Abstract

SUMMARY Grapevine ( Vitis vinifera ) is a high-value fruit crop frequently challenged by cold stress. Here, we identify the E3 ubiquitin ligase Plant U-box 26 (VvPUB26) as a pivotal regulator of the grapevine cold signaling pathway. We demonstrated that the VvPUB26-VvPIF4-VvCBF3 module fine-tunes cold tolerance by integrating light and temperature signals. Overexpression of VvPUB26 ( VvPUB26-OE ) significantly enhances cold tolerance in transgenic plants, whereas RNA interference ( VvPUB26-RNAi ) lines exhibit increased cold sensitivity. Biochemical analysis reveals that VvPUB26 modulates the CBF signaling cascade by interacting with several key cold-related proteins, including VvMYB15, VvICEs, and VvPIF4. Specifically, VvPUB26 facilitates the ubiquitination and subsequent degradation of VvPIF4 and VvMYB15, while simultaneously maintaining the stability of VvICEs protein. Although the VvPIF4 protein typically accumulates under cold stress and darkness, VvPUB26-mediated degradation weakens this stability. Furthermore, VvPIF4 directly binds to the VvCBF3 promoter to repress its transcription, thereby negatively regulating cold tolerance. Overexpression of VvPIF4 ( VvPIF4-OE ) phenocopies the cold-sensitive VvPUB26-RNAi lines, confirming its role as a negative regulator. Our findings establish that the VvPUB26-VvPIF4-VvCBF3 module optimizes grapevine cold adaptation through ubiquitination-dependent protein turnover, effectively linking light-temperature crosstalk to the CBF-COR pathway.