CBL2/4-CIPK31-mediated phosphorylation of raffinose synthase enhances cold tolerance in Campeiostachys nutans and rice

Cold stress constrains global crop productivity. Although raffinose accumulation confers cold tolerance, the regulatory mechanisms governing its biosynthesis remain largely unresolved. Here, we identify a cold-induced raffinose synthase gene (CnRS1) from Campeiostachys nutans—a cold-tolerant perennial grass native to the Qinghai–Tibet Plateau and closely related to cereal crops. Overexpression of CnRS1 significantly enhanced cold tolerance in both C. nutans and transgenic rice (Oryza sativa L.). Using yeast two-hybrid screening, biochemical interaction, and phosphorylation assays, we demonstrated that CnRS1 interacts with and is phosphorylated by the CBL-interacting protein kinase 31 (CnCIPK31) at Ser606, Thr106, and Thr764, thereby enhancing its protein stability. Furthermore, CnCIPK31 interacted with two calcineurin B-like proteins (CnCBL2 and CnCBL4) to form a Ca²⁺-dependent CBL–CIPK complex, which promotes CnRS1 phosphorylation. CnCIPK31 overexpression increased raffinose accumulation and improved cold tolerance, whereas its silencing caused hypersensitivity, which was rescued by exogenous raffinose. Genetic analysis further established that CnCIPK31 acts upstream of CnRS1 to improve plant cold tolerance. Collectively, our study reveals a Ca2+-responsive CnCBL2/4–CnCIPK31–CnRS1 signaling module that integrates calcium signaling with raffinose biosynthesis to enhance cold tolerance, providing insights into alpine plant adaptation and a strategy for improving crop cold resilience.