Multiprotein bridging factor 1 (MBF1) is a transcription factor family playing crucial roles in plant development and stress responses. In this study, we analyzed MBF1 genes in and under abiotic stresses, revealing evolutionary patterns and functional differences. Four genes were identified in and two in , with conserved MBF1 and HTH domains, similar exon/intron structures, and stress-related -elements in their promoters. Subcellular localization showed that MtMBF1a.1 is predominantly localized in the nucleus, while MtMBF1a.2, MtMBF1b, MtMBF1c, and MsMBF1a localize to both the nucleus and cytoplasm. In contrast, MsMBF1c is exclusively localized in the cytoplasm. An expression analysis revealed distinct stress responses: salt stress-induced and expression but repressed and . In contrast, PEG stress did not affect genes but repressed both genes. These findings provide insights into MBF1-mediated stress adaptation and inform strategies for the molecular breeding of stress-tolerant alfalfa.

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