Genome-wide Identification and Expression Pattern Analysis of the Medicago sativa 4-Coumarate: CoA ligase (4CL) Gene Family

4-Coumarate: CoA ligase (4CL) is a pivotal enzyme in the phenylpropanoid metabolic pathway and plays a crucial role in plant growth, development, and stress responses. In this study, a genome-wide analysis was conducted to identify a total of 43 Ms4CL genes ( Ms4CLs ) in 'Xinjiangdaye' ( Medicago sativa ). The fundamental structure, phylogenetic relationships, cis -acting element analysis, and expression patterns of these genes were systematically analysed. The results indicate that Ms4CL genes can be classified into four subfamilies, and subcellular localization predictions suggest that most genes are localized to the plasma membrane. The number of exons ranges from four to thirteen, consistent with the characteristics of most 4CL gene families. The promoter regions are rich in stress response elements associated with MeJA, ABA, and drought, implying that the expression of these genes may be regulated by stress-induced hormonal signals. Collinearity analyses revealed that segmental duplications are the primary mechanisms driving the expansion and functional diversification of this family. Analysis of tissue-specific expression revealed that the four genes Ms4CL8 , Ms4CL22 , Ms4CL37 , and Ms4CL42 exhibit significant tissue-specific expression patterns. An analysis of responses to abiotic stresses indicates that Ms4CL5 and Ms4CL13 are stress-induced genes in response to salt and drought stresses, whereas Ms4CL3 is a stress-repressed gene in response to salt and drought stresses. These three genes exhibit distinct functional differentiation in stress responses. Cis -acting element analysis further revealed promoter enrichment in defense/stress-responsive and phytohormone-related elements, consistent with their roles in environmental adaptation. This study provides a theoretical foundation for elucidating the mechanisms of salt and drought tolerance in alfalfa and supports its genetic improvement.