Amur grape VaMYB4a mediates grapevine cold tolerance via dual regulation of CBF–COR and ABA pathways

1 VaMYB4a integrates CBF-COR and ABA signaling to enhance cold tolerance in grapevines. 2 CRISPR/Cas9 knockout and overexpression studies reveal VaMYB4a 's dual regulatory mechanisms. 3 ABA exerts a dual role, dynamically modulating VaMYB4a-mediated cold tolerance. Cold stress represents a critical constraint on crop productivity, particularly in temperate climates. Despite the established role of abscisic acid (ABA) in cold stress responses, the precise mechanisms through which transcription factors mediate ABA-dependent cold tolerance remain elusive. Here, we identify VaMYB4a , a MYB transcription factor from Vitis amurensis Rupr . ( Amur grape), as a key regulator of cold tolerance. It integrates ABA signaling with the CBF (C-repeat binding factors)- COR (cold-regulated) pathway to orchestrate cold stress adaptation. Through a combination of overexpression and CRISPR/Cas9-mediated knockout lines in Arabidopsis thaliana , grape callus, and Vitis vinifera. L seedlings, we demonstrate that VaMYB4a enhances freezing tolerance by promoting osmotic regulation, ROS (Reactive oxygen species) scavenging, and stomatal closure. VaMYB4a functions as a homo-dimer, with its C-terminal domain being essential for transcriptional activation. Mechanistically, VaMYB4a directly upregulates CBF and COR genes while fine-tuning ABA signaling components such as ABI1 and ABF4 . Notably, ABA exhibits a dual role: enhancing VaMYB4a -mediated freezing tolerance under short-term stress but attenuating its effects during prolonged cold exposure, revealing an intricate regulatory crosstalk between cold and hormonal pathways. Our work not only advances the molecular understanding of cold adaptation but also provides a promising genetic target for developing stress-resilient grape varieties to mitigate the impacts of climate change.