CRISPR/Cas9-Mediated characterization of Kcs6 in wax biosynthesis and drought tolerance of Arabidopsis thaliana

Drought is a major abiotic stress that reduces crop productivity due to global climate change. The plant cuticular wax layer plays a crucial role in reducing non-stomatal water loss and enhancing drought tolerance. KCS6, encoding 3-ketoacyl-CoA synthase 6, is a key enzyme in the biosynthesis of very-long-chain fatty acids (VLCFAs), the main components of cuticular wax. However, its specific function in wax accumulation and drought resistance remains incompletely elucidated. This study aimed to characterize the role of KCS6 in wax biosynthesis and drought tolerance in Arabidopsis thaliana using CRISPR/Cas9-mediated gene knock-out. kcs6 mutants were generated by targeting the first exon of KCS6 to create a loss-of-function allele. Phenotypic analysis, cuticular wax composition, and drought tolerance assays were performed in the mutants compared to wild-type plants. The results showed that kcs6 mutants exhibited a significant reduction in VLCFA content and cuticular wax load. The mutants also displayed a higher rate of water loss and increased sensitivity to drought stress compared to the wild type. These findings provide direct genetic evidence that KCS6 is essential for cuticular wax biosynthesis and for maintaining plant water status under drought conditions. This study is the first to demonstrate, using CRISPR/Cas9-mediated targeted mutagenesis, that disruption of KCS6 directly links altered VLCFA-derived cuticular wax biosynthesis to impaired plant water retention and drought adaptation, providing mechanistic evidence for KCS6 as a promising genetic target for improving crop drought resilience.