Environmental Toxicology and Chemistry, Vol. 33, No. 11, pp. 2429–2437, 2014 # 2014 SETAC Printed in the USA
NANO-SILICON DIOXIDE MITIGATES THE ADVERSE EFFECTS OF SALT STRESS ON CUCURBITA PEPO L. MANZER H. SIDDIQUI, MOHAMED H. AL-WHAIBI, MOHAMMAD FAISAL, and ABDULAZIZ A. AL SAHLI Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia (Submitted 26 March 2014; Returned for Revision 28 April 2014; Accepted 22 July 2014) Abstract: Research into nanotechnology, an emerging science, has advanced in almost all fields of technology. The aim of the present
study was to evaluate the role of nano-silicon dioxide (nano-SiO2) in plant resistance to salt stress through improvement of the antioxidant system of squash (Cucurbita pepo L. cv. white bush marrow). Seeds treated with NaCl showed reduced germination percentage, vigor, length, and fresh and dry weights of the roots and shoots. However, nano-SiO2 improved seed germination and growth characteristics by reducing malondialdehyde and hydrogen peroxide levels as well as electrolyte leakage. In addition, application of nano-SiO2 reduced chlorophyll degradation and enhanced the net photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate, and water use efficiency. The increase in plant germination and growth characteristics through application of nano-SiO2 might reflect a reduction in oxidative damage as a result of the expression of antioxidant enzymes, such as catalase, peroxidase, superoxide dismutase, glutathione reductase, and ascorbate peroxidase. These results indicate that nano-SiO2 may improve defense mechanisms of plants against salt stress toxicity by augmenting the Pn, gs, transpiration rate, water use efficiency, total chlorophyll, proline, and carbonic anhydrase activity in the leaves of plants. Environ Toxicol Chem 2014;33:2429–2437. # 2014 SETAC Keywords: Stress response
Nanoparticles
Plant toxicology
Cucurbita pepo
Soil toxicology
Increasing salinization limits crop productivity in many areas of the world, particularly in semiarid and arid regions. Salinity impairs a number of metabolic and physiological functions in plants, such as water absorption, ion homeostasis, respiration, osmotic balance, and protein and nucleic acid synthesis [15–18]. Salt stress inhibits seed germination and root and shoot growth by limiting the water potential of soil solution, which greatly reduces cell membrane permeability and influx of water to the plant [19,20]. As noted by Almodares et al. [21], the mechanisms involved in cellular damage in plants under high salt conditions during germination are not fully understood. It has recently become extremely important to develop novel strategies to improve the salinity tolerance of plants in semiarid and arid regions. The regulatory effect of silicon on plant growth and development under stress conditions is well documented [22–24]. Therefore, it is important to determine the potential role of nano-SiO2 in plant tolerance to abiotic stress through modulation of the physio-biochemical function of plants. Squash (Cucurbita pepo L.) is an important vegetable that serves as a rich source of protein, carbohydrates, and vitamins [25]. It is important to investigate the potential role of nano-SiO2 in plant tolerance to salt stress and to characterize the ameliorating effect of nano-SiO2 on seed germination, proline content, and antioxidant enzyme activity in squash plants under high salinity.
INTRODUCTION
Nanotechnology has been globally accepted as a modern, advanced technology that could enhance research in many fields [1–3]. The increasing numbers of nanotechnological inventions (nano-devices and materials) have led to novel applications in the fields of biotechnology and agriculture [4]. Nanoparticles have received much attention because of their unique physicochemical properties compared with bulk particles [5]. Nanoparticles act as chemical delivery agents that target molecules to specific cellular organelles in plants [6]. Silica nanoparticles (nano-SiO2) have been used to deliver DNA and chemicals into plant and animal cells and tissues [7]. These nanoparticles are highly hydrophilic, with the potential for surface modifications [8]. Nanobiopesticides made from nanoSiO2 provide a shielding layer to protect pesticides from degradation by ultraviolet light [9]. In addition, nano-SiO2 is used to produce effective fertilizers for crops and to minimize the loss of fertilizer through slow and controlled release, allowing for regulated, responsive, and timely delivery [2,10]. Previous studies have shown that nano-SiO2 significantly enhances seed germination and seedling root growth [11,12]. Silicon is an important element that plays a key role in a number of metabolic and physiological activities in plants [11]. However, few studies have focused on the physiological role of nano-SiO2 in plants. Li et al. [13] showed that foliar application of nano-SiO2 on Indocalamus barbatus improved the contents of soluble protein, free amino acids, and total nitrogen, phosphorus, and potassium; induced activity of the antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD); and suppressed lipid peroxidation. In addition, exogenous application of nano-SiO2 increased the photosynthetic capacity of plants by amplifying gas exchange and chlorophyll fluorescence parameters [14].
MATERIALS AND METHODS
Seed preparation
The present experiment was conducted under laboratory conditions using squash (C. pepo L. cv. white bush marrow) purchased from a local market in Riyadh, Saudi Arabia. Healthy squash seeds were selected, immersed in a 10% sodium hypochlorite solution for 10 min, vigorously rinsed with sterilized double-distilled water, and transferred to a 12-cm Petri dish.
* Address correspondence to [email protected] Published online 26 July 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.2697 2429
2430
Environ Toxicol Chem 33, 2014
Characterization and preparation of nanoparticle suspension for treatment
The SiO2 nanoparticles were purchased from Evonik Industries. The hydrophilic fumed silica (SiO2) commercially known as Aerosil 200 (Evonik Industries) was used, with an average primary particle size of 12 nm and a corresponding surface area of 200 m2/g. The shape and size of the SiO2 nanoparticles were determined by transmission electron microscopy (JEM-2100F; JEOL). Figure 1 shows a typical image of SiO2 nanoparticles; most of the particles are spherical in shape, with smooth surfaces. The average diameter of SiO2 nanoparticles calculated by transmission electron microscopy was approximately 10 nm. A dry SiO2 powder was suspended in deionized water to make the following concentrations: 1.5 g L1, 3.0 g L1, 4.5 g L1, 6.0 g L1, and 7.5 g L1. These solutions were sonicated for 15 min in a probe-type sonicator. Seed treatment and germination
Sterilized healthy seeds were transferred onto 2 sheets of sterilized filter paper in 12-cm Petri dishes. Fifty seeds were placed into each dish. The dishes were arranged in a simple randomized design with a single factor and 5 replicates. The nano-SiO2 and NaCl treatments were applied in the following manner (subscript numbers describe the concentrations in g L1 for nano-SiO2 and in mM for NaCl): 1) SiO0 þ NaCl0 (control), 2) SiO0 þ NaCl120, 3) SiO1.5 þ NaCl120, 4) SiO3.0þ NaCl120, 5) SiO4.5 þ NaCl120, 6) SiO6.0 þ NaCl120, and 7) SiO7.5þ NaCl120. Next, the dishes were sealed with paraffin tape and incubated in the dark at 28 3 8C. The number of germinated seeds was counted every 2 d. After 10 d, seedlings were transferred onto sterile filters in new sterile dishes containing the same concentrations and volumes as the treatments given above. After 15 d, the performance of C. pepo was assessed in terms of root length, shoot length, root fresh weight, shoot fresh weight, root dry weight, shoot dry weight, net photosynthetic rate (Pn), stomatal conductance (gs), chlorophyll content, chlorophyll degradation, electrolyte leakage, malondialdehyde (MDA) content, proline content, and activities of carbonic anhydrase (CA), catalase (CAT), POD, SOD, glutathione reductase (GR), and ascorbate peroxidase (APX). Determination of growth characteristics
The percentage of seed germination was recorded every 2 d for days 4 to 15. The vigor index was calculated by using the
M.H. Siddiqui et al.
following equation [26]: vigor index ¼ (mean root length þ mean shoot length) percentage of germination. After 15 d, the shoot and root lengths were measured using a meter scale. After recording of root and shoot fresh weights, the samples were placed in an oven at 60 8C for 48 h to obtain the dry weight of the roots and shoots. Determination of physio-biochemical characteristics Total chlorophyll concentration. The youngest fully expanded leaves were subjected to extraction using 80% acetone, and the absorbance was measured spectrophotometrically (SPEKOL 1500; Analytik Jena) at 663 nm and 645 nm. The total chlorophyll content was determined by using Arnon’s formula [27]. Proline concentration. The proline concentration was determined spectrophotometrically using the ninhydrin method of Bates et al. [28]. First, we homogenized 300 mg of fresh leaf specimens in 3% sulfosalicylic acid, followed by the addition of 2 mL each of ninhydrin and glacial acetic acid, after which the samples were heated to 100 8C. The mixture was then extracted with toluene, and the free toluene was quantified at 520 nm. MDA concentration. The MDA content was determined according to the method of Heath and Packer [29]. Leaves were weighed, and homogenates containing 10% trichloroacetic acid and 0.65% 2-thiobarbituric acid were heated at 95 8C for 60 min, then cooled to room temperature, and centrifuged at 10 000 g for 10 min. The absorbance of the supernatant was read at 532 nm and 600 nm against a reagent blank. Electrolyte leakage. Electrolyte leakage was used to assess membrane permeability in accordance with Lutts et al. [30]. Samples were washed 3 times with double-distilled water to remove surface contamination, and leaf discs were cut from young leaves and placed in sealed vials containing 10 mL of double-distilled water, followed by incubation on a rotary shaker for 24 h, after which the electrical conductivity of the solution (EC1) was determined. Then the samples were autoclaved at 120 8C for 20 min, and the electrical conductivity was measured again (EC2) after the solution was cooled to room temperature. The electrolyte leakage was defined as EC1/EC2 100 and expressed as percentage. Histochemical detection of hydrogen peroxide (H2O2) localization. The pattern of H2O2 localization in the plant tissues was
determined in hand-cut cross sections stained with a KI/starch reagent containing 4% (w/v) starch and 0.10 M KI [31]. The presence of H2O2 was indicated by a dark color. The stained sections were observed and photographed using a B071 Olympus microscope. Gas exchange parameters
Figure 1. Transmission electron microscopy image of silicon dioxide nanoparticles.
Gas exchange parameters, such as Pn, gs, transpiration rate, and water use efficiency, were recorded using an open system infrared gas analyzer (CID Bio-Science). The youngest healthy expanded leaf of each squash seedling was selected for the measurement of gas exchange parameters. The water use efficiency was quantified as the Pn divided by the transpiration rate. CA activity. The activity of CA (EC 4.2.1.1) was determined using the method of Dwivedi and Randhawa [32]. Leaf samples were cut into small pieces, extracted in cysteine hydrochloride solution, and incubated at 40 8C for 20 min. The pieces were then blotted and transferred to test tubes containing phosphate buffer (pH 6.8), followed by the addition of alkaline bicarbonate solution and bromothymol blue indicator. The test tubes were incubated at 50 8C for 20 min. After the addition of methyl red
Nano-SiO2 mitigates the adverse effects of salt stress
indicator, the reaction solution was titrated against 0.05 N HCl. The results were expressed as mmol CO2 kg1 fresh weight s1. Antioxidant enzyme activity. To determine the activities of antioxidant enzymes, a crude enzyme extract was prepared by homogenizing 500 mg of leaf tissue in extraction buffer (0.5% Triton X-100 and 1% polyvinylpyrrolidone in 100 mM potassium phosphate buffer, pH 7.0) using a chilled mortar and pestle. The homogenate was then centrifuged at 15 000 g for 20 min at 4 8C, and the supernatant was used for the enzymatic assays described below. For the APX assay, the extraction buffer was supplemented with 2 mM ascorbate. All enzyme activities were expressed as milligram of protein per minute. We applied the method of Chance and Maehly [33] to determine POD (EC 1.11.1.7) activity using 5 mL of enzyme reaction solution containing phosphate buffer (pH 6.8), 50 M pyrogallol, 50 mM H2O2, and 1 mL of the enzyme extract diluted 20 times. The assay mixture was incubated for 5 min at 25 8C, and the reaction was terminated by the addition of 0.5 mL of 5% (v/v) H2SO4. Purpurogallin production was measured spectrophotometrically at 420 nm. One unit of POD activity was considered the amount of purpurogallin formed per milligram of protein per minute. The method of Aebi [34] was used to measure CAT (EC 1.11.1.6) activity. The decomposition of H2O2 was measured as the decrease in absorbance at 240 nm. In this assay, 50 mM phosphate buffer (pH 7.8) and 10 mM H2O2 were used in the reaction solution. Activity of SOD (EC 1.15.1.1) was determined based on the inhibition of nitro blue tetrazolium (NBT) photoreduction according to the method of Giannopolitis and Ries [35]. The reaction solution (3 mL) contained 50 mM NBT, 1.3 mM riboflavin, 13 mM methionine, 75 nM ethylenediamine tetraacetic acid (EDTA), 50 mM phosphate buffer (pH 7.8), and 20 mL to 50 mL of enzyme extract. The reaction solution was irradiated under fluorescent light at 75 mM m2 s1 for 15 min. The absorbance at 560 nm was read against a blank (nonirradiated reaction solution). One unit of SOD activity was defined as the amount of enzyme that inhibited 50% of NBT photoreduction. We measured APX (EC 1.11.1.11) activity using the method of Nakano and Asada [36]. The reaction buffer solution contained 50 mM potassium phosphate (pH 7.0), 0.1 mM EDTA, 0.1 mM H2O2, and 0.5 mM ascorbate. The reaction was initiated by the addition of the sample solution to the reaction mixture. The H2O2-dependent oxidation of ascorbate was measured as the decrease in absorbance at 290 nm.
Environ Toxicol Chem 33, 2014
2431
Figure 2. Ameliorating effect of nano-silicon dioxide (nSiO) on (A) seed germination and (B) vigor in squash seedlings. Bars with the same letter are not significantly different at p < 0.05%. S ¼ NaCl.
The ameliorating effect of nano-SiO2 was evaluated on the basis of the growth and physio-biochemical characteristics of C. pepo L. cv. white bush marrow under NaCl stress.
However, when seeds were treated with different concentrations of nano-SiO2, significant differences (p < 0.05) were observed. The application of nano-SiO2 significantly increased the germination percentage and vigor index under salt stress conditions. Application of 6 g L1 of nano-SiO2 increased the germination percentage and vigor index by 77.95% and 285.21%, respectively, compared with the salinity treatment. However, application of 7.5 g L1 and 4.5 g L1 of nano-SiO2 resulted in equal effects on germination percentage. The concentration of 6 g L1 of nano-SiO2 proved to be the best at alleviating salt stress. Similarly, the seedlings exposed to NaCl exhibited reduced root and shoot lengths, as well as reduced root and shoot fresh and dry weights, compared with the control (Figure 3A–C). However, coexposure to NaCl and nano-SiO2 improved root and shoot length, as well as root and shoot fresh and dry weights. Application of 6 g L1 of nano-SiO2 increased root length by 121.99%, shoot length by 105.11%, root fresh weight by 328.72%, shoot fresh weight by 133.98%, root dry weight by 343.95%, and shoot dry weight by 152.87% over the respective NaCl treatments. Application of 7.5 g L1 and 4.5 g L1 of nanoSiO2 showed equal effects on both shoot fresh weight and root dry weight.
Nano-SiO2 effects on morphological characteristics
Effect of nano-SiO2 on physiological and biochemical parameters
In the present study, we observed that salt stress inhibited the seed germination percentage and vigor index (Figure 2A and B).
The MDA content indicated the level of lipid peroxidation, whereas oxidative damage and membrane alterations were
Statistical analysis
Each Petri dish was treated as 1 replicate, and all treatments were repeated 5 times. The data were expressed as the mean standard error and were analyzed statistically using SPSS Ver 17 statistical software. The means were compared statistically using Duncan’s multiple-range test at the level of p < 0.05. RESULTS
2432
Environ Toxicol Chem 33, 2014
M.H. Siddiqui et al.
Figure 3. Ameliorating effect of nano-silicon dioxide (nSiO) on the (A) root and shoot length, (B) root and shoot fresh weight and (C) root and shoot dry weight of squash seedlings. Bars with the same letter are not significantly different at p < 0.05%. S ¼ NaCl.
assessed by electrolyte leakage (Table 1). Salinity had a damaging effect, as revealed by increased MDA levels and electrolyte leakage. However, the addition of nano-SiO2 significantly suppressed lipid peroxidation and electrolyte leakage. Also, all 5 tested concentrations of nano-SiO2 were found to be effective in alleviating the damaging effect of salt stress. The concentration of 6 g L1 of nano-SiO2 reduced the MDA content by 52.23% and electrolyte leakage by 51.87% compared with the NaCl treatment. Histochemical analysis showed greater H2O2 production in the NaCl-treated seedlings than in the control and seedlings treated with nano-SiO2-treated (Figure 4A–C). In the NaCl-treated seedlings, H2O2 was
detected in the vascular bundle and phloem fibers. The intensity of H2O2 production in the xylem and phloem of the vascular bundle and root was high in NaCl-exposed seedlings compared with the control and nano-SiO2-treated seedlings. Application of NaCl improved the proline content (Table 1). However, the proline concentration increased further with increasing levels of nano-SiO2 under salt stress. Application of 6 g L1 of nano-SiO2 increased proline synthesis by 292.93% and 88.35% over the control and NaCl treatment, respectively. Figure 5A–D shows that NaCl treatment significantly decreased Pn, gs, transpiration rate, and water use efficiency values. However, the addition of nano-SiO2 alleviated the
Table 1. Ameliorating effect of nano-SiO2 on the electrolyte leakage (% of total electrolyte leakage) and content of MDA and proline in squash plants Treatmenta Control S S þ nano-SiO1.5 S þ nano-SiO3 S þ nano-SiO4.5 S þ nano-SiO6 S þ nano-SiO7.5
Electrolyte leakage (%)b 15.10 1.04 47.54 1.25 39.10 1.13 32.71 1.45 26.09 0.99 22.88 0.91 28.77 1.71
F A B C D,E E D
MDA content (nmol g1 fresh wt)b 15.11 0.45 49.65 0.88 44.43 0.49 36.42 0.49 31.40 0.42 23.72 0.75 26.32 0.19
Subscript numbers describe nano-SiO concentrations (in g L1). Data followed by the same letters are not significantly different at p < 0.05%. MDA ¼ malondialdehyde; Pro ¼ proline; S¼ salinity; nano-SiO ¼ nano silicon dioxide.
a
b
G A B C D F E
Proline content (mg1 fresh wt)b 10.33 0.62 21.55 0.71 25.43 0.49 27.36 0.84 33.41 0.62 40.59 0.42 36.85 0.71
F E D E C A B
Nano-SiO2 mitigates the adverse effects of salt stress
Environ Toxicol Chem 33, 2014
2433
Figure 4. Histochemical analysis of H2O2 production through starch–KI staining of squash seedling cross sections. (A) Section of control seedling (4 magnification); (B) section of NaCl-treated seedling (4 magnification); (C) Section of NaCl þ nano-SiO6.0-treated seedling (4 magnification). VB ¼ vascular bundle; nano-SiO ¼ nano silicon dioxide. [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]
inhibitory effects of NaCl stress. Application of 6 g L1 of nanoSiO2 proved to be the best at alleviating the adverse effect of NaCl stress. The control and plants treated with 4.5 g L1 nanoSiO2 exhibited statistically equal values for Pn, and gs, as did plants treated with 3 g L1, 4.5 g L1, 6 g L1, or 7.5 g L1 nanoSiO2 for water use efficiency. The control and plants treated with 3 g L1, 4.5 g L1, 6 g L1, and 7.5 g L1 nano-SiO2 showed statistically similar values for the transpiration rate. The data presented in Figure 6A and B reveal that the application of NaCl and nano-SiO2 significantly influenced the total chlorophyll concentration and chlorophyll degradation. Exposure to NaCl caused a decrease in total chlorophyll content and an increase in chlorophyll degradation, whereas application of nano-SiO2 with NaCl improved the total chlorophyll content and reduced chlorophyll degradation compared with NaCltreated seedlings. Application of 6 g L1 of nano-SiO2 increased the total chlorophyll content by 345%, and reduced chlorophyll degradation by 45.13% compared with the NaCl treatment. However, the effects of 6 g L1 of nano-SiO2 were similar to the control treatment for total chlorophyll synthesis. The activity of CA relative to the control was significantly inhibited by the application of NaCl (Figure 6C). However, the addition of nano-SiO2 significantly improved CA activity compared with NaCl treatment. Exposure to nano-SiO2 caused a gradual and concentration-dependent increase in CA activity. Application of 6 g L1 of nano-SiO2 improved CA activity by 26.62% compared with NaCl treatment. Antioxidant enzyme activity (CAT, POD, SOD, GR, and APX) was significantly influenced under all nano-SiO2 concentrations tested (Figure 7). Application of NaCl significantly enhanced the activities of CAT, POD, SOD, GR, and APX, but the increase was even more prominent when nanoSiO2 was added. Application of 6 g L1 of nano-SiO2 relative to the NaCl treatment significantly increased the activities of CAT, POD, SOD, GR, and APX by 26.62%, 40.29%, 39.88%, 65.67%, and 105.51%, respectively. However, the effect of
6 g L1 of nano-SiO2 was equal to that of 7.5 g L1 of nano-SiO2 for POD activity. DISCUSSION
Seed germination is crucial for plant growth and development. In the present study, the application of NaCl significantly inhibited seed germination. These results strongly support the findings of Siddiqui et al. [12] in tomato, Almodares et al. [21] in sorghum, Nyagah and Musyimi [37] in Passiflora edulis, Tobe et al. [38] in Haloxylon ammodendron, Demir et al. [39] in Solanum melongena, and Khajeh-Hosseini et al. [40] in soybean. The inhibitory effect of NaCl might be the result of an osmotic barrier that reduces the ability of the seed to imbibe water, which could result in ion toxicity in the seed embryo [21]. Similarly, in the present study, salinity was observed to reduce the germination rate and vigor index (Figure 2A and B). However, interestingly, application of nano-SiO2 with NaCl significantly enhanced seed germination and vigor index (Figure 2A and B). Haghighi et al. [41] reported similar results. An increase in these attributes with application of nano-SiO2 might reflect the activities of protease and lipase; silicon also improves seedling respiration rates and seed vigor [42]. According to Epstein [43], silicon maintains ion homeostasis and alleviates the toxicity of metals and salt stress. Therefore, the results of the present study suggest that nano-SiO2 provided a suitable environment for the germination of seeds under salinity conditions. The data presented in Figure 3A–C demonstrate a significant effect of nano-SiO2 and NaCl on the root and shoot dry weights, fresh weights, and lengths. The plants generated from seeds treated with NaCl showed reduced growth parameters. Similar results have been observed in many other crops [17,18,44,45]. In the present study, however, nano-SiO2 improved growth characteristics of the squash plants by alleviating the adverse effect of salinity (Figure 3). This enhancement might reflect the physiological roles of nano-SiO2, which is required for
2434
Environ Toxicol Chem 33, 2014
M.H. Siddiqui et al.
Figure 5. Ameliorating effect of nano-silicon dioxide (nSiO) on the (A) net photosynthetic rate (Pn), (B) stomatal conductance (gs), (C) water use efficiency (WUE), and (D) transpiration rate (E) of squash seedlings. Bars with the same letter are not significantly different at p < 0.05%. S ¼ NaCl.
photosynthesis and nitrate assimilation [42,46]. In addition, nano-SiO2 has been implicated in the synthesis of proteins and amino acids and the uptake of nutrients [13], and the strength and rigidity of plants is improved through nano-SiO2 deposition in plant tissues [24]. It was also shown that nano-SiO2 enhanced the growth and physiological characteristics of plants through increasing the production of organic compounds such as proteins, chlorophyll, and phenols, relative to bulk particles [47]. The increase in plant growth characteristics because of nano-SiO2 application might reflect the reduced uptake of Naþ ions and sustained membrane integrity in root cells, as demonstrated by the enhancement of antioxidant systems and reduced oxidative damage. Lipid peroxidation, electrolyte leakage, and H2O2 levels are key parameters that indicate the level of oxidative damage in plants under unfavorable environmental conditions [12]. The increase in electrolyte leakage, MDA, and H2O2 observed in the NaCl-treated seedlings (Table 1 and Figure 4) indicates that NaCl causes cellular dysfunction by enhancing lipid peroxidation through the generation of free radicals [48,49]. However, the seedlings raised from seeds treated with nano-SiO2 in the present study exhibited reduced electrolyte leakage, lipid
peroxidation, and H2O2 levels. These effects might reflect increased activity of antioxidant enzymes (CAT, POD, SOD, GR, and APX; Figure 7A and B). In addition, nano-SiO2 mediates the synthesis of protein, amino acids, and phenols, as well as nutrient uptake [13]. According to Epstein [43] “silicon plays an astonishingly large number of diverse roles in plants, and does so primarily when the plants are under stressful conditions.” Thus, we postulate that the application of nanoSiO2 improves plant tolerance to salt stress. Under stress condition, plants synthesize compatible solutes (glycine betaine and proline) to adjust the osmotic potential within cells. In the present study, the proline content increased relative to that of the control when NaCl was applied (Table 1). This result is consistent with that of previous studies showing increases in proline content in response to salt stress [44,50]. The concentration of proline further increased with the application of nano-SiO2. Proline, a universal osmoprotectant, acts as both an antioxidant and a source of energy [51], and regulates gene expression, leading to osmotic adjustment [52]. Hyperaccumulation of proline increases water uptake by increasing osmotic pressure [50], resulting in enhanced tolerance to salt stress in plants.
Nano-SiO2 mitigates the adverse effects of salt stress
Environ Toxicol Chem 33, 2014
2435
Figure 6. Ameliorating effect of nano-silicon dioxide (nSiO) on the (A) total chlorophyll content, (B) chlorophyll degradation, and (C) carbonic anhydrase activity in squash seedlings. Bars with the same letter are not significantly different at p < 0.05%. S ¼ NaCl.
Photosynthesis is an important process that allows for growth and dry matter production in plants. Under NaCl stress, the observed inhibition of net Pn, gs, transpiration rate, and water use efficiency compared with the respective controls (Figure 5) might reflect the excessive accumulation of Naþ, and Cl, which inhibits stomatal closure, leading to a reduction in internal CO2 availability [52]. A similar result was shown by Shi et al. [53]. Seedlings treated with NaCl showed reduced total chlorophyll contents and CA activity and increased chlorophyll degradation relative to the respective controls (Figure 6). The reduction in total chlorophyll might reflect lipid peroxidation and H2O2 formation in plant cells, which are both significantly affected by NaCl, sodium ion accumulation in chloroplasts, and expression of the chlorophyll-degrading enzyme chlorophyllase [54,55]; thus we observed marked chlorophyll degradation (Figure 6B). The results of the present study showed that application of nanoSiO2 restored the changes in Pn and photosynthetic pigments observed in the seedlings through the inhibition of chlorophyll degradation and the expression of CA enzyme activity (Figure 6). Xie et al. [46] reported that exogenous application of nanoSiO2 improved the photosynthetic activity of mesophyll cells in Indocalamus barbatus. According to Shi et al. [53], the application of silicon improves Pn, gs, transpiration rate, and water use efficiency in rice (Oryza sativa) under salt stress. The degree of alleviation of salt stress and the improvement in the photosynthetic capacity of squash seedlings through the application of nano-SiO2 could be associated with the beneficial
effects of nano-SiO2 on seed germination potential and plant growth (Figures 2 and 3), reflecting the inhibition of photosynthetic pigment degradation and improvement in enzyme activities (Figures 6C and 7). The enhanced CA activity in the seedlings treated with nano-SiO2 might influence the reversible hydration of CO2 and maintain a constant supply of CO2 to RuBisCO to improve chlorophyll synthesis, thereby increasing Pn, gs, and water use efficiency [53]. Reactive oxygen species act as signaling molecules in plant cells and impair macromolecules through interactions with DNA, proteins, and carbohydrates [12,15,56]. Under stress conditions, plants develop a defense mechanism through which they can survive by limiting and detoxifying reactive oxygen species. In the present study, the observed electrolyte leakage, lipid peroxidation, and proline levels in plant tissues were substantially higher in seedlings treated with NaCl (Table 1). However, the application of nano-SiO2 alleviated these negative effects on plant growth by increasing the activity of antioxidant enzymes such as CAT, POD, SOD, GR, and APX (Figure 7A and B). These findings substantiate the results of Li et al. [13], who showed that nano-SiO2 stimulates antioxidant enzyme activity. In addition, Hashemi et al. [57] observed that silicon nutrition reduced the inhibitory effect of salinity on plant growth by reducing the Naþ content, increasing CAT and cell wall peroxidase activities, and maintaining the membrane integrity of root cells, as demonstrated by reduced lipid peroxidation. In the present study, the seedlings were able to maintain steady-state
2436
Environ Toxicol Chem 33, 2014
M.H. Siddiqui et al. Acknowledgment—The present study was supported financially through funding from the Deanship of Scientific Research of King Saud University, Riyadh, Saudi Arabia to Research Group RGPVPP-153. The authors declare that they have no conflicts of interest. REFERENCES
Figure 7. Ameliorating effect of nano-silicon dioxide (nSiO) on the activities of (A) catalase (CAT) and peroxidase (POD) and (B) superoxide dismutase (SOD), glutathione reductase (GR), and ascorbate peroxidase (APX) in squash seedlings. Bars with the same letter are not significantly different at p < 0.05%. S ¼ NaCl.
levels of O2, and H2O2 might have resulted from enhancement of antioxidant enzyme activity in plant cells through the application of nano-SiO2. CONCLUSIONS
Squash seedlings treated with NaCl and nano-SiO2 showed significant changes in growth and physiological characteristics. When seedlings were exposed to salt stress in the present study, the percentage of seed germination, vigor, growth, and physiobiochemical characteristics were decreased, and MDA and H2O2 levels and electrolyte leakage increased. However, nano-SiO2 significantly reduced the adverse effects of salinity by mitigating cellular oxidative damage through the enhancement of free radical scavenging antioxidant enzymes. The effect of nano-SiO2 was dose dependent. The improvements we found in growth parameters, photosynthetic pigments, proline content, and CA activity might underlie the enhanced tolerance of plants to salinity after treatment with nano-SiO. There is insufficient information regarding the positive effects of nano-SiO2 on plant growth and the physiological mechanisms that arise in higher plants under salt stress, but the findings of the present study provide a new platform for exploring the involvement of nanoSiO2 in physiological, biochemical, and molecular activities in plants.
1. Gogos A, Knauer K, Bucheli TD. 2012. Nanomaterials in plant protection and fertilization: Current state, foreseen applications, and research priorities. J Agric Food Chem 60:9781–9792. 2. Nair R, Varghese SH, Nair BG, Maekawa T, Yoshida Y, Kumar DS. 2010. Nanoparticulate material delivery to plants. Plant Sci 179:154– 163. 3. González-Melendi P, Fernández-Pacheco R, Coronado MJ, Corredor E, Testillano PS, Risue~ no M, Marquina C, Ibarra MR, Rubiales D, Perezde-Luque A. 2008. Nanoparticles as smart treatment-delivery systems in plants: Assessment of different techniques of microscopy for their visualization in plant tissues. Ann Bot 101:187–195. 4. Scrinis G, Lyons K. 2007. The emerging nano-corporate paradigm: Nanotechnology and the transformation of nature, food and agri-food systems. Int J Sociol Food Agri 15:22–44. 5. Monica RC, Cremonini R. 2009. Nanoparticles and higher plants. Caryologia 62:161–165. 6. Liu J, He S, Zhang Z, Cao J, Lv P, He S, Cheng G, Joyce DC. 2009. Nano-silver pulse treatments inhibit stem-end bacteria on cut gerbera cv. Ruikou flowers. Postharvest Biol Technol 54:59–62. 7. Torney F, Trewyn BG, Lin VSY, Wang K. 2007. Mesoporous silica nanoparticles deliver DNA and chemicals into plants. Nat Nanotechnol 2:295–300. 8. Nakamura M, Shono M, Ishimura K. 2007. Synthesis, characterization, and biological applications of multifluorescent silica nanoparticles. Anal Chem 79:6507–6514. 9. Chen S, Sun L, Sun T, Chao L, Guo G. 2007. Interaction between cadmium, lead and potassium fertilizer (K2SO4) in a soil-plant system. Environ Geochem Health 29:435–446. 10. Zhang F, Wang R, Xiao Q, Wang Y, Zhang J. 2006. Effects of slow/ controlled-release fertilizer cemented and coated by nano-materials on biology. II. Effects of slow/controlled-release fertilizer cemented and coated by nano-materials on plants. Nanoscience 11:18–26. 11. Bao-Shan L, Shao-qi D, Chun-hui L, Li-jun F, Shu-chun Q, Min Y. 2004. Effect of TMS (nanostructured silicon dioxide) on growth of Changbai larch seedlings. J Forest Res 15:138–140. 12. Siddiqui MH, Al-Whaibi MH, Sakran AM, Ali HM, Basalah MO, Faisal M, Alatar A, Al-Amri AA. 2013. Calcium-induced amelioration of boron toxicity in radish. J Plant Growth Regul 32:61–71. 13. Li B, Tao G, Xie Y, Cai X. 2012. Physiological effects under the condition of spraying nano-SiO2 onto the Indocalamus barbatus McClure leaves. J Nanjing Forest Uni (Natural Sciences Edition) 4:161–164. 14. Yinfeng X, Bo L, Qianqian Z, Chunxia Z, Kouping L, Gongsheng T. 2011. Effects of nano-TiO2 on photosynthetic characteristics of Indocalamus barbatus. J Northeast Forest Uni 39:22–25. 15. Maathuis FJM. 2006. The role of monovalent cation transporters in plant responses to salinity. J Exp Bot 57:1137–1147. 16. Zeng J, Zeng FJ, Arndt SK, Guo HF, Yan HL, Xing WJ, Liu B. 2008. Growth, physiological characteristics and ion distribution of NaCl stressed Alhagi sparsifolia seedlings. Chin Sci Bull 53:169–176. 17. Siddiqui MH, Mohammad F, Khan MMA, Al-Whaibi MH. 2012. Cumulative effect of nitrogen and sulphur on Brassica juncea L. genotypes under NaCl stress. Protoplasma 249:139–153. 18. Siddiqui MH, Al-Whaibi MH, Sakran AM, Basalah MO, Ali HM. 2012. Effect of calcium and potassium on antioxidant system of Vicia fabe L. under cadmium stress. Intt J Mol Sci 13:6604–6619. 19. Malhotra K, Chhabra GK, Jain R, Sharma V, Kumar S. 2013. Drought and salinity tolerant biofuel crops for the Thar Desert. In Tuteja N, Gill S, eds. Plant Acclimation to Environmental Stress. Springer, New York, NY, USA, pp 71–84. 20. Jamil M, Lee DB, Jung KY, Ashraf M, Lee SC, Rha ES. 2006. Effect of salt (NaCl) stress on germination and early seedling growth of four vegetable species. J Cent Eur Agri 7:273–282. 21. Almodares A, Hadi MR, Dosti B. 2007. Effects of salt stress on germination percentage and seedling growth in sweet sorghum cultivars. J Biol Sci 7:1492–1495. 22. Matichenkov VV, Kosobrukhov AA., 2004. Silicon effect on the plant resistance to salt toxicity. Conserving soil and water for society. Proceedings, 13th International Soil Conservation Organization Conference, Brisbane, Australia, July 4-8, 2004, pp 626.
Nano-SiO2 mitigates the adverse effects of salt stress 23. Janislampi KW. 2012. Effect of silicon on plant growth and drought stress tolerance. PhD thesis. Utah State University, Logan, UT, USA. [cited 2013 August 5]. Available from: http://digitalcommons.usu.edu/ etd/1360 24. Ma JF, Yamaji N. 2006. Silicon uptake and accumulation in higher plants. Trends Plant Sci 11:342–397. 25. Vural H, ESs iyok D, Duman I. 2000. Kültür Sebzeleri (Sebze YetiSs tirme). Egean University Press, Bornova, Ïzmir, Turkey (in Turkish). 26. Tao KL, Zheng GH. 1990. Seed Vigor. Science Press, Beijing, China (in Chinese). 27. Arnon DI. 1949. Copper enzymes in isolated chloroplast. Polyphenoloxidases in Beta vulgaris. Plant Physiol 24:1–15. 28. Bates LS, Waldren RP, Teare ID. 1973. Rapid determination of free proline for water-stress studies. Plant Soil 39:205–207. 29. Heath RL, Packer L. 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:189–198. 30. Lutts S, Kinet JM, Bouharmont J. 1995. Changes in plant response to NaCl during development of rice (Oryza sativa L.) varieties differing in salinity resistance. J Exp Bot 46:1843–1852. 31. Olson PD, Varner JE. 1993. Hydrogen peroxide and lignification. Plant J 4:887–892. 32. Dwivedi RS, Randhawa NS. Evaluation of rapid test for hidden hunger of zinc in plants. Plant Soil 40:45–451. 33. Chance B, Maehly AC. 1955. Assay of catalase and peroxidases. Methods Enzymol 2:764–775. 34. Aebi H. 1984. Catalase in vitro. Methods Enzymol 105:121–126. 35. Giannopolitis CN, Ries SK. 1977. Superoxide dismutases: I. Occurrence in higher plants. Plant Physiol 59:309–314. 36. Nakano G, Asada K. 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880. 37. Nyagah AW, Musyimi DM. 2009. Effects of sodium chloride solution stress on germination and growth of passion fruits seedlings. ARPN J Agri Biol Sci 4:49–53. 38. Tobe K, Li X, Omasa K. 2004. Effects of five different salts on seed germination and seedling growth of Haloxylon ammodendron (Chenopodiaceae). Seed Sci Res 14:345–354. 39. Demir I, Mavi K, ozcoban M, Okcu G. 2003. Effect of salt stress on germination and seedling growth in serially harvested aubergine (Solanum melongena L.) seeds during development. Israel J Plant Sci 51:125–131. 40. Khajeh-Hosseini M, Powell AA, Bingham IJ. 2003. The interaction between salinity stress and seed vigour during germination of soyabean seeds. Seed Sci Technol 31:715–725. 41. Haghighi M, Afifipour Z, Mozafarian M. 2012. The effect of N-Si on tomato seed germination under salinity levels. J Biol Environ Sci 6:87–90.
̌
Environ Toxicol Chem 33, 2014
2437
42. Qingfang L, Chengcang M. 2002. Effect of available silicon in soil on cucumber seed germination and seedling growth metabolism. Acta Horticul Sinica 29:433–437. 43. Epstein E. 2009. Silicon: Its manifold roles in plants. Ann Appl Biol 155:155–160. 44. Siddiqui MH, Mohammad F, Khan MN. 2009. Morphological and physio-biochemical characterization of Brassica juncea L. Czern. & Coss. genotypes under salt stress. J Plant Interact 4:67–80. 45. Siddiqui MH, Khan MN, Mohammad F, Khan MMA. 2008. Role of nitrogen and gibberellin (GA3) in the regulation of enzyme activities and in osmoprotectant accumulation in Brassica juncea L. under salt stress. J Agron Crop Sci 194:214–224. 46. Xie Y, Li B, Zhang Q, Zhang C. 2012. Effects of nano-silicon dioxide on photosynthetic fluorescence characteristics of Indocalamus barbatus McClure. J Nanjing Forest Uni (Natural Sciences Edition) 2: 59–63. 47. Suriyaprapha R, Karunakaran G, Yuvakkumar R, Prabu P, Rajendran V, Kannan N. 2012. Growth and physiological responses of maize (Zea mays L.) to porous silica nanoparticles in soil. J Nanopart Res 14: 2–14. 48. EI-Tayeb MA. 2005. Response of barley grains to the interactive effect of salinity and salicylic acid. Plant Growth Regul 45:215–224. 49. Mahajan S, Tuteja N. 2005. Cold, salinity and drought stresses: An overview. Arch Biochem Biophys 444:139–158. 50. Khan MN, Siddiqui MH, Mohammad F, Naeem M. 2012. Interactive role of nitric oxide and calcium chloride in enhancing tolerance to salt stress. Nitric Oxide 27:210–218. 51. Matysik J, Alia Bhalu B, Mohanty P. 2002. Molecular mechanisms of quenching of reactive oxygen species by proline under stress in plants. Curr Sci 82:525–532. 52. Iyer S, Caplan A. 1998. Products of proline catabolism can induce osmotically regulated genes in rice. Plant Physiol 116:203–211. 53. Shi Y, Wang Y, Flowers TJ, Gong H. 2013. Silicon decreases chloride transport in rice (Oryza sativa L.) in saline conditions. J Plant Growth Regul, 170:847–853. 54. Reddy MP, Vora AB. 1986. Salinity induced changes in pigment composition and chlorophyllase activity in wheat. Indian J Plant Physiol 29:331–334. 55. Kyle DJ. 1987. The biochemical basis for photoinhibition of photosystem II. In Kyle DJ, Osmond CB, Artzen CJ, eds Photoinhibition. Elsevier, Amsterdam, The Netherlands, pp 197–226. 56. Foyer CH, Noctor G. 2000. Oxygen processing in photosynthesis: Regulation and signaling. New Phytol 146:359–388. 57. Hashemi A, Abdolzadeh A, Sadeghipour HR. 2010. Beneficial effects of silicon nutrition in alleviating salinity stress in hydroponically grown canola, Brassica napus L., plants. Soil Sci Plant Nutr 56:244– 253.
NANO-SILICON DIOXIDE MITIGATES THE ADVERSE EFFECTS OF SALT STRESS ON CUCURBITA PEPO L. MANZER H. SIDDIQUI, MOHAMED H. AL-WHAIBI, MOHAMMAD FAISAL, and ABDULAZIZ A. AL SAHLI Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia (Submitted 26 March 2014; Returned for Revision 28 April 2014; Accepted 22 July 2014) Abstract: Research into nanotechnology, an emerging science, has advanced in almost all fields of technology. The aim of the present
study was to evaluate the role of nano-silicon dioxide (nano-SiO2) in plant resistance to salt stress through improvement of the antioxidant system of squash (Cucurbita pepo L. cv. white bush marrow). Seeds treated with NaCl showed reduced germination percentage, vigor, length, and fresh and dry weights of the roots and shoots. However, nano-SiO2 improved seed germination and growth characteristics by reducing malondialdehyde and hydrogen peroxide levels as well as electrolyte leakage. In addition, application of nano-SiO2 reduced chlorophyll degradation and enhanced the net photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate, and water use efficiency. The increase in plant germination and growth characteristics through application of nano-SiO2 might reflect a reduction in oxidative damage as a result of the expression of antioxidant enzymes, such as catalase, peroxidase, superoxide dismutase, glutathione reductase, and ascorbate peroxidase. These results indicate that nano-SiO2 may improve defense mechanisms of plants against salt stress toxicity by augmenting the Pn, gs, transpiration rate, water use efficiency, total chlorophyll, proline, and carbonic anhydrase activity in the leaves of plants. Environ Toxicol Chem 2014;33:2429–2437. # 2014 SETAC Keywords: Stress response
Nanoparticles
Plant toxicology
Cucurbita pepo
Soil toxicology
Increasing salinization limits crop productivity in many areas of the world, particularly in semiarid and arid regions. Salinity impairs a number of metabolic and physiological functions in plants, such as water absorption, ion homeostasis, respiration, osmotic balance, and protein and nucleic acid synthesis [15–18]. Salt stress inhibits seed germination and root and shoot growth by limiting the water potential of soil solution, which greatly reduces cell membrane permeability and influx of water to the plant [19,20]. As noted by Almodares et al. [21], the mechanisms involved in cellular damage in plants under high salt conditions during germination are not fully understood. It has recently become extremely important to develop novel strategies to improve the salinity tolerance of plants in semiarid and arid regions. The regulatory effect of silicon on plant growth and development under stress conditions is well documented [22–24]. Therefore, it is important to determine the potential role of nano-SiO2 in plant tolerance to abiotic stress through modulation of the physio-biochemical function of plants. Squash (Cucurbita pepo L.) is an important vegetable that serves as a rich source of protein, carbohydrates, and vitamins [25]. It is important to investigate the potential role of nano-SiO2 in plant tolerance to salt stress and to characterize the ameliorating effect of nano-SiO2 on seed germination, proline content, and antioxidant enzyme activity in squash plants under high salinity.
INTRODUCTION
Nanotechnology has been globally accepted as a modern, advanced technology that could enhance research in many fields [1–3]. The increasing numbers of nanotechnological inventions (nano-devices and materials) have led to novel applications in the fields of biotechnology and agriculture [4]. Nanoparticles have received much attention because of their unique physicochemical properties compared with bulk particles [5]. Nanoparticles act as chemical delivery agents that target molecules to specific cellular organelles in plants [6]. Silica nanoparticles (nano-SiO2) have been used to deliver DNA and chemicals into plant and animal cells and tissues [7]. These nanoparticles are highly hydrophilic, with the potential for surface modifications [8]. Nanobiopesticides made from nanoSiO2 provide a shielding layer to protect pesticides from degradation by ultraviolet light [9]. In addition, nano-SiO2 is used to produce effective fertilizers for crops and to minimize the loss of fertilizer through slow and controlled release, allowing for regulated, responsive, and timely delivery [2,10]. Previous studies have shown that nano-SiO2 significantly enhances seed germination and seedling root growth [11,12]. Silicon is an important element that plays a key role in a number of metabolic and physiological activities in plants [11]. However, few studies have focused on the physiological role of nano-SiO2 in plants. Li et al. [13] showed that foliar application of nano-SiO2 on Indocalamus barbatus improved the contents of soluble protein, free amino acids, and total nitrogen, phosphorus, and potassium; induced activity of the antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD); and suppressed lipid peroxidation. In addition, exogenous application of nano-SiO2 increased the photosynthetic capacity of plants by amplifying gas exchange and chlorophyll fluorescence parameters [14].
MATERIALS AND METHODS
Seed preparation
The present experiment was conducted under laboratory conditions using squash (C. pepo L. cv. white bush marrow) purchased from a local market in Riyadh, Saudi Arabia. Healthy squash seeds were selected, immersed in a 10% sodium hypochlorite solution for 10 min, vigorously rinsed with sterilized double-distilled water, and transferred to a 12-cm Petri dish.
* Address correspondence to [email protected] Published online 26 July 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.2697 2429
2430
Environ Toxicol Chem 33, 2014
Characterization and preparation of nanoparticle suspension for treatment
The SiO2 nanoparticles were purchased from Evonik Industries. The hydrophilic fumed silica (SiO2) commercially known as Aerosil 200 (Evonik Industries) was used, with an average primary particle size of 12 nm and a corresponding surface area of 200 m2/g. The shape and size of the SiO2 nanoparticles were determined by transmission electron microscopy (JEM-2100F; JEOL). Figure 1 shows a typical image of SiO2 nanoparticles; most of the particles are spherical in shape, with smooth surfaces. The average diameter of SiO2 nanoparticles calculated by transmission electron microscopy was approximately 10 nm. A dry SiO2 powder was suspended in deionized water to make the following concentrations: 1.5 g L1, 3.0 g L1, 4.5 g L1, 6.0 g L1, and 7.5 g L1. These solutions were sonicated for 15 min in a probe-type sonicator. Seed treatment and germination
Sterilized healthy seeds were transferred onto 2 sheets of sterilized filter paper in 12-cm Petri dishes. Fifty seeds were placed into each dish. The dishes were arranged in a simple randomized design with a single factor and 5 replicates. The nano-SiO2 and NaCl treatments were applied in the following manner (subscript numbers describe the concentrations in g L1 for nano-SiO2 and in mM for NaCl): 1) SiO0 þ NaCl0 (control), 2) SiO0 þ NaCl120, 3) SiO1.5 þ NaCl120, 4) SiO3.0þ NaCl120, 5) SiO4.5 þ NaCl120, 6) SiO6.0 þ NaCl120, and 7) SiO7.5þ NaCl120. Next, the dishes were sealed with paraffin tape and incubated in the dark at 28 3 8C. The number of germinated seeds was counted every 2 d. After 10 d, seedlings were transferred onto sterile filters in new sterile dishes containing the same concentrations and volumes as the treatments given above. After 15 d, the performance of C. pepo was assessed in terms of root length, shoot length, root fresh weight, shoot fresh weight, root dry weight, shoot dry weight, net photosynthetic rate (Pn), stomatal conductance (gs), chlorophyll content, chlorophyll degradation, electrolyte leakage, malondialdehyde (MDA) content, proline content, and activities of carbonic anhydrase (CA), catalase (CAT), POD, SOD, glutathione reductase (GR), and ascorbate peroxidase (APX). Determination of growth characteristics
The percentage of seed germination was recorded every 2 d for days 4 to 15. The vigor index was calculated by using the
M.H. Siddiqui et al.
following equation [26]: vigor index ¼ (mean root length þ mean shoot length) percentage of germination. After 15 d, the shoot and root lengths were measured using a meter scale. After recording of root and shoot fresh weights, the samples were placed in an oven at 60 8C for 48 h to obtain the dry weight of the roots and shoots. Determination of physio-biochemical characteristics Total chlorophyll concentration. The youngest fully expanded leaves were subjected to extraction using 80% acetone, and the absorbance was measured spectrophotometrically (SPEKOL 1500; Analytik Jena) at 663 nm and 645 nm. The total chlorophyll content was determined by using Arnon’s formula [27]. Proline concentration. The proline concentration was determined spectrophotometrically using the ninhydrin method of Bates et al. [28]. First, we homogenized 300 mg of fresh leaf specimens in 3% sulfosalicylic acid, followed by the addition of 2 mL each of ninhydrin and glacial acetic acid, after which the samples were heated to 100 8C. The mixture was then extracted with toluene, and the free toluene was quantified at 520 nm. MDA concentration. The MDA content was determined according to the method of Heath and Packer [29]. Leaves were weighed, and homogenates containing 10% trichloroacetic acid and 0.65% 2-thiobarbituric acid were heated at 95 8C for 60 min, then cooled to room temperature, and centrifuged at 10 000 g for 10 min. The absorbance of the supernatant was read at 532 nm and 600 nm against a reagent blank. Electrolyte leakage. Electrolyte leakage was used to assess membrane permeability in accordance with Lutts et al. [30]. Samples were washed 3 times with double-distilled water to remove surface contamination, and leaf discs were cut from young leaves and placed in sealed vials containing 10 mL of double-distilled water, followed by incubation on a rotary shaker for 24 h, after which the electrical conductivity of the solution (EC1) was determined. Then the samples were autoclaved at 120 8C for 20 min, and the electrical conductivity was measured again (EC2) after the solution was cooled to room temperature. The electrolyte leakage was defined as EC1/EC2 100 and expressed as percentage. Histochemical detection of hydrogen peroxide (H2O2) localization. The pattern of H2O2 localization in the plant tissues was
determined in hand-cut cross sections stained with a KI/starch reagent containing 4% (w/v) starch and 0.10 M KI [31]. The presence of H2O2 was indicated by a dark color. The stained sections were observed and photographed using a B071 Olympus microscope. Gas exchange parameters
Figure 1. Transmission electron microscopy image of silicon dioxide nanoparticles.
Gas exchange parameters, such as Pn, gs, transpiration rate, and water use efficiency, were recorded using an open system infrared gas analyzer (CID Bio-Science). The youngest healthy expanded leaf of each squash seedling was selected for the measurement of gas exchange parameters. The water use efficiency was quantified as the Pn divided by the transpiration rate. CA activity. The activity of CA (EC 4.2.1.1) was determined using the method of Dwivedi and Randhawa [32]. Leaf samples were cut into small pieces, extracted in cysteine hydrochloride solution, and incubated at 40 8C for 20 min. The pieces were then blotted and transferred to test tubes containing phosphate buffer (pH 6.8), followed by the addition of alkaline bicarbonate solution and bromothymol blue indicator. The test tubes were incubated at 50 8C for 20 min. After the addition of methyl red
Nano-SiO2 mitigates the adverse effects of salt stress
indicator, the reaction solution was titrated against 0.05 N HCl. The results were expressed as mmol CO2 kg1 fresh weight s1. Antioxidant enzyme activity. To determine the activities of antioxidant enzymes, a crude enzyme extract was prepared by homogenizing 500 mg of leaf tissue in extraction buffer (0.5% Triton X-100 and 1% polyvinylpyrrolidone in 100 mM potassium phosphate buffer, pH 7.0) using a chilled mortar and pestle. The homogenate was then centrifuged at 15 000 g for 20 min at 4 8C, and the supernatant was used for the enzymatic assays described below. For the APX assay, the extraction buffer was supplemented with 2 mM ascorbate. All enzyme activities were expressed as milligram of protein per minute. We applied the method of Chance and Maehly [33] to determine POD (EC 1.11.1.7) activity using 5 mL of enzyme reaction solution containing phosphate buffer (pH 6.8), 50 M pyrogallol, 50 mM H2O2, and 1 mL of the enzyme extract diluted 20 times. The assay mixture was incubated for 5 min at 25 8C, and the reaction was terminated by the addition of 0.5 mL of 5% (v/v) H2SO4. Purpurogallin production was measured spectrophotometrically at 420 nm. One unit of POD activity was considered the amount of purpurogallin formed per milligram of protein per minute. The method of Aebi [34] was used to measure CAT (EC 1.11.1.6) activity. The decomposition of H2O2 was measured as the decrease in absorbance at 240 nm. In this assay, 50 mM phosphate buffer (pH 7.8) and 10 mM H2O2 were used in the reaction solution. Activity of SOD (EC 1.15.1.1) was determined based on the inhibition of nitro blue tetrazolium (NBT) photoreduction according to the method of Giannopolitis and Ries [35]. The reaction solution (3 mL) contained 50 mM NBT, 1.3 mM riboflavin, 13 mM methionine, 75 nM ethylenediamine tetraacetic acid (EDTA), 50 mM phosphate buffer (pH 7.8), and 20 mL to 50 mL of enzyme extract. The reaction solution was irradiated under fluorescent light at 75 mM m2 s1 for 15 min. The absorbance at 560 nm was read against a blank (nonirradiated reaction solution). One unit of SOD activity was defined as the amount of enzyme that inhibited 50% of NBT photoreduction. We measured APX (EC 1.11.1.11) activity using the method of Nakano and Asada [36]. The reaction buffer solution contained 50 mM potassium phosphate (pH 7.0), 0.1 mM EDTA, 0.1 mM H2O2, and 0.5 mM ascorbate. The reaction was initiated by the addition of the sample solution to the reaction mixture. The H2O2-dependent oxidation of ascorbate was measured as the decrease in absorbance at 290 nm.
Environ Toxicol Chem 33, 2014
2431
Figure 2. Ameliorating effect of nano-silicon dioxide (nSiO) on (A) seed germination and (B) vigor in squash seedlings. Bars with the same letter are not significantly different at p < 0.05%. S ¼ NaCl.
The ameliorating effect of nano-SiO2 was evaluated on the basis of the growth and physio-biochemical characteristics of C. pepo L. cv. white bush marrow under NaCl stress.
However, when seeds were treated with different concentrations of nano-SiO2, significant differences (p < 0.05) were observed. The application of nano-SiO2 significantly increased the germination percentage and vigor index under salt stress conditions. Application of 6 g L1 of nano-SiO2 increased the germination percentage and vigor index by 77.95% and 285.21%, respectively, compared with the salinity treatment. However, application of 7.5 g L1 and 4.5 g L1 of nano-SiO2 resulted in equal effects on germination percentage. The concentration of 6 g L1 of nano-SiO2 proved to be the best at alleviating salt stress. Similarly, the seedlings exposed to NaCl exhibited reduced root and shoot lengths, as well as reduced root and shoot fresh and dry weights, compared with the control (Figure 3A–C). However, coexposure to NaCl and nano-SiO2 improved root and shoot length, as well as root and shoot fresh and dry weights. Application of 6 g L1 of nano-SiO2 increased root length by 121.99%, shoot length by 105.11%, root fresh weight by 328.72%, shoot fresh weight by 133.98%, root dry weight by 343.95%, and shoot dry weight by 152.87% over the respective NaCl treatments. Application of 7.5 g L1 and 4.5 g L1 of nanoSiO2 showed equal effects on both shoot fresh weight and root dry weight.
Nano-SiO2 effects on morphological characteristics
Effect of nano-SiO2 on physiological and biochemical parameters
In the present study, we observed that salt stress inhibited the seed germination percentage and vigor index (Figure 2A and B).
The MDA content indicated the level of lipid peroxidation, whereas oxidative damage and membrane alterations were
Statistical analysis
Each Petri dish was treated as 1 replicate, and all treatments were repeated 5 times. The data were expressed as the mean standard error and were analyzed statistically using SPSS Ver 17 statistical software. The means were compared statistically using Duncan’s multiple-range test at the level of p < 0.05. RESULTS
2432
Environ Toxicol Chem 33, 2014
M.H. Siddiqui et al.
Figure 3. Ameliorating effect of nano-silicon dioxide (nSiO) on the (A) root and shoot length, (B) root and shoot fresh weight and (C) root and shoot dry weight of squash seedlings. Bars with the same letter are not significantly different at p < 0.05%. S ¼ NaCl.
assessed by electrolyte leakage (Table 1). Salinity had a damaging effect, as revealed by increased MDA levels and electrolyte leakage. However, the addition of nano-SiO2 significantly suppressed lipid peroxidation and electrolyte leakage. Also, all 5 tested concentrations of nano-SiO2 were found to be effective in alleviating the damaging effect of salt stress. The concentration of 6 g L1 of nano-SiO2 reduced the MDA content by 52.23% and electrolyte leakage by 51.87% compared with the NaCl treatment. Histochemical analysis showed greater H2O2 production in the NaCl-treated seedlings than in the control and seedlings treated with nano-SiO2-treated (Figure 4A–C). In the NaCl-treated seedlings, H2O2 was
detected in the vascular bundle and phloem fibers. The intensity of H2O2 production in the xylem and phloem of the vascular bundle and root was high in NaCl-exposed seedlings compared with the control and nano-SiO2-treated seedlings. Application of NaCl improved the proline content (Table 1). However, the proline concentration increased further with increasing levels of nano-SiO2 under salt stress. Application of 6 g L1 of nano-SiO2 increased proline synthesis by 292.93% and 88.35% over the control and NaCl treatment, respectively. Figure 5A–D shows that NaCl treatment significantly decreased Pn, gs, transpiration rate, and water use efficiency values. However, the addition of nano-SiO2 alleviated the
Table 1. Ameliorating effect of nano-SiO2 on the electrolyte leakage (% of total electrolyte leakage) and content of MDA and proline in squash plants Treatmenta Control S S þ nano-SiO1.5 S þ nano-SiO3 S þ nano-SiO4.5 S þ nano-SiO6 S þ nano-SiO7.5
Electrolyte leakage (%)b 15.10 1.04 47.54 1.25 39.10 1.13 32.71 1.45 26.09 0.99 22.88 0.91 28.77 1.71
F A B C D,E E D
MDA content (nmol g1 fresh wt)b 15.11 0.45 49.65 0.88 44.43 0.49 36.42 0.49 31.40 0.42 23.72 0.75 26.32 0.19
Subscript numbers describe nano-SiO concentrations (in g L1). Data followed by the same letters are not significantly different at p < 0.05%. MDA ¼ malondialdehyde; Pro ¼ proline; S¼ salinity; nano-SiO ¼ nano silicon dioxide.
a
b
G A B C D F E
Proline content (mg1 fresh wt)b 10.33 0.62 21.55 0.71 25.43 0.49 27.36 0.84 33.41 0.62 40.59 0.42 36.85 0.71
F E D E C A B
Nano-SiO2 mitigates the adverse effects of salt stress
Environ Toxicol Chem 33, 2014
2433
Figure 4. Histochemical analysis of H2O2 production through starch–KI staining of squash seedling cross sections. (A) Section of control seedling (4 magnification); (B) section of NaCl-treated seedling (4 magnification); (C) Section of NaCl þ nano-SiO6.0-treated seedling (4 magnification). VB ¼ vascular bundle; nano-SiO ¼ nano silicon dioxide. [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]
inhibitory effects of NaCl stress. Application of 6 g L1 of nanoSiO2 proved to be the best at alleviating the adverse effect of NaCl stress. The control and plants treated with 4.5 g L1 nanoSiO2 exhibited statistically equal values for Pn, and gs, as did plants treated with 3 g L1, 4.5 g L1, 6 g L1, or 7.5 g L1 nanoSiO2 for water use efficiency. The control and plants treated with 3 g L1, 4.5 g L1, 6 g L1, and 7.5 g L1 nano-SiO2 showed statistically similar values for the transpiration rate. The data presented in Figure 6A and B reveal that the application of NaCl and nano-SiO2 significantly influenced the total chlorophyll concentration and chlorophyll degradation. Exposure to NaCl caused a decrease in total chlorophyll content and an increase in chlorophyll degradation, whereas application of nano-SiO2 with NaCl improved the total chlorophyll content and reduced chlorophyll degradation compared with NaCltreated seedlings. Application of 6 g L1 of nano-SiO2 increased the total chlorophyll content by 345%, and reduced chlorophyll degradation by 45.13% compared with the NaCl treatment. However, the effects of 6 g L1 of nano-SiO2 were similar to the control treatment for total chlorophyll synthesis. The activity of CA relative to the control was significantly inhibited by the application of NaCl (Figure 6C). However, the addition of nano-SiO2 significantly improved CA activity compared with NaCl treatment. Exposure to nano-SiO2 caused a gradual and concentration-dependent increase in CA activity. Application of 6 g L1 of nano-SiO2 improved CA activity by 26.62% compared with NaCl treatment. Antioxidant enzyme activity (CAT, POD, SOD, GR, and APX) was significantly influenced under all nano-SiO2 concentrations tested (Figure 7). Application of NaCl significantly enhanced the activities of CAT, POD, SOD, GR, and APX, but the increase was even more prominent when nanoSiO2 was added. Application of 6 g L1 of nano-SiO2 relative to the NaCl treatment significantly increased the activities of CAT, POD, SOD, GR, and APX by 26.62%, 40.29%, 39.88%, 65.67%, and 105.51%, respectively. However, the effect of
6 g L1 of nano-SiO2 was equal to that of 7.5 g L1 of nano-SiO2 for POD activity. DISCUSSION
Seed germination is crucial for plant growth and development. In the present study, the application of NaCl significantly inhibited seed germination. These results strongly support the findings of Siddiqui et al. [12] in tomato, Almodares et al. [21] in sorghum, Nyagah and Musyimi [37] in Passiflora edulis, Tobe et al. [38] in Haloxylon ammodendron, Demir et al. [39] in Solanum melongena, and Khajeh-Hosseini et al. [40] in soybean. The inhibitory effect of NaCl might be the result of an osmotic barrier that reduces the ability of the seed to imbibe water, which could result in ion toxicity in the seed embryo [21]. Similarly, in the present study, salinity was observed to reduce the germination rate and vigor index (Figure 2A and B). However, interestingly, application of nano-SiO2 with NaCl significantly enhanced seed germination and vigor index (Figure 2A and B). Haghighi et al. [41] reported similar results. An increase in these attributes with application of nano-SiO2 might reflect the activities of protease and lipase; silicon also improves seedling respiration rates and seed vigor [42]. According to Epstein [43], silicon maintains ion homeostasis and alleviates the toxicity of metals and salt stress. Therefore, the results of the present study suggest that nano-SiO2 provided a suitable environment for the germination of seeds under salinity conditions. The data presented in Figure 3A–C demonstrate a significant effect of nano-SiO2 and NaCl on the root and shoot dry weights, fresh weights, and lengths. The plants generated from seeds treated with NaCl showed reduced growth parameters. Similar results have been observed in many other crops [17,18,44,45]. In the present study, however, nano-SiO2 improved growth characteristics of the squash plants by alleviating the adverse effect of salinity (Figure 3). This enhancement might reflect the physiological roles of nano-SiO2, which is required for
2434
Environ Toxicol Chem 33, 2014
M.H. Siddiqui et al.
Figure 5. Ameliorating effect of nano-silicon dioxide (nSiO) on the (A) net photosynthetic rate (Pn), (B) stomatal conductance (gs), (C) water use efficiency (WUE), and (D) transpiration rate (E) of squash seedlings. Bars with the same letter are not significantly different at p < 0.05%. S ¼ NaCl.
photosynthesis and nitrate assimilation [42,46]. In addition, nano-SiO2 has been implicated in the synthesis of proteins and amino acids and the uptake of nutrients [13], and the strength and rigidity of plants is improved through nano-SiO2 deposition in plant tissues [24]. It was also shown that nano-SiO2 enhanced the growth and physiological characteristics of plants through increasing the production of organic compounds such as proteins, chlorophyll, and phenols, relative to bulk particles [47]. The increase in plant growth characteristics because of nano-SiO2 application might reflect the reduced uptake of Naþ ions and sustained membrane integrity in root cells, as demonstrated by the enhancement of antioxidant systems and reduced oxidative damage. Lipid peroxidation, electrolyte leakage, and H2O2 levels are key parameters that indicate the level of oxidative damage in plants under unfavorable environmental conditions [12]. The increase in electrolyte leakage, MDA, and H2O2 observed in the NaCl-treated seedlings (Table 1 and Figure 4) indicates that NaCl causes cellular dysfunction by enhancing lipid peroxidation through the generation of free radicals [48,49]. However, the seedlings raised from seeds treated with nano-SiO2 in the present study exhibited reduced electrolyte leakage, lipid
peroxidation, and H2O2 levels. These effects might reflect increased activity of antioxidant enzymes (CAT, POD, SOD, GR, and APX; Figure 7A and B). In addition, nano-SiO2 mediates the synthesis of protein, amino acids, and phenols, as well as nutrient uptake [13]. According to Epstein [43] “silicon plays an astonishingly large number of diverse roles in plants, and does so primarily when the plants are under stressful conditions.” Thus, we postulate that the application of nanoSiO2 improves plant tolerance to salt stress. Under stress condition, plants synthesize compatible solutes (glycine betaine and proline) to adjust the osmotic potential within cells. In the present study, the proline content increased relative to that of the control when NaCl was applied (Table 1). This result is consistent with that of previous studies showing increases in proline content in response to salt stress [44,50]. The concentration of proline further increased with the application of nano-SiO2. Proline, a universal osmoprotectant, acts as both an antioxidant and a source of energy [51], and regulates gene expression, leading to osmotic adjustment [52]. Hyperaccumulation of proline increases water uptake by increasing osmotic pressure [50], resulting in enhanced tolerance to salt stress in plants.
Nano-SiO2 mitigates the adverse effects of salt stress
Environ Toxicol Chem 33, 2014
2435
Figure 6. Ameliorating effect of nano-silicon dioxide (nSiO) on the (A) total chlorophyll content, (B) chlorophyll degradation, and (C) carbonic anhydrase activity in squash seedlings. Bars with the same letter are not significantly different at p < 0.05%. S ¼ NaCl.
Photosynthesis is an important process that allows for growth and dry matter production in plants. Under NaCl stress, the observed inhibition of net Pn, gs, transpiration rate, and water use efficiency compared with the respective controls (Figure 5) might reflect the excessive accumulation of Naþ, and Cl, which inhibits stomatal closure, leading to a reduction in internal CO2 availability [52]. A similar result was shown by Shi et al. [53]. Seedlings treated with NaCl showed reduced total chlorophyll contents and CA activity and increased chlorophyll degradation relative to the respective controls (Figure 6). The reduction in total chlorophyll might reflect lipid peroxidation and H2O2 formation in plant cells, which are both significantly affected by NaCl, sodium ion accumulation in chloroplasts, and expression of the chlorophyll-degrading enzyme chlorophyllase [54,55]; thus we observed marked chlorophyll degradation (Figure 6B). The results of the present study showed that application of nanoSiO2 restored the changes in Pn and photosynthetic pigments observed in the seedlings through the inhibition of chlorophyll degradation and the expression of CA enzyme activity (Figure 6). Xie et al. [46] reported that exogenous application of nanoSiO2 improved the photosynthetic activity of mesophyll cells in Indocalamus barbatus. According to Shi et al. [53], the application of silicon improves Pn, gs, transpiration rate, and water use efficiency in rice (Oryza sativa) under salt stress. The degree of alleviation of salt stress and the improvement in the photosynthetic capacity of squash seedlings through the application of nano-SiO2 could be associated with the beneficial
effects of nano-SiO2 on seed germination potential and plant growth (Figures 2 and 3), reflecting the inhibition of photosynthetic pigment degradation and improvement in enzyme activities (Figures 6C and 7). The enhanced CA activity in the seedlings treated with nano-SiO2 might influence the reversible hydration of CO2 and maintain a constant supply of CO2 to RuBisCO to improve chlorophyll synthesis, thereby increasing Pn, gs, and water use efficiency [53]. Reactive oxygen species act as signaling molecules in plant cells and impair macromolecules through interactions with DNA, proteins, and carbohydrates [12,15,56]. Under stress conditions, plants develop a defense mechanism through which they can survive by limiting and detoxifying reactive oxygen species. In the present study, the observed electrolyte leakage, lipid peroxidation, and proline levels in plant tissues were substantially higher in seedlings treated with NaCl (Table 1). However, the application of nano-SiO2 alleviated these negative effects on plant growth by increasing the activity of antioxidant enzymes such as CAT, POD, SOD, GR, and APX (Figure 7A and B). These findings substantiate the results of Li et al. [13], who showed that nano-SiO2 stimulates antioxidant enzyme activity. In addition, Hashemi et al. [57] observed that silicon nutrition reduced the inhibitory effect of salinity on plant growth by reducing the Naþ content, increasing CAT and cell wall peroxidase activities, and maintaining the membrane integrity of root cells, as demonstrated by reduced lipid peroxidation. In the present study, the seedlings were able to maintain steady-state
2436
Environ Toxicol Chem 33, 2014
M.H. Siddiqui et al. Acknowledgment—The present study was supported financially through funding from the Deanship of Scientific Research of King Saud University, Riyadh, Saudi Arabia to Research Group RGPVPP-153. The authors declare that they have no conflicts of interest. REFERENCES
Figure 7. Ameliorating effect of nano-silicon dioxide (nSiO) on the activities of (A) catalase (CAT) and peroxidase (POD) and (B) superoxide dismutase (SOD), glutathione reductase (GR), and ascorbate peroxidase (APX) in squash seedlings. Bars with the same letter are not significantly different at p < 0.05%. S ¼ NaCl.
levels of O2, and H2O2 might have resulted from enhancement of antioxidant enzyme activity in plant cells through the application of nano-SiO2. CONCLUSIONS
Squash seedlings treated with NaCl and nano-SiO2 showed significant changes in growth and physiological characteristics. When seedlings were exposed to salt stress in the present study, the percentage of seed germination, vigor, growth, and physiobiochemical characteristics were decreased, and MDA and H2O2 levels and electrolyte leakage increased. However, nano-SiO2 significantly reduced the adverse effects of salinity by mitigating cellular oxidative damage through the enhancement of free radical scavenging antioxidant enzymes. The effect of nano-SiO2 was dose dependent. The improvements we found in growth parameters, photosynthetic pigments, proline content, and CA activity might underlie the enhanced tolerance of plants to salinity after treatment with nano-SiO. There is insufficient information regarding the positive effects of nano-SiO2 on plant growth and the physiological mechanisms that arise in higher plants under salt stress, but the findings of the present study provide a new platform for exploring the involvement of nanoSiO2 in physiological, biochemical, and molecular activities in plants.
1. Gogos A, Knauer K, Bucheli TD. 2012. Nanomaterials in plant protection and fertilization: Current state, foreseen applications, and research priorities. J Agric Food Chem 60:9781–9792. 2. Nair R, Varghese SH, Nair BG, Maekawa T, Yoshida Y, Kumar DS. 2010. Nanoparticulate material delivery to plants. Plant Sci 179:154– 163. 3. González-Melendi P, Fernández-Pacheco R, Coronado MJ, Corredor E, Testillano PS, Risue~ no M, Marquina C, Ibarra MR, Rubiales D, Perezde-Luque A. 2008. Nanoparticles as smart treatment-delivery systems in plants: Assessment of different techniques of microscopy for their visualization in plant tissues. Ann Bot 101:187–195. 4. Scrinis G, Lyons K. 2007. The emerging nano-corporate paradigm: Nanotechnology and the transformation of nature, food and agri-food systems. Int J Sociol Food Agri 15:22–44. 5. Monica RC, Cremonini R. 2009. Nanoparticles and higher plants. Caryologia 62:161–165. 6. Liu J, He S, Zhang Z, Cao J, Lv P, He S, Cheng G, Joyce DC. 2009. Nano-silver pulse treatments inhibit stem-end bacteria on cut gerbera cv. Ruikou flowers. Postharvest Biol Technol 54:59–62. 7. Torney F, Trewyn BG, Lin VSY, Wang K. 2007. Mesoporous silica nanoparticles deliver DNA and chemicals into plants. Nat Nanotechnol 2:295–300. 8. Nakamura M, Shono M, Ishimura K. 2007. Synthesis, characterization, and biological applications of multifluorescent silica nanoparticles. Anal Chem 79:6507–6514. 9. Chen S, Sun L, Sun T, Chao L, Guo G. 2007. Interaction between cadmium, lead and potassium fertilizer (K2SO4) in a soil-plant system. Environ Geochem Health 29:435–446. 10. Zhang F, Wang R, Xiao Q, Wang Y, Zhang J. 2006. Effects of slow/ controlled-release fertilizer cemented and coated by nano-materials on biology. II. Effects of slow/controlled-release fertilizer cemented and coated by nano-materials on plants. Nanoscience 11:18–26. 11. Bao-Shan L, Shao-qi D, Chun-hui L, Li-jun F, Shu-chun Q, Min Y. 2004. Effect of TMS (nanostructured silicon dioxide) on growth of Changbai larch seedlings. J Forest Res 15:138–140. 12. Siddiqui MH, Al-Whaibi MH, Sakran AM, Ali HM, Basalah MO, Faisal M, Alatar A, Al-Amri AA. 2013. Calcium-induced amelioration of boron toxicity in radish. J Plant Growth Regul 32:61–71. 13. Li B, Tao G, Xie Y, Cai X. 2012. Physiological effects under the condition of spraying nano-SiO2 onto the Indocalamus barbatus McClure leaves. J Nanjing Forest Uni (Natural Sciences Edition) 4:161–164. 14. Yinfeng X, Bo L, Qianqian Z, Chunxia Z, Kouping L, Gongsheng T. 2011. Effects of nano-TiO2 on photosynthetic characteristics of Indocalamus barbatus. J Northeast Forest Uni 39:22–25. 15. Maathuis FJM. 2006. The role of monovalent cation transporters in plant responses to salinity. J Exp Bot 57:1137–1147. 16. Zeng J, Zeng FJ, Arndt SK, Guo HF, Yan HL, Xing WJ, Liu B. 2008. Growth, physiological characteristics and ion distribution of NaCl stressed Alhagi sparsifolia seedlings. Chin Sci Bull 53:169–176. 17. Siddiqui MH, Mohammad F, Khan MMA, Al-Whaibi MH. 2012. Cumulative effect of nitrogen and sulphur on Brassica juncea L. genotypes under NaCl stress. Protoplasma 249:139–153. 18. Siddiqui MH, Al-Whaibi MH, Sakran AM, Basalah MO, Ali HM. 2012. Effect of calcium and potassium on antioxidant system of Vicia fabe L. under cadmium stress. Intt J Mol Sci 13:6604–6619. 19. Malhotra K, Chhabra GK, Jain R, Sharma V, Kumar S. 2013. Drought and salinity tolerant biofuel crops for the Thar Desert. In Tuteja N, Gill S, eds. Plant Acclimation to Environmental Stress. Springer, New York, NY, USA, pp 71–84. 20. Jamil M, Lee DB, Jung KY, Ashraf M, Lee SC, Rha ES. 2006. Effect of salt (NaCl) stress on germination and early seedling growth of four vegetable species. J Cent Eur Agri 7:273–282. 21. Almodares A, Hadi MR, Dosti B. 2007. Effects of salt stress on germination percentage and seedling growth in sweet sorghum cultivars. J Biol Sci 7:1492–1495. 22. Matichenkov VV, Kosobrukhov AA., 2004. Silicon effect on the plant resistance to salt toxicity. Conserving soil and water for society. Proceedings, 13th International Soil Conservation Organization Conference, Brisbane, Australia, July 4-8, 2004, pp 626.
Nano-SiO2 mitigates the adverse effects of salt stress 23. Janislampi KW. 2012. Effect of silicon on plant growth and drought stress tolerance. PhD thesis. Utah State University, Logan, UT, USA. [cited 2013 August 5]. Available from: http://digitalcommons.usu.edu/ etd/1360 24. Ma JF, Yamaji N. 2006. Silicon uptake and accumulation in higher plants. Trends Plant Sci 11:342–397. 25. Vural H, ESs iyok D, Duman I. 2000. Kültür Sebzeleri (Sebze YetiSs tirme). Egean University Press, Bornova, Ïzmir, Turkey (in Turkish). 26. Tao KL, Zheng GH. 1990. Seed Vigor. Science Press, Beijing, China (in Chinese). 27. Arnon DI. 1949. Copper enzymes in isolated chloroplast. Polyphenoloxidases in Beta vulgaris. Plant Physiol 24:1–15. 28. Bates LS, Waldren RP, Teare ID. 1973. Rapid determination of free proline for water-stress studies. Plant Soil 39:205–207. 29. Heath RL, Packer L. 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:189–198. 30. Lutts S, Kinet JM, Bouharmont J. 1995. Changes in plant response to NaCl during development of rice (Oryza sativa L.) varieties differing in salinity resistance. J Exp Bot 46:1843–1852. 31. Olson PD, Varner JE. 1993. Hydrogen peroxide and lignification. Plant J 4:887–892. 32. Dwivedi RS, Randhawa NS. Evaluation of rapid test for hidden hunger of zinc in plants. Plant Soil 40:45–451. 33. Chance B, Maehly AC. 1955. Assay of catalase and peroxidases. Methods Enzymol 2:764–775. 34. Aebi H. 1984. Catalase in vitro. Methods Enzymol 105:121–126. 35. Giannopolitis CN, Ries SK. 1977. Superoxide dismutases: I. Occurrence in higher plants. Plant Physiol 59:309–314. 36. Nakano G, Asada K. 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880. 37. Nyagah AW, Musyimi DM. 2009. Effects of sodium chloride solution stress on germination and growth of passion fruits seedlings. ARPN J Agri Biol Sci 4:49–53. 38. Tobe K, Li X, Omasa K. 2004. Effects of five different salts on seed germination and seedling growth of Haloxylon ammodendron (Chenopodiaceae). Seed Sci Res 14:345–354. 39. Demir I, Mavi K, ozcoban M, Okcu G. 2003. Effect of salt stress on germination and seedling growth in serially harvested aubergine (Solanum melongena L.) seeds during development. Israel J Plant Sci 51:125–131. 40. Khajeh-Hosseini M, Powell AA, Bingham IJ. 2003. The interaction between salinity stress and seed vigour during germination of soyabean seeds. Seed Sci Technol 31:715–725. 41. Haghighi M, Afifipour Z, Mozafarian M. 2012. The effect of N-Si on tomato seed germination under salinity levels. J Biol Environ Sci 6:87–90.
̌
Environ Toxicol Chem 33, 2014
2437
42. Qingfang L, Chengcang M. 2002. Effect of available silicon in soil on cucumber seed germination and seedling growth metabolism. Acta Horticul Sinica 29:433–437. 43. Epstein E. 2009. Silicon: Its manifold roles in plants. Ann Appl Biol 155:155–160. 44. Siddiqui MH, Mohammad F, Khan MN. 2009. Morphological and physio-biochemical characterization of Brassica juncea L. Czern. & Coss. genotypes under salt stress. J Plant Interact 4:67–80. 45. Siddiqui MH, Khan MN, Mohammad F, Khan MMA. 2008. Role of nitrogen and gibberellin (GA3) in the regulation of enzyme activities and in osmoprotectant accumulation in Brassica juncea L. under salt stress. J Agron Crop Sci 194:214–224. 46. Xie Y, Li B, Zhang Q, Zhang C. 2012. Effects of nano-silicon dioxide on photosynthetic fluorescence characteristics of Indocalamus barbatus McClure. J Nanjing Forest Uni (Natural Sciences Edition) 2: 59–63. 47. Suriyaprapha R, Karunakaran G, Yuvakkumar R, Prabu P, Rajendran V, Kannan N. 2012. Growth and physiological responses of maize (Zea mays L.) to porous silica nanoparticles in soil. J Nanopart Res 14: 2–14. 48. EI-Tayeb MA. 2005. Response of barley grains to the interactive effect of salinity and salicylic acid. Plant Growth Regul 45:215–224. 49. Mahajan S, Tuteja N. 2005. Cold, salinity and drought stresses: An overview. Arch Biochem Biophys 444:139–158. 50. Khan MN, Siddiqui MH, Mohammad F, Naeem M. 2012. Interactive role of nitric oxide and calcium chloride in enhancing tolerance to salt stress. Nitric Oxide 27:210–218. 51. Matysik J, Alia Bhalu B, Mohanty P. 2002. Molecular mechanisms of quenching of reactive oxygen species by proline under stress in plants. Curr Sci 82:525–532. 52. Iyer S, Caplan A. 1998. Products of proline catabolism can induce osmotically regulated genes in rice. Plant Physiol 116:203–211. 53. Shi Y, Wang Y, Flowers TJ, Gong H. 2013. Silicon decreases chloride transport in rice (Oryza sativa L.) in saline conditions. J Plant Growth Regul, 170:847–853. 54. Reddy MP, Vora AB. 1986. Salinity induced changes in pigment composition and chlorophyllase activity in wheat. Indian J Plant Physiol 29:331–334. 55. Kyle DJ. 1987. The biochemical basis for photoinhibition of photosystem II. In Kyle DJ, Osmond CB, Artzen CJ, eds Photoinhibition. Elsevier, Amsterdam, The Netherlands, pp 197–226. 56. Foyer CH, Noctor G. 2000. Oxygen processing in photosynthesis: Regulation and signaling. New Phytol 146:359–388. 57. Hashemi A, Abdolzadeh A, Sadeghipour HR. 2010. Beneficial effects of silicon nutrition in alleviating salinity stress in hydroponically grown canola, Brassica napus L., plants. Soil Sci Plant Nutr 56:244– 253.
