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

Abstract Background Alfalfa ( Medicago sativa L.) is globally important forage crop. The survival and consequently the productivity of this leguminous crop is determined by the capacity of the plant to tolerate cold stress, particularly at higher latitudes. However, the molecular mechanisms of how alfalfa defends against cold stress remain unclear. Results In this study, we cloned the candidate gene MsCBF4 from the cold-tolerant alfalfa genotype “Dongnong NO.1”. This gene belongs to the APETALA2/Ethylene response factor (AP2/ERF) family of transcription factors (TFs), which serve as transcriptional activators with a transcription activation domain located at the C-terminal. The protein encoded by MsCBF4 is localized in the cell nucleus. MsCBF4 is induced by cold and expressed in roots, stems, leaves, flowers, and pods, with high expression levels in root and leaf tissues. Arabidopsis thaliana lines that overexpress MsCBF4 demonstrate enhanced cold tolerance during both the seedling and mature stages, and the expression of the gene is thought to improve the physiological scavenging of reactive oxygen species (ROSs), inhibiting cell membrane damage. Furthermore, under freezing stress, the expression levels of key genes involved in abiotic stress resistance are upregulated in transgenic Arabidopsis thaliana . Herein, we describe MsCBF4 is positively regulated by MsERF6 . Conclusion These findings suggest that MsCBF4 can enhance cold tolerance in Arabidopsis thaliana by alleviating oxidative stress, protecting membrane integrity, and upregulating stress-responsive genes.