The alfalfa AP2/ERF transcription factor MsCBF4 enhances frost tolerance in Arabidopsis thaliana

Alfalfa (Medicago sativa L.) is globally important forage crop whose survival and productivity at higher latitudes depend on cold-stress tolerance. However, the molecular mechanisms of how alfalfa defends against freezing stress remain unclear. In this study, the candidate gene MsCBF4 was cloned from the cold-tolerant alfalfa genotype “Dongnong NO.1”. MsCBF4 belongs to the APETALA2/Ethylene Response Factor (AP2/ERF) transcription factor family and functions as a transcriptional activator via a C-terminal activation domain. The encoded protein localized to the nucleus. MsCBF4 is induced by cold and freezing conditions and is expressed in roots, stems, leaves, flowers, and pods, with the highest expression levels in roots and leaves. Overexpression of MsCBF4 in Arabidopsis thaliana seedlings significantly enhanced root length, lateral root number, and fresh weight under cold stress. Lines overexpressing MsCBF4 demonstrated enhanced frost tolerance at both the seedling and mature stages, likely through improved physiological scavenging of reactive oxygen species (ROSs), thereby limiting membrane damage. Furthermore, under freezing stress, the expression levels of key genes involved in abiotic stress resistance were upregulated in transgenic Arabidopsis. The MsCBF4 promoter exhibits transcriptional activity and can be directly activated by cold responses. Yeast one-hybrid (Y1H) and dual-luciferase reporter (Dual-LUC) assays showed that MsERF6 specifically binds to the MsCBF4 promoter and positively regulates its expression. MsCBF4 enhances frost tolerance in Arabidopsis by alleviating oxidative stress, protecting membrane integrity, and upregulating stress-responsive genes.