Efficacy of nano-silicon extracted from rice husk to modulate the physio-biochemical constituents of wheat for ameliorating drought tolerance without causing cytotoxicity

Abstract

Background

Abiotic stresses, like drought, are the major cause of shrinking plant, growth crop yields and quality. Nanotechnology has provided a significant improvement in increasing plant growth and yield of crops under stress conditions. This work assessed the potential of silicon for mitigating the negative effects of drought against wheat. In completely randomized design with three replicates, wheat seedlings grown under three watering levels (100, 60 and 40% of water holding capacity) were treated by silicon dioxide (SiO₂) as a normal or bulk form (Si) and SiO₂ nanoparticles (SiNPs) with concentrations of 100 and 200 mg L⁻¹. SiNPs was extracted from rice husk.

Results

Si and SiNPs treatments are shown to improve the growth of plants and increase the shoots and root weight, relative water content, photosynthetic pigments, and proline in wheat. SiO₂ either normal or nanoparticles at 100 mg L⁻¹ decreased lipid peroxidation as malondialdehyde was reduced. Also, nano-silicon increased free amino acids, antioxidant enzymes while decreased soluble sugars. Cytotoxicity assay proved the safety of nano-silicon usage.

Conclusions

In conclusion, the present study documented the significance of rice husk-extracted nano-silicon at rate of 100 mg L⁻¹ for improving growth and increasing tolerance to drought in wheat grown under water deficit.

1 Background

As reported by FAO, population in the world may exceed nine billion in 2050, and an increase of about 70% in global food is required to fulfill the growing population’s food demand [1,2,3]. After maize, wheat (Triticum aestivum L.) is the crop that is produced the most globally [4] and approximately 30% of the world’s populations rely mostly on wheat as a source of calories [5]. Wheat production reached around 765 million tons worldwide in 2019 [6]. However, the environmental stresses threat the crop productivity.

Critical issues are associated with climatic changes causing detrimental impacts for agricultural production and food security [7,8,9,10]. The concern of climate change is that it stimulates the occurrence of abiotic stresses such as drought, salinity, unfavorable heat, metals toxicity, and nutrient deficit which undoubtedly depress the economic product of crops [11,12,13,14,15]. Under variability of climate, biotic stresses have also pernicious impacts on crop yield and quality [16,17,18,19,20]. Exposure of crop plants to stresses leads to disruption of metabolic processes in plants, which negatively affects crop growth [21,22,23].

Drought, as a major environmental stressor, poses notable risks to crop productivity and global food security, especially as the severity of climate change increases [24, 25]. Water makes up 80–95% of the fresh biomass of the plant body, and it plays an important role in various physiological activities, including plant development, growth, and metabolism [26, 27]. Furthermore, drought reduced wheat yields by 20% on average worldwide owing to adverse impacts on physio-biochemical constituents and crop growth [28]. Climate change is a new phenomenon that frequently causes droughts, which in turn cause water shortages, and ultimately have a fierce impact on wheat yield everywhere in the globe [29].

Silicon (Si) acts a crucial work in plant life cycles and one of Si’s key tasks is to boost plant productivity, particularly in stressful situations. Si increased photosynthesis by exposing leaves to light in a favorable manner to acquire plant stress tolerance [30, 31]. Concerning its relation to stresses, the possible essential functions of Si include improving resistance to infections and pathogens, salt, metal toxicities and drought stress. Wheat is a crop with low salt tolerance and a Si-accumulating species [32]. Most studies on wheat suggest that Si boosts growth and yield during drought [33, 34].

Nanotechnology in agriculture is a developing interdisciplinary topic that plays a favorable effect in boosting plant growth and conferring stress tolerance while having a lot of potential for agricultural applications [35, 36]. Particles in nanoscale (NPs) have exclusive physiological advantages, including larger surface area and smaller volumes than bulk materials, affording them high solubility and rapid cellular transport [37] and stimulating their biological reactions [38, 39].

Silicon (Si) nanoparticles (SiNPs) have a small particle size, making it easy to enter cell membrane and organelles of plant [40]. Silicon dioxide (SiO₂) prepared as nanoparticles signaling molecules has just been shown to be functional regulator of many systems involved in ecological stress [41]. SiNPs might be efficient than bulk silica in reducing the effects of various adverse stresses [42]. Farmers routinely employ pesticides and fertilizers to improve agricultural productivity, posing a serious threat to the environment. As a result, eco-friendly technologies are needed to assist plants in overcoming stress challenges, resulting in optimal crop output and sustainability [43].

Despite the promising results of nanotechnology in agriculture, little information is available about the safety of the nanoparticles of Si. The current research hypothesized that nanoscale Si extracted from rice husk as a natural source of silicon could outperform its regular form in stimulating drought tolerance in wheat without phytotoxicity. Therefore, the effects of rice husk-extracted nano-Si form compared to bulk Si form on physio-biochemical traits and possible cytotoxicity in wheat seedlings under different watering regimes were assessed.

2 Methods

Wheat cv. Misr1 grains were provided from the Agricultural Research Centre, Egypt. Rice husks used for the preparation of silicon dioxide (SiO₂) nanoparticles were gathered from fields et al.-Sharqia governorate. Dry powder of SiO₂ extra pure was purchased from LOBA Chemie (Laboratory Reagents & Fine Chemicals), Mumbai, India. Hydrochloric acid (34%) was supplied by El Nasr Pharmaceutical Chemicals, Abu Zaabal, Egypt.

2.1 Preparation and characterization of silicon nanoparticles

To prepare the dry powder nano-SiO₂ (SiNPs), the husk of rice was grinded frequently until it reached a size of less than 1 mm. Hydrochloric acid (34%) was diluted with distilled water to prepare a (1:1) HCl solution. The grinded rice husk was refluxed with prepared HCl solution with a ratio of 1:10 w/v for 3 h. Reflux with acid solution is done for the removal of iron and partial hydrolysis and removal of hemicellulose, cellulose, and lignin from the rice husk [44, 45]. Subsequently, the treated rice husks were filtered and washed frequently by distilled water, the dried over nightly. Afterward, the dried husks were calcined at 650 °C for 2 h and were exposed suddenly to cooling in air to prevent the crystallization of the formed silica.

The microstructure of the produced silica was investigated using N₂ adsorption–desorption at 77 K using BELSORP MINI X® Automatic surface area and pore size analyzer [46] to determine the specific surface area (S_BET) and pore size distribution (BJH-method). Particle size distribution and zeta potential were measured using Malvern Instruments and the morphology of the prepared silica was checked via a Transmission Electron Microscope (TEM). The phase silica composition was checked by EDX using a JOEL JSM-6510LA and XRD technique using a Philips MPD X’PERT diffractometer, which uses Bragg–Brentano geometry.

2.2 Plant growth conditions

The experiment was performed in a temperature-controlled glasshouse (25 ± 1/18 ± 1 °C) (day/night). At the beginning of December 2022, wheat grains were surface sterilized in 1% (w/v) NaOCl for 10 min, and then washed thrice with sterile distilled water before starting the experiment to prevent fungal contamination. Seven grains were planted in pots 20 cm diameter × 20 cm depth which were filled with 1.5 kg mixed soil (clay: sand 1:2 w/w). Before starting irrigation treatments, the pots were irrigated regularly for maintaining the moisture in soil at 100% water holding capacity (WHC). All pots were irrigated with tap water for 1 week.

2.3 Experimental design and treatment

Dispersions of SiO₂ and SNP were prepared with two concentrations (100 and 200 mg L⁻¹) by dispersing in Hoagland’s solution with a high-power probe-type Sonicator (Ultrasonic Homogenizer, 300 V/T, USA) for 30 min to break up agglomerates for easy spreading of nanoparticles without precipitation. The experiment was factorial and implemented in a completely randomized design with three replicates. Therefore, wheat grains were planted in 45 pots that were arranged as five groups. The first group was irrigated with Hoagland nutrient solution as a control treatment. The second and third groups were irrigated with nutrient solution containing 100 and 200 mg/L⁻¹ bulk SiO₂ (Si100 and Si200). The fourth and fifth groups were irrigated with Hoagland solution with 100 and 200 mg/L⁻¹ SiO₂ nanoparticles (SiNPs100 and SiNPs200). The five groups were divided into three subgroups to be irrigated by 100, 60 and 40% of water holding capacity, WHC (WHC100, WHC60 and WHC40 as well-watered, moderate deficit water and severe deficit water, respectively). After full emergence (7 days from planting), seedlings were regularly irrigated with 2-day interval by the solutions of Hoagland, Si100, Si200, SiNPs100, or SiNPs200 based on WHC100 (no deficit water applied) until the seedlings reached 28-day old. After that, deficit water treatments were started with 2-day interval till seedlings reached 45-day old, where the experiment was terminated.

2.4 Assessments

At 45 days from sowing, the plant samples were taken to determine the fresh weights of shoots and roots, and relative water content (RWC). Also, samples of seedlings were stored at − 20 °C for subsequent biochemical analysis.

2.4.1 Fresh biomass

Wheat seedlings were washed by distilled water, then gently dried by filter paper and separated into shoots and roots. The fresh weights of shoots and roots were evaluated.

2.4.2 Leaf relative water content

Leaf relative water content (RWC) was computed as cited by [47]. Before oven drying small pieces of fresh leaves were weighted. The fresh pieces were soaked in distilled water and incubated for 24 h at 20 °C. After that, the fresh pieces were rapidly and carefully dried using tissue paper after soaking and weighted. The pieces samples were oven-dried for 72 h at 70 °C, and dry weight was determined. RWC% was computed using formula 1.

$$\text{RWC\%}=\frac{\text{Fresh weight }-\text{dry weight}}{\text{Turgid weight}-\text{dry weight}}$$

(1)

2.4.3 Photosynthetic pigments

After extraction by N,N-Dimethyl formamide, leaf chlorophyll a and chlorophyll b using a spectrophotometer at wavelengths of 647 and 665 [48], chlorophyll a, and chlorophyll b concentrations were estimated using formula 2 and 3. At wavelength of 453, carotenoids were estimated [49] using formula 4.

$${\text{Chlorophyll}}\,{\text{a}}\left( {{\text{mg}}\,{\text{g}}^{ - 1} \,{\text{FW}}} \right)\, = \,{12}.{\text{7 A665}} - {2}.{\text{79A647}}$$

(2)

$${\text{Chlorophyll}}\,{\text{b}}\left( {{\text{mg}}\,{\text{g}}^{ - 1} {\text{FW}}} \right)\, = \,{2}0.{\text{7 A647}}\, - \,{4}.{\text{62A665}}$$

(3)

$${\text{Carotenoids }}\left( {{\text{mg}}\,{\text{g}}^{ - 1} \,{\text{FW}}} \right)\, = \,{4}.{\text{2 A453}}\, - \,\left( {0.0{\text{264 Chlorophyll}}\,{\text{a}}\, + \,{\text{Chlorophyll}}\,{\text{b}}} \right)$$

(4)

2.4.4 Osmolytes

2.4.4.1 Proline content

The amount of proline (mg g⁻¹ FW) was measured using the technique described by [50]. 200 μL of the supernatant with 1 mL of the ninhydrin solution were used in the reaction mixture (2.5 g dissolved in 100 mL of ortho-phosphoric acid, acetic acid, and water (15: 60: 25; V.V.V). The reaction took place in a boiling water bath for 1 h. Using 1 ml of toluene to extract the produced dye, it was quantified spectrophotometrically at 515 nm. For calculation the concentration of proline, the standard curve of L-proline was utilized.

2.4.4.2 Fee amino acids

Free amino acid (FAA) content was determined calorimetrically by using ninhydrin solution [51] using glycine as a standard. The produced blue-purple color absorbance was estimated at 570 nm against blank using a spectrophotometer.

2.4.4.3 Reducing sugars

Total reducing sugars were estimated by dinitrosalicylic acid (DNS) method [52]. A spectrophotometer was employed to assess the absorbance at 540 nm. By using a standard curve of glucose, total reducing sugars was estimated.

2.4.5 Malondialdehyde

Lipid peroxidation expressed in malondialdehyde (MDA) concentration (μmol g⁻¹ FW) in plant tissues was measured as specified by [53] using UV–Vis spectrophotometer at 532 nm. The extinction coefficient was 155 mM⁻¹ cm⁻¹.

2.4.6 Antioxidant enzymes

To determine the antioxidant enzyme activity as stress biomarkers, 1 g of the seedlings were homogenized in precooled 100 mM KH₂PO₄ buffer (pH = 7.0) involving 0.1 mM EDTA and 1% polyvinyl pyrrolidone (PVP) (w/v) and centrifuged undercooling (4 °C) at 15,000g for 15 min and supernatant was collected. Based on the method explained by [54], the activity of superoxide dismutase (SOD, EC 1.15.1.1) was assessed. To assess the catalase activity (CAT, EC 1.11.1.6), the method of [55] was exploited. The activity of the enzyme crude extract’s peroxidase (POD, E.C. 1.11.1.7) was measured as stated by [56]. The soluble protein was estimated in the crude extract by using bovine serum albumin as a standard curve to obtain the specific activity of the assessed enzymes.

2.5 Cytotoxicity assay

Mouse normal liver cells, Vero: Green monkey kidney cells were obtained from Nawah Scientific Inc., Mokatam, Cairo, Egypt. The cells were kept in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin at 37 °C in a humidified 5% (v/v) CO₂ atmosphere. To assess the possible cytotoxicity assay of SiNPs, the SRB test was utilized to evaluate the vitality of the cells. In 96-well plates, aliquots of 100 μL cell suspension (5 × 10³ cells) were incubated for 24 h in full medium. Another aliquot of 100μL medium containing drugs at different concentrations was added to treat the cells. Following SiNPs treatment, cells were fixed by incubating at 4 °C for 1 h and changing medium with 150 μL of 10% TCA. After the TCA solution was discarded, the cells were five times cleaned with deionized water. Aliquots of 70 μL SRB solution (0.4% w/v) were added, and the mixture was incubated for 10 min at room temperature in a dark environment. The plates were air-dried for an entire night after being cleaned three times using 1% ethanolic acid. Next, protein-bound SRB stain was dissolved with 150 μL of TRIS (10 mM); the absorbance was then measured at 540 nm using an Infinite F50 microplate reader (TECAN, Switzerland) [57].

2.6 Statistical analysis

Result data were analyzed as a two-way analysis with a probability level of 5% using SPSS for Windows. Duncan’s test (P ≤ 0.05) was exploited to differentiate between the means of treatments.

3 Results

3.1 Characterization of prepared SiNPs

XRD pattern (Fig. 1) shows a diffused broad peak between 2Ө = 15–30, which indicates the amorphic structure of silica produced, and a definitive peak at 2Ө = 26.7 reflecting the presence of partially crystalline silica structure. Also, scanning electron microscope (SEM) and transmission electron microscope (TEM) and images of silicon dioxide nanoparticles (Fig. 1) showed the uniformly distributed silicon dioxide nanoparticles were in the agglomerated form.

Fig. 1

Characterization of Si nanoparticles (SiNPs). a XRD pattern of SiNPs; b Scanning electron microscope image (SEM); c Transmission electron microscopy image (TEM)

As shown in Table 1, the textural characteristics exhibited that the prepared silica nanoparticles (SiNPs) had higher surface area (252.51 m²/g) than the purchased silica (SiO₂) (152.28 m²/g), which indicates smaller particle size and higher activity for SiNPs. The mean pore radius (r_h) for both SiNP and SiO₂ are in the mesopores range (2–50 nm) but SiNPs exhibit narrow mesopores (6.0278 nm). Particle size distribution (PSD) confirms the smaller particle size of SiNPs also the narrower distribution of particle size (180–400 nm) than SiO₂ (97–2500 nm).

Table 1 Textural properties of prepared silicon nanoparticles (SiNPs) and bulk silicon (Si)

3.2 Effect of Si and watering on wheat seedlings

3.2.1 Fresh biomass

Fresh biomasses of wheat seedling shoots and roots as influenced by Si treatments under different watering levels are presented in Table 2. Under well-watered conditions (WHC100), application of SiNPs100 exhibited the heaviest weights in shoots and roots. Specifically, under moderate drought (WHC60), SiNPs100 achieved significant enhancement in shoots fresh weight which amounted to 10.6% compared to no Si supply, and significantly equaling Si100 in this respect. Furthermore, in pots irrigated by WHC40, the treatment of SiNPs100 recorded 1.33-folds in shoots fresh weight and 2.44-folds in roots fresh weight greater that the counterpart control treatment.

Table 2 Fresh weight of wheat seedling shoots and roots as influenced by bulk and nano-silicon concentrations under different watering levels

3.2.2 Relative water content (RWC)

As shown in Fig. 2, there were no obvious variations in RWC of Si-treated wheat plants (except for SI200) compared to the non-treated ones under well water regime (WHC100). The efficiency of Si to enhance RWC was more evident with moderate and severe water deficit. Herein, under WHC60, Si100 surpassed the control treatment by about 5.13%. Furthermore, all Si treatments outperformed the control treatment under WHC40.

Fig. 2

Relative water content (RWC) of wheat seedling leaves as influenced by bulk and nano-silicon concentrations under different watering levels. WHC100, WHC60, WHC40: watering by 100%, 60%, 40% of water holding capacity, respectively; Si100 and Si200: application of bulk silicon at 100 and 200 mg L⁻¹; SiNPs100 and SiNPs200: application of nano-silicon at 100 and 200 mg L⁻¹; Values are the mean of 3 replicates ± standard errors. Different letters of bars indicate that there are significant differences have been distinguished by Duncan’s multiple range test at p ≤ 0.05

3.2.3 Photosynthetic pigments

The combinations of watering and Si treatments revealed significant discrepancies in plant pigments of wheat plants (Fig. 3). In pots which irrigated by WHC100, all Si applications, except SiNPs100 for chlorophyll a, showed significant reductions in wheat plant pigments compared to no Si supply. Unlike, the combinations of Si100 or SiNPs100 × WHC60 and WHC40 and SiNPs200 × WHC40 achieved chlorophyll a content greater than the counterpart control treatments. The most effective practices for stimulating chlorophyll b were SiNPs100 × WHC60 or WHC40 and SiNPs200 × WHC40. Carotenoids showed the maximal increase with Si200 or SiNPs100 × WHC60 and SiNPs200 × WHC40.

Fig. 3

Photosynthetic pigments content of wheat seedling leaves as influenced by bulk and nano-silicon concentrations under different watering levels. WHC100, WHC60, WHC40: watering by 100%, 60%, 40% of water holding capacity, respectively; Si100 and Si200: application of bulk silicon at 100 and 200 mg L⁻¹; SiNPs100 and SiNPs200: application of nano-silicon at 100 and 200 mg L⁻¹; Values are the mean of 3 replicates ± standard errors. Different letters of bars indicate that there are significant differences have been distinguished by Duncan’s multiple range test at p ≤ 0.05

3.2.4 Osmolytes

Osmolytes expressed in proline, free amino acids and reducing sugars were statistically modulated by the Si under different watering regimes (Fig. 4). Si100 and SiNPs200 surpassed the counterpart control treatment under WHC100 by about 63.9 and 72.6%, respectively. SiNPS200 and SiNPs100 were the efficient treatments for increasing proline exceeding their counterpart control treatments under WHC60 and WHC40 by about 133.8 and 25.7%, respectively.

Fig. 4

Proline, free amino acids (FAA) and reducing sugars (RS) of wheat seedling leaves as influenced by bulk and nano-silicon concentrations under different watering levels. WHC100, WHC60, WHC40: watering by 100%, 60%, 40% of water holding capacity, respectively; Si100 and Si200: application of bulk silicon at 100 and 200 mg L⁻¹; SiNPs100 and SiNPs200: application of nano-silicon at 100 and 200 mg L⁻¹; Values are the mean of 3 replicates ± standard errors. Different letters of bars indicate that there are significant differences have been distinguished by Duncan’s multiple range test at p ≤ 0.05

SiNPS200 possessed higher values of free amino acids outperforming the counterpart control treatment by 1.18 and 1.10 times under WHC60 and WHC40, respectively.

As for reducing sugars, all Si treatments showed values lower than the counterpart control treatment with watering by WHC100 and WHC40. However, all Si treatments exhibited statistically similar values to the counterpart control treatment under WHC60, except SiNPs100, which was less in reducing sugars.

3.2.5 Malondialdehyde

Malondialdehyde expresses the oxidation of lipids in the plant cell because of the formation of free radicals inside the cell under drought conditions. Therefore, malondialdehyde is considered a marker of oxidation stress. Compared to the counterpart control treatment, all Si treatments (except for SiNPs200 under WHC40) decreased MDA under severe water deficit (WHC40) and well-watered (WHC100) conditions (Fig. 5). The differences did to reach the level of significance under moderate water deficit (WHC60).

Fig. 5

Malondialdehyde (MDA) of wheat seedling leaves as influenced by bulk and nano-silicon concentrations under different watering levels. WHC100, WHC60, WHC40: watering by 100%, 60%, 40% of water holding capacity, respectively; Si100 and Si200: application of bulk silicon at 100 and 200 mg L⁻¹; SiNPs100 and SiNPs200: application of nano-silicon at 100 and 200 mg L⁻¹; Values are the mean of 3 replicates ± standard errors. Different letters of bars indicate that there are significant differences have been distinguished by Duncan’s multiple range test at p ≤ 0.05

3.2.6 Antioxidant enzymes

Noticeable activity of catalase, peroxidase and super oxide dismutase as influenced by Si treatments was obtained under different watering regimes (Fig. 6). CAT activity increase was more pronounced with SiNPs100 surpassing the other counterpart Si treatments under each of under WHC100, WHC60 and WHC40. Si100 recorded the highest activity of POD and SOD under WHC60, surpassing the other SI treatments. Furthermore, higher activities of POD with SiNPs100 and SiNPs200 as well as SOD with SiNPs200 were obtained under WHC100 as compared to the counterpart control treatments. SiNPs200 × WHC40 (for POD) and SiNPs100 or SiNPs200 × WHC60 (for SOD) recorded fewer values than the counterpart control treatments. Moreover, all Si supplies were statistically at par for increasing SOD under WHC40, outperforming the control treatment.

Fig. 6

Catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) of wheat seedling leaves as influenced by bulk and nano-silicon concentrations under different watering levels. WHC100, WHC60, WHC40: watering by 100%, 60%, 40% of water holding capacity, respectively; Si100 and Si200: application of bulk silicon at 100 and 200 mg L⁻¹; SiNPs100 and SiNPs200: application of nano-silicon at 100 and 200 mg L⁻¹; Values are the mean of 3 replicates ± standard errors. Different letters of bars indicate that there are significant differences have been distinguished by Duncan’s multiple range test at p ≤ 0.05

3.3 Cytotoxicity assay

Based on the findings presented in Fig. 7, the cell viability of Vero and BNL cell lines were evaluated at different concentrations of SiNPs. Cell viability was relatively high at lower concentrations of SiNPs. As the concentration of SiNPs increased, the cell viability decreased. Herein, viability percentages were 99.1, 96.7, 94.8, and 93.1% for BNL cell as well as 100.0, 96.0, 92.8, and 90.3% for Vero cell at 0.08, 0.8, 8, and 80 μg mL⁻¹ of SiNPs, respectively.

Fig. 7

Vero: Green monkey kidney cell (a) and BNL: Mouse normal liver cells (b) viability grown on DMEM medium incubated for 48h. Using different concentrations of silicon dioxide nanoparticles illustrates that IC50 dose is > 50 μg mL⁻¹

4 Discussion

Unfortunately, agriculture is the main sector exposed to drought problems, which negatively affect plant production. Subjecting plants to drought stress can earnestly influence ionic homeostasis, acquisition of water and photosynthesis efficiency [58,59,60,61]. As for ionic homeostasis, it has been documented that the unfavorable conditions such as drought cause detrimental impacts on uptake and utilization of nutrients, influencing plant metabolism [62,63,64,65]. These effects occur owing to reducing leaf water content and cell membrane stability [66,67,68]. However, crop plants have developed different tactics to face various stressed ecosystems while keeping their growth and reproduction [69,70,71,72]. The present study examined the potential of Si, whether as a normal form or nano one, for modulating the drought tolerance in wheat. Despite drought decreased the biomass of shoot and root, Si treatments ameliorated the dry weight during water deficit periods. In addition to being essential for the uptake of water and ions, roots are also necessary for the absorption, synthesis, and movement of a wide variety of solutes. In stressful circumstances, wheat growth can be directly impacted by root features, as the root system is the majorly responsible for uptake of water and ions, and the absorption, synthesis, and movement of a wide variety of solutes [73]. Silicon dioxide nanoparticles are shown to help in water uptake and plant transport. The hydrophilicity of silicon oxide might be attributed to its advantageous properties [74]. These results agree with earlier reports of similar experiments in other plant species, which showed that in non-stressed or osmotic-stressed environments, silicon prepared as nanoscale stimulated the growth of apples [75]. Additionally, it has been demonstrated that applying silicon to drought-stressed rice plants increases root growth [76]. Drought lowered RWC of drought-stressed wheat leaves, while RWC increased when plants received SiNPs because of decreasing the dehydration effect [77]. SiNPs with a concentration of 100 mg L⁻¹ in 100% and 40% of WHC were the efficient practices for increasing RWC. Si’s capacity to keep water during drought could be increased gas exchange features, chlorophyll content, RWC, and water usage effectiveness [78]. Since Si distinctively accumulated under the leaves’ cuticle, forming a double layer of Si-cuticle, transpiration decreased [79]. This agrees with the present study. Owing to the absorption of Si in leaves and the formation of bonds with cell wall constituents, Si supplementation reduced water loss while cell wall integrity stimulated [80]. Under salt-stressed environment (physiological drought), Si addition also boosted plant aquaporin activity and raised the hydraulic conductivity of roots; both improve plant health and raise water levels [81].

SiNPs coordinate numerous plants physiological processes, in particular assimilation of nutrients, carbon dioxide fixation, accumulation of metabolic secondary products and activities of various enzymes under normal or stressed conditions [82,83,84]. Plant pigments degradation, photosynthetic apparatus dysfunction and cellular oxidative blast are considered significant biochemical indicators of deficit water [85, 86]. The biochemical changes under drought could be attributed to the excessively liberation of ROS via handicapping electron transport during photosynthesis process [87,88,89]. On the contrary, the results of the current study clarified that the treatments of SiNPs under deficit water increased the rate of photosynthesis by increasing photosynthetic pigments. The least chlorophyll content during drought conditions was recorded with the blank treatment. Because they make it easier for minerals to be absorbed through the opening of the xylem, supplying SiNPs increased photosynthetic activity [90]. When compared to the control treatment, the content of carotenoid and chlorophyll was significantly reduced with increased stress. The reduction in photosynthetic pigments like chlorophyll, and carotenoids, was likely brought on by the suppression of the ribulose-1, 5-bisphosphate enzyme and structural injury to the chloroplast and photosynthetic machinery [91, 92]. Chlorophyll content, stomata conductance, net photosynthetic rate, intercellular CO₂ concentration and transpiration rate were reduced because of the oxidative stress brought on by drought stress. This decline might be ascribed to pigment photo-oxidation and chlorophyll degradation. Additionally, drought-stressed plants restrict their stomata to stop water loss, inhibit carbon transfer [93], and lower their rubisco and ATP synthase activities [94].

In addition, stable oxygen-evolving processes were generated by nano-sized metal compounds attached to photosynthesis II (PSII), indicating that light-driven electrons were transferred from water molecules to quinone molecules [95]. Accordingly, PSII conjugates could develop into photo-sensors and synthetic photosynthetic apparatuses [95]. Nanoparticles speed up the photosynthetic rate by quickening the formation of plant pigments [96]. SiNPs can enhance photosynthesis by opening PSII reaction centers and stimulating the absorption and utilization of light energy, the transport rate of electron in PSII, and the biosynthesis of chlorophyll and carotenoid [97]. Also, SiNPs can organize the expression of several genes that encode proteins directly included in photosynthesis apparatus [98, 99].

Results of the current study showed that during normal irrigation (100%WHC), the proline level was lower than its level in drought (40%WHC). Proline is a remarkable constituent of osmotic adjustment and is an osmolyte that typically accumulates during stress [100]. However, when using silicon dioxide, it led to a decline in the proline content during drought at 40%WHC. According to one study, silicon supplementation reduced proline accumulation whereas water stress increased the proline content of wheat leaves, which is consistent with proline accumulation being connected as an indication of stress damage in experimental conditions [101]. These osmolytes accumulate in plants, protecting them from increasing oxidative stress, and as protectants against dehydration of the cell and protect macromolecule from denaturation, which is a vital sign of stress tolerance [102, 103]. Under stress, plant cells produce more ROS and have more proline to scavenge them. Si balances proline generation and ROS production to maintain the redox equilibrium, causing stability in the cellular plasma membrane [103, 104]. Additionally, it was discovered that Si accumulation in plants lowers proline levels via shrinking the injury impacts of stress [105].

Using SiNPs at a concentration of 100 mgL⁻¹ with drought at 40% WHC, proline level raised. This is a protective factor, and therefore there is no need to increase some other solutes such as reducing sugars, which led to a decrease in MDA, meaning that despite the presence of drought, it does not stressful, and the evidence for this is a decrease in MDA and reducing sugars. Increasing proline led to an increase in the water content of the plant, which led to an increase in the RWC% compared to the control during drought (40% WHC). Plants tended to accumulate more osmolytes, such as proline under drought stress [106]. During drought stress, endogenous proline was significantly increased when SiNPs were applied exogenously [107]. Proline is used by plants as an osmolyte and as nitrogen provider to prevent denaturation of proteins [108].

It was found that during drought, amino acid content was increased by applying nano-silicon. The tolerant plant group aim to increase their levels of soluble sugars, proline, amino acids, chlorophyll, and antioxidant activities [109]. Silicon supplementation increased protein level in plant seedlings, which provides amino acids and energy [110]. The present work proved that the concentration of soluble sugars increased with 60% WHC and 40% WHC over 100% WHC. This is to help reduce the stress severity, but we find that the treatments decreased the level of soluble sugars relative to the control, and this is evidence of the plant’s tolerance stimulation. Soluble sugars perform a crucial work in drought tolerance by scavenging ROS, controlling stomata activity, protecting essential macromolecules, and preserving the water balance of cells [111]. Furthermore, since it had the potential to promote the antioxidant systems, Si decreased the formation of H₂O₂ and MDA and lowered electrolyte leakage [112]. It has also been observed that SiNPs modify phenylpropanoid and shikimic acid pathway-involved enzymes, causing high accumulation of phenols in leaves [113, 114]. All of these important roles of Si contribute to strengthening plants and increasing their tolerance to various stresses [115]. MDA level is an indicator of oxidative damage of lipids. MDA level was increased in the control during drought (40% WHC). This is evidence of the occurrence of lipids oxidation of because severe drought. In this respect, drought-stressed maize shoots had considerably higher MDA levels than unstressed plants [116]. The injury in plant during drought may be the cause of an increase in MDA levels [117]. When drought-stressed plants were treated with SiNPs, their MDA levels were lower than those of untreated plants. Compared to non-drought plants, plants treated with SiNPs may experience less MDA-induced oxidative stress and membrane damage [118]. This is consistent with the results of our research. However, it was found that the SiNPs at 200 mg L⁻¹ led to a significant increase in MDA during drought (60 and 40% WHC). High concentrations of silica nanoparticles could induce the formation of free radicles affecting the cell membrane functions [119].

Oxidative stress of drought could be suppressed via stimulating enzymatic antioxidants which scavenge ROS [120,121,122,123,124]. As clarified in the current work, generation of oxidative stress stimulated the activity of antioxidant enzymes (CAT, POD, and SOD). The activity of the POD enzyme in well-watered was lower than in drought, and SiNPs at 200 mg L⁻¹ may resulted in that reflect on activity for eliminating hydrogen peroxide. POD activity during dehydration as an antioxidant defense can scavenge ROS to protect cells from damage. By quenching ROS, the organelles create antioxidant defensive system to protect plant cells from oxidative injury [125]. Increased POD, CAT and SOD activities following Si supply in drought-stricken grape plants demonstrate the Si’s capacity to improve the anti-oxidative effectiveness. Additionally, less H₂O₂ is produced, there are less soluble proteins, and more chlorophyll a, b, and total chlorophyll since the chlorophyll is not broken down. Since CAT is the enzyme that scavenges H₂O₂, high levels of H₂O₂ under both moderate and severe drought stress are partially a result of decreased CAT activity. Furthermore, modulation in plant hormones that have important roles for coordinating plant processes and adapting to stresses is another way to respond to stress [126,127,128,129]. Herein, Si altered the endogenous phytohormones of the abiotic stressed plants with mitigation of damages of stress [130].

Regarding the cytotoxicity assessment, low toxicity of SiNPs at various concentrations on mouse normal cells was detected. This could be attributed to the large size (280.6 nm) of the silicon dioxide nanoparticles prepared from rice husk these results agreed with other studies [131, 132]. Increasing SiNPs concentration showed reduced cell viability, refereeing to possible cytotoxicity. It has been found that increased SiNPs rate caused chromosomal injury in cells of mouse and human [133, 134]. Although some studies support our result, it should be mentioned that the phytotoxicity data for SiNPs are still vague and further studies are needed.

5 Conclusions

In conclusion, these results show that the addition of silicon dioxide (SiO₂) and silicon dioxide nanoparticles (SiNPs) extracted from rice husk reduced the negative effects of drought on wheat plant growth. It was found that silicon dioxide nanoparticles with a concentration of 100 mg L⁻¹ is considered the most effective treatment to ameliorate the oxidative stress of drought via increasing the plant biomass, leaves water content, photosynthetic pigments and proline and activity of antioxidant enzymes, while reducing the content of soluble sugars, and malondialdehyde. Although the cytotoxicity study revealed the safety of nano-silicon, more deeply investigations are needed at the molecular level to verify possible alternations in gene expression.