MsAREB1 enhances combined cold and saline–alkali stress tolerance by promoting ascorbic acid biosynthesis in alfalfa

Cold and saline–alkali stress are typical abiotic stresses on forage in middle and high latitudes, and they frequently occur simultaneously, decreasing the yield and quality of forage. Ascorbic acid plays an essential role in reactive oxygen species metabolism in response to abiotic stress. However, the molecular mechanisms of ascorbic acid biosynthesis induced by combined cold and saline–alkali stress remain unclear. This study identified an abscisic acid-responsive element-binding protein/ABRE-binding factors transcription factor (TF), MsAREB1, which was significantly induced by the combined stress and abscisic acid treatment. Under combined stress, MsAREB1 overexpression regulated ascorbic acid biosynthesis and played a role in the defence response to combined stress by positively regulating myo-inositol oxygenase 2 (MsMIOX2) expression. MsAREB1 and MsMIOX2 overexpression improved resistance to combined stress by increasing the ascorbic acid content. In addition, MsILR3, a bHLH TF, interacted with MsAREB1 to form a protein complex, thereby reducing the MsAREB1-induced transcriptional activation of MsMIOX2. This study demonstrates a model for the regulatory mechanism of MsAREB1-mediated regulation of MsMIOX2 expression and ascorbic acid biosynthesis to reduce oxidative damage by combined cold and saline–alkali stress. These results provide insights for improving the resistance of plants to combined cold and saline–alkali stress and lay the foundation for the genetic improvement of stress tolerance in alfalfa.