Characterization of Kcs6 in wax biosynthesis and drought tolerance in Arabidopsis thaliana: Insights from Crispr/Cas9 gene knockout studies

ABSTRACT

3-ketoacyl-CoA synthases (KCSs) are key enzymes of the fatty acid elongation (FAE) complex and are responsible for the production of very long-chain fatty acids (VLCFAs). Among them, KCS5 and KCS6 are known to participate in the elongation of C26 to C28 fatty acids and in cuticular wax biosynthesis. Drought is a major abiotic stress that reduces crop productivity and contributes to food insecurity. Plants are also exposed to several biotic and abiotic stresses, including pathogens, salinity, temperature extremes, and water deficiency. Arabidopsis thaliana is widely used as a model plant for investigating drought-response mechanisms. This study evaluated the role of KCS6 in drought tolerance and wax formation. The KCS6 gene was disrupted using CRISPR/Cas9 genome editing with a designed guide RNA targeting the gene. The construct was introduced into Agrobacterium and subsequently transformed into Arabidopsis using the floral-dip method. Transformation and mutation were confirmed through DNA extraction and PCR analysis. Cuticular wax content was then compared between wild-type plants and KCS6 knockout lines using chloroform extraction. Ten wild-type and ten mutant plants were evaluated. The KCS6 knockout plants showed a 41–51% reduction in wax content compared with the wild type. These results demonstrate that KCS6 contributes substantially to fatty-acid precursor formation and cuticular wax biosynthesis. Loss of KCS6 reduces wax accumulation and may consequently weaken the ability of Arabidopsis plants to tolerate drought and other abiotic stresses.