Silicon and iron nanoparticles protect rice against lead (Pb) stress by improving oxidative tolerance and minimizing Pb uptake

Lead (Pb) is toxic to the development and growth of rice plants. Nanoparticles (NPs) have been considered one of the efficient remediation techniques to mitigate Pb stress in plants. Therefore, a study was carried out to examine the underlying mechanism of iron (Fe) and silicon (Si) nanoparticle-induced Pb toxicity alleviation in rice seedlings. Si–NPs (2.5 mM) and Fe-NPs (25 mg L−1) were applied alone and in combination to rice plants grown without (control; no Pb stress) and with (100 µM) Pb concentration. Our results revealed that Pb toxicity severely affected all rice growth-related traits, such as inhibited root fresh weight (42%), shoot length (24%), and chlorophyll b contents (26%). Moreover, a substantial amount of Pb was translocated to the above-ground parts of plants, which caused a disturbance in the antioxidative enzyme activities. However, the synergetic use of Fe- and Si–NPs reduced the Pb contents in the upper part of plants by 27%. It reduced the lethal impact of Pb on roots and shoots growth parameters by increasing shoot length (40%), shoot fresh weight (48%), and roots fresh weight (31%). Both Si and Fe–NPs synergistic application significantly elevated superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione (GSH) concentrations by 114%, 186%, 135%, and 151%, respectively, compared to plants subjected to Pb stress alone. The toxicity of Pb resulted in several cellular abnormalities and altered the expression levels of metal transporters and antioxidant genes. We conclude that the synergistic application of Si and Fe-NPs can be deemed favorable, environmentally promising, and cost-effective for reducing Pb deadliness in rice crops and reclaiming Pb-polluted soils.


Measurements of Pb concentration in rice seedlings
The Pb contents from roots and shoots of rice seedlings were estimated.First, we carefully rinsed the samples with ultra-high pure ddH 2 O and later dried these samples at 60 ºC until constant weight.Plant samples of about 0.2 g were measured, and HNO 3 -HClO (V: V = 6:2) was placed into a Teflon bottle with plant samples and heated on a hotplate at 180 ºC until 1 mL liquid was left in the tube.Subsequently, samples were diluted, and the final quantity of Pb in the solution was estimated by an Agilent 7700 inductively coupled plasma mass spectrometry (ICP-MS) (Agilent Technologies, CA, USA).

Cytological investigations by transmission electron microscope (TEM)
Cell structure was observed by TEM as described previously 45 .The root tip samples were cut and prefixed in 2.5% glutaraldehyde and then post-fixed in 2% OsO 4 for two hours.Then, specimens were rinsed with PBS, and a series of ethanol solutions with diverse concentrations (30, 50, 70, 80, 90, and 100%) were used to dehydrate and fix in spur resin.Then, a microtome (Leica, Germany) was used to cut ultrathin sections.The cell structure was seen under a TEM equipped with an Oxford INCA Energy TEM 200 EDX system (Philips TECNAI 10, Netherlands).
Semi-thin sections of rice plant roots were made using semi-thin section kits (Technovit7100).The root samples were immersed in FAA solution for 24 h before being dehydrated with different ethanol concentrations 44 .After that, we treated the samples with a semi-permeable agent at 4 °C for 12 h before immersing them in the pure penetrating agent.After solidification, the material was polymerized at 23-37 °C, and slices (4 µ) were cut from embedded blocks using a thin rotary slicer (Leica RM2265) and inspected and photographed using an automatic biomicroscope (Motic BA310).

qRT-PCR analysis
The rice samples were collected after treatments, and RNA Kit was used to isolate total RNA.The NanoDrop spectrophotometer was used to determine the purity of the RNA samples.One µg of RNA was taken to prepare the cDNA library from total RNA with a reagent kit.The primers for each gene were developed by primer 5 software, and all primers' sequences are listed in Table S1.Using a Bio-Rad real-time PCR system and SYBR Green reagent (Takara, Kyoto, Japan), qRT-PCR was performed.Three biological replicates were used for each treatment to quantify the expression levels.Actin was an internal control in the 2 -△△Ct approach used to estimate the relative expression patterns 46 .We calculated the relative expression using the formula F = 2 ^-ΔΔCt , where ΔΔCt = (CT,Target-CT,Act) Ex-(CT,Target-CT,Act) CK.Ex means experiment group, while CK means control.

Statistical analyses
The data, from three independent replications, underwent one-way ANOVA analysis using the SPSS 20.0 program and Graph Pad Prism (version 8.0.2).Bar graphs illustrating the mean and SE for each parameter were constructed.The Tukey test was employed to estimate the least significant difference (LSD) at p < 0.05.Mean values and standard deviations (± SD) from four replications were used for representation.Additionally, using R software, a comprehensive exploration of the data's multivariate patterns was conducted through principal component analysis (PCA).Significance between treatments was determined via an LSD test at a 95% probability level.

Plants biomass and Chlorophyll
We investigated various growth parameters, viz., root fresh weight (RFW), shoot dry weight (SDW), shoot fresh weight (SFW), root dry weight (RDW), and shoot length (SL), as shown in Fig. 1.The results indicated that Pb exposure decreased the SL of rice plants by 23.49% compared to the control plants and RFW, SFW, SDW, and RDW by 18.21%, 30.58%, 30.92%, and 42.10%, respectively.But, the Pb-stressed plants treated with Si and Fe-NPs revealed better performance for all the aforementioned agronomic traits than Pb-stressed plants.Overall, the alone treatment of Fe-and Si-NPs resulted in a much lower increase in all morphological features compared to the combined application of both nanoparticles under a Pb-stressed environment (Fig. 1; Table 1).However, the combined application of Fe-and Si-NPs to the Pb-stressed rice plants increased SL, SFW, RFW, SDW, and RDW by 39.78%, 48.23%, 31.26%,42.33%, and 82.95% as compared to Pb alone, respectively.These outcomes exhibited the constructive influence of Si-and Fe-NPs in improving Pb resistance in rice (Table 1).
The results indicated that Pb exposure decreased carotenoids and chlorophyll a, b contents by 19.19%, 20.50%, and 25.73% in comparison to the control treatment, respectively (Fig. S2).However, the exclusive utilization of Si and Fe-NPs resulted in significant enhancements in the levels of chlorophyll a (27.03% and 24.67%), chlorophyll b (54.57% and 49.49%), and carotenoid (34.29% and 33.46%) under Pb-induced stress, respectively.The synergistic application of Fe-and Si-NPs to Pb-stressed rice plants increased chlorophyll a, b, and carotenoids by 35.48%, 58.57%, and 37.65% compared to Pb alone treatment, respectively.

Si and Fe-NPs reduced Pb uptake to mitigate Pb toxicity
The results indicated that both Si and Fe nanoparticle application in the rice seedlings increased their concentrations in rice plants' tissues (shoot and root).The highest contents of Si and Fe were detected in the roots than in the shoots (Fig. S3).The plants exhibited a notable increase in Pb concentration when subjected to Pb application, as compared to non-stressed plants (Fig. 2).Applying Fe and Si-NPs to Pb-stressed plants significantly decreased Pb concentration in shoot and root samples as opposed to Pb -alone treated rice plants.Our results revealed that Pb decreased by 42.94% and 48.31% under Fe-and Si-NPs treatments in the root, and by 11.08% and 13.08% in shoots relative to Pb -alone treated plants, respectively (Fig. 2).Moreover, the joint application of Fe-and Si-NPs had a more noticeable impact on reducing Pb quantity in roots and shoots, and decreased Pb by 56.43% and 26.86% compared to Pb-stressed plants alone, respectively (Fig. 2).usage resulted in substantial enhancements in SOD activity, with increments of 64.93%, 66.00%, and 114.32%.Furthermore, the activities of POD, CAT, and GSH displayed notable increases.Specifically, Fe-NP treatment led to increments of 156.77%, 85.77%, and 126.58%, while Si-NP treatment resulted in elevations of 159.77%, 88.59%, and 126.34%, respectively.The combined application of both nanoparticles showed even more pronounced effects, with increments of 185.51%, 134.75%, and 150.49% for POD, CAT, and GSH, compared to plants stressed by Pb alone (Fig. 3).

Influence of Si and Fe-NPs on root ultrastructure
The root cortex has a well-maintained cell structure with fine-shaped vacuoles and cell wall under non-stressed conditions.The cell organelles were distorted with disordered and swollen vacuoles under Pb-stressed plants (Fig. 4).In the Pb treatment, cell arrangement was disordered.By contrast, Si and Fe-NP-treated plants retained cell morphology, although some displayed distortion.Compared with Pb-stressed plants, vacuoles, cell wall, and mitochondria were closely organized in Fe-and Si-NPs treated plants.Our results showed that cortical cells breached under Pb exposure, while Fe-and Si-NPs treated plants under Pb exposure-maintained vacuole structure and cell wall that sustained the cell integrity.Semithin sections of only Pb-treated plants revealed anomalies in the pericycle, epidermis, endodermis, vascular cylinder, and cortex.In contrast, co-application of www.nature.com/scientificreports/Fe and Si under Pb exposure revealed fewer abnormalities (Fig. 4).Normal cell structure was seen in rice plants treated with Fe-and Si-NPs.

Gene expression levels of antioxidant and metal transporter genes under Si and Fe-NP and Pb stress
The qRT-PCR measured the relative expression patterns of genes that code for antioxidants.Pb triggered a remarkable decline in the degree of these genes' expression.In contrast, Si and Fe nanoparticle application in the rice seedlings improved these genes' expression patterns under Pb stress (Fig. 5).Moreover, we evaluated the expression patterns of metal transporter genes.For example, OsHMA9 (metal or Pb transporter) displayed the highest expressions under Pb exposure compared to Pb + Si + Fe treatments.In contrast, the lowest expression levels were detected under the combined or alone application of Fe and Si.OsLSi1 (a-Si transporter) showed the highest expression pattern under Pb + Si, followed by Pb + Si + Fe treatment, while the lowest was under Fe and Pb treatments.OsIRT2 (a Fe transporter) expression was higher in treatments with Fe NPs than others, with Pb + Fe treatment having the most significant level followed by Pb + Fe + Si treatment, and Pb and Pb + Si treatment having the lowest level.

Correlation, heatmap, homogeneity, and principal component analysis
According to the grey correlation degree results, the correlation degree among indicators is substantial.All indicators have a correlation degree of more than 0.6 with the content of Pb in the above-ground parts (Fig. S4).The correlation degree of lead content with Fe contents in the shoot, Si contents in the root and shoot, and SOD is high, reaching 0.7.The concentrations of H 2 O 2 and MDA in rice treated with Pb were the highest, indicating that Pb toxicity in rice was significant.But CAT, SOD, POD, GSH, Chl a, and other physiological indices decreased (Fig. 6).Physiological indices such as Chl a, carotenoids, RDW, SL, RFW, and SDW were higher in plants treated with Fe and Si nanoparticles alone or in combination than Pb treated plants.However, these indicators increased slightly with Fe and Pb than Si and Pb.As a result, in this experiment, Fe has a greater ability to mitigate Pb toxicity than Si.Furthermore, Pb content in root and ground parts was lower when treated with Si + Fe + Pb than when treated with Fe and Pb or Si and Pb alone.
According to the principal component analysis, PCA1 accounted for 49.67%, and PCA2 accounted for 32.31%, a total of 81.98% (Fig. S5).The larger the absolute value of Cohen's d, the larger the effect size (Table 2).In the study, we observed significant and substantial differences (large effect sizes) in morphological traits and antioxidant enzyme activities when Si and Fe nanoparticles were applied.The effect sizes observed for the combined application of both Si and Fe nanoparticles were even larger when compared to the effect sizes observed when each nanoparticle was applied individually (sole application).Regarding PCA1, Pb content in roots was significantly increased, while carotenoids, RFW, SDW, and RDW were decreased considerably.In the dimension of PCA2, H 2 O 2 , GSH, and SOD decreased significantly, and H 2 O 2 decreased the most.According to the correlation coefficient, most indicators have a significant association.Pb content was negatively associated with the content of Fe and Si, and the content of Si in the root was negatively linked with Fe content in the ground-parts (Table S2).The concentrations of Pb in root and shoot were positively correlated with a correlation coefficient of 0.996.The correlation coefficient between CAT and SOD was 0.953.Moreover, the Shapiro-Wilk Test (Normality test) results indicate that all data conform to a normal distribution (Table S3).The homogeneity of variance of Levene's Test also shows that the variances are comparable (Table S4).There are no large or small outliers in the data of this experiment.

Discussion
The current findings supported the hypothesis that the synergistic application of Fe-and Si-NPs effectively mitigated Pb stress in rice.This was achieved through the enhancement of enzymatic activities and the reduction of oxidative stress, ultimately resulting in decreased Pb uptake by the plants.Heavy metal stress, particularly Pb, can cause poisonous and environmental pollution.Pb disturbs the enzyme activities that lead to changes in mineral nutrition and membrane permeability, suppressing plants' growth, photosynthesis, and morphological characteristics 47 .Several impacts on plant growth and development, accompanied by noticeable symptoms, including inhibited root development, smaller leaves, and stunted growth, have already been reported under Pb stress 48 .In maize, Pb reduces the primary root growth and decreases the number of branches in seedlings 49 .A previous study showed that Pb toxicity exhibited heavy metal toxicity symptoms such as a brittle texture, stunting, and blackening of roots, severely disturbing the growth of banana (Musa acuminata L.) seedlings 50 .Here, plant growth parameters, including shoot length and biomass accumulation, were suppressed by the excess quantity of Pb in rice seedlings (Fig. 1).The Pb toxicity also inhibited chlorophyll synthesis (Fig. S2) and damaged root ultrastructure.A significantly high content of Pb was found in both roots and shoots of rice.Intriguingly, the introduction of Si-and Fe-NPs had a notable positive impact on various aspects of plant health.Specifically, their application led to a significant enhancement of plant growth parameters, such as shoot length and biomass accumulation.This positive effect on biomass accumulation suggests an overall improvement in the plants' physiological and metabolic activities.Furthermore, the introduction of Si-and Fe-NPs resulted in a substantial reduction in the concentration of Pb detected in both the root and shoot parts of rice plants.This indicates that Si-and Fe-NPs played a crucial role in mitigating the adverse effects of Pb stress.The reduction in Pb content suggests a potential mechanism where Si-and Fe-NPs contribute to limiting the uptake or translocation of Pb within the plant, thus alleviating the negative impacts of Pb on growth and development.
It is well known that Pb prompts phytotoxicity in seedlings and can increase the rate of ROS and oxidative stress, which can change the production of macromolecules, such as nucleic acids, lipids, and proteins in cells 13 .In plant cells, heavy metals triggered by oxidative stress can either repress the production level of antioxidative enzymes or stimulate more ROS 51 .Here, rice seedlings exposed to Pb displayed considerably higher contents of H 2 O 2 and MDA (lipid peroxidation) than the control, and the high rate of lipid peroxidation indicates more damage to lipids (Fig. 3).However, when rice seedlings were treated with a combination of Si-and Fe-NPs along with Pb, the MDA contents were lower compared to control plants.This suggests that the co-application of Si-and Fe-NPs with Pb has a protective effect, mitigating the metal toxicity and inhibiting cell membrane destruction caused by Pb-induced oxidative stress.These findings align with previous research that demonstrated the positive role of NPs in reducing the toxicity of other heavy metals, such as Cd, in rice.Specifically, earlier studies have shown that NPs can effectively reduce the MDA concentration, indicating a potential mechanism through which NPs contribute to alleviating oxidative stress and associated damage in plant cells exposed to heavy metals 44,52 .
The improved antioxidative defense regulatory enzymatic activities, including CAT, SOD, and POD (Fig. 3), will create tolerance in stressed plant cells against severe oxidative cellular damage generated under Pb toxicity in plants through excessive ROS production.SOD acts as the foremost important defense antioxidative enzyme to salvage the extra ROS by regulating the production of superoxide (O 2 − ) 53 .POD also plays a crucial role in scavenging the excess ROS in plant tissues under heavy metal toxicity 54 .Pb heavy metal treatment caused declining SOD enzyme activity in cotton (Gossypium hirsutum L.) seedlings because of high oxidative stress that might not have been enough to reduce the high accumulation of ROS produced by higher MDA contents than their controls.Here, heavy metal-induced cell toxicity caused an increased accumulation of ROS in rice plants under Pb-treatment, reducing the SOD enzyme activity.In the co-exposure scenario of Si-and Fe-NPs along with Pb heavy metals, the increased SOD enzyme activity in rice seedlings suggests a potential mitigating effect on Pbinduced oxidative stress.This elevation in SOD activity can be attributed to the lower levels of ROS observed in the rice seedlings exposed to Si-and Fe-NPs compared to those treated with Pb alone.The presence of Si-and Fe-NPs seems to contribute to a reduced ROS burden within the plant cells, creating an environment with lower oxidative stress.Consequently, the SOD enzyme, being a crucial defense mechanism against excess ROS,  experiences an upregulation in its activity.This increased SOD activity can be seen as a protective response, as it helps the plant cells manage and neutralize the potentially harmful effects of ROS, contributing to an overall enhancement of antioxidative defense mechanisms.Similarly, positive effects of NPs were detected in other plant species, such as barley 55 , rice 52,56,57 , wheat 58,59 , and okra 60 .Co-application of Si-and Fe-NPs with heavy metals triggered the CAT activity, which was also reduced in Pb-stressed plants.We noticed a substantial rise in the CAT activity in the plants with co-exposure of Si-and Fe-NPs under Pb metal treatments than control (Fig. 3).Both POD and CAT enzymes are crucial in reducing ROS accumulation and improving plant cell defense against heavy metal-induced oxidative stress.POD is a significant scavenging enzyme for H 2 O 2 under abiotic stress in plant cells.The co-application of Si and Fe-NPs with Pb heavy metal ions resulted in a notable increase in POD activity.This heightened POD activity indicates a reduction in H 2 O 2 levels within the rice seedlings, showcasing an effective mitigation of oxidative stress induced by Pb exposure.This observation aligns with recent research, such as the study in wheat, which demonstrated that the use of Fe-NPs increased the activity of the POD enzyme under exposure to Cd 39 .The collective presence of Si and Fe-NPs, along with heavy metals, exhibited an enhanced antioxidant activity within the plant tissues compared to seedlings treated solely with Pb.This suggests a synergistic effect between Si and Fe-NPs in alleviating oxidative stress, contributing to improved plant defense mechanisms against the detrimental impacts of heavy metal exposure.
The ROS scavenging systems or GSH can reclaim heavy metals in the vacuole 61,62 .GSH can construct a complex with metals that, by vacuolar compartmentalization, could detoxify the heavy metals 52,61 .Applying Si and Fe-NPs boosted the GSH concentration compared to the Pb treatment alone.Hence, the high activity of GSH helps in the detoxification of Pb.Si and Fe-NPs significantly reduced Pb uptake and translocation to leaves, decreasing Pb toxicity in rice seedlings.We concluded that alleviating oxidative stress in leaves and improving an antioxidant defense mechanism by Si and Fe-NPs may be directly associated with decreased Pb accumulation in leaves.Our results revealed that Fe and Si-NPs enhanced the expression patterns of CAT, SOD, and POD genes in comparison to the control, both alone and in combination (Fig. 5).Pb triggered a remarkable decrease in the degree of these genes' expression, whereas both Si-and Fe-NPs application in the rice seedlings improved the patterns of these genes' expression under Pb stress (Fig. 5).The increased expression of these genes (OsSOD, OsPOD, and OsCAT) were most likely due to the emission of Si and Fe ions from NPs. Importantly, the expression data are consistent with the minimal Pb deposition in rice shoots subjected to Pb + NPs versus Pb treatment.
Several genes are involved in plants' metal and heavy metal metabolism, from transport to bioaccumulation and assimilation 63 .The mechanism was further refined by analyzing the genes involved in Pb absorption and translocation.OsHMA9, is identified as a key transporter gene involved in the uptake and bioaccumulation of Pb 64 .Under normal conditions, OsHMA9 expression is closely linked to the transportation of Pb within plants, indicating its significant role in Pb uptake and transport.Our results revealed that under the sole use of Fe-and Si-NPs treatments, the OsHMA9 expression recovered to control levels, showing that Pb absorption was successfully blocked.The higher expression levels of OsHMA9 in response to Pb exposure suggested its involvement in Pb uptake/transport, which was consistent with prior research that linked OsHMA9 to Pb transportation 64 .metal transporters may also limit Pb ion absorption and accumulation in rice seedlings (Fig. 7).While our study provides valuable insights into the potential of Si and Fe-NPs in mitigating Pb toxicity in rice plants, it is important to acknowledge certain limitations.Firstly, the study primarily focused on the short-term effects, and long-term implications of NPs application, which need further investigation to understand their sustained efficacy and any potential ecological impacts.Additionally, the study was conducted under controlled laboratory conditions, and the translation of these findings to real-world field scenarios requires careful consideration.
The specific mechanisms of NPs interaction with soil microbes and their long-term effects on soil health and ecosystem stability remain areas of uncertainty.Future research should address these limitations by conducting field trials to validate the effectiveness of Si and Fe nanoparticles in actual contaminated soil environments.Furthermore, assessing the potential risks associated with the application of NPs, such as unintended consequences on non-target organisms and soil microbial communities, should be a focus of future investigations.Recommendations include comprehensive field studies, assessing the long-term effects, and incorporating ecological considerations to ensure the safe and effective application of Si and Fe nanoparticles for mitigating heavy metal toxicity in agricultural soils.

Conclusions
Our results showed that Pb stress caused severe damage to rice seedlings, which reduces enzymatic activities and biomass accumulation.The modifications in the POD, CAT, and SOD enzymatic activities of leaves displayed the effects of Pb toxicity, which produce free oxygen radicals.TEM and semi-thin sectioning allowed us to identify many cell abnormalities, such as malformed cell walls, mitochondria, pericycle, epidermis, endodermis, and cortex.However, the synergistic use of

Figure 1 .
Figure 1.Pb-stressed rice seedling growth response to Si and Fe nanoparticles.Small letters above the bar show the significant difference, and results are denoted by the LSD test and mean ± SD at p ≤ 0.05.

Figure 2 .
Figure 2. Concentrations of Pb in rice seedlings under Si and Fe nanoparticles.Small letters above the bar show the significant difference, and results are denoted by the LSD test and mean + SD at p ≤ 0.05.

Figure 3 .
Figure 3. Response of antioxidant system in rice seedlings under Si and Fe nanoparticles.Small letters above the bar show the significant difference, and results are denoted by the LSD test and mean + SD at p ≤ 0.05.

Figure 4 .Figure 5 .
Figure 4. Ultrastructure of root cells of rice under Pb exposure and combined use of Si and Fe nanoparticles.Ultrathin sections of rice seedlings treated with Pb (A-D), Si + Fe (E), and Si + Fe + Pb (F) were observed by TEM.Semi-thin section observation under the application of Pb (G, J, M), Si + Fe (H, K, N), and Si + Fe + Pb (I, L, O).CW: Cell wall; V: Vacuole; ER: Endoplasmic reticulum; Xy: Xylem; Ph: Phloem; En: Endodermis; Ep: Epidermis
Fe-and Si-NPs repressed the lethal effects of Pb in rice seedlings by altering the cellular, metabolic, and morphological traits.The results of Cohen's d and PCA analysis indicated a significant and strong positive correlation between the presence of Fe-and Si-NPs with the observed morphological characteristics and antioxidant activities under Pb toxicity.Both Si and Fe-NPs can suppress oxidative stress and adjust the antioxidant defense system in plants by modulating the expression patterns of metal transporters, such as OsHMA9, OsLSi1, and OsIRT2.These results indicate that the combined use of Si-and Fe NPs can mitigate the harmful effects of Pb exposure on rice plants more effectively than using either kind of nanoparticle alone.Si-and Fe-NPs play the most critical role in reducing Pb concentrations in rice and preventing its translocation from the plant's roots to aerial portions, finally reducing cell damage.
Upon subjecting rice seedlings to Pb application, the levels of ROS, including MDA and H 2 O 2 , experienced increases of 8.59% and 13.74%, respectively.Remarkably, the application of Fe-NPs, Si-NPs, and their combined