Genome-wide analysis of chloride channel-encoding gene family members and identification of CLC genes that respond to Cl/salt stress in upland cotton.

Chloride channels (CLCs) are kinds of anion transport protein family members that are mainly distributed in cell endomembrane systems of prokaryotic and eukaryotic organisms and mediate anion (Cl, as a representative) transport and homeostasis. Some CLC genes have been reported to be involved in Cl/salt tolerance of plants exposed to NaCl stress. Through BLAST in cotton database, a total of 22 CLCs were identified in genomes A and D in upland cotton (Gossypium hirsutum L.), and except for GhCLC6 and GhCLC17, they formed highly similar homologous genes pairs. According to the prediction in PlantCARE database, many cis-acting elements related to abiotic stress responses, including ABREs, AREs, GT-1s, G-boxes, MYBs, MYCs, etc., were found in the promoters of GhCLCs. qRT-PCR revealed that most GhCLC gene expression was upregulated in the roots and leaves of cotton seedlings under salt stress, and those of homologous GhCLC4/15, GhCLC5/16, and GhCLC7/18 displayed more obvious expression. Furthermore, according to leaf virus-induced gene silencing (VIGS) assay and compared with the salt-stressed GhCLC4/15- and GhCLC7/18-silenced cotton plants, the salt-stressed GhCLC5/16-silenced plants displayed relatively better growth with significant increases in both Cl content and Cl/NO ratio in the roots and drop of the same parameters in the leaves. These results indicate that homologous GhCLC5/16, with the highest NaClinduced upregulation of expression and the maximum number of MYC cis-acting elements, might be the key members contributing to cotton Cl/salt tolerance by regulating the transport, interaction and homeostasis of Cl and NO.