Overexpression of MtLOX24 enhances alfalfa resistance to Spodoptera litura via phenylpropanoids and jasmonic acid pathways

Alfalfa ( Medicago sativa ) is a globally important forage crop whose yield, quality, and palatability are compromised by insect feeding. Spodoptera litura is a major pest affecting alfalfa. Lipoxygenases (LOXs) are known to participate in jasmonic acid (JA)-mediated signaling and play key roles in plant defense against insects, pathogens, and mechanical damage. In order to identify the role of alfalfa MtLOX24 in resisting S. litura , resistance evaluations were conducted on wild-type (WT) and overexpression lines (L3). Combined with transcriptome and metabolome analysis, pathways of flavonoid biosynthesis, isoflavonoid biosynthesis, plant hormone signal transduction, starch and sucrose metabolism related pathways were systematic analysis before and after S. litura treatment. Results showed that in leaf and branch-feeding assays, overexpression of MtLOX24 in alfalfa significantly enhanced resistance to S. litura . Integrated transcriptomic and metabolomic analyses identified 6,890 differentially expressed genes (DEGs) and 342 differentially accumulated metabolites (DAMs) between control (WT-c and L3-c) and S. litura -fed groups (WT-s and L3-s). Transcriptome analysis showed that DEGs in MtLOX24- overexpressing plants were enriched in the phenylpropanoid biosynthesis, which includes flavonoid, lignan, anthocyanin, and lignin synthesis. Genes related to flavonoid biosynthesis (e.g., F3H , DFR , ANS , FLS ), isoflavonoid biosynthesis (e.g., IFS , PTS ), and JA signaling (e.g., COI1 , JAZ , MYC2 ) were significantly regulated in response to insect feeding. MtLOX24 was correlated with the JA signaling pathway with S . litura , and JA signaling was activated by treatment with S . litura . Metabolomic analysis revealed alterations in alkanes, phenols, alkaloids, terpenes, monosaccharides, and polysaccharides in the overexpression lines with S . litura . These findings suggest that MtLOX24 enhances resistance to S. litura by regulating both gene expression and metabolite accumulation related to JA and phenylpropanoid pathways. This study highlights MtLOX24 as a promising target for breeding S. litura- resistant alfalfa. • Overexpression of MtLOX24 enhances alfalfa resistance to Spodoptera litura. • The key categories of DEGs and DAMs are phenylpropanoids and jasmonic acid pathways. • DEGs are identified, including F3H, DFR, ANS, COI1, JAZ, MYC2, and others. • The key metabolites such as alkanes, phenols, and alkaloids are screened out.