Aquaporin GhPIP2;1 positively regulates salt tolerance in upland cotton (Gossypium hirsutum L.)

Aquaporins (AQPs) facilitate transmembrane water transport and play a crucial role in plant adaptation to diverse abiotic stress conditions. Plasma membrane Intrinsic Proteins (PIPs) are a crucial subgroup of aquaporins that facilitate water transport across plant cell membrane. However, the role of PIPs under salt stress responses in cotton remains partially unexplored. In this study, we found that the expression level of the aquaporin gene GhPIP2;1 was significantly upregulated under salt stress at three-leaf stage in upland cotton. The overexpression of GhPIP2;1 in Arabidopsis thaliana and virus-induced gene silencing (VIGS) in upland cotton evidenced that GhPIP2;1 positively regulate salt tolerance function. Biochemical analysis including superoxide dismutase (SOD), peroxidase (POD) activities and malondialdehyde (MDA) content measurements indicated that GhPIP2;1 could enhance stress tolerance by regulating the reactive oxygen species (ROS). The Electrophoretic mobility shift assay (EMSA) and dual luciferase reporter assay confirmed that GhNAC072 specifically binds to the promoter region of GhPIP2;1 and enhance the expression of GhPIP2;1. Phenotypic observations and physiological measurements of GhNAC072 overexpressed Arabidopsis and silenced cotton plants demonstrated that GhNAC072 positively responds to salt stress. The expression level of GhPIP2;1 was significantly decreased in GhNAC072-silenced plants, indicating that GhNAC072 enhances salt tolerance by regulating GhPIP2;1 expression. In summary, this study systematically confirmed that GhPIP2;1 may involve in salt tolerance mechanism in cotton and its expression is regulated by GhNAC072. These results will provide gene resources for salt tolerance and enrich the knowledge on the mechanism of salt tolerance in cotton.