VaWRKY65 contributes to cold tolerance through dual regulation of soluble sugar accumulation and reactive oxygen species scavenging in Vitis amurensis

Although the significance of some plant WRKYs in response to cold stress have been identified, the molecular mechanisms of most WRKYs remain unclear in grapevine. In this study, we demonstrate that cold-induced expression of VaBAM3 in Vitis amurensis executes a beneficial role in enhancing resistance by the regulating starch decomposition. VaWRKY65 was identified as an upstream transcriptional activator of VaBAM3 through yeast one-hybrid library screening and validated to directly interacts with the W-box region inside the VaBAM3 promoter. Transgenic Arabidopsis thaliana plants and grapevine roots overexpression VaWRKY65 exhibited improved cold tolerance along with higher BAM activity and soluble sugar levels, whereas opposite changes were observed in VaWRKY65 knockdown lines created by virus-induced gene silencing (VIGS) in grapevine plants and in the knockout wrky65 mutants generated by CRISPR/Cas9 technology in grapevine roots. The transcriptome data show that overexpression of VaWRKY65 led to significant alteration of a diverse set of stress-related genes at the transcriptional level. One of the genes, Peroxidase 36 (VaPOD36) was further verified as a direct target of VaWRKY65. Consistently, VaWRKY65-overexpressing plants had higher VaPOD36 transcript levels and POD activity but a reduced ROS level, while silencing VaWRKY65 results in contrary changes. Collectively, these results reveal that VaWRKY65 enhanced cold tolerance through modulating soluble sugars produced from starch breakdown and ROS scavenging.