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 (Ca2+) concentration assays, correlation analysis, and linear regression analysis, we found that the [Ca2⁺], 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 [Ca2⁺] and CpCBL8 expression. Ca2+ 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 Ca2+-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.

中文翻译：

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Ca2+ 诱导的 CpCBL8-CpCIPK9 模块负向调控冬花冬甜冬眠打破和耐寒


钙调磷酸酶 B 样蛋白 （CBL） 和 CBL 相互作用蛋白激酶 （CIPK） 的复合物是钙感应的关键成分，协调对植物生长、发育以及对生物和非生物胁迫的反应至关重要的各种信号。然而，该模块对寒冷胁迫的响应机制及其在冬甜 （Chimonanthus praecox） 花发育中的作用仍不清楚。通过表达模式分析、钙离子 （Ca2+） 浓度测定、相关性分析和线性回归分析，我们发现冬甜花芽 （FBs） 中 [Ca2⁺] 以及 CpCBL8 和 CpCIPK9 表达水平在开花初期显著降低，当冷却需求达到 570 冷却单位 （CU） 时。值得注意的是，[Ca2⁺] 和 CpCBL8 表达之间存在显著的正相关。Ca2+ 增加了 FBs 中 CpCBL8 和 CpCIPK9 的表达，导致冬甜开花显著延迟。此外，使用异源转化研究 CpCBL8 的功能。过表达 CpCBL8 显著延迟了转基因拟南芥的开花时间，显著降低了耐寒性，并改变了与低温胁迫和花发育相关的内源基因的表达模式。此外，对寒冷诱导的休眠破裂和花芽增大的转录组分析显示，CpCBL8 和 CpCIPK9 受低温负调控，与花发育和寒冷胁迫相关的内源基因的表达模式与拟南芥相似。此外，蛋白质-蛋白质相互作用 （PPI） 分析显示 CpCBL8 和 CpCIPK9 在质膜和细胞核中相互作用。 基于这些发现，我们推测 CpCBL8-CpCIPK9 模块在调节冬甜菊对寒冷胁迫的响应和花的发育中起着至关重要的作用。本研究阐明了 Ca2+ 诱导的 CpCBL8-CpCIPK9 模块下调导致休眠破裂并增强耐寒性的分子机制。本研究为培育具有更高观赏价值和增强耐寒性的新品种提供了有价值的见解。