Characterization of the BPC genes in alfalfa and functional verification of MsBPC10 in salt tolerance

Abstract Background Soil salinization severely restricts the growth and forage yield of Medicago sativa (alfalfa), a globally critical leguminous forage crop, yet the regulatory mechanisms governing its salt tolerance remain incompletely understood-especially the role of the BASIC PENTACYSTEINE (BPC) transcription factor family, which has been implicated in plant stress responses but never systematically investigated in alfalfa’s salt adaptation processes. Results This study presents the genome-wide identification and analysis of the BPC transcription factor family in tetraploid alfalfa (Medicago sativa), revealing 18 MsBPC genes randomly distributed across 14 chromosomes. The encoded proteins are predominantly alkaline and phylogenetically classified into three distinct subgroups. Promoter cis-acting element analysis indicated the presence of multiple elements associated with hormone response, growth and development, and stress adaptation. Tissue-specific expression profiling demonstrated that 7 MsBPC genes were ubiquitously expressed in all six examined tissues, 4 were expressed in only a single tissue, and the remaining genes showed expression in 3-5 tissues, suggesting their potential involvement in regulating various growth and developmental processes in alfalfa. Transcriptome analysis under salt, drought, and cold stress conditions revealed that 12, 11, and 12 MsBPC genes were responsive, respectively. RT-qPCR validation confirmed the differential expression of selected MsBPC genes under salt and drought treatments, reinforcing their roles in abiotic stress responses. Subcellular localization analysis showed that MsBPC5 and MsBPC10 are nuclear-localized, consistent with in silico predictions. Heterologous expression of MsBPC10 in yeast enhanced salt tolerance, supporting its functional role in stress adaptation. Conclusions Overall, this comprehensive study provides foundational insights into the MsBPC gene family and identifies promising candidate genes for enhancing stress resilience in alfalfa breeding programs.