The sodium/hydrogen antiporter (NHX) gene family with the Na + /H + exchange protein domain is a transporter of sodium and hydrogen ions and plays an important role in the response of plants to salt stress. Studying the response of cotton to salt stress through comprehensive identification and analysis of NHX genes in several species and their roles in salt tolerance mechanisms is of great significance. In this study, 23, 24, 12, and 12 NHX genes were identified from Gossypium hirsutum (Gh), G. barbadense, G. arboreum and G. raimondii, respectively. Phylogenetic analysis showed that these genes were mainly divided into three clades with significant subcellular localization, namely, endosome (Endo-class), plasma membrane (PM-class) and vacuole (Vac-class). By analyzing the structure of NHX genes and proteins, each branch of the NHX gene family was found to be structurally conserved, and collinearity analysis showed that NHX genes were mainly expressed through whole genome and segmental duplication. The non-synonymous (Ka)/synonymous (Ks) values showed that the NHX gene family experienced strong purifying selection during long-term evolution. Cis-acting element analysis showed that the NHX gene family may be related to the regulation of abscisic acid (ABA) and methyl jasmonate (MeJA) hormones. Additionally, transcriptomic data analysis and qRT-PCR showed that GhNHXs exhibited different expression patterns in each tissue and under different salinities. These results provide an important reference for us to further understand and analyze the molecular regulation mechanism of cotton NHX genes.

The caleosin (CLO) protein family displays calcium-binding properties and plays an important role in the abiotic stress response. Here, a total of 107 CLO genes were identified in 15 plant species, while no CLO genes were detected in two green algal species. Evolutionary analysis revealed that the CLO gene family may have evolved mainly in terrestrial plants and that biological functional differentiation between species and functional expansion within species have occurred. Of these, 56 CLO genes were identified in four cotton species. Collinearity analysis showed that CLO gene family expansion mainly occurred through segmental duplication and whole-genome duplication in cotton. Sequence alignment and phylogenetic analysis showed that the CLO proteins of the four cotton species were mainly divided into two types: H-caleosins (class I) and L-caleosins (class II). Cis-acting element analysis and quantitative RT–PCR (qRT–PCR) suggested that GhCLOs might be regulated by abscisic acid (ABA) and methyl jasmonate (MeJA). Moreover, transcriptome data and qRT–PCR results revealed that GhCLO genes responded to salt and drought stresses. Under salt stress, gene-silenced plants (TRV: GhCLO06) showed obvious yellowing and wilting, higher malondialdehyde (MDA) content accumulation, and significantly lower activities of superoxide dismutase (SOD) and peroxidase (POD), indicating that GhCLO06 plays a positive regulatory role in cotton salt tolerance. In gene-silenced plants (TRV: GhCLO06), ABA-related genes (GhABF2, GhABI5, and GhNAC4) were significantly upregulated after salt stress, suggesting that the regulation of salt tolerance may be related to the ABA signaling pathway. This research provides an important reference for further understanding and analyzing the molecular regulatory mechanism of CLOs for salt tolerance.

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