GhFLS1 governs quercetin synthesis and the flavonoid pathway to upregulate salt tolerance in cotton

Salinized soil is a potential resource for agricultural development, and cotton can improve and rehabilitate saline-alkali soil. Therefore, identifying, analyzing, and validating salt tolerance genes in cotton are of great significance for remediating saline-alkali land. Flavonol synthase (FLS) plays a key role in flavonol biosynthesis in plant, and it is also important in plant growth, development, and abiotic stress tolerance. However, the function of the FLS gene in cotton under salt stress remains unclear. In this study, the function of GhFLS1 under salt stress was analyzed through combined transcriptomic and metabolomic, exogenous quercetin, 3-3’diaminobenzadine (DAB) and 2-aminoethyl diphyenylborate (DPBA) staining in cotton, and the generation and characterization of GhFLS1- overexpressing lines in Arabidopsis and cotton. Differentially expressed genes and differentially accumulated metabolites were significantly enriched in the flavonoid metabolic pathway. DAB and DPBA staining revealed significant reactive oxygen species (ROS) and flavonol accumulation in cotton leaves under salt stress. Exogenous quercetin application enhanced the salt stress tolerance of cotton. GhFLS1 expression was significantly upregulated in response to salt stress. GhFLS1- overexpressing Arabidopsis exhibited enhanced salt stress tolerance, and ROS accumulation was significantly reduced in GhFLS1 -overexpressing cotton under salt stress. This study presents the first functional characterization of a cotton FLS gene in response to salt stress by overexpression in Arabidopsis and cotton. We integrated transcriptomic and metabolomic, identifying quercetin as the key downstream metabolite, and validated its protective role through exogenous application. These results establish a functional link from GhFLS to quercetin-mediated ROS homeostasis, providing a candidate gene and a metabolic marker for breeding salt-tolerant cotton.