Characterization of the wall-associated kinase (WAK) gene family in Gossypium barbadense reveals the positive role of GbWAK5 in salt tolerance

We characterized the WAK gene family in Gossypium barbadense and revealed the potential function of GbWAK5 in regulating salt tolerance by modulating ion homeostasis. Soil salinization is one of the main factors restricting cotton production. Although the role of the wall-associated kinases (WAKs) in plants has been extensively studied, its response to salt stress in sea-island cotton (Gossypium barbadense L.) has not been reported. Here, we conducted a whole-genome analysis of the WAK gene family in G. barbadense, identifying a total of 70 GbWAK genes, which were classified into five clades. Segmental and tandem duplication events have contributed to the expansion of the GbWAK gene family. A large number of cis-acting elements were predicted in the GbWAK promoter region. Through RNA sequencing, 37 GbWAKs that potentially play a role in cotton's response to salt stress were screened out, among which 10 genes with sustained up-regulated expression were confirmed by quantitative real-time PCR (qRT-PCR). GbWAK5, a member of Clade II, was significantly up-regulated following NaCl treatment and exhibited a typical WAK structure. Subcellular localization indicated that GbWAK5 is localized on the plasma membrane. Virus-induced gene silencing (VIGS) experiments revealed that the knockdown of GbWAK5 resulted in more severe dehydration and wilting in plants compared to the control under NaCl treatment. RNA-seq analysis revealed that several ion transport-related genes were down-regulated in TRV:GbWAK5 plants under salt stress, while TRV:GbWAK5 plants accumulated more Na⁺ and exhibited a higher Na⁺/K⁺ ratio compared to TRV:00 plants. These results offer a comprehensive analysis of the G. barbadense WAK gene family for the first time, and conclude that GbWAK5 is a promising gene for improving cotton's resistance to salt stress.