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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.

Evolution and Stress Responses of CLO Genes and Potential Function of the GhCLO06 Gene in Salt Resistance of Cotton