فصلنامه
نوع مقاله : مقاله پژوهشی
نویسندگان
گروه مهندسی محیطزیست، دانشکده محیطزیست، دانشگاه تهران، تهران، ایران
کلیدواژهها
عنوان مقاله English
نویسندگان English
Introduction
Cadmium (Cd) and lead (Pb) contamination of calcareous soils can restrict wheat production in arid and semi-arid regions. High pH and calcium carbonate reduce metal solubility but cannot fully prevent root uptake or food-chain entry. Both metals impair nutrient acquisition, damage chloroplasts and membranes, reduce photosynthesis, and promote reactive oxygen species (ROS) production, causing oxidative damage and redox imbalance. Thiols mediate plant responses to metal stress. Cysteine (Cys) supplies sulfur for glutathione (GSH) synthesis, while glutathione supports redox buffering, antioxidant reactions, metal detoxification, and phytochelatin production. Evidence for simultaneous foliar application of cysteine and glutathione under Cd–Pb stress in wheat grown in calcareous soil remains limited. This study examined the effects of individual and combined thiol sprays on wheat growth, oxidative damage, glutathione redox status, and Cd and Pb accumulation under Cd–Pb co-contamination.
Materials and Methods
A pot experiment was conducted in a greenhouse at the Faculty of Agriculture, Ferdowsi University of Mashhad. Calcareous clay loam soil was collected from the 0–30 cm layer of agricultural land near Sabzevar, Iran. Soil pH was 7.89 (1:2.5 soil:water), electrical conductivity 2.53 dS m⁻¹, organic matter 1.03%, and calcium carbonate equivalent 24.55%. Diethylenetriaminepentaacetic acid (DTPA)-extractable Cd and Pb were 0.12 and 1.5 mg kg⁻¹, respectively, representing low background levels before contamination.
The experiment used a randomized design with five treatments and three replications (15 pots): control without metals, sprayed with deionized water; heavy metals (HM), with 15 mg Cd kg⁻¹ soil and 300 mg Pb kg⁻¹ soil; HM+Cys, with 0.41 mM L-cysteine foliar spray; HM+GSH, with 2 mM reduced glutathione; and HM+Cys+GSH, receiving 0.41 mM cysteine plus 2 mM glutathione. L-cysteine and reduced glutathione (≥99% purity) were obtained from Sigma–Aldrich.
Cadmium and Pb were added as Cd(NO₃)₂ and Pb(NO₃)₂ to 5 kg soil per pot. Nitrate input was identical among contaminated treatments and considered a common HM component. Soils were mixed and incubated for four weeks near field capacity before planting. Wheat seeds (Triticum aestivum L. cv. Falat) were disinfected in 5% sodium hypochlorite for 10 min, rinsed three times with deionized water, and sown in pots 23 cm in diameter and 21.5 cm high. Eight seeds per pot were thinned to five plants after establishment. Foliar treatments were applied at tillering, stem elongation, and booting; controls received equal volumes of deionized water.
Plants were harvested about 70 days after sowing. Shoot dry biomass was measured after oven drying at 70 °C for 72 h. Total chlorophyll was determined in 80% acetone extracts at 663 and 645 nm following Lichtenthaler (1987). Malondialdehyde (MDA) and Hydrogen peroxide (H₂O₂) were measured at 532 and 390 nm, respectively. Reduced glutathione and oxidized glutathione (GSSG) were assayed in 2% metaphosphoric acid extracts using glutathione reductase, reduced nicotinamide adenine dinucleotide phosphate (NADPH), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and 2-vinylpyridine; the GSH/GSSG ratio was used to assess redox status.
Root and shoot samples were wet-digested with concentrated HNO₃ and H₂O₂, and Cd and Pb concentrations were determined by flame atomic absorption spectrometry (AAS) (Analyst 800, PerkinElmer). Oxidative stress index (OSI), total thiol pool, tolerance index, chlorophyll protection efficiency, and transfer factor were calculated. Data were analyzed by one-way analysis of variance (ANOVA), and means were compared using Duncan’s multiple range test.
Results and Discussion
Combined Cd and Pb stress substantially reduced wheat growth. Shoot dry biomass declined from 12.50 g pot⁻¹ in the control to 7.10 g pot⁻¹ under HM, a 43% reduction. Total chlorophyll decreased from 2.10 to 1.10 mg g⁻¹ fresh weight (47%). All thiol treatments improved both traits. Shoot biomass reached 8.60, 9.60, and 11.00 g pot⁻¹ in HM+Cys, HM+GSH, and HM+Cys+GSH, respectively, while chlorophyll increased to 1.50, 1.70, and 1.90 mg g⁻¹ fresh weight. Tolerance index and chlorophyll protection efficiency showed similar responses. The combined treatment produced the highest values among metal-stressed plants, although neither trait fully reached control values. Oxidative damage increased sharply under HM. MDA increased from 15.00 to 40.00 nmol g⁻¹ fresh weight, while H₂O₂ increased from 4.30 to 12.20 µmol g⁻¹ fresh weight. Cysteine, glutathione, and their combination reduced both indices. MDA concentrations were 32.00, 27.00, and 22.00 nmol g⁻¹ fresh weight in HM+Cys, HM+GSH, and HM+Cys+GSH, respectively; corresponding H₂O₂ values were 9.20, 7.50, and 6.20 µmol g⁻¹ fresh weight. The OSI increased from 0.05 in the control to 0.37 under HM. Cysteine reduced it to 0.17, whereas glutathione alone and the combined treatment both reduced it to 0.10. Therefore, the combined treatment did not exceed glutathione alone for this index, showing that treatment superiority differed among traits.
Cd and Pb strongly affected glutathione redox status. GSH declined from 85.30 to 32.90 nmol mg⁻¹ protein under HM, whereas GSSG increased from 15.20 to 31.00 nmol mg⁻¹ protein. Accordingly, the GSH/GSSG ratio declined from 5.61 to 1.07. Thiol application improved redox status in all treated plants. HM+GSH produced the highest GSH concentration, whereas HM+Cys+GSH produced the highest GSH/GSSG ratio. Under the combined treatment, GSH reached 62.70 nmol mg⁻¹ protein, GSSG decreased to 10.50 nmol mg⁻¹ protein, and the GSH/GSSG ratio reached 6.00. This ratio differed significantly from all stressed treatments but not from the control, indicating near-control redox restoration. Thiols also reduced Cd and Pb accumulation. Under HM, Cd concentrations were 19.0 mg kg⁻¹ in roots and 7.6 mg kg⁻¹ in shoots; under HM+Cys+GSH, they decreased to 10.4 and 4.0 mg kg⁻¹, respectively. Root Pb declined from 25.5 to 12.5 mg kg⁻¹, while shoot Pb declined from 9.8 to 5.5 mg kg⁻¹. Cysteine and glutathione alone also lowered both metals, although differences between HM+GSH and HM+Cys+GSH for shoot Cd and Pb were often not significant.Transfer factors for Cd and Pb remained below 0.5, indicating limited root-to-shoot translocation. Roots retained a substantial proportion of absorbed metals, reducing but not eliminating accumulation in shoots.
Conclusion
Cd–Pb co-contamination reduced wheat shoot biomass and chlorophyll, increased MDA and H₂O₂, and disrupted glutathione redox balance. Foliar cysteine and glutathione alleviated these effects. Their combined application produced the strongest response for most growth, oxidative, redox, and tissue-metal traits, improving biomass and chlorophyll, reducing oxidative damage, restoring GSH/GSSG to a control-comparable value, and lowering Cd and Pb in roots and shoots. Transfer factors remained below 0.5, indicating limited root-to-shoot translocation. Field validation and grain-metal measurements are required before recommending this treatment for food production.
Acknowledgement
The authors acknowledge the Faculty of Agriculture, Ferdowsi University of Mashhad, for providing greenhouse and laboratory facilities for this study.
کلیدواژهها English
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