Ca2+-induced CpCBL8-CpCIPK9 module negatively regulates dormancy breaking and cold tolerance in winter-flowering wintersweet

The complex of calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK) serves as key components in calcium-sensing, orchestrating various signals crucial for plant growth, development, and responses to biotic and abiotic stresses. However, the mechanism underlying the response of this module to cold stress and its role in flower development in wintersweet ( Chimonanthus praecox ) remains unclear. Through expression pattern analysis, calcium ion (Ca 2+ ) concentration assays, correlation analysis, and linear regression analysis, we found that the [Ca 2 ⁺], along with CpCBL8 and CpCIPK9 expression levels in wintersweet flower buds (FBs), significantly decreased during the initial flowering stage when the chilling requirement reached 570 chill units (CU). Notably, there was a significant positive correlation between [Ca 2 ⁺] and CpCBL8 expression. Ca 2+ increased the expression of CpCBL8 and CpCIPK9 in FBs, causing a significant delay in the flowering of wintersweet. Furthermore, the function of CpCBL8 was studied using heterologous transformation. Overexpression of CpCBL8 significantly delayed flowering time and significantly reduced cold tolerance and altered the expression pattern of endogenous genes related to low-temperature stress and flower development in transgenic Arabidopsis thaliana . Additionally, transcriptome analysis of chilling-induced dormancy breaking and flower bud enlargement revealed that CpCBL8 and CpCIPK9 were negatively regulated by cold, and the expression pattern of endogenous genes related to flower development and cold stress in wintersweet were similar to that of in A . thaliana . Moreover, protein–protein interaction (PPI) analysis revealed that CpCBL8 and CpCIPK9 interacted in the plasma membrane and nucleus. On the basis of these findings, we speculated that the CpCBL8-CpCIPK9 module plays a crucial role in regulating responses to cold stress and flower development in wintersweet. This study elucidated molecular mechanisms through which the downregulation of the Ca 2+ -induced CpCBL8-CpCIPK9 module results in dormancy breaking and enhances cold tolerance. This study provides valuable insights for the cultivation of new varieties of wintersweet with increased ornamental value and enhanced cold stress tolerance.