Genome-wide identification of the DREB2 gene family in alfalfa and functional analysis of the MsDREB2-04 gene under abiotic stress

The dehydration-responsive element binding (DREB) subfamily, belonging to the APETALA2/ethylene-responsive element binding (AP2/ERF) superfamily, is crucial for plant growth and development. Despite the identification of DREB genes in numerous plant species, research on the DREB2 family in alfalfa remains incomplete. In this study, we used the alfalfa cultivar "Zhongmu No. 4" to identify and characterize 11 MsDREB2 genes through whole-genome analysis. These genes were unevenly distributed on chromosomes 1 and 2 and on unanchored scaffold chromosomes. A phylogenetic analysis involving Arabidopsis thaliana, Medicago truncatula, and Medicago sativa showed that MsDREB2 members were divided into four clades. Synteny analysis revealed that 45.5% (5 out of 11) of MsDREB2 genes formed segmental duplications, with no tandem duplications observed. The Ka/Ks analysis indicated that some gene pairs underwent purifying and positive selection. Cis-acting elements involved in plant growth, hormone responses, and stress responses were found in the promoters of the MsDREB2 genes. MsDREB2-04 exhibited variable responses to salt and drought stress, determined by qRT-PCR. Under drought stress, the Arabidopsis thaliana dreb2d mutant showed markedly reduced growth. In contrast, MsDREB2-04 overexpression in alfalfa enhanced drought tolerance. Conversely, VIGS silencing of MsDREB2-04 reduced drought tolerance. This study provides a comprehensive identification of the MsDREB2 gene family in alfalfa and confirms the role of MsDREB2-04 in drought stress responses. These findings provide a foundation for functional studies of MsDREB2 genes.