Alfalfa MsJAZ3A as a key factor in JA signal transduction play negative roles in NaHCO3 tolerance

Unraveling the physiological and molecular mechanisms underlying saline-alkali resistance in alfalfa is essential for vegetation restoration in saline-alkali soils. This study investigated phenotypic variations, photosynthetic function, and oxidative damage in Medicago sativa L. under different NaHCO₃ concentrations through physiological analyses combined with RNA-Seq. WGCNA was employed to identify key genes responsive to NaHCO₃ stress. Heterologous transformation of these critical genes in tobacco elucidated their functional roles in NaHCO₃ stress adaptation. The results demonstrated that increasing NaHCO₃ concentrations induced progressive leaf wilting, reduced chlorophyll content, and exacerbated photosynthetic inhibition and oxidative damage. RNA-Seq analysis revealed that differentially expressed genes (DEGs) in alfalfa leaves under NaHCO₃ stress were enriched in pathways associated with photosynthesis, antioxidant activity, and hormone signal transduction. Enhanced antioxidant enzyme activities and elevated levels of methyl jasmonate (MeJA) and its precursor 12-oxo-phytodienoic acid (OPDA) were identified as key adaptive strategies. WGCNA pinpointed MsJAZ3A, a gene in the jasmonate signal transduction pathway, as a potential regulator of NaHCO₃ stress responses. Yeast two-hybrid assays confirmed the interaction between MsJAZ3A and MsMYC2. Overexpression of MsJAZ3A suppressed the growth of yeast and transgenic tobacco under NaHCO₃ stress. Transgenic lines exhibited reduced proline and soluble sugar accumulation, alongside aggravated photosynthetic inhibition and oxidative damage. In conclusion, MsJAZ3A functions as a negative regulator in JA signal transduction by interacting with MsMYC2, thereby modulating alfalfa's adaptation to NaHCO₃ stress. These findings provide novel insights into alfalfa's adaptive mechanisms in saline-alkali environments and propose new targets for genetic improvement of saline-alkali resistance.