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 , a RING-type E3 ubiquitin ligase from , 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 signaling cascade. However, under cold stress, expression is downregulated, leading to the stabilization of VaMYB4a and the activation of signaling. Through a combination of biochemical assays and functional analysis in and grapevine calli, we demonstrate that 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 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.