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.