Natural allelic variation in the CBF2 transcription factor is a pivotal factor controlling cold resistance in potato.

Frost stress poses a serious threat to the potato industry. C-repeat binding factors (CBFs) are key transcription factors involved in plant cold responses and the adaptive evolution of land plants. However, their function and underlying mechanisms in potato remain poorly understood. This study analyzed homologous CBF2 genes from 46 potato genotypes and revealed significant structural variations, including a critical site (site A) that is closely associated with cold tolerance. There are at least 2 site A types, including the cold-tolerant Solanum commersonii type and the cold-sensitive Solanum tuberosum type. Overexpression of ScCBF2 significantly enhanced potato cold tolerance, whereas StCBF2 overexpression had a limited effect. We demonstrated that both ScCBF2 and StCBF2 improve cold resistance by regulating glutathione S-transferase tau (GSTU)- and ZAT10-mediated reactive oxygen species scavenging systems. Notably, ScCBF2 uniquely upregulated Galactinol synthase 3 (GolS3), promoting raffinose biosynthesis. Compared with StCBF2, ScCBF2 exhibited a stronger binding affinity to the GolS3 promoter, resulting in higher transcriptional activation. Overexpression of ScGolS3 increased leaf raffinose content and cold tolerance. Furthermore, we confirmed the critical role of site A in the ScCBF2-GolS3 regulatory pathway. In summary, this study highlights the functional divergence caused by structural variations in CBF2, with differential regulation of GolS3 contributing to cold tolerance. Our work provides insights into the molecular mechanisms underlying cold tolerance in potato and offers potential targets for improving frost resistance in this vital crop.