Functional characterization of the grape VvCAD15-like gene in enhancing cold tolerance via regulation of lignin biosynthesis.

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis, but its role in grapevine cold tolerance is not well understood. Here we identified 23 VvCAD members from the 'Pinot Noir' genome and focused on VvCAD15-like, a Group II member whose expression was induced by cold. Transient overexpression of this gene in grape calli and plantlets increased lignin content, antioxidant enzyme activities (CAT, POD, SOD), and proline level, while decreased MDA, H₂O₂, and membrane permeability. It also upregulated VvCBF1/3 and VvICE1a. Transcriptome data implied potential links between VvCAD15-like and phenylpropanoid, flavonoid, and hormone signaling pathways. Yeast one-hybrid and dual-luciferase reporter assays identified three transcription factors-VvbHLH112, VvbHLH95, and VvNF-YA7-that could bind and activate the VvCAD15-like promoter under cold treatment. Among them, VvbHLH112 had transcriptional activation activity, and VvbHLH95 was closely related to AtICE1. Together, these data indicate that VvCAD15-like promotes cold tolerance possibly through cell wall reinforcement, ROS scavenging, and CBF pathway activation. This is the first report on VvCAD15-like function in grape cold tolerance, revealing its non-canonical role beyond lignin biosynthesis. It should be noted, however, that these results came from transient expression systems, and stable transgenic lines are needed to confirm its function in whole plants. Our work provides useful gene resources for cold tolerance breeding in grapevines.