Background: The TGACG-BINDING FACTORS (TGA) gene family, a key subgroup of bZIP transcription factors, mediates plant stress responses and developmental processes by binding to the as-1 cis-element in target gene promoters to regulate transcriptional activation or repression. Despite its functional significance, systematic characterization of TGA genes in cotton (Gossypium spp.) remains insufficient. Methods: In this study, we performed a comprehensive genome-wide identification and phylogenetic analysis of TGA members across 10 Gossypium species and verified the functions of candidate genes using VIGS technology. Results: A total of 74 TGA homologous genes with conserved DOG1 and bZIP domains were identified. Evolutionary analysis revealed that the cotton TGA gene family can be classified into five distinct branches, suggesting functional diversification. Functional prediction analyses indicated these genes in cotton growth regulation and stress adaptation, potentially through hormone-mediated signaling pathways. Expression profiling demonstrated both tissue-specific expression patterns and salt-stress responsiveness in Gossypium hirsutum TGA genes, and GhTGA2 exhibited the most significant up-regulated expression under salt stress. Virus-induced gene silencing (VIGS)-mediated GhTGA2 silencing significantly reduced the salt tolerance in cotton. Conclusions: The TGA gene family is involved in regulating cotton growth, development, and stress responses, and plays a critical role in mediating salt stress tolerance in cotton. Our results provide mechanistic insights into cotton stress adaptation and establish a valuable genetic resource for developing elite salt-tolerant cotton cultivars, with direct implications for sustainable cotton production.

The SNAC (Stress-responsive NAC) subfamily, a key branch of the conserved NAC transcription factor family, plays a central role in regulating plant stress response. However, systematic characterization of the SNAC family in cotton (Gossypium spp.) remains unclear. Employing a genome-wide screening approach, this study characterized 75 distinct SNAC transcription factor genes across ten Gossypium species, with tetraploid cottons harboring twice as many as their diploid progenitors. Phylogenetic analysis categorized the genes into three subgroups, with members of the same subgroup exhibiting conserved motif compositions and gene structures. Chromosomal localization revealed a conserved distribution pattern of SNAC genes between the Dt and At subgenomes in tetraploid cotton. Genomic collinearity analysis suggested that the primary driver of SNAC family expansion was segmental duplication. Promoter analysis predicted 2974 cis-regulatory elements, including cold- and hormone-responsive motifs, indicating their potential involvement in stress regulation. These GhSNAC genes indicated significant induced expressions under stress conditions, and GhSNAC3D exhibited the most significant up-regulated expression under low temperature stress. Genetic function studies displayed that VIGS-mediated GhSNAC3D-silencing significantly reduced the cold tolerance in cotton. This study systematically analyzed the genomic characteristics of the cotton SNAC family and functionally validated the molecular mechanism of GhSNAC3D-mediated cryogenic response, which lays a foundation for subsequent research on cold resistance in cotton and stress-resistant breeding.

Cotton’s susceptibility to low temperatures makes it a crucial raw resource for the world’s textile industry, yet its cultivation in temperate regions is severely limited. Although plant growth and stress responses depend on receptor-like kinases (RLKs), the functions of the MEDOS (MDS) gene family, which includes genes that encode RLK, are still poorly understood in cotton. In this study, we conducted a genome-wide analysis to systematically investigate the distribution of MDS gene family members in four cotton species. Phylogenetic analysis identified five evolutionary clades of the MDS gene family in cotton. The role of promoter cis-acting elements in hormone signaling and abiotic stress responses was suggested by analysis. Collinearity analysis demonstrated that segmental duplication was the primary driver of family expansion. Gene expression profiling showed that GhMDS11 was significantly upregulated under cold stress. Functional validation through silencing GhMDS11 compromised cold tolerance, confirming its role in stress adaptation. Comparative transcriptome study of silenced plants demonstrated substantial enrichment in pathways associated with hormone signal transduction and fatty acid breakdown. It is speculated that the chain of “hormone synthesis → signal transduction → secondary metabolism” completely presents the transcriptional regulation network and functional response of plants after receptor kinase VIGS. Silencing the GhMDS11 gene in cotton initiates regulatory effects through hormone synthesis, which is amplified via a signal transduction cascade, ultimately affecting secondary metabolism. This comprehensive pathway clearly demonstrates the downstream transcriptional reprogramming and functional changes. This work thoroughly examined the evolutionary traits of the MDS family across four cotton species and clarified the functional and molecular processes of GhMDS11 in improving low-temperature tolerance, laying a solid foundation for further clarifying multidimensional regulatory networks and breeding cold-resistant cotton materials. Simultaneously, our findings pave the way for future research to develop molecular markers, which could potentially shorten the breeding cycle and facilitate the targeted enhancement of cold tolerance in cotton.

Verticillium wilt (VW), caused by the soil-borne fungus Verticillium dahliae, is a major disease that markedly compromises both the yield and fiber quality of cotton. In this study, we explored the function and underlying mechanism of the cotton expansin gene GhEXLB2 in response to VW infection. Expression profiling revealed that members of the GhEXL family exhibit distinct patterns across tissues and under various biotic and abiotic stresses. Notably, GhEXLB2, which encodes an extracellular protein, showed the strongest induction following V. dahliae challenge. Ectopic expression of GhEXLB2 in Arabidopsis thaliana promoted root elongation and root hair formation, and was associated with improved resistance to the pathogen. In contrast, silencing GhEXLB2 in cotton via virus-induced gene silencing (VIGS) led to pronounced vascular browning, increased pathogen recovery, and a lower level of disease resistance. In addition, RNA-seq profiling of GhEXLB2-silenced (VIGS) cotton plants revealed that most differentially expressed genes were enriched in pathways related to phytohormone signaling and plant–pathogen interactions, with salicylic acid (SA) signaling and WRKY transcription factors emerging as central regulatory components. Analysis of the GhEXLB2 promoter further identified multiple cis-acting elements associated with stress and hormone responsiveness. When integrated with protein–protein interaction (PPI) prediction data, these results suggest that GhEXLB2 may be modulated by a network of transcription factors and signaling pathways. Collectively, the evidence supports a positive association between GhEXLB2 and VW resistance. This study provides a framework for understanding expansin functions in cotton defense against VW.