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GhCLCg-1, a Vacuolar Chloride Channel, Contributes to Salt Tolerance by Regulating Ion Accumulation in Upland Cotton

ABSTRACT: 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.

SUBMITTER: Liu W

PROVIDER: S-EPMC8555695 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cotton yields are greatly reduced under high salinity stress conditions, although cotton is considered a moderately salt-tolerant crop. Understanding at the molecular level how cotton responds to salt stress will help in developing salt tolerant varieties. Here, we combined physiological analysis with isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics of seedling leaves of 2 genotypes differing in salinity tolerance to 200 mM (18.3 dS/m) NaCl stress. Salt stress produced significant stress symptoms in the sensitive genotype Nan Dan Ba Di Da Hua (N), including lower relative water and chlorophyll contents and higher relative electrolyte leakage and Na+/K+ ratio in leaf samples, compared with those in the tolerant genotype Earlistaple 7 (Z). A total of 58 differentially abundant salt-responsive proteins were identified. Asp-Glu-Ala-Asp (DEAD)-box ATP-dependent RNA helicase 3 and protochlorophyllide reductase were markedly suppressed after salt treatment, whereas the phosphate-related differentially abundant proteins (DAPs) phosphoethanolamine N-methyltransferase 1 and 14-3-3-like protein E were induced, and all these proteins may play significant roles in salt stress. Twenty-nine salt-responsive proteins were also genotype specific, and 62.1 and 27.6% of these were related to chloroplast and defense responses, respectively. Based on the Arabidopsis thaliana protein interaction database, orthologs of 25 proteins showed interactions in Arabidopsis, and among these, a calmodulin protein was predicted to have 212 functional partners. In addition, the Golgi apparatus and calcium may be important for salt secretion in cotton. Through integrative proteome and transcriptome analysis, 16 DAPs were matched to differentially expressed genes and verified using qRT-PCR. On the basis of these findings, we proposed that some proteins related to chloroplast, ATP, ribosomal, and phosphate metabolism as well as to the Golgi apparatus and calcium may play key roles in the short-term salt stress response of cotton seedling leaves.

Project description:Plants have developed a number of survival strategies which are significant for enhancing their adaptation to various biotic and abiotic stress factors. At the transcriptome level, G-protein-coupled receptors (GPCRs) are of great significance, enabling the plants to detect a wide range of endogenous and exogenous signals which are employed by the plants in regulating various responses in development and adaptation. In this research work, we carried out genome-wide analysis of target of Myb1 (TOM1), a member of the GPCR gene family. The functional role of TOM1 in salt stress tolerance was studied using a transgenic Arabidopsis plants over-expressing the gene. By the use of the functional domain PF06454, we obtained 16 TOM genes members in Gossypium hirsutum, 9 in Gossypium arboreum, and 11 in Gossypium raimondii. The genes had varying physiochemical properties, and it is significant to note that all the grand average of hydropathy (GRAVY) values were less than one, indicating that all are hydrophobic in nature. In all the genes analysed here, both the exonic and intronic regions were found. The expression level of Gh_A07G0747 (GhTOM) was significantly high in the transgenic lines as compared to the wild type; a similar trend in expression was observed in all the salt-related genes tested in this study. The study in epidermal cells confirmed the localization of the protein coded by the gene TOM1 in the plasma membrane. Analysis of anti-oxidant enzymes showed higher concentrations of antioxidants in transgenic lines and relatively lower levels of oxidant substances such as H₂O₂. The low malondialdehyde (MDA) level in transgenic lines indicated that the transgenic lines had relatively low level of oxidative damage compared to the wild types. The results obtained indicate that Gh_A07G0747 (GhTOM) can be a putative target gene for enhancing salt stress tolerance in plants and could be exploited in the future for the development of salt stress-tolerant cotton cultivars.

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GhCLCg-1, a Vacuolar Chloride Channel, Contributes to Salt Tolerance by Regulating Ion Accumulation in Upland Cotton

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