G proteins function directly in cold tolerance of plants. However, the framework of the 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 signal gene. Suppression of CsGPA1 decreased the expression of most of brassinolide-related genes (including CsCDL1) under cold stress. Principal component and linear regression analyses showed that expressions of CsGPA1 and brassinolide-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 Ca 2+ 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.

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