StICE1 activates StCBFs expression to regulate cold stress response in potato (Solanum tuberosum L.) seedlings

Cold stress is a major environmental factor limiting potato yield. Understanding the molecular regulatory networks that govern plant responses to low temperature is therefore crucial for enhancing crop stress tolerance through genetic approaches. In this study, we investigated the functional role and molecular mechanisms of the StICE1 gene in potato under chilling stress, aiming to provide a theoretical basis for the molecular breeding of cold-tolerant varieties. We generated both overexpression (OE) and RNA interference (RNAi) transgenic lines. Cold tolerance among these genotypes was comprehensively evaluated through phenotypic characterization, electrolyte leakage assays, and antioxidant enzyme activity profiling. Genome-wide identification of StICE1 binding sites was performed using DNA affinity purification sequencing (DAP-seq), complemented by electrophoretic mobility shift assays (EMSA), yeast one-hybrid (Y1H) analysis, and dual-luciferase reporter assays to validate downstream target gene interactions. Under cold stress, transgenic OE lines exhibited enhanced tolerance, whereas RNAi lines displayed hypersensitive phenotypes. DAP-seq analysis confirmed that StICE1 directly binds to the promoters of StCBF2-a, StCBF3-a, and StCBF3-b thereby activating their transcription. CBF and COR genes were significantly upregulated in OE lines but downregulated in RNAi lines. Overall, StICE1 augments potato cold tolerance by directly activating CBF expression, initiating a downstream regulatory cascade involving cold-responsive genes (including CORs) that strengthen antioxidant defenses and osmotic homeostasis. EMSA further demonstrated that StCBF2-a binds to the promoter regions, of StCOR15A and StKIN1, confirming its regulatory interaction with these genes. This study elucidates a core regulatory mechanism of the potato StICE-StCBF-StCOR/StKIN1 pathway and provides a foundation for the development of frost-resistant cultivars through precision genome editing.