CPK 27 enhances cold tolerance by promoting flavonoid biosynthesis through phosphorylating HY 5 in tomato

Cold stress is a major environmental challenge affecting the production of crops. Calcium-dependent protein kinases (CDPKs/CPKs) are crucial regulators relaying calcium (Ca²⁺) signals into cellular stress responses. However, the specific mechanisms of CPKs in regulating cold stress signaling are not well understood. In this study, through genetic, physiological and molecular biology assays, we characterized the function of CPK27 in enhancing tomato cold tolerance. We found that CPK27 stimulates flavonoid biosynthesis in a Ca²⁺-dependent manner, which in turn boosts the plant's tolerance. Tomato plants lacking CPK27 (cpk27) showed decreased flavonoid levels under cold stress, accompanied by the increased sensitivity to cold. Activated by cold stress, CPK27 accumulates within the nucleus, where it physically interacts and phosphorylates ELONGATED HYPOCOTYL 5 (HY5) protein at serine23 (S23) and S57 residues, contributing to the cold-induced accumulation of HY5 protein. HY5 directly binds to the promoter regions and stimulates the transcription of flavonoid biosynthesis genes. Further genetic analysis showed that CPK27 acts upstream of HY5, and the flavonoid biosynthesis pathway activated by CPK27 is HY5-dependent. Our study elucidates the regulatory mechanism whereby the CPK27-HY5 molecule integrates cold-triggered Ca²⁺ signals with flavonoid biosynthesis pathways to confer cold stress tolerance, thereby uncovering the key strategy for cold signal transduction.