Phosphorylation-dependent VaMYB4a regulates cold stress in grapevine by inhibiting VaPIF3 and activating VaCBF4

Cold stress severely impacts the quality and yield of grapevine (Vitis L.). In this study, we extend our previous work to elucidate the role and regulatory mechanisms of Vitis amurensis MYB transcription factor 4a (VaMYB4a) in grapevine's response to cold stress. Our results identified VaMYB4a as a key positive regulator of cold stress. We demonstrated that VaMYB4a undergoes phosphorylation by V. amurensis calcineurin B-like (CBL) proteins-interacting protein kinase 18 (VaCIPK18) under cold stress, a process that activates VaMYB4a transcriptional activity. Using chromatin immunoprecipitation sequencing (ChIP-seq). We performed a comprehensive genomic search to identify downstream components that interact with VaMYB4a, leading to the discovery of a basic helix-loop-helix transcription factor, V. amurensis phytochrome-interacting factor 3 (VaPIF3). VaMYB4a attenuated the transcriptional activity of VaPIF3 through a phosphorylation-dependent interaction under cold conditions. Furthermore, VaPIF3, which interacts with and inhibits V. amurensis C-repeat binding factor 4 (VaCBF4, a known positive regulator of cold stress), has its activity attenuated by VaMYB4a, which mediates the modulation of this pathway. Notably, VaMYB4a also interacted with and promoted the expression of VaCBF4 in a phosphorylation-dependent manner. Our study shows that VaMYB4a positively modulates cold tolerance in plants by simultaneously downregulating VaPIF3 and upregulating VaCBF4. These findings provide a nuanced understanding of the transcriptional response in grapevine under cold stress and contribute to the broader field of plant stress physiology.