Nano silica reinforces the tolerance of the wheat plant against drought stress

Nano silica reinforces the tolerance of the wheat plant against drought stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Nano silica reinforces the tolerance of the wheat plant against drought stress Nesma Elsayed, Sadiek Mehasen, Radwan Khalil, Hayder Al-Musawi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5271175/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Silicon nanoparticles have distinctive physicochemical characteristics and improve the plant growth and yield under unfavorable environmental conditions. Therefore, the present investigation was undertaken to study the impact of Nano Silica on drought resistance depending on the Nano-Silica dose and moisture levels. Nano Silica applied at different rates (0.0, 50 and 100 mg/l) and the water regime was 30, 50 and 70% of water holding capacity (WHC). Results indicated that, Si-NPs there was an ability to reduce the drought impact on wheat growth and improve the antioxidant system in plants. Besides, an increment in membrane stability index, chlorophyll, carbohydrates, protein and phenol content of wheat. In addition, spraying or watering of Si-NPs increased wheat tolerance to drought by increasing the activity of antioxidant enzymes; nitrate reductase, phenylalanine ammonia-lyase, catalase, ascorbate peroxidase, glutathione s-transferase and guaiacol peroxidase, as well as reducing the oxidative pressure in leaves which was demonstrated by the diminished electrolyte leakage, malondialdehyde and proline in plant tissue. Data indicated that most of the highest values of the growth parameters and biochemical estimation were recorded for the wheat with application of 100 mg/l nano silica. Overall, this study advanced our understanding of the physiological and biochemical mechanisms underlying drought stress and mitigating its impact using Si-NPs, which may reduce the environmental risks that negatively affect the growth and productivity of agricultural crops globally. Antioxidant enzyme Drought stress Nanoparticles Photosynthetic pigments Proline Silica Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Global warming and climate change have recently increased the frequency and severity of many stresses, which has a direct impact on agricultural yield and quality. The three most significant staple crops in the world, wheat, rice, and maize, account for a sizable portion of daily caloric and protein intake 1 . Wheat holds the top spot among these key grains because of its domestication and role as the world's main staple food crop 2 . Due to adverse surroundings brought on by climate change, which has resulted in the emergence of several abiotic stressors, wheat yield may drop by 6% 3 . Heat and drought have been shown to be the most significant variables affecting crop output and ultimately contributing to food security. Draught-like conditions are becoming more commonplace worldwide due to altered rainfall patterns and a deficiency of precipitation 4 . Drought conditions cause physiological disturbances, physical damage, and biochemical changes in plants that negatively impact crop growth, development, and output. Drought stressors have a complex mechanical effect because they leave behind many impressions. It makes sense that improved knowledge of plant responses to drought stress will influence crop management and modification. Therefore, a comprehensive strategy is required to fully describe and comprehend how drought stress conditions affect plants for increased agricultural production 5 . The unique physiochemical properties of nanoscale silicon particles have applications in a variety of domains, including agriculture, which shows great promise. Because of their unique properties, Si-NPs can withstand agricultural damage from abiotic stressors including climate change (Tripathi et al., 2012). The utilization of Si-NPs in agriculture has the potential to enhance global food security by promoting the development of superior, more productive cultivars 7 . It was discovered that Si-NPs were employed as a weapon in the agricultural sector to resist various environmental stresses, such as dehydration 8 , UVB stress (Tripathi et al., 2017), heavy metal toxicity 10 , and salt stress 11 . Among other creative uses, Si-NPs can be applied as herbicides, insecticides, and fertilizers. Si-NPs may thus improve crops for environmentally friendly farming practices. Silicon, the most prevalent metalloid on Earth and second-most abundant in the crust, is a semi-essential element for plants due to its beneficial effects on environmental stress, making it a valuable resource (Luyckx et al., 2017). This study explores the positive impacts of silicon nanoparticles on plants, highlighting their unique physiological properties and potential use as nanocarriers for various compounds. It also explores the role of silicon nanoparticles in plant development, growth, and productivity, particularly under drought stress, and provides a comprehensive overview of their potential applications. Materials and methods Experimental design Two field experiments were carried out during winter seasons at Moshtohor region, Qalubiya Governorate, Egypt, to study the effect of three water regimes 1- Normal irrigation at tillering stage, elongation stage, heading stage, milk stage and filling stage or irrigation intervals 30 days among irrigations (Five irrigations); 2-Three irrigations. at end tillering stage and starting elongation stage, at end heading stage and starting milk stage and filling stage or irrigation intervals 50 days among irrigations (Three irrigations); 3-Two irrigations at end elongation stage and started heading stage and filling stage or irrigation intervals 70 days between irrigations (Two irrigations), and three silica nanoparticles treatments (Zero nano silica; 50 mg L -1 SiO 2 ; 100 mg L -1 SiO 2 ) on growth parameters, some metabolic activities related with the mechanism of bread wheat c.v. Misr 3. A spilt plot design with four replications was used. The three water regimes were allocated to the main plots and the three silica nanoparticles spraying were arranged in the sub plots. The area of sub-plot was 10.5 m 2 (1/400 fed). SiO 2 nanoparticles were purchased from US Research Nanomaterials. SiO 2 nanoparticles were prepared at two concentrations (50, 100 mg L -1 ) by dissolving in water and dispersed with a high-power probe-type. Electrolyte leakage The total inorganic ions leaked out from the leaves were measured by the method described by Sullivan (1979). In a boiling tube with 10 cm 3 of deionized water, twenty leaf discs were placed. The conductivity meter was used to measure the EC of the tubes after they were heated for 30 minutes at 45 o C (ECa) and 55 o C (ECBs) in a water bath. After the items were cooked for ten minutes at 100 degrees Celsius, the EC was once more reported as Ecco. The following formula was used to determine the electrolyte leakage: Electrolyte leakage (%) \(\:=\frac{\text{E}\text{C}\text{b}-\text{E}\text{C}\text{a}}{\text{E}\text{C}\text{c}}\) X100 Membrane stability index In two sets of ten cm 3 of double-distilled water, 200 mg of fresh leaves were used to determine the MSI. Electrical conductivity C1 was measured after one set was heated to 40°C in a water bath for thirty minutes. As an alternative, the second set's conductivity (C2) was determined after it was boiled for ten minutes at 100 degrees Celsius in a boiling water bath; a conductivity meter was used to test both conductivities. MSI was calculated using the formula described by (Sairam, 1994). MSI (%) = [1 – (C 1 /C 2 ) × 100] Lipid-peroxidation (Malondialdehyde; MDA): Using the Hodges et al . (1999) approach, the degree of lipid peroxidation was expressed in terms of the concentration of malondialdehyde (MDA). Ten milliliters of 5% thiobarbituric acid (TBA) were used to homogenize a 0.2-gram leaf sample. For 10 minutes, the homogenate was centrifuged at 15000 g. 4 ml of 0.5% thiobarbituric acid in 20% TCA was added to a 2 ml portion of the supernatant. The mixture was centrifuged at 10,000 g for 10 minutes after being heated to 95°C for 30 minutes then rapidly cooled in an ice bath. The supernatant's absorbance was measured at 532 nm. The non-specific absorption value at 600 nm was deducted. µg (MDA) g − 1 fresh weight is the MDA content, which was determined using its absorption coefficient of 155 n mole − 1 cm − 1 . Extraction and determination of photosynthetic pigments: Total photosynthetic pigments were extracted and determined according to the method described by Fadeel (1962). The study involved soaking maize leaves in 100% acetone, grinding, adding sodium sulfate and magnesium carbonate, centrifuging, and cleaning to extract pigments. Absorbance measurements were performed using a Perkin-Elmer spectrophotometer, determining carotenoids and chlorophyll amounts (Sestak et al ., 1971). Chlorophyll a = 9.78 E 662 - 0.99 E 644 µg /ml Chlorophyll b = 21.4 E 644 – 4.65 E 662 µg /ml Chlorophyll (a + b) = 5.13 E 662 + 20.41 E 644 µg /ml Carotenoids = 4.69 E 440.5 − 0.268 chlorophyll (a + b) µg/ml Following the preceding formulae, mg pigment g -1 fresh weight was computed from the results, which were expressed as g pigment ml-1 extract. Estimation of Carbohydrate contents. The dried tissues were homogenized with 80% ethanol, heated, filtered, and oven dried to determine soluble sugars, then dissolved in water 18 . To find the soluble sugars, the anthrone sulphuric acid technique 19 was employed. The anthrone reagent, consisting of 100 ml 95% H 2 SO 4 and 0.2 g of anthrone, is made fresh every time. A Pyrex test tube is filled with soluble sugar solution and heated for seven minutes. The blue-green color is measured against a blank, creating a calibration curve. The dry residue from extracted soluble sugars was used to identify polysaccharides. A mixture of 1.5 N sulfuric acid and dry material was heated, prepared for analysis using the anthrone sulfuric acid reagent method, and calibrated using pure glucose 19 . It was computed as the total of the same sample's soluble sugar and polysaccharide contents. For all data, the dry weight was expressed as mg 100 g -1 . Determination of proline : The amount of free proline was calculated using (Bates et al ., 1973). The study involved homogenizing macerated fresh tissue with 3% aqueous sulfosalicylic acid, filtering it, and using a ninhydrin reagent. The mixture was extracted using toluene, and the absorbance was captured at 520 nm. The proline concentration was determined using a standard curve and calculated as mg proline per 100 g − 1 fresh matter. Estimation of protein The amount of total protein was measured using spectrophotometry in accordance with the protocol outlined by Bradford, (1976). Prepare protein samples in test tubes using phosphate-buffered saline. Dilute samples with dye binding solution, blend, and let color take five minutes. Measure absorbance at 595 nm, plot a standard curve, and determine protein content using the absorbance V concentration. Estimation of total phenol Total phenol estimation can be carried out with the Folin- Ciocalteau reagent 22 . The material was ground in 80% ethanol, centrifuged, and dissolved in distilled water. Test tubes were filled with water, Folin-Ciocalteau reagent, and 20% NaCO3 solution. The absorbance at 650 nm was calculated, and a standard curve was created with different catechol concentrations. The amount of phenols in the test sample was calculated from the standard curve and expressed as milligrams per 100 grams of material. Determination of Carbonic anhydrase (CA) . We assessed the CA activity (EC 4.2.1.1) in the leaves using the protocol outlined in 23 . Small sections of the leaf samples were cut and placed in a solution of cysteine hydrochloride. After blotting and transferring the leaf samples into a test tube, 0.2 M NaHCO 3 , phosphate buffer (pH 6.8), bromothymol blue, and methyl red indicator were added. Titrating the reaction against 0.05 N HCl was done. Using fresh mass as a foundation, the enzyme's activity was expressed as mol (CO2) g -1 (FM) s -1 . Determination of Nitrate reductase (NR). The technique established by 24 was used to measure the activity of NR (EC 1.7.1.1). After being sliced into small pieces, the fresh leaf samples were placed in plastic vials with KNO3, isopropa 2h, and phosphate buffer (pH 7.5). N-1-naphthylethylenediamine hydrochloride solutions and sulfanilamide were added after incubation. Using a spectrophotometer (Spectronic 20D, Milton Roy, USA), the absorbance was measured at 540 nm. Based on fresh mass, the enzyme's activity was expressed as nM (NO2)g -1 (FM)s -1 . Determination of phenylalanine ammonia-lyase (PAL): Extraction and assay of PAL (EC 4.3.1.5) were carried out as described by Solecka and Kacperska (2003). The extraction buffer was mixed with liquid-nitrogen tissues, centrifuged, and the supernatant was used to measure PAL activity by using the raw enzyme. The enzyme extract and substrate solution were mixed, and the reaction was stopped by adding 6 N HCl. The absorbance was measured, and the enzyme activity was measured in nmol t-cinnamic acid g − 1 fresh weight at FWT h − 1 . Estimation of antioxidant system Sample preparation was described by 26 . The powder was mixed with phosphate buffer, filtered, and centrifuged. A fresh leaf sample was ground and centrifuged. The supernatant was collected and stored at 4°C for analysis of CAT, APX, POD, and SOD activities. A reaction mixture (3 ml) containing phosphate buffer (50 mM, pH 7.0), 30% (w/v) H 2 O 2 , and 0.5 ml of enzyme extract was used to measure Catalase activity (EC 1.11.1.6.) (Aebi, 1984). According to Havir and McHale (1987), the activity of the CAT enzyme was measured by the decrease in absorbance at 240 nm using a Spectronic 601 UV spectrophotometer as a result of H 2 O 2 consumption. This was expressed as µM H 2 O 2 oxidized g-1 fresh weight min-1. According to 29 , ascorbate peroxidase activity (EC 1. 11. 1. 11) Was measured by observing the drop in absorbance at 290 nm after 1 minute of ascorbic acid oxidation. Using a combination containing 72.7 mM Na-phosphate buffer (pH 6.5), 3.6 mM reduced glutathione, 1 mM 1-chloro-2,4-dinitrobenzene, and enzyme extract, the activity of glutathione-S-transferase (GST; EC 2.5.1.18) was determined by monitoring changes in the absorbance at 340 nm 30 . A UV-VIS 160A spectrophotometer from Shimadzu Corp. in Kyoto, Japan was used to measure the photometrically determined enzyme activities, which were then expressed in nkatal g–1 (fresh mass, FM). As stated by 31 , the guaiacol peroxidase (EC 1.11.1.7.) (G-POD) activity was measured at 470 nm. Enzyme extract, 0.0375% H 2 O 2 , 0.88 mM guaiacol, and 88 mM Na-acetate buffer (pH 5.5) made up the reaction mixture. Results Electrolyte leakage (EL), membrane stability index (MSI) and lipid - peroxidation (Malondialdehyde; MDA): The data recorded in Fig. 1 revealed electrolyte leakage, membrane stability index and lipid-peroxidation which indicate the injury of cell membrane. It is notable from the data that wheat plants offered a different pattern of response in these parameters under drought stress. 50 and 70 days of drought caused a non-significant increase in electrolyte leakage and lipid-peroxidation, as compared with the control plants. A maximum induction in EL and MDA were recorded in the plants exposed to 70 days drought (by 55% % and 58% respectively) over the value of the control plants. On the other hand, the exposure of the wheat plants to drought caused a decline in MSI in the plant exposed to 70 days (~ 23.61%). On the other side, spraying nano silica with different concentrations 50 mg L -1 and 100 mg L -1 treatments improved the MSI (by 7.41% and 13.57% respectively) with reduction in EL and MDA ( by 7.07% and 13.45% respectively) and (by 14.25% and 16.30% respectively) as compared with control plants (30 days). Photosynthetic pigments: The photosynthetically active pigment contents chlorophyll a, chlorophyll b, carotenoids and hence the total pigments which estimated in leaves of wheat plant at filling stage were presented in Fig. 2 . The contents of photosynthetic pigments were a significant decrease with the rise of soil water deficit days as compared with non-stressed plant (30 days). The highest inhibitory effect of water regime on photosynthetic pigments were recorded at 70 days of water regime as compared with non-stressed plant. On the other hand, spraying of nano silica with different concentration 50 mg L -1 and 100 mg L -1 , significantly alleviated the inhibitory effect of soil water deficit on photosynthetic pigment contents (chlorophyll a, chlorophyll b, carotenoids and total pigments) at 70 days of water regime, and induced a significant stimulatory effect on the biosynthesis of the pigment fractions when compared to those of the corresponding control (~ 28.50%, 27.48%, 16.09% and 20.16% respectively) and (~ 62.41%, 48.85%, 34.34% and 48.23% respectively). Carbohydrates content: The pattern of changes in the amounts of various carbohydrate fractions in leaves of wheat plant subjected to drought stress in presence or absence of nano silica are demonstrated in Fig. 3. The data clearly show that drought stress either at 50 days or 70 days caused increases in total soluble sugar, insoluble sugars and total carbohydrate contents compared with those non-stressed plants (30 days). 70 days of water regime was the most harmful drought effect on the determined total soluble sugar, insoluble sugars and total carbohydrate content. However, spraying of nano silica either 50 mg L -1 or 100 mg L -1 on wheat leaves under stress condition showed stimulatory effect on total soluble sugar, insoluble sugars and total carbohydrate contents (by 3.24%, 1.11% and 1.83% respectively) and (by 4.74%, 24.21% and 2.68% respectively) as compared with their reference control plants. Proline content, total protein and total phenol: The effect of drought stress on total protein, proline and total phenol content of wheat leaves in presence or absence of nano silica are given in Fig. 4 . The data clearly indicated that drought stress have a stimulatory effect on the production of proline. In contrast, total protein and total phenol contents were consistently decreased in response to different days of soil water deficit throughout experimental period. The maximum increase value of the examined drought days on the determined proline content were attributed to 70 days stress and evaluated (~ 23.96%), whereas total protein and total phenol content showed a significant decrease (~ 42.83% and 20.47% respectively) in comparison to non-stress plants. On the other hand, spraying of Si-NPs on wheat leaves under stress condition in the case total protein and total phenol also gradually increased with the application of 50 mg L -1 and 100 mg L -1 of Si-NPs, and the maximum value recorded at 50 days of drought stress) by 31.65% and 34.41% respectively) at 50 mg L -1 of Si-NPs at 70 days of soil water deficit as compared with their reference control plants. In contrast, proline content decreased, and maximum decrease notice at 50 days of drought stress (~ 29.13%) at 100 mg L -1 in comparison with their reference control. Carbonic anhydrase (mole CO 2 g -1 F.wt.S -1 ), nitrate reductase (n mole NO 2 g -1 F.wt. S -1 ) and phenylalanine ammonia lyase (M mole t-cinnam g -1 F.wt.min -1 ) The changes in CA, NR and PAL activity of wheat leaves in response to drought stress and / or 50 mg L -1 and 100 mg L -1 of nano silica are represented in Fig. 5 . The data clearly showed that the drought stress 50 days and 70 days have a stimulatory effect on the production of CA and NR activity and inhibitory effect on PAL activity as compared with unstressed plant (30 days). The maximum increase in CA and NR activity were recorded at 70 days of drought and were estimated (by 35.50% and 23.14% respectively); whereas the maximum decrease in PAL (~ 41.67%) as compared with normal irrigation (30 days). Moreover, spraying of Si-NPs with different concentration 50 mg L -1 and 100 mg L -1 on wheat leaves were found to non-significant increase the activity of both CA, NR and PAL activity as compared with those of reference controls at 50 mg L -1 (by 7.10%, 2.9% and 6.43% respectively) in case of application of 100 mg L -1 evaluated (by 10.65%, 11.98% and 31.49% respectively), compared with reference control. On the other hand, spraying of nano silica on wheat leaves under stress condition in the case CA, NR and PAL activity also gradually increased with the application of 100 mg L -1 of nano silica, and the maximum value recorded at 50 days of drought stress (by 18.33%) for CA and at 70 days (by18.12% and 82.46% respectively) for NR and PAL as compared with those of the reference controls. Antioxidant enzymes: The results presented in Fig. 6 demonstrated the effect of different drought stress either alone or in combination with two concentrations 50 mg L -1 and 100 mg L -1 from Si-NPs the activities of the determined antioxidant enzymes: CAT, APX, GST and GPX of wheat leaves. The activity of CAT, APX, GST and GPX showed marked increases in response to drought levels reaching the maximum values in plants exposed to 70 days of drought as compared with that of the un-stressed plant. In which amount of increasing in c CAT, APX, GST and GPX response to 70 days drought were estimated (~ 6.19%, 58.26%, 13.92% and 20.08% respectively) as compared with that of un-stressed wheat plant. The exogenous application of Si-NPs on wheat leaves under stress condition especially 70 days resulted in a non-significant increase in the activity of CAT, APX, GST and GPX; the maximum values were recorded at 100 mg L -1 of Si-NPs (~ 1.86%, 16.48%, 9.11% and 11.82% respectively) as compared with reference control plants. Discussion The plasma membrane is the primary site of ion-specific salt injury is due to salt ion action (Tabaei-Aghdaei et al ., 2000 and Mansour and Salama, 2004). Membrane damage could indirectly be evaluated by measuring solute leakage (Electrolyte leakage) from cells 35 and membrane stability index 36 and peroxidation of membrane lipids (malondialdehyde) 37 . It is clear from the present results that, increasing the drought levels caused increase in electrolyte leakage and lipid – peroxidation levels as indicated by accumulated MAD and caused a decrease in membrane stability index as compared with reference control. It is notable from the data that maximum induction in the maximum values of electrolyte leakage and MDA and the minimum value of membrane stability index were recorded in plant exposed to 70 days water shortage. Electrolyte leakage from plasma membranes has been identified as one of the most important selection criteria for identifying salt-tolerant plants (Ashraf and Ali, 2008). Mahmoudi et al . (2011); Hniličková et al . (2019) and Tanveer et al . (2020) have confirmed that, the EL value increased with increasing salt concentrations in different species. One of the most damaging oxidative effects is the peroxidation of membrane lipids, which results in the production of malondialdehyde (Davey et al ., 2005; Yasar, 2007 and Li et al ., 2013). In this respect, Yasar et al . (2008) and Uzal (2017) recorded that, MDA concentration increased parallel with increased of salt stress in various plants ( bean and pepper) respectively. On contrary the treatment of stressed plants with nano silica with different concentration 50 mg L − 1 and 100 mg L − 1 corrects the stress mediated damage to the plasma membrane, as evident from a non-significant decrease in membrane leakage and lipid peroxidation and non-significant increase in the membrane stability in wheat plants when compared to those of the reference controls. The maximum reduction in ionic leakage and lipid peroxidation along with induction in membrane stability were gained by 100 mg L − 1 of nano silica. Previous research also have shown that silica nanoparticles can increase plant water content under drought conditions 47 . In this regard, it has been shown that nano silica is sedimented in the walls of endodermal cells and, as an apo plastic fluid, helps retaining moisture during drought and salinity stresses 48 . Moreover, silicon can reduce the adverse effects of water shortage by reducing transpiration. These effects have significant role in preserving plant water content and increasing plant drought tolerance 49 . It was observed that the application of Si in rice plants under salinity stress significantly decreased the activities of non-enzymatic MDA and enzymatic antioxidants 50 . 5 reported that, Se/SiO2-NPs sprayed and water sprayed strawberry plants subjected to drought stress revealed that spraying with Se/SiO2-NPs (100 mg L − 1 ) increased MSI and decreased the values of MDA and EL. 51 approved that, nano-silicon application significantly increased MSI, so that the lowest stability index was observed in the control plants without nano-silicon application in Tanacetum parthenium leaves. Photosynthesis is one of the most promising physiological processes contributing to plant growth and productivity of crops for food 52 . Results of the current study showed that, the contents of photosynthetic pigments (chlorophyll a, b, carotenoid and total pigment) were decrease with rising the drought days in wheat plant as compared with non-stressed plant. Severe drought stress inhibits the photosynthesis of plants by changes in chlorophyll content, affecting chlorophyll components and damaging the photosynthetic apparatus. Total chlorophyll content is found to reduce underwater stress conditions. A decrease in chlorophyll content is faster in drought sensitive than in drought tolerant genotypes 53 . The chlorophyll content in flag leaves reflect photosynthetic activity and yields potential of wheat plants. High chlorophyll content in different plant leaves was considered as a favorable trait in crop production under drought stress (Teng et al., 2004 and Hussain et al., 2018). Some studies show that leaf chlorophyll content is positively correlated with photosynthetic capacity. The reduction in plant pigments concentrations may be due to decrease in absorption of some ions as Mg + 2 and Fe + 2 which were involved in chlorophyll biosynthesis under stress conditions and/or as response to an increase in growth inhibitors such as ethylene or abscisic acid (Singer et al ., 1993; El-Sawy, 2009 and Rasmia, 2014). One reason for chlorophyll reduction during drought stress is that water deficit induces the production of active oxygen species which in turn destroys and decreases pigments. On the other hand, chlorophyll molecules decompose within chloroplasts and the thylakoid structure disappears (Cao et al., 2015 and Nxele et al., 2017). In the present work, the decrease in chlorophyll content under drought stressed wheat plant concomitantly with the increase in proline level which led to the suggestion that nitrogen might be shifted to the synthesis of proline instead of chlorophyll 61 . In the present results the applied treatments of nano silica by 50 mg L − 1 and 100 mg L − 1 on wheat plants can alleviate the damage effects of drought stress on pigment contents as compared with those of the reference controls. Silica nanoparticles have great availability and are easily absorbed by plants compared with bulk Si, consequently supporting greater ameliorative impacts under abiotic stresses (Suriyaprabha et al., 2012, Tripathi et al., 2017).The obtained results demonstrated that the application of nano-Si considerably boosted photosynthetic effectiveness and leaf gas exchange in plants under water-deficit stress compared to those that were untreated. Promoting photosynthetic efficiency maintenance could be attributed to optimal stomatal conductance and strong antioxidant activities, which improve plant tolerance to drought stress ( Suriyaprabha et al., 2012; Jia et al., 2021). Xie et al. (2012) reported that, exogenous application of nano- SiO 2 improved the photosynthetic activity of mesophyll cells in Indocalamus barbatus . Previous studies showed that Si and Se increase chlorophyll pigment content in different plants under stress and normal conditions (Cao et al., 2015 and Ahmad et al., 2016). It seems that these elements protect chloroplast structure against severe oxidative damage such as destruction of both grana and stroma lamellae and increase the biosynthesis of photosynthetic pigments by protecting chloroplast enzymes 59 . Probably, these elements act as cofactors in many enzymatic reactions involved in the biosynthetic pathways of the chloroplast (Feng et al., 2013 and Pereira et al., 2018). Carbohydrates which represent one of the main organic constitutes of the dry matter, derived from photosynthesis, were found to be affected by salt stress. Carbohydrates are frequently associated with active osmotic adjustment (Premachandra et al ., 1992 and Zhang and Archbold, 1993). The data obtained by the current study exhibited that marked increases in total soluble sugar, insoluble sugars and total carbohydrate contents in response to drought stress in tested cultivar of wheat plant during the experimental period. Such this accumulation of carbohydrate was recorded by other authors such as Pelleschi et al ., (1997), Bassuony et al . (2008) and Gul et al . (2017). Total soluble sugars are regarded as important osmolytes for osmotic adjustment. Under stressful conditions, total soluble sugar accumulation is a common occurrence. Haq et al . (2011) and Wu et al . (2013) conveyed an increase in total soluble sugars with a progressive escalation in stress, which plays an important role abridged the osmotic potential (Di Martino et al ., 2003). Soluble carbohydrates are other osmotic agents that are involved in osmotic adjustment. Accumulation of these compounds may be associated with drought tolerance 51 . However, some researchers believe that the accumulation of carbohydrates is due to a decrease in their consumption and a decrease in the growth rate under stress conditions 76 . On the other hand, in plants that were treated with silica nanoparticles, the accumulation of soluble sugars was lower, which could be a result of the maintenance of plant growth and health under stress. These results are in contrast with those obtained by 48 they found higher concentration of soluble carbohydrates in silicon treated cucumbers under stress conditions. The data of the current study clearly indicated that drought stress have a stimulatory effect on the production of proline. In contrast, total protein and total phenol contents were consistently decreased in response to different days of soil water deficit throughout experimental period. Whereas application of nano silica with different concentrations 50 mg L -1 and 100 mg L -1 on wheat leaves showed also resulted in significant increases in the amounts of total protein, total phenol contents and decreases in proline content. The endogenous levels of amino acids, carbohydrates, and protein in plants determine the nutritive value of a crop 77 . The degradation of protein under stress condition was supported by the present results which revealed the accumulation of proline content. These results were in good agreement with those of Kasim and Dowidar (2006) and Shah et al . (2021) using several plants. Accumulation of organic solutes like proline is a common response of plants subjected to a stress as a defense mechanism to overcome stress- induced harmful effects (Munns and Tester, 2008). Proline is one of the essential osmo-protectants that play a critical role in osmoregulation in different plants under abiotic stress 81 ; 82 and 83 . The accumulation of proline, in the current study with the rise of drought level are in agreement with the results obtained by Kavi Kishor et al. (2005 ) and Li et al. (2021) whom reported that, proline accumulated in response to several environmental types of stress, to protect the cell by balancing the osmotic strength of cytosol with that vacuole and external environment. In this respect, Arad and Richmond (1976)found that, inhibition of protein synthesis under water stress condition in probably caused by an increase in the RN-ase activity which evidently affect the rate of protein synthesis by destroying the mRNA linking ribosomes 87 . The synthesis of phenolics is generally affected by different biotic or abiotic stresses 88 .It was observed that, the depression in phenolic synthesis was concomitant with the decrease in the synthesis of PAL enzyme under stress condition. This could be explained by that phenylalanine ammonia lyase (PAL) is the first and committed step in the phenyl propanoid pathway and is therefore involved in the biosynthesis of the polyphenol compounds such as flavonoids, phenylpropanoids and lignin in plants (Fritz et al., 1976 and Tanaka et al., 1989). Exogeneous application of silica nanoparticles on wheat plants increased total phenol content under stressed and unstressed conditions. Inorganic elements such as Si play critical roles in determination of organoleptic properties and antioxidative capacity of fruits through adjusting the biosynthetic phenylpropanoid pathway which results in metabolite accumulation 91 . Antioxidants play protective roles against oxidative stress in plants, with free – OH groups attached to the aromatic ring reducing oxidative damage by scavenging ROS and chelating metals. Generally, increases of secondary metabolites contributed to the improvement of cell responses to oxidative stress in addition to antioxidant activity of fruits which improved their quality 92 . Our results in agreement with 5 reported that spraying Se-, SiO2- and especially Se/SiO2-NPs increased the contents of all three above mentioned biochemical parameters such as, total phenolics, and antioxidant activity in fruits of different species by the addition of Si and Se. The total protein content in wheat plants treated with 50 and 100 mg/L of nano silica showed no significant increase with decreasing in proline content as compared to control sample When plant’s cell is under stress signaling pathway in corporation with calcium send signals to nucleus of cell. Due to this signaling, genes expression undergoes changes and because of increasing or decreasing of some genes, plant can resist against stress The result of this change in the genetics, led to changes in the amount and type of special proteins (Amini et al., 2007). These newly synthesized proteins could have diverse functions and may include signal transducers, metabolic enzymes, reactive oxygen species (ROS) scavengers, osmoprotectants, or involved in protein biogenesis ,degradation, cell defense and cell rescue (Lisa et al ., 2011; Kappel et al ., 2020 and Khalil et al ., 2021). Kalteh et al. (2014) reported that proline content in leaves decreased when silica nanoparticles were applied. The results of the current work demonstrated that, drought stress on wheat plant induced the activation of enzymes in the leaves such as carbonic anhydrase, nitrate reductase activity, catalase, Ascorbate peroxidase, Glutation s- transferase, Guaiacol peroxidase and inhibitory effect on phenylalanine ammonia lyase activity as compared to control. In this respect, tolerant plants have evolved different antioxidative mechanisms involving enzymes such as carbonic anhydrase, POD, CAT, PPO and nitrate reductase activity, or other metabolite such as AsA, phenolics and carotenoids to prevent and conteract the increase and effects of ROS 97 and plant adaption to various stresses is associated with metabolic adjustments that lead to the modulation of different enzymes activities 98 . It was observed that, PAL activity in wheat plant decreased gradually by increasing days of drought such reduction was concomitant with a decrease in the leaf total phenols content 99 . Application of nano silica 50 and 100 mg/L were shown to improve the effect of drought and caused a significant increase in the activity of carbonic anhydrase, nitrate reductase, catalase, ascorbate peroxidase, glutathione s- transferase, Guaiacol peroxidase and phenylalanine ammonia lyase in wheat plants, where 100 mg/L from nano silica give the best results on enzymes activity. Silica nanoparticles can act as a stress gen factor in wheat plants and increase the activity of antioxidant enzymes. These enzymes protect plants against toxicity and damage of reactive oxygen 100 . Catalase and ascorbate peroxidase can scavenge H 2 O 2 in plants and therefore, the increase of superoxide dismutase is also predictable. The activity of ascorbate peroxidase was increased in nano silica treatment. Miao et al. (2010) indicated that silica can compensate the effect of potassium shortage in soybean. Ghffar et al. (2015) reported that treatment of bean with nano silica increased the activity of catalase, nitrate reductase and ascorbate peroxidase. A large number of previous investigations on different field crops showed that Si addition enhanced the activity of SOD, CAT, and GPX following various stresses ( Abbasi et al., 2015; Wang & Huang, 2019).The present results are in agreement with Namjoyan et al. (2020) who confirmed that, nano-Si foliar spraying intensified the enzymatic antioxidant activities in sugar beet leaves under drought conditions. Conclusion Nanoparticles have proved their vital role in the agriculture system. In this present work, Nano Silica proved its significant importance for photosynthetic pigments, carbohydrate, total protein, and total phenol and some of antioxidant enzymes etc. carbonic anhydrase, phenylalanine ammonia layase and catalase of wheat plant. All measured parameters were positively affected by Nano Silica having higher values compared to without application of Nano Silica under drought stress. Application of 100 mg/l Nano Silica is the ideal concentration that wheat plants should be treated under water stress which had the highest values of biochemical characteristics. Findings revealed that application of Nano Silica can improve wheat seed yield in the arid region and can be introduced as beneficial fertilizer for foliar application. Declarations Data availability The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. Data declaration The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. References Kizilgeci, F. et al. 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20:23:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5271175/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5271175/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70116994,"identity":"09173b26-f399-408e-923b-d3dfc99d51bf","added_by":"auto","created_at":"2024-11-28 13:31:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":319907,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5271175/v1/da7e3c762ca6c37c51a25d97.png"},{"id":70117000,"identity":"0e22d926-3897-4739-bab0-074f1d08dbe4","added_by":"auto","created_at":"2024-11-28 13:31:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":364831,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5271175/v1/fbde2ebd20e203756337082a.png"},{"id":70115441,"identity":"be010d5d-e39a-4f3d-b73c-2065e506af10","added_by":"auto","created_at":"2024-11-28 13:15:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":339157,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5271175/v1/d636df0d988f4ad2995d6ff1.png"},{"id":70115445,"identity":"20f37137-e9c0-40ac-a584-5548ff58e3d9","added_by":"auto","created_at":"2024-11-28 13:15:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":337548,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5271175/v1/922aa69bbad41eda7895d2d2.png"},{"id":70116141,"identity":"06ed6004-c571-4322-a321-f75ec4ab67dc","added_by":"auto","created_at":"2024-11-28 13:23:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":307691,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5271175/v1/ae6a891d048cbfe9db5e3120.png"},{"id":70117876,"identity":"4610a9b3-0ec7-46b2-bdd4-c6867c1e2f79","added_by":"auto","created_at":"2024-11-28 13:39:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":447418,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5271175/v1/2d9c12bbb07d940f7f25445f.png"},{"id":72189345,"identity":"eb331819-7f6f-4cf0-911d-13faeed7c7b3","added_by":"auto","created_at":"2024-12-23 14:02:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2976154,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5271175/v1/1a61c81b-0f2d-4c2a-8a9d-d7f0f87f5d79.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nano silica reinforces the tolerance of the wheat plant against drought stress","fulltext":[{"header":"Background","content":"\u003cp\u003eGlobal warming and climate change have recently increased the frequency and severity of many stresses, which has a direct impact on agricultural yield and quality. The three most significant staple crops in the world, wheat, rice, and maize, account for a sizable portion of daily caloric and protein intake \u003csup\u003e1\u003c/sup\u003e. Wheat holds the top spot among these key grains because of its domestication and role as the world's main staple food crop \u003csup\u003e2\u003c/sup\u003e. Due to adverse surroundings brought on by climate change, which has resulted in the emergence of several abiotic stressors, wheat yield may drop by 6% \u003csup\u003e3\u003c/sup\u003e. Heat and drought have been shown to be the most significant variables affecting crop output and ultimately contributing to food security. Draught-like conditions are becoming more commonplace worldwide due to altered rainfall patterns and a deficiency of precipitation \u003csup\u003e4\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDrought conditions cause physiological disturbances, physical damage, and biochemical changes in plants that negatively impact crop growth, development, and output. Drought stressors have a complex mechanical effect because they leave behind many impressions. It makes sense that improved knowledge of plant responses to drought stress will influence crop management and modification. Therefore, a comprehensive strategy is required to fully describe and comprehend how drought stress conditions affect plants for increased agricultural production \u003csup\u003e5\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe unique physiochemical properties of nanoscale silicon particles have applications in a variety of domains, including agriculture, which shows great promise. Because of their unique properties, Si-NPs can withstand agricultural damage from abiotic stressors including climate change (Tripathi et al., 2012). The utilization of Si-NPs in agriculture has the potential to enhance global food security by promoting the development of superior, more productive cultivars \u003csup\u003e7\u003c/sup\u003e. It was discovered that Si-NPs were employed as a weapon in the agricultural sector to resist various environmental stresses, such as dehydration \u003csup\u003e8\u003c/sup\u003e, UVB stress (Tripathi et al., 2017), heavy metal toxicity \u003csup\u003e10\u003c/sup\u003e, and salt stress \u003csup\u003e11\u003c/sup\u003e. Among other creative uses, Si-NPs can be applied as herbicides, insecticides, and fertilizers. Si-NPs may thus improve crops for environmentally friendly farming practices.\u003c/p\u003e \u003cp\u003eSilicon, the most prevalent metalloid on Earth and second-most abundant in the crust, is a semi-essential element for plants due to its beneficial effects on environmental stress, making it a valuable resource (Luyckx et al., 2017). This study explores the positive impacts of silicon nanoparticles on plants, highlighting their unique physiological properties and potential use as nanocarriers for various compounds. It also explores the role of silicon nanoparticles in plant development, growth, and productivity, particularly under drought stress, and provides a comprehensive overview of their potential applications.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eExperimental design\u003c/h2\u003e\n \u003cp\u003eTwo field experiments were carried out during winter seasons at Moshtohor region, Qalubiya Governorate, Egypt, to study the effect of three water regimes 1- Normal irrigation at tillering stage, elongation stage, heading stage, milk stage and filling stage or irrigation intervals 30 days among irrigations (Five irrigations); 2-Three irrigations. at end tillering stage and starting elongation stage, at end heading stage and starting milk stage and filling stage or irrigation intervals 50 days among irrigations (Three irrigations); 3-Two irrigations at end elongation stage and started heading stage and filling stage or irrigation intervals 70 days between irrigations (Two irrigations), and three silica nanoparticles treatments (Zero nano silica; 50 mg L\u003csup\u003e-1\u003c/sup\u003e SiO\u003csub\u003e2\u003c/sub\u003e; 100 mg L\u003csup\u003e-1\u003c/sup\u003e SiO\u003csub\u003e2\u003c/sub\u003e) on growth parameters, some metabolic activities related with the mechanism of bread wheat c.v. Misr 3. A spilt plot design with four replications was used. The three water regimes were allocated to the main plots and the three silica nanoparticles spraying were arranged in the sub plots. The area of sub-plot was 10.5 m\u003csup\u003e2\u003c/sup\u003e (1/400 fed). SiO\u003csub\u003e2\u003c/sub\u003e nanoparticles were purchased from US Research Nanomaterials. SiO\u003csub\u003e2\u003c/sub\u003e nanoparticles were prepared at two concentrations (50, 100 mg L\u003csup\u003e-1\u003c/sup\u003e) by dissolving in water and dispersed with a high-power probe-type.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eElectrolyte leakage\u003c/h3\u003e\n\u003cp\u003eThe total inorganic ions leaked out from the leaves were measured by the method described by Sullivan (1979). In a boiling tube with 10 cm\u003csup\u003e3\u003c/sup\u003e of deionized water, twenty leaf discs were placed. The conductivity meter was used to measure the EC of the tubes after they were heated for 30 minutes at 45\u003csup\u003eo\u003c/sup\u003eC (ECa) and 55\u003csup\u003eo\u003c/sup\u003eC (ECBs) in a water bath. After the items were cooked for ten minutes at 100 degrees Celsius, the EC was once more reported as Ecco. The following formula was used to determine the electrolyte leakage:\u003c/p\u003e\n\u003cp\u003eElectrolyte leakage (%) \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:=\\frac{\\text{E}\\text{C}\\text{b}-\\text{E}\\text{C}\\text{a}}{\\text{E}\\text{C}\\text{c}}\\)\u003c/span\u003e\u003c/span\u003eX100\u003c/p\u003e\n\u003ch3\u003eMembrane stability index\u003c/h3\u003e\n\u003cp\u003eIn two sets of ten cm\u003csup\u003e3\u003c/sup\u003e of double-distilled water, 200 mg of fresh leaves were used to determine the MSI. Electrical conductivity C1 was measured after one set was heated to 40\u0026deg;C in a water bath for thirty minutes. As an alternative, the second set\u0026apos;s conductivity (C2) was determined after it was boiled for ten minutes at 100 degrees Celsius in a boiling water bath; a conductivity meter was used to test both conductivities. MSI was calculated using the formula described by (Sairam, 1994).\u003c/p\u003e\n\u003ch3\u003eMSI (%) = [1 \u0026ndash; (C\u003csub\u003e1\u003c/sub\u003e/C\u003csub\u003e2\u003c/sub\u003e) \u0026times; 100]\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eLipid-peroxidation (Malondialdehyde; MDA):\u003c/h2\u003e\n \u003cp\u003eUsing the Hodges \u003cem\u003eet al\u003c/em\u003e. (1999) approach, the degree of lipid peroxidation was expressed in terms of the concentration of malondialdehyde (MDA). Ten milliliters of 5% thiobarbituric acid (TBA) were used to homogenize a 0.2-gram leaf sample. For 10 minutes, the homogenate was centrifuged at 15000 g. 4 ml of 0.5% thiobarbituric acid in 20% TCA was added to a 2 ml portion of the supernatant. The mixture was centrifuged at 10,000 g for 10 minutes after being heated to 95\u0026deg;C for 30 minutes then rapidly cooled in an ice bath. The supernatant\u0026apos;s absorbance was measured at 532 nm. The non-specific absorption value at 600 nm was deducted. \u0026micro;g (MDA) g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003efresh weight is the MDA content, which was determined using its absorption coefficient of 155 n mole\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eExtraction and determination of photosynthetic pigments:\u003c/h2\u003e\n \u003cp\u003eTotal photosynthetic pigments were extracted and determined according to the method described by Fadeel (1962). The study involved soaking maize leaves in 100% acetone, grinding, adding sodium sulfate and magnesium carbonate, centrifuging, and cleaning to extract pigments. Absorbance measurements were performed using a Perkin-Elmer spectrophotometer, determining carotenoids and chlorophyll amounts (Sestak \u003cem\u003eet al\u003c/em\u003e., 1971).\u003c/p\u003e\n \u003cp\u003eChlorophyll a\u0026thinsp;=\u0026thinsp;9.78 E\u003csub\u003e662\u003c/sub\u003e- 0.99 E\u003csub\u003e644\u003c/sub\u003e \u0026micro;g /ml\u003c/p\u003e\n \u003cp\u003eChlorophyll b\u0026thinsp;=\u0026thinsp;21.4 E\u003csub\u003e644\u003c/sub\u003e \u0026ndash; 4.65 E\u003csub\u003e662\u003c/sub\u003e \u0026micro;g /ml\u003c/p\u003e\n \u003cp\u003eChlorophyll (a\u0026thinsp;+\u0026thinsp;b)\u0026thinsp;=\u0026thinsp;5.13 E\u003csub\u003e662\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;20.41 E\u003csub\u003e644\u003c/sub\u003e \u0026micro;g /ml\u003c/p\u003e\n \u003cp\u003eCarotenoids\u0026thinsp;=\u0026thinsp;4.69 E\u003csub\u003e440.5\u003c/sub\u003e \u0026minus;\u0026thinsp;0.268 chlorophyll (a\u0026thinsp;+\u0026thinsp;b) \u0026micro;g/ml\u003c/p\u003e\n \u003cp\u003eFollowing the preceding formulae, mg pigment g\u003csup\u003e-1\u003c/sup\u003e fresh weight was computed from the results, which were expressed as g pigment ml-1 extract.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eEstimation of Carbohydrate contents.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe dried tissues were homogenized with 80% ethanol, heated, filtered, and oven dried to determine soluble sugars, then dissolved in water \u003csup\u003e18\u003c/sup\u003e. To find the soluble sugars, the anthrone sulphuric acid technique \u003csup\u003e19\u003c/sup\u003e was employed. The anthrone reagent, consisting of 100 ml 95% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and 0.2 g of anthrone, is made fresh every time. A Pyrex test tube is filled with soluble sugar solution and heated for seven minutes. The blue-green color is measured against a blank, creating a calibration curve.\u003c/p\u003e\n \u003cp\u003eThe dry residue from extracted soluble sugars was used to identify polysaccharides. A mixture of 1.5 N sulfuric acid and dry material was heated, prepared for analysis using the anthrone sulfuric acid reagent method, and calibrated using pure glucose \u003csup\u003e19\u003c/sup\u003e. It was computed as the total of the same sample\u0026apos;s soluble sugar and polysaccharide contents. For all data, the dry weight was expressed as mg 100 g\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cstrong\u003eDetermination of proline\u003c/strong\u003e:\u003c/div\u003e\n\u003cp\u003eThe amount of free proline was calculated using (Bates \u003cem\u003eet al\u003c/em\u003e., 1973). The study involved homogenizing macerated fresh tissue with 3% aqueous sulfosalicylic acid, filtering it, and using a ninhydrin reagent. The mixture was extracted using toluene, and the absorbance was captured at 520 nm. The proline concentration was determined using a standard curve and calculated as mg proline per 100 g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fresh matter.\u003c/p\u003e\n\u003ch3\u003eEstimation of protein\u003c/h3\u003e\n\u003cp\u003eThe amount of total protein was measured using spectrophotometry in accordance with the protocol outlined by Bradford, (1976). Prepare protein samples in test tubes using phosphate-buffered saline. Dilute samples with dye binding solution, blend, and let color take five minutes. Measure absorbance at 595 nm, plot a standard curve, and determine protein content using the absorbance V concentration.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eEstimation of total phenol\u003c/h2\u003e\n \u003cp\u003eTotal phenol estimation can be carried out with the Folin- Ciocalteau reagent \u003csup\u003e22\u003c/sup\u003e. The material was ground in 80% ethanol, centrifuged, and dissolved in distilled water. Test tubes were filled with water, Folin-Ciocalteau reagent, and 20% NaCO3 solution. The absorbance at 650 nm was calculated, and a standard curve was created with different catechol concentrations. The amount of phenols in the test sample was calculated from the standard curve and expressed as milligrams per 100 grams of material.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eDetermination of Carbonic anhydrase (CA)\u003c/strong\u003e.\u003c/p\u003e\n \u003cp\u003eWe assessed the CA activity (EC 4.2.1.1) in the leaves using the protocol outlined in \u003csup\u003e23\u003c/sup\u003e. Small sections of the leaf samples were cut and placed in a solution of cysteine hydrochloride. After blotting and transferring the leaf samples into a test tube, 0.2 M NaHCO\u003csub\u003e3\u003c/sub\u003e, phosphate buffer (pH 6.8), bromothymol blue, and methyl red indicator were added. Titrating the reaction against 0.05 N HCl was done. Using fresh mass as a foundation, the enzyme\u0026apos;s activity was expressed as mol (CO2) g\u003csup\u003e-1\u003c/sup\u003e(FM) s\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eDetermination of Nitrate reductase (NR).\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe technique established by\u003csup\u003e24\u003c/sup\u003e was used to measure the activity of NR (EC 1.7.1.1). After being sliced into small pieces, the fresh leaf samples were placed in plastic vials with KNO3, isopropa 2h, and phosphate buffer (pH 7.5). N-1-naphthylethylenediamine hydrochloride solutions and sulfanilamide were added after incubation. Using a spectrophotometer (Spectronic 20D, Milton Roy, USA), the absorbance was measured at 540 nm. Based on fresh mass, the enzyme\u0026apos;s activity was expressed as nM (NO2)g\u003csup\u003e-1\u003c/sup\u003e(FM)s\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eDetermination of phenylalanine ammonia-lyase (PAL):\u003c/h2\u003e\n \u003cp\u003eExtraction and assay of PAL (EC 4.3.1.5) were carried out as described by Solecka and Kacperska (2003). The extraction buffer was mixed with liquid-nitrogen tissues, centrifuged, and the supernatant was used to measure PAL activity by using the raw enzyme. The enzyme extract and substrate solution were mixed, and the reaction was stopped by adding 6 N HCl. The absorbance was measured, and the enzyme activity was measured in nmol t-cinnamic acid g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fresh weight at FWT h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eEstimation of antioxidant system\u003c/h2\u003e\n \u003cp\u003eSample preparation was described by \u003csup\u003e26\u003c/sup\u003e. The powder was mixed with phosphate buffer, filtered, and centrifuged. A fresh leaf sample was ground and centrifuged. The supernatant was collected and stored at 4\u0026deg;C for analysis of CAT, APX, POD, and SOD activities. A reaction mixture (3 ml) containing phosphate buffer (50 mM, pH 7.0), 30% (w/v) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and 0.5 ml of enzyme extract was used to measure \u003cstrong\u003eCatalase activity\u003c/strong\u003e (EC 1.11.1.6.) (Aebi, 1984). According to Havir and McHale (1987), the activity of the CAT enzyme was measured by the decrease in absorbance at 240 nm using a Spectronic 601 UV spectrophotometer as a result of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e consumption. This was expressed as \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e oxidized g-1 fresh weight min-1.\u003c/p\u003e\n \u003cp\u003eAccording to \u003csup\u003e29\u003c/sup\u003e, \u003cstrong\u003eascorbate peroxidase activity\u003c/strong\u003e (EC 1. 11. 1. 11) Was measured by observing the drop in absorbance at 290 nm after 1 minute of ascorbic acid oxidation.\u003c/p\u003e\n \u003cp\u003eUsing a combination containing 72.7 mM Na-phosphate buffer (pH 6.5), 3.6 mM reduced glutathione, 1 mM 1-chloro-2,4-dinitrobenzene, and enzyme extract, the activity of \u003cstrong\u003eglutathione-S-transferase\u003c/strong\u003e (GST; EC 2.5.1.18) was determined by monitoring changes in the absorbance at 340 nm \u003csup\u003e30\u003c/sup\u003e. A UV-VIS 160A spectrophotometer from Shimadzu Corp. in Kyoto, Japan was used to measure the photometrically determined enzyme activities, which were then expressed in nkatal g\u0026ndash;1 (fresh mass, FM). As stated by \u003csup\u003e31\u003c/sup\u003e, the \u003cstrong\u003eguaiacol peroxidase\u003c/strong\u003e (EC 1.11.1.7.) (G-POD) activity was measured at 470 nm. Enzyme extract, 0.0375% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 0.88 mM guaiacol, and 88 mM Na-acetate buffer (pH 5.5) made up the reaction mixture.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eElectrolyte leakage (EL), membrane stability index (MSI) and lipid - peroxidation (Malondialdehyde; MDA):\u003c/h2\u003e \u003cp\u003eThe data recorded in \u003cb\u003eFig.\u0026nbsp;1\u003c/b\u003e revealed electrolyte leakage, membrane stability index and lipid-peroxidation which indicate the injury of cell membrane. It is notable from the data that wheat plants offered a different pattern of response in these parameters under drought stress. 50 and 70 days of drought caused a non-significant increase in electrolyte leakage and lipid-peroxidation, as compared with the control plants. A maximum induction in EL and MDA were recorded in the plants exposed to 70 days drought (by 55% % and 58% respectively) over the value of the control plants. On the other hand, the exposure of the wheat plants to drought caused a decline in MSI in the plant exposed to 70 days (~\u0026thinsp;23.61%). On the other side, spraying nano silica with different concentrations 50 mg L\u003csup\u003e-1\u003c/sup\u003e and 100 mg L\u003csup\u003e-1\u003c/sup\u003e treatments improved the MSI (by 7.41% and 13.57% respectively) with reduction in EL and MDA ( by 7.07% and 13.45% respectively) and (by 14.25% and 16.30% respectively) as compared with control plants (30 days).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePhotosynthetic pigments:\u003c/h2\u003e \u003cp\u003eThe photosynthetically active pigment contents chlorophyll a, chlorophyll b, carotenoids and hence the total pigments which estimated in leaves of wheat plant at filling stage were presented in \u003cb\u003eFig.\u0026nbsp;2\u003c/b\u003e. The contents of photosynthetic pigments were a significant decrease with the rise of soil water deficit days as compared with non-stressed plant (30 days). The highest inhibitory effect of water regime on photosynthetic pigments were recorded at 70 days of water regime as compared with non-stressed plant. On the other hand, spraying of nano silica with different concentration 50 mg L\u003csup\u003e-1\u003c/sup\u003e and 100 mg L\u003csup\u003e-1\u003c/sup\u003e, significantly alleviated the inhibitory effect of soil water deficit on photosynthetic pigment contents (chlorophyll a, chlorophyll b, carotenoids and total pigments) at 70 days of water regime, and induced a significant stimulatory effect on the biosynthesis of the pigment fractions when compared to those of the corresponding control (~\u0026thinsp;28.50%, 27.48%, 16.09% and 20.16% respectively) and (~\u0026thinsp;62.41%, 48.85%, 34.34% and 48.23% respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCarbohydrates content:\u003c/h2\u003e \u003cp\u003eThe pattern of changes in the amounts of various carbohydrate fractions in leaves of wheat plant subjected to drought stress in presence or absence of nano silica are demonstrated in \u003cb\u003eFig.\u0026nbsp;3.\u003c/b\u003e The data clearly show that drought stress either at 50 days or 70 days caused increases in total soluble sugar, insoluble sugars and total carbohydrate contents compared with those non-stressed plants (30 days). 70 days of water regime was the most harmful drought effect on the determined total soluble sugar, insoluble sugars and total carbohydrate content. However, spraying of nano silica either 50 mg L\u003csup\u003e-1\u003c/sup\u003e or 100 mg L\u003csup\u003e-1\u003c/sup\u003e on wheat leaves under stress condition showed stimulatory effect on total soluble sugar, insoluble sugars and total carbohydrate contents (by 3.24%, 1.11% and 1.83% respectively) and (by 4.74%, 24.21% and 2.68% respectively) as compared with their reference control plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eProline content, total protein and total phenol:\u003c/h2\u003e \u003cp\u003eThe effect of drought stress on total protein, proline and total phenol content of wheat leaves in presence or absence of nano silica are given in \u003cb\u003eFig.\u0026nbsp;4\u003c/b\u003e. The data clearly indicated that drought stress have a stimulatory effect on the production of proline. In contrast, total protein and total phenol contents were consistently decreased in response to different days of soil water deficit throughout experimental period. The maximum increase value of the examined drought days on the determined proline content were attributed to 70 days stress and evaluated (~\u0026thinsp;23.96%), whereas total protein and total phenol content showed a significant decrease (~\u0026thinsp;42.83% and 20.47% respectively) in comparison to non-stress plants. On the other hand, spraying of Si-NPs on wheat leaves under stress condition in the case total protein and total phenol also gradually increased with the application of 50 mg L\u003csup\u003e-1\u003c/sup\u003e and 100 mg L\u003csup\u003e-1\u003c/sup\u003e of Si-NPs, and the maximum value recorded at 50 days of drought stress) by 31.65% and 34.41% respectively) at 50 mg L\u003csup\u003e-1\u003c/sup\u003e of Si-NPs at 70 days of soil water deficit as compared with their reference control plants. In contrast, proline content decreased, and maximum decrease notice at 50 days of drought stress (~\u0026thinsp;29.13%) at 100 mg L\u003csup\u003e-1\u003c/sup\u003e in comparison with their reference control.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCarbonic anhydrase (mole CO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eg\u003c/b\u003e\u003csup\u003e\u003cb\u003e-1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eF.wt.S\u003c/b\u003e\u003csup\u003e\u003cb\u003e-1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e), nitrate reductase (n mole NO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eg\u003c/b\u003e\u003csup\u003e\u003cb\u003e-1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eF.wt. S\u003c/b\u003e\u003csup\u003e\u003cb\u003e-1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e) and phenylalanine ammonia lyase (M mole t-cinnam g\u003c/b\u003e\u003csup\u003e\u003cb\u003e-1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eF.wt.min\u003c/b\u003e\u003csup\u003e\u003cb\u003e-1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe changes in CA, NR and PAL activity of wheat leaves in response to drought stress and / or 50 mg L\u003csup\u003e-1\u003c/sup\u003e and 100 mg L\u003csup\u003e-1\u003c/sup\u003e of nano silica are represented in \u003cb\u003eFig.\u0026nbsp;5\u003c/b\u003e. The data clearly showed that the drought stress 50 days and 70 days have a stimulatory effect on the production of CA and NR activity and inhibitory effect on PAL activity as compared with unstressed plant (30 days). The maximum increase in CA and NR activity were recorded at 70 days of drought and were estimated (by 35.50% and 23.14% respectively); whereas the maximum decrease in PAL (~\u0026thinsp;41.67%) as compared with normal irrigation (30 days). Moreover, spraying of Si-NPs with different concentration 50 mg L\u003csup\u003e-1\u003c/sup\u003e and 100 mg L\u003csup\u003e-1\u003c/sup\u003e on wheat leaves were found to non-significant increase the activity of both CA, NR and PAL activity as compared with those of reference controls at 50 mg L\u003csup\u003e-1\u003c/sup\u003e (by 7.10%, 2.9% and 6.43% respectively) in case of application of 100 mg L\u003csup\u003e-1\u003c/sup\u003e evaluated (by 10.65%, 11.98% and 31.49% respectively), compared with reference control.\u003c/p\u003e \u003cp\u003e On the other hand, spraying of nano silica on wheat leaves under stress condition in the case CA, NR and PAL activity also gradually increased with the application of 100 mg L\u003csup\u003e-1\u003c/sup\u003e of nano silica, and the maximum value recorded at 50 days of drought stress (by 18.33%) for CA and at 70 days (by18.12% and 82.46% respectively) for NR and PAL as compared with those of the reference controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant enzymes:\u003c/h2\u003e \u003cp\u003eThe results presented in \u003cb\u003eFig.\u0026nbsp;6\u003c/b\u003e demonstrated the effect of different drought stress either alone or in combination with two concentrations 50 mg L\u003csup\u003e-1\u003c/sup\u003e and 100 mg L\u003csup\u003e-1\u003c/sup\u003e from Si-NPs the activities of the determined antioxidant enzymes: CAT, APX, GST and GPX of wheat leaves. The activity of CAT, APX, GST and GPX showed marked increases in response to drought levels reaching the maximum values in plants exposed to 70 days of drought as compared with that of the un-stressed plant. In which amount of increasing in c CAT, APX, GST and GPX response to 70 days drought were estimated (~\u0026thinsp;6.19%, 58.26%, 13.92% and 20.08% respectively) as compared with that of un-stressed wheat plant. The exogenous application of Si-NPs on wheat leaves under stress condition especially 70 days resulted in a non-significant increase in the activity of CAT, APX, GST and GPX; the maximum values were recorded at 100 mg L\u003csup\u003e-1\u003c/sup\u003e of Si-NPs (~\u0026thinsp;1.86%, 16.48%, 9.11% and 11.82% respectively) as compared with reference control plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe plasma membrane is the primary site of ion-specific salt injury is due to salt ion action (Tabaei-Aghdaei \u003cem\u003eet al\u003c/em\u003e., 2000 and Mansour and Salama, 2004). Membrane damage could indirectly be evaluated by measuring solute leakage (Electrolyte leakage) from cells \u003csup\u003e35\u003c/sup\u003e and membrane stability index \u003csup\u003e36\u003c/sup\u003e and peroxidation of membrane lipids (malondialdehyde) \u003csup\u003e37\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt is clear from the present results that, increasing the drought levels caused increase in electrolyte leakage and lipid \u0026ndash; peroxidation levels as indicated by accumulated MAD and caused a decrease in membrane stability index as compared with reference control. It is notable from the data that maximum induction in the maximum values of electrolyte leakage and MDA and the minimum value of membrane stability index were recorded in plant exposed to 70 days water shortage.\u003c/p\u003e \u003cp\u003eElectrolyte leakage from plasma membranes has been identified as one of the most important selection criteria for identifying salt-tolerant plants (Ashraf and Ali, 2008). Mahmoudi \u003cem\u003eet al\u003c/em\u003e. (2011); Hniličkov\u0026aacute; \u003cem\u003eet al\u003c/em\u003e. (2019) and Tanveer \u003cem\u003eet al\u003c/em\u003e. (2020) have confirmed that, the EL value increased with increasing salt concentrations in different species. One of the most damaging oxidative effects is the peroxidation of membrane lipids, which results in the production of malondialdehyde (Davey \u003cem\u003eet al\u003c/em\u003e., 2005; Yasar, 2007 and Li \u003cem\u003eet al\u003c/em\u003e., 2013). In this respect, Yasar \u003cem\u003eet al\u003c/em\u003e. (2008) and Uzal (2017) recorded that, MDA concentration increased parallel with increased of salt stress in various plants ( bean and pepper) respectively. On contrary the treatment of stressed plants with nano silica with different concentration 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 100 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corrects the stress mediated damage to the plasma membrane, as evident from a non-significant decrease in membrane leakage and lipid peroxidation and non-significant increase in the membrane stability in wheat plants when compared to those of the reference controls. The maximum reduction in ionic leakage and lipid peroxidation along with induction in membrane stability were gained by 100 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of nano silica.\u003c/p\u003e \u003cp\u003ePrevious research also have shown that silica nanoparticles can increase plant water content under drought conditions \u003csup\u003e47\u003c/sup\u003e. In this regard, it has been shown that nano silica is sedimented in the walls of endodermal cells and, as an apo plastic fluid, helps retaining moisture during drought and salinity stresses \u003csup\u003e48\u003c/sup\u003e. Moreover, silicon can reduce the adverse effects of water shortage by reducing transpiration. These effects have significant role in preserving plant water content and increasing plant drought tolerance \u003csup\u003e49\u003c/sup\u003e. It was observed that the application of Si in rice plants under salinity stress significantly decreased the activities of non-enzymatic MDA and enzymatic antioxidants \u003csup\u003e50\u003c/sup\u003e. \u003csup\u003e5\u003c/sup\u003ereported that, Se/SiO2-NPs sprayed and water sprayed strawberry plants subjected to drought stress revealed that spraying with Se/SiO2-NPs (100 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) increased MSI and decreased the values of MDA and EL. \u003csup\u003e51\u003c/sup\u003e approved that, nano-silicon application significantly increased MSI, so that the lowest stability index was observed in the control plants without nano-silicon application in \u003cem\u003eTanacetum parthenium\u003c/em\u003e leaves.\u003c/p\u003e \u003cp\u003ePhotosynthesis is one of the most promising physiological processes contributing to plant growth and productivity of crops for food \u003csup\u003e52\u003c/sup\u003e. Results of the current study showed that, the contents of photosynthetic pigments (chlorophyll a, b, carotenoid and total pigment) were decrease with rising the drought days in wheat plant as compared with non-stressed plant. Severe drought stress inhibits the photosynthesis of plants by changes in chlorophyll content, affecting chlorophyll components and damaging the photosynthetic apparatus. Total chlorophyll content is found to reduce underwater stress conditions. A decrease in chlorophyll content is faster in drought sensitive than in drought tolerant genotypes \u003csup\u003e53\u003c/sup\u003e. The chlorophyll content in flag leaves reflect photosynthetic activity and yields potential of wheat plants. High chlorophyll content in different plant leaves was considered as a favorable trait in crop production under drought stress (Teng et al., 2004 and Hussain et al., 2018).\u003c/p\u003e \u003cp\u003eSome studies show that leaf chlorophyll content is positively correlated with photosynthetic capacity. The reduction in plant pigments concentrations may be due to decrease in absorption of some ions as Mg\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e and Fe\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e which were involved in chlorophyll biosynthesis under stress conditions and/or as response to an increase in growth inhibitors such as ethylene or abscisic acid (Singer \u003cem\u003eet al\u003c/em\u003e., 1993; El-Sawy, 2009 and Rasmia, 2014). One reason for chlorophyll reduction during drought stress is that water deficit induces the production of active oxygen species which in turn destroys and decreases pigments. On the other hand, chlorophyll molecules decompose within chloroplasts and the thylakoid structure disappears (Cao et al., 2015 and Nxele et al., 2017).\u003c/p\u003e \u003cp\u003eIn the present work, the decrease in chlorophyll content under drought stressed wheat plant concomitantly with the increase in proline level which led to the suggestion that nitrogen might be shifted to the synthesis of proline instead of chlorophyll \u003csup\u003e61\u003c/sup\u003e. In the present results the applied treatments of nano silica by 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 100 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e on wheat plants can alleviate the damage effects of drought stress on pigment contents as compared with those of the reference controls.\u003c/p\u003e \u003cp\u003eSilica nanoparticles have great availability and are easily absorbed by plants compared with bulk Si, consequently supporting greater ameliorative impacts under abiotic stresses (Suriyaprabha et al., 2012, Tripathi et al., 2017).The obtained results demonstrated that the application of nano-Si considerably boosted photosynthetic effectiveness and leaf gas exchange in plants under water-deficit stress compared to those that were untreated. Promoting photosynthetic efficiency maintenance could be attributed to optimal stomatal conductance and strong antioxidant activities, which improve plant tolerance to drought stress \u003cb\u003e(\u003c/b\u003eSuriyaprabha et al., 2012; Jia et al., 2021). Xie et al. (2012) reported that, exogenous application of nano- SiO\u003csub\u003e2\u003c/sub\u003e improved the photosynthetic activity of mesophyll cells in \u003cem\u003eIndocalamus barbatus\u003c/em\u003e. Previous studies showed that Si and Se increase chlorophyll pigment content in different plants under stress and normal conditions (Cao et al., 2015 and Ahmad et al., 2016). It seems that these elements protect chloroplast structure against severe oxidative damage such as destruction of both grana and stroma lamellae and increase the biosynthesis of photosynthetic pigments by protecting chloroplast enzymes \u003csup\u003e59\u003c/sup\u003e. Probably, these elements act as cofactors in many enzymatic reactions involved in the biosynthetic pathways of the chloroplast (Feng et al., 2013 and Pereira et al., 2018).\u003c/p\u003e \u003cp\u003eCarbohydrates which represent one of the main organic constitutes of the dry matter, derived from photosynthesis, were found to be affected by salt stress. Carbohydrates are frequently associated with active osmotic adjustment (Premachandra \u003cem\u003eet al\u003c/em\u003e., 1992 and Zhang and Archbold, 1993). The data obtained by the current study exhibited that marked increases in total soluble sugar, insoluble sugars and total carbohydrate contents in response to drought stress in tested cultivar of wheat plant during the experimental period. Such this accumulation of carbohydrate was recorded by other authors such as Pelleschi \u003cem\u003eet al\u003c/em\u003e., (1997), Bassuony \u003cem\u003eet al\u003c/em\u003e. (2008) and Gul \u003cem\u003eet al\u003c/em\u003e. (2017). Total soluble sugars are regarded as important osmolytes for osmotic adjustment. Under stressful conditions, total soluble sugar accumulation is a common occurrence. Haq \u003cem\u003eet al\u003c/em\u003e. (2011) and Wu \u003cem\u003eet al\u003c/em\u003e. (2013) conveyed an increase in total soluble sugars with a progressive escalation in stress, which plays an important role abridged the osmotic potential (Di Martino \u003cem\u003eet al\u003c/em\u003e., 2003).\u003c/p\u003e \u003cp\u003eSoluble carbohydrates are other osmotic agents that are involved in osmotic adjustment. Accumulation of these compounds may be associated with drought tolerance \u003csup\u003e51\u003c/sup\u003e. However, some researchers believe that the accumulation of carbohydrates is due to a decrease in their consumption and a decrease in the growth rate under stress conditions \u003csup\u003e76\u003c/sup\u003e. On the other hand, in plants that were treated with silica nanoparticles, the accumulation of soluble sugars was lower, which could be a result of the maintenance of plant growth and health under stress. These results are in contrast with those obtained by \u003csup\u003e48\u003c/sup\u003e they found higher concentration of soluble carbohydrates in silicon treated cucumbers under stress conditions.\u003c/p\u003e \u003cp\u003eThe data of the current study clearly indicated that drought stress have a stimulatory effect on the production of proline. In contrast, total protein and total phenol contents were consistently decreased in response to different days of soil water deficit throughout experimental period. Whereas application of nano silica with different concentrations 50 mg L\u003csup\u003e-1\u003c/sup\u003e and 100 mg L\u003csup\u003e-1\u003c/sup\u003e on wheat leaves showed also resulted in significant increases in the amounts of total protein, total phenol contents and decreases in proline content. The endogenous levels of amino acids, carbohydrates, and protein in plants determine the nutritive value of a crop \u003csup\u003e77\u003c/sup\u003e. The degradation of protein under stress condition was supported by the present results which revealed the accumulation of proline content. These results were in good agreement with those of Kasim and Dowidar (2006) and Shah \u003cem\u003eet al\u003c/em\u003e. (2021) using several plants. Accumulation of organic solutes like proline is a common response of plants subjected to a stress as a defense mechanism to overcome stress- induced harmful effects (Munns and Tester, 2008).\u003c/p\u003e \u003cp\u003eProline is one of the essential osmo-protectants that play a critical role in osmoregulation in different plants under abiotic stress \u003csup\u003e81\u003c/sup\u003e; \u003csup\u003e82\u003c/sup\u003eand \u003csup\u003e83\u003c/sup\u003e. The accumulation of proline, in the current study with the rise of drought level are in agreement with the results obtained by Kavi Kishor et al. (2005\u003cb\u003e)\u003c/b\u003e and Li et al. (2021) whom reported that, proline accumulated in response to several environmental types of stress, to protect the cell by balancing the osmotic strength of cytosol with that vacuole and external environment. In this respect, Arad and Richmond (1976)found that, inhibition of protein synthesis under water stress condition in probably caused by an increase in the RN-ase activity which evidently affect the rate of protein synthesis by destroying the mRNA linking ribosomes \u003csup\u003e87\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe synthesis of phenolics is generally affected by different biotic or abiotic stresses \u003csup\u003e88\u003c/sup\u003e.It was observed that, the depression in phenolic synthesis was concomitant with the decrease in the synthesis of PAL enzyme under stress condition. This could be explained by that phenylalanine ammonia lyase (PAL) is the first and committed step in the phenyl propanoid pathway and is therefore involved in the biosynthesis of the polyphenol compounds such as flavonoids, phenylpropanoids and lignin in plants (Fritz et al., 1976 and Tanaka et al., 1989).\u003c/p\u003e \u003cp\u003eExogeneous application of silica nanoparticles on wheat plants increased total phenol content under stressed and unstressed conditions. Inorganic elements such as Si play critical roles in determination of organoleptic properties and antioxidative capacity of fruits through adjusting the biosynthetic phenylpropanoid pathway which results in metabolite accumulation \u003csup\u003e91\u003c/sup\u003e. Antioxidants play protective roles against oxidative stress in plants, with free \u0026ndash; OH groups attached to the aromatic ring reducing oxidative damage by scavenging ROS and chelating metals. Generally, increases of secondary metabolites contributed to the improvement of cell responses to oxidative stress in addition to antioxidant activity of fruits which improved their quality \u003csup\u003e92\u003c/sup\u003e. Our results in agreement with \u003csup\u003e5\u003c/sup\u003e reported that spraying Se-, SiO2- and especially Se/SiO2-NPs increased the contents of all three above mentioned biochemical parameters such as, total phenolics, and antioxidant activity in fruits of different species by the addition of Si and Se.\u003c/p\u003e \u003cp\u003eThe total protein content in wheat plants treated with 50 and 100 mg/L of nano silica showed no significant increase with decreasing in proline content as compared to control sample When plant\u0026rsquo;s cell is under stress signaling pathway in corporation with calcium send signals to nucleus of cell. Due to this signaling, genes expression undergoes changes and because of increasing or decreasing of some genes, plant can resist against stress The result of this change in the genetics, led to changes in the amount and type of special proteins (Amini et al., 2007). These newly synthesized proteins could have diverse functions and may include signal transducers, metabolic enzymes, reactive oxygen species (ROS) scavengers, osmoprotectants, or involved in protein biogenesis ,degradation, cell defense and cell rescue (Lisa \u003cem\u003eet al\u003c/em\u003e., 2011; Kappel \u003cem\u003eet al\u003c/em\u003e., 2020 and Khalil \u003cem\u003eet al\u003c/em\u003e., 2021). Kalteh et al. (2014) reported that proline content in leaves decreased when silica nanoparticles were applied.\u003c/p\u003e \u003cp\u003eThe results of the current work demonstrated that, drought stress on wheat plant induced the activation of enzymes in the leaves such as carbonic anhydrase, nitrate reductase activity, catalase, Ascorbate peroxidase, Glutation s- transferase, Guaiacol peroxidase and inhibitory effect on phenylalanine ammonia lyase activity as compared to control. In this respect, tolerant plants have evolved different antioxidative mechanisms involving enzymes such as carbonic anhydrase, POD, CAT, PPO and nitrate reductase activity, or other metabolite such as AsA, phenolics and carotenoids to prevent and conteract the increase and effects of ROS \u003csup\u003e97\u003c/sup\u003e and plant adaption to various stresses is associated with metabolic adjustments that lead to the modulation of different enzymes activities \u003csup\u003e98\u003c/sup\u003e. It was observed that, PAL activity in wheat plant decreased gradually by increasing days of drought such reduction was concomitant with a decrease in the leaf total phenols content \u003csup\u003e99\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eApplication of nano silica 50 and 100 mg/L were shown to improve the effect of drought and caused a significant increase in the activity of carbonic anhydrase, nitrate reductase, catalase, ascorbate peroxidase, glutathione s- transferase, Guaiacol peroxidase and phenylalanine ammonia lyase in wheat plants, where 100 mg/L from nano silica give the best results on enzymes activity.\u003c/p\u003e \u003cp\u003eSilica nanoparticles can act as a stress gen factor in wheat plants and increase the activity of antioxidant enzymes. These enzymes protect plants against toxicity and damage of reactive oxygen \u003csup\u003e100\u003c/sup\u003e. Catalase and ascorbate peroxidase can scavenge H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in plants and therefore, the increase of superoxide dismutase is also predictable. The activity of ascorbate peroxidase was increased in nano silica treatment. Miao et al. (2010) indicated that silica can compensate the effect of potassium shortage in soybean. Ghffar et al. (2015) reported that treatment of bean with nano silica increased the activity of catalase, nitrate reductase and ascorbate peroxidase. A large number of previous investigations on different field crops showed that Si addition enhanced the activity of SOD, CAT, and GPX following various stresses \u003cb\u003e(\u003c/b\u003eAbbasi et al., 2015; Wang \u0026amp; Huang, 2019).The present results are in agreement with Namjoyan et al. (2020) who confirmed that, nano-Si foliar spraying intensified the enzymatic antioxidant activities in sugar beet leaves under drought conditions.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eNanoparticles have proved their vital role in the agriculture system. In this present work, Nano Silica proved its significant importance for photosynthetic pigments, carbohydrate, total protein, and total phenol and some of antioxidant enzymes etc. carbonic anhydrase, phenylalanine ammonia layase and catalase of wheat plant. All measured parameters were positively affected by Nano Silica having higher values compared to without application of Nano Silica under drought stress. Application of 100 mg/l Nano Silica is the ideal concentration that wheat plants should be treated under water stress which had the highest values of biochemical characteristics. Findings revealed that application of Nano Silica can improve wheat seed yield in the arid region and can be introduced as beneficial fertilizer for foliar application.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKizilgeci, F. \u003cem\u003eet al.\u003c/em\u003e Normalized difference vegetation index and chlorophyll content for precision nitrogen management in durum wheat cultivars under semi-arid conditions\u003cspan dir=\"RTL\"\u003e\u0026rlm;\u003c/span\u003e. \u003cem\u003emdpi.com\u003c/em\u003e\u003cem\u003e\u003cspan dir=\"RTL\"\u003e\u0026rlm;\u003c/span\u003e\u003c/em\u003e\u003cspan dir=\"RTL\"\u003e (2021\u003c/span\u003e) doi:10.3390/su13073725.\u003c/li\u003e\n\u003cli\u003eIqbal, M. 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Plant.\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 1\u0026ndash;16 (2020).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antioxidant enzyme, Drought stress, Nanoparticles, Photosynthetic pigments, Proline, Silica","lastPublishedDoi":"10.21203/rs.3.rs-5271175/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5271175/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSilicon nanoparticles have distinctive physicochemical characteristics and improve the plant growth and yield under unfavorable environmental conditions. Therefore, the present investigation was undertaken to study the impact of Nano Silica on drought resistance depending on the Nano-Silica dose and moisture levels. Nano Silica applied at different rates (0.0, 50 and 100 mg/l) and the water regime was 30, 50 and 70% of water holding capacity (WHC). Results indicated that, Si-NPs there was an ability to reduce the drought impact on wheat growth and improve the antioxidant system in plants. Besides, an increment in membrane stability index, chlorophyll, carbohydrates, protein and phenol content of wheat. In addition, spraying or watering of Si-NPs increased wheat tolerance to drought by increasing the activity of antioxidant enzymes; nitrate reductase, phenylalanine ammonia-lyase, catalase, ascorbate peroxidase, glutathione s-transferase and guaiacol peroxidase, as well as reducing the oxidative pressure in leaves which was demonstrated by the diminished electrolyte leakage, malondialdehyde and proline in plant tissue. Data indicated that most of the highest values of the growth parameters and biochemical estimation were recorded for the wheat with application of 100 mg/l nano silica. Overall, this study advanced our understanding of the physiological and biochemical mechanisms underlying drought stress and mitigating its impact using Si-NPs, which may reduce the environmental risks that negatively affect the growth and productivity of agricultural crops globally.\u003c/p\u003e","manuscriptTitle":"Nano silica reinforces the tolerance of the wheat plant against drought stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-28 13:15:16","doi":"10.21203/rs.3.rs-5271175/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1ef88fdd-17e6-4d4c-ab0c-60c074098df9","owner":[],"postedDate":"November 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-23T13:54:05+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-28 13:15:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5271175","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5271175","identity":"rs-5271175","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}