Identification and preliminary functional characterization of MsPAL2, a key salt-tolerance gene during seed germination in Medicago sativa

Alfalfa ( Medicago sativa ) is an important legume forage crop. Salt stress severely impairs seed germination, compromising field establishment and subsequent seedling growth. However, the key genes underlying alfalfa seed responses to salt stress during germination remain largely unidentified, hindering targeted genetic improvement of salt tolerance within the genus Medicago . In this study, transcriptomic sequencing was performed on germinated and ungerminated seeds of alfalfa treated with 150 mmol/L NaCl, using the ‘Zhongmu No. 4’ reference genome. Through integrated KEGG pathway enrichment, gene expression fold-change analysis, and Gene Set Enrichment Analysis (GSEA), we identified the gene MsPAL2 (Msa1079410), which encodes the key rate-limiting enzyme in phenylpropanoid metabolism, as a critical salt-tolerance gene. Functional characterization via virus-induced gene silencing (VIGS) revealed that silencing MsPAL2 did not affect seed germination under normal conditions but significantly reduced the germination rate under salt stress. These findings suggest that MsPAL2 acts as a positive regulator of alfalfa seed germination under salt stress. Promoter analysis and AlphaFold3 prediction collectively revealed MYB-binding cis -elements and identified MYB transcription factors as candidate upstream regulators of MsPAL2 , laying the groundwork for elucidating its regulatory network. Phenylpropanoid metabolism constitutes a key mechanism underlying salt tolerance in alfalfa. Through transcriptome analysis, we identified MsPAL2 , a phenylpropanoid pathway gene, as a critical regulator of alfalfa seed germination under salt stress, providing valuable genetic resources for molecular breeding of salt-tolerant alfalfa.