Characterization of With-No-Lysine Kinase Family Genes and Roles of CaWNK6 in the Heat Tolerance of Pepper (Capsicum annuum L.).

With-No-Lysine (WNK) kinases constitute a subgroup within the serine/threonine protein kinase family, characterized by the absence of a catalytic lysine residue in the kinase subdomain II located in their N-terminal region. These kinases play critical roles in regulating plant growth, development, and responses to abiotic stressors. However, members of the WNK and their responses to heat stress in pepper (Capsicum annuum L.) remain unexplored. In the present study, we identified eleven WNK genes within the genome of pepper cultivar 'UCD-10X-F1' and designated them CaWNK1 to CaWNK11 according to their chromosomal positions. Comprehensive analyses were conducted to elucidate their phylogenetic relationships, chromosomal distribution, collinearity, gene structure, protein properties, and cis-acting elements within promoter regions. The findings revealed that the CaWNK gene family segregates into five distinct subgroups. Comparative genomic analysis identified eleven and nine segmental duplication gene pairs between pepper and tomato and between pepper and Arabidopsis, respectively. Within the pepper genome, two pairs of segmentally duplicated genes and two pairs of tandemly repeated genes were also detected. The CaWNK gene sequences in pepper exhibited a high degree of conservation; however, variations were observed in the number of introns and exons. Analysis of the promoter regions revealed an abundance of cis-acting elements associated with growth and development, stress responses, and hormone regulation. Subsequent transcriptomic analyses demonstrated that CaWNK genes displayed tissue-specific expression patterns and differential expression levels following treatments with exogenous plant hormones and abiotic stresses. Notably, the expression of CaWNK6 was significantly up-regulated under heat stress conditions. To elucidate the functional role of CaWNK6, virus-induced gene silencing (VIGS) was employed to suppress its expression in pepper seedlings. Silencing of CaWNK6 resulted in disrupted tissue architecture, stomatal closure, and diminished heat tolerance. These phenotypic changes correlated with excessive accumulation of reactive oxygen species (ROS), reduced activity of antioxidant enzymes, and down-regulation of heat shock factor (HSF) genes in the silenced plants. Collectively, these findings offer valuable insights into the functional roles of CaWNK genes and hold significant implications for the development of novel heat-tolerant pepper cultivars.