Overexpression of the Medicago falcata NAC transcription factor MfNAC3 enhances cold tolerance in Medicago truncatula

•We cloned and identified a gene, MfNAC3, which encodes a NAC-type transcriptional factor from M. falcate.•MfNAC3 was induced under salt, drought and cold stress.•Overexpression of MfNAC3 was shown to enhance cold tolerance in M. truncatula.•We provided evidence that MfNAC3 is a positive regulator of MtCBF4.•Overexpression of MtNAC3 did not enhance cold tolerance in M. truncatula. Cold stress is the main factor underlying the reduction in productivity of Medicago. Medicago falcata and Medicago truncatula are two subspecies of Medicago, whose geographic adaption is limited by water, salinity and temperature. However, the regulatory signaling pathway under cold stress in Medicago is unclear. In this study, we identified a gene, MfNAC3, induced under salt, drought and cold stress. By generating the overexpression lines of MfNAC3, we observed a typical cold-resistant phenotype under both cold-acclimated and non-acclimated conditions, featured by an increased survival rate and significantly higher expression levels of the cold-responsive genes MtCBFs and MtCASs. Further investigations revealed that this gene encodes a NAC-type transcriptional factor that is localized in the nucleus and exhibits transcription activity. By performing an electrophoretic mobility shift assay, we found that MfNAC3 could bind to the CATGTG and CACG motifs in the promoter region of MtCBF4. Taken together, our results demonstrate that MfNAC3 exerts a positive role in cold response and provide evidence that MfNAC3 is a positive regulator of MtCBF4.