The transcription factor VaNAC72-regulated expression of the VaCP17 gene from Chinese wild Vitis amurensis enhances cold tolerance in transgenic grape (V. vinifera)

Papain-like cysteine proteases (PLCP) play diverse roles in plant biology. In our previous studies, a VaCP17 gene from the cold-tolerant Vitis amurensis accession 'Shuangyou' was isolated and its role in cold tolerance was preliminarily verified in Arabidopsis. Here, we confirmed the function of VaCP17 in cold tolerance by stably overexpressing VaCP17 in the cold-sensitive Vitis vinifera cultivar 'Thompson Seedless' and transiently silencing VaCP17 in 'Shuangyou' leaves. The results showed that overexpression of VaCP17 improved the cold tolerance in 'Thompson Seedless' as manifested by reduced electrolyte leakage and malondialdehyde accumulation, chlo-rophyll homeostasis, increased antioxidant enzymes (superoxide dismutase, peroxidase, and catalase) activitiy, and rapid up-regulation of stress-related genes (VvKIN2, VvRD29B, and VvNCED1) compared with wild-type line. Conversely, RNA interfere-mediated knockdown of VaCP17 in 'Shuangyou' leaves resulted in opposite physio-logical and biochemical responses and exacerbated leaves wilting compared with control. Subsequently, by yeast one-hybrid, dual-luciferase assays, and transient overexpression of VaNAC72 in 'Shuangyou' leaves, a VaCP17-interacting protein VaNAC72 was confirmed to promote the expression of VaCP17 under cold stress, which depends on abscisic acid, methyl jasmonate, and salicylic acid signaling. By yeast two-hybrids, bimolecular fluorescence complementation and luciferase complementation assays, it was found that VaNAC72 could form homodimers or heterodimers with VaCBF2. Furthermore, co-expression analysis confirmed that VaNAC72 works synergistically with VaCBF2 or VaCP17 to up-regulate the expression of VaCP17. In conclusion, the study revealed that the VaNAC72-VaCP17 module positively regulated cold tolerance in grapevine, and this knowledge is useful for further revealing the cold-tolerance mechanism of V. amurensis and grape molecular breeding.