The analysis of changes in antioxidant enzyme activity and gene expression caused by lead contamination in <i>Azolla caroliniana</i>

Heavy metal contamination in aquatic ecosystems poses serious environmental and health risks. <i>Azolla caroliniana</i>, a promising candidate for phytoremediation, has the potential to absorb heavy metals like lead <b>(</b>Pb<b>).</b> However, limited information is available on the enzymatic and genetic responses of <i>A. caroliniana</i> under Pb stress. This study investigates the plant's phytoremediation capacity by analyzing antioxidant enzyme activity and gene expression under lead (II) acetate [Pb(C₂H₃O₂)₂] concentrations (0, 500, 750, 1,000 µM) over three time points (days 2, 4, and 6). The results showed that with increasing Pb concentration, antioxidant enzyme activity increased. Chlorophyll content increased by 25% at 1,000 µM Pb, whereas carotenoid and anthocyanin levels decreased by 233% and 30%, respectively. Total protein content declined by 90%. Additionally, SOD and CAT activities increased by 28%, while APX activity rose by 25%. Gene expression analysis revealed that genes associated with antioxidant enzymes <i>CAT</i> (94% decrease), <i>APX</i> (64% decrease), <i>SOD</i> (40% decrease), <i>GR</i> (8% increase), and <i>PPO</i> (93% decrease) as well as anthocyanin biosynthesis genes <i>C4H</i> (56% decrease) and <i>CHS</i> (87% decrease) were significantly downregulated at the highest Pb concentration in the later stages, indicating a critical adaptation phase. Observed gene expression fluctuations in the later stages may result from <i>A. caroliniana's</i> dynamic stress response, where initial upregulation of antioxidant defense genes suggests an attempt to mitigate oxidative stress, followed by metabolic adjustments leading to variations in gene expression levels. Lead uptake peaked on day 2 but significantly declined by 42% on day 6, likely due to cellular saturation, activation of detoxification mechanisms, or lead translocation into the growth medium. These findings highlight the potential of <i>A. caroliniana</i> as an effective phytoremediator for Pb-contaminated water bodies.