Comprehensive transcriptome and metabolome analysis revealing the molecular and metabolic basis of methyl jasmonate response in alfalfa

Methyl jasmonate (MeJA), as a plant signaling molecule and growth regulator, can regulate plant physiological indicators and synthesis of metabolites. Exogenous MeJA application can alter the resistance to abiotic and biotic stress in alfalfa ( Medicago sativa ). However, there are still challenges and difficulties in practical applications. This study identified differentially abundant metabolites (DAMs) and related pathways in alfalfa by treating the cultivated variety “Zhongtian No. 3” with different concentrations of MeJA. Based on the measurement of morphological and physiological indicators, combined with metabolomic and transcriptomic analyses, key gene functions regulating these metabolic changes were further screened and analyzed. Physiological results showed that with increasing MeJA concentrations, alfalfa leaves became more yellowed, and the activities of POD and PAL, as well as the contents of soluble sugars, starch, total phenols, and flavonoids, significantly increased. SOD activity only changed significantly at high concentrations, while OPC content slightly increased at low concentrations and significantly decreased at high concentrations. Based on leaf sectioning and staining observations, the thickness of leaves, palisade tissue, and spongy tissue significantly decreased when treated with MeJA at concentrations of 0.15 mmol/L and 0.3 mmol/L, while there were no significant differences at a concentration of 0.075 mmol/L. Metabolomic analysis revealed that a total of 3,166 DAMs were detected in alfalfa MeJA application, primarily including flavonoids, terpenoids, amino acids and their derivatives. Transcriptomic analysis reveals that seven phenylpropanoid-related genes ( C4H , F3H , CYP93B16 , CYP75B1 , CYP81E1 / E7 , IFS , CYP81E9 ) were transcriptionally activated, and hormone-related genes including GH3 , JAZ , COI-1 , CAM , GS2 , AHA1 / 2 , EBF1 / 2 and PR1 were consistently induced, indicating engagement of the jasmonate signaling cascade. This study clarifies how MeJA modulates key secondary metabolites and the underlying molecular mechanism in alfalfa. Collectively, these outcomes lay a theoretical basis for utilizing MeJA induction to increase functional metabolite contents, optimize forage nutritive value and breed stress-resistant alfalfa materials.