Amur Grape VaMYB4a-VaERF054-Like Module Regulates Cold Tolerance Through a Regulatory Feedback Loop

Cold stress can limit the growth and development of grapevines, which can ultimately reduce productivity. However, the mechanisms by which grapevines respond to cold stress are not yet fully understood. Here, we characterized an APETALA2/ethylene response factor (AP2/ERF) which was shown to be a target gene of our previously identified VaMYB4a from Amur grape. We further investigated the molecular interactions between VaMYB4a and VaERF054-like transcription factors in grapes and their role in cold stress tolerance. Our results demonstrated that VaMYB4a directly binds to and activates the VaERF054-like gene promoter, leading to its enhanced expression. Moreover, we also explored the influence of ethylene precursors and inhibitors on VaERF054-like expression and grape cold tolerance. Our findings indicate that VaERF054-like contribute to cold tolerance in grapes through modulation of the ethylene pathway and the CBF signal pathway. Overexpression of VaERF054-like in Vitis vinifera 'Chardonnay' calli and transgenic grape lines resulted in increased freezing stress tolerance, confirming its role in the cold stress response. We further confirmed the interaction between VaMYB4a and VaERF054-like in vivo and in vitro. The co-transformation of VaMYB4a and VaERF054-like in grape calli demonstrates a synergistic interaction, enhancing the cold tolerance through a regulatory feedback mechanism. Our finding provides new insights into grape cold tolerance mechanisms, potentially contributing to the development of cold-resistant grape varieties.