Alleviation of lead toxicity and enhancement of wheat (Triticum aestivum L.) yield and antioxidant capacity using silicon and humic acid amendments in contaminated soil

Abstract

Lead (Pb) contaminates agricultural soils, endangering human health, plant life, and the global food supply. Several strategies have been explored to mitigate Pb toxicity. Silicon (Si) can be used to increase crop production and sustain crop growth under heavy-metal stress. A field trial was conducted to assess the influence of two Si fertilizer sources on wheat yield, Pb accumulation, and antioxidant activities in a Pb-contaminated field. Si was applied as Ca silicate and burnt rice husk at 75 and 150 kg ha⁻¹, respectively, either individually or in combination with humic acid (10 mg kg⁻¹ of soil), as compared with the control (Pb-contaminated soil only). The addition of both Si sources reduced Pb stress; however, Ca silicate at 75 kg ha⁻¹ + humic acid increased the plant height (23.22%), total chlorophyll content (24.16%), and grain yield (32.20%) compared with the control. Notably, both Si sources reduced Pb accumulation in roots as well as Pb translocation to the shoots and grains. Both Si fertilizers also reduced oxidative stress in wheat and increased superoxide dismutase and peroxidase activities. However, the outcome of synthetic Si fertilizer (Ca silicate + humic acid) was more pronounced than that of natural Si fertilizer (burnt rice husk + humic acid). Significant reductions of 22.39%, 25.19%, and 46.55% in Pb concentrations were observed in the roots, shoots, and grains, respectively, in response to synthetic Si fertilizer. Thus, the use of Si fertilizer (Ca silicate at 75 kg ha⁻¹ + humic acid) was a more viable strategy for enhancing wheat productivity by improving the antioxidant defense system and reducing Pb accumulation in food crops.

Study showed that Si amendments, particularly Ca silicate with humic acid, improved wheat yield while reducing the Pb accumulation in roots, shoots and grains by enhancing the antioxidant activities under Pb contaminated soil conditions.

1. Introduction

Heavy metal contamination has become a global environmental hazard due to industrial and agricultural activities.¹ Among various toxic metals, lead (Pb) is one of the most concerning metals because of its high persistence and toxicity.² It is a major contributor to environmental contamination and occurs primarily as a result of human activities. Pb-contaminated soil has unfavorable effects on plants as well as humans in multiple ways. It can disrupt hemoglobin production; long-term Pb exposure can cause renal problems, high blood pressure in adults, and deficits in children's natural and psychological development.³

Lead (Pb) is strongly adsorbed by soil particles;⁴ however, changes in soil physicochemical properties, including fluctuations in soil organic matter breakdown, pH and moisture content, can alter Pb solubility and enhance Pb uptake by plants.⁵ Elevated Pb concentrations (80–240 mg kg⁻¹) in soils may disrupt healthy plant growth, limit photosynthesis, and alter plant water and nutrient stability.⁶ Pb enters humans via the food chain and damages the brain and digestive system, ultimately causing major health and safety concerns.⁷ Pb has a threshold level of 30 mg kg⁻¹ (reported as a reference value) in many agricultural soils; however, Pb guidelines and critical concentrations vary among jurisdictions, and elevated Pb concentrations may adversely affect root and shoot growth, chlorophyll content, and plant productivity.^8,9 Many important metabolic processes in plants grown in Pb-contaminated soil are disrupted, resulting in reduced photosynthesis, root and shoot growth, and biomass production.¹⁰ Furthermore, metal toxicity limits physiological events, such as photosynthesis, respiration, transpiration, and cell elongation, resulting in reduced biomass.¹¹ Pb toxicity delays plant growth and causes oxidative stress.¹²

Silicon (Si) is one of the most prevalent elements in the Earth's crust and accounts for 60–70% of the soil mass as silicates.¹³ Although Si is not essential for plant growth and development, it protects plants from abiotic stressors and diseases.¹⁴ Latest studies have highlighted the potential of Si fertilizers as climate-smart agricultural inputs capable of improving crop resilience under changing environmental conditions.¹⁵ Si has been identified as a valuable element because of its immediate effects on the development of several plant species and its ability to reduce metal toxicity.¹⁶ Reduced Pb toxicity from Si application is associated with lower Pb absorption and oxidative stress,¹⁷ as well as increased uptake of important nutrients, such as N, P, Zn, Fe, Mg, and Mn, and enhanced antioxidant capacity. These mechanisms work together to protect photosynthesis from the effects of Pb, thereby increasing biomass.¹⁸ Si also helps plants cope with the negative effects of metals through various mechanisms, including changes in metal uptake. It has a strong capability to convert soluble and replaceable metals in soil into insoluble chemical complexes through several mechanisms, including reduced translocation, metal requisite to cell walls, and coprecipitation with Si.¹⁹ Si also stimulates root exudates, which chelate metals and reduce metal absorption by plants.²⁰

Wheat (Triticum aestivum L.) is the primary food crop for more than half of the global population.²¹ It is an excellent source of vital nutrients, such as protein, vitamins (vitamin B), phytochemicals, and dietary fiber.²² Limited information is available on the use of natural and synthetic Si fertilizers under field conditions to reduce Pb toxicity in wheat by decreasing Pb mobility in soil. Therefore, in the current study, we hypothesized that Si fertilizers could effectively reduce Pb toxicity in wheat by decreasing Pb accumulation, improving plant development and antioxidant capacity, and lowering human health risks. The current study aimed to assess the role of two Si fertilizer sources in (1) improving wheat productivity, (2) reducing Pb uptake and accumulation in plant tissues, and (3) strengthening the antioxidant defense system of wheat under Pb-contaminated soil conditions. The findings of this study will contribute to the development of sustainable nutrient management strategies aimed at mitigating heavy-metal stress, improving wheat productivity, and reducing Pb accumulation in wheat grain.

2. Materials and methods

2.1. Field selection and characterization of Pb-contaminated soil

The current experiment was performed in a farmer's field (Malik Zarai Farm, Industrial Area, Faisalabad, Punjab, Pakistan) during the winter season of 2021–22. Soil contamination may arise from several sources, such as fertilizers, irrigation water, and pollution from nearby factories and cities. In addition, three chemical production facilities are located within 5 km of the test site and may be sources of contamination. The soil had been contaminated by municipal and industrial sewage sludge for 8 years. The Pb concentration in the soil was 303 mg kg⁻¹. The physicochemical properties of the soil are described in Table 1. Soil samples were collected from a depth of 0–20 cm from the experimental plots. Soil samples were collected from three different points within each plot and thoroughly homogenized to obtain a representative composite sample for analysis. The reported values in Table 1 are presented as mean ± standard deviation. Various soil properties were measured using standard procedures. The experimental soil had a pH of 5.80, an EC of 725 µS cm⁻¹, dissolved organic C of 25.34 mg kg⁻¹, and a sandy clay loam texture. Fig. 1 depicts the daily maximum and minimum temperatures and rainfall distribution during the trial. The total rainfall during the wheat growing season was 97.04 mm.

Table 1. Basic properties of the lead (Pb)-contaminated field.

Characteristics	Values
Soil texture class	Clay loam
Sand (%)	43
Clay (%)	26
Silt (%)	31
Soil pH	5.80 ± 0.41
EC (µS cm−1)	725 ± 4.70
C : N ratio	35.45 ± 0.28
Dissolved organic C (mg kg−1)	25.34 ± 2.45
Water holding capacity (g g−1)	0.19 ± 0.04
Available N (mg kg−1)	22 ± 0.88
Available P (mg kg−1)	3.71 ± 0.74
Available K (mg kg−1)	109.24 ± 2.85
Total lead (Pb) (mg kg−1)	303 ± 10.35

Fig. 1. Climatic factors in the experimental region during the growing period of 2021–2022. The existing climatic statistics were obtained from weather station 416 300 (latitude: 31.43; longitude: 73.1; elevation: 184 m).

2.2. Experimental design and treatments

The field trial was conducted using a randomized complete block design (RCBD) with four blocks (field replicates). Each treatment was assigned to one plot within each block, resulting in four independent field plots per treatment. Each field plot was considered the experimental unit for all agronomic, yield, Pb uptake, and biochemical measurements. The present study consisted of five distinct amendment regimes: T₁: control (Pb-contaminated soil only), T₂: T₁ + Ca silicate as a source of Si at 75 kg ha⁻¹, T₃: T₁ + burnt rice husk as a source of Si at 150 kg ha⁻¹, T₄: T₁ + Ca silicate as a source of Si at 75 kg ha⁻¹ + humic acid at 10 mg kg⁻¹ of soil, and T₅: T₁ + burnt rice husk as a source of Si at 150 kg ha⁻¹ + humic acid at 10 mg kg⁻¹ of soil. Si fertilization with calcium silicate contained 80% SiO₂, 12% CaO, 2% MgO, and 6% other trace minerals, with no detectable Pb. Burnt rice husk (BRH) contained 75% SiO₂, 2.95% K₂O, 0.82% CaO, 0.36% MgO, 0.79% Fe₂O₃, 0.59% P₂O₅, 0.52% MnO and 18.97% unreported constituents. The BRH had a pH of 7.8, a total calcium carbonate equivalent of 9.86%, and an effective calcium carbonate equivalent of 6.27%, indicating a slightly alkaline reaction and measurable, although relatively modest, liming potential. It was prepared by limited-oxygen pyrolysis at 600 °C for 60 minutes. The material was crushed, sieved (less than 2 mm), and kept in closed containers until use. To avoid phytotoxicity, two application rates of Si (inorganic: 75 kg ha⁻¹ and organic: 150 kg ha⁻¹) were selected from earlier studies, wherein these rates were demonstrated to be physiologically and biochemically relevant for examination and suitable for field trials. The humic acid used was purchased from Syngenta® under the name “Enrich,” and it contained humic acid (400 g kg⁻¹) and potash (70 g kg⁻¹). As the humic acid product also contained potash, the responses of the humic-acid-containing treatments may reflect the combined contribution of humic acid and the accompanying potash. Humic acid was incorporated into selected amendment regimes to evaluate the combined response of the corresponding amendment treatments on Pb immobilization and wheat performance. The required quantity of humic acid was calculated based on the net plot area (5 × 2 m = 10 m²), incorporation depth (0–20 cm), and soil bulk density (1.25 g cm⁻³). The soil volume within each plot was calculated as 10 m² × 0.20 m = 2.0 m³. Considering a bulk density of 1.25 g cm⁻³ (equivalent to 1250 kg m⁻³), the estimated soil mass within the incorporation layer was 2500 kg per plot. At the target humic acid application rate of 10 mg per kg soil, the required amount was therefore 2500 × 10 = 25 000 mg, equivalent to 25 g of humic acid per plot.

2.3. Experimental protocol

Seeds of the wheat variety “Faisalabad-2008” were obtained from the Ayub Agricultural Research Institute (AARI), Faisalabad, Pakistan. The natural Si fertilizer source was burnt rice husk, while the synthetic Si fertilizer source was calcium silicate. Plots were demarcated and cleared of debris prior to seedbed preparation. Two to three cultivations were performed, and planking was done to prepare the seedbed. The required amounts of Ca silicate, burnt rice husk and humic acid were calculated for each plot and thoroughly mixed with the soil. These amendments were evenly spread over the plots and incorporated into the top 0 to 20 cm of soil using a rotavator. The soil was moistened to field capacity and incubated for 7–14 days prior to sowing. A 0.5 m buffer distance was maintained between plots to provide physical separation between treatments. Humic acid and other amendments were weighed separately for each plot and applied uniformly within the boundaries of their respective treatment plots. During application and soil incorporation, particular care was taken to prevent the amendment material from being displaced into adjacent plots. Soil incorporation was restricted to the designated plot area, thereby minimizing lateral movement of amended soil between treatments. The seeds (100 kg ha⁻¹) were planted in rows 22.5 cm apart. The net plot size was 5 × 2 m². At planting, basal fertilizers were equally applied to all plots at the rates of 50 kg ha⁻¹ for phosphorus, 25 kg ha⁻¹ for nitrogen and 30 kg ha⁻¹ for potassium. At the beginning and end of tillering, two doses of urea (25 kg ha⁻¹) were applied. Wheat was sown on November 24, 2021, and harvested on April 22, 2022, after maturity. Weeds were treated using both pre- and post-emergence herbicides, while insects were controlled using best management practices for wheat. Irrigation was done as per the requirements of each crop.

2.4. Measurement of chlorophyll content and agronomic parameters

The total chlorophyll content was determined spectrophotometrically using fresh leaf samples weighing 0.2 g, which were cut into small pieces and thoroughly ground in a mortar and pestle with 5 to 10 mL of 80% acetone to extract the pigments. Next, the homogenate was centrifuged at 3000 to 5000 rpm for 5 to 10 minutes (or filtered through Whatman No. 1 paper), the clear supernatant was collected, the remaining pellet was washed with additional solvent, and the final combined extract volume (V) was brought up to 10 mL or 25 mL using 80% acetone. Finally, the spectrophotometer was blanked with pure 80% acetone, sample absorbance was recorded at 645 nm and 663 nm, and the total chlorophyll concentration was calculated using the formula:Total chlorophyll content (mg g⁻¹) = [20.2(A₆₄₅) + 8.02(A₆₆₃)] × V/(1000 × W)where W represents the fresh tissue weight in grams.

When the crop matured, harvesting of grains was done. The collected samples were air-dried and then placed in an oven at 65 ± 5 °C for 72 hours and ground using a grinding mill. The agronomic attributes were measured following standard practices and included plant height (cm), spike length (cm), number of grains per spike, 1000-grain weight (g) and grain yield (kg ha⁻¹).

2.5. Determination of Si concentration in the roots and shoots of wheat

Roots and shoots were collected at physiological maturity (final harvest) for the determination of metal concentrations. The Si content was extracted from wheat using the Tiron extraction method.²³ To summarize, 50 mg of each plant sample (roots or shoots) was combined with 30 mL of 0.1 M Tiron [4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt] at a pH of 10.5. The polypropylene tubes were then placed in an 85 °C water bath and agitated horizontally for two hours. Once the tubes had cooled, 10 mL of the mixture was transferred to previously labeled 40 mL vials, and 10 mL of 30% H₂O₂ was added to remove the Tiron. The resulting solution was re-shaken in a water bath at 85 °C for about 30 minutes until the colour faded. The Si concentration was determined following filtration through a 0.2 µm filter by inductively coupled plasma optical emission spectroscopy (Jobin-Yvon, Ultima-C).

2.6. Determination of Pb concentrations in the roots, shoots and grains of wheat

At maturity, wheat plants were carefully uprooted, and roots were separated from the shoots. Roots were thoroughly washed with tap water, followed by deionized water, while shoots and mature grains were rinsed with deionized water to remove surface contaminants. Grains were separated from spikes and dried at 65–70 °C to a constant weight, and all tissues were finely ground. A 0.25 g sample was digested with 4 mL of concentrated HNO₃ (70%; Fluka, Buchs, Switzerland) at 95 °C for 6 h in a digestion block. After cooling, the digests were filtered through 0.2 µm cellulose nitrate membranes (Whatman) and diluted to 100 mL with ultrapure water. Procedural blanks were processed with each digestion batch. Pb concentrations were determined using a Shimadzu AA-6200 atomic absorption spectrophotometer equipped with a Pb hollow-cathode lamp and operated in flame atomization mode. Calibration was performed using certified Pb standards covering the expected concentration range, with calibration blanks and verification standards included to monitor analytical stability. Pb concentrations were calculated on a dry-weight basis and expressed as mg kg⁻¹.

2.7. Calculation of electrolyte leakage (%)

The method described in ref. 24 was applied to calculate the electrolyte leakage. After 21 days, fully grown upper leaves were cut into 5 mm lengths and transferred to test tubes filled with 8 mL of deionized and sanitized water. The initial electrical conductivity of the medium (EC₁) was recorded after two hours of incubation in a water bath incubator at 32 °C. Samples were then autoclaved for 20 minutes at 121 °C to remove all electrolytes and subsequently cooled to 25 °C. The electrical conductivity (EC₂) was then calculated again and recorded as per the following equation:EL = EC₁/EC₂ × 100

2.8. Antioxidant activities

The leaf samples were crushed in a pestle and mortar in a phosphate buffer solution (pH 7.8, 0.01 M) and centrifuged at 13 000×g for 10 min. Following centrifugation, the supernatant was tested for the presence of antioxidant enzymes using the following procedure. Superoxide dismutase (SOD) activity was assessed in a reaction solution containing 20 µM riboflavin, 130 mM methionine, 75 µM NBT, 100 µM EDTA-NA2, and an enzyme extract. The absorbance was recorded at 560 nm. Peroxidase (POD) activity was measured according to the procedure outlined in ref. 25. A 25-µL enzyme extract was combined with 1% guaiacol (v/v) and 10 mM H₂O₂ in 50 mM Tris buffer (pH 7.0) to make a total volume of 750 µL. Differences in absorbance related to guaiacol oxidation (ε = 25.5 mM⁻¹ cm⁻¹) were recorded at 470 nm.

2.9. Statistical analysis

The statistical analysis of the collected data was performed using Statistix 10.0 software. Analysis of variance (ANOVA) was performed according to the randomized complete block design, with treatment considered a fixed effect and block/replication included in the model to account for field variability. The individual field plot was considered the experimental unit, with four independent field replicates per treatment. The effects of Si treatments on wheat growth, yield, Pb accumulation, and biochemical attributes were evaluated. Treatment means were compared using Tukey's HSD test at p ≤ 0.05.²⁶ For biochemical measurements, samples from each of the four field plots per treatment represented independent biological replicates. Each sample was measured in triplicate for analytical precision, and the three technical measurements were averaged to generate one plot-level observation. Therefore, technical triplicates were not considered independent replicates in the statistical analysis. Data were graphically presented using SigmaPlot 12.5 and BioRender software.

3. Results

3.1. Growth and yield attributes of wheat

Both Si fertilizer sources enhanced wheat growth and yield (Fig. 2A–E). The tallest plants and longest spikes were observed under T₄, followed by T₂. However, the lowest values for plant height (66.03 cm) and spike length were observed under T₁ (control) (Fig. 2A). In terms of grain attributes, significant increases in the number of grains spike⁻¹ (80), 1000-grain weight (35.65 g), and grain yield (2814.3 kg ha⁻¹) were recorded under T₄, followed by T₂ and T₅. However, the synthetic Si fertilizer (T₄) resulted in 10%, 9%, and 4.05% increases of number of grains spike⁻¹, 1000-grain weight, and grain yield, respectively, compared with those obtained with the natural Si fertilizer (T₅). Compared with T₁ (control), T₄ resulted in significant increases of 25.86% and 32.20% in 1000-grain weight and grain yield, respectively.

Fig. 2. Effects of synthetic and natural Si fertilizer sources on wheat plant height (A), spike length (B), number of grains per spike (C), 1000-grain weight (D), grain yield (E), and total chlorophyll content (F) under Pb contamination. Four biological replicates were used, and the data are presented as mean ± SE (n = 4). Different letters indicate statistically significant differences among treatments according to ANOVA, followed by the HSD test at P < 0.05. T₁: control (Pb-contaminated soil only), T₂: T₁ + Ca silicate as a source of Si at 75 kg ha⁻¹, T₃: T₁ + burnt rice husk as a source of Si at 150 kg ha⁻¹, T₄: T₁ + Ca silicate as a source of Si at 75 kg ha⁻¹ + humic acid at 10 mg kg⁻¹ of soil, and T₅: T₁ + burnt rice husk as a source of Si at 150 kg ha⁻¹ + humic acid at 10 mg kg⁻¹ of soil.

3.2. Chlorophyll and Si uptake

The application of synthetic Si fertilizer significantly improved the total chlorophyll content (Fig. 2F). T₄ had the highest total chlorophyll content (1.862 mg g⁻¹), followed by T₂, with a total chlorophyll content of 1.782 mg g⁻¹. The total chlorophyll content of T₄ increased significantly by 24.16% compared with that of the control (T₁). As expected, both Si fertilizers considerably augmented the Si concentration in the roots and shoots of wheat (Fig. 3A and B). The Si concentration in the shoots increased significantly with both Si sources (synthetic and natural), with synthetic Si fertilizer being more effective than natural Si fertilizer. Among all tested treatments, T₄ had the highest Si concentrations in shoots (6.04 g per kg DW) and roots (6.28 g per kg DW), followed by T₂ and T₅. Notably, the Si concentrations in the shoots (61.42%) and roots (63.60%) were substantially greater in T₄ than in the control (T₁).

Fig. 3. Effects of synthetic and natural Si fertilizer sources on wheat Si concentration in roots (A), Si concentration in shoots (B), Pb concentration in roots (C), Pb concentration in shoots (D), and Pb concentration in grains (E) under Pb contamination. Four biological replicates were used, and the data are presented as mean ± SE (n = 4). Different letters indicate statistically significant differences among treatments according to ANOVA, followed by the HSD test at P < 0.05. T₁: control (Pb-contaminated soil only), T₂: T₁ + Ca silicate as a source of Si at 75 kg ha⁻¹, T₃: T₁ + burnt rice husk as a source of Si at 150 kg ha⁻¹, T₄: T₁ + Ca silicate as a source of Si at 75 kg ha⁻¹ + humic acid at 10 mg kg⁻¹ of soil, and T₅: T₁ + burnt rice husk as a source of Si at 150 kg ha⁻¹ + humic acid at 10 mg kg⁻¹ of soil.

3.3. Pb concentrations in roots, shoots, and grains

A significant reduction in the Pb concentrations in plant roots, shoots, and grains was noted under both synthetic and natural Si fertilizer sources (Fig. 3C–E). The Pb concentrations increased in the roots, shoots, and grains in response to the Pb toxicity in the soil. The lowest Pb concentrations in the roots (14.21 mg per kg DW), shoots (0.950 mg per kg DW), and grains (0.031 mg per kg DW) were detected under T₄. Compared with T₁, T₄ resulted in 43.74% and 41.89% decreases in the Pb concentration in the roots and shoots, respectively, indicating a significant positive impact of Si application under Pb stress.

3.4. Electrolyte leakage and antioxidant activities

Both Si sources had a significant effect on electrolyte leakage in wheat (Fig. 4A). The highest electrolyte leakage was observed when no Si fertilizer source was applied (T₁). In contrast, both natural and synthetic Si fertilizer sources reduced the electrolyte leakage; however, the lowest value (29.50%) was recorded under T₄. The SOD and POD activities of wheat treated with Si fertilizers increased to different degrees (Fig. 4B and C). Among all tested treatments, T₄ had the highest SOD (306.75 units per g FW) and POD (1310.8 units per g FW) activities, followed by T₂. However, significant reductions in both the SOD and POD activities were observed under T₁ (control). Importantly, compared with T₁ (control), the SOD and POD activities in T₄ were significantly greater (by 32.84% and 39.57%, respectively).

Fig. 4. Effects of synthetic and natural Si fertilizer sources on wheat: (A) electrolyte leakage and (B) superoxide dismutase (SOD), and (C) peroxidase (POD) activities under Pb contamination. Four biological replicates were used, and the data are presented as mean ± SE (n = 4). Different letters indicate statistically significant differences among treatments according to ANOVA, followed by the HSD test at P < 0.05. T₁: control (Pb-contaminated soil only), T₂: T₁ + Ca silicate as a source of Si at 75 kg ha⁻¹, T₃: T₁ + burnt rice husk as a source of Si at 150 kg ha⁻¹, T₄: T₁ + Ca silicate as a source of Si at 75 kg ha⁻¹ + humic acid at 10 mg kg⁻¹ of soil, and T₅: T₁ + burnt rice husk as a source of Si at 150 kg ha⁻¹ + humic acid at 10 mg kg⁻¹ of soil.

3.5. Correlation analysis

The Pearson correlation analysis of treatment means (n = 5 treatments) revealed significant relationships among all tested traits of wheat (Fig. 5). Plant height (PH), number of grains per spike (NGPS), spike length (LOS), 1000-grain weight (KGW), grain yield (GY), and total chlorophyll content (TCC) were positively correlated with Si accumulation in the shoots (SiS) and roots (SiR). In contrast, Pb accumulation in the roots (PbR), shoots (PbS), and grains (PbG) showed significant negative correlations with these growth and yield attributes. Electrolyte leakage (EL) was positively correlated with Pb accumulation and negatively correlated with growth, yield, chlorophyll content, and Si accumulation. Antioxidant enzymes, such as peroxidase (POD) and superoxide dismutase (SOD), were negatively correlated with Pb accumulation and EL, suggesting that enhanced antioxidant defenses were associated with reduced Pb-induced oxidative stress and membrane damage. The results suggest that increased Si accumulation was associated with improved growth, physiological performance, and yield, whereas elevated Pb accumulation and membrane damage adversely affected wheat productivity.

Fig. 5. Correlation matrix showing the relationships among agronomic characteristics (PH, LOS, NGPS, KGW, GY), chlorophyll content (TCC), Si accumulation (SiS, SiR), Pb accumulation (PbS, PbG, PbR), electrolyte leakage (EL), and antioxidant enzyme activities (SOD, POD). The correlation coefficient determines the colour intensity, with positive correlations represented by purple and negative correlations by brown. *p < 0.05, **p < 0.01, ***p < 0.001.

4. Discussion

Currently, metal stress is one of the main ecological problems facing agricultural soils, resulting from geological sources, extensive industrialization, and specific farming practices.²⁷ In addition, human activities, such as mining and smelting,²⁸ rapid industrialization,²⁹ irrigation with untreated wastewater, atmospheric deposition, waste disposal,³⁰ and the use of pesticide and fertilizer, are major contributors to the increasing concentrations of various toxic pollutants in soil.³¹ According to ref. 32, plant roots can absorb Pb from soil, translocate it into vegetative tissues, and subsequently introduce it into the food chain, eventually transferring it to consumers and posing health risks to humans.

Lead (Pb) contamination significantly inhibited wheat plant growth. Both Si sources significantly improved wheat growth, as indicated by plant height and spike length, thereby mitigating the adverse effects of Pb stress. The reduced plant growth under Pb stress may be attributed to impaired nutrient uptake and oxidative damage.² Likewise, several physiological processes in plants are adversely affected by Pb stress.^17,33 The observed decreases in plant height and spike length induced by heavy-metal stress were consistent with earlier research on other crops, including sunflower,³⁴ maize,³⁵ and chickpea.³⁶ Similarly, ref, 37 reported that Pb stress substantially reduced plant height and shoot length in cotton plants at both tested Pb concentrations (50 and 100 µM) compared with the control, whereas Si supplementation improved cotton growth under Pb stress.

In our study, the increase in wheat yield and its attributes observed with Si application may have been due to improved photosynthetic activity and nutrient availability. The increase in 1000-grain weight with Si supplementation may be associated with increased photosynthetic activity and efficient translocation of photosynthates. This may have enhanced carbohydrate accumulation and increased the number of filled grains, ultimately contributing to higher grain yield. Si application may increase grain production in heavy-metal-stressed plants because it affects the absorption and transport of many mineral elements that are essential for vital plant functions.³⁸ Our findings were consistent with those of ref. 39, who reported that Si play a significant role in increasing the grain number per rice panicle. Compared with the control plants, the Si-treated plants produced more grain because of improvements in growth, photosynthetic capacity, and nutritional balance. Grain yield and biomass production are adversely affected by heavy-metal stress in major field crops.^40,41

In the present study, the application of both tested Si sources increased the chlorophyll index in plants under Pb stress. Si treatment may have contributed to the increas in the chlorophyll index of wheat plants under Pb stress, thereby supporting photosynthetic activity. The increase in photosynthesis may be due to reduced oxidative stress and improved nutrient availability.^42,43 Additionally, Si may actively contributes to improving crop light-use efficiency, which may enhance the chlorophyll content in wheat.^40,44 Previous studies have reported that chlorophyll formation is greatly reduced by heavy-metal stress.^45,46 The concentration of Si in the shoots and roots of wheat plants increased with increasing Si addition in Pb-contaminated soil. Si likely mitigated the adverse effects of Pb on wheat plants, and higher concentrations of Si absorbed by plant roots may have resulted in greater Si translocated to the shoots, possibly through xylem-mediated transport.²¹ These findings are consistent with previous studies, indicating that the addition of Si in the form of NPs increases the Si concentration in plants.^42,47 Similarly, increasing Si concentrations in the growing medium increased Si accumulation in plant roots and leaves.⁴³

Our results indicated that the application of both Si sources reduced Pb accumulation in wheat roots, shoots, and grains. Moreover, Pb translocation from roots to the aerial parts (shoots and grains) of wheat was inhibited by Si fertilizer sources. The possible mechanisms by which Si inhibits metal transport in plants can be described mainly in two ways: first, Si reduces metal transfer from roots to shoots by promoting lignin deposition in cell walls and enhancing the immobilization of metal ions within cell walls;⁴⁸ second, Si co-precipitates with hazardous metal ions or forms complexes with them.⁴⁹ Similarly, ref. 50 reported that, compared with control plants, Pb concentrations in rice grains were 21.3–40.9% lower, while those in shoots were 28.6–54.0% lower. Our findings are corroborated by ref. 51, who established that Si supplementation prevents the translocation of Pb from rice roots to aboveground parts and reduces Pb toxicity in rice grains.

Heavy-metal stress causes oxidative injury in plants by enhancing ROS production.⁵² In the present study, electrolyte leakage (EL) decreased in response to Si sources, and the reduction was more distinct with the synthetic Si fertilizer source. By preserving cell membrane integrity under heavy-metal stress, Si reduces EL levels.⁵³ Plants have established a self-defense system that includes antioxidant enzymes, among other mechanisms, to counteract oxidative stress and other types of stresses.⁵⁴ According to ref. 37, Pb stress leads to a marked increase in membrane damage and EL, whereas Si treatment reduces EL at both Pb concentrations (50 µM and 100 µM).

In the present study, a decrease in SOD and POD activities was observed under Pb stress. The direct interaction between heavy metals and proteins may be responsible for this decline in enzyme activity, resulting in impaired enzyme function, as proposed by ref. 55. The use of Si greatly increased POD and SOD activities. The decrease in antioxidant activities without Si may be due to greater oxidative stress in the plants, as indicated by the increased EL in wheat shoots. Consistent with our results, ref. 37 reported that the inclusion of Si had a substantial positive effect on SOD and POD activities under Pb stress. These authors proposed that adding Si may significantly improve the ability of cotton seedlings to mitigate oxidative damage under Pb stress. Si strongly increased SOD and POD activities in wheat leaves under heavy-metal stress.^17,56 However, the mechanisms underlying the effects of Si in the soil and within plant cells are illustrated in Fig. 6. These findings imply that the effects of Si on the antioxidant system in plants under heavy-metal stress may be complex, and further research is needed.

Fig. 6. Conceptual illustration showing (a) potential sources of Pb contamination in the field and Si fertilization for soil amendment; (b) proposed mechanisms by which Si may alleviate Pb-induced toxicity in wheat plants at the cellular level.

5. Conclusion

Both synthetic and natural Si fertilizers efficiently alleviated the toxic effects of Pb on wheat, mainly by promoting wheat growth and grain yield. Moreover, the addition of Si in the form of either synthetic or natural silicate protected wheat plants from Pb stress by reducing Pb uptake by roots, accumulation in shoots, and translocation to grains. Both Si fertilizer sources alleviated oxidative stress by enhancing the activities of antioxidant enzymes, such as POD and SOD. However, the effect of Ca silicate as a synthetic Si fertilizer was more pronounced than that of the natural Si fertilizer. These findings suggest that the addition of Si fertilizers to Pb-contaminated soil can help reduce Pb uptake and grain Pb accumulation while improving wheat productivity under heavy-metal stress.

Author contributions

Conceptualization: M. K. S., M. A. T.; writing – original draft: M. K. S.; A. G., M. M.; supervision: M. A. T; formal analysis: A. G., M. M.; methodology: M. A. T.; data curation: M. K. S.; writing – review & editing: H. W., B. M. A. B.; visualization: H. W., B. M. A. B.

Conflicts of interest

The authors declare no competing interests.

Funding

The authors extend their gratitude to the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Kingdom of Saudi Arabia, for funding the publication of this work under the Ambitious Researcher program (KFU265011).

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request. All data generated or analyzed during this study are included in the manuscript.