The critical role of CmCIPK1-CmRbohD1/D2 complexes in generating H2O2 signals for enhancing salt tolerance in pumpkins

Pumpkin is of great economic importance not only as food resources but also as the most widely used rootstock to graft cucurbit crops. Pumpkin rootstock improves salt tolerance of cucumber scion through respiratory burst oxidase homolog protein D (CmRbohD)-mediated reactive oxygen species (ROS) burst, which further enhances Na+ export and K+ uptake. RbohD activation requires calcium signaling. However, the underlying mechanism remains largely unknown. In this study, we discovered two RbohD members from pumpkin involved in the ROS burst at the early stage of salt stress. CmRbohD1 and CmRbohD2 were functionally redundant and double mutation significantly impaired salt tolerance in pumpkin. Overexpression of CmRbohD1 and CmRbohD2 mitigated salinity-induced damage and maintained a relatively low Na+ content and high K+ content. We screened the potential calcineurin B-like interacting protein kinases (CmCIPKs) which could bind with CmRbohD1 and CmRbohD2. Our results revealed that two RbohDs formed complexes specifically with CmCIPK1, thereby bursting ROS production. Overexpression of CmCIPK1 promoted the early ROS burst under salt stress condition and maintained a relatively balanced Na+/K+ homeostasis. Altogether, we proposed a CmCIPK1-CmRbohD1/D2 complex for pumpkin salt stress signaling transduction, which regulates the ROS burst and Na+/K+ homeostasis. Our findings offer unprecedented insights into the general mechanism of pumpkin salt tolerance.