Science of the Total Environment 472 (2014) 834–841

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Single-bilayer graphene oxide sheet impacts and underlying potential mechanism assessment in germinating faba bean (Vicia faba L.) Naser A. Anjum a, Neetu Singh b, Manoj K. Singh b, Iqbal Sayeed c, Armando C. Duarte a, Eduarda Pereira a, Iqbal Ahmad a,d,⁎ a

Department of Chemistry and Centre for Environmental & Marine Studies (CESAM), University of Aveiro, 3810-193 Aveiro, Portugal Department of Mechanical Engineering and Centre for Mechanical Technology & Automation (TEMA), University of Aveiro, Aveiro 3810-193, Portugal Department of Emergency Medicine, Brain Research Laboratory, 1365B Clifton Road NE, Suite 5100, Emory University, Atlanta, GA 30322, USA d Department of Biology and Centre for Environmental & Marine Studies (CESAM), University of Aveiro, 3810-193 Aveiro, Portugal b c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Graphene oxide (GO) impacted Vicia faba both positively and negatively. • GO (1600 N 200 N 100 mg GO L− 1) elevated oxidative stress but lowered its metabolism. • GO (800 N 400 mg GO L− 1) enhanced H2O2 scavenging and improved V. faba health status. • V. faba-root-polypeptide patterns substantiated GO-positive and -negative impacts. • Results imply 800 N 400 mg GO L− 1-safe nature and also warrant further studies.

a r t i c l e

i n f o

Article history: Received 17 June 2013 Received in revised form 1 November 2013 Accepted 3 November 2013 Available online 15 December 2013 Keywords: Graphene oxide Vicia faba Phytotoxicity Oxidative stress Polypeptide pattern

a b s t r a c t This study investigates the impact of different single-bilayer graphene oxide sheet (hereafter ‘graphene oxide’, GO; size: 0.5–5 μm) concentrations (0, 100, 200, 400, 800 and 1600 mg L−1) and underlying potential mechanisms in germinating faba bean (Vicia faba L.) seedlings. The study revealed both positive and negative concentration-dependent GO-effects on V. faba. Significant negative impacts of GO concentrations (ordered by magnitude of effect: 1600 N 200 N 100 mg GO L−1) were indicated by decreases in growth parameters and the activity of H2O2-decomposing enzymes (ascorbate peroxidase, APX; catalase, CAT), and by increases in the levels of electrolyte leakage (EL), H2O2, and lipid and protein oxidation. The positive impacts of 400 and 800 mg GO L−1 included significant improvements in V. faba health status indicated by decreased levels of EL, H2O2, and lipid and protein oxidation, and by enhanced H2O2-decomposing APX and CAT activity, and increased proline and seed-relative water content. V. faba seedlings-polypeptide patterns strongly substantiated these GOconcentration effects. Overall, the positive effects of these two GO concentrations (800 N 400 mg L−1) on V. faba seedlings indicate their safe nature and allow to suggest further studies. © 2013 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: Department of Biology and Centre for Environmental & Marine Studies (CESAM), University of Aveiro, 3810-193 Aveiro, Portugal. Tel.: + 351 234401527x24907; fax: +351 234370084. E-mail address: [email protected] (I. Ahmad). 0048-9697/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2013.11.018

N.A. Anjum et al. / Science of the Total Environment 472 (2014) 834–841

1. Introduction Single-bilayer graphene oxide sheet (hereafter termed ‘graphene oxide’, GO; size: 0.5–5 μm) is a water-soluble derivative of graphene (a novel one-atom-thick two-dimensional nanoparticle composed of a single layer of sp2-bonded carbon atoms) and has been widely used as a precursor of graphene-based nanoparticle composites (Geim, 2009; Xu and Wang, 2012). Extensive anchoring of engineered GO onto the surface of river sand is used to produce effective adsorbents for the removal of heavy metal ions, pesticides and natural dyes (Sreeprasad et al., 2011; Gupta et al., 2012). Owing to the wide use of GO-based adsorbents in environmental cleanup, it is clear that organisms are widely exposed to GO and GO can thus be transferred to the human/animal food chain. Therefore, the ultimate effects of GO-based materials on both the environment and biota (including plants) are of prime concern. Plants are the essential base component of all ecosystems and they are vital for devising and implementing sustainable mitigation or control measures against pollution by engineered nanoparticles (Navarro et al., 2008; Ma et al., 2010). However, to date, studies on interaction of crop plants and engineered GO are scarce (Begum et al., 2011; Anjum et al., 2013a), and GO phytotoxicity studies have yielded only speculative and unsubstantiated results reporting only minor or no effects on higher plants (Begum et al., 2011). Two recent reports reflect a partial mechanism underlying GO impact on plants in which GO-induced reactive oxygen species (ROS) generation (Begum et al., 2011) and glutathione (GSH) redox system impairments (Anjum et al., 2013a) were found to be the major factors controlling plant responses to GO. Being sedentary in nature, plants have limited mechanisms to avoid exposure to environmental contaminants. GO impacts on plants have been reported to be dependent on plant species/genotypes and/or time/duration of exposure (Begum et al., 2011). Nanoparticles have been extensively reported to impair plant growth, photosynthesis and its related variables, and also to interfere with plant–water–nutrient pathways in different plant models (Lin and Xing, 2008; Asli and Neumann, 2009; Dimkpa et al., 2012; Zhao et al., 2012). High levels of ROS have also been found in different plant species exposed to a number of nanoparticles including ZnO (Lin and Xing, 2008), TiO2 (Asli and Neumann, 2009), CuO and ZnO (Dimkpa et al., 2012), CeO2 (Zhao et al., 2012; Rico et al., 2013) and GO (Begum et al., 2011). • A number of reports indicate that ROS (such as O•− 2 , OH , and H2O2) induce oxidative damage in bio-molecules including nucleic acids, proteins and membrane lipids leading to weakening of membrane integrity, elevated electrolyte leakage (EL), and eventually to cell and plant death (Gill and Tuteja, 2010; Anjum et al., 2012, 2013b). Thus, the tissue levels of H2O2 (a predominant toxic intermediate produced during the oxidative burst), lipid peroxidation products (thiobarbituric acid reactive substances, TBARS) and the EL have been extensively reported as indicators of oxidative injury status in stressed plants (Gill and Tuteja, 2010; Anjum et al., 2012). Although the significance of ascorbate peroxidases (APX, EC 1.11.1.11) and catalase (CAT, EC 1.11.1.6), important enzymes for the scavenging of H2O2, has been demonstrated in different plant species exposed to non-nano-sized materials (reviewed by Gill and Tuteja, 2010; Anjum et al., 2012) and CeO2 nanoparticles (Rico et al., 2013), the literature contains no report on this mechanism in GO-exposed plants. The accumulation of osmolytes (such as excess free proline) has been extensively reported in different plant species where these compounds help plants to survive under various stress conditions by controlling various physiological functions viz., osmotic adjustment, sub-cellular structure stabilization, and free radical scavenging (Sharma and Dietz, 2006). Additionally, plant polypeptide patterns have been reported to be modulated under biotic and abiotic stress exposure which can be easily visualized using sodium dodecyl sulfate–polyacrylamide-gel electrophoresis (SDS-PAGE) (Sobkowiak and Deckert, 2006; Ahsan et al., 2007). As a fundamental step toward proteomic studies the SDS-PAGE of plant proteins

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has also been shown to yield important information for understanding the molecular mechanisms of stress responses and developing transgenic plants with enhanced tolerance to stress (Ahsan et al., 2007). In the light of views thus far available on the GO phytotoxicity and tolerance mechanisms (Begum et al., 2011) and the known differential impairments in cellular GSH redox system components in higher plants under GO exposure (Anjum et al., 2013a), it was hypothesized that cross-talk among oxidative stress parameters and their metabolizing enzymes, and the modulation of seedlings polypeptide patterns may illuminate the basic mechanisms underlying sensitivity to GO concentrations in the food crop — faba bean (Vicia faba L.). To test this hypothesis, the present study aimed: (i) to investigate the impact of GO on the growth traits (germination rate, root length) and water relations (measured as seed-relative water content); (ii) to assess cell membrane permeability (measured as EL), oxidative stress and its metabolism (measured as lipid and protein oxidation, H2O2 content and the activity of its decomposing enzymes — APX and CAT), and osmolyte level (measured as proline content); and (iii) to explore the potential relationship between GO-mediated anomalies with the correlative modulation of seedlings polypeptide patterns. V. faba was chosen as a model plant system and the germinating seedling as a model test plant stage for this study because V. faba is among the most common food crops and a primary dietary legume for humans and animals. It contributes about 33% of the dietary protein nitrogen needs of humans (Popelka et al., 2004). Germination and/or seedling — an early stage of growth and a complex physiological process in plants are widely used for evaluation of environmental-contaminants-phytotoxicity (Markwiese et al., 2001; Ahsan et al., 2007; Anjum et al., 2013a). 2. Materials and methods 2.1. Graphene oxide test solution preparation Having characterized the as-synthesized GO (Anjum et al., 2013a), the test solution was prepared following the procedure described elsewhere (Anjum et al., 2013a). In brief, GO test concentrations (0, 100, 200, 400, 800 and 1600 mg GO L−1) were prepared from a stock graphene aqueous suspension using freshly prepared deionized water and subsequently vortexed for 20 s and sonicated for 2 × 20 s with a 20 s interval. GO test solutions were neutralized using an aqueous 0.1 mol L−1 NaOH solution to achieve the pH values (6.3–6.5) favorable to plant growth (Begum et al., 2011). 2.2. Seed culture conditions and treatments V. faba seeds were cultured and treated following the procedure described elsewhere (Anjum et al., 2013a). In brief, healthy and equalsized V. faba seeds were surface sterilized by immersion in 10% NaClO solution for 10 min (USEPA, 1996) and subsequent vigorous rinsing with sterilized double-distilled water. V. faba seeds were sown (10 seeds for each treatment) in five replicates on Petri dishes with 90-mm filter paper (Whatman No. 1) round strips, soaked in 4 ml solution with GO concentrations (0, 100, 200, 400, 800 and 1600 mg L−1), covered with a lid and incubated in the dark at 23 ± 2 °C until germination (Ahsan et al., 2007). The seeds were considered germinated when the root measured at least 20 mm in length for controls (USEPA, 1996). Roots were excised with sharp sterile blades, and estimations for both germination and growth trait were performed. Roots were either used fresh for biochemical estimations or dipped into liquid nitrogen and stored at −80 °C for further assays. 2.3. Seedlings growth and relative water content The number of germinated seeds at each GO concentration was counted to estimate percent germination. Root length was measured using a meter scale. The relative water content (RWC) of the seeds

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2.4.1. Membrane lipids and permeability, cellular proteins and osmolytes As a marker of cell membrane lipid peroxidation, the content of TBARS was measured following the method of Dhindsa et al. (1981) as adapted and described by Anjum et al. (2013b). Briefly, fresh root tissues (0.25 g) were ground in liquid nitrogen, mixed with 0.73% 2-thiobarbituric acid in 12% trichloroacetic acid (TCA), incubated for 30 min in boiling water, ice-cooled and centrifuged at 1000 ×g for 10 min at 4 °C. The absorbance in the supernatant was recorded at 532 nm in the UV–Vis plate reader (Spectra Max 384, Molecular Devices). The rate of lipid peroxidation was expressed as nanomoles of TBARS formed per gram of fresh weight using a molar extinction coefficient of 1.55 × 105 M−1 cm−1. The potential impact of GO on the plant cell membrane permeability was assayed by measuring the EL following the method of Anjum et al. (2013b). In brief, fresh root tissues (0.25 g) were placed in glass vials containing 10 ml deionized water. The vials were covered with plastic caps and placed on a shaker at 25 °C for 6 h and the electrical conductivity (EC) of the solution was measured (EC1) using an electrical conductivity meter (WTW Cond 330i/SET, Weilheim, Germany). Subsequently, the same vials were kept in a water bath shaker at 90 °C for 2 h, cooled, and then EC2 was measured. EL was expressed as EL = EC1/EC2 × 100. Based on the reaction of carbonyl groups with 2,4dinitrophenylhydrazine (DNPH), the level of protein oxidation was estimated following the method of Levine et al. (1994) as described elsewhere (Anjum et al., 2013b). Briefly, protein extract was obtained by homogenization of root tissues (0.25 g) in a phosphate buffer (25 mM, pH 7.0). After centrifuging at 2000 ×g, the supernatants (200 μl) were combined with 300 μl of 10 mM DNPH prepared in 2 M HCl. After 1 h incubation at room temperature, the proteins were precipitated with 10% (w/v) TCA and the pellets were washed three times with 500 μl of ethanol/ethyl acetate (1/1, v/v). The pellets were finally dissolved in 6 M guanidine hydrochloride in 20 mM potassium phosphate buffer (pH 2.3). The reactive carbonyl content was calculated by absorbance at 370 nm, using the extinction coefficient for aliphatic hydrazones (22.1 mmol−1 cm−1) and expressed as nanomoles of reactive carbonyl g−1 fresh weight. For the estimation of the GO-mediated modulation of osmolyte (proline) level, root tissues (0.25 g) were homogenized with 3% aqueous sulfosalicylic acid and centrifuged at 3000 ×g for 10 min. Supernatant proline content was estimated using the method of Bates et al. (1973) and was expressed as μmol g−1 fresh weight. 2.4.2. H2O2 content and its decomposing enzymes Root-H2O2 content was determined following the method of Loreto and Velikova (2001) as described elsewhere (Dipierro et al., 2005). In brief, fresh root tissues (0.25 g) were homogenized in 2 ml of 0.1% (w/v) TCA. The homogenate was centrifuged at 12,000 ×g for 15 min and 0.5 ml of the supernatant was mixed with 0.5 ml of 10 mM K-phosphate buffer pH 7.0 and 1 ml of 1 M KI. The H2O2 content of the supernatant was evaluated by comparing its absorbance at 390 nm with a standard calibration curve. For H2O2-decomposing enzyme assays, fresh root tissues (0.25 g) were homogenized in K-phosphate buffer (100 mM, pH 7.0) containing 0.5% (v/v) Triton X-100 and 1% (w/v) polyvinylpyrrolidone with pre-

2.5. Protein extraction, quantification and characterization Root tissues (0.25 g) were homogenized with phosphate buffer (25 mM, pH 7.0) using chilled mortar and pestle. The homogenate was centrifuged at 15,000 ×g for 20 min at 4 °C. Quantification of protein in the supernatant was done by Bradford protein assay (Bradford, 1976) with bovine serum albumin as a standard. Proteins were characterized using sodium dodecyl sulfate–polyacrylamide-gel electrophoresis (SDS-PAGE). Five percent and 12.5% acrylamide was used for stacking and resolving gels respectively (Laemmli, 1970). Electrophoresed gel was stained with Coomassie Brilliant Blue R-250 solution

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chilled mortar and pestle. The homogenate was centrifuged at 15,000 ×g for 20 min at 4 °C. The supernatant was used for the CAT activity assay; whereas, the homogenizing buffer was supplemented with 2 mM ascorbate for the APX activity assay. In brief, the activity of APX was determined as described by Nakano and Asada (1981) by monitoring the ascorbate decomposition per minute at 25 °C and was calculated using an extinction coefficient of 2.8 mM− 1 cm− 1. CAT activity was determined following the method of Aebi (1984) by monitoring the disappearance of H2O2 at 240 nm and calculated using extinction coefficient 0.036 mM− 1 cm− 1 and expressed as μmol of H2O2 g−1 fresh weight min−1.

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was measured by determining the fractional water content (FWC) using the equation FWC = Seed Fresh Weight (SFW) − Seed Dry Weight (SDW) / Seed Fresh Weight (SFW) (Purcell and Sinclair, 1995; Ali et al., 2005). It was assumed that seeds at 12 h of soaking were fully turgid and FWC was used to estimate the seed turgid weight (STW) for each treatment using the equation STW = SDW / (1-FWC). Subsequently, the seed RWC was calculated using the equation: RWC = SFW − SDW / STW − SDW. Weights were recorded using an analytical balance (Mettler Toledo, USA).

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Graphene oxide concentrations (mg L-1) Fig. 1. Morphology (A), germination rate (B), root length (C) and relative water content (D) of Vicia faba seeds exposed to graphene oxide (GO) concentrations (0, 100, 200, 400, 800 and 1600 mg L−1). Values represent the means of five replicates (± standard deviation) from each of three independent experiments. Significant differences are: avs. 0; b vs. 100; cvs. 200; dvs. 800; evs. 1600.

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and subsequently destained in the same solution without dye. The proteins fractionated into bands were scanned using the Molecular Imager Gel Doc XR + System (Bio-Rad) and analyzed with Quantity One software version 4.6.3 (Bio-Rad, Hercules, CA).

2.6. Statistical analysis SPSS (Predictive Analytics Software statistics 18) for Windows was used for statistical analysis. One-way analysis of variance (ANOVA) followed by pairwise multiple comparisons employing the Tukey test was used to detect significant data among treatments. The data are expressed as mean values ± SD of three independent experiments with at least five replicates for each. The significance level was set at P ≤ 0.05.

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3. Results 3.1. Seedlings growth and relative water content With exposures to 100, 200 and 1600 mg concentrations of GO L−1 (vs. control), the seed germination rate and root length significantly decreased, whereas these parameters significantly increased with 800 mg GO L−1 (vs. control). Comparison of the effects of different GO-concentrations revealed a significant increase (vs. 100, 200 and 1600 mg GO L−1) and decrease (vs. 800 mg GO L−1) in germination rate with 400 mg GO L−1. The same GO concentration (400 mg L−1) was also associated with a significant increase (vs. 200 mg GO L− 1) and decrease (vs. 800 mg GO L−1) in root length. With respect to GOmediated changes in RWC, the RWC of seeds significantly decreased with 100, 200 and 1600 mg of GO L−1 (vs. control) but significantly increased with 400 and 800 mg GO L−1. Comparisons among GO concentration effects revealed both significant increase (vs. 100, 200 and 1600 GO L−1) and decrease with 400 mg GO L−1 (vs. 800 mg GO L− 1) (Fig. 1A–D).

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Graphene oxide concentrations (mg L-1) Fig. 2. Lipid peroxidation (A), membrane permeability (B), protein oxidation (C) and osmolyte level (D) in Vicia faba sprouted roots exposed to graphene oxide (GO) concentrations (0, 100, 200, 400, 800 and 1600 mg L−1). Values represent the means of five replicates (± standard deviation) from each of three independent experiments. Significant differences are: avs. 0; bvs. 100; cvs. 200; dvs. 800; evs. 1600.

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Fig. 3. H2O2 content (A) and the activity of its decomposing enzyme ascorbate peroxidase (APX) (B) and catalase (CAT) (C) in Vicia faba sprouted roots exposed to graphene oxide (GO) concentrations (0, 100, 200, 400, 800 and 1600 mg L−1). Values represent the means of five replicates (± standard deviation) from each of three independent experiments. Significant differences are: avs. 0; bvs. 100; cvs. 200; dvs. 800; evs. 1600.

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order of effect magnitude, 1600, 200 and 100 mg GO L−1, whereas significant decreases in the activity of H2O2-decomposing enzymes APX and CAT were seen with the same GO concentrations (vs. control). GO concentration effect comparisons revealed significant decrease in the content of H2O2 and increase in the activity of APX and CAT maximally with 800 mg GO L−1 followed by 400 mg GO L− 1 (vs. 100, 200, and 1600 mg GO L− 1). GO concentrations-mediated changes in V. faba root osmolyte levels were indicated by a significant decrease in proline content with 100, 200 and 1600 mg GO L−1 (vs. control), and a significant increase at 800 and to a lesser extent 400 mg GO L−1 (vs. control). GO concentration effect comparisons revealed significant increase (vs. 0, 100, 200, 1600 mg GO L− 1) and decrease (vs. 800 mg GO L−1) in proline content with 400 mg GO L−1 (Figs. 2A–D and 3A–C). 3.3. Polypeptide patterns Visualization of the SDS-PAGE intensity of V. faba seedlings protein bands (approx. 80, 75, 55, 40, 35 and 15 kDa) revealed gradual decreases with 100, 200 and 1600 mg GO L−1. In contrast, the intensity of these bands increased with 400 and 800 mg GO L−1 when compared with the control V. faba seedlings. Interestingly, compared to 400 mg GO L− 1, the intensity of these protein bands increased with 800 mg GO L−1. An extra band of approx. 7–8 kDa was perceptible only with 800 mg GO L−1 (Fig. 4). 4. Discussion In our previous study, we reported that GO induced impairments in the cellular GSH redox system were the major cause of V. fabadifferential susceptibility towards its concentrations (Anjum et al., 2013a). Herein, the least explored potential impact of GO concentrations on cellular H2O2 level, cell membrane permeability/integrity, and the levels of cellular proteins and osmolyte (proline) has been studied as a further step in this context. In addition, the modulation of H2O2-decomposing enzymes and polypeptide patterns has also been cross-talked. 4.1. Seedlings growth and water relations Plants are extensively used for the development of a comprehensive toxicity profile of various environmental stressors including nanoparticles (Navarro et al., 2008; Ma et al., 2010). In particular, V. faba seed germination rate and root elongation have been extensively used for acute phytotoxicity tests (Ahsan et al., 2007; Wang et al., 2011; Anjum et al., 2013a). The present investigation found a GO concentration-dependent

impact on both the intake of water by seeds (known as ‘imbibition’, the phase 1 of an important physiological process called ‘germination’) (Kordan, 1992) and growth traits (measured as seed germination rate and root length). These observations corroborate reports on the other nanoparticles and plant species (Lin and Xing, 2008; Asli and Neumann, 2009). However, in contrast to an earlier graphene–plant interaction study in plants other than V. faba (Begum et al., 2011), in the present study, GO concentrations (800 and 400 mg L−1) significantly improved V. faba seed RWC, germination rate and root length. Although the mechanism responsible for these effects is unclear, we speculate that compared to 100, 200 and 1600 mg GO L−1, the concentrations of 400 and 800 mg GO L−1 activated the hydration of V. faba seeds (i) by influencing the seed coat-hydrophilic groups (\NH3, \OH and/or \COOH) of proteins and carbohydrates, as reported earlier in 28-homobrassinolide and/or potassium-treated Cicer arietinum seeds (Ali et al., 2005); and/or (ii) by influencing the seed coat lipid content and/or the wax (Zeng et al., 2005; Wu et al., 2012). Previous processes might have attracted dipolar water molecules to form a hydrated shell around these molecules, affected the strength of hydrophobic interactions between GO concentrations and the seed coat, facilitated seed coat swelling, and finally increased seed RWC and improved growth traits (germination rate and root length). Nevertheless, enhanced utilization of water by Brassica napus and Raphanus sativus exposed to nanoparticles and/or a mixture of nanoparticles other than GO has been reported to facilitate increased germination rate and root length (Lin and Xing, 2007; Wu et al., 2012). 4.2. Oxidative stress traits, its metabolism and osmolyte level ROS including H2O2 are produced in plants as normal byproducts of various metabolic pathways; however, various environmental stressors lead to excessive production of ROS and their reaction products causing progressive oxidative damage and ultimately cell death (Gill and Tuteja, 2010; Anjum et al., 2012). Our finding that GO mediates enhanced H2O2 accumulation in a concentration manner (1600 N 200 N 100 mg GO L−1) is in close agreement with earlier studies considering graphene (Begum et al., 2011) and other nanoparticle-treated plant species (Nel et al., 2006; Tan et al., 2009; Dimkpa et al., 2012). GO-mediated enhanced H2O2-accumulation was also accompanied by significantly decreased activity of two important H2O2-decomposing enzymes APX and CAT and severe damages to cell membranes (in terms of increased TBARS) causing a significant increase in EL in V. faba roots exposed to these GO concentrations (in order of effect magnitude, 1600 N 200 N 100 mg GO L−1). However, significantly enhanced APX and CAT activity with 800 and 400 mg GO L−1 helped root tissues to keep tight control over H2O2 accumulation to an optimum and/or metabolically relevant level that was earlier believed to be an important factor for the activation of plant response to varied stress factors (Desikan et al., 2001; Campos et al., 2003). Nevertheless, elevated activity of both APX and CAT has been reported to tightly control cellular H2O2 level and efficiently avert H2O2-mediated lipid peroxidation as well as the EL in plants (Gill and Tuteja, 2010; Anjum et al., 2012). With respect to GO-mediated membrane lipid peroxidation and protein oxidation, GO-concentrations with the following order of impact: 1600 N 200 N 100 mg GO L−1 severely oxidized root cell lipids and proteins measured in terms of significant increase in TBARS and reactive carbonyl contents, respectively. The oxidation of proteins and membrane lipids has been considered of paramount significance in the environmental toxicology of nanoparticles because it can provide fundamental insights into both toxicity mechanisms and biomarker discovery (Dowling and Sheehan, 2006; Dimkpa et al., 2012; Zhao et al., 2012). On the one hand, low accumulation and cross-membrane mobility of H2O2 (a known factor for lipid and protein oxidation) as a result of enhanced activities of H2O2-decomposing enzymes (APX and CAT) can explain 400 and 800 mg GO L− 1-mediated decreased membrane lipid peroxidation but increased protein stability. On the other hand, possible

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strengthening of the thiol–disulfide bonds by 400 and 800 mg GO L−1 may also help the stabilization of proteins as indicated by low reactive carbonyl content. GO concentration-mediated decrease (in order of magnitude of effect: 1600 N 200 N 100 mg GO L−1) in the level of proline (a well-known osmolyte) is in close agreement with Sharma et al. (2012) where the authors showed a continuous decrease in free proline content in silver nanoparticle-treated B. juncea. Most importantly, since the occurrence of 400 and 800 mg GO L−1 helped V. faba roots to efficiently scavenge ROS such as H2O2 which in turn modulated both cell membrane permeability in terms of decreased EL and the oxidation of proteins by strengthening the peptide chain, significantly increased proline accumulation due to these concentrations is promising. There are now numerous studies reporting a vital positive role of elevated

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proline accumulation in plant tissues of silver nanoparticle-exposed B. juncea (Sharma et al., 2012) and under varied stress conditions (Sharma and Dietz, 2006; Gill and Tuteja, 2010), where prolinemediated positive effects in plants were attributed largely to its antioxidant property and its actions as a stabilizer of plasma membrane and some macromolecules (Jain et al., 2001). 4.3. Polypeptide patterns Knowledge of stress-responsive proteins is critical for understanding the molecular mechanisms of plant tolerance to various stressors. However, to date, no polypeptide pattern analysis of GO tolerance in food crops has been published. To address this lack, the present study

Graphene oxide concentrations (mg L-1) In order of negative effect magnitude

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Polypeptide patterns Fig. 5. Schematic presentation of graphene oxide (GO) concentrations (100, 200, 400, 800 and 1600 mg L−1) negative and positive impacts on Vicia faba seedlings and summary of underlying potential mechanisms. See text for details.

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separated sprouted root proteins using SDS-PAGE to advance the discussion on the potential mechanisms underlying GO-concentrationaccrued anomalies in V. faba roots. Polypeptide disappearance (in order of decreasing impact: 1600 N 200 N 100 mg GO L− 1) and reappearance (in order of decreasing impact: 800 N 400 mg GO L−1) of protein bands showing polypeptides of approximately 80, 75, 55, 40, 35 and 15 kDa molecular weights reflect V. faba's GO stress adaptation strategy at the protein level. We have also found that V. faba root proteins were clearly responsive to GO concentrations, either upregulating or down-regulating their expression. Our results agree with Munoz et al. (1997), Unni and Rao (2001) and Parida et al. (2004) who reported salinity-mediated disappearance and re-appearance of proteins of molecular weights of 22–68 kDa in Prosopsis, Rhizobium, and Bruguiera gymnorrhiza, respectively. Furthermore, the potential involvement of 55, 40, 35 and 15 kDa polypeptides in the oxidative stress is corroborated by our findings that 1600, 200 and 100 mg GO L−1 increased H2O2 and TBARS levels, the EL, and reactive carbonyls, and diminished activity of APX and CAT. Regarding the re-appearance of the 80, 75, 55, 40, 35 and 15 kDa polypeptides in response to 800 and 400 mg concentrations of GO L− 1, it is likely that these two GO concentrations significantly induced the protein synthesis. In addition, the appearance of a new polypeptide (≈7–8 kDa) with 800 mg GO L−1 clearly suggests the involvement of this protein in the adaptive tolerance mechanism that responds to GO toxicity in V. faba roots. Polypeptides between 4 and 8 kDa are known to represent cysteine (Cys)-rich metallothioneins (MTs) which have been extensively reported and reviewed to function as antioxidants and also to chelate varied metals via their Cys-thiol groups (Cobbett and Goldsbrough, 2002; Hassinen et al., 2011). Improved growth, seed RWC, decreased oxidative stress traits but enhanced H2O2-decomposing enzyme activity and osmolyte (proline) levels further support this conclusion (Fig. 5). 5. Conclusions This study shows that GO impacted V. faba both positively, at concentrations of 800 and 400 mg GO L−1 and negatively at 1600, 200 and 100 mg GO L−1. The 400 and 800 mg GO L−1 mediated significant improvements in V. faba health status through increased seed-imbibition (in terms of increased seed RWC) and enhanced H2O2-decomposing enzyme (APX, CAT) activity, and osmolyte (proline) levels. These effects cumulatively decreased oxidative stress (in terms of decreased H2O2 and TBARS content), ensured improved cell membrane integrity (in terms of low percent EL) and reduced protein oxidation (in terms of low reactive carbonyls), leading ultimately to improved V. faba health, reflected in increasing seed germination and root elongation. Conflicts of interests The authors declare that there are no conflicts of interest. Acknowledgments Financial support from both the Portuguese Foundation for Science and Technology (FCT) through Post-Doctoral grant to N.A. Anjum (SFRH/BPD/84671/2012) and from the Aveiro University Research Institute/CESAM is gratefully acknowledged. References Aebi H. Catalase in vitro. Methods Enzymol 1984;105:121–30. Ahsan N, Lee SH, Lee DG, Lee H, Lee SW, Bahk JD, et al. Physiological and protein profiles alternation of germinating rice seedlings exposed to acute cadmium toxicity. C R Biol 2007;330:735–46. Ali B, Hayat S, Ahmad A. Response of germinating seeds of Cicer arietinum to 28-homobrassinolide and/or potassium. Gen Appl Plant Physiol 2005;31:55–63.

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