Genome-wide analysis of MsGDSL gene family reveals MsGDSL69 as a key regulator of salt stress responses in alfalfa.

GDSL esterase/lipase (GDSL) is a type of hydrolase with lipolysis activity. It can regulate the lipid metabolism process in plants and participate in the regulation of abiotic stress, among which salt stress is one of the main types of abiotic stress it regulates. Alfalfa (Medicago sativa) is an excellent forage crop characterized by high protein content, rich nutrition, but its growth was affected by soil salinization. In this study, a total of 148 MsGDSL genes were identified in alfalfa and subjected to systematic bioinformatic analysis. Phylogenetic analysis revealed that alfalfa MsGDSL and AtGDSL proteins were resolved into five distinct subgroups. When MsGDSL and GmGDSL proteins were analyzed together, all sequences clustered into six subgroups. Cis-element analysis revealed abundant abiotic stress-responsive and hormone-responsive elements in their promoter regions. Collinearity analysis indicated that segmental duplication events contributed predominantly to the expansion of the MsGDSL gene family. Transcriptome and RT-qPCR analyses demonstrated that the expression of MsGDSLs was responsive to salt stress. MsGDSL69 was strongly upregulated by salt stress, prompting an in-depth functional investigation of this gene. Under salt stress, Overexpression of MsGDSL69 in Arabidopsis thaliana (A. thaliana) increased germination rate, survival rate, and catalase (CAT) activity by 1.3-, 1.43-, and 1.41-fold over wild-type (WT), respectively, whereas the gdsl mutant showed reductions to 0.8-, 0.57-, and 0.72-fold of WT. Furthermore, MsGDSL69-silenced alfalfa exhibited markedly impaired growth and physiological activity compared with control plants. CAT and SOD activities were significantly decreased (by 55% and 12%, respectively), whereas the contents of H2O2 and malondialdehyde (MDA) were substantially increased (by 73% and 20%, respectively). Under salt stress conditions, the expression levels of CAT, SOD, and LOX were significantly altered in both MsGDSL69-overexpressing Arabidopsis lines and MsGDSL69-silenced alfalfa lines, suggesting that MsGDSL69 may participate in oxidative stress responses by activating the transcription of antioxidant-related genes. These results demonstrate that MsGDSL69 functions as a positive regulator of plant salt tolerance. This study systematically identified the MsGDSL gene family and analyzed their expression patterns under salt stress, with a particular focus on the role of MsGDSL69 in salt stress responses. This study provides theoretical insights for breeding salt-tolerant alfalfa cultivars.