Article pubs.acs.org/JAFC
CeO2 and ZnO Nanoparticles Change the Nutritional Qualities of Cucumber (Cucumis sativus) Lijuan Zhao,†,‡ Jose R. Peralta-Videa,†,‡,§ Cyren M. Rico,†,‡ Jose A. Hernandez-Viezcas,† Youping Sun,∥ Genhua Niu,∥ Alia Servin,† Jose E. Nunez,† Maria Duarte-Gardea,⊥ and Jorge L. Gardea-Torresdey*,†,‡,§ †
Chemistry Department, The University of Texas at El Paso, 500 West University Avenue, El Paso, Texas 79968, United States UC Center for Environmental Implications of Nanotechnology (UC CEIN), The University of Texas at El Paso, 500 West University Avenue, El Paso, Texas 79968, United States § Environmental Science and Engineering PhD Program, The University of Texas at El Paso, 500 West University Avenue, El Paso, Texas 79968, United States ∥ Texas AgriLife Research Center at El Paso, Texas A&M University System, 1380 A & M Circle, El Paso, Texas 79927, United States ⊥ Department of Public Health Sciences, The University of Texas at El Paso, 500 West University Avenue, El Paso, Texas 79968, United States ‡
S Supporting Information *
ABSTRACT: There is lack of information about the effects of nanoparticles (NPs) on cucumber fruit quality. This study aimed to determine possible impacts on carbohydrates, proteins, mineral nutrients, and antioxidants in the fruit of cucumber plants grown in soil treated with CeO2 and ZnO NPs at 400 and 800 mg/kg. Fourier transform infrared spectroscopy (FTIR) was used to detect changes in functional groups, while ICP-OES and μ-XRF were used to quantify and map the distribution of nutrient elements, respectively. Results showed that none of the ZnO NP concentrations affected sugars; however at 400 mg/kg, CeO2 and ZnO NPs increased starch content. Conversely, CeO2 NPs did not affect starch content but impacted nonreducing sugar content (sucrose). FTIR data showed changes in the fingerprint regions of 1106, 1083, 1153, and 1181, indicating that both NPs altered the carbohydrate pattern. ZnO NPs did not impact protein fractionation; however, CeO2 NPs at 400 mg/kg increased globulin and decreased glutelin. Both CeO2 and ZnO NPs had no impact on flavonoid content, although CeO2 NPs at 800 mg/ kg significantly reduced phenolic content. ICP-OES results showed that none of the treatments reduced macronutrients in fruit. In case of micronutrients, all treatments reduced Mo concentration, and at 400 mg/kg, ZnO NPs reduced Cu accumulation. μXRF revealed that Cu, Mn, and Zn were mainly accumulated in cucumber seeds. To the best of the authors’ knowledge this is the first report on the nutritional quality of cucumber fruit attributed to the impact of CeO2 and ZnO NPs. KEYWORDS: CeO2 NPs, ZnO NPs, Cucumber, Fruit nutritional quality, Carbohydrates, Proteins
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INTRODUCTION The rapid development of nanotechnologies has encouraged the production of engineered nanoparticles (NPs) worldwide. Very likely, a portion of these NPs will reach agricultural soils after end-user application, accidentally, or through their direct use in the delivery of agricultural products.1−3 Therefore, it is important to elucidate the behavior of NPs in terrestrial ecosystems and their interaction with plants, especially edible plants. ZnO and CeO2 NPs are among the most common metal oxide NPs in use, therefore the transport and toxicity of those NPs in plants is being intensely studied.1 Zinc is among the 18 essential elements for plant growth and development, while Ce is not considered essential.4 In addition, CeO2 NPs has lower dissolution compared to ZnO NPs;5 thus, as evident from previous reports, the contribution of Ce ions to the observed response is expected to be low. Schwabe et al. observed translocation of CeO2 from root to shoot in pumpkin (Cucurbita maxima Gelber Zentner) plants.6 Zhang et al. also reported that a portion of the CeO2 found in cucumber (Cucumis sativus) shoot had a different form compared to the CeO2 found in root.7 The chance of translocation of NPs into the edible tissues and their possible impact on food safety and © 2014 American Chemical Society
quality are a main public concern. However, up to now, very few studies have been conducted through the entire life cycle of a plant. Accordingly, the knowledge regarding the bioaccumulation of NPs in edible tissue and the impact on fruit quality remains largely unknown. Wang et al. conducted a life cycle study and revealed that CeO2 NPs entered into the tomato fruit.8 In a subsequent study, they found that the seeds collected from CeO2 NP treated tomato plants were smaller and weaker compared to control, indicating that the next generation was impacted.9 In another life cycle study, by using μ-XRF and μ-XANES technique, Hernandez-Viezcas et al. reported the presence of CeO2 NPs in soybean pods.10 In our most recent study, we found that Ce accumulated in cucumber fruit of plants exposed to 800 mg/kg CeO2 NPs in greenhouse conditions.11 It is still unknown whether Ce/CeO2 NPs influence the plant metabolism and nutrient element transport in plants. Kole et al. Received: Revised: Accepted: Published: 2752
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Table 1. Effects of CeO2 and ZnO Nanoparticles on Sugar Content of Cucumber Fruita reducing sugar control CeO2 400 mg/kg CeO2 800 mg/kg ZnO 400 mg/kg ZnO 800 mg/kg a
3.72 3.11 5.35 4.98 4.50
± ± ± ± ±
0.27ab 0.34b 0.56a 0.64ab 0.42ab
nonreducing sugar 23.3 ± 3.00b 8.31 ± 0.96c 37.2 ± 4.05a 33.9 ± 2.41ab 30.4 ± 0.74ab
total sugar 27.0 11.4 42.5 38.9 34.9
± ± ± ± ±
2.73b 1.09c 4.39a 3.04ab 0.96ab
starch 70.9 90.7 90.6 110.8 81.0
± ± ± ± ±
11.3b 5.9 ab 4.5ab 2.6a 2.9b
Data are average of four replicates ± standard deviation.
showed the first evidence that fullerol greatly increased bitter melon water content and fruit yield and improved fruit quality by increasing the phytomedicine content.12 Servin et al. reported an increase in phosphorus and potassium concentration in cucumber fruit of plants exposed to 750 mg/kg of TiO2 NPs.13 In addition, Rico et al. reported that CeO2 at 500 mg/kg soil modified the quality of rice grains.14 Cucumber is one of the very low calorie vegetables. In addition it contains many nutrient elements, including Ca, Fe, Mg, P, K, Na, Mn, S, and Zn. According to USDA National Nutrient Database, 100 g of fresh cucumber provides as much as 147 mg of K, which is an important intracellular electrolyte.15 In addition, cucumber contains phenolic compounds (pcoumaric, caffeic, and ferulic acids and p-coumaric acid methyl ester), and those compounds act as protective scavengers against oxygen-derived free radicals and reactive oxygen species (ROS) that play a role in cardiovascular disease prevention and anticancer activity.16−18 The aim of this study was to study the effect of ZnO and CeO2 NPs on the nutritional quality of cucumber fruit. Carbohydrates, proteins, antioxidants, and mineral nutrient were analyzed by using several biochemical and spectroscopic techniques.
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extraction was repeated three times, and all the supernatants were combined. Total soluble sugar content was measured following the method of Dubois et al.20 while reducing sugar content was determined according to the procedure of Nelson-Somogyi.21 The nonreducing sugar content was reported as the difference between total and reducing sugars. Determination of Starch in Fruit. The starch extraction was performed following the method of Verma and Dubey.19 The residue from sugar extraction was dried at 80 °C for 24 h. To the dried residue was added 2 mL of DI water, and the mixture was boiled in a water bath for 15 min. The suspension was cooled to room temperature, and 2 mL of concentrated H2SO4 was added. The suspension was stirred for 15 min, and the final volume was adjusted to 10 mL using water. The supernatant was centrifuged at 3000g for 20 min, and the extraction was repeated using 50% H2SO4. The supernatants were combined and diluted with water to the 50 mL mark. The starch content was quantified following the method of Dubois et al.20 Protein Fractionation. Protein extraction and fractionation was conducted according to Chen and Bushak.22 Dried cucumber fruit (500 mg) was extracted sequentially with 8 mL each of water, 0.5 mol/ L NaCl, 70% ethanol, and 0.05 M acetic acid. The extracted protein in each step was labeled as albumin, globulin, prolamin, and glutelin fractions, respectively. The protein content was quantified by the Bradford method.23 Total Phenolics and Total Flavonoids. Total phenolic content was determined by the spectrophotometric method followed by Dewanto et al.24 At room temperature, Folin−Ciocalteu phenol reagent (0.2 mL) was added to each sample (0.1 mL). After 1 min, 3 mL of 5% Na2CO3 solution was added to the mixture and the mixture was allowed to stand for 1 h in a dark place. The absorbance was measured at 760 nm using a Perkin-Elmer Lambda 14 UV/Vis Spectrometer (single-beam mode, Perkin-Elmer, Uberlinger, Germany). Total phenolic contents of the sample were expressed on a fresh weight basis as mg of gallic acid equivalent (GAE) g−1. Total flavonoid content of cucumber fruit was measured using the method of Jia et al.25 Briefly, to 500 μL of each sample was added 75 μL of 5% NaNO2. After 5 min, to the mixture were sequentially added 150 μL 10% AlCl3, 500 μL 1 M NaOH, and 275 μL of DI water. The absorbance was measured at 510 nm using a spectrophotometer. The total flavonoid content was expressed as catechin equivalents. Fourier Transform Infrared (FTIR) Spectra Acquisition. The dried cucumber fruits were ground, and the powder was analyzed using FTIR spectroscopy (Perkin-Elmer, Spectrum 100, Universal ATR Sampling Accessory) in a range of 650−3950 cm−1. The second derivative spectrum was collected using Spectrum software (Version 6.0.2.0025, Perkin-Elmer, Shelton, CT). A total of 3 replicates per treatment were used for the analysis. μ-XRF Analysis. Fruits were carefully washed with DI water to eliminate any surface contaminants. Then, they were transversally cut and frozen in liquid nitrogen for 30 min. The samples were fixed with Tissue Tek (Sakura Finetek USA, Torrance, CA) and sectioned with a cryomicrotome (Triangle Biomedical Sciences, Durham, NC) at −20 °C to a thickness of 30 μm. Subsequently, the samples were mounted onto Kapton tape and freeze-dried for 1 h in a Labconco freeze-dryer (FreeZone 4.5, Kansas City, MO) with operating conditions of −53 °C and 0.140 mBar pressure. X-ray fluorescence imaging was obtained at beamline 10-2 at the Stanford Synchrotron Radiation Lightsource (Menlo Park, CA). Standard operating conditions were 3 GeV beam energy and 80−
MATERIALS AND METHODS
Characterization of NPs. The CeO2 and ZnO NPs (Meliorum Technologies, NY, U.S.) used in this study were obtained from the University of California Center for Environmental Implications of Nanotechnology. According to the supplier, the primary size for those two NPs is 10 nm. The characterization of both NPs, including hydrodynamic size, ζ-potential, and pH, were previously reported by Keller et al.5 Stock solutions were prepared by adding 1000 mg of nanoparticles to 1.0 L of deionized water. The dispersed nanoparticles were sonicated for 30 min in a water bath and immediately applied to the substrate. Experimental Design and Growth Conditions. CeO2 NPs and ZnO NPs were used, separately, at 400 and 800 mg/kg. Pots containing substrate without the addition of NPs were included as a control. Each treatment had four replicate (pots), and each pot contained two plants. More details regarding the experimental design, soil, and greenhouse conditions were introduced in previous a study.11 Mineral Content. Dried cucumber fruits were ground to pass a 40mesh screen (0.425 mm) with a stainless Wiley mill (Thomas Scientific, Swedesboro, NJ). Subsequently, the samples were digested with a mixture of H2O2 and plasma pure HNO3 (v/v: 4:1) using microwave oven (CEM Corp, Mathews, NC, U.S.).14 The standard reference materials NIST 1547 and 1570a were also digested and analyzed as samples. The recoveries for all elements were between 90 and 99%. The macro- and micronutrient elements were analyzed using inductively coupled plasma optical emission spectrometry (PerkinElmer Optima 4300 DV). Determination of Total Soluble and Reducing Sugars. The total soluble sugars were extracted by the method of Verma and Dubey.19 One hundred grams of dried cucumber tissue was ground in 10 mL of 80% ethanol and then boiled (80 °C) in a water bath for 30 min. After cooling to room temperature, the extracts were centrifuged at 22000g (Thermo Scientific, Soruall T1, U.S.) for 20 min. The 2753
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Figure 1. FTIR spectra of cucumber fruit treated with 0, 400, and 800 mg/kg ZnO NPs and 400 and 800 mg/kg CeO2 NPs. Spectra are means of three replicates.
Figure 2. Second derivative of FTIR band in the region of 800−1800 cm−1. 100 mA beam current. Incident X-ray energy was set to 10 keV, and a Si (111) monochromator was used. SMAK27 software was used for data analyses. Data Analysis. The treatments from two NPs at two concentrations and four replicate/treatments were allocated in a completely random design. All data including sugars, starch, protein, mineral nutrients, and antioxidant were analyzed by using PROC GLM in SAS software (Version 9.1.3, SAS Institute, Cary, NC).
sugars content was significantly increased, compared to control. Typically, sugars are a source of carbon and energy.28 On the other hand, sugars such as glucose, fructose, and sucrose also act as signaling molecules in plant responses under biotic and abiotic stresses.29,30 Among them, sucrose contributes to stressrelated responses.31 It has been reported that some of the defense-related genes were upregulated in rice leaves when sucrose was fed to the roots.32 In this study, the upregulation of sucrose by CeO2 NPs was a possible indicator of stress. In contrast, ZnO NPs at both concentrations did not impact the reducing and nonreducing sugar contents (Table 1). Leaves are plant’s main photosynthetic organs. Sugar and starch are synthesized in the chloroplasts of leaves and are translocated from leaves to fruits. Previous studies reported that reduced photosynthetic rate led to a decrease in the concentration of soluble sugars, and often starch accumulation in leaves.33 In our study, the photosynthetic rate in leaves was not decreased by ZnO NPs.11 This explained well that ZnO NPs did not change the carbohydrate composition in fruit.
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RESULTS AND DISCUSSION Effects of NPs on Fruit Carbohydrates (Sugar and Starch) Content. Sugars, starches, and fiber are main components of carbohydrates in cucumber fruit. Among them, “soluble sugars and starch are important parameters to evaluate the quality of fruits.”26,27 As shown in Table 1, reducing sugars (glucose and fructose) were not modified by CeO2 NPs. This implies that the sweet taste of cucumber fruit was not affected. However, the nonreducing sugar (sucrose) was impacted by CeO2 NPs. At 400 mg/kg treatment, the content of nonreducing sugars was significantly decreased compared to control, while at 800 mg/kg, the nonreducing 2754
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Table 2. Effect of CeO2 and ZnO NPs on Protein Fractionation and Antioxidant Content of Cucumber Fruita albumin control CeO2 400 mg/kg CeO2 800 mg/kg ZnO 400 mg/kg ZnO 800 mg/kg a
2.32 6.82 5.31 5.68 4.93
± ± ± ± ±
0.83 0.91 1.61 0.74 0.47
globulin a a a a a
0.75 1.32 0.44 1.28 0.86
± ± ± ± ±
0.11 0.17 0.07 0.08 0.04
prolamin b a b a ab
2.50 2.61 2.84 3.57 3.68
± ± ± ± ±
glutelin
0.15 0.54 0.11 0.15 0.21
a a a a a
0.20 0.07 0.25 0.18 0.39
± ± ± ± ±
0.03 0.02 0.00 0.04 0.01
flavonoid
phenolic b c b bc a
0.689 0.662 0.517 0.557 0.582
± ± ± ± ±
0.027 0.041 0.043 0.026 0.016
a ab b ab ab
0.537 0.596 0.403 0.505 0.426
± ± ± ± ±
0.056 0.076 0.047 0.003 0.003
a a a a a
Data are average of four replicates ± standard deviation.
Table 3. Effect of CeO2 and ZnO Nanoparticle on Mineral Content of Cucumber Fruit (mg/kg)a K control CeO2 400 mg/kg CeO2 800 mg/kg ZnO 400 mg/kg ZnO 800 mg/kg a
60224 64018 58837 60225 56533
Ca a a a a a
7249 8907 8436 6900 6988
Mg a a a a a
3349 3934 3452 3956 3583
S c ab c a bc
3443 3538 3260 3344 3481
P a a a a a
1136 1174 1134 1087 1059
Na a a a a a
589 580 522 527 544
Fe a a a a a
12.7 a 14.6 a 9.34 a 14.6 a 6.41 a
Cu 62.6 60.5 57.3 47.2 50.7
a a ab c bc
Mn 25.2 28.1 24.4 32.0 27.1
b ab b a b
Mo 22.9 a 5.48 c 9.93 bc 13.8 b 10.7 bc
Zn 36.1 38.3 37.7 61.3 89.1
c c c b a
Data are average of four replicates ± standard deviation.
Effects of NPs on Fruit Protein Fractions and Antioxidant Content. Although cucumber is a low protein vegetable, protein is an important factor contributing to the sweet taste of cucumber fruit.38 The effect of ZnO and CeO2 NPs on the four soluble protein fractions is presented in Table 2. Generally, albumin and prolamin fractions are the predominant proteins in cucumber fruit, which represents 84%−92% of the fruit protein. Globulin and glutelin fractions are present to a lesser extent. Results showed that neither CeO2 nor ZnO NPs significantly affected albumin and prolamin fractions. However, compared to control, globulin content was significantly increased by CeO2 NPs at 400 mg/kg treatment. Glutelin content was also significantly decreased by CeO2 NPs at 400 mg/kg. Those results indicated that CeO2 NPs at 400 mg/kg changed the protein content in fruit. CeO2 NPs at 800 mg/kg did not affect any of the protein fractions. More studies are needed in order to explain the different response at 400 and 800 mg/kg treatments. ZnO NPs at 400 mg/kg treatment increased globulin content while those at 800 mg/kg treatment increased glutelin content. Du et al. reported that salt stress increased the soluble protein content of cucumber seedlings.39 Several proteins such as heat shock protein, catalase, and glutathione S-transferase are related to stress response.40 Those proteins were upregulated under abiotic or biotic stress. It is possible that protein increased as a response to NP induced stress. Phenolic compounds including flavonoids and phenolic acids are the most frequently examined group of antioxidants, and they are highly correlated with the total antioxidant activity.41,42 This is an important trait in cucumber because phenolic compounds haven been associated with a reduction in breast and prostate cancer risks.43 As shown in Table 2, none of the CeO2 and ZnO concentrations impacted flavonoid content. However, CeO2 NPs at 800 mg/kg significantly decreased the phenolic content. As a result of this treatment there was an increase in total sugar content but a decrease in phenolic content. This indicates that, at this concentration, CeO2 NP impacted the nutritional quality of the fruit. Effects of NPs on the Fruit Mineral Composition. The uptake of nutrients by plant roots is affected by abiotic and biotic stressors including soil composition, cation exchange capacity, pH of the soil solution, microorganisms, and metal immobilization in root cell walls, among others.44,45 Cucumber
Starch is another important carbohydrate in fruits. From Table 1, it seems that all NP treatments increased the starch content compared to control, but only at 400 mg/kg ZnO NPs, the difference was significantly increased. Previous reports showed that copper stress induced the overaccumulation of starch and sucrose in cucumber plants.34 Another report indicates that salinity stress stimulated the starch accumulation in chloroplasts and resulted in increased accumulation of starch and total sugars in Thellungiella halophile leaves.35 This could indicate that starch and sucrose increased in cucumber fruit as a result of stress caused by the NPs. FTIR Analysis. As a rapid and noninvasive tool, Fourier transform infrared spectroscopy is used to identify changes in functional groups in plant tissues. FTIR spectra of cucumber fruit from plants treated with CeO2 and ZnO NPs are shown in Figure 1. As seen in this figure, the band area of the carbohydrate region (900−1200 cm−1) was reduced by all NP treatments, compared with the control. Furthermore, the difference between NP treatments and control can be seen in the lignin area (1635 cm−1). The data also showed band differences in control and NP treated plants in the lipid region located between 2840 and 2960 cm−1. In this region, control and 400 mg/kg CeO2 NP treatments were similar; however, the 800 mg/kg CeO 2 NP treatment and both ZnO NP concentrations reduced this band area. In addition, it was observed that the band areas at 1350−1420 cm−1 and 1520− 1650 cm−1 decreased in all NP treatments. The spectrum at 400 mg/kg CeO2 NPs was similar to that of the control, while those of the other three treatments were apparently lower than that of the control. For a better understanding of the fingerprint of carbohydrates, second derivatives of spectra were collected at the 800− 1800 cm−1 region. The plots of the second derivative spectra are shown in Figure 2. Carbohydrates (glucose, fructose, and sucrose) showed intense characteristic bands in the fingerprint region (900−1400 cm−1).36,37 It is clear from the spectra that the bands at 1181, 1153, 1106, and 1083 cm−1 were lower in the NP treatments, compared to control. According to Duarte et al.,37 spots 1153 and 1083 cm−1 are related to sucrose while 1108 cm−1 corresponds to fructose and glucose. Although it is difficult to quantify based on those bands, the spectra revealed that NPs changed the carbohydrate pattern in cucumber fruit. 2755
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Figure 3. μ-XRF temperature map from a cross cut cucumber fruit showing normalized K, Ca, Fe, Cu, Mn, and Zn intensities. The image corresponds to control fruit. Red represents higher intensity, and dark blue represents the absence of the element.
that ZnO NPs at 200 mg/kg soil and above increased the concentration of Zn in roots and shoots of corn plants.54 At all concentrations, both the CeO2 and ZnO NPs significantly reduced molybdenum concentration in cucumber fruit (Table 3). Reductions were in the range of 57 to 76% for CeO2 NPs and about 40 to 53% for the ZnO NP treatments. Previous studies have shown that Mo is taken up by plants as MoO42− .55 Reports indicate that Mo uptake is reduced by sulfate and increased by phosphate.56 In addition, previous work with alfalfa showed that Mo is complexed with malate.55 As shown in Table 3, P and S were at the same levels in control and NP treated plants; thus, we cannot conclude about the role of P or S in Mo uptake. Perhaps the NPs altered the production of organic acids like citrate or malate, and then, they reduced Mo uptake. Studies about the effects of CeO2 and ZnO NPs on organic acid production are currently being performed in order to explain the uptake of some micronutrients. The reduction in Mo in CeO2 and ZnO NPs treated cucumber might not represent a concern to human health as it has been documented that most people in the U.S. consume more than sufficient amounts of this trace mineral in their diets.48 Manganese was increased by ZnO NPs at 400 mg/kg. A previous study showed that 34% of Zn/ZnO NPs was bound to Mn−Fe oxides.57 At this time we cannot explain why this could increase Mn uptake. Additionally, we do not know the effect ZnO NPs produce in Nramp transporters, which are associated with Mn uptake by plants.58 The increase of Mn in ZnO NPs treatment might not represent a health concern especially in diets that include the consumption of foods high in phytic acid (beans, seeds, nuts, whole grains, and soy products), oxalic acid
is a source of vitamins, potassium, magnesium, and phosphorus required for human nutrition.46 The influence of both the CeO2 and ZnO NPs on the cucumber fruit mineral content is shown in Table 3. As seen in this table, from all macronutrients analyzed, only Mg showed significant differences in the NP treated plants. At 400 mg/kg, both the CeO2 and ZnO NPs increased Mg in cucumber fruit by 17% and 18%, respectively (p ≤ 0.05). Magnesium is an essential element for human health. It is obtained from foods and concentrates in bones and muscle.47 Magnesium participates in multiple functions including energy production, protein synthesis, cell membranes and chromosomes, ion transport across cell membrane, and cell migration and interacts with protein, fiber, vitamin D, calcium, and zinc.48 Magnesium is essential in plants as it is the core of the chlorophyll molecule. Mg2+ enters the root cortex cells through the apoplast.49 However, it is not clear how Mg2+ enters the symplast and then the xylem apoplast.50 To this end, we cannot explain why the CeO2 NPs increased Mg translocation to cucumber fruit. However, other NPs like CuO NPs have been found to increase the production of indole-3-acetic acid in bacteria,51 and this growth hormone induces the expression of two aquaporins in tomato (Lycopersicon esculentum Mill.). Magnesium has been found to be transported through the rca channel in wheat root.52 Thus, it is possible that the CeO2 NPs promote the expression of aquaporins or other channels, like the MRS2, which is Mg2+ transporter to the shoot, enhancing Mg2+ uptake.53 CeO2 and ZnO NPs changed the fruit profile of micronutrients Cu, Mo, Mn, and Zn. As expected, Zn was increased at both ZnO NP concentrations. Previous reports have shown 2756
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Table 4. Comparison of Effects of CeO2 NPs and ZnO NPs on Cucumber Fruit Qualitya soluble sugar CeO2 NPs 400 mg/kg CeO2 NPs 800 mg/kg ZnO NPs 400 mg/kg ZnO NPs 800 mg/kg a
starch
− +
protein fractionation
phenolic
flavonoid
globulin +; glutelin − − +
globulin + glutelin +
mineral Mg +, Mo − Mo − Mg, Mn +; Cu, Mo − Cu, Mo −
+ means positive impact; − means negative impact; blank means no impact.
CeO2 NPs not only decreased Mo concentration, they also altered the nonreducing sugars and phenolic content and they also changed the fractionation of proteins (Table 4). This suggests that CeO2 NPs impacted the fruit flavor and reduced the antioxidant capacity. Furthermore, as previously reported,11 the fruits were grouped by size as large, medium, and small and the total weight of each group was recorded. The fresh weight of large cucumbers was reduced by 31.4% by CeO2 NPs at 800 mg/kg (Table S1 in the Supporting Information). More studies have to be performed in order to determine the effects of NPs on other parameters such as lignin and pectin that can influence fruit weight. The above-mentioned results suggest that, at the concentration tested, both CeO2 and ZnO NPs impacted the nutritional quality of cucumber.
(spinach, cabbage, and sweet potatoes), and tannins (tea), compounds that might slightly inhibit manganese absorption. Intake of minerals including iron, calcium, and phosphorus have been found to limit retention of manganese.48 Copper was also reduced by ZnO NPs. Previous studies have shown that ZnO NPs dissociate releasing Zn ions into the medium,59 and according to Alloway, Zn decreases the uptake of Cu because both metals are taken up by the same transporter.60 Some experts propose that Cu deficiency rather than excess Cu increases the risk of cardiovascular diseases.48 More studies are needed in order to understand the effects of CeO2 and ZnO NPs at the root surface interface and their effect on nutrient uptake by plants. Micro-XRF Analysis of Mineral Localization in Cucumber Fruit. ICP-OES data only reflect total mineral concentration in cucumber fruit. In order to detect whether NPs altered the mineral distribution pattern, the μ-XRF technique was applied. Because of the limited beam time, only two macronutrients Ca and K and four micronutrients including Fe, Cu, Mn, and Zn were analyzed. The mineral distribution between CeO2 treated and control plants was very similar. This indicates that CeO2 NPs did not change the nutrient element distribution in cucumber plant. The microXRF intensity map of the cucumber fruit cross-section is shown in Figure 3. In general, K and Ca were localized in skin, pulp, and seeds, while iron was almost evenly distributed in the whole cross section. Figure 3 also shows that Cu, Zn, and Mn were more concentrated in seeds and at lower concentration in the skin. As revealed by ICP-OES results, Cu concentration was reduced by ZnO NPs. Furthermore, μ-XRF results indicate that Cu is mainly present in cucumber seeds (Figure 3). Therefore, the next generation could also be impacted. Cu is an essential element for plant growth because they participate in many important physiological processes such as photosynthesis and respiration.61 It has been reported that soaking seeds in Cu sulfate solution may enhance plant growth and development and, even, improve the nutritional quality of fruit.62 Thus, a reduction in Cu in cucumber seeds suggests that ZnO NPs may have a negative impact on the growth of next plant generation. More studies are needed in order to solve this knowledge gap. Conclusions. In summary, at the concentration tested, the ZnO NPs did not show negative impact on carbohydrate, protein, and antioxidant contents compared to control. To some extent, ZnO NPs increased starch and protein content, which might result in an increased caloric value of a food that is typically characterized by its low caloric content. However, it is not clear if the increased starch and protein contents are indicators of stress induced by ZnO NPs. Both ZnO NP treatments significantly decreased the concentration of the micronutrients Cu and Mo. Since both Cu and Mo are important minerals in plant tissues and participate in many processes, the quality of fruit in the next generation of cucumber plants may be impacted.
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ASSOCIATED CONTENT
S Supporting Information *
Table providing average weight of large, medium, and small cucumber fruits harvested from plants grown in CeO2 NP amended soil. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Phone: 915-747-5359. Fax: (915) 747-5748. Funding
This material is based upon work supported by the National Science Foundation and the Environmental Protection Agency under Cooperative Agreement Number DBI-0830117. The authors also acknowledge USDA Grant No. 2011-38422-30835 and NSF Grant No. CHE-0840525. J.L.G.-T. 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 No. W11NF-10-2-0076, Subgrant 13-7. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors 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.
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REFERENCES
(1) Rico, C. M.; Majumdar, S.; Duarte-Gardea, M.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L. Interaction of nanoparticles with edible plants and their possible implications in the food chain. J. Agric. Food Chem. 2011, 59, 3485−3498. (2) Priester, J. H.; Ge, Y.; Mielke, R. E.; Horst, A. M.; Cole Moritz, S.; Espinosa, K.; Gelb, J.; Walker, S. L.; Nisbet, R. M.; Schimel, J. P.;
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Palmer, R. G.; Hernandez-Viezcas, J. A.; Zhao, L.; Gardea-Torresdey, J. L.; Holden, P. A. Soybean susceptibility to manufactured nanomaterials with evidence for food quality and soil fertility interruption. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 2451−2456. (3) Hong, J.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L. Nanomaterials in agricultural production: benefits and possible threats? In Sustainable Nanotechnology and the Environment: Advances and Achievements; Sharma, V., Shamin, A. N., Eds.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013; Vol. 1124, pp 73−90. (4) Diatloff, E.; Smith, F. W.; Asher, C. J. Effects of lanthanum and cerium on the growth and mineral nutrition of corn and mungbean. Ann. Bot. 2008, 101, 971−982. (5) Keller, A. A.; Wang, H.; Zhou, D.; Lenihan, H. S.; Cherr, G.; Cardinale, B. J.; Miller, R.; Ji, Z. Stability and aggregation of metal oxide nanoparticles in natural aqueous matrices. Environ. Sci. Technol. 2010, 344, 1962−1967. (6) Schwabe, F.; Schulin, R.; Limbach, L. K.; Stark, W.; Burge, D. Influence of two types of organic matter on interaction of CeO2 nanoparticles with plants in hydroponic culture. Chemosphere 2013, 91, 512−520. (7) Zhang, P.; Ma, Y.; Zhang, Z.; He, X.; Zhang, J.; Guo, Z.; Tai, R.; Zhao, Y.; Chai, Z. Biotransformation of ceria nanoparticles in cucumber plants. ACS Nano 2012, 6, 9943−9950. (8) Wang, Q.; Ma, X.; Zhang, W.; Pei, H.; Chen, Y. The impact of cerium oxide nanoparticles on tomato (Solanum lycopersicum L.) and its implications for food safety. Metallomics 2012, 4, 1105−1112. (9) Wang, Q.; Ebbs, S. D.; Chen, Y.; Ma, X. Trans-generational impact of cerium oxide nanoparticles on tomato plants. Metallomics 2013, 5, 753−759. (10) Hernandez-Viezcas, J. A.; Castillo- Michel, H.; Andrews, J. C.; Cotte, M.; Rico, C. M.; Peralta-Videa, J. R.; Priester, J. H.; Holden, P. A.; Gardea-Torresdey, J. L. In situ synchrotron fluorescence mapping and coordination of CeO2 and ZnO nanoparticles in soil cultivated soybean (Glycine max). ACS Nano 2013, 26, 1415−1423. (11) Zhao, L.; Sun, Y.; Hernandez-Viezcas, J. A.; Servin, A.; Hong, J.; Niu, G.; Peralta-Videa, J. R.; Duarte-Gardea, M.; Gardea-Torresdey, J. L. Influence of CeO2 and ZnO nanoparticles on cucumber physiological markers and bioaccumulation of Ce and Zn: A life cycle study. J. Agric. Food Chem. 2014, 61, 11945−11951. (12) Kole, C.; Kole, P.; Randunu, K. M.; Choudhary, P.; Podila, R.; Ke, P. C.; Rao, A. M.; Marcus, R. K. Nanobiotechnology can boost crop production and quality: first evidence from increased plant biomass, fruit yield and phytomedicine content in bitter melon (Momordica charantia). BMC Biotechnol. 2013, 13, 37 DOI: 10.1186/ 1472-6750-13-37. (13) Servin, A. D.; Morales, M. I.; Castillo-Michel, H.; HernadezViezcas, J. A.; Munoz, B.; Zhao, L.; Nunez, J. E.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L. Synchrotron verification of TiO2 accumulation in cucumber fruit: A possible pathway of TiO2 nanoparticle transfer from soil into the food chain. Environ. Sci. Technol. 2013, 47, 11592− 11598. (14) Rico, C.; Morales, M.; Barrios, A.; McCreary, R.; Hong, J.; Lee, W.; Nunez, J.; Peralta-Videa, J.; Gardea-Torresdey, J. L. Effect of cerium oxide nanoparticles on the quality of rice (Oryza sativa L.) grains. J. Agric. Food Chem. 2013, 61, 11278−11285. (15) USDA National Nutrient Data Base (http://www.fns.usda.gov). (16) Daayf, F.; Ongena, M.; Boulanger, R.; Hadrami, I. E.; Belanger, R. R. Induction of Phenolic compounds in two cultivars of cucumber by treatment of healthy and powdery mildew-infected plants with extracts of Reynoutria sachalinensis. J. Chem. Ecol. 2000, 26, 1579− 1593. (17) Yao, L. H.; Jiang, Y. M.; Shi, J.; Tomas-Barberan, F. A.; Dalta, N.; Singanusong, R.; Chen, S. S. Flavonoids in food and their health effect. Plant Foods Hum. Nutr. 2004, 59, 113−122. (18) Del Rio, D.; Rodriguez-Mateos, A.; Spencer, J.; Tognolini, M.; Borges, G.; Crozier, A. Dietary (poly) phenolics in human health: Structures, bioavailability, and evidence of protective effects against chronic diseases. Antioxid. Redox Signaling 2013, 18, 1118−1892.
(19) Verma, S.; Dubey, R. S. Effect of cadmium on soluble sugars and enzymes of their metabolism in rice. Biol. Plant. 2001, 44, 117−123. (20) Dubois, M.; Gilles, K. A.; Hamilton, J. K.; Rebers, P. A.; Smith, F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 1956, 26, 350−356. (21) Somogyi, M. Notes on sugar determination. J. Biol. Chem. 1952, 195, 19−23. (22) Chen, C. H.; Bushuk, W. Nature of proteins in triticale and its parental species 1. Solubility characteristic and amino acid composition of endosperm proteins. Can. J. Plant Sci. 1970, 50, 9−14. (23) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248−254. (24) Dewanto, V.; Wu, X.; Adonm, K. K.; Liu, R. H. Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. J. Agric. Food. Chem. 2002, 50, 3010−3014. (25) Jia, Z.; Tang, M.; Wu, J. The determination of flavoid contents in mulberry and they scavenging effects on superoxide radicals. Food Chem. 1999, 64, 555−559. (26) Ruiz, J. M.; Romero, L. Commercial yield and quality of fruits of cucumber plants cultivated under greenhouse conditions: response to increases in nitrogen fertilization. J. Agric. Food Chem. 1998, 46, 4171− 4173. (27) Ho, L. C. The mechanism of assimilate partitioning and carbohydrate compartmentation in fruit in relation to the quality and yield of tomato. J. Exp. Bot. 1996, 47, 1239−1243. (28) Koch, K. Sucrose metabolism: regulatory mechanisms and pivotal roles in sugar sensing and plant development. Curr. Opin. Plant Biol. 2004, 7, 235−246. (29) Levitz, S. M. Interactions of Toll-like receptors with fungi. Microbes Infect. 2004, 6, 1351−1355. (30) Zaragoza, O.; Rodrigues, M. L.; De Jesus, M.; Frases, S.; Dadachova, E.; Casadevall, A. The capsule of the fungal pathogen Cryptococcus neoformans. Adv. Appl. Microbiol. 2009, 68, 133−216. (31) Moghaddam, M. R. B.; Ende, W. V. D. Sugars and plant innate immunity. J. Exp. Bot. 2012, DOI: 10.1093/jxb/ers129. (32) Gómez-Ariza, J.; Campo, S.; Rufat, M.; Estopa, M.; Messeguer, J.; San Segundo, B.; Coca, M. Sucrose-mediated priming of plant defence responses and broad-spectrum disease resistance by overexpression of the maize pathogenesis-related PRms protein in rice plants. Mol. Plant−Microbe Interact. 2007, 20, 832−842. (33) Goodman, R. N.; Kiraly, Z.; Wood, K. R. The Biochemistry and Physiology of Plant Disease; University of Missouri Press: Columbia, 1986. (34) Alaoui-Sosse, B.; Genet, P.; Vinit-Dunand, F.; Toussaint, M.; Epron, D.; Badot, P. Effect of copper on growth in cucumber plants (Cucumis sativus) and its relationships with carbohydrate accumulation and changes in ion contents. Plant Sci. 2004, 166, 1213−1218. (35) Wang, X.; Chang, L.; Wang, B.; Wang, D.; Li, P.; Wang, L.; Yi, X.; Huang, Q.; Peng, M.; Guo, A. Comparative proteomics of Thellungiella halophila leaves from plants subjected to salinity reveals the importance of chloroplastic starch and soluble sugars in halophyte salt tolerance. Mol. Cell. Proteomics 2013, 12, 2174−2195. (36) Leopold, L. F.; Leopold, N.; Diehl, H.; Socaciu, C. Quantification of carbohydrates in fruit juices using FTIR spectroscopy and multivariate analysis. Spectroscopy 2011, 26, 93−104. (37) Duarte, I. F.; Barros, A.; Delgadillo, I.; Almeida, C.; Gil, A. A. M. Application of FTIR spectroscopy for the quantification of sugars in Mango juice as a function of ripening. J. Agric. Food. Chem. 2002, 50, 3104−3111. (38) Gajc-Wolska, J.; Szwacka, M.; Malepszy, S. The evaluation of cucumber fruit quality (Cucumis sativus L.) transgenic line with thaumatin gene. Folia Hort. 2005, 17, 23−28. (39) Du, C.; Fan, H.; Guo, S.; Tezuka, T. Applying spermidine for differential responses of antioxidant enzymes in cucumber subjected to short-term salinity. J. Am. Soc. Hortic. Sci. 2010, 135, 18−24. (40) Chan, Z. Proteomic responses of fruits to environmental stresses. Front. Plant Sci. 2013, DOI: 10.3389/fpls.2012.00311. 2758
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CeO2 and ZnO Nanoparticles Change the Nutritional Qualities of Cucumber (Cucumis sativus) Lijuan Zhao,†,‡ Jose R. Peralta-Videa,†,‡,§ Cyren M. Rico,†,‡ Jose A. Hernandez-Viezcas,† Youping Sun,∥ Genhua Niu,∥ Alia Servin,† Jose E. Nunez,† Maria Duarte-Gardea,⊥ and Jorge L. Gardea-Torresdey*,†,‡,§ †
Chemistry Department, The University of Texas at El Paso, 500 West University Avenue, El Paso, Texas 79968, United States UC Center for Environmental Implications of Nanotechnology (UC CEIN), The University of Texas at El Paso, 500 West University Avenue, El Paso, Texas 79968, United States § Environmental Science and Engineering PhD Program, The University of Texas at El Paso, 500 West University Avenue, El Paso, Texas 79968, United States ∥ Texas AgriLife Research Center at El Paso, Texas A&M University System, 1380 A & M Circle, El Paso, Texas 79927, United States ⊥ Department of Public Health Sciences, The University of Texas at El Paso, 500 West University Avenue, El Paso, Texas 79968, United States ‡
S Supporting Information *
ABSTRACT: There is lack of information about the effects of nanoparticles (NPs) on cucumber fruit quality. This study aimed to determine possible impacts on carbohydrates, proteins, mineral nutrients, and antioxidants in the fruit of cucumber plants grown in soil treated with CeO2 and ZnO NPs at 400 and 800 mg/kg. Fourier transform infrared spectroscopy (FTIR) was used to detect changes in functional groups, while ICP-OES and μ-XRF were used to quantify and map the distribution of nutrient elements, respectively. Results showed that none of the ZnO NP concentrations affected sugars; however at 400 mg/kg, CeO2 and ZnO NPs increased starch content. Conversely, CeO2 NPs did not affect starch content but impacted nonreducing sugar content (sucrose). FTIR data showed changes in the fingerprint regions of 1106, 1083, 1153, and 1181, indicating that both NPs altered the carbohydrate pattern. ZnO NPs did not impact protein fractionation; however, CeO2 NPs at 400 mg/kg increased globulin and decreased glutelin. Both CeO2 and ZnO NPs had no impact on flavonoid content, although CeO2 NPs at 800 mg/ kg significantly reduced phenolic content. ICP-OES results showed that none of the treatments reduced macronutrients in fruit. In case of micronutrients, all treatments reduced Mo concentration, and at 400 mg/kg, ZnO NPs reduced Cu accumulation. μXRF revealed that Cu, Mn, and Zn were mainly accumulated in cucumber seeds. To the best of the authors’ knowledge this is the first report on the nutritional quality of cucumber fruit attributed to the impact of CeO2 and ZnO NPs. KEYWORDS: CeO2 NPs, ZnO NPs, Cucumber, Fruit nutritional quality, Carbohydrates, Proteins
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INTRODUCTION The rapid development of nanotechnologies has encouraged the production of engineered nanoparticles (NPs) worldwide. Very likely, a portion of these NPs will reach agricultural soils after end-user application, accidentally, or through their direct use in the delivery of agricultural products.1−3 Therefore, it is important to elucidate the behavior of NPs in terrestrial ecosystems and their interaction with plants, especially edible plants. ZnO and CeO2 NPs are among the most common metal oxide NPs in use, therefore the transport and toxicity of those NPs in plants is being intensely studied.1 Zinc is among the 18 essential elements for plant growth and development, while Ce is not considered essential.4 In addition, CeO2 NPs has lower dissolution compared to ZnO NPs;5 thus, as evident from previous reports, the contribution of Ce ions to the observed response is expected to be low. Schwabe et al. observed translocation of CeO2 from root to shoot in pumpkin (Cucurbita maxima Gelber Zentner) plants.6 Zhang et al. also reported that a portion of the CeO2 found in cucumber (Cucumis sativus) shoot had a different form compared to the CeO2 found in root.7 The chance of translocation of NPs into the edible tissues and their possible impact on food safety and © 2014 American Chemical Society
quality are a main public concern. However, up to now, very few studies have been conducted through the entire life cycle of a plant. Accordingly, the knowledge regarding the bioaccumulation of NPs in edible tissue and the impact on fruit quality remains largely unknown. Wang et al. conducted a life cycle study and revealed that CeO2 NPs entered into the tomato fruit.8 In a subsequent study, they found that the seeds collected from CeO2 NP treated tomato plants were smaller and weaker compared to control, indicating that the next generation was impacted.9 In another life cycle study, by using μ-XRF and μ-XANES technique, Hernandez-Viezcas et al. reported the presence of CeO2 NPs in soybean pods.10 In our most recent study, we found that Ce accumulated in cucumber fruit of plants exposed to 800 mg/kg CeO2 NPs in greenhouse conditions.11 It is still unknown whether Ce/CeO2 NPs influence the plant metabolism and nutrient element transport in plants. Kole et al. Received: Revised: Accepted: Published: 2752
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Table 1. Effects of CeO2 and ZnO Nanoparticles on Sugar Content of Cucumber Fruita reducing sugar control CeO2 400 mg/kg CeO2 800 mg/kg ZnO 400 mg/kg ZnO 800 mg/kg a
3.72 3.11 5.35 4.98 4.50
± ± ± ± ±
0.27ab 0.34b 0.56a 0.64ab 0.42ab
nonreducing sugar 23.3 ± 3.00b 8.31 ± 0.96c 37.2 ± 4.05a 33.9 ± 2.41ab 30.4 ± 0.74ab
total sugar 27.0 11.4 42.5 38.9 34.9
± ± ± ± ±
2.73b 1.09c 4.39a 3.04ab 0.96ab
starch 70.9 90.7 90.6 110.8 81.0
± ± ± ± ±
11.3b 5.9 ab 4.5ab 2.6a 2.9b
Data are average of four replicates ± standard deviation.
showed the first evidence that fullerol greatly increased bitter melon water content and fruit yield and improved fruit quality by increasing the phytomedicine content.12 Servin et al. reported an increase in phosphorus and potassium concentration in cucumber fruit of plants exposed to 750 mg/kg of TiO2 NPs.13 In addition, Rico et al. reported that CeO2 at 500 mg/kg soil modified the quality of rice grains.14 Cucumber is one of the very low calorie vegetables. In addition it contains many nutrient elements, including Ca, Fe, Mg, P, K, Na, Mn, S, and Zn. According to USDA National Nutrient Database, 100 g of fresh cucumber provides as much as 147 mg of K, which is an important intracellular electrolyte.15 In addition, cucumber contains phenolic compounds (pcoumaric, caffeic, and ferulic acids and p-coumaric acid methyl ester), and those compounds act as protective scavengers against oxygen-derived free radicals and reactive oxygen species (ROS) that play a role in cardiovascular disease prevention and anticancer activity.16−18 The aim of this study was to study the effect of ZnO and CeO2 NPs on the nutritional quality of cucumber fruit. Carbohydrates, proteins, antioxidants, and mineral nutrient were analyzed by using several biochemical and spectroscopic techniques.
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extraction was repeated three times, and all the supernatants were combined. Total soluble sugar content was measured following the method of Dubois et al.20 while reducing sugar content was determined according to the procedure of Nelson-Somogyi.21 The nonreducing sugar content was reported as the difference between total and reducing sugars. Determination of Starch in Fruit. The starch extraction was performed following the method of Verma and Dubey.19 The residue from sugar extraction was dried at 80 °C for 24 h. To the dried residue was added 2 mL of DI water, and the mixture was boiled in a water bath for 15 min. The suspension was cooled to room temperature, and 2 mL of concentrated H2SO4 was added. The suspension was stirred for 15 min, and the final volume was adjusted to 10 mL using water. The supernatant was centrifuged at 3000g for 20 min, and the extraction was repeated using 50% H2SO4. The supernatants were combined and diluted with water to the 50 mL mark. The starch content was quantified following the method of Dubois et al.20 Protein Fractionation. Protein extraction and fractionation was conducted according to Chen and Bushak.22 Dried cucumber fruit (500 mg) was extracted sequentially with 8 mL each of water, 0.5 mol/ L NaCl, 70% ethanol, and 0.05 M acetic acid. The extracted protein in each step was labeled as albumin, globulin, prolamin, and glutelin fractions, respectively. The protein content was quantified by the Bradford method.23 Total Phenolics and Total Flavonoids. Total phenolic content was determined by the spectrophotometric method followed by Dewanto et al.24 At room temperature, Folin−Ciocalteu phenol reagent (0.2 mL) was added to each sample (0.1 mL). After 1 min, 3 mL of 5% Na2CO3 solution was added to the mixture and the mixture was allowed to stand for 1 h in a dark place. The absorbance was measured at 760 nm using a Perkin-Elmer Lambda 14 UV/Vis Spectrometer (single-beam mode, Perkin-Elmer, Uberlinger, Germany). Total phenolic contents of the sample were expressed on a fresh weight basis as mg of gallic acid equivalent (GAE) g−1. Total flavonoid content of cucumber fruit was measured using the method of Jia et al.25 Briefly, to 500 μL of each sample was added 75 μL of 5% NaNO2. After 5 min, to the mixture were sequentially added 150 μL 10% AlCl3, 500 μL 1 M NaOH, and 275 μL of DI water. The absorbance was measured at 510 nm using a spectrophotometer. The total flavonoid content was expressed as catechin equivalents. Fourier Transform Infrared (FTIR) Spectra Acquisition. The dried cucumber fruits were ground, and the powder was analyzed using FTIR spectroscopy (Perkin-Elmer, Spectrum 100, Universal ATR Sampling Accessory) in a range of 650−3950 cm−1. The second derivative spectrum was collected using Spectrum software (Version 6.0.2.0025, Perkin-Elmer, Shelton, CT). A total of 3 replicates per treatment were used for the analysis. μ-XRF Analysis. Fruits were carefully washed with DI water to eliminate any surface contaminants. Then, they were transversally cut and frozen in liquid nitrogen for 30 min. The samples were fixed with Tissue Tek (Sakura Finetek USA, Torrance, CA) and sectioned with a cryomicrotome (Triangle Biomedical Sciences, Durham, NC) at −20 °C to a thickness of 30 μm. Subsequently, the samples were mounted onto Kapton tape and freeze-dried for 1 h in a Labconco freeze-dryer (FreeZone 4.5, Kansas City, MO) with operating conditions of −53 °C and 0.140 mBar pressure. X-ray fluorescence imaging was obtained at beamline 10-2 at the Stanford Synchrotron Radiation Lightsource (Menlo Park, CA). Standard operating conditions were 3 GeV beam energy and 80−
MATERIALS AND METHODS
Characterization of NPs. The CeO2 and ZnO NPs (Meliorum Technologies, NY, U.S.) used in this study were obtained from the University of California Center for Environmental Implications of Nanotechnology. According to the supplier, the primary size for those two NPs is 10 nm. The characterization of both NPs, including hydrodynamic size, ζ-potential, and pH, were previously reported by Keller et al.5 Stock solutions were prepared by adding 1000 mg of nanoparticles to 1.0 L of deionized water. The dispersed nanoparticles were sonicated for 30 min in a water bath and immediately applied to the substrate. Experimental Design and Growth Conditions. CeO2 NPs and ZnO NPs were used, separately, at 400 and 800 mg/kg. Pots containing substrate without the addition of NPs were included as a control. Each treatment had four replicate (pots), and each pot contained two plants. More details regarding the experimental design, soil, and greenhouse conditions were introduced in previous a study.11 Mineral Content. Dried cucumber fruits were ground to pass a 40mesh screen (0.425 mm) with a stainless Wiley mill (Thomas Scientific, Swedesboro, NJ). Subsequently, the samples were digested with a mixture of H2O2 and plasma pure HNO3 (v/v: 4:1) using microwave oven (CEM Corp, Mathews, NC, U.S.).14 The standard reference materials NIST 1547 and 1570a were also digested and analyzed as samples. The recoveries for all elements were between 90 and 99%. The macro- and micronutrient elements were analyzed using inductively coupled plasma optical emission spectrometry (PerkinElmer Optima 4300 DV). Determination of Total Soluble and Reducing Sugars. The total soluble sugars were extracted by the method of Verma and Dubey.19 One hundred grams of dried cucumber tissue was ground in 10 mL of 80% ethanol and then boiled (80 °C) in a water bath for 30 min. After cooling to room temperature, the extracts were centrifuged at 22000g (Thermo Scientific, Soruall T1, U.S.) for 20 min. The 2753
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Figure 1. FTIR spectra of cucumber fruit treated with 0, 400, and 800 mg/kg ZnO NPs and 400 and 800 mg/kg CeO2 NPs. Spectra are means of three replicates.
Figure 2. Second derivative of FTIR band in the region of 800−1800 cm−1. 100 mA beam current. Incident X-ray energy was set to 10 keV, and a Si (111) monochromator was used. SMAK27 software was used for data analyses. Data Analysis. The treatments from two NPs at two concentrations and four replicate/treatments were allocated in a completely random design. All data including sugars, starch, protein, mineral nutrients, and antioxidant were analyzed by using PROC GLM in SAS software (Version 9.1.3, SAS Institute, Cary, NC).
sugars content was significantly increased, compared to control. Typically, sugars are a source of carbon and energy.28 On the other hand, sugars such as glucose, fructose, and sucrose also act as signaling molecules in plant responses under biotic and abiotic stresses.29,30 Among them, sucrose contributes to stressrelated responses.31 It has been reported that some of the defense-related genes were upregulated in rice leaves when sucrose was fed to the roots.32 In this study, the upregulation of sucrose by CeO2 NPs was a possible indicator of stress. In contrast, ZnO NPs at both concentrations did not impact the reducing and nonreducing sugar contents (Table 1). Leaves are plant’s main photosynthetic organs. Sugar and starch are synthesized in the chloroplasts of leaves and are translocated from leaves to fruits. Previous studies reported that reduced photosynthetic rate led to a decrease in the concentration of soluble sugars, and often starch accumulation in leaves.33 In our study, the photosynthetic rate in leaves was not decreased by ZnO NPs.11 This explained well that ZnO NPs did not change the carbohydrate composition in fruit.
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RESULTS AND DISCUSSION Effects of NPs on Fruit Carbohydrates (Sugar and Starch) Content. Sugars, starches, and fiber are main components of carbohydrates in cucumber fruit. Among them, “soluble sugars and starch are important parameters to evaluate the quality of fruits.”26,27 As shown in Table 1, reducing sugars (glucose and fructose) were not modified by CeO2 NPs. This implies that the sweet taste of cucumber fruit was not affected. However, the nonreducing sugar (sucrose) was impacted by CeO2 NPs. At 400 mg/kg treatment, the content of nonreducing sugars was significantly decreased compared to control, while at 800 mg/kg, the nonreducing 2754
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Table 2. Effect of CeO2 and ZnO NPs on Protein Fractionation and Antioxidant Content of Cucumber Fruita albumin control CeO2 400 mg/kg CeO2 800 mg/kg ZnO 400 mg/kg ZnO 800 mg/kg a
2.32 6.82 5.31 5.68 4.93
± ± ± ± ±
0.83 0.91 1.61 0.74 0.47
globulin a a a a a
0.75 1.32 0.44 1.28 0.86
± ± ± ± ±
0.11 0.17 0.07 0.08 0.04
prolamin b a b a ab
2.50 2.61 2.84 3.57 3.68
± ± ± ± ±
glutelin
0.15 0.54 0.11 0.15 0.21
a a a a a
0.20 0.07 0.25 0.18 0.39
± ± ± ± ±
0.03 0.02 0.00 0.04 0.01
flavonoid
phenolic b c b bc a
0.689 0.662 0.517 0.557 0.582
± ± ± ± ±
0.027 0.041 0.043 0.026 0.016
a ab b ab ab
0.537 0.596 0.403 0.505 0.426
± ± ± ± ±
0.056 0.076 0.047 0.003 0.003
a a a a a
Data are average of four replicates ± standard deviation.
Table 3. Effect of CeO2 and ZnO Nanoparticle on Mineral Content of Cucumber Fruit (mg/kg)a K control CeO2 400 mg/kg CeO2 800 mg/kg ZnO 400 mg/kg ZnO 800 mg/kg a
60224 64018 58837 60225 56533
Ca a a a a a
7249 8907 8436 6900 6988
Mg a a a a a
3349 3934 3452 3956 3583
S c ab c a bc
3443 3538 3260 3344 3481
P a a a a a
1136 1174 1134 1087 1059
Na a a a a a
589 580 522 527 544
Fe a a a a a
12.7 a 14.6 a 9.34 a 14.6 a 6.41 a
Cu 62.6 60.5 57.3 47.2 50.7
a a ab c bc
Mn 25.2 28.1 24.4 32.0 27.1
b ab b a b
Mo 22.9 a 5.48 c 9.93 bc 13.8 b 10.7 bc
Zn 36.1 38.3 37.7 61.3 89.1
c c c b a
Data are average of four replicates ± standard deviation.
Effects of NPs on Fruit Protein Fractions and Antioxidant Content. Although cucumber is a low protein vegetable, protein is an important factor contributing to the sweet taste of cucumber fruit.38 The effect of ZnO and CeO2 NPs on the four soluble protein fractions is presented in Table 2. Generally, albumin and prolamin fractions are the predominant proteins in cucumber fruit, which represents 84%−92% of the fruit protein. Globulin and glutelin fractions are present to a lesser extent. Results showed that neither CeO2 nor ZnO NPs significantly affected albumin and prolamin fractions. However, compared to control, globulin content was significantly increased by CeO2 NPs at 400 mg/kg treatment. Glutelin content was also significantly decreased by CeO2 NPs at 400 mg/kg. Those results indicated that CeO2 NPs at 400 mg/kg changed the protein content in fruit. CeO2 NPs at 800 mg/kg did not affect any of the protein fractions. More studies are needed in order to explain the different response at 400 and 800 mg/kg treatments. ZnO NPs at 400 mg/kg treatment increased globulin content while those at 800 mg/kg treatment increased glutelin content. Du et al. reported that salt stress increased the soluble protein content of cucumber seedlings.39 Several proteins such as heat shock protein, catalase, and glutathione S-transferase are related to stress response.40 Those proteins were upregulated under abiotic or biotic stress. It is possible that protein increased as a response to NP induced stress. Phenolic compounds including flavonoids and phenolic acids are the most frequently examined group of antioxidants, and they are highly correlated with the total antioxidant activity.41,42 This is an important trait in cucumber because phenolic compounds haven been associated with a reduction in breast and prostate cancer risks.43 As shown in Table 2, none of the CeO2 and ZnO concentrations impacted flavonoid content. However, CeO2 NPs at 800 mg/kg significantly decreased the phenolic content. As a result of this treatment there was an increase in total sugar content but a decrease in phenolic content. This indicates that, at this concentration, CeO2 NP impacted the nutritional quality of the fruit. Effects of NPs on the Fruit Mineral Composition. The uptake of nutrients by plant roots is affected by abiotic and biotic stressors including soil composition, cation exchange capacity, pH of the soil solution, microorganisms, and metal immobilization in root cell walls, among others.44,45 Cucumber
Starch is another important carbohydrate in fruits. From Table 1, it seems that all NP treatments increased the starch content compared to control, but only at 400 mg/kg ZnO NPs, the difference was significantly increased. Previous reports showed that copper stress induced the overaccumulation of starch and sucrose in cucumber plants.34 Another report indicates that salinity stress stimulated the starch accumulation in chloroplasts and resulted in increased accumulation of starch and total sugars in Thellungiella halophile leaves.35 This could indicate that starch and sucrose increased in cucumber fruit as a result of stress caused by the NPs. FTIR Analysis. As a rapid and noninvasive tool, Fourier transform infrared spectroscopy is used to identify changes in functional groups in plant tissues. FTIR spectra of cucumber fruit from plants treated with CeO2 and ZnO NPs are shown in Figure 1. As seen in this figure, the band area of the carbohydrate region (900−1200 cm−1) was reduced by all NP treatments, compared with the control. Furthermore, the difference between NP treatments and control can be seen in the lignin area (1635 cm−1). The data also showed band differences in control and NP treated plants in the lipid region located between 2840 and 2960 cm−1. In this region, control and 400 mg/kg CeO2 NP treatments were similar; however, the 800 mg/kg CeO 2 NP treatment and both ZnO NP concentrations reduced this band area. In addition, it was observed that the band areas at 1350−1420 cm−1 and 1520− 1650 cm−1 decreased in all NP treatments. The spectrum at 400 mg/kg CeO2 NPs was similar to that of the control, while those of the other three treatments were apparently lower than that of the control. For a better understanding of the fingerprint of carbohydrates, second derivatives of spectra were collected at the 800− 1800 cm−1 region. The plots of the second derivative spectra are shown in Figure 2. Carbohydrates (glucose, fructose, and sucrose) showed intense characteristic bands in the fingerprint region (900−1400 cm−1).36,37 It is clear from the spectra that the bands at 1181, 1153, 1106, and 1083 cm−1 were lower in the NP treatments, compared to control. According to Duarte et al.,37 spots 1153 and 1083 cm−1 are related to sucrose while 1108 cm−1 corresponds to fructose and glucose. Although it is difficult to quantify based on those bands, the spectra revealed that NPs changed the carbohydrate pattern in cucumber fruit. 2755
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Figure 3. μ-XRF temperature map from a cross cut cucumber fruit showing normalized K, Ca, Fe, Cu, Mn, and Zn intensities. The image corresponds to control fruit. Red represents higher intensity, and dark blue represents the absence of the element.
that ZnO NPs at 200 mg/kg soil and above increased the concentration of Zn in roots and shoots of corn plants.54 At all concentrations, both the CeO2 and ZnO NPs significantly reduced molybdenum concentration in cucumber fruit (Table 3). Reductions were in the range of 57 to 76% for CeO2 NPs and about 40 to 53% for the ZnO NP treatments. Previous studies have shown that Mo is taken up by plants as MoO42− .55 Reports indicate that Mo uptake is reduced by sulfate and increased by phosphate.56 In addition, previous work with alfalfa showed that Mo is complexed with malate.55 As shown in Table 3, P and S were at the same levels in control and NP treated plants; thus, we cannot conclude about the role of P or S in Mo uptake. Perhaps the NPs altered the production of organic acids like citrate or malate, and then, they reduced Mo uptake. Studies about the effects of CeO2 and ZnO NPs on organic acid production are currently being performed in order to explain the uptake of some micronutrients. The reduction in Mo in CeO2 and ZnO NPs treated cucumber might not represent a concern to human health as it has been documented that most people in the U.S. consume more than sufficient amounts of this trace mineral in their diets.48 Manganese was increased by ZnO NPs at 400 mg/kg. A previous study showed that 34% of Zn/ZnO NPs was bound to Mn−Fe oxides.57 At this time we cannot explain why this could increase Mn uptake. Additionally, we do not know the effect ZnO NPs produce in Nramp transporters, which are associated with Mn uptake by plants.58 The increase of Mn in ZnO NPs treatment might not represent a health concern especially in diets that include the consumption of foods high in phytic acid (beans, seeds, nuts, whole grains, and soy products), oxalic acid
is a source of vitamins, potassium, magnesium, and phosphorus required for human nutrition.46 The influence of both the CeO2 and ZnO NPs on the cucumber fruit mineral content is shown in Table 3. As seen in this table, from all macronutrients analyzed, only Mg showed significant differences in the NP treated plants. At 400 mg/kg, both the CeO2 and ZnO NPs increased Mg in cucumber fruit by 17% and 18%, respectively (p ≤ 0.05). Magnesium is an essential element for human health. It is obtained from foods and concentrates in bones and muscle.47 Magnesium participates in multiple functions including energy production, protein synthesis, cell membranes and chromosomes, ion transport across cell membrane, and cell migration and interacts with protein, fiber, vitamin D, calcium, and zinc.48 Magnesium is essential in plants as it is the core of the chlorophyll molecule. Mg2+ enters the root cortex cells through the apoplast.49 However, it is not clear how Mg2+ enters the symplast and then the xylem apoplast.50 To this end, we cannot explain why the CeO2 NPs increased Mg translocation to cucumber fruit. However, other NPs like CuO NPs have been found to increase the production of indole-3-acetic acid in bacteria,51 and this growth hormone induces the expression of two aquaporins in tomato (Lycopersicon esculentum Mill.). Magnesium has been found to be transported through the rca channel in wheat root.52 Thus, it is possible that the CeO2 NPs promote the expression of aquaporins or other channels, like the MRS2, which is Mg2+ transporter to the shoot, enhancing Mg2+ uptake.53 CeO2 and ZnO NPs changed the fruit profile of micronutrients Cu, Mo, Mn, and Zn. As expected, Zn was increased at both ZnO NP concentrations. Previous reports have shown 2756
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Table 4. Comparison of Effects of CeO2 NPs and ZnO NPs on Cucumber Fruit Qualitya soluble sugar CeO2 NPs 400 mg/kg CeO2 NPs 800 mg/kg ZnO NPs 400 mg/kg ZnO NPs 800 mg/kg a
starch
− +
protein fractionation
phenolic
flavonoid
globulin +; glutelin − − +
globulin + glutelin +
mineral Mg +, Mo − Mo − Mg, Mn +; Cu, Mo − Cu, Mo −
+ means positive impact; − means negative impact; blank means no impact.
CeO2 NPs not only decreased Mo concentration, they also altered the nonreducing sugars and phenolic content and they also changed the fractionation of proteins (Table 4). This suggests that CeO2 NPs impacted the fruit flavor and reduced the antioxidant capacity. Furthermore, as previously reported,11 the fruits were grouped by size as large, medium, and small and the total weight of each group was recorded. The fresh weight of large cucumbers was reduced by 31.4% by CeO2 NPs at 800 mg/kg (Table S1 in the Supporting Information). More studies have to be performed in order to determine the effects of NPs on other parameters such as lignin and pectin that can influence fruit weight. The above-mentioned results suggest that, at the concentration tested, both CeO2 and ZnO NPs impacted the nutritional quality of cucumber.
(spinach, cabbage, and sweet potatoes), and tannins (tea), compounds that might slightly inhibit manganese absorption. Intake of minerals including iron, calcium, and phosphorus have been found to limit retention of manganese.48 Copper was also reduced by ZnO NPs. Previous studies have shown that ZnO NPs dissociate releasing Zn ions into the medium,59 and according to Alloway, Zn decreases the uptake of Cu because both metals are taken up by the same transporter.60 Some experts propose that Cu deficiency rather than excess Cu increases the risk of cardiovascular diseases.48 More studies are needed in order to understand the effects of CeO2 and ZnO NPs at the root surface interface and their effect on nutrient uptake by plants. Micro-XRF Analysis of Mineral Localization in Cucumber Fruit. ICP-OES data only reflect total mineral concentration in cucumber fruit. In order to detect whether NPs altered the mineral distribution pattern, the μ-XRF technique was applied. Because of the limited beam time, only two macronutrients Ca and K and four micronutrients including Fe, Cu, Mn, and Zn were analyzed. The mineral distribution between CeO2 treated and control plants was very similar. This indicates that CeO2 NPs did not change the nutrient element distribution in cucumber plant. The microXRF intensity map of the cucumber fruit cross-section is shown in Figure 3. In general, K and Ca were localized in skin, pulp, and seeds, while iron was almost evenly distributed in the whole cross section. Figure 3 also shows that Cu, Zn, and Mn were more concentrated in seeds and at lower concentration in the skin. As revealed by ICP-OES results, Cu concentration was reduced by ZnO NPs. Furthermore, μ-XRF results indicate that Cu is mainly present in cucumber seeds (Figure 3). Therefore, the next generation could also be impacted. Cu is an essential element for plant growth because they participate in many important physiological processes such as photosynthesis and respiration.61 It has been reported that soaking seeds in Cu sulfate solution may enhance plant growth and development and, even, improve the nutritional quality of fruit.62 Thus, a reduction in Cu in cucumber seeds suggests that ZnO NPs may have a negative impact on the growth of next plant generation. More studies are needed in order to solve this knowledge gap. Conclusions. In summary, at the concentration tested, the ZnO NPs did not show negative impact on carbohydrate, protein, and antioxidant contents compared to control. To some extent, ZnO NPs increased starch and protein content, which might result in an increased caloric value of a food that is typically characterized by its low caloric content. However, it is not clear if the increased starch and protein contents are indicators of stress induced by ZnO NPs. Both ZnO NP treatments significantly decreased the concentration of the micronutrients Cu and Mo. Since both Cu and Mo are important minerals in plant tissues and participate in many processes, the quality of fruit in the next generation of cucumber plants may be impacted.
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ASSOCIATED CONTENT
S Supporting Information *
Table providing average weight of large, medium, and small cucumber fruits harvested from plants grown in CeO2 NP amended soil. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Phone: 915-747-5359. Fax: (915) 747-5748. Funding
This material is based upon work supported by the National Science Foundation and the Environmental Protection Agency under Cooperative Agreement Number DBI-0830117. The authors also acknowledge USDA Grant No. 2011-38422-30835 and NSF Grant No. CHE-0840525. J.L.G.-T. 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 No. W11NF-10-2-0076, Subgrant 13-7. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors 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.
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