Abstract
BACKGROUND: The Phytochrome-interacting factors (PIFs) are members of the basic helix-loop-helix (bHLH) transcription factor family and play essential roles in plant growth, development, and stress response. While the PIF gene family has been extensively studied in various plant species, there is limited information available regarding their presence in high-quality perennial legume alfalfa (Medicago sativa). RESULTS: In this study, we identified 29, 9, and 27 genes in Xinjiang Daye, Zhongmu No. 1, and Zhongmu No. 4 alfalfa varieties, respectively. These genes are unevenly distributed on 5 chromosomes of Xinjiang Daye, 3 chromosomes of Zhongmu No. 1, and 7 chromosomes of Zhongmu No. 4, with 2 genes not located on any chromosome. Phylogenetic analysis revealed that the MsPIFs genes clustered into five different branches (PIF I to PIF V), with members within the same subfamily sharing conserved motifs and displaying similar exon-intron distribution patterns. Gene duplication analysis indicated that segmental duplications facilitated the expansion and evolution of the alfalfa PIF gene family. Functional cis-element analysis of the MsPIFs genes promoter regions identified elements related to light, hormones, development, and response to abiotic stresses. The expression levels of the majority of MsPIF family genes significantly varied under drought, high temperature, and combined stress conditions, notably showing pronounced responses in genes MsPIF4, MsPIF6, and MsPIF9 to these stressors. CONCLUSION: We systematically identified and classified the members of the MsPIFs gene family, analyzing their chromosomal locations and genetic structure. These MsPIFs are crucial in alfalfa's responses to drought and high-temperature stress.