Soil and freshwater salinization is increasingly becoming a problem worldwide and has adversely affected plant growth. However, most of the related studies have focused on sodium ion (Na+) stress, with relatively little research on chloride ion (Cl–) stress. Here, we found that upland cotton (Gossypium hirsutum) plants accumulated Cl– and exhibited strong growth inhibition under NaCl or KCl treatment. Then, a chloride channel gene (GhCLCg-1) was cloned from upland cotton. Phylogenetic and sequence analyses indicated that GhCLCg-1 was highly homologous to AtCLCg and also have conserved voltage_CLC and CBS domains. The subcellular localization assay showed that GhCLCg-1 was localized on the vacuolar membrane. Gene expression analyses revealed that the expression of GhCLCg-1 increased rapidly in cotton in response to chloride stress (NaCl or KCl), and the transcript levels increased as the chloride stress intensified. The overexpression of GhCLCg-1 in Arabidopsis thaliana changed the uptake of ions with a decrease of the Na+/K+ ratios in the roots, stems, and leaves, and enhanced salt tolerance. In contrast, silencing GhCLCg-1 in cotton plants increased the Cl– contents in the roots, stems, and leaves and the Na+/K+ ratios in the stems and leaves, resulting in compromised salt tolerance. These results provide important insights into the toxicity of chloride to plants and also indicate that GhCLCg-1 can positively regulates salt tolerance by adjusting ion accumulation in upland cotton.

Soil salinization, which is primarily due to excessive Na+ levels, is a major abiotic stress adversely affecting plant growth and development. The Na+/H+ antiporter (NHX) is a transmembrane protein mediating the transport of Na+ or K+ and H+ across the membrane to modulate the ionic balance of plants in response to salt stress. Research regarding NHXs has mainly focused on the vacuolar-type NHX family members. However, the biological functions of the endosomal-type NHXs remain relatively uncharacterized. In this study, 22 NHX family members were identified in Gossypium hirsutum. A phylogenetic analysis divided the GhNHX genes into two categories, with 18 and 4 in the vacuolar and endosomal groups, respectively. The chromosomal distribution of the NHX genes revealed the significant impact of genome-wide duplication during the polyploidization process on the number of GhNHX genes. Analyses of gene structures and conserved motifs indicated that GhNHX genes in the same phylogenetic cluster are conserved. Additionally, the salt-induced expression patterns confirmed that the expression levels of most of the GhNHX genes are affected by salinity. Specifically, in the endosomal group, GhNHX4A expression was substantially up-regulated by salt stress. A yeast functional complementation test proved that GhNHX4A can partially restore the salt tolerance of the salt-sensitive yeast mutant AXT3. Silencing GhNHX4A expression decreased the resistance of cotton to salt stress because of an increase in the accumulation of Na+ in stems and a decrease in the accumulation of K+ in roots. The results of this study may provide the basis for an in-depth characterization of the regulatory functions of NHX genes related to cotton salt tolerance, especially the endosomal-type GhNHX4A. Furthermore, the presented data may be useful for selecting appropriate candidate genes for the breeding of new salt-tolerant cotton varieties.

Histone modification is an important epigenetic modification that controls gene transcriptional regulation in eukaryotes. Histone methylation is accomplished by histone methyltransferase and can occur on two amino acid residues, arginine and lysine. JumonjiC (JmjC) domain-containing histone demethylase regulates gene transcription and chromatin structure by changing the methylation state of the lysine residue site and plays an important role in plant growth and development. In this study, we carried out genome-wide identification and comprehensive analysis of JmjC genes in the allotetraploid cotton species Gossypium hirsutum. In total, 50 JmjC genes were identified and in G. hirsutum, and 25 JmjC genes were identified in its two diploid progenitors, G. arboreum and G. raimondii, respectively. Phylogenetic analysis divided these JmjC genes into five subfamilies. A collinearity analysis of the two subgenomes of G. hirsutum and the genomes of G. arboreum and G. raimondii uncovered a one-to-one relationship between homologous genes of the JmjC gene family. Most homologs in the JmjC gene family between A and D subgenomes of G. hirsutum have similar exon-intron structures, which indicated that JmjC family genes were conserved after the polyploidization. All G. hirsutumJmjC genes were found to have a typical JmjC domain, and some genes also possess other special domains important for their function. Analysis of promoter regions revealed that cis-acting elements, such as those related to hormone and abiotic stress response, were enriched in G. hirsutum JmjC genes. According to a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis, most G. hirsutumJmjC genes had high abundance expression at developmental stages of fibers, suggesting that they might participate in cotton fiber development. In addition, some G. hirsutumJmjC genes were found to have different degrees of response to cold or osmotic stress, thus indicating their potential role in these types of abiotic stress response. Our results provide useful information for understanding the evolutionary history and biological function of JmjC genes in cotton.

Vacuolar sodium/proton (Na+/H+) antiporters (NHXs) can stabilize ion contents to improve the salt tolerance of plants. Here, GhNHX3D was cloned and characterized from upland cotton (Gossypium hirsutum). Phylogenetic and sequence analyses showed that GhNHX3D belongs to the vacuolar-type NHXs. The GhNHX3D-enhanced green fluorescent protein (eGFP) fusion protein localized on the vacuolar membrane when transiently expressed in Arabidopsis protoplasts. The quantitative real-time PCR (qRT-PCR) analysis showed that GhNHX3D was induced rapidly in response to salt stress in cotton leaves, and its transcript levels increased with the aggravation of salt stress. The introduction of GhNHX3D into the salt-sensitive yeast mutant ATX3 improved its salt tolerance. Furthermore, silencing of GhNHX3D in cotton plants by virus-induced gene silencing (VIGS) increased the Na+ levels in the leaves, stems, and roots and decreased the K+ content in the roots, leading to greater salt sensitivity. Our results indicate that GhNHX3D is a member of the vacuolar NHX family and can confer salt tolerance by adjusting the steady-state balance of cellular Na+ and K+ ions.