Grape CIPK18 acts as a positive regulator of CBF cold signaling pathway by modulating ROS homeostasis

Cold temperatures trigger a diverged Ca2+ decoding paradigm that includes the proteins CBL (Calcineurin B-like proteins) and CIPK (CBL-interacting protein kinases). Here, putative CBL and CIPK genes were surveyed in cold-tolerant wild Amur grape (Vitis amurensis), designated as VaCBLs and VaCIPKs, respectively. The transcripts of 8 VaCBLs and 19 VaCIPKs encoded in this genome were confirmed, among which VaCIPK18 physically interacted with all 8 VaCBLs through the C-terminal regulatory domain (NAF), indicating that it may serve as a node regulator. VaCIPK18 was ubiquitously expressed in various tissues and was highly induced by cold, drought, salt, and ABA. The NAF domain of VaCIPK18 was required for functional homodimerization in yeast as well as in planta. Complementation tests confirmed that VaCIPK18 rescued the cold sensitivity of ArabidopsisAtcipk3 mutant, in which the expressions of CBFs were suppressed and is sensitive to cold and freezing stresses. In addition, overexpression of VaCIPK18 enhanced the cold tolerance by upregulating the C-repeat binding transcription factor (CBF) transcriptional pathway under normal or cold conditions, and reducing reactive oxygen species production. Thus, our study demonstrates that VaCIPK18 positively regulates cold signaling at least partly through the direct transcriptional control of cold-regulated CBFs and modulation of ROS homeostasis. Moreover, VaCIPK18 was found to interact with VaMYB4a in vivo and in vitro. Our findings provide evidence that VaCIPK18 functions as a key node in the CBL-CIPK network and may interact with VaMYB4a to contribute to cold response in grape plants.