Research Article pubs.acs.org/journal/ascecg
Titanium Dioxide Nanoparticles Alleviate Tetracycline Toxicity to Arabidopsis thaliana (L.) Hong Liu,†,‡,# Chuanxin Ma,‡,§,# Guangcai Chen,‡,∥ Jason C. White,§ Zonghua Wang,⊥ Baoshan Xing,*,‡ and Om Parkash Dhankher*,‡
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Fujian Provincial Key Laboratory of Soil Environmental Health and Regulation, College of Resources and Environmental Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China ‡ Stockbridge School of Agriculture, University of Massachusetts, Amherst, Massachusetts 01003, United States § Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station, New Haven, Connecticut 06504, United States ∥ Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, Zhejiang 311400, China ⊥ State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou 350002, China S Supporting Information *
ABSTRACT: Arabidopsis thaliana (L.) Heynh. was used as a model plant to investigate the biochemical and molecular response upon coexposures to tetracycline (TC) and titanium oxide nanoparticles (TiO2 NPs). Results showed that 1 mg/L TC severely reduced A. thaliana biomass by 33.3% as compared with the control; however, the presence of 50 and 100 mg/L TiO2 NPs alleviated TC toxicity, increasing fresh biomass by 45% and 28%, respectively, relative to the TC alone treatment. The presence of TC notably decreased Ti accumulation in both shoots and roots. Antioxidant enzyme activity, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POD), in A. thaliana shoots and roots indicated that TC significantly increased the activity of reactive oxygen species (ROS) scavengers. However, in the coexposure treatments, TiO2 NPs reduced antioxidant enzyme activity back to the control levels. The relative expression of genes encoding sulfur assimilation and glutathione biosynthesis pathways was separately measured in shoots and roots. Interestingly, the relative expressions of adenylytransferase (APT), adenosine-5′phosphosulfate reductase (APR), and sulfite reductase (SiR) in the roots across all three treatments (TC alone, TiO2 NPs alone, and TC × TiO2 NPs treatment) were 2−3.5-fold higher than the control. The expression of γ-glutamylecysteine synthetase (ECS) and glutathione synthetase (GS) was increased in A. thaliana treated with either TiO2 NPs or TC alone. At harvest, almost 93% reduction of the pod biomass was evident in the TC alone treatment as compared with the control; however, TiO2 NPs increased the pod biomass by 300% in the coexposed plants relative to the TC alone treatment. These findings provide important information for understanding the interactions of metal-based NPs and cocontaminants such as antibiotics in plant systems. KEYWORDS: Arabidopsis thaliana, Titanium oxide Nanoparticles, Tetracycline, Cocontamination, Molecular response, Crop quality
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tries.1,5 Since soils and water bodies are considered the primary sinks for environmental pollutants, concerns have been raised over the adverse impacts of antibiotics on microbial, plant, and animal communities.1,6 According to the European Union, if the environmental concentration of antibiotics exceeds 10 μg/kg, the further assessment of the specific chemical compound should be conducted. The estimated concentrations of TC and TC derivatives could be in the range 450−900 μg/kg, which was
INTRODUCTION Antibiotics have been widely used in agriculture and livestock industries for the purposes of growth enhancement and disease prevention.1 Most antibiotics are water-soluble and are poorly absorbed by livestock; approximately 40−90% of the antibiotics are excreted through urine or feces.2 In the United States, approximately 51 tons of antibiotics were used, and almost 79% of antibiotics (equals to 13540000 kg) were applied in the raising of livestock annually.3 Similarly, in China, approximately 210,000 tons of antibiotics are produced annually and more than three-quarters are used in animal husbandry.4 Antibiotics can be released to the environment from pharmaceutical wastes, wastewater treatment facilities, and livestock indus© 2017 American Chemical Society
Received: December 7, 2016 Revised: January 23, 2017 Published: February 24, 2017 3204
DOI: 10.1021/acssuschemeng.6b02976 ACS Sustainable Chem. Eng. 2017, 5, 3204−3213
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In the present study, we chose Arabidopsis thaliana (L.) Heynh. as a target plant and hydroponically exposed it to TiO2 NPs and TC. In order to comprehensively understand the defense mechanism and impacts of cocontaminants on crop yield, the physiological and molecular responses of A. thaliana upon coexposure to TiO2 NPs and TC were investigated. The resulting impact on biomass, Ti uptake, chlorophyll content, protein content, and pod formation was determined. Additionally, the activities of the main reactive oxygen species (ROS) scavengers, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POD), were measured in A. thaliana shoots and roots across all treatments. At the molecular level, the relative expression of genes involved in sulfur assimilation, glutathione biosynthesis, as well as stress-related genes, was analyzed in shoots and roots of A. thaliana. To our knowledge, this is the first report on addressing the role of metal-based NPs in alleviating the toxicity of antibiotics to plants. Our findings provided the important information on the potential risks of antibiotics and metal-based NPs to the agricultural crops in terms of crop yield and quality if such impacts on A. thaliana could translate to the real crops.
approximately 45−90-fold higher than the European Union regulation.7 Thus, it is necessary to investigate the TC toxicity to terrestrial plants with the presence of other emerging contaminants in the environment. Sulfonamides (SA) and tetracycline (TC) are among the most widely used antibiotic groups due to their broad inhibition of microorganisms, protozoa, and other parasite populations.8 Once the antibiotics are discharged into the environment, it is likely that plant species will accumulate the residues from soil. In fact, a large body of the present studies has focused on antibiotic uptake in terrestrial plants. A recent study reported that common vegetable crops, including cucumber (Cucumis sativa L.), tomato (Solanum lycopersicum L.), and lettuce (Lactuca sativa L.), could accumulate both SA and TC in the different plant tissues upon exposures to various concentrations (0−20 mg/ kg) for 45 days.9 Separately, Topal et al. found that the concentrations of TC and its degradation products in Phragmites australis significantly decreased from root to leaf.10 Additionally, the relative accumulation of TC and its degradation products was as follows: 4-epitetracycline > tetracycline >4-epianhydrotetracycline > anhydrotetracycline.10 Pot experiments with soybean grown in 105 mg/kg oxytetracycline contaminated saline soil showed that the antibiotic was only accumulated in the roots and no translocation was evident to the shoots.11 The type of antibiotic will also clearly impact the uptake levels by plants. For example, oxy-TC and TC at a 50 mg/L exposure had similar root concentration factors (RCF≈2100) in treated rice over a period of 15 days of exposure, whereas a significantly higher value of RCF (approximately 2700) was evident for chlor-TC at 11 days of exposure.12 Pan et al. suggested that antibiotic bioaccumulation along the food chain should not be neglected, but also noted that the concentrations (TC is 2100 μg) in vegetable crops are significantly lower than the minimum therapeutic dose (20− 200 mg).13 Importantly, few mechanistic studies investigating the basis of plant toxicity and response are available in the literature, and as such, understanding of these processes remains inadequate. Engineered nanomaterials (ENMs) have been increasingly applied in various fields, such as agriculture, food manufacturing, biomedicals, electronics, and renewable energy.14 Titanium oxide (TiO2) is one of the commonly used nanoparticles (NPs); more than 3000 tons of TiO2 NPs are produced annually, and more than half is used in personal care products.15,16 In agriculture, ENMs can be used to detect pathogens, to more effectively deliver pesticides and fertilizers, and to monitor soil conditions.17 Due to their nanoscale size, ENMs may pose potential risks in agricultural systems despite the positive impacts on crops.18 Recent laboratory studies have demonstrated that the presence of ENMs can result in root elongation inhibition, biomass decrease, low photosynthetic efficiency, developmental delay, and unique molecular effects of unknown consequence.19−22 Plant defense mechanisms upon exposure to different types of metal-based NMs have been recently reviewed by Ma et al.14 Antioxidant enzyme activity in terrestrial plants played an essential role in detoxifying nanoparticle-induced phytotoxicity.23,24 In addition, studies at the molecular level also provide insight on responses of stressrelated genes in plants upon nanoparticle exposures.25 Given the complex nature of agricultural systems, the likelihood of coexposure to metal oxide nanoparticles and pharmaceutical compounds such as antibiotics is quite high; however, little work has been done in this area.
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MATERIALS AND METHODS
Experimental design. Concentration optimization. Titanium oxide nanoparticles (TiO2 NPs) were purchased from US Research Nanomaterials, Inc. The size of TiO2 NPs was ranging from 5 to 15 nm, and the form of TiO2 NPs was rutile. Tetracycline (≥98%) was obtained from Sigma-Aldrich. Twenty-five surface sterilized seeds of A. thaliana ecotype Columbia (Col-0) were placed on half strength (1/ 2×) Murashige and Skoog (MS) semisolid medium amended with different concentrations of TiO2 NPs ranging from 0 to 500 mg/L. Similarly, sterilized seeds were grown in 1/2× MS semisolid medium amended with different concentrations of TC (0−5 mg/L). Seeds were stratified at 4 °C for 24 h prior to transfer to a controlled plant growth chamber with 16 h of light and 8 h of dark at 22 and 8 °C, respectively. At harvest, total fresh biomass was used to determine the toxicity of TiO2 NPs and TC; the results are shown in Figure S1. Hydroponic system. A. thaliana was grown in vermiculite for 21 days, and the seedlings were then transferred to a hydroponic system as shown in Figure S2 and were allowed to acclimatize for 5 days. The doses of TiO2 and TC in the hydroponic system were determined from the concentration optimization test described above. As shown in Figure S1A, lower concentration of TiO2 NPs (50 mg/L) significantly enhanced plant growth; no significant difference was observed at 100 mg/L TiO2 NPs; and the fresh biomass was greatly reduced as TiO2 NPs concentrations were increased to 200 mg/L. Thus, these three exposures of TiO2 NPs (50, 100, and 200 mg/L) were chosen for the hydroponic experiment. The identical method was applied to determine the appropriate TC concentrations in the hydroponic experiment. TC exposure had no impact on fresh biomass until 1 and 5 mg/L, where fresh biomass was reduced by 21.7% and 59.6% relative to the control, respectively (Figure S1B). The three doses of TC chosen were 1, 5, and 10 mg/L. In total, there are 16 treatments (Table S1), including 9 different coexposure treatments, three single analyte controls for each TiO2 NPs and TC exposure, and one control with 1/2X Hoagland’s solution only. A number of six replicates were applied in each treatment. A. thaliana seedlings were exposed to different concentrations of TiO2 NPs and TC for 12 days. At harvest, the roots were thoroughly rinsed with deionized water three times and root length and total fresh biomass were measured. Plant tissues were stored at −80 °C until further analysis. Chlorophyll content. The total chlorophyll content in A. thaliana was measured as described by Lichtenthaler et al.26 Briefly, 50 mg of fresh leaves were collected and cut into pieces (less than 1 cm); 10 mL of 95% ethanol was then used to extract the total chlorophyll. All samples were kept in the dark for 3 days to avoid chlorophyll 3205
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ACS Sustainable Chemistry & Engineering degradation. The absorbance was measured at 664.2 and 648.6 nm with a UV−vis spectrophotometer (Agilent 8453, Santa Clara, CA). Chlorophyll a, chlorophyll b, and the total chlorophyll were calculated by the following equations: Chla = 13.36A664.2 − 5.19A648.6 (1), Chlb = 27.43A648.6 − 8.12A664.2 (2), and Total chlorophyll = Chla + Chlb (3). Ti and nutrient element contents. A. thaliana shoots and roots were separately freeze-dried and then were ground to a fine powder. Approximately 50 mg of shoot tissues or 10 mg of root tissues were digested in sulfuric acid (H2SO4) and hydrogen peroxide (H2O2) following the method described by Short et al. (1996) and Wei et al. (2015) with slight modification.27,28 Detailed information is provided in the Supporting Information. The samples were measured using inductively coupled plasma optical emission spectrometry (ICPOES).29 Total protein content. The Bradford reagent (Sigma-Aldrich, St. Louis, MO) was used to measure the total protein content in plant tissues.30 A sample of 50 mg of A. thaliana shoots or roots was extracted in 2 mL of 10 mM Tris-HCl (pH 7.2) solution. The mixture was then centrifuged at 2683g for 20 min at 4 °C. One hundred microliters of supernatant was used to react with 1000 μL of Bradford reagent for 15 min at ambient temperature, and then the absorbance was measured at 595 nm with a UV−vis spectrophotometer (Agilent 8453, Santa Clara, CA). Antioxidant enzyme assays. Fresh root and shoot tissues were homogenized in liquid nitrogen to a fine powder. A 0.5 g sample of homogenized tissue was then vigorously mixed with 5 mL of extraction buffer for 5 min using a vortex mixer. The mixture was centrifuged at 2683g for 20 min at 4 °C, and the supernatant was used to measure antioxidant enzyme activity, including SOD, CAT, APX, and POD. The modified protocol for each antioxidant enzyme assay is provided in the Supporting Information.31−33 Gene expression measurement by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Shoot and root tissues were separately homogenized in liquid nitrogen prior to RNA isolation. Protocols for total RNA isolation, cDNA synthesis, and gene expression using qRT-PCR were described by Ma et al.25 Briefly, RNeasy plant mini kits were used to isolate total RNA, with the concentration being quantified by NanoDrop spectrophotometry (ThermoScientific, West Palm Beach, FL). A Verso cDNA synthesis kit was used to synthesize cDNA, and the gene-specific primer was designed using Primer Quest (Integrated DNA Technologies, Coralville, IA). A complete list of primer sequences is provided in Table S2. Information for the qTR-PCR amplification program can be found in the Supporting Information. Relative quantities (2−ΔΔCt method) were used to calculate the transcription level of each gene. Statistical analysis. A one-way analysis of variance (One-way ANOVA) followed by Duncan’s multiple comparison test was used to determine the statistical significance of each parameter across treatments, except the qRT-PCR assay, in which a Student t test was applied to determine the statistical significance for each gene. In the figures for each assay, values followed by different letters are significantly different at p ≤ 0.05.
Figure 1. Physiological effects of TiO2 NPs and TC on A. thaliana. (A) Images of A. thaliana coexposed to TiO2 NPs and TC; (B) Root length; (C) Total fresh biomass. Data are mean ± standard error of five to six replicates. Values of each parameter followed by different letters indicate that the data points are significantly different at p ≤ 0.05.
The additions of 50 and 100 mg/L TiO2 NPs significantly alleviated the phytotoxicity, elevating the fresh biomass in 1 mg/L TC treated A. thaliana back to the level of the control group (Figure 1C). This result suggests that TiO2 NPs could counteract the TC toxicity to A. thaliana at the lower antibiotic doses. As the concentrations of TC were increased to 5 and 10 mg/L, although there was a trend of fresh biomass increase, the results were statistically insignificant. One of the possible explanations is that the TiO2 NPs interact with the TC outside of the plant, preventing exposure at a micro-/nanolevel, but that this process is saturated when the exposure dose of TC reached 1 mg/L. Ti uptake and nutrient element contents in A. thaliana upon exposure to TiO2 NPs and TC. The contents of Ti and other essential nutrients were measured in A. thaliana shoots and roots treated with TiO2 NPs and TC (Figure S4 and Table S3). The presence of 10 mg/L TC significantly reduced Ti accumulation in the roots and decreased Ti translocation to the shoots. In 200 mg/L TiO2 NPs alone treatment, the root Ti content was 47140.55 mg/kg over 12 days exposure, while this value was decreased by 24.81% in the coexposure treatment. A similar trend showing
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RESULTS AND DISCUSSION Growth of A. thaliana upon exposure to TiO2 NPs and TC. Different concentrations of TiO2 NPs did not significantly affect plant growth in Figure 1A and Figure S3. Although 1 mg/ L TC exhibited negative effects on growth as compared with the control group (Figure 1A), the presence of TiO2 NPs seemed to visibly alleviate TC phytotoxicity. At harvest, root length and total fresh biomass were measured (Figure 1B and C). The root length was significantly reduced in the 10 mg/L TC exposure; no difference was evident in the coexposure treatments, except the treatments of 1 mg/L TC × 100 and 200 mg/L TiO2 NPs; in both cases, root length was reduced by 22% and 17%, respectively, as compared with the TC control. The total fresh biomass in all three TC alone treatments was reduced by approximately 30% relative to the control group. 3206
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Figure 2. Antioxidant enzyme activity of A. thaliana shoots coexposed to TiO2 NPs and TC. Parts A−D represent SOD, CAT, APX, and POD activities, respectively. Data are mean ± standard error of three to five replicates. Values of each antioxidant enzyme activity followed by different letters indicate that the data points are significantly different at p ≤ 0.05.
the total chlorophyll contents in all treatments (Figure S5). In the TC alone treatments, the chlorophyll content was not significantly affected as compared with the control group (1/2X Hoagland’s solution). Interestingly, among all 9 coexposure treatments, the total chlorophyll content was significantly lowered as compared to the control group, except the treatment with 1 mg/L TC × 50 mg/L TiO2 NPs, where no effect was evident. A large number of studies have reported that metalbased NPs could alter the photosynthetic output of plants, including changes to chloroplast structure, total chlorophyll content, net photosynthetic rate, and chlorophyll fluorescence.24,31,35,36 The physiological effects of antibiotics on plant growth have also been reported by Ahmed et al.9 The presence of 5 and 20 mg/L SA and TC severely inhibited tomato, cucumber, and lettuce growth health, as determined by biomass, plant height, total root surface area, and chlorophyll content (SPAD). However, this phytotoxicity was not dosedependent; 10 mg/L antibiotic exposure had equivalent or less impact on plant growth than did the control and lower exposures.9 Our present work is aligned with the published results in that both NPs and TC could significantly alter the chlorophyll content, although single analyte exposure to TC at concentrations up to 10 mg/L exerted no such effect. Additional study is necessary to characterize the mechanistic basis for these observations. Figure S6 shows that both TiO2 NPs and TC, either as single analyte exposures or under cocontaminant conditions, could alter the total protein content in A. thaliana shoots and roots. For example, in the TiO2 NPs alone treatments, the total protein content in 100 mg/L NPs treated shoots was increased by 26%, while, at 200 mg/L, the protein content was reduced by more than 20% relative to the control. However, the similar results were not evident in the roots, where TiO2 had no effect on protein content. In the TC alone treatments, 1 mg/L TC significantly elevated the protein content in both A. thaliana roots and shoots, whereas higher concentrations (10 mg/L) significantly reduced protein levels by 34.6% and 23.8% in the
that the presence of TC lowered the Ti content in the shoots was also evident. Along with the Ti analysis, we also investigated whether the additions of TiO2 NPs and TC could alter the contents of essential nutrients in A. thaliana shoots and roots (Table S3). The results indicated that both NPs and antibiotic did not change the contents of macronutrients, including Mg, P, and K, in A. thaliana roots. The root Ca contents were significantly decreased by approximately 50% upon exposure to TiO2 NPs, TC, and TiO2 NPs × TC. In the shoots, the levels of all macronutrients in 200 mg/L TiO2 NPs alone treatment were similar to the control; however, the presence of TC in either single analyte treatment or cotreatment resulted in 25.9−29.1% and 28.3−48.5% increases of P and K, respectively, relative to their corresponding control. Similar to the levels of macronutrients, TiO2 NPs had much less impact on the contents of micronutrients in both shoots and roots of A. thaliana. However, the presence of 10 mg/L TC notably altered the nutrient distribution in the shoots. For example, in the TC alone treatment, the shoot Fe content was decreased by 39.4% as compared to the control; 49.1% elevation of the Zn content in A. thaliana shoots was also found. In A. thaliana roots, TC lowered the Mn content by 186.84% relative to the control. Ma et al. (2016) reported that both cerium oxide (CeO2) NPs and indium oxide (In2O3) NPs could significantly decrease the Fe and Mn contents in A. thaliana roots.29 Other metal-based NPs, such as neodymium oxide (Nd2O3) NPs, could also alter the essential nutrient contents in the terrestrial plants. For example, Chen et al. (2016) found that Nd2O3 NPs could notably decrease the levels of macronutrients, such as Mg and K, in pumpkin roots relative to the control.34 However, our results suggested that TiO2 NPs had less impact on nutrient alteration as compared with other metal-based NPs. Total chlorophyll and total protein content in A. thaliana upon exposure to TiO2 NPs and TC. Chlorophyll is an important parameter to assess abiotic stressor toxicity to plants. At harvest, the fresh leaf tissues were used to determine 3207
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Figure 3. Antioxidant enzyme activity of A. thaliana roots coexposed to TiO2 NPs and TC. Parts A−D represent SOD, CAT, APX, and POD activities, respectively. Data are mean ± standard error of three to five replicates. Values of each antioxidant enzyme activity followed by different letters indicate that the data points are significantly different at p ≤ 0.05.
43.5% greater than the control group. Upon exposure to 10 mg/L TC alone, SOD activities were increased to 2- and 1.9fold more than controls in the shoots and the roots, respectively. The coexposure treatments exhibited different effects on SOD activities in the two tissues. In the shoots, the SOD activities were similar as compared with their respective TC control, except the 50 and 100 mg/L TiO2 NPs coexposure treatments at 10 mg/L TC, where SOD activity was reduced back to the no analyte control level. On the contrary, in the roots, the SOD activities in the coexposure treatments were highly induced as compared to their respective TC control. For example, at 1 and 5 mg/L TC, TiO2 NPs exposure at all concentrations significantly increased SOD activities (not dose dependent) in A. thaliana roots as compared to their respective TC control. However, at 10 mg/L TC, SOD activities at 50 and 100 mg/L exposures were equivalent to the TC alone control, but 200 mg/L TiO2 NPs exposure resulted in significantly higher SOD activity. CAT and APX are two main antioxidant enzymes that are capable of breaking down H2O2 to H2O and O2. As shown in Figures 2B and 3B, the CAT activity in the roots was almost ten times more than that in the shoots. The CAT activity in the shoots treated with the highest exposure dose of either TiO2 NPs alone or TC alone was approximately 170- and 150-fold that of the control, respectively, whereas the root CAT levels were unaffected relative to the control. Notably, the CAT level response of the plant upon coexposure was consistently different when comparing the shoot and root tissues. In the coexposure treatments at 1 mg/L TC, TiO2 coexposure had no impact on CAT activity in the shoots. Conversely, in the roots, CAT levels decreased in a dose-dependent fashion with TiO2 NPs exposure. At 5 mg/L TC, elevations of the shoot CAT activities were found at all three TiO2 NPs treatments relative to their TC control group. However, no such effect was evident in the roots, where CAT levels were significantly decreased at the low and medium TiO2 doses. Similarly, in the treatment with 10 mg/L TC × 200 mg/L TiO2 NPs, the shoot CAT activity was increased by approximately 65% as compared with
shoots and the roots, respectively. The total protein contents in the coexposure treatments exhibited a dose-dependent yet consistent response. For example, as TiO2 NPs exposure doses increased, the protein contents in 1 mg/L TC treated shoots were elevated at both 100 mg/L and 200 mg/L TiO2 NPs. In the coexposure treatments of 10 mg/L × different concentrations of TiO2 NPs, the shoot protein contents increased at 50 and 100 mg/L TiO2 NPs, and then decreased at 200 mg/L. A similar pattern was also evident in the coexposure treatments of A. thaliana roots. Plant proteins play essential roles in plant growth (root elongation), carbohydrate transport, the photosynthesis system, and the defense mechanism.37 Reduced levels of protein were evident in metal-based NPs treated plants in previous studies.37,38 In our study, a higher concentration of TC led to the significantly lower protein content in the A. thaliana roots, while exposure to TiO2 NPs alone had no impact on the protein content. As the protein content is a critical property, it may be highly useful to figure out how to reasonably and effectively apply NPs and antibiotics to maximize (or at least not reduce) crop quality. So far, the studies on food quality and security of crops upon coexposure to metal-based NPs and antibiotics are scarce. However, the available information from single contaminant exposure studies, along with our coexposure work herein, suggests that the potential risks posed by these emerging contaminants under coexposure scenarios should be further investigated. Antioxidant enzyme activities in A. thaliana roots and shoots. Antioxidant defense is one of the most essential mechanisms of plants to alleviate the toxicity from exposure to xenobiotics in the environment.39,40 In order to understand the detoxification process in A. thaliana upon coexposures of TiO2 NPs and TC, the activities of ROS scavengers, including SOD, CAT, APX, and POD, were separately determined in A. thaliana shoots and roots (Figure 2 and 3). SOD is capable of converting O2•− to H2O2 and O2. Different concentrations of TiO2 NPs (alone) had no impact on SOD activities in the shoots and roots (Figures 2A and 3A), except the treatment with 50 mg/L TiO2 NPs, where the root SOD levels were 3208
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Relative expressions of stress-related genes in A. thaliana roots and shoots. Upon exposure to TiO2 NPs and TC, the relative expression of three important genes involved in the sulfur assimilation pathway, including sulfate adenylytransferase (APT), adenosine-5′-phosphosulfate reductase (APR), and sulfite reductase (SiR), was determined (Figure 4). The
their respective TC and TiO2 NPs control; no difference was evident in the roots. The APX activities in the shoots and the roots of A. thaliana are shown in Figures 2C and 3C, respectively. TC exposure alone had no impact on the shoot or the root APX activities. The results suggested that the root APX activities were more sensitive to TiO2 than the one in the shoots. Notably, the presence of 100 and 200 mg/L TiO2 NPs significantly elevated the root APX activities to 3.5- and 3-fold of the control, respectively; no significant effects were evident in the shoots. The impacts of coexposure on the APX levels in the roots and shoots were generally minimal. However, roots exposed to 5 mg/L TC × 100 mg/L TiO2 NPs had 5-fold greater APX activities; similarly, in the shoots at 5 mg/L TC × 200 mg/L TiO2 NPs, the APX levels were significantly increased. Conversely, at 10 mg/L TC, 100 and 200 mg/L TiO2 significantly suppressed the shoot APX levels, but no such effects were noted in the roots. POD is another antioxidant enzyme that can scavenge the free radicals induced by abiotic stresses in plants. In the TC alone or the TiO2 NPs alone treatments, a significantly high level of POD activity was evident in both shoots and roots treated with 10 mg/L TC, while this elevation was only found in the roots treated with 200 mg/L TiO2 NPs. Among the coexposure treatments, the common result of POD activity in both shoots and roots was that the addition of TiO2 NPs significantly reduced the POD activities at 10 mg/L TC, regardless of the TiO2 NPs concentrations (Figure 2D and 3D). Due to the complexity of the cocontaminant interactions at different exposure doses and over time, more intensive investigation may be required in order to elucidate the antioxidant defense mechanism in plants. Abiotic stress such as metal-based NPs can produce excessive amounts of ROS in terrestrial plants, subsequently causing significant oxidative stress.41−44 Excessive amounts of ROS, acting as signaling molecules, will trigger an antioxidant defense mechanism in the plant so as to detoxify the free radicals that have been generated. Ma et al. (2015) reviewed the roles of antioxidant enzymes in detoxifying NPs-induced toxicity in plants and noted that the magnitude of the antioxidant enzyme response can vary significantly with NPs type, exposure time, and plant species.14 In the study, exposure to TiO2 NPs elevated the activities of CAT, APX, as well as POD in A. thaliana. Conversely, less is known about the antioxidant response of plants to antibiotic exposures. Liu et al. exposed Phragmites australis to different concentrations of an antibiotic mixture including ciprofloxacin, oxy-TC, and SA for 62 days. At harvest, analysis of SOD, CAT, and POD activities suggests that the antibiotics significantly inhibited the enzyme activities at exposures as low as 1 mg/L and that the inhibition is typically dose-dependent.45 There were significant differences in the antioxidant response of the shoots and roots upon coexposure to TC and TiO2 NPs. Within a tissue, TiO2 NPs often significantly affected the enzyme levels in the coexposure scenarios, but consistent trends in the changes were difficult to find. Due to the complexity of interactions between TC and TiO2 NPs, the changes of each antioxidant enzyme activity may vary as a function of the exposure concentration of each analyte, or some unknown impact from other factors such as plant root exudation. In order to thoroughly understand plant antioxidant enzyme response upon antibiotic and NP coexposure, one must also likely consider the role of TC metabolites and TiO2 NPs biotransformation, both of which could significantly impact plant exposure and response.
Figure 4. Relative expression of genes involved in the sulfur assimilation pathway in A. thaliana treated with TiO2 NPs and TC. Parts A−C represent the expression levels of APT, APR, and SiR in A. thaliana shoots and roots upon exposure to TiO2 NPs and TC, respectively. Data are mean ± standard error of three replicates.
relative expression of the genes encoding ATP and SiR in A. thaliana shoots did not significantly change (less than 1.5-fold) in the treated groups relative to the control (Figure 4A and C), but significant down-regulation of APR was evident in both the TC alone and the coexposure treatment (Figure 4B). Interestingly, in A. thaliana roots, the relative expression of APT in all three treatments was significantly increased (3- to 3.5-fold) over the control. Similar upregulation of APR and SiR was also evident in the roots across the three treatments. The glutathione (GSH) biosynthesis pathway is one of the most essential pathways in plants for defense against abiotic stresses, including heavy metal, metal-based NPs, cold, drought, 3209
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Figure 5. Relative expression of stress related genes in A. thaliana treated with TiO2 NPs and TC. Parts A−C represent expression levels of CS, ECS, and GS, respectively. Parts D−F represent expression levels of GST, GR, and MDAR, respectively. Data are mean ± standard error of three replicates.
and heat.25,46,47 Sulfide is the precursor of cysteine in the GSH biosynthesis pathway. The relative expressions of the genes encoding cysteine synthase (CS), γ-glutamylecysteine synthetase (ECS), and GSH synthetase (GS) were measured in A. thaliana shoots and roots treated with TiO2 NPs, TC, and TiO2 NPs × TC (Figure 5A−C). In the shoots, TC alone caused a more than 2-fold decrease in the relative expression of CS, ECS, and GS as compared to the control, whereas no difference in expression was observed in either the TiO2 NPs alone or the coexposure treatment. Conversely, upregulation of ECS and GS was evident in A. thaliana roots treated with either TiO2 NPs alone or TC alone; there was no change in the relative expression of the three genes in the coexposure treatment. The responses of other stress-related genes, including glutathione S-transferase (GST), glutathione reductase (GR), and monodehydroascorbate reductase (MDAR), were also investigated (Figure 5D−F). Upon exposure to TiO2 NPs and TC, downregulation of GST was observed in both A. thaliana shoots and roots; this was particularly notable in the TC alone and the coexposure treatment, where 5- and 2-fold decreases in GST relative expression were noted, respectively (Figure 5D). No significant change of GR relative expression was observed across all three treatments, except the coexposure treatment in the roots, where GR was upregulated relative to the control
(Figure 5E). Similar to the GST relative expression, the gene encoding MDAR in the shoots was downregulated by more than 2-fold in response to exposure to TC alone and the coexposure treatment (Figure 5F). Although slight decreases in MDAR in the roots were also evident, these changes were less than 50% relative to the control. Sulfur is one of the most important macronutrients and plays a critical role in plant growth and viability.48−50 Our previous study indicated that the relative expression of the genes involved in sulfur assimilation was highly up-regulated upon exposure to CeO2 and In2O3 NPs.25 Importantly, this response is not specific to metal-based NPs; upregulation will occur in response to other abiotic stresses, such as metalloid exposure. For example, upon exposure to arsenate, the expression levels of genes involved in sulfate metabolism were highly induced in Abyssinian mustard.51 Sulfide is a precursor of cysteine, which is critical to the first step of the GSH biosynthesis pathway. Under the abiotic stress, upregulation of important genes involved in the GSH biosynthesis pathway indicated that plant defense mechanisms have been activated. Previous studies have demonstrated that the GSH metabolic pathway, as measured by GSH and its derivatives, could greatly enhance plant tolerance to metal-based NPs;36 evidence for upregulation of GSH biosynthesis related genes was also found in our previous 3210
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Figure 6. Effects of coexposure on A. thaliana pod formation. (A) Image of pods exposed to TiO2 NPs and TC; (B) pod number; (C) pod biomass. Data are mean ± standard error of four replicates. Values of each parameter followed by different letters indicate that the data points are significantly different at p ≤ 0.05.
study.25 Our current work suggests that the sulfur assimilation pathway was highly activated given the expression levels of APT, APR, and SiR in the roots across all three treatments. Although the upregulation of CS, ECS, and GS in the roots was also evident in the single analyte TiO2 NPs or TC treatment, no difference was found in the coexposure treatments relative to controls. The potential explanation of sulfur responses upon TC exposure needs to be further investigated, and future study of TC metabolites in plants may help us thoroughly understand the role of the sulfur assimilation pathway in detoxifying TC effects. Other stress-related genes were also evaluated, including GST, GR, and MDAR, in A. thaliana tissues. All three genes are involved in the ascorbate-glutathione cycle in plants and play essential roles in scavenging excess amounts of ROS induced by abiotic stresses. Others have reported similar glutathione related effects with other NPs; both Ag NPs and ZnO NPs could induce GST expressions by approximately 10- and 3.5fold in A. thaliana.52,53 However, in our study, the downregulations of GST and GR were evident in TiO2 NP treated A. thaliana shoots, which suggests that regulation of stress related genes varied under the abiotic stress conditions induced by different types of metal-based NPs. Pod numbers and biomass of A. thaliana treated with TiO2 NPs and TC. At harvest, the total number of pods in each A. thaliana plant and the total pod biomass were recorded. As shown in Figure 6A, 10 mg/L TC severely reduced plant growth and inhibited the pod formation, in terms of both pod size and number (Figure S7). The addition of TiO2 NPs partially alleviated the adverse effects and significantly enhanced plant growth. Although 200 mg/L TiO2 NPs still reduced the total number of pods and the pod biomass by 23% and 30%, respectively, approximately 100% and 300% increases in pod number and biomass were observed upon coexposure, as compared with the TC alone treatment (Figure 6B and C). The results further suggest that both TiO2 NPs and TC could separately affect food quality and yield; however, upon
coexposure, TiO2 NPs counteract the TC-induced toxicity and subsequently enhance plant biomass and yield. Given the concerns over food quality and safety, it is clear that long-term study of metal-based NPs effects, including interactions with cocontaminants, on crop edible tissues is necessary. Some relevant work has been published. For example, Zhao et al. demonstrated that exposure to CeO2 NPs had no impact on nutrient levels in tested corncobs.54 However, CeO2 NPs and related species were detected in soybean edible tissues;55 similarly, TiO2 NPs were also detected in cucumber fruit.24 In addition, several studies have reported trophic transfer of metal-based NPs in model terrestrial systems and have demonstrated potential contamination of the food chain.56,57 Although far from conclusive, these limited number of studies do suggest that NPs accumulation in food does present a potential risk to human health. Similarly, Ahmed et al. reported that antibiotics could accumulate in the edible portions of vegetables such as cucumber, lettuce, and tomato.9 Our results indicate that both xenobiotic substances significantly inhibited pod formation and biomass in A. thaliana, although, upon coexposure, the addition of TiO2 NPs partially alleviated the TC-induced toxicity. Taken together, the present study found that the addition of TiO2 NPs could reduce the TC toxicity to A. thaliana in terms of fresh biomass, total number of pods, as well as pod yield. The relative expressions of genes encoding the sulfur assimilation pathway were strongly up-regulated in A. thaliana upon exposure to TiO2 NPs and TC, indicating the sulfur assimilation pathway plays important roles in detoxification of xenobiotic substances. The impacts on A. thaliana pod formation and pod yield imply the potential risks of both substances to agricultural crop yield and food safety, as the likelihood of such negative effects could translate to the real crop. Clearly, a detailed assessment of the realistic exposure and risk associated with coexposure to NPs and antibiotics is needed and could have significant implications for food safety and consumer health. 3211
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(7) Kim, K.-R.; Owens, G.; Kwon, S.-I.; So, K.-H.; Lee, D.-B.; Ok, Y. S. Occurrence and environmental fate of veterinary antibiotics in the terrestrial environment. Water, Air, Soil Pollut. 2011, 214 (1−4), 163− 174. (8) Baran, W.; Adamek, E.; Ziemiańska, J.; Sobczak, A. Effects of the presence of sulfonamides in the environment and their influence on human health. J. Hazard. Mater. 2011, 196, 1−15. (9) Ahmed, M. B. M.; Rajapaksha, A. U.; Lim, J. E.; Vu, N. T.; Kim, I. S.; Kang, H. M.; Lee, S. S.; Ok, Y. S. Distribution and accumulative pattern of tetracyclines and sulfonamides in edible vegetables of cucumber, tomato, and lettuce. J. Agric. Food Chem. 2015, 63 (2), 398−405. (10) Topal, M. Uptake of tetracycline and degradation products by Phragmites australis grown in stream carrying secondary effluent. Ecological Engineering 2015, 79, 80−85. (11) Boonsaner, M.; Hawker, D. W. Accumulation of oxytetracycline and norfloxacin from saline soil by soybeans. Sci. Total Environ. 2010, 408 (7), 1731−1737. (12) Boonsaner, M.; Hawker, D. W. Investigation of the mechanism of uptake and accumulation of zwitterionic tetracyclines by rice (Oryza sativa L.). Ecotoxicol. Environ. Saf. 2012, 78, 142−147. (13) Pan, M.; Wong, C. K.; Chu, L. Distribution of antibiotics in wastewater-irrigated soils and their accumulation in vegetable crops in the Pearl River Delta, Southern China. J. Agric. Food Chem. 2014, 62 (46), 11062−11069. (14) Ma, C.; White, J. C.; Dhankher, O. P.; Xing, B. Metal-based nanotoxicity and detoxification pathways in higher plants. Environ. Sci. Technol. 2015, 49 (12), 7109−7122. (15) Keller, A. A.; Lazareva, A. Predicted releases of engineered nanomaterials: from global to regional to local. Environ. Sci. Technol. Lett. 2014, 1 (1), 65−70. (16) Weir, A.; Westerhoff, P.; Fabricius, L.; Hristovski, K.; von Goetz, N. Titanium dioxide nanoparticles in food and personal care products. Environ. Sci. Technol. 2012, 46 (4), 2242−2250. (17) Asli, S.; Neumann, P. M. Colloidal suspensions of clay or titanium dioxide nanoparticles can inhibit leaf growth and transpiration via physical effects on root water transport. Plant, Cell Environ. 2009, 32 (5), 577−584. (18) Ma, X.; Geiser-Lee, J.; Deng, Y.; Kolmakov, A. Interactions between engineered nanoparticles (ENPs) and plants: phytotoxicity, uptake and accumulation. Sci. Total Environ. 2010, 408 (16), 3053− 3061. (19) Lee, W.-M.; Kwak, J. I.; An, Y.-J. Effect of silver nanoparticles in crop plants Phaseolus radiatus and Sorghum bicolor: Media effect on phytotoxicity. Chemosphere 2012, 86 (5), 491−499. (20) Yoon, S.-J.; Kwak, J. I.; Lee, W.-M.; Holden, P. A.; An, Y.-J. Zinc oxide nanoparticles delay soybean development: A standard soil microcosm study. Ecotoxicol. Environ. Saf. 2014, 100 (0), 131−137. (21) Kim, S.; Lee, S.; Lee, I. Alteration of Phytotoxicity and Oxidant Stress Potential by Metal Oxide Nanoparticles in Cucumis sativus. Water, Air, Soil Pollut. 2012, 223 (5), 2799−2806. (22) Pagano, L.; Servin, A. D.; De La Torre-Roche, R.; Mukherjee, A.; Majumdar, S.; Hawthorne, J.; Marmiroli, M.; Maestri, E.; Marra, R. E.; Isch, S. M. Molecular Response of Crop Plants to Engineered Nanomaterials. Environ. Sci. Technol. 2016, 50 (13), 7198−7207. (23) Rico, C. M.; Morales, M. I.; McCreary, R.; Castillo-Michel, H.; Barrios, A. C.; Hong, J.; Tafoya, A.; Lee, W.-Y.; Varela-Ramirez, A.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L. Cerium Oxide Nanoparticles Modify the Antioxidative Stress Enzyme Activities and Macromolecule Composition in Rice Seedlings. Environ. Sci. Technol. 2013, 47 (24), 14110−14118. (24) Servin, A. D.; Morales, M. I.; Castillo-Michel, H.; HernandezViezcas, 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 (20), 11592−11598. (25) Ma, C.; Chhikara, S.; Xing, B.; Musante, C.; White, J. C.; Dhankher, O. P. Physiological and molecular response of Arabidopsis
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.6b02976. Additional information on the experimental design, the hydroponic setup, details for each antioxidant enzyme assay, metal analysis, qPCR primers, figures of Ti uptake, total chlorophyll and total protein content, a table of nutrient element content, as well as images of A. thaliana cotreated with TiO2 NPs and TC (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Om Parkash Dhankher:
[email protected]; Phone: 413545-0062;. *Baoshan Xing:
[email protected]; Phone: 413-545-5212; Fax: 413-577-0242. ORCID
Om Parkash Dhankher: 0000-0003-0737-6783 Present Addresses
(H.L. and Z.W.) No. 15 Shangxiadian Road, Cangshan District, Fuzhou, Fujian 350002, China. (C.M. and J.W.) 123 Huntington Street, New Haven, Connecticut 06504, United States. (G.C.) 73 Daqiao Road, Fuyang, Hangzhou 311400, China. (O.P.D. and B.X.) 161 Holdsworth Way, Amherst, Massachusetts 01003, United States. Author Contributions #
H.L. and C.M. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by USDA-AFRI (2011-6700630181) and USDA-NIFA Hatch program (MAS 00475 and MAS 00401). H.L. gratefully acknowledges the support from China Scholarship Council (201207870010) to study at University of Massachusetts, Amherst.
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REFERENCES
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