Citrate accumulation mediated through GhCS6 enhances antioxidant modulation under Cd²⁺ stress in cotton

Citrate synthase (CS) catalyzes the biosynthesis of citrate (CA), a tricarboxylic acid metabolite that serves as a pivotal biochemical regulator in plant cellular metabolism. This investigation conducted systematic genome-wide identification of CS family, integrating structural, phylogenetic relationships, collinearity analysis, cis-acting elements and expression analysis. GhCS6 protein is localized in the mitochondria. The pivotal cadmium-responsive regulatory candidate gene GhCS6 was identified, followed by its silencing mediation through virus-induced gene silencing (VIGS), silenced plants displaying phenotypic evidence of enhanced Cd²⁺ susceptibility. Simultaneously, CA content in the silenced plants was also significantly decreased, which reduced the chelating capacity for Cd²⁺ and consequently exacerbates the accumulation of Cd²⁺. The silenced plants showed an imbalance in the reactive oxygen species (ROS) scavenging system, as evidenced by lower superoxide dismutase (SOD) activity and a marked increase in H₂O₂ content, causing increased oxidative damage. This oxidative stress further compromised the integrity of cellular membranes, resulting in elevated malondialdehyde (MDA) content and compromised cellular structure. These changes ultimately impaired photosynthetic efficiency and plant growth and development. This study contributes significantly to understanding the function of the GhCS6 in plant Cd²⁺ tolerance, and identifies potential gene targets for breeding Cd²⁺ tolerant cotton varieties.