Overexpression of a Malus baccata NAC Transcription Factor Gene MbNAC40 Increases Cold Tolerance in Arabidopsis

As a primary apple rootstock extensively cultivated across Northeast China, Malus baccata (L.) Borkh. constantly suffers from low temperature, drought and salt damage throughout its growth cycle. NAC transcription factors take critical parts in coordinating plant adaptation to various adverse environments, yet relevant functional research on this woody rootstock species remains limited. In this work, a NAC family gene designated MbNAC40 was cloned from M. baccata, and its biological function was explored via heterologous transformation in Arabidopsis thaliana. Sequence analysis revealed that the open reading frame of MbNAC40 spans 1467 bp and encodes a 488-amino-acid protein targeted to the cell nucleus. After 24 h cold acclimation at 4 °C, plants were subjected to −4 °C freezing stress for 12 h, then recovered at 25 °C for 3 d. Transgenic Arabidopsis lines overexpressing MbNAC40 exhibited much stronger cold tolerance relative to wild-type plants. Transgenic materials retained more chlorophyll and synthesized more osmoprotective proline, accompanied by enhanced activity of three antioxidant enzymes (SOD, POD and CAT). Meanwhile, multiple stress-marker genes (AtCAT1, AtCSD1, AtAPX6, and AtP5CS1) associated with ROS and osmotic adjustment were significantly activated in overexpression lines. Taken together, these results confirm that MbNAC40 acts as a positive modulator of cold resistance in Arabidopsis, which provides a valuable gene resource for molecular breeding of cold-resistant M. baccata rootstock varieties.