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

TL;DR

In hydroponically grown harmel, Pb stress reduced growth and increased oxidative markers, while citric acid (CA) and EDTA altered antioxidant enzymes and Pb distribution; 2.5 mM CA/EDTA generally improved tolerance, whereas 5 mM EDTA was toxic. CA-assisted phytoextraction is proposed as safer, but the authors state findings need soil-based validation.

Key findings

• Pb (5 and 25 mg L−1) reduced shoot length by 16% and 38% vs untreated; 2.5 mM CA + 5 mg L−1 Pb increased shoot and root length by 69% and 35% and shoot/root dry weight by 72% and 6% vs controls/Pb alone. At 25 mg L−1 Pb, MDA and other aldehydes rose by 36% and 76% vs control; 2.5 mM CA or EDTA lowered MDA by 33%/38% and aldehydes by 15%/12% vs Pb alone.
• CA and EDTA raised ascorbate, dehydroascorbate, glutathione, and protein. Pb5 + EDTA 2.5/5 mM increased ascorbate by 76%/99% and DHA by 160%/34% vs Pb alone; Pb5 + CA 2.5/5 mM increased ascorbate by 34%/27% and DHA by 26%/147%. Pb5 and Pb25 decreased leaf protein by 19% and 46%; Pb5 + CA5 increased protein 31% and Pb5 + EDTA2.5 increased it 28% vs Pb alone.
• Antioxidant enzymes responded differently: at 25 mg L−1 Pb, CAT, SOD, and GPX increased by 35.3%, 156.5%, and 17.8% vs control. Highest CAT/SOD occurred in Pb25 + EDTA5 (+355.7%/+174.9% over control); APX and LOX peaked in Pb5 + CA5 (+272.4% and +399.0% over control). GPX generally decreased with CA/EDTA; Pb0 + EDTA5 was 81.9% lower than Pb25 alone.
• Roots stored most Pb. Pb25 increased shoot/root Pb by 39%/226% vs Pb5. 2.5 mM CA + Pb5 reduced shoot/root Pb by 79%/14% vs control, while 2.5 mM EDTA + Pb5 increased shoot/root Pb by 317%/108% vs corresponding control. TF was highest in Pb5 + EDTA5 and exceeded one vs Pb alone. Growth traits correlated positively (r = 0.73–1.00), root Pb negatively with growth (r = −0.53 to −0.77), shoot Pb weakly (r = −0.22 to −0.46), and TF weakly/negatively (r = −0.14 to −0.28); PCA PC1/PC2 explained 36.41%/19.23%.

Methods

• Hydroponic CRD with 15 treatment combinations: Pb (0, 5, 25 mg L−1), CA (0, 2.5, 5 mM), EDTA (0, 2.5, 5 mM); n = 3 replicates, one pot with six uniform seedlings each; *P. harmala* seeds from Koshk Pb/Zn mining site, grown 40 days then treated 14 days (harvest reported at 61 days of age).
• Measured root/shoot length and dry biomass; Pb by FAAS after acid digestion; TF as shoot Pb/root Pb; MDA/aldehydes; ASC/DHA/GSH; protein by Bradford; CAT, APX, GPX, SOD, LOX activities.
• Two-way ANOVA with Tukey HSD (p < 0.05) in SPSS v26; Pearson correlations and heatmaps; PCA in Python/scikit-learn with seaborn/matplotlib.

Limitations

• Stated: hydroponic findings require validation for extrapolation to soil conditions, and chelator doses need further optimization; the conclusion calls for soil-based validation.
• Evident from text: only one species and one metallicolous population from Koshk were tested, with n = 3 biological replicates and a short 14-day treatment, limiting generalizability.
• Evident from text: some treatment labels/units are inconsistent (e.g., GSH section says “5 mM and 25 mM Pb”; Pb uptake section says “5 mg L−1 Pb soil” in a hydroponic study), which complicates interpretation.