VaMIEL1-mediated ubiquitination of VaMYB4a orchestrates cold tolerance through integrated transcriptional and oxidative stress pathways in grapevine

Cold stress poses a significant threat to viticulture, particularly under the increasing pressures of climate change. In this study, we identified <i>VaMIEL1</i>, a RING-type E3 ubiquitin ligase from <i>Vitis amurensis</i>, as a negative regulator of cold tolerance. Under normal temperature conditions, VaMIEL1 facilitates the ubiquitination and subsequent proteasomal degradation of the cold-responsive transcription factor VaMYB4a, thereby attenuating its regulatory role in the <i>CBF-COR</i> signaling cascade. However, under cold stress, <i>VaMIEL1</i> expression is downregulated, leading to the stabilization of VaMYB4a and the activation of <i>CBF-COR</i> signaling. Through a combination of biochemical assays and functional analysis in <i>Arabidopsis thaliana</i> and grapevine calli, we demonstrate that <i>VaMIEL1</i> overexpression reduces cold tolerance, as evidenced by increased oxidative stress, excessive reactive oxygen species (ROS) accumulation, and downregulated expression of cold-responsive genes. Conversely, silencing of <i>VaMIEL1</i> enhances cold tolerance by stabilizing VaMYB4a and boosting antioxidant defenses. These findings uncover a previously unrecognized regulatory mechanism by which VaMIEL1 modulates cold tolerance through transcriptional and oxidative stress pathways, offering potential targets for the development of climate-resilient grapevine cultivars and other crops.