The APT1-NACsa3-GGP1 Module Enhances Salt Tolerance and Regulates Ascorbic Acid Biosynthesis in Medicago

The unclear salt tolerance mechanism of alfalfa (Medicago sativa) is a major constraint to its molecular design breeding. Here, we identified a novel ATAF subfamily transcription factor, MtNACsa3, in the closely related diploid Medicago truncatula, and elucidated its function in salt tolerance. Phenotypic analysis of the MtNACsa3 mutants and overexpression lines showed its positive role in salt tolerance. Under salt or oxidative stress, MtNACsa3 relocates from the plasma membrane to the nucleus mediated by the de-S-acylation of Acyl-Protein Thioesterase 1 (MtAPT1). MtNACsa3 up-regulates the expression of GDP-L-galactose phosphorylase 1 (MtGGP1), enhancing ascorbic acid (AsA) synthesis to scavenge H₂O₂. Furthermore, we created MtNACsa3 exogenous transgenic alfalfa, which has higher AsA content and higher biomass than the recipient Zhongmu No.1 under salt stress. The APT1-NACsa3-GGP1 module not only enriches our understanding of the mechanism of salt tolerance, but also provides valuable genetic resources for salt-tolerant and AsA-rich alfalfa breeding.