The study was performed in an open greenhouse with an artificial rain shelter at Jiangxi Agricultural University Nanchang, China (28°46′ N, 115°36′ E) in 2024. This site has a sub-tropical humid climate with an annual temperature of 17°C and an annual precipitation of 1,741 mm. The soil for the experiment was taken from the experiment field, which has a silty loam texture with an acid pH of 5.45, total nitrogen (TN; 1.62 g kg⁻¹), and available phosphorus (AP) and potassium (AK) contents of 31.22 and 114.33 mg kg⁻¹, respectively. Pots were filled with 10 kg of dry soil, and Pb was applied according to the specific treatment plan. The source of Pb was PbCl₂, and it was mixed with and stabilized for 1 month. Thereafter, soil from every pot was taken, BC was added, and soil pots were filled again; water was applied to five 25-day-old seedlings sown in every pot. Rice straw was collected, and BC was prepared via pyrolysis of straws for 8 hours at 600°C. The biochar contained a significant amount of carbon (611 g kg⁻¹), a cation exchange capacity of 10.13 cmol kg⁻¹, and a nitrogen concentration of 3.98 g kg⁻¹ and had an alkaline pH of 9.72. Furthermore, BC was also subjected to scanning electron microscopy analysis. Biochar used in the current study showed porous structures with rough surfaces and aromatic structures with a high degree of amorphous mass disorder ( Figure 1 ).

The study included the following treatments: control, Pb stress (300 mg kg⁻¹), Pb stress (300 mg kg⁻¹) + BC (2%), Pb stress (300 mg kg⁻¹) + MT (30 µM), and Pb stress (300 mg kg⁻¹) + BC (2%) + MT (30 µM). The study was conducted in a completely randomized design (CRD) with three replications. The pots were regularly monitored, and a water depth of 3–4 cm was maintained throughout the growing period. The foliar spraying of MT was conducted after 20 days of transplanting. Foliar spraying was performed using a hand sprayer until the plants became fully wet. The plants were fully acclimatized after 20 days of transplantation, and they developed a sufficient area for the absorption of MT. Therefore, MT was applied after the 20th day of transplanting.

The plant samples were collected at the flag leaf stage (45 days after transplanting) for the analysis of physiological and biochemical traits. Leaves were collected and weighed to determine fresh weight (FW), and then they were water-soaked for 24 hours and weighed again to determine turgid weight (TW). Then, these leaves were oven-dried (24°C) and weighed to determine the dry weight (DW), and finally, relative water content (RWC) was determined with the following equation as suggested by Chattha et al. (2022): (FW − DW)/(TW − DW) × 100. For assessing electrolyte leakage (EL), rice leaves were incubated at 25°C for 30 minutes, and electrical conductivity (EC₁) was measured. Then, these leaves were again incubated for 24 hours at 90°C, EC₂ was measured, and finally, EL was determined following the procedures of Chattha et al. (2022) and using the following formula: EL = EL₁/EC₂ × 100. To determine photosynthetic pigments, 0.5-g leaf samples were ground in 80% acetone solution and centrifuged (10,000 rpm) to obtain the supernatant. Then, the concentrations of chlorophyll-a, chlorophyll-b, and carotenoid were estimated by reading absorbance at 663, 645, and 480 nm, respectively (Lichtenthaler, 1987) with the following formulas:

Here, W indicates the weight of the sample, while V is the volume of the supernatant.

Fresh leaf samples (0.5 g) were ground in trichloroacetic acid (TCA; 5%) solution and centrifuged (10,000 rpm) for 15 minutes to collect the supernatant. Subsequently, 1 mL of the supernatant was combined with 1 mL of potassium iodide buffer (PIB) and 100 µL of phosphate buffer, and the absorbance was measured at 390 nm (Velikova et al., 2000). The malondialdehyde (MDA) concentration was determined according to the protocols of Rao and Sresty (2000). Fresh leaf samples (0.5 g) were ground using TCA (5%) solution and centrifuged (15,000 rpm) for 15 minutes, and MDA was estimated by measuring absorbance (532 nm). For total soluble protein (TSP), fresh leaves (0.5 g) were ground in 5 mL of potassium phosphate buffer (PPB; 50 mM) and homogenized (12,000 rpm) for 15 minutes. Then, 3 mL of Bradford reagent and 100 µL of plant extract were combined and placed at room temperature conditions, and absorbance was measured (590 nm; Bradford, 1976). For free amino acids (FAA), the supernatant was obtained by grinding 0.5-g fresh leaves in PPB. The 1 mL of collected supernatant and 1 mL of both ninhydrin and pyridine were mixed and incubated (90°C), and absorbance (570 nm) was measured by following the procedures of Hamilton and VanSlyke (1943).

The fresh leaves (0.5 g) of rice were ground in chilled PPB (50 mM) and centrifuged (12,000 rpm) at 4°C, after which the supernatant was collected. To measure ascorbate peroxidase (APX), 100 µL of enzyme extract was taken; 700 µL of buffer (50 mM), H₂O₂ (6.1 mM), and ascorbic acid (0.5 M) was added; and later, absorbance was read at 290 nm (Asada and Takahashi, 1987). For catalase (CAT), 0.1 mL of supernatant was added with 2.5 mL of buffer and 0.1 mL of H₂O₂, and absorbance was measured at 240 nm (Chance and Maehly, 1955). For peroxidase (POD), 100 µL enzyme extract was mixed with 700 µL PPB and 100 µL H₂O₂ (300 mM), and the absorbance was measured at 470 nm to determine POD activity (Chance and Maehly, 1955). For superoxide dismutase (SOD), enzyme extract (50 μL) was mixed with 25 mL of PPB, H₂O₂ (400 µL), Triton (100 µL), and 50 µL of riboflavin and nitro blue tetrazolium (NBT), and the absorbance was measured at 560 nM (Zhang, 1992).

The plant samples were ground and digested with HNO₃:HClO₄ (2:1) on a hot plate (Jones and Case, 1990), and the Pb concentration was measured via atomic absorption spectrometry. The concentration of N was measured using the Kjeldahl procedure, whereas P was assessed using a spectrophotometer. Moreover, calcium (Ca), potassium (K), and magnesium (Mg) concentrations in roots and shoots were determined using a flame photometer. A soil and water paste (1:5) was made, and a pH meter was used to measure the soil pH. Soil organic carbon (SOC) was measured via the sulfuric acid–potassium dichromate external heating technique. The concentration of available phosphorous (AP) was measured using the sodium bicarbonate extraction procedure following the Olsen method (Olsen et al., 1982). The soil available potassium (AK) was determined via the ammonium acetate extraction technique (Helmke and Sparks, 1996). The soil TN concentration was assessed using the Kjeldahl method as suggested by Bao (2000). To determine the soil Pb concentration, the soil samples were digested with HClO₄ and HNO₃ at 160°C. Then, the samples were allowed to cool and filtered, and the Pb concentration was assessed via atomic absorption spectrophotometry. The biological accumulation coefficient (BAC) and translocation factors (TFs) were determined via the procedures of Malik et al. (2010). Different fractions of Pb were assessed via the sequential extraction technique based on the European Community Bureau of Reference (BCR) (Xiong et al., 2018). This method measures the following fractions of Pb: exchangeable, reducible, oxidizable, and residual.

Five plants were harvested at the maturity stage (90 days after transplanting) to measure growth and yield traits. The height of five plants from each pot was measured using a measuring tape, and then the average was taken. The same plants were carefully uprooted, the roots and shoots were separated, and the roots were washed carefully to remove the soil. The root length was measured in centimeters using a measuring tape. The roots were weighed on an electric balance to determine their fresh weight. Then, roots were oven-dried (65°C) until constant weight to determine their dry weight (g). The plants from the weighed pot were harvested and weighed to determine biomass yield, and later, grains were separated and weighed to determine the grain yield. Both grain and biomass yields were measured in grams. Furthermore, 100 grains were taken to determine the grain weight (g), and the harvest index was determined as the ratio of grain to biomass yield.

Fresh root samples were collected, and iron plaque formation on the roots was determined via the ascorbic citrate acetic (ACA) extraction technique. For this purpose, an ACA mixture containing ascorbic acid (3 g), 5 mL of sodium acetate (10%), and 40 mL of sodium citrate (0.3 M) was prepared. Thereafter, 1-g root sample was collected, placed in the prepared mixture, and shaken (280 rpm) for 3 hours at 25°C. The mixture was subsequently filtered into 100-mL flasks, and the roots were carefully washed three times. The eluent obtained after washing the roots was transferred to the same 100-mL flasks, and the volume was increased to 100 mL by adding the water. The concentrations of Fe and Pb were determined via atomic absorption spectrophotometry.

The plant samples (100 mg) were collected, and RNA was extracted using an RNA extraction kit (MiniBEST; Takara, China) following the protocols of the manufacturer. Thereafter, both the quality and concentration of RNA were determined using a NanoDrop spectrophotometer. A transcription kit (HiScript^® III RT SuperMix) was used to convert the RNA into cDNA. To perform real-time PCR, 11 μg of extracted RNA was reverse-transcribed into cDNA using a Dispelling RT, SuperMix kit. Later, qRT–PCR was performed using a SuperReal PreMix Plus (SYBR Green) kit (Tiangen, Takara, China; FP205–2). Additionally, OsActin was used as a reference gene, and relative gene expression was calculated using the methods of Livak and Schmittgen (2001). The copper/zinc SOD was targeted from the SOD gene family, and in the case of POD, the POD from the peroxidase family was targeted. Moreover, CAT was targeted from the catalase–peroxidase family, and APX was targeted from the ascorbate peroxidase family. The details of the primers used in the study are given in Supplementary Table S1 .

The data were analyzed by one-way analysis of variance to determine the impact of different treatments. Moreover, differences among means were analyzed using Tukey’s honestly significant difference (HSD) test, Sigma-plot 10 was used for figure preparation, and principal component analysis (PCA) and correlation matrix were plotted in RStudio.

Pb toxicity decreased chlorophyll, carotenoid, and RWC ( Table 1 ). However, BC and MT applications offset this decrease and caused a marked improvement in the chlorophyll contents and RWC ( Table 1 ). The co-application of BC and MT enhanced Chl-a, Chl-b, and carotenoid concentrations by 37.74%, 84.85%, and 30.02%, respectively, in Pb-polluted soil ( Table 1 ). Biochar alone increased Chl-a, Chl-b, and carotenoid synthesis by 32.07%, 57.57%, and 14.86%, respectively, while MT alone increased Chl-a, Chl-b, and carotenoid synthesis by 32.07%, 57.57%, and 14.86%, respectively ( Table 1 ). Leaf RWC was significantly decreased by 33.48% than control in Pb-contaminated soil. However, BC and MT increased the leaf RWC, and the co-application of BC and MT showed more promising results than their individual application. The co-application of BC and MT increased RWC by 22.41%, while the individual BC and MT applications increased RWC by 13.18% and 8.13%, respectively ( Table 1 ).

The results revealed that plants growing in Pb-polluted soil faced a serious increase in EL, MDA, and H₂O₂ production. Nevertheless, BC and MT profoundly decreased the EL, MDA, and H₂O₂ production. EL, MDA, and H₂O₂ were increased by 192%, 93%, and 173%, respectively, under Pb stress than control ( Table 2 ). BC+MT appreciably reduced EL, MDA, and H₂O₂ by 124%, 67.39%, and 74%, respectively ( Table 2 ). Further, BC alone decreased EL, MDA, and H₂O₂ production by 74.33%, 32.75%, and 45.94%, respectively, while MT alone decreased EL, MDA, and H₂O₂ synthesis by 63.92%, 40.24%, and 29.25%, respectively ( Table 2 ). The results depicted that Pb toxicity decreased TSP and FAA, while the synthesis of proline was increased in Pb stress. Further, BC and MT increased TSP, FAA, and proline synthesis ( Table 2 ). The combined application of BC and MT increased TSP, FAA, and proline synthesis by 47.09%, 17.45%, and 32%, respectively ( Table 2 ). We also noted that BC alone increased TSP, FAA, and proline synthesis by 34.69%, 17.60%, and 17.71%, respectively, while MT alone increased TSP, FAA, and proline synthesis by 16.32%, 8.04%, and 0.37%, respectively ( Table 2 ). Antioxidant activities were slightly increased in Pb-contaminated soil; however, BC, MT, and BC+MT enhanced the activities of all the antioxidants ( Table 2 ). The co-application of BC and MT enhanced APX, CAT, POD, and SOD activities by 26.47%, 65.51%, 93.10%, and 65.53%, respectively. Moreover, the individual BC application increased APX, CAT, POD, and SOD activities by 15.03%, 44.20%, 65.51%, and 46.46%, respectively, while the individual MT application increased by 8%, 37.30%, 20.68%, and 24.92%, respectively ( Table 2 ).

The presented results indicated that the Pb concentration significantly increased in rice roots and shoots growing in Pb-polluted soil ( Figure 2 ). The maximum Pb concentration in the roots (139.67 mg kg⁻¹) and shoots (96.37 mg kg⁻¹) was detected in Pb-contaminated soil. BC+MT reduced Pb accretion in plant tissues, and the lowest root (104.80 mg kg⁻¹) and shoot (55.77 mg kg⁻¹) Pb were detected with BC+MT. Thereafter, BC showed better results, and it had the lowest root (116.70 mg kg⁻¹) and shoot (76.40 mg kg⁻¹) Pb concentrations, while MT alone had higher root (122.80 mg kg⁻¹) and shoot (70.60 mg kg⁻¹) Pb concentrations. We observed that Pb toxicity seriously decreased the tissue nutrient concentrations. The lowest root (9.67, 6.48, and 15.40 mg kg⁻¹) and shoot (11.73, 9.81, and 18.54 mg kg⁻¹) N, P, and K concentrations were found under Pb stress ( Table 3 ). Biochar and MT increased the uptake of nutrients and resulted in better concentrations of these nutrients in roots and shoots. The co-application of BC and MT substantially enhanced root N, P, and K concentrations by 67.11%, 98.61%, and 100%. Further, BC enhanced N, P, and K concentrations by 53.46%, 68.05%, and 71.62%, respectively, while MT alone increased the root N, P, and K concentrations by 29.16%, 46.14%, and 48.63%, respectively ( Table 3 ). The results also revealed that BC+MT enhanced shoot N, P, and K concentrations by 81.50%, 96.63%, and 108.78%, respectively. BC alone enhanced shoot N, P, and K concentrations by 62.23%, 59.73%, and 83.44%, and MT alone enhanced shoot N, P, and K concentrations by 53.70%, 31.80%, and 58.41%, respectively ( Table 3 ). Pb toxicity also decreased the root and shoot Ca and Mg concentrations. The presented results indicated that the lowest root (41.40 and 33.93 mg kg⁻¹) and shoot (56.40 and 44.43 mg kg⁻¹) Ca and Mg contents were observed in Pb-contaminated soil, and the maximum root (70.03 and 62.47 mg kg⁻¹) and shoot Ca (89.31 and 73.13 mg kg⁻¹) concentrations in Pb-polluted soil were detected with the co-application of BC and MT and thereafter with the BC alone and MT alone applications ( Table 2 ).

The presented results revealed that BC and MT applications significantly decreased the TF and BAC of Pb ( Figure 3 ). The maximum TF (0.693) and BAC (0.673) were observed in Pb-contaminated soil, followed by the MT alone (0.575 and 0.560) and BC alone (0.656 and 0.608) applications, and the lowest TA (0.533) and BAC (0.560) were observed with the combined BC and MT application ( Figure 3 ).

The maximum soil Pb concentration (158.50 mg kg⁻¹) was found in Pb-contaminated soil, and the lowest soil Pb (99.67 mg kg⁻¹) was observed with the co-application of BC and MT, followed by BC alone (113.63 mg kg⁻¹) and MT alone (126.38 mg kg⁻¹) ( Figure 2 ). Different treatments induced a slight change in soil pH; however, BC and MT applications more effectively increased soil pH ( Figure 4 ). N, P, and K availability was significantly decreased by 116.04, 84.88, and 78.69%, respectively, than the control. Nevertheless, the combined BC and MT application enhanced N, P, and K availability by 74.25%, 52.07%, and 45.81%, respectively, in Pb-polluted soil ( Figure 4 ). Furthermore, BC alone enhanced soil N, P, and K availability by 35.16%, 47.28, and 31.72%, respectively, while MT alone enhanced soil, P, and K availability by 20.27%, 31.30%, and 21.55%, respectively ( Figure 4 ). Soil organic concentration was significantly increased with BC application. The maximum SOC concentration (36.14 mg kg⁻¹) in Pb-contaminated soil was obtained with the combined BC and MT application, followed by the BC alone (31.38 mg kg⁻¹) and MT alone (25.11 mg kg⁻¹) applications ( Figure 4 ). BC+MT showed a significant impact on the Pb fractions ( Figure 5 ). The results indicated that the BC+MT application significantly increased the residual and oxidizable forms of Pb, while BC+MT decreased the extractable and reducible forms of Pb ( Figure 5 ). The maximum reducible and extractable fractions of Pb were observed in Pb-contaminated soil, whereas the lowest fractions were detected in soil that received BC+MT ( Figure 5 ).

Root length (RL), root fresh weight (RFW), and root dry weight (RDW) were decreased by 28.27%, 113.17%, and 57.45%, respectively, under Pb stress ( Table 3 ). Biochar, MT, and their combination significantly increased the RL, RFW, and RDW of rice plants ( Table 3 ). The co-application of BC and MT increased RL, RFW, and RDW by 11.22%, 82.68%, and 45.56%, respectively; BC alone enhanced RL, RFW, and RDW by 3.12%, 55.03%, and 29.23%, respectively; MT alone enhanced RL, RFW, and RDW by 6.73%, 26.35%, and 14.51%, respectively ( Table 3 ). The results also indicated that shorter plants (79 cm) with fewer tillers/plant (TPP) (7.67) and less 100-kernel weight (HKW) (2.01 g) were observed in Pb-polluted soil without any amendment, and taller plants (96 cm) with higher TPP (9.67) and HKW (3.67 g) were observed in Pb-contaminated soil with the co-application of BC and MT, followed by the BC application and then the MT alone application ( Table 3 ). Pb toxicity also caused a reduction of 74.40%, 40.15%, and 24.40%, respectively, in grain yield (GY), biological yield (BY), and harvest index (HI) of rice crops ( Table 3 ). Biochar, MT, and their combination appreciably increased the GY, BY, and HI of rice crops. The results indicated an increase of 54.39%, 33.30%, and 14.65% in GY, BY, and HI, respectively, with the combined BC and MT application in Pb-contaminated soil ( Table 3 ). Moreover, BC alone enhanced GY, BY, and HI by 30.67%, 21.98%, and 4.16%, respectively, while MT alone enhanced GY, BY, and HI by 15.27%, 8.46%, and 0.73%, respectively ( Table 3 ).

The results regarding the concentrations of Pb and Fe in the ACA extracts of the Fe plaque are given in Figure 6 . The results revealed that the concentration of Pb significantly increased Pb in roots grown in Pb-polluted soil, while the application of both MT and BC significantly decreased the Pb concentration (50.14%) in ACA extracts of the Fe plaque of rice roots, while BC alone and MT alone decreased the Pb concentration by 18.69% and 10.60%, respectively ( Figure 6 ). Notably, Fe concentration was significantly increased with BC and MT; however, a more significant increase (67.70%) in Fe concentration in ACA extracts of Fe plaque was observed with the co-application of BC and MT, followed by BC alone (57.21%) and MT (26.06%) ( Figure 6 ).

The results indicated that BC, MT, and their combined application significantly impacted the expression of antioxidant defense genes (OsAPX, OsCAT, OsPOX, and OsSOD) and metal transporter genes (OsHMA9 and OsNRAMP5). Pb toxicity decreased the expression of antioxidant defense genes; however, BC+MT significantly enhanced the expression of all the antioxidant genes ( Figure 7 ). Co-applying BC+MT increased OsAPX, OsCAT, OsPOX, and OsSOD expression by 121%, 57.14%, 85.32%, and 140%, respectively, as compared to control ( Figure 7 ). Moreover, BC alone increased OsAPX, OsCAT, OsPOX, and OsSOD expression by 106.50%, 41.07%, 49.80%, and 123.75%, respectively, while MT alone increased OsAPX, OsCAT, OsPOX, and OsSOD expression by 96%, 33.92%, 55.98%, and 100%, respectively, compared to control ( Figure 7 ). The application of BC and MT also decreased the expression of the metal transporter genes, and more promising results were seen with the BC+MT application ( Figure 7 ). Co-applying BC+MT decreased the expression of OsHMA9 and OsNRAMP5 by 96.19% and 83%, respectively, under Pb stress compared to control ( Figure 7 ). Moreover, BC alone decreased OsHMA9 and OsNRAMP5 expression by 27.19% and 43.62%, respectively, while MT alone decreased OsHMA9 and OsNRAMP5 expression by 24.43% and 24.15%, respectively, than the control ( Figure 7 ).

The PCA biplot explains 97.48% of the total variance, with PC1 accounting for 74.33% and PC2 accounting for 23.15%. The treatments exhibited distinct separation, with the control group clustering on the negative side of PC1, which correlated with growth parameters such as RL, BY, and HI. In contrast, Pb stress grouped on the positive side of PC1, which is closely associated with oxidative stress markers like MDA, EL, and H₂O₂. The Pb+BC and Pb+MT treatments showed moderate separation, with Pb+BC associated with antioxidant enzyme activity (APX, POD, and SOD) and Pb+MT linked to higher H₂O₂ levels. The combined treatment of Pb+BC+MT clustered centrally, indicating strong associations with antioxidant enzymes and proline, suggesting enhanced stress mitigation. Vector analysis revealed an inverse relationship between growth parameters and stress indicators, underscoring the effectiveness of BT+MT in alleviating Pb-induced stress and enhancing plant growth and physiological responses ( Figure 8 ).

The correlation matrix for growth parameters is depicted in Figure 9a . A strong and significant positive correlation was observed among all growth traits, including RL, RFW, RDW, PH, TPP, HKW, BY, GY, and HI. The r values ranged from 0.86 to 1.00, indicating a very close association among these parameters. Notably, RDW, BY, and GY showed particularly strong correlations (r > 0.97) with HI, highlighting their importance as determinants of harvest index performance. The correlation among physiological traits is depicted in Figure 9b . Chl-a, Chl-b, and carotenoids were strongly positively correlated (r = 0.93–1.00). The RWC was negatively correlated with MDA (r = −0.50), EL (r = −0.58), and H₂O₂ (r = −0.52), indicating that increased oxidative stress results in reduced water retention. Antioxidant enzymes, such as APX, CAT, POD, and SOD, exhibited weak to moderate correlations, with oxidative stress markers having r values ranging from −0.11 to 0.37. Proline (Pro) showed a weak negative association with EL and oxidative stress markers (r = −0.17 to −0.25).

R-Pb and S-Pb were strongly correlated with N, K P, Mg, and Ca levels in plant tissues. Root and shoot Pb concentrations were positively correlated with R-K, R-P, S-K, and S-P (r > 0.90), indicating a coordinated uptake pattern. In contrast, R-Pb exhibited a weak negative correlation with N uptake (r = −0.26; Figure 9c ). These patterns suggest that Pb accumulation may disrupt specific nutrient dynamics, particularly N. The relationships among soil properties are illustrated in Figure 9d . The soil Pb concentration was negatively correlated with the soil pH (r = −0.54), indicating that acidic conditions increase Pb bioavailability. SOC and TN contents were weakly negatively associated with the Pb fraction (r = −0.31 to −0.46). Exchangeable and reducible Pb fractions were strongly correlated (r = 0.78) and positively associated with total soil Pb content (r = 0.73). Interestingly, AP and AK were moderately positively correlated with the soil Pb concentration (r = 0.57).