G protein functions directly in cold tolerance of plants. However, the framework of Gα subunit in regulating cold tolerance remains to be explored. Here, we used protein interaction techniques to elucidate cold-related pathways regulated by CsGPA1. Suppression of CsGPA1 decreased the cold tolerance of cucumber. Further protein interaction experiments showed that CsGPA1 interacted with Csa_4G663630.1 located in the cell membrane and nucleus and with CsCOR413PM2 located in the cell membrane. Csa_4G663630.1 was named CsCDL1 due to its 71% protein sequence similarity to AtCDL1, a positive brassinolide (BR) signal gene. Suppression of CsGPA1 decreased most of the expression of BR-related genes (including CsCDL1) under cold stress. Principal component and linear regression analysis showed that expression of CsGPA1 and BR-related genes were positively correlated. Suppression of CsCOR413PM2 also decreased cold tolerance of cucumber. The expression and protein content of CsCOR413PM2 and CsGPA1 in CsGPA1-RNAi and CsCOR413PM2-RNAi lines were determined under cold tolerance. Only CsGPA1 silencing affected the expression and protein content of CsCOR413PM2 during cold stress. Moreover, suppression of CsGPA1 or CsCOR413PM2 decreased Ca2+ influx at low temperature and then decreased the expression of CsICE-CsCBF. These results indicated that the CsGPA1-CsCOR413PM2-Ca2+ axis regulated the expression of CsICE-CsCBF during cold stress. In conclusion, Our results provide the first framework of CsGPA1 in regulating cold tolerance of cucumber, laying the foundation for further mechanistic studies of cold tolerance for Gα in cucumber.

中文翻译：

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CsGPA1调控黄瓜耐寒性的机制

G蛋白直接在植物的耐寒性中起作用。然而，Gα亚基调节耐寒性的框架仍有待探索。在这里，我们使用蛋白质相互作用技术来阐明由 CsGPA1 调节的与寒冷相关的途径。CsGPA1的抑制降低了黄瓜的耐寒性。进一步的蛋白质相互作用实验表明，CsGPA1 与位于细胞膜和细胞核中的 Csa_4G663630.1 以及位于细胞膜中的 CsCOR413PM2 相互作用。Csa_4G663630.1 被命名为 CsCDL1，因为它与 AtCDL1（一种阳性油菜素内酯 (BR) 信号基因）具有 71% 的蛋白质序列相似性。CsGPA1的抑制降低了冷应激下大部分BR相关基因（包括CsCDL1）的表达。主成分和线性回归分析表明CsGPA1与BR相关基因的表达呈正相关。CsCOR413PM2的抑制也降低了黄瓜的耐寒性。在耐寒条件下测定了 CsGPA1-RNAi 和 CsCOR413PM2-RNAi 系中 CsCOR413PM2 和 CsGPA1 的表达和蛋白质含量。只有 CsGPA1 沉默影响冷应激期间 CsCOR413PM2 的表达和蛋白质含量。此外，CsGPA1 或 CsCOR413PM2 的抑制降低了低温下 Ca2+ 的流入，然后降低了 CsICE-CsCBF 的表达。这些结果表明，CsGPA1-CsCOR413PM2-Ca2+ 轴在冷应激期间调节 CsICE-CsCBF 的表达。总之，我们的研究结果提供了第一个 CsGPA1 调控黄瓜耐寒性的框架，