co-immunoprecipitation (CoIP) in yeast in vitro, and using the firefly luciferase complementation imaging (LCI) system in vivo. Additionally, the co-localization of VvANR and VvLEA in Arabidopsis thaliana protoplasts also provids the essential conditions for their interaction. The different expressions of VvANR and VvLEA depended both on the age of the grape berries and on the duration of cold treatment. These findings provide primary evidence for proteinregulation and the potential formation of multi-enzyme complex of VvANR, as well as of the PA biosynthesis.
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