Alleviatory effects of Silicon on the morphology, physiology, and antioxidative mechanisms of wheat (Triticum aestivum L.) roots under cadmium stress in acidic nutrient solutions

Silicon (Si), as a quasi-essential element, has a vital role in alleviating the damaging effects of various environmental stresses on plants. Cadmium (Cd) stress is severe abiotic stress, especially in acidic ecological conditions, and Si can demolish the toxicity induced by Cd as well as acidic pH on plants. Based on these hypotheses, we demonstrated 2-repeated experiments to unfold the effects of Si as silica gel on the root morphology and physiology of wheat seedling under Cd as well as acidic stresses. For this purpose, we used nine treatments with three levels of Si nanoparticles (0, 1, and 3 mmol L−1) derived from sodium silicate (Na2SiO3) against three concentrations of Cd (0, 50, and 200 µmol L−1) in the form of cadmium chloride (CdCl2) with three replications were arranged in a complete randomized design. The pH of the nutrient solution was adjusted at 5. The averages of three random replications showed that the mutual impacts of Si and Cd in acidic pH on wheat roots depend on the concentrations of Si and Cd. The collective or particular influence of low or high levels of Si (1 or 3 mM) and acidic pH (5) improved the development of wheat roots, and the collective influence was more significant than that of a single parallel treatment. The combined effects of low or high concentrations of Cd (50 or 200 µM) and acidic pH significantly reduced root growth and biomass while increased antioxidants, and reactive oxygen species (ROS) contents. The incorporation of Si (1 or 3 mmol L−1) in Cd-contaminated acidic nutrient solution promoted the wheat root growth, decreased ROS contents, and further increased the antioxidants in the wheat roots compared with Cd single treatments in acidic pH. The demolishing effects were better with a high level of Si (3 mM) than the low level of Si (1 Mm). In conclusion, we could suggest Si as an effective beneficial nutrient that could participate actively in several morphological and physiological activities of roots in wheat plants grown under Cd and acidic pH stresses.

Scientific Reports | (2021) 11:1958 | https://doi.org/10.1038/s41598-020-80808-x www.nature.com/scientificreports/ were filled with a solution of 3/4L Hoagland's solution 38 (See Online appendix heading 1). The nutrient solution was replaced every 3 days interval. The 1-week-old wheat seedlings were transplanted in half-strength Hoagland's solution. The Hoagland solution was exchanged with full-strength after 20 days of transplantation till the end of the experiment, and the pH of the growing media was modified to 5 (acidic) with 81% phosphoric acid using a PHS-29A pH meter. The treatments were given after 20 days of transplanting for 3 weeks (21 days). Silicon (Si) nanoparticles (See Online appendix heading 2) made by sodium silicate (Na 2 SiO 3 ) was added with the rate of 0, 1, and 3 mmol L −1 while, Cadmium (Cd) in the form of cadmium chloride (CdCl 2 ) was supplemented with the rate of 0, 50 and 200 µmol L −1 in the nutrient solution of acidic pH (5). Si treatments were introduced when seedlings were 27 days old; the 0, 1, and 3 mmol L −1 Si solutions with neutral pH were attained by mixing a sufficient amount of Na 2 SiO 3 in the double-distilled water. The acidic pH (5) was maintained three times a day with 0.1 M HCl using a PHS-29A pH meter. After every 3 days, the Hoagland solution was renewed to sustain the pH value, and double-distilled water was added to keep the volume of the container. The treatments were also re-introduced with the renewal of nutrient solution.
The pots were then distributed into nine treatments/groups each of 3 pots. In group one plant were exposed at Cd 50 µmol L −1 and not received any concentration of Si. In second and third groups of pots, plants were exposed at Cd 50 along with Si 1 and 3 mmol L −1 in the Hoagland solution, respectively. In group 4, pots were exposed at Cd 200 µmol L −1 and not received any concentration of Si. In group 5 and 6 pots were exposed at Cd 200 µmol L −1 along with Si 1 and 3 mmol L −1 in Hoagland solution, respectively. In group 7, pots were exposed at Si 1 mmol L −1 and not received any concentration of Cd. In group 8, pots were exposed at Si 3 mmol L −1 and not received any concentration of Cd. In a group, nine pots were exposed at Hoagland solution and not received any concentration of Cd and Si (control). Three replications for each treatment were used in both experiments. The pots were randomly placed inside the control room. Both experiments were performed in natural conditions at an ambient temperature of 22 to 30 °C during daytime and 15-20 °C during nighttime. After 120 days of transplantation, all the plants were sampled. Root samples for extraction of the enzyme assays were immediately frozen in liquid nitrogen and stored at − 80 °C.

Determination of root biomass and root morphological traits.
Plants were sampled after 120 days of germination for assessment of root biomass and root morphological traits, including root tips, average root diameter, root volume, and root length. Root fresh weight was measured immediately after harvesting the plants' samples. "Took two root samples from each replication and placed in an oven at 105 C for 30 min, and then kept at 80ºC for 24 h to get constant weight and measured the dry weight" 26 . Remaining root parameters such as root width, average root diameter, root tips, and root length were measured using a root automated scan tool ( Biochemical analysis. "Enzymatic antioxidants (superoxide dismutase: SOD, catalase: CAT, guaiacol peroxidase: POD), non-enzymatic antioxidants (proline), lipid peroxidation contents in terms of malondialdehyde (MDA), and reactive oxygen species in term of hydrogen peroxide (H 2 O 2 ) in three random replications of root samples of wheat plants were assessed by using the kits of Beijing Solarbio Science & Technology Co., Ltd ((http://www.solar bio.com). Briefly, 0.5 g fresh samples of roots were milled with the help of a motor and pestle and standardized in 0.05 M phosphate buffer with pH 7.8 under chilled condition. The standardized mixture was centrifuged at 12,000 rpm for 10 min at 4 °C after sieving through four layers of muslin cloth. The activity of CAT was assessed by the following formula: SQ = Sample Quantity, OD control = absorption of light in control, OD test = absorption of light in test samples.
After mixing all reagents in the standardized mixture, the supernatant was again centrifuged at 3500 rpm for 10 min. The light diameter of 1 cm was adjusted to zero by double streaming water. OD was measured at 420 nm wavelength. The activity of POD was measured by the following equation: V t = Total volume of the reaction liquid, SQ = Sample Quantity, RT = Reaction time, OD control = absorption of light in control, OD test = absorption of light in test samples.
After mixing all reagents in a standardized mixture, the supernatant was placed at room temperature for 10 min. SOD was measured at 550 nm wavelength. The activity of SOD was measured by the following equation: V t = Total volume of the reaction liquid, SQ = Sample Quantity, OD control = absorption of light in control, OD test = absorption of light in test samples.
The level of lipid peroxidation in the leaf tissue was assessed by measuring the contents of malondialdehyde (MDA), a by-product of lipid peroxidation. Briefly, 0.2-0.5 g weighted fresh samples of roots were milled with the help of a motor and added 2 ml 10% TCA and a small amount of quartz sand, ground to homogenate, add × 1000 www.nature.com/scientificreports/ 3 ml TCA, further ground. The homogenized sample was centrifuged at 12,000 rpm for 10 min. Took 2 ml supernatant, added 0.67% TBA, mixed and boiled for 15 min in 100 ºC water bath. Cooled the sample at room temperature and centrifuged again. Absorption values of samples were measured at 532 nm, 600 nm, and 450 nm, respectively. The activity of MDA was measured by the following formula: V t = Total volume of the reaction liquid, SQ = Sample Quantity. Proline was also assessed by using the kit of Beijing Solarbio Science & Technology Co., Ltd. The following formula was used to measure the proline contents: where CoD = the coefficient of dilution in the pre-treatment process.
Similarly, H 2 O 2 was also assessed by using the kit of Beijing Solarbio Science & Technology Co., Ltd. The following formula was used to measure the H 2 O 2 contents: CoD = the coefficient of dilution in the pre-treatment process, C st = Concentration of standard, OD st = Absorption of standard sample" 8 .
Ascorbic acid (AsA) contents were measured by the method of Mukherjee and Choudhuri 39 . We measurement AsA contents in the extract of trichloroacetic acid combined with dinitrophenylhydrazine and thiourea. Subsequently, the mixture was boiled for 15 min in a water bath (HWS-28), cooled at 27ºC, added H 2 SO 4, and absorbance was measured at 530 nm with the help of a spectrophotometer (TU-1810).
Meanwhile, glutathione (GHS) concentration in roots was assessed by the technique of Griffith 40 . For the measurement of GSH first, we neutralized metaphosphoric acid with sodium citrate. Analysis of three replicates of each treatment we made 700 μl of 0.3 mM NADPH, 100 μl of 6 mM 5, 5′-dithiol-bis-2-nitro benzoic acid, and 100 μl distilled water and100 μl of extract. In the end, GSH reductase (10 μl of 50 Units ml -1 ) was added, and a spectrophotometer (TU-1810) was used to detect the absorbance at 412 nm to calculate GSH contents from a standard arc.
Relative water contents (RWC) in the roots of wheat plants were assessed by using the method of WEATHERLEY 41 with some modification of Osman and Rady 42 . Similarly, the method of Premachandra et al. 43 with some modifications by Rady, et al. 16 was used to measure membrane stability index (MSI).
The method of Irigoyen, et al. 44 was used to measure total soluble sugar (TSS) contents in oven-dried roots of wheat plants. Took 0.2 g of dried root sample and homogenized with 5 ml 96% (v/v) ethanol, and then eroded with 5 ml 70% (v/v) ethanol. The extract was centrifuged with a centrifuge (TGL-18 M) at 3500 × g for 10 min, and then stored the supernatant at 4 °C. The water bath (HWS-28) was used to boil the 0.1 ml extract of ethanol along with 3 ml anthrone reagent [150 mg anthrone + 100 ml of sulphuric acid; 72%, v/v] for 10 min. The absorption was measured with a spectrophotometer at 625 nm, after cooling the supernatant at room temperature.
Determination of Cd, Si, and nutrient elements in plant tissues. "The contents of micronutrients (nitrogen: N, phosphorus: P, and potassium: K), macronutrients (calcium: Ca, magnesium: Mg, and zinc: Zn), heavy metal Cd contents, and beneficial elements Si contents were assessed by inductively coupled plasma mass spectroscopy (ICP-MS, Agilent, and 7700 X, USA) after being oven-dried by following the methods of previous researchers 8 (See Online appendix heading 3 and 4).
Statistical analysis. "The data were processed and analyzed using the SPSS 21.0 (SPSS, Chicago, IL), and all the graphs were made using the Sigma plot 12.5 software packages. The means of the three random replicates of 2-repeated experiments were subjected to analysis of variance (ANOVA), and multiple comparisons were performed using Duncan's multiple range test (DMR) at P < 0.05 8 .

Results
Effect of silicon and cadmium on root biomass. The effect of silicon (Si) and cadmium (Cd) on root fresh and dry weight of wheat plants grown under acidic nutrient solution is shown in Fig. 1. The means of three random replicates of 2-repeated experiments showed that Cd concentrations in acidic nutrient solution significantly reduced root fresh and dry weight as compared to the control. Root fresh weight for Cd50 and Cd200 was decreased by 10% and 37%, respectively, than that of control. Similarly, root dry weight for Cd 50 and 200 µmol L −1 was reduced by 14% and 47%, respectively, than that of control. The application of Si in Cd contaminated acidic nutrient solution significantly alleviated Cd stress by increasing root fresh and dry weight (Fig. 1). Si application with 1 and 3 mmol L −1 along with Cd 50 and 200 µmol L -1 increased fresh root weight by 21%, 73%, and 52%, 87%, respectively, as parallel to alone Cd 50 and Cd 200. Similarly, Si 1 and 3 mmol L −1 along with Cd50 and Cd200 increased root dry weight by 14%, 84%, and 28%, 98%, respectively, as parallel to alone Cd50 and Cd200.  Fig. 1. The means of three random replicates of 2-repeated experiments showed that all levels of Cd in an acidic nutrient environment significantly increased root volume and diameter than that of control. Cd with the concentration of 50 and 200 µmol L −1 increased root volume by 37% and 54% and increased root diameter by 20% and 59%, respectively, than that of control (acidic pH). The addition of Si in Cd-contaminated acidic solution significantly alleviated Cd toxicity by further increasing root volume and diameter ( Fig. 1). Si 1 mmol L −1 , along with Cd50 and Cd200, significantly increased root volume by 27% and 22% and increased root diameter by 20% and 18%, respectively, as parallel to alone Cd50 and Cd200. Similarly, Si 3 mmol L −1 , along with Cd50 and Cd200, significantly increased root volume by 40% and 42% and increased root diameter by 54% and 35%, respectively, as compared with Cd50 and Cd200. The most significant

Effect of silicon and cadmium on total root length and root tips. The effects of Si and Cd on
wheat plants in terms of root length and root tips are shown in Fig. 1. The means of three random replicates of 2-repeated experiments showed that all levels of Cd concentration in an acidic nutrient solution significantly decreased root length and root tips than that of control ( Fig. 1). Cd concentration with 50 and 200 µmol La −1 significantly reduced root length by 39% and 47% and decreased root tips by 65% and 73%, respectively, than that of control (acidic pH 5). The addition of Si in Cd-contaminated acidic nutrient solution significantly encountered the adverse effects of both Cd and acidic stress by elevating root length and root tips. Si 1 mmol L −1 , along with Cd50 and Cd200, significantly increased root length by 101% and 38%, and increased root tips by 105% and 126%, respectively, as paralleled to alone Cd50 and Cd200. Similarly, Si 3 mmol L −1 , along with Cd50 and Cd200, increased root length by 169% and 124%, and increased root tips by 270% and 237%, respectively, as paralleled with alone Cd50 and Cd200. Moreover, Si single application with a preference of 3 mmol L −1 in an acidic solution also led to a significant increase in root length and root tips as paralleled to control (Fig. 1).

Effect of silicon and cadmium on enzymatic antioxidant and protein contents. The effect of Cd
and Si on enzymatic (catalase; CAT, superoxide dismutase; SOD, peroxidase; POD) antioxidants in the roots of wheat plants grown in the acidic nutrient solution are shown in Table 1. The means of three random replicates of 2 back to back experiments showed that all levels of Cd concentrations (50 and 200 µmol L −1 ) significantly increased the contents of antioxidants in the roots of wheat plants as paralleled to control (Table 1). Cd with the concentrations of 50 and 200 µmol L −1 significantly increased CAT by 52% and 112%, SOD by 22% and 32%, and POD by 51% and 139%, respectively, in the roots of wheat plants as paralleled to control. The addition of Si with the preference of 3 mmol L −1 in Cd-contaminated acidic solution significantly encountered both Cd and acidic toxicities by further elevating enzymatic antioxidant contents in roots of wheat plants. Si 3 mmol L −1 along with Cd50 and Cd200 significantly increased CAT contents by 145% and 277%, SOD contents by 60% and 61%, and POD contents by 89% and 63%, respectively as paralleled to alone Cd50 and Cd200. Moreover, Si alone application in highly acidic nutrient solution also led to significant increased enzymatic antioxidants in the roots of wheat plants ( Table 1). The estimation of protein contents also indicated that Si treatments significantly effect on the roots of wheat plants with and without Cd stress ( Table 1). The recorded data showed that protein contents in roots increased with the decreased in Cd concentration from 200 to 50 µmol L -1 . The maximum protein contents were observed when plants were treated with 3 mmol L −1 Si alone, while minimum protein contents were observed when plants were grown under 200 µM toxicity of Cd. As paralleled to control and Cd-treated-plants, soluble protein contents were highest in both levels of Si (1 and 3 mmol L −1 ). Similarly, protein contents in Si, along with Cd treated plants, were also greater as paralleled to Cd alone (50 and 200 µmol L −1 ) ( Table 1).
Effect of silicon and cadmium on non-enzymatic antioxidants. The effect of Cd and Si on nonenzymatic (ascorbic acid; AsA and glutathione; GHS) antioxidants in wheat roots grown in the acidic growing medium, as shown in Table 1. The means of three random replicates of 2-repeated experiments showed that all levels of Cd concentrations (50 and 200 µmol L -1 ) significantly increased the non-enzymatic antioxidants contents in the roots of wheat plants as paralleled to control (Table 1). Cd with the concentrations of 50 and 200 µmol L −1 significantly increased AsA by 67% and 106%, and GHS by 44% and 83%, respectively, in the roots of wheat plants as related to control. The application of Si with the preference of 3 mmol L -1 in Cd-contaminated  www.nature.com/scientificreports/ acidic solution significantly encountered both Cd and acidic toxicities by further elevating non-enzymatic antioxidant contents in roots of wheat plants. Si 3 mmol L −1 along with Cd50 and Cd200 significantly increased AsA contents by 83% and 80% and GHS contents by 55% and 48%, respectively, as related to alone Cd50 and Cd200. Moreover, Si alone application in highly acidic nutrient solution also led to significant increased non-enzymatic antioxidants in the roots of wheat plants ( Table 1).

Effect of silicon and cadmium on osmoprotectants.
The effect of Cd and Si on osmoprotectants (total soluble sugars; TSS and proline) in the wheat roots grown in the acidic nutrient solution is shown in Table 1. The means of three random replicates of two back to back experiments revealed that all levels of Cd concentrations (50 and 200 µmol L -1 ) increased the contents of osmoprotectants in the roots of wheat plants as related to control (Table 1). Cd with the concentrations of 50 and 200 µmol L −1 significantly increased TSS by 92% and 167%, and proline by 19% and 66%, respectively, in the roots of wheat plants as related to control. The application of Si with the preference of 3 mmol L −1 in Cd-contaminated acidic solution significantly encountered both Cd and acidic toxicities by further elevating osmoprotectants contents in roots of wheat plants. Si 3 mmol L −1 , along with Cd50 and Cd200, significantly increased TSS contents by 121% and 33% and proline contents by 82% and 62%, respectively, as related to alone Cd50 and Cd200. Moreover, Si alone application in highly acidic nutrient solution also leads to increase osmoprotectants in the roots of wheat plants ( Table 1).

Effect of silicon and cadmium on reactive oxygen species (ROS) and malondialdehyde contents. The effect of Cd and Si on ROS in terms of hydrogen peroxide (H 2 O 2 ) and lipid peroxidation in terms
of malondialdehyde (MDA) contents in the roots of wheat plants grown in the acidic growing media is shown in Table 1. The means of three random replicates of two repeated experiments displayed that all levels of Cd concentrations (50 and 200 µmol L -1 ) remarkably increased the contents of ROS in the wheat roots as related to control (Table 1). Cd with the concentrations of 50 and 200 µmol L -1 significantly increased MDA by 46% and 175%, and H 2 O 2 by 53% and 116%, respectively, in the wheat roots as paralleled to control. The application of Si with the preference of 3 mmol L −1 in Cd-contaminated acidic solution significantly encountered both Cd and acidic toxicities by decreasing ROS contents in roots of wheat plants. Si 3 mmol L −1 , along with Cd50 and Cd200, significantly decreased MDA contents by 53% and 47% and H 2 O 2 contents by 66% and 50%, respectively, as related to alone Cd50 and Cd200. Moreover, Si alone application in highly acidic nutrient solution also led to remarkably reduced H 2 O 2 and MDA contents in the roots of wheat plants (Table 1).

Tissue-specific silicon concentration in roots. The concentration of Si in the roots of wheat plants
with and without Cd exposure in the acidic nutrient solution is shown in Table 2. The means of three random replicates of two back to back experiments showed that Si concentration was increased in root tissues with the rise of Cd level in the acidic growing media. There was an antagonistic correlation with the concentration of Cd and the accumulation of Si in the wheat roots. Si concentration in Si1 + Cd200 was 17% higher as compared with Si1 + Cd50. Similarly, Si concentration in Si3 + Cd200 was 26% higher as compared with Si3 + Cd50 (Table 2).

Tissue-specific cadmium concentration in roots. Cd accumulation in roots of wheat plants with and
without Si application is shown in Table 2. Without Si application, Cd accumulation in roots was enhanced with the rise of the Cd level from 50 to 200 µmol L −1 . While Si (1 and 3 mmol L −1 ) addition in acidic nutrient solution significantly hindered Cd uptake, translocate, and accumulation in wheat plants. But most significant results were recorded at Si 3 mmol L −1 compared to Si 1 mmol L −1 against both levels of Cd (50 and 200 µM). Si at the concentration of 1 mmol L -1 along with Cd50 and Cd200 reduced Cd accumulation by 15% and 23% in roots as compared to alone Cd50 and Cd200. Similarly, Si at the level of 3 mmol L −1 along with Cd50 and Cd200 reduced Cd accumulation by 68% and 41% in roots as parallel to alone Cd50 and Cd200 (Table 2). Table 2. Effect of Si and Cd on nitrogen (N) (mg g −1 ), phosphorus (P) (mg g −1 ), potassium (K) (mg g −1 ), calcium (Ca) (g kg −1 ), magnesium (Mg) (g kg −1 ), zinc (Zn) (mg kg −1 ), cadmium (Cd) (mg kg −1 ), and silicon (Si) (mg kg −1 ), contents in the roots of wheat plants. The values are expressed as the mean ± S.D. (n = 3). The different superscript letters within a column indicate significant differences at P < 0.005.
Treatments N (mg g −1 ) P (mg g −1 ) K (mg g −1 ) Ca (g kg −1 ) Mg (g kg −1 ) Zn (mg kg −1 ) Cd (mg kg −1 ) Si (mg kg −1 )  Table 2). The addition of Si with the preference of 3 mmol L −1 in Cd-contaminated acidic solution significantly reversed the toxicity of both Cd and acidic pH toxicities by elevating the concentrations of all essential nutrients. Si 3 mmol L −1 along with Cd50 and Cd200 significantly increased N concentrations by 50% and 66%, P concentrations by 49% and 205%, K concentrations by 61% and 75%, Ca concentrations by 63% and 65%, Mg concentrations by 21% and 59%, and Zn concentrations by 65% and 158%, respectively as parallel to Cd50 and Cd200. Si single application with a preference of 3 mmol L −1 in an acidic solution also led to a significant increase in concentrations of all recorded essential nutrients as parallel to control (Table 2).

Discussion
The root physiological and morphological traits display significant alterations in response to various environmental stresses [45][46][47] , and our findings showed the influence of Cd and Si on roots morphology and physiology of wheat crop grown in acidic nutrient solutions. First, the roots of wheat crops treated with moderate and high levels of Cd (50 or 200 µmol L −1 ) along with acidic pH (5) showed significant decreased growth and development of roots against control (acidic pH). Notably, the root morphological and physiological traits of wheat seedlings simultaneously treated with a low or moderate concentration of Si (1 and 3 mM) and low or high levels of Cd (50 and 200 µmol L −1 ) in acidic environmental conditions were enhanced than that of wheat seedlings treated to the parallel Cd and low pH particular treatments. Second, treatment with a small and moderate concentration of Si (1 and 3 mmol L −1 ) had positive effects on root morphology and physiology as compared with low pH single treatment (Fig. 1); besides, the biomass and phenotype of roots of wheat seedlings exposed under high concentration of Si (3 mmol L −1 ) along with acidic pH were significantly enhanced than that of roots treated with Si low concentration (1 mM) and the roots treated with both levels of Cd (50 and 200 µmol L −1 ) along with low pH. These findings showed that the inhibitory effects of acidic pH and low and high concentrations of Cd (50 and 200 µM) on the root morphology and physiology were alleviated by the treatment of Si (Fig. 1). Third, the effect of Si application on root morphology and physiology strongly correlated with the concentration of Si (Fig. 1), and the two way ANOVA results shown an apparent collaboration among Si and Cd concentrations along with acidic pH (Fig. 1 and Table 1). Root growth and development directly influence the root morphology and physiology. Our study demonstrated that the root physiology and morphology treated with Cd along with acidic pH is negatively correlated with the biomass of the root (Fig. 1). Our research is the consistency of previous findings where Cd concentration inhibited 50% root growth by increasing root hairs near the root tip in numerous plant species 5,48 . Moreover, previous findings have shown that Cd concentrations increased root diameter without causing necrosis 49,50 . Our study demonstrated the same results where low and high concentrations of Cd initiated a remarkable increase in average root diameter and root volume. At the same time, decrease average root biomass, root length, and the number of lateral roots (Fig. 1). The increased diameter and volume of Cd-treated root of wheat seedlings might be due to the high accumulation of Cd in the root parenchyma cells, which play a functional role in enhancing resistance to the radical flow of Cd. It was in the line of previous findings where a high accumulation of Cd often recorded in roots than shoots 7 . While Si application in our experiment significantly alleviated Cd toxicity by further increasing roots diameter and volume (Fig. 1). In our study, low and moderate concentrations of Si (1 and 3 mM) traps more level of Cd in root through vacuolar sequestrations, which were reflected by a further increase in root average diameter and volume (Fig. 1). The same results were recorded by Greger et al. 34 , where Si traps more concentration of Cd in root cell walls.
Si plays a central role in plant development, particularly under unfavorable conditions like heavy metal and acidic stresses 47,51,52 . Previous findings have shown different effects on plant growth and yield with various levels of Si concentrations 52 . The recorded data of the present findings indicate that Si accumulation in the roots of wheat seedlings grown under Si treaded acidic nutrient solution is entirely associated with root fresh and dry weights (Fig. 1). The application of Si (1 or 3 mM) in acidic nutrient solution boosted up root growth-related parameters, and it improved Si root accumulation in wheat seedlings against the corresponding acidic pH single treatment (Fig. 1). Though, the incorporation of Si 3 mM (high concentration) upregulated root morphology and physiological traits compared with Si 1 mM (low concentration) because Si deposition in wheat roots increases with the increase of Si amount in growth solution and also increases the roots capability to absorb Si 53 . The present study also supports the conclusion of previous findings ( Fig. 1 and Table 2). It has been reported by previous researchers that silica deposition in nature is strongly correlated with many chemicals, environmental factors, including, pH, silica concentrations, temperature, as well as the presence of other polymers, small compounds in a nutrient solution, and different cations and anions 54 . Acidic pH could release the toxicity perceived in roots of wheat seedlings treated with a high Si concentration (3 Mm) (Fig. 1), which might be due to a fact of an acidic pH prevents Si installation 54 . Though, this hypothesis still needs to be more deliberate.
The present study demonstrated that the root physiology and morphology treated with Si in the acidic nutrient solution is strongly associated with the root biomass (Fig. 1). Root development and yield are strongly affected by essential nutrient uptake and accumulation 45,55 . The vital nutrients like; N, P, K, Ca, Mg, and Zn influenced strongly on plant growth. K is a crucial element to the various metabolic reaction and organic plant structuring Scientific Reports | (2021) 11:1958 | https://doi.org/10.1038/s41598-020-80808-x www.nature.com/scientificreports/ due to its significant contribution to enzyme activation, protein synthesis, and photosynthesis reaction. Ca contributes to maintaining cell wall configuration and membrane performance 56 . Moreover, Ca deals with the physical and biological responses of plants by transduction of stress signals under various stresses 57 . The negative impact of acidic pH on seed emergent, growth, and root morphology and physiology could be improved by the incorporation of Ca. The previous researchers have shown that on behalf of Ca requirements, and different plants often show different levels of tolerance to acidic pH 58 . As an important component of chlorophyll and an enzyme cofactor, Mg plays a crucial role in carbon fixation and photosynthetic intensity 59 . Micronutrients, like Zn, play an important role in the organic plant structure, biochemical reactions, and metabolic activity of plants 60 . The results of previous findings have demonstrated that macro and microelements can affect plant growth and development to varying extents 61,62 . Therefore, the balanced amounts of nutrient elements are essential for adequate growth and survival of wheat plants under worse conditions of Cd and acidic pH. Several studies have shown that acidic pH inhibits the uptake and utilization of essential nutrients and thereby affects plant optimum growth 46,47 . Our study herein supports the conclusion of previous findings, but toxicities triggered by Cd and acidic pH were strongly encountered by the incorporation of Si. Specifically, treatment with Cd (200 µM) along with acidic pH (5) significantly diminished the root biomass (FW and DW) of wheat plants, significantly hindered the uptake of N, P, K, Ca, Mg, and Si, and visibly improved the concentrations of Zn and Cd ( Table 2). The addition of Si (1 or 3 mM) in acidic nutrient solution significantly amplified the wheat root biomass (F.W. and D.W.) and reassured any severe rises or falls (Fig. 1). Our findings demonstrated that Si in the wheat roots or Si treatment significantly disturbed the root uptake and utilization of essential nutrients and well-maintained the relative equilibrium of the mineral elements in roots ( Table 2). Similar conclusions have been drawn in previous findings 51,63 , and changed concentrations of mineral nutrients might establish a tool for the hindering or upregulating root growth 10,61 .
Reactive oxygen species (ROS) in terms of hydrogen peroxide (H 2 O 2 ) and peroxidation of membrane lipids in terms of MDA contents caused by the environmental stresses can be caused the irrevocable cellular injury through their intensive oxidative behavior 9 , which endorse variations in normal morphological and physiological structure of roots that enhance resistance 64 . In the current study, herein, a parallel response was recorded in the roots of wheat seedlings grown under low or high concentrations of Cd (50 or 200 µM) in the acidic growing media (Table 1). Moreover, the addition of Si in Cd contaminated acidic nutrient solution remarkably reduced the contents of H 2 O 2 and MDA in roots of wheat seedlings (Table 1). Previous studies have demonstrated that the incorporation of Si reduced the H 2 O 2 and MDA contents in plants under hostile conditions of stresses 31 , demonstrating that Si ameliorates oxidative burst tempted by acidic pH as well as Cd toxicity.
Although the overproduction of ROS can cause cell loss, it is needed to regulate ROS contents to a safe level in plants. Plants have enzymatic (superoxide dismutase; SOD, catalase; CAT, and peroxidase; POD) and nonenzymatic (glutathione; GHS, and ascorbic acid; AsA) antioxidants and osmoprotectants (total soluble sugar; TSS and proline) to remove ROS contents to confer resistance to stress 10,46 . In enzymatic antioxidants, SOD is more affective in demolishing ROS 65 . Our experimental results demonstrated that SOD activity was remarkably improved in the roots of wheat seedlings grown in low or high concentrations of Cd (50 or 200 µM) in an acidic nutrient solution, the incorporation of Si in Cd-contaminated acidic nutrient solution further improved SOD activity than that of the parallel Cd single treatment (Table 1), demonstrating improved alleviation of oxidative stress. Parallel results have revealed in earlier researchers that Si treatment demolishes Cd stress by improving the functioning of antioxidants in both roots and shoots of plants 31,66 . CAT, as a crucial antioxidant enzyme, participates in the deduction of harmful peroxides in root cells through the breakdown of H 2 O 2 compounds into H 2 O and O 2 . A significant increase in CAT contents in roots of wheat seedlings under low or high concentrations of Cd in the acidic nutrient solution (50 or 200 µM) was reported in the recent findings (Table 1), and an equivalent improvement was stated in previous results 31,67 . However, Si moderate or high concentration (1 or 3 mM) significantly reversed the adverse effects of Cd toxicity by further increasing in CAT contents in the roots of wheat plants under Cd exposure as compared with corresponding Cd single treatment (Table 1), representing that Si improves the antioxidant or scavenger contents and thereby decreases free radicals of oxidants, as was formerly reported 45,46,63 . POD also involves demolishing H 2 O 2 in below-ground parts of various plant species 10,47 . In the current study, POD contents were improved in the roots of wheat plants grown under low or high concentrations of Cd (50 or 200 µM) in acidic nutrient solution, the addition of Si moderate or severe concentrations (1 or 3 mM) in Cd-contaminated acidic nutrient solution further increased POD activity (Table 1).
In the non-enzymatic antioxidative ROS-scavenging mechanism, AsA, as a frontline of defense in the intervention with exterior oxidant injury 68 , plays a central role in demolishing H 2 O 2 69 . In our study, herein, AsA flow in roots of wheat seedlings was enhanced with the treatment of low or high level of Cd (50 or 200 µM) in an acidic nutrient solution, the addition of Si moderate or high concentrations (1 or 3 mM) in Cd-contaminated acidic nutrient solution further enhanced AsA flow in roots of wheat plants, indicated Si demolishing effect to Cd and acidic pH morphological impacts (Table 1). GSH displays a central role in cell tolerance to metal stresses by regulating redox imbalance and by minimizing the free ion cellular concentration above and below-ground parts of plants 70 . Our experimental data demonstrated that GSH activity was increased in the roots of wheat plants grown under Cd low or high concentrations (50 or 200 µM), the addition of Si moderate or high concentrations ( 1 or 3 mM) in Cd-contaminated acidic nutrient solution further increased GSH activity (Table 1), indicated Si role in regulating redox imbalance.
Osmoprotectants, in terms of total soluble sugar (TSS) and proline contents, maintain the homeostasis of ROS in both root and shoot cells, consequently protects the plants from various stresses 71 . TSS as an important osmoprotectant provides membrane protection by regulating the osmotic adjustment and by scavenging toxic ROS under various abiotic stresses 72 . Proline plays an essential role in stress tolerance by stabilizing the redox status of plant cells 73 . Moreover, proline is considered as an antioxidant to scavenge ROS and reserve the intercellular pool, a key redox buffer for cells 74 . In our study, herein, the activity of TSS and proline was increased in the roots of wheat plants treated with low and high concentrations of Cd (50 and 200 µM) along with acidic pH, Scientific Reports | (2021) 11:1958 | https://doi.org/10.1038/s41598-020-80808-x www.nature.com/scientificreports/ the addition of Si in Cd-contaminated acidic nutrient solution further increased the activity of TSS and proline, indicated the Si role to preserver the intercellular pool to stabilize the redox status of cells (Table 1). It has been recommended in prior findings that Si is an effective quasi-essential element that could participate actively in several morphological, physiological, and structural reactions in higher plants grown under biotic and abiotic environmental strains 31,75 , and the results of our study supported this assumption. In summary, on the one hand, Si incorporated with safe concentrations (1 and 3 mM) significantly improves the morphology and physiology of the roots of wheat seedlings, increases the activities of CAT, SOD, POD, AsA, GSH, TSS, and proline in roots of wheat plants, affects the uptake and consumption of mineral nutrients, and alleviates the acidic pH toxicity. On the other hand, Si addition also improves the combined toxicities of acidic pH and Cd concentrations in roots of wheat seedlings by demolishing ROS contents and by increasing the activities of enzymatic and non-enzymatic antioxidants and osmoprotectant contents.

Data availability
The data that support the findings of this study are presented in this manuscript. Raw data of enzymatic study and plasma mass spectroscopy in this research will be available on request after acceptance of the paper.