Comparative transcriptome analysis and functional verification revealed that GhSAP6 negatively regulates salt tolerance in upland cotton

Owing to the scarcity of cultivable land in China, the agricultural sector is primarily focused on grain and oil crops. Simultaneously, the cultivation of cotton has gradually shifted towards regions characterized by elevated soil salinity levels. Additionally, the mechanism behind cotton's ability to tolerate salt remains elusive. In this study, we identified the Z9807 genotype as highly tolerant to salt stress, exhibiting superior leaf wilting resistance, antioxidant activity, catalase activity, K+/Na+ ratio, and growth compared to the salt-sensitive ZJ0102. Comparative transcriptome analysis revealed marked differences in salt stress responses between Z9807 and ZJ0102. This study identified a considerable number of differentially expressed genes associated with salt tolerance across multiple time points. By integration of QTL and GWAS mapping data, we successfully identified 621 candidate genes associated with salt tolerance. Weighted gene correlation network analysis exhibited three co-expression modules related to salt-tolerant Z9807 samples, ultimately identifying 15 core salt-tolerant candidate genes. We also conducted in-depth research on the salt tolerance of the stress-associated protein (SAP) GhSAP6 (GhSAP6_At and GhSAP6_Dt homologs). Results revealed that these candidate genes may inhibit salt tolerance through Virus-Induced Gene Silencing (VIGS) and transgenic overexpression assays conducted in Arabidopsis thaliana. Furthermore, we used yeast two-hybrid and luciferase assay experiments to confirm the ubiquitin degradation pathway between selected interacting proteins and verified the interaction with RAD23C. This study will provide new insights into the mechanisms related to salt tolerance in upland cotton. •Transcriptome, QTL and GWAS mapping data led to the identification of 621 candidate genes associated with salt tolerance.•Fifteen core salt tolerance candidate genes were identified by weighted gene correlation network analysis (WGCNA).•GhSAP6 has been shown to negatively regulate the salt tolerance of upland cotton.•Yeast two-hybrid test confirmed GhSAP6-RAD23C interaction, potentially in ubiquitination pathway.