Synergistic modulation of antioxidant enzymes by citric acid and EDTA to enhance lead tolerance and phytoextraction efficiency in harmel

Lead (Pb) induces physiological, morphological, and metabolic effects in plants. The exogenous application of ethylenediaminetetraacetic acid (EDTA) and citric acid (CA) is a common method to enhance the phytoextraction of Pb from plants. However, the impact of these treatments on harmel's ability to tolerate and accumulate Pb remains unclear. This study aimed to investigate the effects of CA (0, 2.5, and 5 mM) and EDTA (0, 2.5, and 5 mM) on physiological growth responses, antioxidant enzymes, as well as lead translocation factor (TF) and uptake of harmel under Pb stress (0, 5, and 25 mg L^-1). The results indicated that Pb treatment significantly reduced growth parameters in harmel plants. The application of 5 mM EDTA adversely affected plant growth, while the 2.5 and 5 mM CA treatments enhanced it. The application of 2.5 mM CA and EDTA significantly decreased lipid peroxidation and improved biomass production in Pb-stressed plants. The diversity of enzymatic activity in the 2.5 mM EDTA treatments and both CA concentrations was greater than in the control and the 5 mM EDTA treatment. CA and EDTA primarily increased the levels of ascorbate, dehydroascorbate, glutathione, and protein in the leaves, thereby enhancing the Pb tolerance of harmel plants. Furthermore, CA and EDTA significantly elevated TF values, with the highest amount observed in Pb 5 + EDTA 5, compared to plants treated with Pb alone. Correlation analysis, visualized through a heat map, revealed strong positive relationships among all growth indicators and a consistently strong negative correlation between root Pb concentration and plant growth, underscoring root Pb accumulation as the primary constraint on development. Conversely, shoot Pb concentration showed weaker associations, and TF exhibited no consistent correlation with growth suppression. Principal Component Analysis (PCA) effectively distinguished Pb-stressed from treated plants and confirmed the ameliorative effects of CA and EDTA. The PCA further highlighted distinct physiological response patterns induced by the two chelating agents, suggesting different underlying mechanisms of action. Overall, CA and EDTA improved Pb tolerance in harmel by modulating antioxidant defense systems and influencing Pb distribution, offering promising tools for enhancing phytoremediation potential under Pb-contaminated conditions.