Titanium Dioxide Nanoparticles Alleviate Tetracycline Toxicity to

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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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ACS Sustainable Chemistry & Engineering

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Titanium dioxide nanoparticles alleviate tetracycline toxicity to Arabidopsis thaliana

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(L.)

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Hong Liu1, 2, †, Chuanxin Ma2, 3, †, Guangcai Chen2, 4, Jason C. White3, Zonghua Wang5,

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Baoshan Xing2, * and Om Parkash Dhankher 2, *

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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 2 Stockbridge School of Agriculture, University of Massachusetts, Amherst, Massachusetts 01003, United States 3 Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station, New Haven, Connecticut 06504, United States 4 Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, Zhejiang 311400, China 5 State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou 350002, China

†

These authors contributed equally to this work. Corresponding authors: * Om Parkash Dhankher: [email protected]; Phone: 413-545-0062; * Baoshan Xing: [email protected]; Phone: 413-545-5212; Fax: 413-577-0242. Mailing address: H. L.: 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

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ABSTRACT Arabidopsis thaliana (L.) Heynh. was used as a model plant to investigate the

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biochemical and molecular response upon co-exposures to tetracycline (TC) and titanium

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oxide nanoparticles (TiO2 NPs). Results showed that 1 mg/L TC severely reduced A.

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thaliana biomass by 33.3% as compared with the control; however, the presence of 50

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and 100 mg/L TiO2 NPs alleviated TC toxicity, increasing fresh biomass by 45% and

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28%, respectively, relative to the TC alone treatment. The presence of TC notably

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decreased Ti accumulation in both shoots and roots. Antioxidant enzyme activity,

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including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX),

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peroxidase (POD), in A. thaliana shoots and roots indicated that TC significantly

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increased the activity of reactive oxygen species (ROS) scavengers. However, in the co-

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exposure treatments, TiO2 NPs reduced antioxidant enzyme activity back to the control

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levels. The relative expression of genes encoding sulfur assimilation and glutathione

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biosynthesis pathways was separately measured in shoots and roots. Interestingly, the

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relative expressions of adenylytransferase (APT), adenosine-5′-phosphosulfate reductase

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(APR), and sulfite reductase (SiR) in the roots across all three treatments (TC alone, TiO2

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NPs alone, and TC×TiO2 NPs treatment) were 2-3.5 fold higher than the control. The

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expression of γ-glutamylecysteine synthetase (ECS) and glutathione synthetase (GS) was

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increased in A. thaliana treated with either TiO2 NPs or TC alone. At harvest, almost 93%

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reduction of the pod biomass was evident in the TC alone treatment as compared with the

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control; however, TiO2 NPs increased the pod biomass by 300% in the co-exposed plants

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relative to the TC alone treatment. These findings provide important information for

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understanding the interactions of metal-based NPs and co-contaminants such as

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antibiotics in plant systems.

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Keywords: Arabidopsis thaliana, titanium oxide nanoparticles, tetracycline, co-

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contamination, molecular response, crop quality

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INTRODUCTION

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Antibiotics have been widely used in agriculture and livestock industries for the purposes

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of growth enhancement and disease prevention.1 Most antibiotics are water-soluble and

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are poorly absorbed by livestock; approximately 40-90% of the antibiotics are excreted

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through urine or feces.2 In the United States, approximately 51 tons of antibiotics were

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used, and almost 79% of antibiotics (equals to 13540000 kg) were applied in the raising

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of livestock annually.3 Similarly, in China, approximately 210,000 tons of antibiotics are

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produced annually and more than three quarters are used in animal husbandry.4

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Antibiotics can be released to the environment from pharmaceutical wastes, wastewater

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treatment facilities, and livestock industries.1, 5 Since soils and water bodies are

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considered the primary sinks for environmental pollutants, concerns have been raised

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over the adverse impacts of antibiotics on microbial, plant, and animal communities.1, 6

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According to the European Union, if the environmental concentration of antibiotics

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exceeds 10 µg/kg, the further assessment of the specific chemical compound should be

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conducted. The estimated concentrations of TC and TC derivatives could be in a range of

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450-900 µg/kg, which was approximately 45-90 folds higher than the European Union

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regulation.7 Thus, it is necessary to investigate the TC toxicity to the terrestrial plants

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with the presence of other emerging contaminants in environment. Sulfonamides (SA)

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and tetracycline (TC) are among the most widely used antibiotic groups due to their

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broad inhibition of microorganisms, protozoa, and other parasite populations.8 Once the

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antibiotics are discharged into the environment, it is likely that plant species will

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accumulate the residues from soil. In fact, a large body of the present studies has focused

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on antibiotic uptake in terrestrial plants. A recent study reported that common vegetable

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crops, including cucumber (Cucumis sativa L.), tomato (Solanum lycopersicum L.), and

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lettuce (Lactuca sativa L.), could accumulate both SA and TC in the different plant

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tissues upon exposures to various concentrations (0-20 mg/kg) for 45 day.9 Separately,

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Topal et al found that the concentrations of TC and its degradation products in

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Phragmites australis significantly decreased from root to leaf.10 Additionally, the relative

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accumulation of TC and its degradation products was as follows: 4-epitetracycline >

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tetracycline > 4-epianhydrotetracycline > anhydrotetracycline.10 Pot experiments with

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soybean grown in 105 mg/kg oxytetracycline contaminated saline soil showed that the

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antibiotic was only accumulated in the roots and no translocation was evident to the

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shoots.11 The type of antibiotic will also clearly impact the uptake levels by plants. For

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example, oxy-TC and TC at a 50 mg/L exposure had the similar root concentration

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factors (about 2100) in treated rice over a period of 15 d exposure, whereas a

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significantly higher value of RCF (approximately 2700) was evident for chlor-TC at 11 d

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of exposure.12 Pan et al. suggested that antibiotic bioaccumulation along the food chain

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should not be neglected, but also noted that the concentrations (TC is 2100 µg) in

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vegetable crops are significantly lower than the minimum therapeutic dose (20-200mg).13

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Importantly, few mechanistic studies investigating the basis of plant toxicity and response

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are available in the literature and as such, understanding of these processes remains

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inadequate.

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Engineered nanomaterials (ENMs) have been increasingly applied in various fields, such

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as agriculture, food manufacturing, biomedicals, electronics, and renewable energy.14

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Titanium oxide (TiO2) is one of the commonly used nanoparticles (NPs); more than 3000

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tons of TiO2 NPs are produced annually, and more than half is used in personal care

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products.15,16 In agriculture, ENMs can be used to detect pathogens, more effectively

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deliver pesticides and fertilizers, and to monitor soil conditions.17 Due to their nanoscale

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size, ENMs may pose potential risks in agriculture system despite of the positive impacts

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on crops.18 Recent laboratory studies have demonstrated that the presence of ENMs can

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result in root elongation inhibition, biomass decrease, low photosynthetic efficiency,

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developmental delay, and unique molecular effects of unknown consequence.19, 20, 21, 22

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Plant defense mechanisms upon exposure to different types of metal-based NMs have

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been recently reviewed by Ma et al.14 Antioxidant enzyme activity in terrestrial plants

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played an essential role in detoxifying nanoparticle-induced phytotoxicity.23, 24 In

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addition, studies at the molecular level also provide insight on responses of stress-related

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genes in plants upon nanoparticle exposures.25 Given the complex nature of agricultural

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systems, the likelihood of co-exposure to metal oxide nanoparticles and pharmaceutical

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compounds such as antibiotics is quite high; however, little work has been done in this

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area.

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In the present study, we chose Arabidopsis thaliana (L.) Heynh. as a target plant and

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hydroponically exposed it to TiO2 NPs and TC. In order to comprehensively understand

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the defense mechanism and impacts of co-contaminants on crop yield, the physiological

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and molecular responses of A. thaliana upon co-exposure to TiO2 NPs and TC were

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investigated. The resulting impact on biomass, Ti uptake, chlorophyll content, protein

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content, and pod formation was determined. Additionally, activities of main reactive

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oxygen species (ROS) scavengers including superoxide dismutase (SOD), catalase

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(CAT), ascorbate peroxidase (APX), and peroxidase (POD) were measured in A. thaliana

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shoots and roots across all treatments. At the molecular level, the relative expression of

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genes involved in sulfur assimilation, glutathione biosynthesis, as well as stress-related

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genes, were analyzed in shoots and roots of A. thaliana. To our knowledge, this is the

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first report on addressing the role of metal-based NPs in alleviating the toxicity of

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antibiotics to plants. Our findings provided the important information for the potential

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risks of antibiotics and metal-based NPs to the agricultural crops in terms of crop yield

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and quality if such impacts on A. thaliana could translate to the real crops.

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MATERIALS AND METHODS

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Experimental design.

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Concentration optimization. Titanium oxide nanoparticles (TiO2 NPs) were purchased

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from US Research Nanomaterials, Inc. The size of TiO2 NPs was ranging from 5 to 15

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nm, and the form of TiO2 NPs was rutile. Tetracycline (≥98%) was obtained from Sigma-

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Aldrich. Twenty-five surface sterilized seeds of A. thaliana ecotype Columbia (Col-0)

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were placed on half strength (1/2x) Murashige and Skoog (MS) semisolid medium

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amended with different concentrations of TiO2 NPs ranging from 0 to 500 mg/L.

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Similarly, sterilized seeds were grown in 1/2x MS semisolid medium amended with

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different concentrations of TC (0-5 mg/L). Seeds were stratified at 4 °C for 24 hours

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prior to transfer to a controlled plant growth chamber with 16 h light and 8 h dark at 22

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and 8 °C, respectively. At harvest, total fresh biomass was used to determine the toxicity

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of TiO2 NPs and TC; the results are shown in Figure S1.

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Hydroponic system. A. thaliana was grown in vermiculite for 21 days, and the

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seedlings were then transferred to a hydroponic system as shown in Figure S2 and were

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allowed to acclimatize for 5 days. The doses of TiO2 and TC in the hydroponic system

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were determined from the concentration optimization test described above. As shown in 7



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Figure S1A, lower concentration of TiO2 NPs (50 mg/L) significantly enhanced plant

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growth; no significant difference was observed at 100 mg/L TiO2 NPs; and the fresh

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biomass was greatly reduced as TiO2 NPs concentrations were increased to 200 mg/L.

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Thus, these three exposures of TiO2 NPs (50, 100, and 200 mg/L) were chosen for the

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hydroponic experiment. The identical method was applied to determine the appropriate

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TC concentrations in the hydroponic experiment. TC exposure had no impact on fresh

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biomass until 1 and 5 mg/L, where fresh biomass was reduced by 21.7% and 59.6%

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relative to the control, respectively (Figure S1B). The three doses of TC chosen were 1, 5,

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and 10 mg/L. In total, there are 16 treatments (Table S1), including 9 different co-

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exposure treatments, three single analyte controls for each TiO2 NPs and TC exposure,

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and one control with 1/2X Hoagland’s solution only. A number of six replicates were

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applied in each treatment. A. thaliana seedlings were exposed to different concentrations

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of TiO2 NPs and TC for 12 days. At harvest, the roots were thoroughly rinsed with

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deionized water three times and root length and total fresh biomass were measured. Plant

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tissues were stored at -80 °C until further analysis.

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Chlorophyll content. The total chlorophyll content in A. thaliana was measured as

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described by Lichtenthaler et al.26 Briefly, 50 mg of fresh leaves were collected and cut

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into pieces (less than 1 cm); 10 mL 95% ethanol was then used to extract the total

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chlorophyll. All samples were kept in dark for 3 days to avoid chlorophyll degradation.

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The absorbance was measured at 664.2 nm and 648.6 nm by a UV-Vis spectrophotometer

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(Agilent 8453, Santa Clara, CA). Chlorophyll a, chlorophyll b and the total chlorophyll

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were calculated by the following equations: Chla=13.36A664.2-5.19A648.6 (1),

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Chlb=27.43A648.6-8.12A664.2 (2), and Total chlorophyll=Chla + Chlb (3).

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Ti and nutrient element contents. A. thaliana shoots and roots were separately freeze-

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dried and then were ground to find powder. Approximately 50 mg of shoot tissues or 10

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mg of root tissues were digested in sulfuric acid (H2SO4) and hydrogen peroxide (H2O2)

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following the method as described by Short et al. (1996) and Wei et al. (2015) with slight

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modification.27, 28 The detailed information is provided in the supporting information. The

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samples were measured using inductively coupled plasma optical emission spectrometry

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(ICP-OES).29

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Total protein content. The Bradford reagent (Sigma Aldrich, St. Louis, MO) was used

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to measure the total protein content in plant tissues.30 A sample of 50 mg of A. thaliana

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shoots or roots was extracted in 2 mL of 10 mM Tris-HCl (pH 7.2) solution. The mixture

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was then centrifuged at 2683 ×g for 20 min at 4 °C. One hundred µL supernatant was

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used to react with 1000 µL of Bradford reagent for 15 min at ambient temperature, and

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then the absorbance was measured at 595 nm by a UV-Vis spectrophotometer (Agilent

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8453, Santa Clara, CA).

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Antioxidant enzyme assays. Fresh root and shoot tissues were homogenized in liquid

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nitrogen to fine powder. A 0.5 g sample of homogenized tissue was then vigorously

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mixed with 5 mL extraction buffer for 5 min using a vortex mixer. The mixture was

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centrifuged at 2683 ×g for 20 min at 4 °C, and the supernatant was used to measure

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antioxidant enzyme activity, including SOD, CAT, APX, and POD. The modified

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protocol for each antioxidant enzyme assay is provided in the supporting information.31, 32,

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Gene expression measurement by quantitative reverse transcription polymerase

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chain reaction (qRT-PCR). Shoot and root tissues were separately homogenized in 9



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liquid nitrogen prior to RNA isolation. Protocols for total RNA isolation, cDNA

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synthesis, and gene expression using qRT-PCR were described in Ma et al.25 Briefly,

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RNeasy plant mini kits were used to isolate total RNA, with the concentration being

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quantified by NanoDrop spectrophotometry (ThermoScientific, West Palm Beach, FL). A

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Verso cDNA synthesis kit was used to synthesize cDNA and the gene-specific primer

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was designed using Primer Quest (Integrated DNA Technologies, Coralville, IA). A

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complete list of primer sequences is provided in Table S2. Information for qTR-PCR

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amplification program can be found in the supplementary information. Relative quantities

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(2−∆∆Ct method) were used to calculate the transcription level of each gene.

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Statistical analysis. A one-way analysis of variance (One-way ANOVA) followed by

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Duncan’s multiple comparison test was used to determine statistical significance of each

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parameter across treatments, except qRT-PCR assay, in which a Student t-test was

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applied to determine statistical significance for each gene. In the figures for each assay,

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values followed by different letters are significantly different at p ≤ 0.05.

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RESULTS AND DISCUSSION

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1. Growth of A. thaliana upon exposure to TiO2 NPs and TC

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Different concentrations of TiO2 NPs did not significantly affect plant growth in Figure

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1A and Figure S3. Although 1 mg/L TC exhibited negative effects on growth as

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compared with the control group (Figure 1A), the presence of TiO2 NPs seemed to

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visibly alleviate TC phytotoxicity. At harvest, root length and total fresh biomass were

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measured (Figure 1B and C). The root length was significantly reduced in the 10 mg/L

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TC exposure; no difference was evident in the co-exposure treatments, except the

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treatments of 1 mg/L TC × 100 and 200 mg/L TiO2 NPs; in both cases, root length was

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reduced by 22% and 17%, respectively, as compared with the TC control. The total fresh

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biomass in all three TC alone treatments was reduced by approximately 30% relative to

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the control group. The additions of 50 and 100 mg/L TiO2 NPs significantly alleviated

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the phytotoxicity, elevating the fresh biomass in 1mg/L TC treated A. thaliana back to the

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level of the control group (Figure 1C). This result suggests that TiO2 NPs could

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counteract the TC toxicity to A. thaliana at the lower antibiotic doses. As the

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concentrations of TC were increased to 5 and 10 mg/L, although there was a trend of

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fresh biomass increase, the results were statistically insignificant. One of the possible

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explanations is that the TiO2 NPs interact with the TC outside of the plant, preventing

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exposure at a micro/nano level, but that this process is saturated when exposure dose of

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TC reached to 1 mg/L.

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2. Ti uptake and nutrient element contents in A. thaliana upon exposure to TiO2 NPs

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and TC

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The contents of Ti and other essential nutrients were measured in A. thaliana shoots and

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roots treated with TiO2 NPs and TC (Figure S4 and Table S3). The presence of 10 mg/L

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TC significantly reduced Ti accumulation in the roots and decreased Ti translocation to

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the shoots. In 200 mg/L TiO2 NPs alone treatment, the root Ti content was 47140.55

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mg/kg over 12 days exposure, while this value was decreased by 24.81% in the co-

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exposure treatment. Similar trend showing the presence of TC lowered the Ti content in

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the shoots was also evident. Along with the Ti analysis, we also investigated whether the

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additions of TiO2 NPs and TC could alter the contents of essential nutrients in A. thaliana

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shoots and roots (Table S3). The results indicated that both NPs and antibiotic did not

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change the contents of macronutrients, including Mg, P, and K, in A. thaliana roots. The

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root Ca contents were significantly decreased by approximately 50% upon exposure to

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TiO2 NPs, TC, and TiO2 NPs × TC. In the shoots, the levels of all macronutrients in 200

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mg/L TiO2 NPs alone treatment were similar to the control, however, the presence of TC

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in either single treatment or co-treatment resulted in 25.9-29.1% and 28.3-48.5 %

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increases of P and K, respectively, relative to their corresponding control. Similar to the

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levels of macronutrients, TiO2 NPs had very less impact on the contents of micronutrients

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in both shoots and roots of A. thaliana. However, the presence of 10 mg/L TC notably

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altered the nutrient distribution in the shoots. For example, in the TC alone treatment, the

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shoot Fe content was decreased by 39.4% as compared to the control; 49.1% elevation of

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the Zn content in A. thaliana shoots was also found. In A. thaliana roots, TC lowered the

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Mn content by 186.84% relative to the control. Ma et al. (2016) reported that both cerium

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oxide (CeO2) NPs and indium oxide (In2O3) NPs could significantly decrease the Fe and

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Mn contents in A. thaliana roots.29 Other metal-based NPs, such as neodymium oxide

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(Nd2O3) NPs, could also alter the essential nutrient contents in the terrestrial plants. For

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example, Chen et al. (2016) found that Nd2O3 NPs could notably decrease the levels of

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macronutrients, such as Mg and K, in pumpkin roots relative to the control.34 However,

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our results suggested that the presence of TiO2 NPs had less impact on nutrient alteration

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as compared with other metal-based NPs.

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3. Total chlorophyll and total protein content in A. thaliana upon exposure to TiO2

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NPs and TC

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Chlorophyll is an important parameter to assess abiotic stressor toxicity to plants. At

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harvest, the fresh leaf tissues were used to determine the total chlorophyll contents in all

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treatments (Figure S5). In the TC alone treatments, the chlorophyll content was not

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significantly affected as compared with the control group (1/2X Hoagland’s solution).

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Interestingly, among all 9 co-exposure treatments the total chlorophyll content was

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significantly lowered as compared to the control group, except the treatment with 1mg/L

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TC + 50 mg/L TiO2 NPs, where no effect was evident. A large number of studies have

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reported that metal-based NPs could alter the photosynthetic output of plants, including

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changes to chloroplast structure, total chlorophyll content, net photosynthetic rate, and

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chlorophyll fluorescence. 24, 31, 35, 36 The physiological effects of antibiotics on plant

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growth have also been reported by Ahmed et al.9 The presence of 5 and 20 mg/L SA and

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TC severely inhibited tomato, cucumber, and lettuce growth health, as determined by

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biomass, plant height, total root surface area, and chlorophyll content (SPAD). However,

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this phytotoxicity was not dose-dependent; 10 mg/L antibiotic exposure had equivalent or

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less impact on plant growth than did the control and lower exposures.9 Our present work

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is aligned with the published results in that both NPs and TC could significantly alter the

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chlorophyll content, although single analyte exposure to TC at concentrations up to 10

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mg/L exerted no such effect. Additional study is necessary to characterize the

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mechanistic basis for these observations.

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Figure S6 shows that both TiO2 NPs and TC, either as single analyte exposures or under

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co-contaminant conditions, could alter the total protein content in A. thaliana shoots and

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roots. For example, in the TiO2 NPs alone treatments, the total protein content in 100

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mg/L NPs treated shoots was increased by 26%, while at 200 mg/L, the protein content

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was reduced by more than 20% relative to the control. However, the similar results were

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not evident in the roots, where TiO2 had no effect on protein content. In the TC alone

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treatments, 1 mg/L TC significantly elevated the protein content in both A. thaliana roots

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and shoots, whereas higher concentrations (10mg/L) significantly reduced protein levels

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by 34.6% and 23.8% in the shoots and the roots, respectively. The total protein contents

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in the co-exposure treatments exhibited a dose-dependent yet consistent response. For

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example, as TiO2 NPs exposure doses increased, the protein contents in 1 mg/L TC

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treated shoots were elevated at both 100 mg/L and 200 mg/L TiO2 NPs. In the co-

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exposure treatments of 10 mg/L × different concentrations of TiO2 NPs, the shoot protein

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contents increased at 50 and 100 mg/L TiO2 NPs, and then decreased at 200 mg/L.

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Similar pattern was also evident in the co-exposure treatments of A. thaliana roots. Plant

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proteins play the essential roles in plant growth (root elongation), carbohydrate transport,

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photosynthesis system, and defense mechanism.37 Reduced levels of protein were evident

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in metal-based NPs treated plants in previous studies.37, 38 In our study, a higher

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concentration of TC led to the significantly lower protein content in the A. thaliana roots,

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while exposure to TiO2 NPs alone had no impact on the protein content. As the protein

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content is a critical property, it may be highly useful to figure out how to reasonably and

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effectively apply NPs and antibiotics in to maximize (or at least not reduce) crop quality.

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So far, the studies on food quality and security of crops upon co-exposure to metal-based

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NPs and antibiotics are scarce. However, the available information from single

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contaminant exposure studies, along with our co-exposure work herein, suggests that the

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potential risks posed by these emerging contaminants under co-exposure scenarios should

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be further investigated.

14


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4. Antioxidant enzyme activities in A. thaliana roots and shoots

315

Antioxidant defense is one of the most essential mechanisms of plants to alleviate the

316

toxicity from exposure to xenobiotics in the environment.39, 40 In order to understand the

317

detoxification process in A. thaliana upon co-exposures of TiO2 NPs and TC, the

318

activities of ROS scavengers, including SOD, CAT, APX, and POD, were separately

319

determined in A. thaliana shoots and roots (Figure 2 and 3). SOD is capable of

320

converting to O2 to H2O2 and O2. Different concentrations of TiO2 NPs (alone) had no

321

impact on SOD activities in the shoots and roots (Figure 2A and 3A), except the

322

treatment with 50 mg/L TiO2 NPs, where the root SOD levels were 43.5% greater than

323

the control group. Upon exposure to 10 mg/L TC alone, SOD activities were increased to

324

2- and 1.9-fold more than controls in the shoots and the roots, respectively. The co-

325

exposure treatments exhibited different effects on SOD activities in the two tissues. In the

326

shoots, the SOD activities were similar as compared with their respective TC control,

327

except the 50 and 100 mg/L TiO2 NPs co-exposure treatments at 10 mg/L TC, where

328

SOD activity was reduced back to the no analyte control level. On the contrary, in the

329

roots, the SOD activities in the co-exposure treatments were highly induced as compared

330

to their respective TC control. For example, at 1 and 5 mg/L TC, TiO2 NPs exposure at

331

all concentrations significantly increased SOD activities (not dose dependent) in A.

332

thaliana roots as compared to their respective TC control. However, at 10 mg/L TC, SOD

333

activities at 50 and 100 mg/L exposures were equivalent to the TC alone control, but 200

334

mg/L TiO2 NPs exposure resulted in significantly higher SOD activity.

335

CAT and APX are two main antioxidant enzymes that are capable of breaking down

336

H2O2 to H2O and O2. As shown in Figure 2B and 3B, the CAT activity in the roots were

·−

15



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337

almost ten times more than in the shoots. The CAT activity in the shoots treated with the

338

highest exposure dose of either TiO2 NPs alone or TC alone was approximately 170 and

339

150-fold of the control, respectively, whereas the root CAT levels were unaffected

340

relative to the control. Notably, the CAT level response of the plant upon co-exposure

341

was consistently different when comparing the shoot and root tissues. In the co-exposure

342

treatments at 1 mg/L TC, TiO2 co-exposure had no impact on CAT activity in the shoots.

343

Conversely, in the roots, CAT levels decreased in a dose-dependent fashion with TiO2

344

exposure. At 5 mg/L TC, elevations of the shoot CAT activities were found at all three

345

TiO2 NPs treatments relative to their TC control group. However, no such effect was

346

evident in the roots, where CAT levels were significantly decreased at the low and

347

medium TiO2 doses. Similarly, in the treatment with 10mg/L TC × 200 mg/L TiO2 NPs,

348

the shoot CAT activity was increased by approximately 65% as compared with their

349

respective TC and TiO2 NPs control, no difference was evident in the roots. The APX

350

activities in the shoots and the roots of A. thaliana are shown in Figure 2C and 3C,

351

respectively. TC exposure alone had not impact on the shoot or root APX activities. The

352

results suggested that the root APX activities were sensitive to TiO2 than the one in the

353

shoots. Notably, the presence of 100 and 200 mg/L TiO2 NPs significantly elevated the

354

root APX activities to 3.5- and 3-fold of the control, respectively; no significant effects

355

were evident in the shoots. The impact of co-exposure on APX levels in the roots and

356

shoots were generally minimal. However, roots exposed to 5 mg/L TC × 100 mg/L TiO2

357

NPs had 5-fold greater APX activities; similarly, in the shoots at 5 mg/L TC × 200 mg/L

358

TiO2 NPs, APX levels were significantly increased. Conversely, at 10 mg/L TC, 100 and

16


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359

200 mg/L TiO2 significantly suppressed the shoot APX levels, but no such effects were

360

noted in the roots.

361

POD is another antioxidant enzyme that can scavenge the free radicals induced by abiotic

362

stresses in plants. In the TC alone or the TiO2 NPs alone treatments, significantly high

363

level of POD activity was evident in both shoots and roots treated with 10 mg/L TC;

364

while this elevation was only found in the roots treated with 200 mg/L TiO2 NPs. Among

365

the co-exposure treatments, the common result of POD activity in both shoots and roots

366

was that the addition of TiO2 NPs significantly reduced the POD activities at 10 mg/L TC,

367

regardless of the TiO2 NPs concentrations (Figure 2D and 3D). Due to the complexity of

368

the co-contaminant interactions at different exposure doses and over time, more intensive

369

investigation may require in order to elucidate the antioxidant defense mechanism in

370

plants.

371

Abiotic stress such as metal-based NPs can produce excessive amounts of ROS in

372

terrestrial plants, subsequently causing significant oxidative stress.41 42, 43, 44 Excessive

373

amounts of ROS, acting as signaling molecules, will trigger antioxidant defense

374

mechanism in the plant so as to detoxify the free radicals that have been generated. Ma et

375

al. (2015) reviewed the roles of antioxidant enzymes in detoxifying NPs-induced toxicity

376

in plants and noted that the magnitude of the antioxidant enzyme response can vary

377

significantly with NPs types, exposure time and plant species.14 In the study, exposure to

378

TiO2 NPs elevated the activities of CAT, APX, as well as POD in A. thaliana.

379

Conversely, less is known about the antioxidant response of plants to antibiotic exposures.

380

Liu et al. exposed Phragmites australis to different concentrations of an antibiotic

381

mixture including ciprofloxacin, oxy-TC, and SA for 62 days. At harvest, analysis of 17



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382

SOD, CAT and POD activities suggests that the antibiotics significantly inhibited the

383

enzyme activities at exposures as low as 1 mg/L and that the inhibition is typically dose-

384

dependent.45 There were significant differences in the antioxidant response of the shoots

385

and roots upon co-exposure to TC and TiO2 NPs. Within a tissue, TiO2 NPs often

386

significantly affected the enzyme levels in the co-exposure scenarios but consistent trends

387

in the changes were difficult to find. Due to the complexity of interactions between TC

388

and TiO2 NPs, the changes of each antioxidant enzyme activity may vary as a function of

389

the exposure concentration of each analyte, or some unknown impact from other factors

390

such as plant root exudation. In order to thoroughly understand plant antioxidant enzyme

391

response upon antibiotic and NP co-exposure, one must also likely consider the role of

392

TC metabolites and TiO2 NPs biotransformation, both of which could significantly

393

impact plant exposure and response.

394

5. Relative expressions of stress-related genes in A. thaliana roots and shoots

395

Upon exposure to TiO2 NPs and TC, the relative expression of three important genes

396

involved in the sulfur assimilation pathway, including sulfate adenylytransferase (APT),

397

adenosine-5′-phosphosulfate reductase (APR), and sulfite reductase (SiR), was

398

determined (Figure 4). The relative expression of the genes encoding ATP and SiR in A.

399

thaliana shoots did not significantly change (less than 1.5-fold) in the treated groups

400

relative to the control (Figure 4A and C), but significant down-regulation of APR was

401

evident in both the TC alone and the co-exposure treatment (Figure 4B). Interestingly, in

402

A. thaliana roots, the relative expression of APT in all three treatments was significantly

403

increased (3 to 3.5-fold) over the control. Similar upregulation of APR and SiR were also

404

evident in the roots across the three treatments.

18


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405

The glutathione (GSH) biosynthesis pathway is one of the most essential pathways in

406

plants for defense against abiotic stresses, including heavy metal, metal-based NPs, cold,

407

drought, heat.25, 46, 47 Sulfide is the precursor of cysteine in the GSH biosynthesis pathway.

408

The relative expressions of the genes encoding cysteine synthase (CS), γ-

409

glutamylecysteine synthetase (ECS), and GSH synthetase (GS) were measured in A.

410

thaliana shoots and roots treated with TiO2 NPs, TC, and TiO2 NPs × TC (Figure 5A-C).

411

In the shoots, TC alone caused a more than 2-fold decrease in the relative expression of

412

CS, ECS and GS as compared to the control, whereas no difference in expression was

413

observed in either the TiO2 NPs alone or the co-exposure treatment. Conversely,

414

upregulation of ECS and GS was evident in A. thaliana roots treated with either TiO2

415

NPs alone or TC alone; there was no change in the relative expression of the three genes

416

in the co-exposure treatment.

417

Responses of other stress-related genes including glutathione S-transferase (GST),

418

glutathione reductase (GR), and monodehydroascorbate reductase (MDAR), were also

419

investigated (Figure 5D-F). Upon exposures to TiO2 NPs and TC, downregulation of

420

GST were observed in both A. thaliana shoots and roots; this was particularly notable in

421

the TC alone and the co-exposure treatment, where 5- and 2-fold decreases in GST

422

relative expression was noted, respectively (Figure 5D). No significant change of GR

423

relative expression was observed across all three treatments, except the co-exposure

424

treatment in the roots, where GR was upregulated relative to the control (Figure 5E).

425

Similar to the GST relative expression, the gene encoding MDAR in the shoots was

426

downregulated by more than 2-fold in response to exposure to TC alone and the co-

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

427

exposure treatment (Figure 5F). Although slight decreases in MDAR in the roots were

428

also evident, these changes were statistically insignificant.

429

Sulfur is one of the most important macronutrients and plays a critical role in plant

430

growth and viability.48, 49, 50 Our previous study indicated that the relative expression of

431

the genes involved in sulfur assimilation was highly up-regulated upon exposure to CeO2

432

and In2O3 NPs.25 Importantly, this response is not specific to metal-based NPs;

433

upregulation will occur in response to other abiotic stresses, such as metalloid exposure.

434

For example, upon exposure to arsenate, the expression levels of genes involved in

435

sulfate metabolism were highly induced in Abyssinian mustard.51 Sulfide is a precursor

436

of cysteine, which is critical to the first step of the GSH biosynthesis pathway. Under the

437

abiotic stress, upregulation of important genes involved in the GSH biosynthesis pathway

438

indicate that plant defense mechanisms have been activated. Previous studies have

439

demonstrated that the GSH metabolic pathway, as measured by GSH and its derivatives,

440

could greatly enhance plant tolerance to metal-based NPs;36 evidence for upregulation of

441

GSH biosynthesis related genes were also found in our previous study.25 Our current

442

work suggests that the sulfur assimilation pathway was highly activated given the

443

expression levels of APT, APR, and SiR in the roots across all three treatments. Although

444

the upregulation of CS, ECS, and GS in the roots was also evident in the single analyte

445

TiO2 NPs or TC treatment, no difference was found in the co-exposure treatments relative

446

to controls. The potential explanation of sulfur responses upon on TC exposure needs to

447

be further investigated and future study of TC metabolites in plants may help us

448

thoroughly understand the role of sulfur assimilation pathway in detoxifying TC effects.

449

Other stress-related genes were also evaluated, including GST, GR, and MDAR, in A.

20


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450

thaliana tissues. All three genes involve in ascorbate-glutathione cycle in plants and play

451

the essential roles in scavenging excess amounts of ROS induced by abiotic stresses.

452

Others have reported similar glutathione related effects with other NPs; both Ag NPs and

453

ZnO NPs could induce GST expressions by approximately 10- and 3.5-fold in A.

454

thaliana.52, 53 However, in our study, the downregulations of GST and GR were evident in

455

TiO2 NPs treated A. thaliana shoots, which suggests that regulations of stress related

456

genes varied under the abiotic stress conditions induced by different types of metal-based

457

NPs.

458

6. Pod numbers and biomass of A. thaliana treated with TiO2 NPs and TC

459

At harvest, the total number of pods in each A. thaliana plant and the total pod biomass

460

were recorded. As shown in Figure 6A, 10 mg/L TC severely reduced plant growth and

461

inhibited the pod formation, both in terms of pod size and numbers (Figure S7). The

462

addition of TiO2 NPs partially alleviated the adverse effects and significantly enhanced

463

plant growth. Although 200 mg/L TiO2 NPs still reduced the total number of pods and the

464

pod biomass by 23% and 30%, respectively, approximate 100% and 300% increases in

465

pod number and biomass were observed upon co-exposure as compared with the TC

466

alone treatment (Figure 6B and C). The results further suggest that both TiO2 NPs and TC

467

could separately affect food quality and yield; however, upon co-exposure TiO2 NPs

468

counteract the TC-induced toxicity and subsequently enhance plant biomass and yield.

469

Given the concerns over food quality and safety, it is clear that long-term study of metal-

470

based NPs effects, including interactions with co-contaminants, on crop edible tissues is

471

necessary. Some relevant work has been published. For example, Zhao et al.

472

demonstrated that exposure to CeO2 NPs had no impact on nutrient levels in tested 21



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473

corncobs.54 However, CeO2 NPs and related species were detected in soybean edible

474

tissues;55 similarly, TiO2 NPs were also detected in cucumber fruit.24 In addition, several

475

studies have reported trophic transfer of metal-based NPs in model terrestrial systems and

476

have demonstrated potential contamination of the food chain.56, 57 Although far from

477

conclusive, these limited number of studies do suggest that NPs accumulation in food

478

does present a potential risk to human health. Similarly, Ahem et al reported that

479

antibiotics could accumulate in the edible portions of vegetables such as cucumber,

480

lettuce, and tomato.9 Our results indicate that both xenobiotic substances significantly

481

inhibited pod formation and biomass in A. thaliana, although upon co-exposure, the

482

addition of TiO2 NPs partially alleviated the TC-induced toxicity.

483

Taken together, the present study found that the addition of TiO2 NPs could reduce the

484

TC toxicity to A. thaliana in terms of fresh biomass, total number of pod, as well as pod

485

yield. The relative expressions of genes encoding sulfur assimilation pathway were

486

strongly up-regulated in A. thaliana upon exposure to TiO2 NPs and TC, indicating the

487

sulfur assimilation pathway plays important roles in detoxification of xenobiotic

488

substances. The impacts on A. thaliana pod formation and pod yield imply the potential

489

risks of both substances to agricultural crop yield and food safety as likelihood of such

490

negative effects could translate to the real crop. Clearly, a detailed assessment of the

491

realistic exposure and risk associated with co-exposure to NPs and antibiotics is needed

492

and could have significant implications for food safety and consumer health.

493

ASSOCIATE CONTENTS

494

Supporting information

22


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495

Additional information on the experimental design, the hydroponic setup, details for each

496

antioxidant enzyme assay, metal analysis, qPCR primers, figures of Ti uptake, total

497

chlorophyll and total protein content, a table of nutrient element content, as well as

498

images of A. thaliana co-treated with TiO2 NPs and TC, is provided in the supplementary

499

information.

500

ACKNOWLEDGEMENTS

501

This research was supported by USDA-AFRI (2011-67006-30181) and USDA-NIFA

502

Hatch program (MAS 00475 and MAS 00401). H. L. gratefully acknowledges the

503

support from China Scholarship Council (201207870010) to study at University of

504

Massachusetts, Amherst.

505

REFERENCES:

506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529

1. Sarmah, A. K.; Meyer, M. T.; Boxall, A. B., A global perspective on the use, sales, exposure pathways, occurrence, fate and effects of veterinary antibiotics (VAs) in the environment. Chemosphere 2006, 65 (5), 725-759. 2. Zhang, X.; Li, Y.; Liu, B.; Wang, J.; Feng, C.; Gao, M.; Wang, L., Prevalence of veterinary antibiotics and antibiotic-resistant Escherichia coli in the surface water of a livestock production region in northern China. PloS one 2014, 9 (11), e111026. 3. Hollis , A.; Ahmed , Z., Preserving Antibiotics, Rationally. New England Journal of Medicine 2013, 369 (26), 2474-2476. 4. Luo, Y.; Xu, L.; Rysz, M.; Wang, Y.; Zhang, H.; Alvarez, P. J., Occurrence and transport of tetracycline, sulfonamide, quinolone, and macrolide antibiotics in the Haihe River Basin, China. Environmental science & technology 2011, 45 (5), 1827-1833. 5. Blackwell, P. A.; Kay, P.; Boxall, A. B., The dissipation and transport of veterinary antibiotics in a sandy loam soil. Chemosphere 2007, 67 (2), 292-299. 6. Boxall, A.; Fogg, L.; Blackwell, P.; Blackwell, P.; Kay, P.; Pemberton, E.; Croxford, A., Veterinary medicines in the environment. In Reviews of environmental contamination and toxicology, Springer: 2004; pp 1-91. 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. Journal of hazardous materials 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

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579

edible vegetables of cucumber, tomato, and lettuce. Journal of agricultural and food chemistry 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. Science of the Total Environment 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.). Ecotoxicology and environmental safety 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. Journal of agricultural and food chemistry 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. Environmental science & technology 2015, 49 (12), 7109-7122. 15. Keller, A. A.; Lazareva, A., Predicted releases of engineered nanomaterials: from global to regional to local. Environmental Science & Technology Letters 2013, 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. Environmental science & technology 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 & environment 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. Science of the total environment 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), 491499. 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. Ecotoxicology and Environmental Safety 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. Environmental Science & Technology 2016, 50 (13), 71987207. 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. Environmental Science & Technology 2013, 47 (24), 14110-14118. 24. Servin, A. D.; Morales, M. I.; Castillo-Michel, H.; Hernandez-Viezcas, 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. Environmental Science & Technology 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 thaliana (L.) to nanoparticle cerium and indium oxide exposure. ACS Sustainable Chemistry & Engineering 2013, 1 (7), 768-778.

24


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Page 25 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629


26. Lichtenthaler, H. K., [34] Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. In Methods in Enzymology, Academic Press: 1987; Vol. Volume 148, pp 350382. 27. Short, F.; Gorton, P.; Wiseman, J.; Boorman, K., Determination of titanium dioxide added as an inert marker in chicken digestibility studies. Animal feed science and technology 1996, 59 (4), 215-221. 28. Wei, Y.; Bie, Z.; Luo, X.; Lu, W.; Liu, M.; Ji, S., Determination of titanium dioxide in tipping paper by wet digestion-ICP-MS. Chinese Tobacco Science 2015, 36 (1), 10-13. 29. Ma, C.; Liu, H.; Guo, H.; Musante, C.; Coskun, S. H.; Nelson, B. C.; White, J. C.; Xing, B.; Dhankher, O. P., Defense mechanisms and nutrient displacement in Arabidopsis thaliana upon exposure to CeO 2 and In 2 O 3 nanoparticles. Environmental Science: Nano 2016, 3 (6), 13691379. 30. Kruger, N. J., The Bradford method for protein quantitation. In Basic protein and peptide protocols, Springer: 1994; pp 9-15. 31. Zhao, L.; Peng, B.; Hernandez-Viezcas, J. A.; Rico, C.; Sun, Y.; Peralta-Videa, J. R.; Tang, X.; Niu, G.; Jin, L.; Varela-Ramirez, A., Stress response and tolerance of Zea mays to CeO2 nanoparticles: Cross talk among H2O2, heat shock protein, and lipid peroxidation. ACS nano 2012, 6 (11), 9615-9622. 32. Dixit, V.; Pandey, V.; Shyam, R., Differential antioxidative responses to cadmium in roots and leaves of pea (Pisum sativum L. cv. Azad). Journal of Experimental Botany 2001, 52 (358), 1101-1109. 33. Jing, G.; Huang, H.; Yang, B.; Li, J.; Zheng, X.; Jiang, Y., Effect of pyrogallol on the physiology and biochemistry of litchi fruit during storage. Chemistry Central Journal 2013, 7 (1), 19. 34. Chen, G.; Ma, C.; Mukherjee, A.; Musante, C.; Zhang, J.; White, J. C.; Dhankher, O. P.; Xing, B., Tannic acid alleviates bulk and nanoparticle Nd2O3 toxicity in pumpkin: a physiological and molecular response. Nanotoxicology 2016, 10 (9), 1243-1253. 35. Ma, C.; Rui, Y.; Liu, S.; Li, X.; Xing, B.; Liu, L., Phytotoxic mechanism of nanoparticles: destruction of chloroplasts and vascular bundles and alteration of nutrient absorption. Scientific reports 2015, 5. 36. Ma, C.; Chhikara, S.; Minocha, R.; Long, S.; Musante, C.; White, J. C.; Xing, B.; Dhankher, O. P., Reduced Silver Nanoparticle Phytotoxicity in Crambe abyssinica with Enhanced Glutathione Production by Overexpressing Bacterial γ-Glutamylcysteine Synthase. Environmental science & technology 2015, 49 (16), 10117-10126. 37. Krishnaraj, C.; Jagan, E.; Ramachandran, R.; Abirami, S.; Mohan, N.; Kalaichelvan, P., Effect of biologically synthesized silver nanoparticles on Bacopa monnieri (Linn.) Wettst. plant growth metabolism. Process Biochemistry 2012, 47 (4), 651-658. 38. Larue, C.; Castillo-Michel, H.; Sobanska, S.; Cécillon, L.; Bureau, S.; Barthès, V.; Ouerdane, L.; Carrière, M.; Sarret, G., Foliar exposure of the crop Lactuca sativa to silver nanoparticles: evidence for internalization and changes in Ag speciation. Journal of hazardous materials 2014, 264, 98-106. 39. Mittler, R., Oxidative stress, antioxidants and stress tolerance. Trends in plant science 2002, 7 (9), 405-410. 40. Mittler, R.; Vanderauwera, S.; Gollery, M.; Van Breusegem, F., Reactive oxygen gene network of plants. Trends in Plant Science 2004, 9 (10), 490-498. 41. Carocho, M.; Ferreira, I. C. F. R., A review on antioxidants, prooxidants and related controversy: Natural and synthetic compounds, screening and analysis methodologies and future perspectives. Food and Chemical Toxicology 2013, 51 (0), 15-25. 42. Nair, P. M. G.; Chung, I. M., Physiological and molecular level effects of silver nanoparticles exposure in rice (Oryza sativa L.) seedlings. Chemosphere 2014, 112, 105-113.

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43. Chen, J.; Liu, X.; Wang, C.; Yin, S.-S.; Li, X.-L.; Hu, W.-J.; Simon, M.; Shen, Z.-J.; Xiao, Q.; Chu, C.-C., Nitric oxide ameliorates zinc oxide nanoparticles-induced phytotoxicity in rice seedlings. Journal of hazardous materials 2015, 297, 173-182. 44. Faisal, M.; Saquib, Q.; Alatar, A. A.; Al-Khedhairy, A. A.; Hegazy, A. K.; Musarrat, J., Phytotoxic hazards of NiO-nanoparticles in tomato: A study on mechanism of cell death. Journal of Hazardous Materials 2013, 250–251 (0), 318-332. 45. Liu, L.; Liu, Y.-h.; Liu, C.-x.; Wang, Z.; Dong, J.; Zhu, G.-f.; Huang, X., Potential effect and accumulation of veterinary antibiotics in Phragmites australis under hydroponic conditions. Ecological Engineering 2013, 53, 138-143. 46. Dhankher, O. P.; Li, Y.; Rosen, B. P.; Shi, J.; Salt, D.; Senecoff, J. F.; Sashti, N. A.; Meagher, R. B., Engineering tolerance and hyperaccumulation of arsenic in plants by combining arsenate reductase and γ-glutamylcysteine synthetase expression. Nature biotechnology 2002, 20 (11), 1140-1145. 47. Cobbett, C. S.; May, M. J.; Howden, R.; Rolls, B., The glutathione-deficient, cadmiumsensitive mutant,cad2–1, ofArabidopsis thalianais deficient in γ-glutamylcysteine synthetase. The Plant Journal 1998, 16 (1), 73-78. 48. Leustek, T.; Saito, K., Sulfate transport and assimilation in plants. Plant physiology 1999, 120 (3), 637-644. 49. Takahashi, H.; Kopriva, S.; Giordano, M.; Saito, K.; Hell, R., Sulfur assimilation in photosynthetic organisms: molecular functions and regulations of transporters and assimilatory enzymes. Annual review of plant biology 2011, 62, 157-184. 50. Na, G.; Salt, D. E., The role of sulfur assimilation and sulfur-containing compounds in trace element homeostasis in plants. Environmental and Experimental Botany 2011, 72 (1), 1825. 51. Paulose, B.; Kandasamy, S.; Dhankher, O. P., Expression profiling of Crambe abyssinica under arsenate stress identifies genes and gene networks involved in arsenic metabolism and detoxification. BMC plant biology 2010, 10 (1), 108. 52. Landa, P.; Vankova, R.; Andrlova, J.; Hodek, J.; Marsik, P.; Storchova, H.; White, J. C.; Vanek, T., Nanoparticle-specific changes in Arabidopsis thaliana gene expression after exposure to ZnO, TiO2, and fullerene soot. Journal of Hazardous Materials 2012, 241–242 (0), 55-62. 53. Kaveh, R.; Li, Y.-S.; Ranjbar, S.; Tehrani, R.; Brueck, C. L.; Van Aken, B., Changes in Arabidopsis thaliana Gene Expression in Response to Silver Nanoparticles and Silver Ions. Environmental Science & Technology 2013, 47 (18), 10637-10644. 54. Zhao, L.; Sun, Y.; Hernandez-Viezcas, J. A.; Hong, J.; Majumdar, S.; Niu, G.; DuarteGardea, M.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L., Monitoring the Environmental Effects of CeO2 and ZnO Nanoparticles Through the Life Cycle of Corn (Zea mays) Plants and in Situ µXRF Mapping of Nutrients in Kernels. Environmental Science & Technology 2015, 49 (5), 29212928. 55. Hernandez-Viezcas, J. A.; Castillo-Michel, H.; Andrews, J. C.; Cotte, M.; Rico, C.; Peralta-Videa, J. R.; Ge, Y.; Priester, J. H.; Holden, P. A.; Gardea-Torresdey, J. L., In Situ Synchrotron X-ray Fluorescence Mapping and Speciation of CeO2 and ZnO Nanoparticles in Soil Cultivated Soybean (Glycine max). ACS Nano 2013, 7 (2), 1415-1423. 56. De la Torre Roche, R.; Servin, A.; Hawthorne, J.; Xing, B.; Newman, L. A.; Ma, X.; Chen, G.; White, J. C., Terrestrial Trophic Transfer of Bulk and Nanoparticle La2O3 Does Not Depend on Particle Size. Environmental science & technology 2015, 49 (19), 11866-11874. 57. Hawthorne, J.; De la Torre Roche, R.; Xing, B.; Newman, L. A.; Ma, X.; Majumdar, S.; Gardea-Torresdey, J.; White, J. C., Particle-size dependent accumulation and trophic transfer of cerium oxide through a terrestrial food chain. Environmental science & technology 2014, 48 (22), 13102-13109.

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Figure 1. Physiological effects of TiO2 NPs and TC on A. thaliana. (A) Images of A. thaliana co-exposed 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. Figure 2. Antioxidant enzyme activity of A. thaliana shoots co-exposed to TiO2 NPs and TC. Figure 2A-D represents SOD, CAT, APX, and POD activities, respectively. Data are mean ± standard error of three to five replicates. Values of each antioxidant enzyme activities followed by different letters indicate that the data points are significantly different at p≤0.05. Figure 3. Antioxidant enzyme activity of A. thaliana roots co-exposed to TiO2 NPs and TC. Figure 3A-D represents SOD, CAT, APX, and POD activities, respectively. Data are mean ± standard error of three to five replicates. Values of each antioxidant enzyme activities followed by different letters indicate that the data points are significantly different at p≤0.05. Figure 4. Relative expression of genes involved in the sulfur assimilation pathway in A. thaliana treated with TiO2 NPs and TC. Figure 4A-C represents 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. Figure 5. Relative expression of stress related genes in A. thaliana treated with TiO2 NPs and TC. Figure 5A-C represents expression levels of CS, ECS, and GS, respectively. Figure 5D-F represents expression levels of GST, GR, and MDAR, respectively. Data are mean ± standard error of three replicates. Figure 6. Effects of co-exposure 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.

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A

Root length (cm)

727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755

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A 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849

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Total number of pods

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200mg/L 10mg/L TiO2 NPs TiO2 NPs TC ×TC


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936

For Table of Contents Use Only

937

Title: Titanium dioxide nanoparticles alleviate tetracycline toxicity to Arabidopsis

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thaliana (L.)

939

Hong Liu†, Chuanxin Ma†, Guangcai Chen, Jason C. White, Zonghua Wang, Baoshan

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Xing* and Om Parkash Dhankher*

941 942

†

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Synopsis: Investigation of the effects of co-contaminations of TiO2 NPs and tetracycline

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on Arabidopsis thaliana at the biochemical and molecular levels.

*

These authors contributed equally to this work. Corresponding authors

945 946 947 948 949 950 951 TOC graphic 952 953 954 955 956 957 958 959

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Titanium Dioxide Nanoparticles Alleviate Tetracycline Toxicity to

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