Soil salinization conditions seriously restrict cotton yield and quality. Related studies have shown that the DUF4228 proteins are pivotal in plant resistance to abiotic stress. However, there has been no systematic identification and analysis of the DUF4228 gene family in cotton and their role in abiotic stress. In this study, a total of 308 DUF4228 genes were identified in four Gossypium species, which were divided into five subfamilies. Gene structure and protein motifs analysis showed that the GhDUF4228 proteins were conserved in each subfamily. In addition, whole genome duplication (WGD) events and allopolyploidization might play an essential role in the expansion of the DUF4228 genes. Besides, many stress-responsive (MYB, MYC) and hormone-responsive (ABA, MeJA) related cis-elements were detected in the promoters of the DUF4228 genes. The qRT-PCR results showed that GhDUF4228 genes might be involved in the response to abiotic stress. VIGS assays and the measurement of relative water content (RWC), Proline content, POD activity, and malondialdehyde (MDA) content indicated that GhDUF4228-67 might be a positive regulator of cotton response to salt stress. The results in this study systematically characterized the DUF4228s in Gossypium species and will provide helpful information to further research the role of DUF4228s in salt tolerance.

Abiotic stress, such as drought and salinity stress, seriously inhibit the growth and development of plants. Therefore, it is vital to understand the drought and salinity resistance mechanisms to enable cotton to provide more production under drought and salt conditions. In this study, we identified 8806 and 9108 differentially expressed genes (DEGs) through a comprehensive analysis of transcriptomic data related to the PEG-induced osmotic and salt stress in cotton. By performing weighted gene co-expression network analysis (WGCNA), we identified four co-expression modules in PEG treatment and five co-expression modules in salinity stress, which included 346 and 324 predicted transcription factors (TFs) in these modules, respectively. Correspondingly, whole genome duplication (WGD) events mainly contribute to the expansion of those TFs. Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) analyses revealed those different modules were associated with stress resistance, including regulating macromolecule metabolic process, peptidase activity, transporter activity, lipid metabolic process, and responses to stimulus. Quantitative RT-PCR analysis was used to confirm the expression levels of 15 hub TFs in PEG6000 and salinity treatments. We found that the hub gene GhWRKY46 could alter salt and PEG-induced drought resistance in cotton through the virus-induced gene silencing (VIGS) method. Our results provide a preliminary framework for further investigation of the cotton response to salt and drought stress, which is significant to breeding salt- and drought-tolerant cotton varieties.