Genome-wide identification of the WRKY transcription factors family and regulation of metabolites under cold stress in Astragalus membranaceus

Abstract Background WRKY transcription factors (TFs) are important transcriptional regulators in plants, with their members widely involved in plant growth and development as well as responses to abiotic stresses. However, researches on WRKY genes in the medicinal plant A. membranaceus are scarce. Specifically, the roles of AmWRKYs in cold stress adaptation and their regulatory effects on flavonoid biosynthesis, which determines both medicinal quality and stress resistance, remain largely unexplored. Given its high economic value and extreme sensitivity to cold in its main cultivation regions, identifying key regulators of its cold tolerance is crucial for genetic improvement. Result In this study, 94 AmWRKY were identified based on genome analysis, distributed across 8 chromosomes. AmWRKYs are structurally conserved, all carrying the core conserved domain "WRKYGQK" and classified into 6 subgroups. Cis-acting elements responsive to plant growth and development, abiotic stress, and hormone responses were identified in the promoter regions. Additionally, the transcriptome and metabolome data under cold stress were analyzed, and a co-expression and metabolite association network of AmWRKY genes was constructed. Sixteen AmWRKY transcription factors showed dynamic expression under cold stress, among which AmWRKY22/24/44/65 were continuously upregulated, indicating their core roles in cold adaptation. Co-expression network analysis revealed the synergistic effects of AmWRKY with AP2/ERF, MYB, and NAC transcription factors, forming a regulatory module integrating hormone signaling, antioxidant pathways, and circadian rhythm regulation. Metabolomics analysis indicated that AmWRKY24/44 expression was positively correlated with the upregulation of key flavonoid biosynthesis genes ( AmCHS , AmFLS ) and the accumulation of nine cold-responsive flavonoids. These findings suggest a new regulatory pathway of AmWRKY24/44 → flavonoid biosynthesis → cold resistance, linking secondary metabolism with environmental adaptation. Conclusion This study reveals a novel regulatory pathway—“ AmWRKY24/44 ” → flavonoid biosynthesis → cold resistance—in A. membranaceus , providing deeper mechanistic insights into how WRKY transcription factors modulate secondary metabolism under cold stress. These findings offer a valuable theoretical foundation for genetic improvement of cold tolerance in this medicinally important species.