Liu2017 TiO2 Tetracycline Arabidopsis

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Article
Titanium dioxide nanoparticles alleviate
tetracycline toxicity to Arabidopsis thaliana (L.)
Hong Liu, Chuanxin Ma, Guangcai Chen, Jason C. White, Baoshan Xing, and Om Parkash Dhankher
ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/
acssuschemeng.6b02976 • Publication Date (Web): 24 Feb 2017
Downloaded from http://pubs.acs.org on March 3, 2017
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Page 1 of 34 ACS Sustainable Chemistry & Engineering
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1 Titanium dioxide nanoparticles alleviate tetracycline toxicity to Arabidopsis thaliana
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6 2 (L.)
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8 3 Hong Liu1, 2, †, Chuanxin Ma2, 3, †, Guangcai Chen2, 4, Jason C. White3, Zonghua Wang5,
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4 Baoshan Xing2, * and Om Parkash Dhankher 2, *
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13 5 Fujian Provincial Key Laboratory of Soil Environmental Health and Regulation, College
14 6 of Resources and Environmental Sciences, Fujian Agriculture and Forestry University,
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16 7 Fuzhou 350002, China
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Stockbridge School of Agriculture, University of Massachusetts, Amherst,
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19 9 Massachusetts 01003, United States
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10 Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station,
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22 11 New Haven, Connecticut 06504, United States
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12 Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang,
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25 13 Zhejiang 311400, China
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14 State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian
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28 15 Agriculture and Forestry University, Fuzhou 350002, China
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32 †
18 These authors contributed equally to this work.
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34 19 Corresponding authors:
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35 20 Om Parkash Dhankher: parkash@umass.edu; Phone: 413-545-0062;
36 *
37 21 Baoshan Xing: bx@umass.edu; Phone: 413-545-5212; Fax: 413-577-0242.
38 22
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23 Mailing address:
41 24 H. L.: No. 15 Shangxiadian Road, Cangshan District, Fuzhou, Fujian 350002, China
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25 C. M. and J. W.: 123 Huntington Street, New Haven, Connecticut 06504, United States
44 26 G. C.: 73 Daqiao Road, Fuyang, Hangzhou 311400, China
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27 O. P. D. and B. X.: 161 Holdsworth Way, Amherst, Massachusetts 01003, United States
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ACS Sustainable Chemistry & Engineering Page 2 of 34
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34 ABSTRACT
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5 35 Arabidopsis thaliana (L.) Heynh. was used as a model plant to investigate the
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7 36 biochemical and molecular response upon co-exposures to tetracycline (TC) and titanium
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9 37 oxide nanoparticles (TiO2 NPs). Results showed that 1 mg/L TC severely reduced A.
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12 38 thaliana biomass by 33.3% as compared with the control; however, the presence of 50
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14 39 and 100 mg/L TiO2 NPs alleviated TC toxicity, increasing fresh biomass by 45% and
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40 28%, respectively, relative to the TC alone treatment. The presence of TC notably
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19 41 decreased Ti accumulation in both shoots and roots. Antioxidant enzyme activity,
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21 42 including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX),
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43 peroxidase (POD), in A. thaliana shoots and roots indicated that TC significantly
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26 44 increased the activity of reactive oxygen species (ROS) scavengers. However, in the co-
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28 45 exposure treatments, TiO2 NPs reduced antioxidant enzyme activity back to the control
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31 46 levels. The relative expression of genes encoding sulfur assimilation and glutathione
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33 47 biosynthesis pathways was separately measured in shoots and roots. Interestingly, the
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35 48 relative expressions of adenylytransferase (APT), adenosine-5′-phosphosulfate reductase
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38 49 (APR), and sulfite reductase (SiR) in the roots across all three treatments (TC alone, TiO2
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40 50 NPs alone, and TC×TiO2 NPs treatment) were 2-3.5 fold higher than the control. The
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51 expression of γ-glutamylecysteine synthetase (ECS) and glutathione synthetase (GS) was
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45 52 increased in A. thaliana treated with either TiO2 NPs or TC alone. At harvest, almost 93%
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47 53 reduction of the pod biomass was evident in the TC alone treatment as compared with the
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50 54 control; however, TiO2 NPs increased the pod biomass by 300% in the co-exposed plants
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52 55 relative to the TC alone treatment. These findings provide important information for
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54 56 understanding the interactions of metal-based NPs and co-contaminants such as
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57 57 antibiotics in plant systems.
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58 Keywords: Arabidopsis thaliana, titanium oxide nanoparticles, tetracycline, co-
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6 59 contamination, molecular response, crop quality
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62 INTRODUCTION
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6 63 Antibiotics have been widely used in agriculture and livestock industries for the purposes
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8 64 of growth enhancement and disease prevention.1 Most antibiotics are water-soluble and
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65 are poorly absorbed by livestock; approximately 40-90% of the antibiotics are excreted
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13 66 through urine or feces.2 In the United States, approximately 51 tons of antibiotics were
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15 67 used, and almost 79% of antibiotics (equals to 13540000 kg) were applied in the raising
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18 68 of livestock annually.3 Similarly, in China, approximately 210,000 tons of antibiotics are
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20 69 produced annually and more than three quarters are used in animal husbandry.4
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22 70 Antibiotics can be released to the environment from pharmaceutical wastes, wastewater
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25 71 treatment facilities, and livestock industries.1, 5 Since soils and water bodies are
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27 72 considered the primary sinks for environmental pollutants, concerns have been raised
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73 over the adverse impacts of antibiotics on microbial, plant, and animal communities.1, 6
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33 74 According to the European Union, if the environmental concentration of antibiotics
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35 75 exceeds 10 µg/kg, the further assessment of the specific chemical compound should be
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38 76 conducted. The estimated concentrations of TC and TC derivatives could be in a range of
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40 77 450-900 µg/kg, which was approximately 45-90 folds higher than the European Union
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42 78 regulation.7 Thus, it is necessary to investigate the TC toxicity to the terrestrial plants
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45 79 with the presence of other emerging contaminants in environment. Sulfonamides (SA)
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47 80 and tetracycline (TC) are among the most widely used antibiotic groups due to their
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81 broad inhibition of microorganisms, protozoa, and other parasite populations.8 Once the
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52 82 antibiotics are discharged into the environment, it is likely that plant species will
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54 83 accumulate the residues from soil. In fact, a large body of the present studies has focused
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84 on antibiotic uptake in terrestrial plants. A recent study reported that common vegetable
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85 crops, including cucumber (Cucumis sativa L.), tomato (Solanum lycopersicum L.), and
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6 86 lettuce (Lactuca sativa L.), could accumulate both SA and TC in the different plant
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8 87 tissues upon exposures to various concentrations (0-20 mg/kg) for 45 day.9 Separately,
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88 Topal et al found that the concentrations of TC and its degradation products in
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13 89 Phragmites australis significantly decreased from root to leaf.10 Additionally, the relative
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15 90 accumulation of TC and its degradation products was as follows: 4-epitetracycline >
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18 91 tetracycline > 4-epianhydrotetracycline > anhydrotetracycline.10 Pot experiments with
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20 92 soybean grown in 105 mg/kg oxytetracycline contaminated saline soil showed that the
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22 93 antibiotic was only accumulated in the roots and no translocation was evident to the
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25 94 shoots.11 The type of antibiotic will also clearly impact the uptake levels by plants. For
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27 95 example, oxy-TC and TC at a 50 mg/L exposure had the similar root concentration
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96 factors (about 2100) in treated rice over a period of 15 d exposure, whereas a
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32 97 significantly higher value of RCF (approximately 2700) was evident for chlor-TC at 11 d
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34 98 of exposure.12 Pan et al. suggested that antibiotic bioaccumulation along the food chain
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37 99 should not be neglected, but also noted that the concentrations (TC is 2100 µg) in
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39 100 vegetable crops are significantly lower than the minimum therapeutic dose (20-200mg).13
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41 101 Importantly, few mechanistic studies investigating the basis of plant toxicity and response
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44 102 are available in the literature and as such, understanding of these processes remains
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46 103 inadequate.
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104 Engineered nanomaterials (ENMs) have been increasingly applied in various fields, such
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52 105 as agriculture, food manufacturing, biomedicals, electronics, and renewable energy.14
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54 106 Titanium oxide (TiO2) is one of the commonly used nanoparticles (NPs); more than 3000
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107 tons of TiO2 NPs are produced annually, and more than half is used in personal care
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108 products.15,16 In agriculture, ENMs can be used to detect pathogens, more effectively
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6 109 deliver pesticides and fertilizers, and to monitor soil conditions.17 Due to their nanoscale
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8 110 size, ENMs may pose potential risks in agriculture system despite of the positive impacts
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111 on crops.18 Recent laboratory studies have demonstrated that the presence of ENMs can
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13 112 result in root elongation inhibition, biomass decrease, low photosynthetic efficiency,
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15 113 developmental delay, and unique molecular effects of unknown consequence.19, 20, 21, 22
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18 114 Plant defense mechanisms upon exposure to different types of metal-based NMs have
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20 115 been recently reviewed by Ma et al.14 Antioxidant enzyme activity in terrestrial plants
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22 116 played an essential role in detoxifying nanoparticle-induced phytotoxicity.23, 24 In
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25 117 addition, studies at the molecular level also provide insight on responses of stress-related
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27 118 genes in plants upon nanoparticle exposures.25 Given the complex nature of agricultural
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119 systems, the likelihood of co-exposure to metal oxide nanoparticles and pharmaceutical
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32 120 compounds such as antibiotics is quite high; however, little work has been done in this
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34 121 area.
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38 122 In the present study, we chose Arabidopsis thaliana (L.) Heynh. as a target plant and
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40 123 hydroponically exposed it to TiO2 NPs and TC. In order to comprehensively understand
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42 124 the defense mechanism and impacts of co-contaminants on crop yield, the physiological
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45 125 and molecular responses of A. thaliana upon co-exposure to TiO2 NPs and TC were
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47 126 investigated. The resulting impact on biomass, Ti uptake, chlorophyll content, protein
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127 content, and pod formation was determined. Additionally, activities of main reactive
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52 128 oxygen species (ROS) scavengers including superoxide dismutase (SOD), catalase
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54 129 (CAT), ascorbate peroxidase (APX), and peroxidase (POD) were measured in A. thaliana
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130 shoots and roots across all treatments. At the molecular level, the relative expression of
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131 genes involved in sulfur assimilation, glutathione biosynthesis, as well as stress-related
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6 132 genes, were analyzed in shoots and roots of A. thaliana. To our knowledge, this is the
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8 133 first report on addressing the role of metal-based NPs in alleviating the toxicity of
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134 antibiotics to plants. Our findings provided the important information for the potential
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13 135 risks of antibiotics and metal-based NPs to the agricultural crops in terms of crop yield
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15 136 and quality if such impacts on A. thaliana could translate to the real crops.
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19 137 MATERIALS AND METHODS
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21 138 Experimental design.
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23 139 Concentration optimization. Titanium oxide nanoparticles (TiO2 NPs) were purchased
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26 140 from US Research Nanomaterials, Inc. The size of TiO2 NPs was ranging from 5 to 15
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28 141 nm, and the form of TiO2 NPs was rutile. Tetracycline (≥98%) was obtained from Sigma-
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142 Aldrich. Twenty-five surface sterilized seeds of A. thaliana ecotype Columbia (Col-0)
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33 143 were placed on half strength (1/2x) Murashige and Skoog (MS) semisolid medium
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35 144 amended with different concentrations of TiO2 NPs ranging from 0 to 500 mg/L.
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38 145 Similarly, sterilized seeds were grown in 1/2x MS semisolid medium amended with
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40 146 different concentrations of TC (0-5 mg/L). Seeds were stratified at 4 °C for 24 hours
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42 147 prior to transfer to a controlled plant growth chamber with 16 h light and 8 h dark at 22
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45 148 and 8 °C, respectively. At harvest, total fresh biomass was used to determine the toxicity
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47 149 of TiO2 NPs and TC; the results are shown in Figure S1.
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150 Hydroponic system. A. thaliana was grown in vermiculite for 21 days, and the
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53 151 seedlings were then transferred to a hydroponic system as shown in Figure S2 and were
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55 152 allowed to acclimatize for 5 days. The doses of TiO2 and TC in the hydroponic system
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153 were determined from the concentration optimization test described above. As shown in
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154 Figure S1A, lower concentration of TiO2 NPs (50 mg/L) significantly enhanced plant
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6 155 growth; no significant difference was observed at 100 mg/L TiO2 NPs; and the fresh
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8 156 biomass was greatly reduced as TiO2 NPs concentrations were increased to 200 mg/L.
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157 Thus, these three exposures of TiO2 NPs (50, 100, and 200 mg/L) were chosen for the
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13 158 hydroponic experiment. The identical method was applied to determine the appropriate
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15 159 TC concentrations in the hydroponic experiment. TC exposure had no impact on fresh
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18 160 biomass until 1 and 5 mg/L, where fresh biomass was reduced by 21.7% and 59.6%
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20 161 relative to the control, respectively (Figure S1B). The three doses of TC chosen were 1, 5,
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22 162 and 10 mg/L. In total, there are 16 treatments (Table S1), including 9 different co-
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25 163 exposure treatments, three single analyte controls for each TiO2 NPs and TC exposure,
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27 164 and one control with 1/2X Hoagland’s solution only. A number of six replicates were
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165 applied in each treatment. A. thaliana seedlings were exposed to different concentrations
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32 166 of TiO2 NPs and TC for 12 days. At harvest, the roots were thoroughly rinsed with
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34 167 deionized water three times and root length and total fresh biomass were measured. Plant
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37 168 tissues were stored at -80 °C until further analysis.
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40 169 Chlorophyll content. The total chlorophyll content in A. thaliana was measured as
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42 170 described by Lichtenthaler et al.26 Briefly, 50 mg of fresh leaves were collected and cut
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45 171 into pieces (less than 1 cm); 10 mL 95% ethanol was then used to extract the total
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47 172 chlorophyll. All samples were kept in dark for 3 days to avoid chlorophyll degradation.
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173 The absorbance was measured at 664.2 nm and 648.6 nm by a UV-Vis spectrophotometer
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52 174 (Agilent 8453, Santa Clara, CA). Chlorophyll a, chlorophyll b and the total chlorophyll
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54 175 were calculated by the following equations: Chla=13.36A664.2-5.19A648.6 (1),
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176 Chlb=27.43A648.6-8.12A664.2 (2), and Total chlorophyll=Chla + Chlb (3).
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177 Ti and nutrient element contents. A. thaliana shoots and roots were separately freeze-
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6 178 dried and then were ground to find powder. Approximately 50 mg of shoot tissues or 10
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8 179 mg of root tissues were digested in sulfuric acid (H2SO4) and hydrogen peroxide (H2O2)
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180 following the method as described by Short et al. (1996) and Wei et al. (2015) with slight
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13 181 modification.27, 28 The detailed information is provided in the supporting information. The
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15 182 samples were measured using inductively coupled plasma optical emission spectrometry
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18 183 (ICP-OES).29
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21 184 Total protein content. The Bradford reagent (Sigma Aldrich, St. Louis, MO) was used
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23 185 to measure the total protein content in plant tissues.30 A sample of 50 mg of A. thaliana
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26 186 shoots or roots was extracted in 2 mL of 10 mM Tris-HCl (pH 7.2) solution. The mixture
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28 187 was then centrifuged at 2683 ×g for 20 min at 4 °C. One hundred µL supernatant was
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188 used to react with 1000 µL of Bradford reagent for 15 min at ambient temperature, and
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33 189 then the absorbance was measured at 595 nm by a UV-Vis spectrophotometer (Agilent
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35 190 8453, Santa Clara, CA).
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39 191 Antioxidant enzyme assays. Fresh root and shoot tissues were homogenized in liquid
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41 192 nitrogen to fine powder. A 0.5 g sample of homogenized tissue was then vigorously
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43 193 mixed with 5 mL extraction buffer for 5 min using a vortex mixer. The mixture was
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46 194 centrifuged at 2683 ×g for 20 min at 4 °C, and the supernatant was used to measure
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48 195 antioxidant enzyme activity, including SOD, CAT, APX, and POD. The modified
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55 198 Gene expression measurement by quantitative reverse transcription polymerase
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199 chain reaction (qRT-PCR). Shoot and root tissues were separately homogenized in
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200 liquid nitrogen prior to RNA isolation. Protocols for total RNA isolation, cDNA
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6 201 synthesis, and gene expression using qRT-PCR were described in Ma et al.25 Briefly,
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8 202 RNeasy plant mini kits were used to isolate total RNA, with the concentration being
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203 quantified by NanoDrop spectrophotometry (ThermoScientific, West Palm Beach, FL). A
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13 204 Verso cDNA synthesis kit was used to synthesize cDNA and the gene-specific primer
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15 205 was designed using Primer Quest (Integrated DNA Technologies, Coralville, IA). A
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18 206 complete list of primer sequences is provided in Table S2. Information for qTR-PCR
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20 207 amplification program can be found in the supplementary information. Relative quantities
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22 208 (2−∆∆Ct method) were used to calculate the transcription level of each gene.
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26 209 Statistical analysis. A one-way analysis of variance (One-way ANOVA) followed by
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28 210 Duncan’s multiple comparison test was used to determine statistical significance of each
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211 parameter across treatments, except qRT-PCR assay, in which a Student t-test was
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33 212 applied to determine statistical significance for each gene. In the figures for each assay,
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35 213 values followed by different letters are significantly different at p ≤ 0.05.
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38 214
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40 215 RESULTS AND DISCUSSION
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42 216 1. Growth of A. thaliana upon exposure to TiO2 NPs and TC
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45 217 Different concentrations of TiO2 NPs did not significantly affect plant growth in Figure
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47 218 1A and Figure S3. Although 1 mg/L TC exhibited negative effects on growth as
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219 compared with the control group (Figure 1A), the presence of TiO2 NPs seemed to
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52 220 visibly alleviate TC phytotoxicity. At harvest, root length and total fresh biomass were
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54 221 measured (Figure 1B and C). The root length was significantly reduced in the 10 mg/L
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222 TC exposure; no difference was evident in the co-exposure treatments, except the
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223 treatments of 1 mg/L TC × 100 and 200 mg/L TiO2 NPs; in both cases, root length was
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6 224 reduced by 22% and 17%, respectively, as compared with the TC control. The total fresh
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8 225 biomass in all three TC alone treatments was reduced by approximately 30% relative to
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226 the control group. The additions of 50 and 100 mg/L TiO2 NPs significantly alleviated
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13 227 the phytotoxicity, elevating the fresh biomass in 1mg/L TC treated A. thaliana back to the
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15 228 level of the control group (Figure 1C). This result suggests that TiO2 NPs could
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18 229 counteract the TC toxicity to A. thaliana at the lower antibiotic doses. As the
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20 230 concentrations of TC were increased to 5 and 10 mg/L, although there was a trend of
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22 231 fresh biomass increase, the results were statistically insignificant. One of the possible
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25 232 explanations is that the TiO2 NPs interact with the TC outside of the plant, preventing
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27 233 exposure at a micro/nano level, but that this process is saturated when exposure dose of
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234 TC reached to 1 mg/L.
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33 235 2. Ti uptake and nutrient element contents in A. thaliana upon exposure to TiO2 NPs
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35 236 and TC
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38 237 The contents of Ti and other essential nutrients were measured in A. thaliana shoots and
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40 238 roots treated with TiO2 NPs and TC (Figure S4 and Table S3). The presence of 10 mg/L
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42 239 TC significantly reduced Ti accumulation in the roots and decreased Ti translocation to
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45 240 the shoots. In 200 mg/L TiO2 NPs alone treatment, the root Ti content was 47140.55
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47 241 mg/kg over 12 days exposure, while this value was decreased by 24.81% in the co-
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242 exposure treatment. Similar trend showing the presence of TC lowered the Ti content in
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52 243 the shoots was also evident. Along with the Ti analysis, we also investigated whether the
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54 244 additions of TiO2 NPs and TC could alter the contents of essential nutrients in A. thaliana
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245 shoots and roots (Table S3). The results indicated that both NPs and antibiotic did not
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246 change the contents of macronutrients, including Mg, P, and K, in A. thaliana roots. The
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6 247 root Ca contents were significantly decreased by approximately 50% upon exposure to
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8 248 TiO2 NPs, TC, and TiO2 NPs × TC. In the shoots, the levels of all macronutrients in 200
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249 mg/L TiO2 NPs alone treatment were similar to the control, however, the presence of TC
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13 250 in either single treatment or co-treatment resulted in 25.9-29.1% and 28.3-48.5 %
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15 251 increases of P and K, respectively, relative to their corresponding control. Similar to the
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18 252 levels of macronutrients, TiO2 NPs had very less impact on the contents of micronutrients
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20 253 in both shoots and roots of A. thaliana. However, the presence of 10 mg/L TC notably
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22 254 altered the nutrient distribution in the shoots. For example, in the TC alone treatment, the
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25 255 shoot Fe content was decreased by 39.4% as compared to the control; 49.1% elevation of
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27 256 the Zn content in A. thaliana shoots was also found. In A. thaliana roots, TC lowered the
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257 Mn content by 186.84% relative to the control. Ma et al. (2016) reported that both cerium
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32 258 oxide (CeO2) NPs and indium oxide (In2O3) NPs could significantly decrease the Fe and
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34 259 Mn contents in A. thaliana roots.29 Other metal-based NPs, such as neodymium oxide
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37 260 (Nd2O3) NPs, could also alter the essential nutrient contents in the terrestrial plants. For
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39 261 example, Chen et al. (2016) found that Nd2O3 NPs could notably decrease the levels of
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41 262 macronutrients, such as Mg and K, in pumpkin roots relative to the control.34 However,
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44 263 our results suggested that the presence of TiO2 NPs had less impact on nutrient alteration
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46 264 as compared with other metal-based NPs.
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48 265
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51 266 3. Total chlorophyll and total protein content in A. thaliana upon exposure to TiO2
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53 267 NPs and TC
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268 Chlorophyll is an important parameter to assess abiotic stressor toxicity to plants. At
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6 269 harvest, the fresh leaf tissues were used to determine the total chlorophyll contents in all
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8 270 treatments (Figure S5). In the TC alone treatments, the chlorophyll content was not
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271 significantly affected as compared with the control group (1/2X Hoagland’s solution).
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13 272 Interestingly, among all 9 co-exposure treatments the total chlorophyll content was
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15 273 significantly lowered as compared to the control group, except the treatment with 1mg/L
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18 274 TC + 50 mg/L TiO2 NPs, where no effect was evident. A large number of studies have
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20 275 reported that metal-based NPs could alter the photosynthetic output of plants, including
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22 276 changes to chloroplast structure, total chlorophyll content, net photosynthetic rate, and
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25 277 chlorophyll fluorescence. 24, 31, 35, 36 The physiological effects of antibiotics on plant
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27 278 growth have also been reported by Ahmed et al.9 The presence of 5 and 20 mg/L SA and
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279 TC severely inhibited tomato, cucumber, and lettuce growth health, as determined by
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32 280 biomass, plant height, total root surface area, and chlorophyll content (SPAD). However,
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34 281 this phytotoxicity was not dose-dependent; 10 mg/L antibiotic exposure had equivalent or
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37 282 less impact on plant growth than did the control and lower exposures.9 Our present work
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39 283 is aligned with the published results in that both NPs and TC could significantly alter the
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41 284 chlorophyll content, although single analyte exposure to TC at concentrations up to 10
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44 285 mg/L exerted no such effect. Additional study is necessary to characterize the
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46 286 mechanistic basis for these observations.
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287 Figure S6 shows that both TiO2 NPs and TC, either as single analyte exposures or under
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52 288 co-contaminant conditions, could alter the total protein content in A. thaliana shoots and
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54 289 roots. For example, in the TiO2 NPs alone treatments, the total protein content in 100
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290 mg/L NPs treated shoots was increased by 26%, while at 200 mg/L, the protein content
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291 was reduced by more than 20% relative to the control. However, the similar results were
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6 292 not evident in the roots, where TiO2 had no effect on protein content. In the TC alone
7
8 293 treatments, 1 mg/L TC significantly elevated the protein content in both A. thaliana roots
9
10
11
294 and shoots, whereas higher concentrations (10mg/L) significantly reduced protein levels
12
13 295 by 34.6% and 23.8% in the shoots and the roots, respectively. The total protein contents
14
15 296 in the co-exposure treatments exhibited a dose-dependent yet consistent response. For
16
17
18 297 example, as TiO2 NPs exposure doses increased, the protein contents in 1 mg/L TC
19
20 298 treated shoots were elevated at both 100 mg/L and 200 mg/L TiO2 NPs. In the co-
21
22 299 exposure treatments of 10 mg/L × different concentrations of TiO2 NPs, the shoot protein
23
24
25 300 contents increased at 50 and 100 mg/L TiO2 NPs, and then decreased at 200 mg/L.
26
27 301 Similar pattern was also evident in the co-exposure treatments of A. thaliana roots. Plant
28
29
302 proteins play the essential roles in plant growth (root elongation), carbohydrate transport,
30
31
32 303 photosynthesis system, and defense mechanism.37 Reduced levels of protein were evident
33
34 304 in metal-based NPs treated plants in previous studies.37, 38 In our study, a higher
35
36
37 305 concentration of TC led to the significantly lower protein content in the A. thaliana roots,
38
39 306 while exposure to TiO2 NPs alone had no impact on the protein content. As the protein
40
41 307 content is a critical property, it may be highly useful to figure out how to reasonably and
42
43
44 308 effectively apply NPs and antibiotics in to maximize (or at least not reduce) crop quality.
45
46 309 So far, the studies on food quality and security of crops upon co-exposure to metal-based
47
48 310 NPs and antibiotics are scarce. However, the available information from single
49
50
51 311 contaminant exposure studies, along with our co-exposure work herein, suggests that the
52
53 312 potential risks posed by these emerging contaminants under co-exposure scenarios should
54
55
56
313 be further investigated.
57
58
59
60
14
1
2
3
314 4. Antioxidant enzyme activities in A. thaliana roots and shoots
4
5
6 315 Antioxidant defense is one of the most essential mechanisms of plants to alleviate the
7
8 316 toxicity from exposure to xenobiotics in the environment.39, 40 In order to understand the
9
10
11
317 detoxification process in A. thaliana upon co-exposures of TiO2 NPs and TC, the
12
13 318 activities of ROS scavengers, including SOD, CAT, APX, and POD, were separately
14
15 319 determined in A. thaliana shoots and roots (Figure 2 and 3). SOD is capable of
16
17 ·−
18 320 converting to O2 to H2O2 and O2. Different concentrations of TiO2 NPs (alone) had no
19
20 321 impact on SOD activities in the shoots and roots (Figure 2A and 3A), except the
21
22
23 322 treatment with 50 mg/L TiO2 NPs, where the root SOD levels were 43.5% greater than
24
25 323 the control group. Upon exposure to 10 mg/L TC alone, SOD activities were increased to
26
27
28
324 2- and 1.9-fold more than controls in the shoots and the roots, respectively. The co-
29
30 325 exposure treatments exhibited different effects on SOD activities in the two tissues. In the
31
32 326 shoots, the SOD activities were similar as compared with their respective TC control,
33
34
35 327 except the 50 and 100 mg/L TiO2 NPs co-exposure treatments at 10 mg/L TC, where
36
37 328 SOD activity was reduced back to the no analyte control level. On the contrary, in the
38
39 329 roots, the SOD activities in the co-exposure treatments were highly induced as compared
40
41
42 330 to their respective TC control. For example, at 1 and 5 mg/L TC, TiO2 NPs exposure at
43
44 331 all concentrations significantly increased SOD activities (not dose dependent) in A.
45
46 332 thaliana roots as compared to their respective TC control. However, at 10 mg/L TC, SOD
47
48
49 333 activities at 50 and 100 mg/L exposures were equivalent to the TC alone control, but 200
50
51 334 mg/L TiO2 NPs exposure resulted in significantly higher SOD activity.
52
53
54
55
335 CAT and APX are two main antioxidant enzymes that are capable of breaking down
56
57 336 H2O2 to H2O and O2. As shown in Figure 2B and 3B, the CAT activity in the roots were
58
59
60
15
1
2
3
337 almost ten times more than in the shoots. The CAT activity in the shoots treated with the
4
5
6 338 highest exposure dose of either TiO2 NPs alone or TC alone was approximately 170 and
7
8 339 150-fold of the control, respectively, whereas the root CAT levels were unaffected
9
10
11
340 relative to the control. Notably, the CAT level response of the plant upon co-exposure
12
13 341 was consistently different when comparing the shoot and root tissues. In the co-exposure
14
15 342 treatments at 1 mg/L TC, TiO2 co-exposure had no impact on CAT activity in the shoots.
16
17
18 343 Conversely, in the roots, CAT levels decreased in a dose-dependent fashion with TiO2
19
20 344 exposure. At 5 mg/L TC, elevations of the shoot CAT activities were found at all three
21
22 345 TiO2 NPs treatments relative to their TC control group. However, no such effect was
23
24
25 346 evident in the roots, where CAT levels were significantly decreased at the low and
26
27 347 medium TiO2 doses. Similarly, in the treatment with 10mg/L TC × 200 mg/L TiO2 NPs,
28
29
348 the shoot CAT activity was increased by approximately 65% as compared with their
30
31
32 349 respective TC and TiO2 NPs control, no difference was evident in the roots. The APX
33
34 350 activities in the shoots and the roots of A. thaliana are shown in Figure 2C and 3C,
35
36
37 351 respectively. TC exposure alone had not impact on the shoot or root APX activities. The
38
39 352 results suggested that the root APX activities were sensitive to TiO2 than the one in the
40
41 353 shoots. Notably, the presence of 100 and 200 mg/L TiO2 NPs significantly elevated the
42
43
44 354 root APX activities to 3.5- and 3-fold of the control, respectively; no significant effects
45
46 355 were evident in the shoots. The impact of co-exposure on APX levels in the roots and
47
48 356 shoots were generally minimal. However, roots exposed to 5 mg/L TC × 100 mg/L TiO2
49
50
51 357 NPs had 5-fold greater APX activities; similarly, in the shoots at 5 mg/L TC × 200 mg/L
52
53 358 TiO2 NPs, APX levels were significantly increased. Conversely, at 10 mg/L TC, 100 and
54
55
56
57
58
59
60
16
1
2
3
359 200 mg/L TiO2 significantly suppressed the shoot APX levels, but no such effects were
4
5
6 360 noted in the roots.
7
8
9 361 POD is another antioxidant enzyme that can scavenge the free radicals induced by abiotic
10
11
12 362 stresses in plants. In the TC alone or the TiO2 NPs alone treatments, significantly high
13
14 363 level of POD activity was evident in both shoots and roots treated with 10 mg/L TC;
15
16 364 while this elevation was only found in the roots treated with 200 mg/L TiO2 NPs. Among
17
18
19 365 the co-exposure treatments, the common result of POD activity in both shoots and roots
20
21 366 was that the addition of TiO2 NPs significantly reduced the POD activities at 10 mg/L TC,
22
23 367 regardless of the TiO2 NPs concentrations (Figure 2D and 3D). Due to the complexity of
24
25
26 368 the co-contaminant interactions at different exposure doses and over time, more intensive
27
28 369 investigation may require in order to elucidate the antioxidant defense mechanism in
29
30
31
370 plants.
32
33
34 371 Abiotic stress such as metal-based NPs can produce excessive amounts of ROS in
35
36 372 terrestrial plants, subsequently causing significant oxidative stress.41 42, 43, 44 Excessive
37
38
39 373 amounts of ROS, acting as signaling molecules, will trigger antioxidant defense
40
41 374 mechanism in the plant so as to detoxify the free radicals that have been generated. Ma et
42
43 375 al. (2015) reviewed the roles of antioxidant enzymes in detoxifying NPs-induced toxicity
44
45
46 376 in plants and noted that the magnitude of the antioxidant enzyme response can vary
47
48 377 significantly with NPs types, exposure time and plant species.14 In the study, exposure to
49
50
378 TiO2 NPs elevated the activities of CAT, APX, as well as POD in A. thaliana.
51
52
53 379 Conversely, less is known about the antioxidant response of plants to antibiotic exposures.
54
55 380 Liu et al. exposed Phragmites australis to different concentrations of an antibiotic
56
57
58
381 mixture including ciprofloxacin, oxy-TC, and SA for 62 days. At harvest, analysis of
59
60
17
1
2
3
382 SOD, CAT and POD activities suggests that the antibiotics significantly inhibited the
4
5
6 383 enzyme activities at exposures as low as 1 mg/L and that the inhibition is typically dose-
7
8 384 dependent.45 There were significant differences in the antioxidant response of the shoots
9
10
11
385 and roots upon co-exposure to TC and TiO2 NPs. Within a tissue, TiO2 NPs often
12
13 386 significantly affected the enzyme levels in the co-exposure scenarios but consistent trends
14
15 387 in the changes were difficult to find. Due to the complexity of interactions between TC
16
17
18 388 and TiO2 NPs, the changes of each antioxidant enzyme activity may vary as a function of
19
20 389 the exposure concentration of each analyte, or some unknown impact from other factors
21
22 390 such as plant root exudation. In order to thoroughly understand plant antioxidant enzyme
23
24
25 391 response upon antibiotic and NP co-exposure, one must also likely consider the role of
26
27 392 TC metabolites and TiO2 NPs biotransformation, both of which could significantly
28
29
393 impact plant exposure and response.
30
31
32
33 394 5. Relative expressions of stress-related genes in A. thaliana roots and shoots
34
35 395 Upon exposure to TiO2 NPs and TC, the relative expression of three important genes
36
37
38 396 involved in the sulfur assimilation pathway, including sulfate adenylytransferase (APT),
39
40 397 adenosine-5′-phosphosulfate reductase (APR), and sulfite reductase (SiR), was
41
42 398 determined (Figure 4). The relative expression of the genes encoding ATP and SiR in A.
43
44
45 399 thaliana shoots did not significantly change (less than 1.5-fold) in the treated groups
46
47 400 relative to the control (Figure 4A and C), but significant down-regulation of APR was
48
49
401 evident in both the TC alone and the co-exposure treatment (Figure 4B). Interestingly, in
50
51
52 402 A. thaliana roots, the relative expression of APT in all three treatments was significantly
53
54 403 increased (3 to 3.5-fold) over the control. Similar upregulation of APR and SiR were also
55
56
57
404 evident in the roots across the three treatments.
58
59
60
18
1
2
3
405 The glutathione (GSH) biosynthesis pathway is one of the most essential pathways in
4
5
6 406 plants for defense against abiotic stresses, including heavy metal, metal-based NPs, cold,
7
8 407 drought, heat.25, 46, 47 Sulfide is the precursor of cysteine in the GSH biosynthesis pathway.
9
10
11
408 The relative expressions of the genes encoding cysteine synthase (CS), γ-
12
13 409 glutamylecysteine synthetase (ECS), and GSH synthetase (GS) were measured in A.
14
15 410 thaliana shoots and roots treated with TiO2 NPs, TC, and TiO2 NPs × TC (Figure 5A-C).
16
17
18 411 In the shoots, TC alone caused a more than 2-fold decrease in the relative expression of
19
20 412 CS, ECS and GS as compared to the control, whereas no difference in expression was
21
22 413 observed in either the TiO2 NPs alone or the co-exposure treatment. Conversely,
23
24
25 414 upregulation of ECS and GS was evident in A. thaliana roots treated with either TiO2
26
27 415 NPs alone or TC alone; there was no change in the relative expression of the three genes
28
29
416 in the co-exposure treatment.
30
31
32
33 417 Responses of other stress-related genes including glutathione S-transferase (GST),
34
35 418 glutathione reductase (GR), and monodehydroascorbate reductase (MDAR), were also
36
37
38 419 investigated (Figure 5D-F). Upon exposures to TiO2 NPs and TC, downregulation of
39
40 420 GST were observed in both A. thaliana shoots and roots; this was particularly notable in
41
42 421 the TC alone and the co-exposure treatment, where 5- and 2-fold decreases in GST
43
44
45 422 relative expression was noted, respectively (Figure 5D). No significant change of GR
46
47 423 relative expression was observed across all three treatments, except the co-exposure
48
49
424 treatment in the roots, where GR was upregulated relative to the control (Figure 5E).
50
51
52 425 Similar to the GST relative expression, the gene encoding MDAR in the shoots was
53
54 426 downregulated by more than 2-fold in response to exposure to TC alone and the co-
55
56
57
58
59
60
19
1
2
3
427 exposure treatment (Figure 5F). Although slight decreases in MDAR in the roots were
4
5
6 428 also evident, these changes were statistically insignificant.
7
8
9 429 Sulfur is one of the most important macronutrients and plays a critical role in plant
10
11
12 430 growth and viability.48, 49, 50 Our previous study indicated that the relative expression of
13
14 431 the genes involved in sulfur assimilation was highly up-regulated upon exposure to CeO2
15
16 432 and In2O3 NPs.25 Importantly, this response is not specific to metal-based NPs;
17
18
19 433 upregulation will occur in response to other abiotic stresses, such as metalloid exposure.
20
21 434 For example, upon exposure to arsenate, the expression levels of genes involved in
22
23 435 sulfate metabolism were highly induced in Abyssinian mustard.51 Sulfide is a precursor
24
25
26 436 of cysteine, which is critical to the first step of the GSH biosynthesis pathway. Under the
27
28 437 abiotic stress, upregulation of important genes involved in the GSH biosynthesis pathway
29
30
31
438 indicate that plant defense mechanisms have been activated. Previous studies have
32
33 439 demonstrated that the GSH metabolic pathway, as measured by GSH and its derivatives,
34
35 440 could greatly enhance plant tolerance to metal-based NPs;36 evidence for upregulation of
36
37
38 441 GSH biosynthesis related genes were also found in our previous study.25 Our current
39
40 442 work suggests that the sulfur assimilation pathway was highly activated given the
41
42 443 expression levels of APT, APR, and SiR in the roots across all three treatments. Although
43
44
45 444 the upregulation of CS, ECS, and GS in the roots was also evident in the single analyte
46
47 445 TiO2 NPs or TC treatment, no difference was found in the co-exposure treatments relative
48
49
446 to controls. The potential explanation of sulfur responses upon on TC exposure needs to
50
51
52 447 be further investigated and future study of TC metabolites in plants may help us
53
54 448 thoroughly understand the role of sulfur assimilation pathway in detoxifying TC effects.
55
56
57
449 Other stress-related genes were also evaluated, including GST, GR, and MDAR, in A.
58
59
60
20
1
2
3
450 thaliana tissues. All three genes involve in ascorbate-glutathione cycle in plants and play
4
5
6 451 the essential roles in scavenging excess amounts of ROS induced by abiotic stresses.
7
8 452 Others have reported similar glutathione related effects with other NPs; both Ag NPs and
9
10
11
453 ZnO NPs could induce GST expressions by approximately 10- and 3.5-fold in A.
12
13 454 thaliana.52, 53 However, in our study, the downregulations of GST and GR were evident in
14
15 455 TiO2 NPs treated A. thaliana shoots, which suggests that regulations of stress related
16
17
18 456 genes varied under the abiotic stress conditions induced by different types of metal-based
19
20 457 NPs.
21
22
23 458 6. Pod numbers and biomass of A. thaliana treated with TiO2 NPs and TC
24
25
26 459 At harvest, the total number of pods in each A. thaliana plant and the total pod biomass
27
28 460 were recorded. As shown in Figure 6A, 10 mg/L TC severely reduced plant growth and
29
30
31
461 inhibited the pod formation, both in terms of pod size and numbers (Figure S7). The
32
33 462 addition of TiO2 NPs partially alleviated the adverse effects and significantly enhanced
34
35 463 plant growth. Although 200 mg/L TiO2 NPs still reduced the total number of pods and the
36
37
38 464 pod biomass by 23% and 30%, respectively, approximate 100% and 300% increases in
39
40 465 pod number and biomass were observed upon co-exposure as compared with the TC
41
42 466 alone treatment (Figure 6B and C). The results further suggest that both TiO2 NPs and TC
43
44
45 467 could separately affect food quality and yield; however, upon co-exposure TiO2 NPs
46
47 468 counteract the TC-induced toxicity and subsequently enhance plant biomass and yield.
48
49
50
469 Given the concerns over food quality and safety, it is clear that long-term study of metal-
51
52
53 470 based NPs effects, including interactions with co-contaminants, on crop edible tissues is
54
55 471 necessary. Some relevant work has been published. For example, Zhao et al.
56
57
58
472 demonstrated that exposure to CeO2 NPs had no impact on nutrient levels in tested
59
60
21
1
2
3
473 corncobs.54 However, CeO2 NPs and related species were detected in soybean edible
4
5
6 474 tissues;55 similarly, TiO2 NPs were also detected in cucumber fruit.24 In addition, several
7
8 475 studies have reported trophic transfer of metal-based NPs in model terrestrial systems and
9
10
11
476 have demonstrated potential contamination of the food chain.56, 57 Although far from
12
13 477 conclusive, these limited number of studies do suggest that NPs accumulation in food
14
15 478 does present a potential risk to human health. Similarly, Ahem et al reported that
16
17
18 479 antibiotics could accumulate in the edible portions of vegetables such as cucumber,
19
20 480 lettuce, and tomato.9 Our results indicate that both xenobiotic substances significantly
21
22 481 inhibited pod formation and biomass in A. thaliana, although upon co-exposure, the
23
24
25 482 addition of TiO2 NPs partially alleviated the TC-induced toxicity.
26
27
28 483 Taken together, the present study found that the addition of TiO2 NPs could reduce the
29
30
31
484 TC toxicity to A. thaliana in terms of fresh biomass, total number of pod, as well as pod
32
33 485 yield. The relative expressions of genes encoding sulfur assimilation pathway were
34
35 486 strongly up-regulated in A. thaliana upon exposure to TiO2 NPs and TC, indicating the
36
37
38 487 sulfur assimilation pathway plays important roles in detoxification of xenobiotic
39
40 488 substances. The impacts on A. thaliana pod formation and pod yield imply the potential
41
42 489 risks of both substances to agricultural crop yield and food safety as likelihood of such
43
44
45 490 negative effects could translate to the real crop. Clearly, a detailed assessment of the
46
47 491 realistic exposure and risk associated with co-exposure to NPs and antibiotics is needed
48
49
492 and could have significant implications for food safety and consumer health.
50
51
52
53 493 ASSOCIATE CONTENTS
54
55 494 Supporting information
56
57
58
59
60
22
1
2
3
495 Additional information on the experimental design, the hydroponic setup, details for each
4
5
6 496 antioxidant enzyme assay, metal analysis, qPCR primers, figures of Ti uptake, total
7
8 497 chlorophyll and total protein content, a table of nutrient element content, as well as
9
10
11
498 images of A. thaliana co-treated with TiO2 NPs and TC, is provided in the supplementary
12
13 499 information.
14
15
16 500 ACKNOWLEDGEMENTS
17
18
19 501 This research was supported by USDA-AFRI (2011-67006-30181) and USDA-NIFA
20
21 502 Hatch program (MAS 00475 and MAS 00401). H. L. gratefully acknowledges the
22
23 503 support from China Scholarship Council (201207870010) to study at University of
24
25
26 504 Massachusetts, Amherst.
27
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679
58 680
59 681
60
26
1
2
3
682 Figure Captions:
4
5
6 683 Figure 1. Physiological effects of TiO2 NPs and TC on A. thaliana. (A) Images of A.
7 684 thaliana co-exposed to TiO2 NPs and TC; (B) Root length; (C) Total fresh biomass. Data
8 685 are mean ± standard error of five to six replicates. Values of each parameter followed by
9 686 different letters indicate that the data points are significantly different at p≤0.05.
10
11
687
12 688 Figure 2. Antioxidant enzyme activity of A. thaliana shoots co-exposed to TiO2 NPs and
13 689 TC. Figure 2A-D represents SOD, CAT, APX, and POD activities, respectively. Data are
14 690 mean ± standard error of three to five replicates. Values of each antioxidant enzyme
15 691 activities followed by different letters indicate that the data points are significantly
16
692 different at p≤0.05.
17
18 693
19 694 Figure 3. Antioxidant enzyme activity of A. thaliana roots co-exposed to TiO2 NPs and
20 695 TC. Figure 3A-D represents SOD, CAT, APX, and POD activities, respectively. Data are
21 696 mean ± standard error of three to five replicates. Values of each antioxidant enzyme
22 697 activities followed by different letters indicate that the data points are significantly
23
24
698 different at p≤0.05.
25 699
26 700 Figure 4. Relative expression of genes involved in the sulfur assimilation pathway in A.
27 701 thaliana treated with TiO2 NPs and TC. Figure 4A-C represents expression levels of
28 702 APT, APR, and SiR in A. thaliana shoots and roots upon exposure to TiO2 NPs and TC,
29
703 respectively. Data are mean ± standard error of three replicates.
30
31 704
32 705 Figure 5. Relative expression of stress related genes in A. thaliana treated with TiO2 NPs
33 706 and TC. Figure 5A-C represents expression levels of CS, ECS, and GS, respectively.
34 707 Figure 5D-F represents expression levels of GST, GR, and MDAR, respectively. Data
35 708 are mean ± standard error of three replicates.
36
37 709
38 710 Figure 6. Effects of co-exposure on A. thaliana pod formation. (A) Image of pods
39 711 exposed to TiO2 NPs and TC; (B) Pod number; (C) Pod biomass. Data are mean ±
40 712 standard error of four replicates. Values of each parameter followed by different letters
41 713 indicate that the data points are significantly different at p≤0.05.
42
43
714
44 715
45 716
46 717
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48 719
49
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725
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1
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727
4
5 728 A
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15 737
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738
18 739
19 740
20 741
21 20.0
742 B 0 50 100 200 mg/L TiO2 NPs
22 743 18.0 de
23 de de de
744 16.0 cde
24 bcd
745 ab abc
Root length (cm)
25 14.0 abc
746 ab ab ab ab ab
26 a
27 747 12.0 a
28 748 10.0
29
30
749 8.0
31 750
6.0
32 751
33 752 4.0
34 753 2.0
35 754
36 0.0
37 755 TC 0 TC 1 TC 5 TC10
38 3.5
39 756 C d 0 50 100 200 mg/L TiO2 NPs
d
40
3.0 cd cd cd
41
42 bc
Fresh biomass (g)
43 2.5
ab ab
44 ab a ab
a a
45 2.0 a a a
46
47 1.5
48
49 1.0
50
51 0.5
52
53
54
0.0
55 TC 0 TC 1 TC 5 TC10
56
57 Figure 1
58
59
60
28
1
2
3
4
5 757
6 e e
758 A 0 50 100 B de cd
7 759 200 mg/L TiO2 NPs
8 bc
9 760 bc
cde
10 761 de cd b
11 762 cd
bcd cde bc bcd bc bc
12 763 de a bc abab a
13 764
14 a
15
765
16 766
a a a a a
17 767 a a a
18 768
19 769
20 770
21
22 771
C D
23 772 d i
24 773 cdbcd
25 774 bcd bcd h
26 775 g
g g
27 g
abc abc abc
28 abc f
ef ef
29 ab ab ab ab de cde e
30 a a a
abc
a abbcd
31
32
33
34
35
36
37
38
39
40 Figure 2
41
42
43
44
45
29
46 ACS Paragon Plus Environment
47
48
1
2
3
4
5 776 e
6 777 A 0 50 100 B
7 g
778 200 mg/L TiO2 NPs
8
9 779 d
780 cd d
10 f
11 781 ef ef
de de abc
12 782 cd bc abc
bc cd bc bc cd cd ab
13 783 bc
14 ab a ab ab ab
15
784 a a
a
16 785 a
17 786 a
18 787
19 788
20 789
21 d D k
790 C
22
23 791 j
792 c
24 hi i gh
25 793 fg
bc cdef efg
26 794 ab bcdcde cde def bcd abc
27 ab a ab
28
795 ab
796 ab ab
29 ab
797 a a a a a
30 a a
31 798
32 799
33 800
34
35 801
36 802
37 803
38 804 Figure 3
39
40
41
42
43
44
45
30
46 ACS Paragon Plus Environment
47
48
1
2
3
A Control TiO2 NPs TC *
805 TiO2 NPs × TC
4
5 806 * **
6 807
7 808
8 809
9 810
10
11 811
12 812
13 813
14 814
15 815
16
17
816
18 817
19 818
20 819 B **
21 820
22 821
23
24 822
25 823
26 824
27 ** **
825
28 826
29
30
827
31 828
32 829 * **
33 830
34 831
35 832
36 C
833 ** **
37 **
38 834
39 835
40 836
41 837
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838
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45 840
46 841
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843
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50 844
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54 848
55
56
849 Figure 4
57
58
59
60
31
1
2
3
850
4 D
851 A Control TiO2 NPs
5
852 TC TiO2 NPs × TC
6
7 853
8 854
9 * ** **
855 **
10
11 856
**
12 857
13 858 ** **
*
14 859
15 860
16
861 E
17 B * **
18 862
19 863
20 864 *
21 865
22 866
23 *
24 867
25 868 *
26 869
27 870
28 871
29 F
30
872 C
31 873 **
32 874
33 875 ** **
34 *
876
35 * **
877 *
36
37 878
38 879 *
39 880
40 881
41 882
42
43
883
44 884
45 885
46 886
47 887
48 Figure 5
888
49
50 889
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32
1
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890
4
5 891
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8 894 A
9 895
10
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13 898
14 899
15 900
16
17
901
18 902
19 903
20 904
21 905
22 906 120.0 0.35
23 B d C
24 907 0.30 d
Pod biomass (g/plant)

100.0
Total number of pods
25 908 0.25
c
26 909 80.0 c
27 910 0.20
28 60.0
911 b 0.15
29
30
912 40.0 b
a 0.10
31 913
32 914 20.0 0.05 a
33 915 0.0 0.00
34 916 200mg/L 10mg/L TiO2 NPs 200mg/L 10mg/L TiO2 NPs
Control Control
35 917 TiO2 NPs TC ×TC TiO2 NPs TC ×TC
36
37 918
38 919
39 920
40
Figure 6
921
41 922
42
43
923
44 924
45 925
46 926
47 927
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57 935
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936 For Table of Contents Use Only
4
5
6 937 Title: Titanium dioxide nanoparticles alleviate tetracycline toxicity to Arabidopsis
7
8 938 thaliana (L.)
9
10
11
939 Hong Liu†, Chuanxin Ma†, Guangcai Chen, Jason C. White, Zonghua Wang, Baoshan
12
13 940 Xing* and Om Parkash Dhankher*
14
15 †
941 These authors contributed equally to this work.
16 *
17 942 Corresponding authors
18
19 943 Synopsis: Investigation of the effects of co-contaminations of TiO2 NPs and tetracycline
20
21
22
944 on Arabidopsis thaliana at the biochemical and molecular levels.
23
24 945
25
26 946
27
28
29 947
30
31 948
32
33 949
34
35
36 950
37
38 951
39 TOC graphic
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952
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45 954
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47
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50 956
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52 957
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55 958
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34

Liu2017 TiO2 Tetracycline Arabidopsis

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