Genome-wide identification and expression profiling of the ARF transcription factor gene family in Astragalus membranaceus under cold stress

Cold stress is a major abiotic factor that limits plant growth, development, and geographical distribution, especially impacting medicinal plants from cold or high-latitude regions. Auxin response factors (ARFs) are key transcriptional regulators in the auxin signaling pathway and play essential roles in plant development and adaptation to environmental stresses. However, in medicinal plants such as Astragalus membranaceus , a cold-adapted species valued for its bioactive compounds. Unlike model plants where ARF functions are well-characterized, the extent to which ARFs mediate cold acclimation in medicinal species, and whether this regulation correlates with metabolic adjustments affecting quality is unknown. Furthermore, the balance between growth maintenance and stress defense under low temperatures is likely orchestrated by auxin signaling, yet the specific ARF members involved have not been identified in this system. A genome-wide analysis identified 40 AmARF genes in Astragalus membranaceus . These genes were characterized by physicochemical properties, chromosomal localization, phylogeny, gene structure, conserved motifs, cis-regulatory elements, and protein structure. Phylogenetic and synteny analyses identified tandem and segmental duplications as the main drivers of family expansion. Ka/Ks ratios for duplicated gene pairs ranged from 0.149 to 0.293, all below 1. Transcriptome analysis under cold stress revealed 22 differentially expressed AmARFs, with distinct expression patterns among members, and these results were validated by qRT-PCR for six selected genes. Promoter analysis detected low-temperature-responsive (LTR) and hormone-related cis-elements in most cold-responsive AmARFs . Metabolomic correlation analysis further showed that 52 significantly upregulated metabolites, predominantly lipids (29) and soluble sugars were strongly correlated with the expression of core AmARFs (|r| > 0.8). In this study, we performed the first genome-wide identification and cold-stress expression analysis of the ARF gene family in Astragalus membranaceus . The unique contributions include: (i) the first genome-wide identification of ARF genes in this cold-region medicinal plant; (ii) elucidation of expansion patterns driven by tandem/segmental duplications under strong purifying selection; and (iii) construction of a transcriptome-metabolome correlation network that generates testable hypotheses for cold adaptation mechanisms. The findings enhance our understanding of the functional diversity of ARF transcription factors in medicinal plants and offer valuable candidate genes and a theoretical foundation for future research into cold tolerance mechanisms.