The CCCH zinc-finger protein contains a typical C3H-type motif widely existing in plants, and it plays an important role in plant growth, development, and stress responses. In this study, a CCCH zinc-finger gene, GhC3H20, was isolated and thoroughly characterized to regulate salt stress in cotton and Arabidopsis. The expression of GhC3H20 was up-regulated under salt, drought, and ABA treatments. GUS activity was detected in the root, stem, leaves, and flowers of ProGhC3H20::GUS transgenic Arabidopsis. Compared with the control, the GUS activity of ProGhC3H20::GUS transgenic Arabidopsis seedlings under NaCl treatment was stronger. Through the genetic transformation of Arabidopsis, three transgenic lines of 35S-GhC3H20 were obtained. Under NaCl and mannitol treatments, the roots of the transgenic lines were significantly longer than those of the wild-type (WT) Arabidopsis. The leaves of the WT turned yellow and wilted under high-concentration salt treatment at the seedling stage, while the leaves of the transgenic Arabidopsis lines did not. Further investigation showed that compared with the WT, the content of catalase (CAT) in the leaves of the transgenic lines was significantly higher. Therefore, compared with the WT, overexpression of GhC3H20 enhanced the salt stress tolerance of transgenic Arabidopsis. A virus-induced gene silencing (VIGS) experiment showed that compared with the control, the leaves of pYL156-GhC3H20 plants were wilted and dehydrated. The content of chlorophyll in pYL156-GhC3H20 leaves was significantly lower than those of the control. Therefore, silencing of GhC3H20 reduced salt stress tolerance in cotton. Two interacting proteins (GhPP2CA and GhHAB1) of GhC3H20 have been identified through a yeast two-hybrid assay. The expression levels of PP2CA and HAB1 in transgenic Arabidopsis were higher than those in the WT, and pYL156-GhC3H20 had expression levels lower than those in the control. GhPP2CA and GhHAB1 are the key genes involved in the ABA signaling pathway. Taken together, our findings demonstrate that GhC3H20 may interact with GhPP2CA and GhHAB1 to participate in the ABA signaling pathway to enhance salt stress tolerance in cotton.

Salt stress is a major abiotic stressor that can severely limit plant growth, distribution, and crop yield. DNA-binding with one finger (Dof) is a plant-specific transcription factor that plays a crucial role in plant growth, development, and stress response. In this study, the function of a Dof transcription factor, GhDof1.7, was investigated in upland cotton. The GhDof1.7 gene has a coding sequence length of 759 base pairs, encoding 252 amino acids, and is mainly expressed in roots, stems, leaves, and inflorescences. Salt and abscisic acid (ABA) treatments significantly induced the expression of GhDof1.7. The presence of GhDof1.7 in Arabidopsis may have resulted in potential improvements in salt tolerance, as suggested by a decrease in H₂O₂ content and an increase in catalase (CAT) and superoxide dismutase (SOD) activities. The GhDof1.7 protein was found to interact with GhCAR4 (C2-domain ABA-related 4), and the silencing of either GhDof1.7 or GhCAR4 resulted in reduced salt tolerance in cotton plants. These findings demonstrate that GhDof1.7 plays a crucial role in improving the salt tolerance of upland cotton and provide insight into the regulation of abiotic stress response by Dof transcription factors.

Background: Cyclic nucleotide-gated ion channels (CNGCs) are calcium-permeable channels that participate in a variety of biological functions, such as signaling pathways, plant development, and environmental stress and stimulus responses. Nevertheless, there have been few studies on CNGC gene family in cotton. Results: In this study, a total of 114 CNGC genes were identified from the genomes of 4 cotton species. These genes clustered into 5 main groups: I, II, III, IVa, and IVb. Gene structure and protein motif analysis showed that CNGCs on the same branch were highly conserved. In addition, collinearity analysis showed that the CNGC gene family had expanded mainly by whole-genome duplication (WGD). Promoter analysis of the GhCNGCs showed that there were a large number of cis-acting elements related to abscisic acid (ABA). Combination of transcriptome data and the results of quantitative RT–PCR (qRT–PCR) analysis revealed that some GhCNGC genes were induced in response to salt and drought stress and to exogenous ABA. Virus-induced gene silencing (VIGS) experiments showed that the silencing of the GhCNGC32 and GhCNGC35 genes decreased the salt tolerance of cotton plants (TRV:00). Specifically, physiological indexes showed that the malondialdehyde (MDA) content in gene-silenced plants (TRV:GhCNGC32 and TRV:GhCNGC35) increased significantly under salt stress but that the peroxidase (POD) activity decreased. After salt stress, the expression level of ABA-related genes increased significantly, indicating that salt stress can trigger the ABA signal regulatory mechanism. Conclusions: we comprehensively analyzed CNGC genes in four cotton species, and found that GhCNGC32 and GhCNGC35 genes play an important role in cotton salt tolerance. These results laid a foundation for the subsequent study of the involvement of cotton CNGC genes in salt tolerance.

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.

As one of the most important factors in alternative splicing (AS) events, serine/arginine-rich (SR) proteins not only participate in the growth and development of plants but also play pivotal roles in abiotic stresses. However, the research about SR proteins in cotton is still lacking. In this study, we performed an extensive comparative analysis of SR proteins and determined their phylogeny in the plant lineage. A total of 169 SR family members were identified from four Gossypium species, and these genes could be divided into eight distinct subfamilies. The domain, motif distribution and gene structure of cotton SR proteins are conserved within each subfamily. The expansion of SR genes is mainly contributed by WGD and allopolyploidization events in cotton. The selection pressure analysis showed that all the paralogous gene pairs were under purifying selection pressure. Many cis-elements responding to abiotic stress and phytohormones were identified in the upstream sequences of the GhSR genes. Expression profiling suggested that some GhSR genes may involve in the pathways of plant resistance to abiotic stresses. The WGCNA analysis showed that GhSCL-8 co-expressed with many abiotic responding related genes in a salt-responding network. The Y2H assays showed that GhSCL-8 could interact with GhSRs in other subfamilies. The subcellular location analysis showed that GhSCL-8 is expressed in the nucleus. The further VIGS assays showed that the silencing of GhSCL-8 could decrease salt tolerance in cotton. These results expand our knowledge of the evolution of the SR gene family in plants, and they will also contribute to the elucidation of the biological functions of SR genes in the future.