Identification and analysis of salt-responsive F-box genes in upland cotton and the potential role of GhFBX83 in salt stress tolerance through regulating ROS homeostasis

The F-box family is among the largest gene families in plants and plays an extremely important role in diverse life processes, including salt responses. Most F-box proteins regulate stress responses through ubiquitin-mediated protein degradation as components of SCF (SKP1-CUL1-F-box) ubiquitin ligase complexes. However, knowledge of cotton F-box (GhFBX) genes and their roles in salt tolerance remains limited. In this study, we performed a comprehensive analysis of the response of GhFBX genes to salt stress in cotton. 160 salt-responsive GhFBX genes, which showed transcriptional expression differences in response to salt, were defined based on transcriptome data analysis, and their conserved domains, phylogenetic relationships, and chromosome locations were characterized. In accordance with the C-terminal conserved domains, the SR GhFBX genes were divided into 10 subfamilies. Phylogenetic analysis revealed that genes with the same C-terminal structural domains were mostly clustered together. The promoter regions of the SR GhFBX genes mainly contain three types of cis-elements, namely, hormone-responsive elements, light-responsive elements, and stress-related elements. To clarify how GhFBX and GhASK proteins interact, we selected 6 SR GhFBX genes and 4 SKP1 genes for interaction analysis using a yeast two-hybrid (Y2H) assay. The results revealed that the SR GhFBX proteins can interact with various GhSKP1-like proteins. In addition, the SR gene GhFBX83 was selected for gene silencing via VIGS. The results revealed that silencing GhFBX83 increased proline (PRO) levels, significantly reduced hydrogen peroxide (H₂O₂) and superoxide anion (O₂⁻) levels, and significantly increased the activities of superoxide dismutase (SOD) and peroxidase (POD) compared to the controls, thereby enhancing salt tolerance. These findings indicate that GhFBX83 acts as a negative regulator in the response to salt stress. The research identified and analysed 160 salt-responsive (SR) GhFBX genes in Gossypium hirsutum (upland cotton) and GhFBX83 was found to negatively regulate salt tolerance by promoting the accumulation of reactive oxygen species (ROS). This study provides valuable insights into the molecular basis of cotton salt tolerance and lays a foundation for further functional studies on cotton salt resistance breeding.