Functional characterization of Arabidopsis PQT3 homolog in cotton reveals as a potential candidate for redox homeostasis and abiotic stress resistance

• GhPQT3 identified as a conserved E3 ligase in cotton. • CRISPR-Cas12a knockout enhanced stress tolerance. • Mutants showed higher APX and GPX expression and activity. • Improved redox balance reduced oxidative stress damage in mutant plants. • GhPQT3 is a target for climate-resilient cotton breeding and other crops. Abiotic stresses like drought and salinity severely impact cotton productivity by triggering excessive accumulation of reactive oxygen species (ROS), leading to oxidative damage. In this study, we functionally characterized GhPQT3 , a homolog of Paraquat tolerance 3 from the Arabidopsis thaliana E3 ligase family, and its role in regulating oxidative stress responses. Computational analysis identified two GhPQT3 homologs in the Gossypium hirsutum , showing high sequence conservation with Arabidopsis and rice PQT3 . Protein sequence alignment, phylogenetic analysis, conserved motif, and domain annotation confirmed that GhPQT3 shares structural and functional similarity with its dicot homologs. CRISPR-Cas12 mediated knock out mutants were generated for functional characterization of the protein. Functional studies revealed that GhPQT3 acts as a negative regulator of antioxidant defenses, and its suppression in mutant lines Ghpqt3-4 and Ghpqt3-6 significantly enhanced tolerance to drought and salinity. These mutant lines exhibited elevated gene expression and enzymatic activity of APX and GPX especially Ghpqt3-6 , which consistently maintained higher antioxidant activity and reduced oxidative damage. The DPPH radical scavenging assay, quantification of chlorophyll content and leaf necrosis further confirmed improved cellular integrity, improved redox homeostasis. These findings establish GhPQT3 as a promising target for genetic improvement, offering a robust strategy to develop stress-resilient cotton cultivars capable of withstanding climate-induced abiotic stresses.