OES studies

Microchemical Journal 134 (2017) 54–61

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Microchemical Journal journal homepage: www.elsevier.com/locate/microc

Effect of ZnO nanoparticles on corn seedlings at different temperatures; X-ray absorption spectroscopy and ICP/OES studies Martha Laura López-Moreno a,b,⁎, Guadalupe de la Rosa c, Gustavo Cruz-Jiménez d, Laura Castellano c, Jose R. Peralta-Videa b,e,f, Jorge L. Gardea-Torresdey b,e,f a

University of Puerto Rico at Mayagüez, P.O. Box 9019, Mayagüez 00681-901, Puerto Rico Chemistry Department, The University of Texas at El Paso, El Paso, TX 79968, United States Division of Science and Engineering, University of Guanajuato, Loma del Bosque, 103, Col. Lomas del Campestre, C.P. 37150, Guanajuato, Mexico d Division of Natural and Exact Sciences, Campus Guanajuato, University of Guanajuato, Col. N. Alta s/n, Guanajuato, C.P. 36050, Mexico e Environmental Science and Engineering PhD Program, the University of Texas at El Paso, 500 West Univ. Ave, El Paso, TX 79968, United States f University of California Center for Environmental Implications of Nanotechnology (UC CEIN), The University of Texas at El Paso, El Paso, TX 79968, United States b c

a r t i c l e

i n f o

Article history: Received 1 May 2017 Accepted 11 May 2017 Available online 12 May 2017 Keywords: Zea mays XAS ZnO nanoparticles Zn uptake Oxidative stress Nanotoxicity

a b s t r a c t Previous studies have shown that ZnO nanoparticles (NPs) affect corn germination and root growth. However, there is a lack of information about the effects of these NPs at different temperatures. In this study, corn seedlings were exposed to ZnO NPs (24 ± 3 nm) at 0–1600 mg L−1 and ionic Zn 0–250 mg L−1 for 15 days. Germination, root elongation, Zn uptake and oxidation state, enzyme activity and protein expression were analyzed. At 20 and 25 °C, 400 mg ZnO NPs L−1 significantly reduced the germination (40 and 53% respectively), while no effect of Zn2+ was observed. Temperature and Zn concentration affected root growth. At 20 °C, ZnO at 50, 400, and 1600 mg L−1 reduced root growth by 18, 47, and 26% respectively. At 25 °C, 100 and 800 mg L−1 increased root growth by 22 and 27%, while at 30 °C, 100 mg L−1 reduced root growth by 42%. At 30 °C, 0.1 mg L−1 of Zn2+ increased the growth by 50%. Zn accumulation in seedlings was significant only at 1600 mg L−1 ZnO and 250 mg L−1 Zn2+. Ascorbate peroxidase activity increased by 24 and 57% under exposure to ZnO at 400 and 1600 mg L−1 at 25 °C. At any temperature the XAS analyses showed presence on NPs in roots. Exposure to ZnO NPs did not show changes in protein expression; however, a protein band with molecular weight of 85 kDa decreased its expression at 30 °C, while a protein of 75 kDa increased its expression at 30 °C. This study suggests that temperature may alter the way the ZnO NPs interact with plants. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Nanomaterials (NMs) have been fundamental to the development of nanotechnologies. Different NMs have been synthesized in recent years to be used in personal care products, industry, medicine and agriculture [1]. Although the number of nanomaterials and nanoproducts steadily increase in the market, their interactions with living organisms are not well understood. Thu, rules to properly manage and dispose of NMs and nanoproducts after end user application are still missing. Plants are exposed to NMs, either intentional or unintentional. Intentional exposure includes the use of nano-enabled pesticides and fertilizers in agricultural activities [2–5]. Previous reports indicate that nanoparticulate ZnO and MgO have the potential to be used to combat pathogenic fungi including Alternaria alternate, Fusarium oxysporum, Rhizopus stolonifer and Mucor plumbeus [6]. Other reports suggest the use of nano⁎ Corresponding author at: University of Puerto Rico at Mayagüez, P.O. Box 9019, Mayagüez 00681-901, Puerto Rico. E-mail address: [email protected] (M.L. López-Moreno).

http://dx.doi.org/10.1016/j.microc.2017.05.007 0026-265X/© 2017 Elsevier B.V. All rights reserved.

fertilizers to increase nutrient availability to plants, avoiding excessive input of chemical elements into the environment. However, maximum benefits/threats from ions released by nanoparticles (NPs) into plant cells, compared with those from common fertilizers, are not well documented [7]. Zinc and boron nano-fertilizers have been reported to increase fruit yield and quality of pomegranate (Punica granatum cv. Ardestani) without affecting the physical characteristics of fruit [8]. ZnO NPs have also been used as fungicide in agriculture. Ghosh et al., [9] evaluated the toxicity of ZnO NPs in Allium cepa, Nicotiana tabacum, and Vicia faba. The authors observed that ZnO NPs promoted cell death in root of A. cepa and showed higher toxicity, compared with the Zn bulk form. Corn (Zea mays) is one of the main crops in the world, with a production of 780 metric tons per year. USA, China, Mexico, and Argentina are the top corn producers for staple food and Brazil for ethanol production. According to the USDA, N 90 million acres were planted with corn in 2015 [10]. However, crop production is affected by genetic or environmental factors. Ambient temperature, light, water quality and quantity, and contaminants, are among the last. Temperature plays a very

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important role in plant growth, since warming or cooling conditions affect plant-microbe interactions, nutrient availability and transport inside plant tissues, and flowering time, among others [11]. It is well known that temperature is one of the most critical factors affecting seed germination [12,13]. Kumar et al. [14] reported the optimal germination temperature for Kalmegh (Andrographis paniculata Wall. Ex Nees) was 25 °C, in comparison to 20 and 30 °C. If, besides temperature, seeds are exposed to additional abiotic stress such as metals or metal oxide nanoparticles, the emergence of radicle can be prevented [15,16]. Although many reports have described the effects of NMs on seed germination to the best of the authors' knowledge, there are no reports on the effect of temperature and ZnO NPs as abiotic stress elicitors on germination and growth of corn plants. In this study, corn seeds were exposed to ZnO NPs at 0–1600 mg L−1 and Zn2 + from Zn(NO3)2 0– 250 mg L−1 and set at 20, 25, and 30 °C. In this study, X-ray Absorption Spectroscopy (XAS) and Inductively Plasma Optical Emission Spectroscopy (ICP/OES) techniques were used to evaluate Zn uptake, oxidative stress, as well as the oxidation state of Zn in ZnO NPs exposed to corn plants. 2. Materials and methods ZnO NPs were obtained from Melliorum technologies (Rochester, NY). Previous characterization of NPs was reported by Keller et al. [17]. Zinc nitrate Zn(NO3)2 99% purity was purchased from Alfa Aesar. 2.1. Preparation of ZnO NP suspensions and Zn2+ solutions Suspensions of ZnO NPs were prepared in Millipore water at the following concentrations: 0, 50, 100, 200, 400, 800, and 1600 mg L−1. The suspensions were sonicated for 30 min to avoid aggregation according to Lin and Xing [18]. The pH of these solutions was about 7.0 ± 0.1. Solutions of Zn2+ were prepared from Zn(NO3)2 at 0, 0.05, 0.5, 5, 10, 50, and 250 mg Zn2 + L−1 in Millipore water and the pH was adjusted to 7.0 ± 0.2. 2.2. Germination experiments Corn (Golden variety) kernels were set in a 4% NaClO4 solution for disinfection for 30 min and rinsed three times with sterilized Millipore water. Filter paper was used as the inert material for germination. The paper was cut to fit the Petri dishes and sterilized to avoid contamination. Ten kernels were placed between two pieces of paper and watered with 5 mL of ZnO or Zn2+ suspension/solution [18]. Two milliliters of antimycotic/antibiotic solution (A5955, Sigma Aldrich) were added to the top of the second filter paper. Petri dishes were covered with aluminum paper and placed at 20, 25, and 30 °C. According to USEPA [19], seeds were allowed to germinate until about a 65% of the root controls were at least 5 mm long. Percent of germination was calculated in every treatment and every temperature. Seedlings were rinsed with 0.01 M HNO3 and Millipore water to eliminate any surface metal. Root and stem length of 10 seedlings per replicate were measured and oven dried at 70 °C for two days. The average weight was calculated on a 10-seedling basis. A second set of treatments at same temperatures and concentrations were placed for oxidative stress experiments. 2.3. Quantification of Zn in corn seedlings After 15 d of exposure to different treatments and ZnO and Zn+ 2 concentrations, seedlings were digested on a CEM microwave oven (CEM Corporation Mathews, NC; USA) with 3 mL of plasma pure HNO3 according to USEPA 3051 method [20]. Samples were diluted to 25 mL with Millipore water and Zn content was measured by ICP/OES Perkin Elmer Optima 4300 DV (Perkin-Elmer Optima 4300 DV, Shelton, CT). A blank and a standard were read every ten samples for QC/QA purposes.

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2.4. Determination of catalase specific activity Catalase activity was determined following the procedure previously described by Gallego et al. [21]. Corn seedlings exposed to different treatments for 15 d were homogenized in 0.1 M KH2PO4 buffer at pH 7.4 ± 0.1 and then centrifuged at 9000 rpm for 10 min in a refrigerated centrifuge (MWP Med. Instruments, Warsaw, Polland).The supernatant was placed in a quartz cuvette with 10 mM H2O2 in phosphate buffer and the absorbance was recorded at 240 nm using a UV/VIS Spectrophotometer (UV–Visible Spectrophotometer, Evolution 60S, Thermo Scientific, China). 2.5. Determination of ascorbate peroxidase activity Ascorbate peroxidase was determined according to the procedure previously described by Murgia et al. [22], with slight modifications. Seedlings were homogenized in a solution containing 0.1 M KH2PO4 (buffer at pH 7.4 ± 0.1), 25 mM ascorbate and 17 mM H2O2. The mixture was homogenized and the absorbance was recorded at 265 nm in a UV/VIS Spectrometer (UV–Visible Spectrophotometer, Evolution 60S, Thermo Scientific, China). Bovine serum albumin (BSA) was used as a standard to quantify the protein content in corn seedlings. 2.6. X-ray absorption spectroscopy experiments (XAS) Corn seedlings exposed to 1600 mg ZnO L−1 and 250 mg Zn2+ L−1 for 15 d were frozen in liquid nitrogen and lyophilized on a Labconco FreeZone 4.5 freeze-dryer at − 53 °C and 0.140 mBar pressure for 3 days. Powdered dry tissues were placed on aluminum sample holders and covered with Mylar© Tape. XAS experiments were done at beam Line 7–3 at the Stanford Synchrotron Radiation Laboratory (SSRL). A Canberra 29-element array germanium detector was used to monitor Zn Kα fluorescence spectra. The standard operating conditions of the beam line were 3 GeV beam energy, a 50–100 mA beam current, and a Si (220) φ 90 monochromator. Spectra from samples were calibrated with spectra from Zn foil at the time of data collection. Data analysis was done using Athena software [23]. Zinc edge energy was calibrated using the edge position of an internal zinc foil with edge energy of 9665 eV. AUTOBK algorithm was used in spectra background subtraction according to Newville et al. [24]. Edge-step data normalization was determined by a linear preedge subtraction and regression of a quadratic polynomial beyond the edge. Polynomial difference is extrapolated to E0 and used as normalization constant in the relationship χ (E) = [μ(E) − μ0(E)] / μ0(E0). Normalized data is obtained after subtraction of the curvature of the regressed quadratic and the difference in slope between the post- and pre-edge polynomials after the edge. Main parameters used for fine tune normalization and background removal were: edge-step = 0.83, normalization range 150–663, k-weight = 2, and E shift = 1. 2.7. SDS-PAGE analysis Total protein was extracted from corn seedlings exposed to different concentrations of ZnO NPs and Zn2+. Protein concentration of each extract was determined using a Bradford assay. Furthermore, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out as described by Laemmli [25]. Briefly, the corn seedling extracts (15 μg of protein) were mixed with loading buffer, subjected to electrophoresis using 12% polyacrylamide gels at a constant voltage (90 V). Molecular weight standards were obtained from BIO-RAD. Gels were stained with Coomasie Brilliant Blue R-250. 2.8. Statistical analysis Data from all experiments was reported as mean ± Standard Deviation (SD). A one way ANOVA analysis followed by a Tukey's H.S.D. was

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used to determine statistical differences between means. Minitab Express 17.0 (State College PA, USA) was used to perform the analysis. Statistical significance was based on probabilities of p ≤ 0.05.

concentrations mitigated the stress caused by temperature. Further studies need to be done to determine the role of Zn2 + ions in ABA synthesis. 3.2. Effect of temperature, ZnO NPs, and Zn2+ on root growth

3. Results and discussion

Effects of Zn and temperature treatments on seed germination are shown in Fig. 1. As seen in Fig. 1A, at 30 °C there were no effects; however at 25 °C, 400 mg ZnO NP L−1 significantly reduced the germination by about 53% compared with the respective controls. At 20 °C, the 400 and 1600 mg L− 1 treatments significantly reduced the germination, compared with controls (47 and 26% respectively). De la Rosa et al. [26] reported that germination of cucumber seeds exposed to 1600 mg L−1 of ZnO NPs increased 10%; however, alfalfa and tomato reduced their germination percentage by 40 and 20% respectively. Fig. 1B shows the results for Zn2+ exposure, supplied as Zn (NO3)2. These concentrations were chosen according to the Zn2+ concentrations found in the ZnO NPs suspensions used in the present study. This figure shows that none of the ionic treatments interfered with the germination. Thus, very likely, the effect observed with ZnO NP treatments were due to the NPs presence and not to the Zn ions. Deng et al., [15] demonstrated the effect of temperature and essential and non-essential heavy metals on corn seed germination. They found that trace concentrations of Cu2+, Cd2+, and Hg2+ as well as high temperatures (40 °C), affected seed dormancy. Stress caused by non-essential metals and temperature can increase production of reactive oxygen species (ROS) and reverse the abscisic acid (ABA) role in seed germination decreasing stomatal activity and producing seed dormancy [27]. However, essential metals such as Cu2+ at low concentrations (0.1 mM) may increase seed germination by stimulating ROS production and decreasing ABA accumulation in seeds. Zinc is an essential metal which acts as co-factor of a large number of enzymes. It could be possible that Zn at low

Fig. 2A shows the effect of ZnO NPs and temperature on corn seedlings root growth. It is interesting to note that the response to the three temperatures followed similar patterns (a sort of cosine form), with higher increase/reduction at different NP concentrations. At 20 °C, 50, 400, and 1600 mg ZnO NP L−1 treatments produced similar reductions 18, 47, and 26% respectively. Fig. 2B shows that the elongation of roots under exposure to ionic Zn followed a similar pattern, compared to NP exposure. At 25 °C, 1 and 5 mg L−1 of Zn2 + the root elongation was statistically reduced compared with control (40 and 33% respectively). Several researchers have studied the effect of the soil temperature and heavy metals on the germination and root growth of plants [15,28,29]. They stated that soil temperature plays a very important role in the root plant development because root system processes can be modified or inhibited. Inadequate soil temperature will result in alterations of root length, branching, nutrient, and water uptake, among others. On the other hand, excess of heavy metals can cause detrimental effects on seed germination and plant growth. Lipids and polypeptide composition in plasma membranes also change with temperature and HM uptake (specifically phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid) modifying protein conformation, proton transport and ATPase activity [30,31]. Nevertheless, effect of NPs such as ZnO at different temperatures on the corn root growth has not been evaluated. ZnO nanoparticles have been reported to induce toxicity in some plant species because of the release of Zn2+ ions and nanoparticle agglomeration. Triphaty et al. [32] mentioned that the probability of NP aggregation increased with the increase in concentration, being the smaller one more prone to dissolve than the larger ones. Thus, the difference in concentration between 800 and 1600 affected the aggregation and

Fig. 1. Germination percentage in corn seeds exposed to: A) ZnO NPs, and B) Zn provided as Zn(NO3)2 at □ 20 °C; ■ 25 °C; and 30 °C. Means followed by the same letter are not statistically different and * indicates a significant difference (Tukey's test p ≤ 0.05). Error bars indicate ±S.E.

Fig. 2. Root length in corn seedlings exposed to different Temperatures (□ 20 °C; ■ 25 °C; and 30 °C) and germinated in A) ZnO NPs and B) Zn provided as Zn(NO3)2. Means followed by the same letter and * are not significantly different (Tukey's test p ≤ 0.05). Error bars indicate ±S.E.

3.1. Effect of temperature, ZnO NPs, and Zn+2 on seed germination

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dissolution, resulting in different effects on root elongation. Since the concentration of Zn2+ ions was similar in the 2 NP suspensions (25.62 ± 0.1 and 24.17 ± 0.05 for 800 and 1600 mg L− 1), we hypothesize that the difference was due to the size of the aggregates that could cause a physical interference in water and nutrient uptake. The particular response in seed germination and root growth observed at 400 mg L−1 cannot be explained with the data gathered and further experiments are needed in order to clarify these effects. As we mentioned before, at 20 °C, none of the Zn2+ concentrations significantly affected the elongation of corn roots. Conversely, at 30 °C there was a significant increase in root length of plants exposed to 0.1 mg L−1. At 0.1 mg L−1 of Zn corn roots grew 18.6 ± 7.0, 9.3 ± 8.0, and 44.0 ± 8.3% more than control roots at 20, 25, and 30 °C, respectively. Li and co-workers [33] corroborated a relationship between Zn concentration and cell viability in the root tips from wheat plants. They found that excess Zn promoted loss of cell viability on root tips, ending in a lignification of root cell walls that prevented their growth. It is possible that this occurred under exposure to 30 °C, where there was a clear tendency to root reduction, compared with those treatments at 20 and 25 °C. 3.3. Effect of temperature, ZnO NPs, and Zn2+ on corn biomass production According to plant species, plant biomass can be altered by temperature changes because of the decrease in the nutrient uptake by roots [34]. In this study, none of the ZnO NPs and Zn2+ ions concentrations, at any of the three temperatures, affected the biomass production (data not shown). However, Rao and Shekhawat [35] reported a decrease in Brassica juncea biomass exposed to ZnO NPs in a range of (0– 1500 mg ZnO L−1). The authors stated that biomass production is a primary indicator of ZnO NP toxicity, since the NPs in the root vascular system decrease the uptake of water, nutrients, and plant tissues growth. However, these authors did not mention the temperature where plants were grown. 3.4. Zn uptake in corn seedlings exposed to ZnO NPs and Zn2+ ions at different temperatures Zinc concentrations in root seedlings exposed to the ZnO NPs and Zn2 + treatment concentrations at the three temperatures are shown in Figs. 3A-B. As seen in Fig. 3A, root Zn slightly increase, at the three temperatures, under exposure up to 800 mg L−1 of ZnO NPs. However, root Zn in plant exposed to 1600 mg L− 1 was significantly higher, at the three temperatures, compared with control and the other treatments. This could be due to the present of particles attached to the root surface that were not removed by the washing process. It could also be due to the rot absorption of NPs and Zn 2 + ions. The concentration of Zn2 + resulted from the dissolution of ZnO NPs in the suspension of 1600 mg L− 1 was approximately 25 mg Zn L − 1 (data not shown).This suggests that ZnO NPs within the, plus the Zn ions from dissolution, contributed to the high Zn concentration found in this treatment [36]. Fig. 3B shows the concentration of Zn found in plants exposed to ionic Zn at the three temperatures. As seen in this figure, only seedlings exposed to the highest concentration treatment (250 mg L−1) showed significantly higher root Zn. However, the temperature was not a factor that determines the uptake of Zn by corn roots.

Fig. 3. Zn concentration in corn kernels germinated in A)ZnO and B) Zn(NO3)2 at □ 20 °C; ■ 25 °C; and 30 °C. Means followed by the same letter and * are not significantly different (Tukey's test p ≤ 0.05). Error bars indicate ± S.E.

and temperatures of 20, 25, and 30 °C. As shown in this figure, only numerical increments or decrement were recorded. Srivastava et al. [39] reported a higher CAT and APOX activities in leaves of sugarcane exposed to high temperatures (40 °C). Lukatkin et al. [40] also stated that response of plants to low temperatures may reduce the activity of several enzymes. Fig. 5 displays the APOX enzymatic activity for corn seedlings exposed to ZnO and Zn2+ treatments at different temperatures. It can be observed that APOX follow the same behavior than CAT in ZnO NP treatments at 20, 25, and 30 °C (Fig. 5A, B, and C), and Zn+2 treatments (Fig. 7D, E, and F). However, only seedlings exposed to 25 °C and ZnO NPs showed statistical differences in APOX activity (Fig. 5B). This figure shows that APOX activity increased as the concentration of ZnO NPs increased; however, the differences were statistically significant only at 400 and 1600 mg L−1, compares with control. Since the exposure to 250 mg L− 1 of ionic Zn at 25 °C did not produced significant effects, and the Zn ions released by the NPs were 25 mg L−1, we strongly believe that the effects was due to the presence of the NPs. APOX is found in chloroplasts and cytosol and its affinity for H2O2 is higher that CAT enzyme, which is found at the peroxisomes (Km for APOX = 20–50 μM, Km for CAT = 40–60 mM) [41]. It is clear than CAT is less efficient removing H2O2 than APOX (detoxification). Several studies have been reported metal oxide NPs such as ZnO, CuO, and NiO among others produced more ROS inside plant tissues than their bulk counterparts [42]. 3.6. XANES studies of seedlings exposed to ZnO NPs and Zn2+

3.5. CAT and APOX activities in corn seedlings exposed to ZnO NPs and Zn2+ ions at different temperatures Increase or decrease in enzyme activities result in changes in seed viability and vigor influencing plant growth and development [37,38]. It is well known that ROS are produced inside plant tissues as response to biotic or abiotic stress. Fig. 4 shows the CAT enzymatic activity in corn seedlings exposed to different concentrations of ZnO NPs and Zn2 +

XANES spectra from corn seedlings exposed to 250 ppm of Zn2+ and 1600 ppm of ZnO NP is shown in Fig. 6(a-b). From these figures, it can be seen that temperature did not affect Zn speciation in corn seedlings in both treatments (ZnO NP and Zn2+). Zn is present in plant tissues as Zn2+ resembling the spectra of Zn (NO3)2. It is suggested that Zn2+ inside plant tissues likely comes from the biotransformation of ZnO NP [43,44]. Some researchers reported that Zn in plant tissues has been

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Fig. 4. Catalase specific activities in corn seedlings exposed to different concentrations of A) ZnO NP at 20 °C, B) ZnO NP at 25 °C, C) ZnO NP at 30 °C, D) Zn(NO3)2 at 20 °C, E) Zn(NO3)2 at 25 °C, and F) Zn(NO3)2 at 30 °C. Data are average from extracts of corn seedlings. Means followed by the same letter are not significantly different (Tukey's test p ≤ 0.05). Error bars represent ±S.E.

found to be tetrahedrally coordinated and complexed to carbonyl and hydroxyl groups [45]. Salt et al. [46] also reported that Zn in plant tissues of the hyperaccumulator Thlaspi caerulescens transported through plant xylem as Zn2+ and coordinated to the neutral ring and amino nitrogens and to the oxygen from a carboxyl group to form Zn(His)2 complex. Castillo-Michel et al., [47] stated that ligand coordination between atoms promoted changes in the white line position (edge) by about 1 eV. They conclude that Zn\\S bonds have edges at lower energies than Zn-(O/N)

bonds. A broad XANES and EXAFS study done by Liu et al., [33] revealed that Zn2+ is complexed to O ligands in leaves and stems of Sedum alfredii plants, specifically to malic acid. On the other hand, citric acid plays a role in Zn2+ complexation in plant roots. The authors associate plant detoxification from Zn2+ levels to the vacuole sequestration of Zn-malic acid complexes mainly in leaves of Sedum alfredii plants. However, further deep EXAFS analysis need to be done in corn seedlings in order to clarify Zn biotransformation mechanisms inside plant tissues.

Fig. 5. Ascorbate peroxidase specific activities in corn seedlings exposed to different concentrations of A) ZnO NP at 20 °C, B) ZnO NP at 25 °C, C) ZnO NP at 30 °C, D) Zn(NO3)2 at 20 °C, E) Zn(NO3)2 at 25 °C, and F) Zn(NO3)2 at 30 °C. Data are average from extracts of corn seedlings. Error bars represent ±S.E.

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Fig. 6. XANES K-edge spectra (9665 eV) of reference materials (ZnO NP and Zn(NO3)2) and corn seedlings exposed to A) 250 mg L−1 Zn(NO3)2 and B) 1600 mg L−1 of ZnO at 20, 25, and 30 °C.

3.7. Protein profile analysis by SDS-PAGE Total protein was extracted from corn seedling exposed to different concentrations of ZnO NPs and Zn2+ and analyzed by SDS-PAGE (Figs. 7 and 8). No visual changes in the protein profiles were observed between corn seedling exposed to increasing of ZnO NPs concentrations and different temperatures (Fig. 7). Fig. 8 shows a protein profile between 25 and 30 °C in corn seedlings exposed to different Zn2+ concentrations with no changes observed. However, we can visualize a protein band with molecular weight of 85 kDa at 25 °C, which decreases its expression at 30 °C. In addition, a protein of 75 kDa increases its expression at 30 °C. Zhao el al. [48] reported an increase in Heat-shock proteins (HSP) when corn seedlings were exposed to different concentrations of CeO2 NPs. The authors stated that CeO2 NPs seems to remain in the

nano form inside corn seedlings reaching the chloroplasts and producing an overexpression of some HSP. However, these researchers did not have data from different temperature studies. Results reported in the literature correlate ROS production in plant cells (due to environmental stress) to the increase in gene expression [49,50]. Alharby et al., [50] reported an increase in mRNA levels of SOD and GPX in tomato plants exposed to salinity. These researchers observed that ZnO NPs (at 15 and 30 mg L−1) have a positive effect on reducing H2O2 levels in tomato cultivars and suggest that this may be due to the increase in key nutrient uptake (such as nitrogen), which plays an important role in protein synthesis, metabolic pathways, and ion homeostasis. Changes in molecular markers were produced in plants exposed to ZnO NPs. Authors stated that SDS-PAGE results showed the appearance of protein bands at 74.991, 25.801, and 19.059 kDa in

Fig. 7. SDS-PAGE analysis of protein extracted from corn seedling grown under different concentrations of ZnO NPs at 25 °C and 30 °C. Lines 1: control; 2: 100 mg L−1; 3: 400 mg L−1; 4: 1600 mg L−1; MM: molecular mass marker.

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Fig. 8. SDS-PAGE analysis of protein extracted from corn seedling grown under different concentrations of Zn2+ at 25 °C and 30 °C. Lines 1: control; 2: 1 mg L−1; 3: 25 mg L−1; 4: 250 mg L−1; MM: molecular mass marker.

tomato cultivars. These results suggested that plants counteract abiotic stress increasing protein synthesis as well as adjusting some biochemical processes. Moreover, Nair and Chung [51] reported that Arabidopsis thaliana seedlings treated with ZnO NPs (20–200 mg L−1) induced AtKC1 and AtCHX17 gene expression in roots and shoots. These genes are responsible for maintaining mineral homeostasis in Arabidopsis thaliana plants. The authors concluded that exposure to ZnO NPs instead of Zn2+ from salts can be a very good alternative for plant nutrition due to the fact that mechanisms of Zn uptake and translocation as well as genes regulation are very different when seedlings are exposed to NPs or ions. Further experiments need to be done in order to provide more information about the effect of ZnO NPs on protein synthesis. 4. Conclusions Results from this study shown that the ambient temperature can modify the effects of ZnO NPs exposure to plants. In addition, it seems that the response is also associated with exposure the concentration. For example, at 20 °C, 400 and 1600 mg L− 1 significantly reduced seed germination, while at 25 °C, only 400 mg L− 1 caused an effect. On the other hand, none of the Zn2+ (0–250 mg L−1) affected the germination, which strongly suggest that the effects were due to the NPs. Different effects were observed in root growth. The 400 mg L−1 treatment produced significant root growth reductions only at 20 and 25 °C, while at 800 mg L−1, 20 and 25 °C produced significant increases. Responses observed with Zn2+ treatments strongly suggest that the observed effects with ZnO were mostly due to the presence of the particles. Although Zn in NP exposed roots was not in the nano form, and the amount measured higher than in Zn2+ exposed plants, there was no significant oxidative stress, except for APOX at 25 °C, at the highest concentrations. Moreover, The NP exposure did not alter protein expression, which was observed under Zn2+ treatments. This corroborate that the effects of ZnO NPs in plants depend on the interaction with environmental factors such as temperature. Acknowledgements This material is based upon work supported by the National Science Foundation and the Environmental Protection Agency under Cooperative Agreement Number DBI-1266377. Any opinions, findings, and

conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation or the Environmental Protection Agency. This work has not been subjected to EPA review and no official endorsement should be inferred. The authors also acknowledge the USDA grant 2016-67021-24985 and the NSF Grants EEC-1449500, CHE-0840525 and DBI-1429708. Partial funding was provided by the NSF ERC on Nanotechnology-Enabled Water Treatment (EEC-1449500). This work was also supported by Grant 2G12MD007592 from the National Institutes on Minority Health and Health Disparities (NIMHD), a component of the National Institutes of Health (NIH). J. L. Gardea-Torresdey acknowledges the Dudley family for the Endowed Research Professorship and the Academy of Applied Science/US Army Research Office, Research and Engineering Apprenticeship program (REAP) at UTEP, grant #W11NF-10-2-0076, sub-grant 13-7, and the LERR and STARs programs of the University of Texas System. References [1] Bradley, R. 2015. The great big question about really tiny materials. (Fortune.com 193-200). [2] A.D. Servin, J.C. White, Nanotechnology in agriculture: next steps for understanding engineered nanoparticle exposure and risk, NanoImpact 1 (2016) 9–12. [3] P. Liou, F.X. Nayigiziki, F. Kong, A. Mustapha, M. Lin, Cellulose nanofibers coated with silver nanoparticles as a SERS platform for detection of pesticides in apples, Carbohydr. Polym. 157 (2017) 643–650. [4] T. Tolaymat, A. Genaidy, W. Abdelraheem, D. Dionysiou, C. Andersen, The effects of metallic engineered nanoparticles upon plant systems: an analytic examination of scientific evidence, Sci. Total Environ. 579 (2017) 93–106. [5] D. Wang, Q. Chen, H. Huo, S. Bai, G. Cai, W. Lai, J. Lin, Efficient separation and quantitative detection of Listeria monocytogenes based on screen-printed interdigitated electrode, urease and magnetic nanoparticles, Food Control 73 (2017) 555–561. [6] A.H. Wani, M.A. Shah, A unique and profound effect of MgO and ZnO nanoparticles on some plant pathogenic fungi, J. Appl. Pharm. Sc.i 2 (3) (2012) 40–44. [7] R. Liu, R. Lal, Potentials of engineered nanoparticles as fertilizers for increasing agronomic productions, Sci. Total Environ. 514 (2015) (2015) 131–139. [8] S. Davarpanah, A. Tehranifar, G. Davarynejad, J. Abadía, R. Khorasani, Effects of foliar applications of zinc and boron nano-fertilizers on pomegranate (Punica granatum cv. Ardestani) fruit yield and quality, Sci. Hortic. 210 (2016) 57–64. [9] M. Ghosh, A. Jana, S. Sinha, M. Jothiramajayam, A. Nag, A. Chakraborty, A. Mukherjee, A. Mukherjee, Effects of ZnO nanoparticles in plants: cytotoxicity, genotoxicity, deregulation of antioxidant defenses, and cell-cycle arrest, Mutat. Res. 807 (2016) 25–32. [10] https://www.ers.usda.gov/topics/crops/corn/background.aspx. [11] R.K. Mall, A. Gupta, G. Sonkar, Effect of Climate Change on Agricultural Crops in: Current Developments in Biotechnology and Bioengineering: Crop Modification, Nutrition, and Food Production, Elsevier B.V., 2017 23–46.

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