A Hierarchical VvbHLH30‐VvERF70‐VvACS2 Module Orchestrates Ethylene Biosynthesis and Cold Adaptation in Grapevine

Ethylene is a key gaseous phytohormone that plays crucial roles in regulating plant growth, development and stress responses. However, ethylene-associated biosynthetic and transcriptional regulatory mechanisms governing cold-adaptation responses in plants remain poorly understood. In this work, genome-wide analysis from grapevines (Vitis vinifera) identified nine ACS family members, of which VvACS2, VvACS4 and VvACS6 exhibited the most dynamic transcriptional responses to cold stress and were chosen for functional validation. CRISPR-Cas9-mediated knockout and overexpression experiments revealed that VvACS2 is the major contributor to ethylene biosynthesis during cold stress in grapevine roots. Screening time-course cold treatment data from Vitis vinifera and Vitis amurensis roots identified VvERF70 and VvbHLH30 as the only two TFs, among six candidates, that directly regulate VvACS2 expression. Overexpression and CRISPR-Cas9-mediated knockout of VvERF70 or VvbHLH30 in roots further confirmed their contribution to enhanced ethylene production and cold tolerance under low-temperature treatment. Furthermore, the induction of VvACS2 was greatly enhanced when VvERF70 dimerized with VvbHLH30. Notably, VvbHLH30 further positively regulates ethylene biosynthesis under cold stress by interacting with VvERF70 and binding to its promoter. Taken together, we define a hierarchical transcriptional regulatory network where the VvbHLH30-VvERF70-VvACS2 module is pivotal for ethylene biosynthesis and underpins grapevine cold tolerance. This work provides new mechanistic insights into cold adaptation mechanisms and offers novel strategies to mitigate frost damage in agricultural crops.