Type One Protein Phosphatase 4aD Negatively Regulates Cotton (Gossypium hirsutum) Salt Tolerance by Inhibiting the Phosphorylation of Kinases That Respond to Abscisic Acid

Salinity is one of the major factors limiting the growth, development, and yield of cotton. Although the mechanisms of cotton tolerance to salt stress have been studied, the regulatory roles and mechanisms of protein kinases and phosphatases in cotton salt response remain poorly understood. Here, we identify Type One Protein Phosphatase 4aD (<i>GhTOPP4aD</i>), belonging to the Type One Protein Phosphatase (TOPP) family, as a negative regulator in cotton salt stress response. To reveal the post-translational modification mechanism by which <i>GhTOPP4aD</i> regulates salt stress response in cotton, phosphoproteome analysis was performed. A total of 6055 phosphoproteins with 12,608 phosphosites were identified. In VIGS-Ctrl plants, there were 935 upregulated and 35 downregulated phosphoproteins, while there were 1026 upregulated and 89 downregulated phosphoproteins in VIGS-<i>GhTOPP4aD</i> plants after NaCl treatment. Moreover, a class of tyrosine kinases responsive to abscisic acid (ABA) was significantly enriched at upregulated, differentially phosphorylated sites that were induced by NaCl in <i>GhTOPP4aD</i>-silenced plants, suggesting that these proteins could be regulated by dephosphorylation mediated by <i>GhTOPP4aD</i> in response to salt stress. Among them, Raf-like Kinase 36 (GhRAF36), FERONIA (GhFER), and Lysin Motif-containing Receptor-like Kinase 3 (GhLYK3) interacted with GhTOPP4aD and their kinase activities were inhibited by GhTOPP4aD. VIGS-<i>GhRAF36</i>, VIGS-<i>GhFER</i>, and VIGS-<i>GhLYK3</i> plants were sensitive to salt stress, suggesting that these kinases may play important roles in the regulation of cotton salt stress response mediated by GhTOPP4aD. These studies provide new insights into the mechanisms of cotton salt stress tolerance and the potential molecular targets for breeding salt-tolerant cotton varieties.