Genome-wide identification and comprehensive characterization of the ABF/AREB transcription factor family in Medicago sativa L. and their responsiveness to abiotic stress

Abscisic acid (ABA) is a key phytohormone regulating plant responses to abiotic stress, with ABA-responsive element binding factor/ABA-responsive element binding protein (ABF/AREB) transcription factors serving as central mediators in ABA signaling pathways. Despite their importance, a systematic characterization of this gene family in the economically important forage crop Medicago sativa L. (alfalfa) has been lacking. In this study, we carried out genome-wide identification of ABF/AREB family members in alfalfa using integrated homology-based and domain-based search strategies. Subsequent analyses included phylogenetic classification, examination of the gene structures and conserved motifs, prediction of promoter cis-regulatory elements, and expression profiling using public RNA sequencing (RNA-seq) datasets, complemented by experimental validation under drought, salt, and cold stress conditions. In total, 46 MsABF genes were identified and phylogenetically grouped into seven distinct clades. Family expansion appears to have been driven primarily by segmental duplication events. Promoter analysis uncovered 826 cis-regulatory elements, exhibiting distinct distributions across phylogenetic groups. Expression analysis revealed both tissue-preferential patterns and significant induction of specific members, such as MsABF18, MsABF20, and MsABF25, under multiple abiotic stresses. This study firstly provides the genome-wide overview of the MsABF gene family in alfalfa, elucidating its genomic organization, evolutionary dynamics, and transcriptional responses to environmental challenges. Our findings establish a valuable genomic resource and identify candidate genes for future functional studies aimed at understanding and improving stress adaptation in alfalfa.