Growth and Physiological Responses of Wheat Seedlings to Cadmium Alone and in Combination with SiO2 Nanoparticles

Citation :Egypt. Acad. J. Biolog. Sci. ( C. physiology and Molecular biology ) Vol.7(2)pp25-39(2015) Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Physiology & molecular biology journal is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers that elucidateimportant biological, chemical, or physical mechanisms of broad physiological significance. www.eajbs.eg.net Provided for non-commercial research and education use.


Cadmium toxicities
Wheat seedlings SiO 2 nanoparticles alleviating effects Heavy metals are the major environmental pollutants, mainly in areas with high anthropogenic activities.In this study, the effects of three cadmium (Cd) concentrations (30, 60, and 120 mg - 1 l) alone and in combination with two concentrations of SiO 2 nanoparticles (10 and 100 mg -1 l) on growth and some physiological parameters of wheat (Triticum aestivum) seedlings were investigated.Cadmium treatments decreased the fresh and dry weight of roots, shoots, chlorophyll, carotenoid and total protein contents of the leaf tissues significantly.It also increased the amount of proline, lipid peroxidation and catalase activity of wheat seedling.The toxic effects of cadmium ions on growth and physiological activities of wheat seedlings were reduced in the presence of SiO 2 nanoparticles.their age and developmental stage, pH, specific metals, concentrations and chemical forms (Keller et al. 2003).One of the criteria to evaluate the toxicity of heavy metals on plants growth and development is to treat plants with these metals at the early stages of growth, especially during seed germination and early seedlings growth (Li et al. 2005).
Cadmium is highly soluble in water and it is not an essential element for plants.Its effects on higher plants by several ways: blocking signaling receptors (Beyersmann and Hechtenberg 1997;Monroe and Halvorsen 2006), inhibiting enzymes action e.g.kinase, phosphatase, nitrate reductase, and ATP synthatase.The inhibiting enzymes, involved in photosynthetic CO 2 fixation (Herbette et al. 2006;Sharma and Dubey 2005), inducing oxidative stresses (Schützendübel and Polle 2002), disturb the metabolic activities including leaves chlorosis, growth inhibition, roots tips necrosis, cells membrane permeability, roots ions uptake and transport to shoots (Påhlsson 1989;Sanita di Toppi and Gabbrielli 1999).
According to Arnon and Stout (Arnon and Stout 1939), silicon is not classified as an essential element for all higher plants, but it is one of the beneficial element for number of plants species, that improves their growth and resistance to biotic and abiotic stresses (Ma et al. 2001;Ma 2004;Okuda and Takahashi 1965).In most plants especially monocots, it acts as physic mechanical barrier, by depositing on the walls of epidermal cells, vascular tissues, of the stem, leaf sheath and controlling plants physiological activities (Ma 2004;Reynolds et al. 2009).
In the present study, we evaluated some of the physiological parameters of young wheat seedlings affected by cadmium alone and in combination with SiO 2 nanoparticles.

Plant materials and growth conditions
Seeds of wheat (Triticum aestivum L. var.Chamran) were obtained from Zarghan Agricultural Research Center, Iran.They were surface sterilized by soaking in 5% (w/v) sodium hypochlorite for 10 minutes and washed three times with distilled water then air-dried on filter papers.Seeds were allowed to germinate in the dark at 25°C on moist filter papers.Twenty of five-day old seedlings were transferred into small plastic containers filled with perlite and Hoagland nutrient solution (pH 6.2).Wheat seedlings were grown in growth chamber set at 16 h/8 h light-dark periods.Three replicates were used for each treatment.

Cadmium chloride and SiO 2 nanoparticles treatments
SiO 2 nanoparticles with average sizes of 20 nm and 99.5% purity were purchased from US Research Nanomaterials, lnc.(USA).Using Hoagland nutrient solution as solvent, two concentrations (10 and 100 mg/L) of SiO 2 nanoparticles were prepared.The dissolved particles were dispersed by a high-power probe-type Sonicator (Misonix, Q Sonica LLC, Newton, USA) for 30 minutes.Cadmium was used as CdCl 2 (Sigma-Aldrich).Wheat seedlings (21-day old) were collected one week after the beginning of treatments, washed with double-distilled water and used for analyses.Roots and shoots fresh and dry weight, chlorophyll and carotenoid pigments, catalase activity, lipid peroxidation, proline and leaves total protein contents were the analyzed parameters.

Seedlings fresh and dry weight
After washing with distilled water, wheat seedlings were blotted dry on tissue papers, their fresh weights, were measured and dried at 70 C for 48 h for dry weight analysis.

Photosynthetic Pigment Measurement
The contents of photosynthetic pigments were determined using Lichtenthaler and Wellburn methods (Wellburn and Lichtenthaler 1984).Fresh leaf tissue (200 mg) was weighed and powdered using liquid nitrogen.After adding 80% acetone, the volume was brought to 25 ml.This solution was centrifuged at 4800 rpm for 20 min.The supernatant were used for measuring the chlorophyll a, b, and carotenoid.Absorbance of the clear supernatant was read at 645 nm (chlorophyll b), 663 nm (chlorophyll a), and 470 nm (carotenoid).

Protein determination
Soluble protein was quantified according to Bradford (Bradford 1976).Samples were homogenized in 0.1 M Naphosphate buffer (pH 7; 1:5 w/v).After adding the reagent, absorbance was recorded at 595 nm and the concentration was calculated using a calibration curve made with bovine serum albumin.Protein concentrations were determined after realizing a standard curve.

Proline determination
Free proline content was measured by the method of Bates (Bates et al. 1973).Fresh leaf tissue (100 mg) was homogenized in 3% (w/v) sulphosalicylic acid and proline was estimated by ninhydrin reagent (0.125 g of ninhydrin in 2 ml orthophosphoric acid 6 M, and 3 ml of acetic acid).The earned chromophore was extracted from liquid phase by toluene and remarking the organic layer at 520 nm.Proline concentrations were determined after realizing a standard curve.

Catalase determination
Catalase (CAT) activity was determined by decomposition of H 2 O 2 and measured by a decrease in absorbance at 240 nm (Aebi 1984).The reaction mixture contained 200 mM KPO 4 buffer (pH 7.0), 30 mM H 2 O 2 and enzyme extract.Catalase activity was calculated using Aebi formula and H 2 O 2 decomposed g -1 FW min -1 was defined as a unit of CAT.

Lipid peroxidation
The lipid peroxidation in the leaf tissue was measured by malondialdehyde (MDA).Malondialdehyde was assayed by Thiobarbituric acid reactive substances contents (Heath and Packer 1968).

Statistical analysis
The experimental designs were randomized in a complete block and each reporte value corresponds the average of three repeats.The raw data were imported into Microsoft Excel 2007 program for calculations and graphic representation.SPSS (version 16.0) software was used for analysis of variance.Quantitative changes of parameters were analyzed by analysis of variance (one-way ANOVA, corresponds), using Duncan's multiple range tests at P≤0.05 to find out significant differences among treatments.The results are presented as the means ± standard deviation (SD).

Plant growth
After one-week of exposure, three Cd concentrations alone and in combination with two concentrations of SiO 2 nanoparticles of fresh and dry weight of root and shoot of T. aestivum L. were measured (Table 1 & 2 and,  Figures 1 & 2).A clear and significant growth inhibition was observed.
Based on the current results almost in all treatments, roots and shoot fresh and dry weight of wheat seedlings decreased significantly with increase in Cd concentrations, comparing with control.The amounts of root plus shoot fresh weight, in cadmium treated plants decreased approximately 28, 40 and, 54%, and, root plus shoot dry weight, decreased 31, 48 and, 64%, at 30, 60, and, 120 mg -1 l CdCl 2 concentrations comparing with the control, respectively.Based on our findings, concentration of SiO 2 nanoparticles at 50 and 100 mg -1 l in combination with Cd treatment, partially reduced the adverse effects of cadmium ions on wheat seedlings.Alleviated effects of SiO 2 nanoparticles in 50 mg -1 l concentrations were fairly more than 100 mg -1 l.In general, Cd in combination with SiO 2 nanoparticles increased root plus shoot fresh weight, approximately 36 and 28%, in 50 mg-l l concentration and, 45 and 39%, in 100 mg-l l concentration compared to the Cd alone.Reduction in the biomass of wheat in all Cd concentrations, supports the outcomes of previous studies regarding the effect of heavy metals on other plants.
Reduction in the biomass of wheat in all concentration of Cd as a result of our study supported the outcomes of found in the study of the effect of heavy metals on other plant (Glick 2003;de Albuquerque Lima et al. 2011;Påhlsson 1989;Peralta et al. 2001).
There is also evidence for alleviation effects of silicon and silicon oxide in some biotic and abiotic stresses as it has been reported earlier (Eraslan et al. 2008;Li et al. 2004;Masarovič et al. 2012;Mohaghegh et al. 2011;Nwugo and Huerta 2008;Savvas et al. 2007).

Content of photosynthetic pigments
The responses of photosynthetic pigments (chlorophyll a, b and carotenoid) in wheat are presented in table 3 and figures 3-4.Significant decrease in chlorophyll a, b and carotenoid contents in wheat seedlings (one-week exposed to Cd treatments) was observed in comparison with control.Chlorophyll a decreased approximately 34, 40 and 54%, chlorophyll b, 47, 58 and, 72%, chlorophyll a plus b, 38, 45 and, 59%, carotenoids, 31, 37 and, 50%, at 30, 60, and, 120 mg -1 l CdCl 2 concentrations, compared to the control, respectively.Combination of Cd and SiO 2 nanoparticles 50 and 100 mg -1 l concentrations, partially reduced the effects of cadmium ions on photosynthetic pigments.In general, Cd and SiO 2 nanoparticles combination at 50 and 100 mg -l l concentrations increased chlorophyll a, approximately 37 and 15%, respectively compared to Cd alone.

Cadmium
SiO 2 combination nanoparticles at 50 mg -l l increased chlorophyll b, approximately 29%, but at 100 mg -l l decreased chlorophyll b, approximately 13%, compared to Cd alone.This combination at 50 and 100 mg -l l concentrations increased of chlorophyll a and b, approximately 34 and 7%, respectively compared to Cd alone.

Contents of proline
Proline contents of treated and untreated wheat are shown in Table 4 and Figure 5. Increase in cadmium concentration, caused the significant raise of the proline content of leaf, compared to the control.The maximum increase in proline content was observed at 120 mg -1 l of cadmium concntration.Proline contents of cadmium treated plants increased approximately 15, 18 and, 21%, at 30, 60, and, 120 mg -1 l CdCl 2 concentrations respectively compared to the control.
Also, proline contents in combination with SiO 2 nanoparticles at concentrations of 50 and 100 mg -1 l, partially reduced than Cd alone.In general, Cd in combination with SiO 2 nanoparticles at 50 and 100 mg -l l concentration lead to decrease of MDA approximately 5% and 9% respectively, compared to Cd alone.Proline as an amino acid is an important osmolyte which accumulates in a broad range of organisms from bacteria to higher plants, after exposure to abiotic stress, for adapting to divers environmental stresses especially drought, cold, salinity, high temperature, nutrient lack, and exposure to heavy metals (Ashraf and Foolad 2007b).

Lipid peroxidation
The effect Cd alone and in combination with SiO 2 amounts of lipid peroxidation is meaningful (Table 4 and Figure 6).The amounts of MDA formation indicate the level of free radical production and lipid peroxidation (Dexter et al. 1989;Mak and Weglicki 1988).We realize the smallest amounts of lipid peroxidation was on control and the most of it was at 120 mg -l l concentration of CdCl 2 .Amounts of MDA formation of cadmium treated plants increased approximately 28, 59 and, 68%, at 30, 60, and, 120 mg -1 l CdCl 2 concentration compared to control, respectively.
Increase of lipid peroxidation in Cd alone treatments is significantly more than that of combined with SiO 2 nanoparticles.Apart from the control sample, the lowest amounts of MDA were showed in 120 mg -l l concentration of Cd in combination with SiO 2 nanoparticles at 50 mg -l l concentration.In general, Cd in combination with SiO 2 nanoparticles at 50 and 100 mg -l l concentration leads to decrease of MDA approximately 13% and 23% respectively, compared to Cd alone.MDA contents is a product of lipid peroxidation and has been considered as an indicator of oxidative damage and peroxidation of membrane lipids in plants (Nacif de Abreu and Mazzafera 2005;Xu et al. 2006).The cell membrane is usually the main site of attack by any heavy metal in a plant cell.In the current experiments, we observed significant increase in MDA concentration with increasing the CdCl 2 concentration that indicates the negative effect of heavy metals on membrane integrity and permeability.The free radicals produced by heavy metals, can attack the unsaturated fatty acid side chains of membrane lipids, and cause formation of lipid hydroperoxides (Halliwell and Chirico 1993).The similar result was obtained by previous investigators (Gallego et al. 1996;Ghosh et al. 2010;Panda et al. 2003;Sayes et al. 2005;Zhang et al. 2007).

Catalase activity
Significant increasing of catalase activity was observed in response to the increase of Cd concentrations (Table 4 and Figure 7).The highest value of catalase activity was recorded at 120 mg - 1 l CdCl 2 .Increase of cadmium concentration, significantly enhance the catalase activity of wheat leaf compared with the control.Amounts of catalase activity of cadmium treated plants increased about 60, 100 and, 120%, at 30, 60, and, 120 mg -1 l CdCl 2 concentration compared with the control, respectively.Also, catalase activity in all concentrations of cadmium in combination with SiO 2 nanoparticles at 50 and 100 mg -1 l concentrations, moderately reduced than Cd alone.In general, Cd in combination with SiO 2 nanoparticles at 50 and 100 mg -l l concentration decreased of MDA approximately 3% and 12% respectively, compared to Cd alone.The activities of antioxidant enzyme have generally increased during abiotic stress, e.g.chilling, drought, high temperature, salt, and heavy metal stress (Baker and Orlandi 1995;Mittler 2006) and correlated with enhanced cellular protection of reactive oxygen species.Catalase is an important antioxidant which protects plants by suppressing oxidative injury and assist as a reactive species scavenger.This result were in agreement with that of the previous researchers (Du et al. 2011;Gallego et al. 1996;Krishnaraj et al. 2012;Zhang et al. 2007).

Contents of total protein
Increase in CdCl 2 concentrations led to the reduction of total protein contents, compared to control sample (Table 4 and Figure 8).Total protein contents of cadmium treated plants decreased approximately 22, 50 and, 56%, at 30, 60, and, 120 mg -1 l CdCl 2 concentration compared to the control, respectively.Also, total protein contents in samples which treatment by in combination with SiO 2 nanoparticles moderately increased than Cd alone.In general, Cd in combination with SiO 2 nanoparticles at 50 and 100 mg -l l concentrations lead to increase in protein contents about 33% and 11% respectively compared Cd alone.Under heavy metals and nanoparticles, oxidative stresses results the generation of reactive oxygen species and degeneration of protein (Choi and Hu 2008;Rana 2008;Wan et al. 2012;Xia et al. 2008).The results showed that AgNO 3 has more negative effects than AgNPs, and in some cases, no significant difference was found between AgNPs at low concentration and control.This result confirms the previous studies.Both dissolved silver and AgNPs lead to the production of reactive oxygen species, However, the later have direct toxic effects without dissolution (Yin et al. 2011).The toxicity of AgNPs to plants is obvious, while their negative effects and mechanisms on higher plants have not been completely characterized (Jiang et al. 2012).

CONCLUSION
The inhibitory effect of Cd on plant physiology and growth has been reported by several authors.Our study was focused on the potential effect of Cd alone and in combination with SiO 2 nanoparticles on wheat.Exposure of wheat plants to Cd on the whole, caused a significant decrease in fresh and dry weight of root and shoot, photosynthetic pigments and, protein of leaf and a significant increasing of proline, lipid peroxidation and, catalase activity.Almost in all cases, Cd in combination with SiO 2 nanoparticles improves the negative effects.The results of the previous and the present studies, revealed the negative aspects and toxicity problems in plants when they exposed to cadmium ions.Studies about SiO 2 nanoparticles on plants is infrequent, therefore for better understanding the effects of silicon oxide nanoparticles on plant exposed to heavy metal stress, further experiments should be performed.

Fig. 1 .
Fig. 1.Shoot, root and shoot + root fresh weights (mg) in T. aestivum plants treatment with Cd alone and in combination with SiO 2 nanoparticles.

Fig. 3 :
Fig. 3: Chlorophyll a, b, and chlorophyll a+b (mg g -1 FW) in T. aestivum plants treatment with Cd alone and in combination with SiO 2 nanoparticles.

Fig. 4 :
Fig. 4: Carotenoids (mg g -1 FW) in T. aestivum plants treatment with Cd alone and in combination with SiO 2 nanoparticles.

Fig. 5 :
Fig. 5: Proline in T. aestivum plants treatment with Cd alone and in combination with SiO 2 nanoparticles.Proline alleviates metal toxicity by acting as a metal chelator (Sharma and Dubey 2005), functioning as detoxification of reactive oxygen species (ROS) such as hydroxyl radical, singlet oxygen (Szabados and Savoure 2010) and osmoprotectant (Ashraf and Foolad 2007a; Tamayo and Bonjoch 2001), acting as a protection of the enzymes against denaturation and stabilization of protein synthesis (Sanchez-Partida et al. 1992; Shah and Dubey 1997).In addition, proline supports mitochondrial oxidative phosphorylation for protecting natural generation of ATP (Ashraf and Foolad 2007b; Siripornadulsil et al. 2002) and acts as an inhibitor of lipid peroxidation (Hara et al. 2003; Mehta and Gaur 1999).Our results were congruent with the previous investigators (Jiang et al. 2012; John et al. 2009; Kastori et al. 1992; Mehta and Gaur 1999).Lipid peroxidationThe effect Cd alone and in combination with SiO 2 amounts of lipid peroxidation is meaningful (Table4and Figure6).The amounts of MDA formation indicate the level of free radical production and lipid peroxidation(Dexter et al. 1989; Mak and Weglicki  1988).We realize the smallest amounts

Fig. 6 :
Fig. 6: Lipid peroxidation in T. aestivum plants treatment with Cd alone and in combination with SiO 2 nanoparticles.

Fig. 7 :
Fig. 7: Catalase activity in T. aestivum plants treatment with Cd alone and in combination with SiO 2 nanoparticles.

Fig. 8 :
Fig. 8: Protein in T. aestivum plants treatment with Cd alone and in combination with SiO 2 nanoparticles.

Table 1 :
Shoot, root and shoot + root fresh weights (mg) in T. aestivum plants subjected to three Cd concentrations alone and in combination with two concentrations of SiO 2 nanoparticles treatments for 7 days period.

Table 2 :
Shoot, root and shoot + root dry weights (mg) in T. aestivum plants subjected to Cd concentrations alone and in combination with two concentrations of SiO 2 nanoparticles treatments for 7 days period.

Table 3 :
Chlorophyll a, b, total chlorophylls and carotenoids (mg g -1 FW) in T. aestivum plants subjected to Cd concentrations alone and in combination with two concentrations of SiO 2 nanoparticles treatments for 7 days period.

Table 4 :
Proline, lipid peroxidation, catalase and total protein in T. aestivum plants subjected to Cd concentrations alone and in combination with two concentrations of SiO 2 nanoparticles treatments for 7 days period.Values are means of three replicates ± SD per treatment.Means in each column followed by different letters are significantly different (p ≤ 0.05).