The pea (Pisum sativum L.) variety used in this study was Zhongwan 6 (ZW6). In this study, the Hoagland nutrient solution hydroponics method was employed for plant tissue culture and drug treatment. Pea seeds with complete grains and uniform sizes were carefully selected, followed by germination promotion at 28 °C in moist sand. Once the pea seedling roots reached a length of 0.5–1 cm (typically within 1–2 d), seedlings exhibiting similar growth patterns were chosen. The sand adhering to the root surface was subsequently rinsed with distilled water before being transferred into a 1/4-strength Hoagland nutrient solution (Xu et al., 2013). The hydroponic box was then placed on a light culture rack for seedling cultivation under specific conditions (light/dark: 18/6 h, temperature: 25/16 °C, humidity: 60%, light intensity: 20000 lux). During seedling cultivation, the nutrient mixture was replaced every 3 days. Ten-day-old pea seedlings were exposed to Cd (9 μM, cadmium chloride (CdCl₂) was used as the source of Cd) or nSiO₂ (50 mg/L) for 12 d. The selected concentrations of Cd and nSiO₂ used in this experiment have been previously investigated and reported in existing studies (Głowacka et al., 2019; Ghosh et al., 2022). After 12 d of treatment, a range of physiological and biochemical analyses were conducted. Some samples were quickly frozen in liquid nitrogen after sampling and stored at -80 °C for later use.

In this study, nSiO₂ (purity 99%, size 50 ± 5 nm) was purchased from McLean Biotechnology Co., Ltd. Numerous studies have employed X-ray diffraction (XRD) to analyze the crystallinity of nSiO₂, Fourier-transform infrared spectroscopy (FTIR) to detect its surface functional groups, and high-resolution transmission electron microscopy (HR-TEM) to characterize the shape, size, and morphology of nSiO₂ in aqueous environments. XRD measurements were conducted on a Rigaku SmartLab diffractometer using Cu Kα radiation (λ = 1.5406 Å), with a scanning range from 10° to 80° (2θ), a step size of 0.02°, and a scanning speed of 2° per minute. The FTIR analysis was performed using a Thermo Scientific Nicolet iS10 spectrometer, with scans acquired over the range of 4000–400 cm⁻¹ at a resolution of 4 cm⁻¹, accumulating 32 scans per sample (Cui et al., 2017; Ghosh et al., 2022; Liu et al., 2025). nSiO₂ was suspended in sterile deionized water (ddH₂O), stirred for 2 h, and homogenized by ultrasonication at 40 kHz for 60 min until the NPs were evenly distributed, as described previously (Cui et al., 2017; Zou et al., 2022). The final working concentration of nSiO₂ for plant treatment was 50 mg/L.

Plant growth parameters (including plant height, primary root (PR) length, leaf dry weight (DW), root DW, lateral root number, chlorophyll content and photosynthetic parameters) were measured after 12 d of Cd, nSiO₂ or combined treatment. The roots were placed in a scanning dish, the pea root system was scanned via a scanner (EPSON Perfection V800 Photo), and the results were analyzed via WinRHIZO (Pro2016A). The chlorophyll content was measured via SPAD 502 (Minolta, Japan). At least three independent biological replicates were performed, with 25 plants measured in each treatment group.

Total protein was extracted in potassium phosphate buffer (50 mM, pH 7.8) on ice. After centrifugation (15 min, 15000 rpm, 4 °C), the supernatant was removed for determination of SOD, CAT, and POD activities. SOD activity was measured as described by Gong et al. (2014) with a spectrophotometer. Briefly, samples were homogenized in ice-cold 50 mM phosphate buffer (pH 7.4). The assay system contained 50 mM phosphate buffer (pH 7.8), 100 μM EDTA, 50 mM xanthine, 24 μM NBT, and sample. The reaction was initiated by adding 50 mU/mL xanthine oxidase and incubated at 25 °C for 20 min. The absorbance of the formazan product was read at 560 nm using a spectrophotometer (Shimadzu UV-1800) (n = 3). One unit of SOD activity was defined as the amount of enzyme causing 50% inhibition of NBT reduction. Activity was normalized to protein concentration determined by BCA assay and expressed as U/mg protein. CAT activity was measured following Sun et al. (2020). The reaction mixture contained 50 mM phosphate buffer (pH 7.0), 30 mM H₂O₂, and enzyme extract. The decomposition of H₂O₂ was monitored by the decrease in absorbance at 240 nm for 1 min using a spectrophotometer (Shimadzu UV-1800) (n = 3). One unit of CAT activity was defined as the amount of enzyme that decomposed 1 μmol H₂O₂ per minute and was expressed as U/min/mg protein. POD activity was determined according to Sun et al. (2020). The assay contained 50 mM phosphate buffer (pH 7.0), 10 mM guaiacol, 5 mM H₂O₂, and enzyme extract. The increase in absorbance due to tetraguaiacol formation was recorded at 470 nm for 2 min using a spectrophotometer (Shimadzu UV-1800) (n = 3). One unit of POD activity was defined as the amount of enzyme that caused an increase of 0.01 in absorbance per minute and was expressed as U/min/mg protein.

Hydrogen peroxide (H₂O₂) content was assayed as described by Gong et al. (2014) using a spectrophotometer. Briefly, samples were homogenized in cold acetone. The reaction mixture contained extract, 0.1% titanium sulfate, and 0.2 M H₂SO₄. After incubation at 25 °C for 10 min, the absorbance was measured at 415 nm using a spectrophotometer (Shimadzu UV-1800) (n = 3). H₂O₂ content was quantified against a standard curve and expressed as μmol/g fresh weight.

In this study, the TTC method was employed to quantify root activity. Initially, 0.2 g of fresh pea root was accurately weighed and placed in a triangular bottle. A solution containing 0.5% TTC and 0.1 M phosphate buffer (pH=7.5) was subsequently added in equal volumes and thoroughly mixed with the sample before being incubated at 37 °C for 1 h. Following the incubation period, termination of the reaction was achieved by adding 1 M H₂SO₄ solution to the triangular bottle. The pretreated roots were then ground with ethyl acetate via a mortar, after which the absorbance value of the resulting liquid extract was measured at 485 nm following volume standardization (n = 3). Root activity was quantified on the basis of the reduction intensity of TTC.

The leaves (No. 2–3 pairs) of pea plants subjected to different treatments were carefully selected, and the relevant photosynthetic data were measured via LI-6800 portable photosynthesis system (LI-COR) under natural light conditions. The intensity of photosynthetically active radiation (PAR) was set at 500 μmol·m^-2·s^-1, while the relative humidity (RH) was maintained at 50%. Measurements were taken for the net photosynthetic rate (Pn, μmol·m^-2·s^-1), stomatal conductance (Gsw, mol·m^-2·s^-1), and transpiration rate (Tr, mmol H₂O·m^-2·s^-1). After 3 h in darkness to culture the pea plants, the chlorophyll fluorescence indices of the leaves were subsequently determined via pulse-modulated fluorometry equipment (Image-PAMM, Walz, Germany) with 25 plants measured in each treatment group.

After treatment, the roots and leaves of the pea plants in each group were collected. The samples were soaked in 1 mM EDTA solution for 30 min and then rinsed 5 times with ddH₂O. The samples were subsequently fixed for 15 min at 105 °C and dried to a constant weight at 70 °C. The dried sample was ground and digested with HNO₃ according to the methods of Sun et al. (2020). We initially measured a 0.2 g sample, which was subsequently transferred to a clean digestion tube for nitric acid (HNO₃) digestion. The resulting solution was subsequently filtered through a microporous filter membrane (pore size 0.22 µm) before being transferred into a sample vial for measurement purposes. The contents of Cd, zinc (Zn), copper (Cu), magnesium (Mg), iron (Fe) and silicon (Si) were determined via inductively coupled plasma–mass spectrometry (ICP–MS). Each experiment was repeated three times.

Ten-day-old pea seedlings were exposed to Cd, nSiO, or a combination of both. After a 2-day treatment and culture period, the roots and leaves of the peas in each treatment group were collected. Total RNA was extracted by grinding frozen samples with liquid nitrogen for subsequent transcriptomic high-throughput sequencing and qRT–PCR experiments (n = 3). The obtained transcriptome data were subjected to a bioinformatics analysis process provided by BMKCloud (www.biocloud.net). Differential expression analysis and functional annotation of differentially expressed genes (DEGs) via Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed on the basis of gene expression levels in different sample groups. Gene reads represent gene expression levels, with fragments per kilobase of transcript per million fragments mapped (FPKM) values commonly used as a measure for convenience.

Genes exhibiting significantly different expression levels across samples are referred to as DEGs. During the process of detecting differentially expressed genes, the screening criteria were set as a fold change ≥ 2 and the FDR < 0.01. The fold change represents the ratio of expression between two samples, whereas the false discovery rate (FDR) is derived from correcting the significance p value (p-value), indicating the level of significance for differences observed. To facilitate comparison, the fold changes were paired and represented as log₂FC values. The log₂FC transformation was applied to normalize the gene expression data. The screening criteria for differentially expressed genes included a |log₂(fold change)| ≥1 and q value ≤ 0.05.

Three independent biological replicates were used for each experiment in our study. The relevant figures were graphed with the software GraphPad Prism 8 and enhanced with Adobe Photoshop 2019 CC. The experimental results are shown as the means ± standard errors (SEs). The significance of differences was analyzed via Student’s t test (IBM SPSS Statistics 23.0). The asterisk indicates P < 0.05. One-way ANOVA with Tukey’s test was used to compare multiple groups. Different lowercase letters represent significant differences at P < 0.05.

The effects of Cd on the growth of pea seedlings were analyzed in this study. The application of Cd adversely affected various growth parameters of the seedlings, resulting in reductions of 49.43% in plant height (compared with the control 105.89 ± 7.21 mm), 34.81% in primary root length (compared with the control 89.15 ± 5.77 mm), 27.03% in lateral root number (compared with the control 37 ± 1.33 roots), 39.87% in leaf dry weight (DW) (compared with the control 0.3958 ± 0.0230 g), and 24.83% in root DW (compared with the control 0.1180 ± 0.0058 g) ( Figures 1A–F ). In contrast, exposure to nSiO₂ significantly increased the height of pea plants compared with those in the control group ( Figures 1A, F ). Furthermore, exposure to nSiO₂ under Cd stress conditions stimulated the growth of pea seedlings. As shown in Figures 1A–E , the Cd+nSiO₂ treatment group presented increases of 33.93% in primary root length, 25.00% in lateral root number, 29.18% in leaf DW, and 17.41% in root DW compared with those of the seedlings treated with Cd alone. Cd stress not only hindered leaf expansion in pea plants but also decreased the leaf count ( Figure 1A ). Notably, exposure to nSiO₂ significantly increased the growth and development of pea leaves compared with those in the control group ( Figure 1A ). Exposure to nSiO₂ under Cd stress conditions significantly promoted leaf growth ( Figures 1A, D ). Ultimately, exposure to nSiO₂ increased Cd tolerance in pea seedlings, thereby increasing their overall growth and development.

On the other hand, Cd toxicity significantly inhibited the activity of pea seedling roots by 26.48% ( Figure 1G ). Compared with the control, exposure to nSiO₂ had no significant effect on the root activity of pea seedlings ( Figure 1G ). When pea seedlings were exposed to nSiO₂, the activity of pea seedling roots increased under Cd toxicity. As shown in Figure 1G , the group treated with ‘Cd+nSiO₂’ presented a notable increase in root activity of 15.52% compared with that in the Cd-only treatment group. The activity of pectin methylesterase (PME) is crucial for protecting plant root tips from stress (Chen et al., 2018). Therefore, we investigated PME activity in pea root tips subjected to Cd toxicity, nSiO₂ exposure, or combined Cd+nSiO₂ treatment. Cd toxicity increased PME activity in pea seedling root tips by 22.85% ( Figure 1H ). Compared with the control, exposure to nSiO₂ did not significantly affect PME activity in pea seedling root tips ( Figure 1H ). In contrast, exposure to nSiO₂ significantly reduced PME activity in pea seedling root tips under Cd stress conditions. As depicted in Figure 1H , the ‘Cd+nSiO₂’ group presented a substantial 14.23% decrease in PME activity in pea seedling root tips compared with the Cd treatment alone group. Moreover, Cd toxicity strongly inhibited both seed germination and radicle growth in peas; however, exposure to nSiO₂ increased pea seed germination and radicle growth under Cd stress conditions ( Figure 1I ).

The accumulation of ROS serves as a critical indicator of plant responses to both biotic and abiotic stresses. In this study, we examined ROS accumulation in pea seedlings exposed to Cd toxicity, nSiO₂ exposure, or a combination of both. Fluorescence staining for total ROS indicated a significant increase in ROS levels in the roots of pea seedlings under Cd toxicity ( Figure 2A ). NBT staining analysis of pea seedling roots revealed elevated superoxide anion (O₂¯) levels under Cd toxicity ( Figure 2B ). Exposure to nSiO₂ resulted in a reduction in ROS accumulation in pea roots under Cd stress ( Figures 2A, B ). Quantitative analysis revealed that under Cd stress, O₂¯ and hydrogen peroxide (H₂O₂) levels in pea seedlings increased by 151.24%, 119.57%, 150.51%, and 73.96% in roots and leaves, respectively ( Figures 2C, D ; Supplementary Figures S1A, B ). However, exposure to nSiO₂ did not affect the levels of O₂¯ or H₂O₂ in either the roots or leaves of pea seedlings compared with those in the control group ( Figures 2C, D ; Supplementary Figures S1A, B ). Notably, in the Cd+nSiO₂ group, the levels of O₂¯ and H₂O₂ in the root and leaf tissues were reduced by 31.68%, 39.9%, 39.69%, and 59.97%, respectively, compared with those in the Cd treatment alone group ( Figures 2C, D ; Supplementary Figures S1A, B ).

We subsequently investigated the activity of antioxidant system-related enzymes in pea seedlings subjected to Cd toxicity, nSiO₂ exposure, or Cd+nSiO₂ interactive treatment. These findings revealed that Cd triggered an increase in SOD activity in both the roots and leaves of pea plants. Specifically, compared with the control, Cd toxicity significantly increased SOD activity by 11.69% in pea seedling roots ( Figure 2E ). Conversely, when exposed to nSiO₂ under Cd stress, SOD activity decreased by 5.30% compared with that under Cd treatment alone ( Figure 2E ). Similarly, compared with the control, Cd toxicity led to a significant increase of 23.71% in SOD activity in the leaves of pea seedlings ( Supplementary Figure S1C ). However, when exposed to nSiO₂ under Cd stress, SOD activity was reduced by 9.88% compared with that under Cd alone ( Supplementary Figure S1C ).

On the other hand, compared with the control, exposure to Cd significantly increased the CAT activity in the roots and leaves of pea plants by 212.53% and 137.81%, respectively ( Figure 2F ; Supplementary Figure S1D ). However, compared with the Cd treatment alone, the Cd+nSiO₂ interaction treatment resulted in decreases in CAT activity of 54.12% and 30.15% in the roots and leaves of pea seedlings, respectively ( Figure 2F ; Supplementary Figure S1D ). In addition, compared with the control, exposure to Cd significantly increased POD activity in the roots and leaves of pea plants by 26.71% and 87.57%, respectively ( Figure 2G ; Supplementary Figure S1E ). However, compared with the Cd treatment alone, the Cd+nSiO₂ interaction treatment resulted in decreases in POD activity of 14.23% and 28.98% in the roots and leaves of pea seedlings, respectively ( Figure 2G ; Supplementary Figure S1E ).

The degree of oxidative damage and lipid peroxidation can be evaluated by the malondialdehyde (MDA) content in plants (Barclay and McKersie, 1994). We measured MDA levels in pea seedlings. Compared with that in the control plants, the content of MDA in the roots and leaves of pea plants significantly increased by 167.94% and 293.25%, respectively, under Cd stress ( Figure 2H ; Supplementary Figure S1F ). Under Cd stress, the MDA content in the roots and leaves of pea seedlings treated with Cd+nSiO₂ decreased significantly, by 30.07% and 60.90%, respectively, compared with that in the roots and leaves of pea seedlings treated with Cd alone ( Figure 2H ; Supplementary Figure S1F ). Additionally, the relative electrical conductivity of pea seedlings can serve as an indicator of cell membrane permeability. Our results revealed that, compared with the control, Cd stress significantly increased the relative electrical conductivity of the roots and leaves of pea plants by 48.97% and 59.86%, respectively ( Figure 2I ; Supplementary Figure S1G ). Compared with Cd treatment alone, treatment with Cd+nSiO₂ resulted in a decrease in the relative electrical conductivity of pea seedling roots and leaves by 31.37% and 31.10%, respectively (( Figure 2I ; Supplementary Figure S1G ).

By observing the phenotype of pea plants, we found that Cd toxicity significantly impeded the growth and development of pea leaves ( Figure 1A ). We subsequently analyzed the relevant parameters of photosynthetic efficiency in pea seedlings subjected to Cd toxicity, nSiO₂ exposure, or Cd+nSiO₂ interactive treatment. The results indicated that a significant decrease in the photosynthetic rate of pea seedlings was affected by Cd toxicity ( Figures 3A–D ). Specifically, Cd toxicity led to substantial reductions of 45.23%, 40.63%, 57.39%, and 52.55% in the net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and total conductance to CO₂ (gtc), respectively, compared with those of the control ( Figures 3A–D ). However, under Cd stress, the combined treatment with Cd+nSiO₂ increased the Pn, Tr, Gs, and gtc values by approximately 13.84%, 43.86%, 53.73%, and 48.31%, respectively, compared with those under Cd treatment alone ( Figures 3A–D ).

We then detected and analyzed the chlorophyll fluorescence-related parameters of the pea seedlings in each group. Compared with the control, Cd toxicity significantly reduced the maximal photosystem II (PSII) activity parameter (Fv/Fm) of pea seedlings by 16.93% ( Figures 3E, F ). Compared with the Cd alone treatment, the Cd+nSiO₂ interaction treatment increased the Fv/Fm of the pea seedlings to 16.99% ( Figures 3E, F ). Conversely, compared with the control, Cd toxicity significantly increased the photochemical quenching coefficient (qP) and qL of pea seedlings by 20.37% and 73.98%, respectively ( Figures 3E, G, H ). Compared with Cd alone, the Cd+nSiO₂ interaction treatment decreased the qP and qL of pea seedlings by 16.13% and 40.96%, respectively ( Figures 3E, G, H ).

The mineral element content of the pea seedlings was determined via inductively coupled plasma emission spectrometry (ICP–MS). Compared with Cd stress alone, the Cd+nSiO₂ interaction treatment significantly reduced the Cd content in both the roots and leaves of pea seedlings by 22.24% and 67.88%, respectively ( Figure 4A ; Supplementary Figure S2A ). All three independent biological replicates produced consistent results, confirming the reproducibility of the findings. Moreover, compared with the control, Cd toxicity increased the accumulation level of Fe in both the roots and leaves of pea plants by 44.06% and 42.51%, respectively ( Figure 4B ; Supplementary Figure S2B ), with a significant increase observed specifically in the roots. However, there was no significant effect on Fe accumulation in pea seedlings subjected to the Cd+nSiO₂ interaction treatment compared with those subjected to Cd toxicity alone ( Figure 4B ; Supplementary Figure S2B ).

On the other hand, compared with the control, Cd toxicity significantly suppressed the accumulation of Cu, Mg, Zn, and Si in the roots of pea plants by 70.01%, 30.17%, 78.68%, and 49.55%, respectively ( Figures 4C–F ). Under Cd stress, the combined treatment of Cd+nSiO₂ had no significant effect on the accumulation levels of Cu, Mg or Zn in pea seedlings ( Figures 4C–E ). Moreover, compared with the control, Cd toxicity inhibited the accumulation of Cu, Mg and Zn in the leaves of pea plants by 74.85%, 29.62%, and 54.05%, respectively ( Supplementary Figures S2C–E ). Under Cd stress, the Cd+nSiO₂ interaction treatment promoted the accumulation of Cu, Mg and Zn in pea seedlings ( Supplementary Figures S2C–E ), but this difference did not reach statistical significance.

To explore the molecular mechanism underlying the alleviation of Cd toxicity to pea seedlings by nSiO₂ and elucidate the potential regulatory pathway of the nSiO₂-mediated response to Cd stress in pea, we performed transcriptome sequencing analysis on pea seedlings exposed to Cd toxicity, nSiO₂ treatment, or Cd+nSiO₂ interactive treatment. Each group of samples was subjected to three biological replicates, and the statistics of the filter quality control results are presented in Supplementary Table S1 . After filtering the original sequencing data, we obtained a total of 151.26 Gb of clean data. The percentage of Q30 bases in all the transcriptome samples was 91.37% or greater, indicating acceptable data quality for each sample ( Supplementary Table S1 ).

We then generated an FPKM box plot to visualize the gene expression of each transcriptome sample ( Supplementary Figure S3 ). The discrete distribution of gene expression in the transcriptome samples of each group directly reflects the overall gene expression of different samples ( Supplementary Figure S3 ). Additionally, we evaluated the dispersion of samples within each treatment group via principal component analysis (PCA). In Figures 5A, B , PC1 on the x-axis represents the first principal component, with its percentage indicating its contribution to sample differences. The contribution values of the first principal component in the roots and leaves of the pea seedlings to the sample difference were 30.18% and 34.33%, respectively ( Figures 5A, B ). On the y-axis, PC2 represents the second principal component. The contribution values of the second principal component in the roots and leaves of the pea seedlings to the sample difference were 14.70% and 12.60%, respectively ( Figures 5A, B ). These results indicate that the similarity between the transcriptome repeats is high, which meets the requirements of transcriptome data analysis and can be used for subsequent differentially expressed genes (DEGs) analysis.

The Venn diagram provides a visual representation of the common and unique genes detected in each transcriptome sample. Figures 5C, D show Venn diagrams depicting gene detection between four treated transcriptome samples from pea roots and leaves, respectively. A total of 20,979 genes were detected in pea roots ( Figure 5C ). Among these genes, 18,297 coexpressed genes were unrelated to exogenous treatment. Additionally, 908 genes whose expression was specifically associated with only Cd toxicity (the Cd toxicity and Cd+nSiO₂ treatment groups) were identified. Furthermore, 171 genes were specifically expressed in response to the Cd+nSiO₂ treatment ( Figure 5C ). Similarly, a total of 18,847 genes were detected in pea leaves ( Figure 5D ). Among these genes, 15,753 coexpressed genes were not related to exogenous treatment. Moreover, there were 1,033 genes whose expression was exclusively associated with Cd toxicity (the Cd toxicity and Cd+nSiO₂ treatment groups). Finally, 278 genes were specifically expressed in response to the Cd+nSiO₂ treatment ( Figure 5D ).

The DEGs (|log2FC|≥1 and q value ≤ 0.05) were subsequently subjected to further analysis among the various pea seedling treatment groups. Comparative analysis revealed 1682 and 3530 DEGs in the roots and leaves of pea seedlings, respectively ( Figures 5E, F ). Compared with those in the control, a total of 1386 DEGs (including 451 upregulated and 935 downregulated) and 3624 DEGs (including 1606 upregulated and 2018 downregulated) were identified in the roots and leaves of pea seedlings under Cd toxicity (Cd/control), respectively ( Figures 5E, F ). In addition, 34 DEGs (including 13 upregulated DEGs and 21 downregulated DEGs) and 422 DEGs (including 88 upregulated DEGs and 334 downregulated DEGs) were identified in the roots and leaves of pea seedlings treated with nSiO₂ (nSiO₂/control) ( Figures 5E, F ). A total of 1220 DEGs (including 219 upregulated and 1001 downregulated) and 348 DEGs (including 17 upregulated and 331 downregulated) were identified in the roots and leaves of pea seedlings treated with Cd+nSiO₂ (Cd+nSiO₂/Control), respectively ( Figures 5E, F ). In addition, compared with those under the Cd treatment alone, 22 DEGs (3 upregulated and 19 downregulated) and 812 DEGs (158 upregulated and 654 downregulated) were identified in the roots and leaves of peas under the Cd+nSiO₂ treatment (Cd+nSiO₂/Cd), respectively ( Figures 5E, F ).

We subsequently used the K-means algorithm to perform cluster analysis on 1682 DEGs identified in the root samples of the four groups of pea seedlings under different treatments. The clustering results revealed that these DEGs were clustered into 6 gene clusters ( Figure 6A ). The number of DEGs in cluster 1, cluster 2, cluster 3, cluster 4, cluster 5 and cluster 6 in the roots of the pea seedlings was 315, 250, 386, 268, 203 and 260, respectively ( Figure 6A ). As shown in Figure 6B , Cd toxicity significantly induced gene expression in cluster 1, cluster 2, cluster 3, and cluster 5 in the roots of pea seedlings. In contrast, Cd toxicity significantly inhibited gene expression in Clusters 4 and 6 ( Figures 6A, B ). Further analysis revealed that the expression of genes in cluster 4 slightly increased under nSiO₂ exposure, significantly decreased under Cd toxicity, and slightly increased under the Cd+nSiO₂ interactive treatment (compared with that under Cd toxicity alone) ( Figure 6B ). The expression trends of these genes were consistent with the growth and development phenotypes of the pea seedlings in the four treatment groups ( Figure 6B ; Figures 1A–G ). On the other hand, the expression of genes in cluster 5 essentially remained unchanged under nSiO₂ exposure, significantly increased under Cd toxicity, and significantly decreased under the Cd+nSiO₂ interactive treatment (compared with that under Cd toxicity alone) ( Figure 6B ). The expression trends of these genes were opposite those of the growth and development phenotypes of the pea seedlings in the four treatment groups ( Figure 6B ; Figures 1A–G ).

On the other hand, we used the K-means algorithm to perform cluster analysis on 3530 DEGs identified from pea seedling leaf samples in the four treatment groups. The clustering results revealed that these DEGs were clustered into 6 gene clusters ( Figure 6C ). The number of DEGs in cluster 1, cluster 2, cluster 3, cluster 4, cluster 5 and cluster 6 in the leaves of pea seedlings was 616, 812, 670, 536, 651 and 245, respectively ( Figure 6C ). As shown in Supplementary Figure S4 , Cd toxicity significantly induced gene expression in cluster 1, cluster 3, cluster 4 and cluster 6 pea leaves. In contrast, Cd toxicity significantly inhibited gene expression in cluster 2 and cluster 5 ( Supplementary Figure S4 ). Further analysis revealed that the expression of genes in clusters 2 and 5 significantly decreased under nSiO₂ exposure, significantly decreased under Cd toxicity, and significantly increased under the Cd+nSiO₂ interactive treatment (compared with that under Cd toxicity alone) ( Supplementary Figure S4 ). The change trend of DEG expression was similar to the change trend of the growth and development phenotypes of pea seedlings in the four treatment groups ( Supplementary Figure S4 ; Figures 1A–G ). In contrast, the expression of genes in cluster 3 and cluster 4 increased under nSiO₂ exposure, significantly increased under Cd toxicity, and significantly decreased under the Cd+nSiO₂ interactive treatment (compared with that under Cd toxicity alone) ( Supplementary Figure S4 ). The change trend of DEG expression contrasted with the change trend of the growth and development phenotypes of pea seedlings in each treatment group ( Supplementary Figure S4 ; Figures 1A–G ).

On the basis of the above results, KEGG enrichment analysis was performed on 268 DEGs from cluster 4 in pea seedling roots ( Figure 6C ; Figure 7A ). The results revealed that the DEGs in root cluster 4 were significantly enriched in alanine, aspartate and glutamate metabolism; arginine biosynthesis; the phenylpropanoid biosynthesis pathway; etc. ( Figure 7A ). The 203 DEGs in cluster 5 of pea seedling roots were significantly enriched in starch and sucrose metabolism, carotenoid biosynthesis, nitrogen metabolism, arginine biosynthesis and other pathways ( Figure 7B ). On the other hand, KEGG enrichment analysis was performed on 670 DEGs from cluster 3 in pea seedling leaves ( Supplementary Figure S4 ; Supplementary Figure S5A ). The results revealed that DEGs in leaf cluster 3 of pea seedlings were significantly enriched in the biosynthesis of amino acids, starch and sucrose metabolism, fatty acid metabolism, and the flavonoid biosynthesis pathway ( Supplementary Figure S5A ). The 536 DEGs in cluster 4 of pea seedling leaves were significantly enriched in phenylpropanoid biosynthesis, starch and sucrose metabolism, amino acid biosynthesis, endocytosis and other pathways ( Supplementary Figure S5B ).

Further analysis revealed that the LOC127094975 and LOC127093608 genes were enriched in the alanine, aspartate and glutamate metabolism pathways in pea seedling root cluster 4. These proteins play important regulatory roles in the conversion of L-glutamate to L-glutamine ( Figure 8A ). The heatmap shows that the expression of these genes is reduced under Cd stress, thus inhibiting this metabolic process. However, the expression of these genes in the roots of peas increased after exposure to nSiO₂ under Cd stress ( Figure 8A ). The expression trends of these genes were consistent with the growth and development phenotypes of the pea seedlings in the four treatment groups ( Figure 8A ; Figures 1A–G ). In root cluster 5 of pea seedlings, two genes, LOC127126644 and LOC127125929, were significantly enriched in the alanine, aspartate and glutamate metabolism pathways ( Figure 8B ). They play crucial roles in regulating the conversion of 2-oxoglutamate to L-glutamate. The heatmap clearly shows that the expression levels of these genes increase under Cd stress, thereby facilitating this metabolic process. Conversely, exposure to nSiO₂ under Cd stress led to a decrease in the expression levels of these genes in pea roots ( Figure 8B ). The pattern of gene expression changes contrasted with the trend of growth and developmental phenotypic changes in pea seedlings under the four treatments ( Figure 8B ; Figures 1A–G ).

On the basis of these previous results, KEGG enrichment analysis was performed on 670 DEGs from Cluster 3 in the leaves of pea seedlings ( Supplementary Figure S4A ). The results revealed that the DEGs in cluster 3 of pea seedling roots were significantly enriched in the ribosome, biosynthesis of amino acids, anthocyanin biosynthesis, starch and sucrose metabolism, flavonoid biosynthesis and other pathways ( Supplementary Figure S4A ). A total of 536 DEGs from cluster 4 pea seedling leaves were significantly enriched in glutathione metabolism, phenylpropanoid biosynthesis, starch and sucrose metabolism, and the endocytosis pathway ( Supplementary Figure S4B ).

Further analysis revealed that the genes enriched in the starch and sucrose metabolism pathways in leaf cluster 3 of pea seedlings were as follows: LOC127076145, LOC127108237, LOC127081761, LOC127118385, LOC127119689, LOC127132519, LOC127127306, LOC127128158, LOC127128624, LOC127076688, LOC127082706, LOC127085359, LOC127080507, LOC127083424, LOC127084279, LOC127091213, LOC127096584, LOC127108448, LOC127 106106, LOC127106154 and LOC127108192 ( Figure 8C ). These genes encode granule-bound starch synthase 1, B-S glucosidase 44, alpha-glucan phosphorylase 2, O-glycosyl hydrolase family 17 protein, fructokinase-like 2, metal ion-binding protein, endo-beta-mannase 5, O-glycosyl hydrolase family 17 protein, alpha-amylase-like 3,6-fructan exohydrolase, O-glycosyl hydrolase family 17 protein, APL4, disproportionate enzyme, glycosyl hydrolase 9B13, sucrose synthase 6, glycosyl hydrolase 9B13, carbohydrate-binding X8 domain superfamily protein, isoamylase 1, alpha-glucan phosphorylase 1, plasasmaodesmata callose-binding protein 5, and reduced beta amylase 1, respectively. These genes regulate the conversion pathway of sucrose to fructose and glucose ( Figure 8C ). The expression of these genes increased under Cd stress, thus promoting this metabolic process. Under Cd stress, nSiO₂ exposure decreased the expression levels of these genes in pea leaves. The expression trend of these genes was opposite to that of the growth and development phenotypes of pea seedlings in the four groups ( Figure 8C ; Figures 1A–G ).