Characterization of the WRKY family transcription factors in Astragalus membranaceus and their expression under drought stress.

Drought and other abiotic stressors exert significant impacts on plant growth, development, and yield. WRKY transcription factors (TFs) are pivotal in regulating plant responses to such stresses. This study conducted a genome-wide identification of the Astragalus membranaceus (A. membranaceus) WRKY TF family, aiming at providing essential stress-tolerance genes for molecular breeding in A. membranaceus. Using bioinformatics approaches, including phylogenetic analysis, gene structure analysis, protein conserved motif prediction, and promoter cis-acting element examination, we identified 76 WRKY TF family members unevenly distributed across eight chromosomes. Phylogenetic analysis categorized the 76 AmWRKY proteins into three major classes: 14 in Class I, 52 in Class II, and 10 in Class III. Analysis of conserved structural domains revealed that similarities among domains of the same class AmWRKYs, with a predominance of members containing the conserved "WRKYGQK" heptapeptide domain and zinc finger structure, despite a few members had conserved domain variants. Gene structure analysis demonstrated that AmWRKY genes include 1-6 exons and 1-5 introns. GO and KEGG analyses revealed the differentially expressed WRKY genes are primarily transcription regulators involved in stress response pathways, with significant enrichment in DNA-binding activities, nuclear localization, and plant-pathogen/MAPK signaling. Transcriptome and qRT-PCR analyses indicated that several WRKY TFs, including AmWRKY8, were involved in the response to drought stress. Furthermore, it was confirmed that AmWRKY8 is localized exclusively in the nucleus and possesses transcriptional activation activity. Protein interaction analysis revealed that AmWRKY8 coordinates drought stress responses by interacting with key regulators of hormone signaling (AtWRKY70, ERF6), mitochondrial stress (NAC017), and developmental processes (HBI1). These findings are useful in further investigations into the regulatory role of this this TF in abiotic stress responses.