Effects of nanoTiO2 on tomato plants under different irradiances

Environmental Pollution 255 (2019) 113141

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Effects of nanoTiO2 on tomato plants under different irradiances Jung Aa Ko, Yu Sik Hwang* Environmental Fate & Exposure Research Group, Korea Institute of Toxicology, 17 Jegok-gil, Jinju, 52834, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 January 2019 Received in revised form 19 June 2019 Accepted 29 August 2019 Available online 9 September 2019

In this study, we investigated the physiological and photochemical influences of nanoTiO2 exposure on tomato plants (Lycopersicum esculentum Mill.). Tomato plants were exposed to 100 mg L
1 of nanoTiO2 for 90 days in a hydroponic system. Light irradiances of 135 and 550 mmolphoton m
2 s
1 were applied as environmental stressors that could affect uptake of nanoTiO2. To quantify nanoTiO2 accumulation in plant bodies and roots, we used transmission electron microscopy, energy-dispersive X-ray spectroscopy, inductively coupled plasma mass spectrometry, and X-ray powder diffraction. Phenotypic and physiological influences such as color change, growth rate, fruit productivity, pigment concentration, and enzyme activity (SOD, CAT, APX) were monitored. We observed numerous effects caused by high irradiance and nanoTiO2 exposure, such as rapid chlorophyll decrease, increased anthocyanin and carotenoid concentrations, high enzymatic activity, and an approximately eight-fold increase in fruit production. Moreover, light absorption in the nanoTiO2-treated tomato plants, as measured by a ultravioletevisible light spectrometer, increased by a factor of approximately 19, likely due to natural pigments that worked as sensitizers, and this resulted in an ~120% increase in photochemical activities on A, ФPSII, ФCO2, gsw, and E. © 2019 Elsevier Ltd. All rights reserved.

This paper has been recommended for acceptance by Wei Shi. Keywords: Titanium dioxide nanoparticle Irradiance change Fruit productivity Photochemical activity Infrared gas-exchange analyzer

1. Introduction Environmental conditions are key factors determining crop yields and the quality of fruits in agriculture. In the past, temperature, quality of water and soil, irradiance, and microorganisms have been regarded as major factors for successful cultivation practices. However, due to growing concerns regarding consumer well-being, and the fact that plants often exhibit high pollutantuptake capacities, environmental pollutants are receiving an increasing share of the attention. Rapid industrial development introduced various pollutants such as heavy metals (Yi et al., 2011), pesticides (Daly et al., 2007), persistent organic pollutants (Ren et al., 2018), and nanoparticles (Amde et al., 2017; Gottschalk et al., 2009) into the soil, air, and water. A strong emphasis has been placed on nanoparticles as a new type of pollutant because of their high usage, rapid distribution through commercial products, and physicochemical activities. Nanoparticles have the potential to create side effects if they accumulate in the human body, where cellular uptake is facilitated due to their small size and large surface area.

* Corresponding author. E-mail address: [email protected] (Y.S. Hwang). https://doi.org/10.1016/j.envpol.2019.113141 0269-7491/© 2019 Elsevier Ltd. All rights reserved.

Many types of engineered nanoparticles (ENPs) have been synthesized and applied to various purposes. Titanium dioxide (TiO2) is among the most commonly used ENPs because of its photochemical properties. Specifically, it scatters ultraviolet light due to a high reflective index (Smijs and Pavel, 2011) and has a photocatalytic ability based on a high redox activity due to electrons and holes (e
/hþ) on the nanoTiO2 surface (Schneider et al., 2014). TiO2 is widely used in cosmetics to prevent skin damage and as an industrial photocatalyst (Liu et al., 2008; Xiao et al., 2013). Zinc oxide (ZnO) is widely used for similar purposes, but it transforms easily from particles into ions depending on environmental conditions (Bian, 2011). Because it is difficult for nanoTiO2 to dissolve without a strong chemical reaction and can therefore exist in nature with its physicochemical properties, it has attracted great attention as an environmental pollutant. According to Keller and Lazareva (2014), the predicted concentration of nanoTiO2 in wastewater treatment plant effluent and biosolids in San Francisco Bay was 4e70 mg L
1 and 400 to 1000 mg kg
1, respectively. This is higher than levels reported for other nanoparticles such as ZnO, silicon dioxide (SiO2), carbon nanotube (CNTs), and aluminum oxide (Al2O3), which are present in effluent and biosolids at 0.005e10 mg L
1 and 0.3e300 mg kg
1, respectively. Many researchers have studied the fate of nanoTiO2 in living organisms as well as in environmental systems. Plants are

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often used as a model to investigate the effects of nanoTiO2 because they are cultivated in soil and are producers in the food chain. Furthermore, influences caused by the photocatalytic activity of nanoTiO2 can be studied because plants also use light as an energy source. For example, Conway et al. (2015) and Gao et al. (2013) reported decreased photochemical activities associated with photosynthetic rate, nonphotochemical quenching, and the quantum yield of CO2 assimilation when plants were exposed to nanoTiO2. However, Hong et al. (2005) demonstrated improved photochemical efficiency through the reduction rate of FeCy and photophosphorylation, and Li et al. (2015) reported that increased uptake of nitrogen and magnesium ions promoted chlorophyllase production. Numerous papers have reported that nanoTiO2 can influence DNA expression, root and shoot elongation, seed germination, biomass, concentration of H2O2, and enzyme activity for reactive oxygen species (ROS) in diverse plant species (Tan et al., 2018b; Du et al., 2017; Ma et al., 2017). There is great interest in the effects of nanoTiO2 accumulation on photochemistry and other physiological aspects in plants. For this study, we chose a tomato plant model in consideration of the high global consumption of tomatoes and a convenient life-cycle period. Tomato plants were grown in a nutrient solution instead of soil so that the effects of translocated nanoTiO2 could be investigated more clearly. Two different illumination conditions were provided, low light (LL) and high light (HL), on the assumption that higher irradiance will enhance nanoTiO2 uptake, along with other nutrients and water, due to increased water evaporation on the leaves. A higher accumulation of nanoTiO2 under HL conditions was expected to have a significant effect on plant physiology and photochemistry compared with LL conditions. The study was continued for 90 days, during which various physical and physiological changes on the model plants were monitored to determine if any were altered by nanoTiO2 under different light irradiances.

to light. Further sonication was performed for approximately 5 min in a water bath to prevent precipitation before adding the suspension to the nutrient solution. 2.3. Tomato growth and harvest Tomato seeds (Lycopersicum esculentum Mill., Asia Seed Co., Ltd., Korea) were germinated overnight with DI. Five seeds were then planted in a pot using gardening soil (mixed culture soil, Europot, Korea). Approximately 30 mL of water was supplied each day, and the tomato plants were cultivated for 15 days under 12 h light/12 h dark, 60% humidity, 24  C, and a photosynthetic photon flux density of 135 mmolphoton m
2 s
1. After cultivation in soil, 12 young tomato plants for each treatment were transplanted into 7 L of 50% Hoagland solution after removing the soil attached to the roots. The exposure concentration of 100 mg L
1 was selected at intermediate concentration level to produce a toxic effects between that of the low and high concentration. The Hoagland solutions with or without nanoTiO2 were topped to 1 L per a week for 30 days and 2 L per a week after 30 days. Tomato plants were cultivated for 90 days in the hydroponic system with different light intensities in the chamber: 135 mmol
2
1 s for low light (LL) and 550 mmolphoton m
2 s
1 for high photon m light (HL) under 12 h light/12 h dark, 60% humidity, and 24  C. Four tomato plants were harvested at the 30th, 60th, and 90th days for phenotypic and physiological analysis. Harvested plants were separated into leaf, stem, root, and fruit portions. Collected samples except fruit were severed into 1 cm2 pieces under liquid nitrogen treatment, and stored in a deep freezer at
70  C. In the case of fruit, we collected every tomato on the 90th day regardless of size, color, and shape to determine fruit yield. Fruits that turned completely red before the 90th day were collected and stored in a deep freezer at
70  C without incision. Sample procedures for elemental analysis are discussed in the Supplementary Material.

2. Experiment 2.4. Sample preparation for TEM and EDS analyses 2.1. Materials Hoagland solution materials such as KNO3 (99.0%), Ca(NO3)2$4H2O (98.5%), MgSO4$7H2O (99.5%), KH2PO4 (99.0%), MnCl2$4H2O (99.0%), CuSO4$5H2O (99.5%), H2MoO4 (87.0%), and ZnSO4$7H2O (99.5%) were purchased from Sigma-Aldrich. (USA); concentrations are described in Supplementary Table 1. A nanotitanium dioxide (nanoTiO2) mixture of rutile and anatase, which have 10e30 nm diameters according to the manufacturer, was purchased from Sky Spring (7918DL, Sky Spring Nanomaterials, USA). Other materials such as nitroblue tetrazolium (NBT, 98%; Sigma-Aldrich, USA), L-methionine (98%; Sigma-Aldrich, USA), riboflavin (98%; Sigma, USA), ethylenediaminetetraacetic acid (EDTA, 99.4e100.6%; Sigma-Aldrich, USA), sodium L-ascorbate (BioXtra,  99%; Sigma, USA), glutaraldehyde (25% in H2O; SigmaAldrich, USA), osmium tetroxide (OsO4, 4% in H2O; Sigma-Aldrich, USA), propylene oxide (99%; Sigma-Aldrich, USA), Epon 812 embedding medium (Sigma-Aldrich, USA), and Bradford reagent (Sigma, USA) were obtained from Sigma-Aldrich and Sigma, and phosphate buffer (0.1 mM, pH 7.4) was purchased from Tech & Innovation (Korea). 2.2. Preparation of nanoTiO2 stock suspension A nanoTiO2 stock suspension was prepared with deionized water (DI) and adjusted to 1000 mg L
1. The suspension was sonicated for 3 h with 5 s pulses per min at 70 W to increase homogeneity. The prepared stock suspension was stored at room temperature and covered with aluminum foil to prevent exposure

Leaves that were harvested at 90 days under HL were used for ultra-corrected-energy-filtered transmission electron microscopy and energy-dispersive X-ray spectroscopy analysis (Libra 200 HT Mc Cs, ZEISS, Germany). For the fixation, fresh leaves were cut into 1 mm2 pieces, 2.5% glutaraldehyde was added, and the samples were stored overnight at 4  C. Samples were washed with icecooled 1X phosphate buffer (3 times), added to 1% OsO4 for 1 h at 4  C, and then washed with ice-cooled 1X phosphate buffer (3 times). Dehydration employed a series of ethanol concentrations (50, 60, 70, 80, 90, and 100%) for 20 min each, followed by three repeats with 100% ethanol and 100% propylene oxide for 30 min (2 times). For embedding, propylene oxide and an embedding medium were used at ratios of 3:1, 1:1, and 1:3 for 5 h, after which 100% Epon 812 resin was added. The samples were stored overnight and incubated at 70  C for embedding polymerization. To obtain ultra-thin sections, ultra-microtome (Ultracut UCT, Leica, Austria) was used, embedded blocks were sliced into 90 nm sections that were then mounted on carbon grids, and post-staining was performed with uranyl acetate for 10 min and lead citrate for 5 min. Accumulated nanoTiO2 in the plant cells were observed using UCET-TEM and EDS was used for elemental analysis of the observed nanoparticles. 2.5. Enzyme assays To evaluate stress levels, enzyme activities for superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) were analyzed using 0.5 g of leaves or roots homogenized with 5 mL of

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50 mM phosphate buffer with a pre-cooled mortar and pestle. During the process, temperatures were kept at 4  C to prevent enzyme digestion. The obtained homogenate was separated with a centrifuge (Himac CT15RE, Koki Holdings Co., Ltd) at 21,500 g and 4  C for 20 min, the supernatant was discarded, and the product was stored in 2 mL Eppendorf tube in an ice-filled box for further enzymatic analysis. SOD (EC 1.15.1.1) activity was detected as described by Zhang et al. (2007) using a method by Beauchamp and Fridovich (1971) with minor modification; reduced sample volume to 50 mL and increased reaction time to 15 min. The reagent mixture was adjusted to 3 mL with 50 mM phosphate buffer, 13 mM methionine, 75 mM NBT, 10 mM EDTA, and 50 mL of supernatant. Next, 2 mM of riboflavin was added, and the vials were irradiated with fluorescent lamps for 15 min. After the reaction, absorbance was measured at 560 nm using an ultravioletevisible light (UV/Vis) spectrometer (Lamda25, PerkinElmer, USA). One unit of SOD activity was calculated based on the amount that inhibited photoreduction of NBT by 50%. The concentration of protein in each supernatant was measured with a Bradford assay, and bovine serum albumin (Sigma-Aldrich, USA) was used as a standard. Experimental procedures for APX and CAT are described in the Supplementary Materials. 2.6. Pigment analysis For chlorophyll and carotenoid analysis, 0.5 g of leaves were homogenized with 5 mL of pure acetone. The resulting extracts were centrifuged at 2900 g for 10 min, and the chlorophyll and carotenoids in the supernatant were analyzed with a UV/Vis spectrometer in a range of 350e700 nm. Concentration was calculated based on Lichtenthaler's method, with absorbance at 645 and 660 nm for Chl. a and b, respectively, and at 470 nm for carotenoids (Lichtenthaler and Buschmann, 2001). To detect anthocyanin, 0.5 g of leaves were homogenized with 5 mL methanol:HCl (99:1,v/v), and the homogenate was centrifuged at 2900 g for 10 min. The absorbance (A) of the supernatant was measured and the concentration was then calculated using equation A530e0.24 A653 and an extinction coefficient of 33,000 L mol
1 cm
1 (Murray and Hackett, 1991; Neill et al., 2002). All pigment extract processes were performed with a mortar and pestle pre-cooled to 4  C.

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two (**), and three asterisks (***), respectively. 3. Results and discussion 3.1. Confirmation of accumulated nanoTiO2 in plants Details of nanoTiO2's physical properties, including size and zeta potential, and images are supplied in Suppl. Fig. 1 and Suppl. Table 2. Confirmation of accumulated nanoTiO2 in plants helps explain observed phenotypic and physiological influences and whether they were triggered by translocated nanoparticles or high irradiance. To detect uptake of nanoparticles by plant cells, electron microscopy is the most frequently used tool as it can provide visible evidence of nanoparticle accumulation in living bodies. Transmission electron microscopy (TEM) was applied to confirm the presence of nanoTiO2 in plant cells. To obtain convincing evidence for nanoTiO2 translocation from roots to leaves and to confirm nanoTiO2 accumulation at organelles in the leaf, samples were prepared with leaves collected from 90-day HL-exposed plants. The presence of nanoTiO2 in plant leaves was confirmed using TEM, as shown in Fig. 1a and Suppl. Fig. 2. Translocated nanoparticles were widely distributed in various organelles, such as chloroplasts, mitochondria, cell walls, and cytosol. Furthermore, a clear lattice structure was observed in magnified TEM images of the crystalline structure of the synthesized nanoparticles (Fig. 1b). We also found a chloroplast that exhibited unstacked thylakoid of grana and significant structural damage from HL treatment, as shown in Fig. 1a and Suppl. Fig. 2. This may be due to concentration of photosystem II (PSII) and light-harvesting complex II (LHCII) in the stacked grana (Kirchhoff, 2013; Yamamoto, 2016). Although analysis with TEM can provide information on nanoTiO2 accumulation, fake nanoparticles similar in shape and size to nanoTiO2 can confuse the process. Qualitative EDS analysis was therefore suggested to remove any uncertainty caused by fake nanoparticles. Significant titanium peaks appeared only in nanoTiO2-treated tomato leaves, along with peaks associated with other elements such as C, Cu, Ca, and Si (Fig. 1c). No recognizable peaks for titanium were found in control samples. Further qualitative and quantitative analysis with XRD and ICP-MS are discussed in the Supplementary Material. 3.2. Phenotypic effects

2.7. Photosynthetic ability Photosynthetic efficiency and parameters for photochemistry of plant were analyzed with a portable infrared gas-exchange analyzer (IRGA, LI-6400XT, LI-COR, Inc., USA). We selected 30-day samples of tomato plants under LL because LL-Con and LL-nanoTiO2 recorded similar chlorophyll concentrations. A leaf was placed on the sensor head, and parameters such as assimilation rate (A), quantum yield of PS II (ФPSII), quantum yield of CO2 (ФCO2), transpiration rate (E), stomatal conductance (gsw), ambient concentration of CO2 (Ca), intercellular concentration of CO2 (Ci), and electron transport rate (ERT) were recorded using the following settings: 1000 mmolphoton m
2 s
1 for ParIn and 400 mmolCO2 mol
1 for reference CO2 chamber. 2.8. Statistical analysis The results are shown as mean and standard deviation (SD) for n ¼ 4e7. Statistical analysis was performed with one-way or twoway analysis of variance (ANOVA), and Bonferroni correction was used for the post hoc analysis. The p-values less than 0.05 (p < 0.05), 0.01 (p < 0.01), and 0.005 (p < 0.005) are represented with one (*),

For analysis of growth rate, tomato plants were divided into two parts: body (stem and leaves) and roots. As a common effect, tomato plants exposed to nanoTiO2 showed enhanced body mass compared with the control, but the difference was not significant because of the high SD (Fig. 2a). In contrast, body length was statistically significantly (p < 0.01) affected by irradiance, ~20.1 cm for LL, which is approximately 2.1 times longer than the ~9.6 cm seen for HL. Based on this result, the body weight-to-length ratio showed a higher value at HL (3.0e3.2) than at LL (1.3e1.6), which represents greater leaf and branch production under HL than under LL. In the roots, light intensity as well as nanoTiO2 treatment showed a weak effect on root extension, but no statistical significance was observed (Fig. 2b). After the vegetative stage, yellow flowers appeared on all the tomato plants; however, fruit productivity varied with the culture conditions. Generally, tomato plants under HL showed faster growth than under LL, with first flowering occurring approximately 7e10 days earlier under HL. In the case of LL, only a few flowers produced fruit owing to flowers dropping before or after pollination. Consequently, a lower fruit yield (Fig. 2c) was recorded with LL compared with HL (i.e., 0 and 1 ± 1 for LL-Con and HL-Con,

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Fig. 1. Microscopic evidence of nanoTiO2 accumulation in leaf cells (grown under HL for 90 days) by STEM analysis. (a) TEM image for confirmation of translocated nanoTiO2 (CW: cell wall, M: mitochondria, and Chl.: chloroplast) and unstacked thylakoid of grana indicated with arrows, (b) magnified image of nanoTiO2, and (c) EDS spectrum of translocate nanoTiO2.

respectively, in fruit per plant). With irradiance change, there was a noteworthy yield difference (p < 0.01) with 0.5 ± 0.55 and 4 ± 1 fruit per plant for LL-nanoTiO2 and HL-nanoTiO2, respectively. NanoTiO2 treatment also resulted in a four-fold increase (p < 0.05) in fruit production despite the same light irradiance of HL. Other studies have shown similar improvement in growth rates as well as
r flower, fruit, and seed production under stressful conditions (Kola  kov
and Sen a, 2008; Wada et al., 2010). According to Takeno (2016), this “stress-induced flowering” is a species-preserving strategy for unsuitable environments. Various pathways are reportedly involved in stress-induced development, including regulation of gene expression of FLOWERING LOCUS T (FT) (Boss et al., 2004) and production of stress substances for signal delivery such as ROS, salicylic acid, and nitric oxide, among others (Martínez et al., 2004; Kohli et al., 2013). Here, we observed accelerated fruit production under high irradiance, and ROS signaling is suspected to be a major cause. As shown in TEM images (Fig. 1a and Suppl. Fig. 2), significantly damaged chloroplasts were found due to photooxidative stress. Chloroplasts contribute to various cellular functions, including photosynthesis, production of phytohormones and antioxidants, and control of the redox state of the cell (Joyard et al., 2009). However, during leaf senescence, structural damage and degradation of chlorophyll weaken chloroplast activity. This results in increased ROS levels due to an imbalance between ROS production and elimination as the growth period, and consequently

the unremoved ROS, work as signaling molecules for activating gene expression for flower and fruit development (Xia et al., 2015). In contrast to HL results, the presence of nanoTiO2 enhanced this phenotypic effect, but the change was not significant because of the dominating effect of strong irradiation. 3.3. Monitoring of physiological influence 3.3.1. Pigment concentration Chlorophyll is a pigment in chloroplasts that plays a central role photochemical reactions. Chlorophyll concentrations decreased constantly with all treatments d even in the tomato plants without any pollutants dbecause of aging, as shown in Fig. 3. Furthermore, a noticeable reduction in total chlorophyll was found under HL, i.e., the total concentration of chlorophyll was 1888 mg g
1 for LL-Con and 1365 mg g
1 for HL-Con at 90 days, and only 66% (p < 0.05) and 59% (p < 0.005) of chlorophyll remained in leaves compared to 30-day levels. The decline was amplified by approximately 9% with nanoTiO2 treatments as well as HL. Among all measurements, the lowest total chlorophyll concentration was detected in tomato plants grown for 90 days with HL and nanoTiO2 treatment. During the analysis, we recognized notable changes in the pigment ratio of Chl. a and Chl. b with respect to irradiance. For example, reduction of the ratio between Chl. a and Chl. b corresponded with a growth period in the case of LL, with about 0.38 (p < 0.005) and 0.28

Fig. 2. Growth rate of harvested tomato plants under different cultural conditions. Weight and length of (a) body (stem and leaves) and (b) roots, (c) fruit productivity (NH; not harvested), and (d) phenotype images were obtained from 90-day tomato plants. Two-way ANOVA and Bonferroni post hoc were used to determine statistical significance. P values are represented by asterisk(s), p < 0.05 (*), p < 0.01 (**), and p < 0.005 (***).

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Fig. 3. Chlorophyll concentration and chlorophyll ratio change with respect to culture period. The bar graph shows concentrations of Chl. a and b (colored for Chl. a and white for Chl. b), and the dot graph exhibits the Chl. a to Chl. b ratio. Statistical significance was analyzed with one-way ANOVA and Bonferroni post hoc based on the culture condition. P values are represented with asterisk(s), p < 0.05 (*), p < 0.01 (**), and p < 0.005 (***).

(p < 0.05) decreases for LL-Con and LL-nanoTiO2, respectively. In contrast, a gradual increase in the ratio of approximately 0.2 was noted with HL regardless of the presence of nanoTiO2. In general, the chlorophyll ratio decreased naturally because of photo-induced damage to Chl. a, which is involved in direct light absorption. An increase in the ratio of Chl. a to Chl. b means more intensive reduction of Chl. b than Chl. a. Chlorophyll is an acknowledged indicator of stress because of its high sensitivity to growth conditions. It has also been reported that controlling the concentration of Chl. a and b is an observable response to stress. Chl. a is a crucial pigment for photochemistry in catching and transferring light energy, Chl. b works to stabilize the binding protein of Chl. a, and sufficient Chl. a can be supplied via interconversion with Chl. b (Tanaka and Tanaka, 2011). On this basis, we consider that weak enhancement of Chl. a occurred through the chlorophyll cycle, although the increase was less significant than the reduction of chlorophyll. Leaf pigments carotenoids and anthocyanin were investigated along with chlorophyll. Distinct differences in concentrations were revealed for nanoTiO2 and irradiance as shown in Fig. 4, indicating a response against the stressors in the order of HL-Con  HL-nanoTiO2 > LL-nanoTiO2 > LL-Con for carotenoids and HL-nanoTiO2 > LLnanoTiO2 > HL-Con > LL-Con for anthocyanin. In plants, carotenoids function as supplements for the photochemical reaction, i.e., the antenna system of carotenoids helps catch more light of blue-green wavelengths (450e570 nm) (Havaux, 2014). Furthermore, carotenoids facilitate the dissipation of triplet chlorophyll (3Chl.*) and singlet oxygen (1O2) at Chl. a by quenching excess energy (Young and Frank, 1996). Anthocyanin has a different function; it acts as a sunscreen in the mesophyll by reducing transmitted irradiance through the cells, working as an ROS-scavenger, especially for H2O2 (Landi et al., 2015). Because of the defensive ability of carotenoids and anthocyanin, their concentrations were elevated in response to stressors, helping the plants cope with damage from strong irradiance and ROS. However, a distinct response was presented to each type of stressor. In the case of carotenoids, there was a statistically significant increase in concentration in response to changes in light intensity (LL to HL), 359 (p < 0.005) and 252 (p < 0.05) mg g
1 increases for control leaves and nanoTiO2 treated leaves, respectively, and only a minor alteration caused by nanoTiO2 exposure. Anthocyanin

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Fig. 4. Concentration for (a) carotenoids and (b) anthocyanin in tomato leaves at 90 days. Two-way ANOVA and Bonferroni post hoc were used to determine statistical significance, and P values are represented with asterisk(s), p < 0.05 (*), p < 0.01 (**), and p < 0.005 (***).

showed a stronger response to nanoTiO2 than to irradiance, with the concentration increasing ~0.041 mM g
1 for light change (LL to HL) and ~0.110 (p < 0.005) mM g
1 for nanoTiO2 exposure (control to nanoTiO2). This suggests that increased irradiance resulted in greater activation of carotenoids, improving light absorption by supporting Chl. a, because the level of Chl. a was reduced significantly owing to photo-damage and aging. Meanwhile anthocyanin was influenced by the accumulation of nanoTiO2 because anthocyanin can remove ROS produced by nanoTiO2.

3.3.2. Enzymatic activity Enzyme activities for SOD, APX, and CAT were monitored to estimate oxidative stress (graphs for APX and CAT are shown in Suppl. Fig. 5). Each enzyme has a different function as an ROS scavenger, i.e., SOD changes O2_- to H2O2, and APX and CAT convert H2O2 into the stable molecules of H2O and O2 (Mittler, 2002). In this study, the root was an organ directly exposed to high concentrations of nanoTiO2. Hence, stronger enzymatic activities were observed in root tissues than in leaves, ~4.49 times higher activity in roots compared with leaves, as shown in Fig. 5a. Moreover, there was a statistically significant increase in activity in a positive correlation against the stressors with 78.1, 112.6, 177.2, and 197.0 U mg
1 in roots for LL-Con, LL-nanoTiO2, HL-Con, and HLnanoTiO2, respectively, because of the damage driven by direct physical collision as well as nanoTiO2-produced ROS. Furthermore, light intensity also had a notable effect on the SOD level of roots because strong irradiation increases water evaporation from the leaves, which leads to higher water uptake through the root. However, leaves showed weaker SOD activity than roots, notwithstanding direct irradiance. NanoTiO2 exposure led to a noticeable elevation of ~75% in enzyme activity in leaves compared with the control, likely due to increased redox reactions of nanoTiO2; a wider range of light can be absorbed if nanoTiO2 is presented along with natural pigments, with a similar mechanism to that of a dye-sensitized solar cell (Ludin et al., 2014; Calogero et al., 2012). In particular, we expected that anthocyanin would act as a major sensitizer for translocated nanoTiO2 in leaves because it is synthesized in the cytoplasm, unlike other pigments such as chlorophyll and carotenoids, which are

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Fig. 5. SOD enzyme activity for (a) roots and (b) leaves under different culture conditions based on growth periods of 30, 60, and 90 days. Two-way ANOVA and Bonferroni post hoc were used to determine statistical significance in respect to the culture period. P values are represented with asterisk(s), p < 0.05 (*), p < 0.01 (**), and p < 0.005 (***).

produced in chloroplast. Consequently, sensitized nanoTiO2 produced more O2_- by receiving transferred electrons from the excited pigments (S*), resulting in higher SOD activity than when nanoTiO2 is absent, as illustrated in Fig. 6. Meanwhile, exposure to nanoTiO2 and HL showed reduced enhancement of ~16%, due to dominant stress and damage driven by strong irradiance rather than nanoTiO2. Additionally, the graph for leaves (Fig. 5b) shows a similar trend from LL-nanoTiO2, HL-Con, and HL-nanoTiO2, with the highest value observed at 60 days. The reason is not clear, but we expected a close correlation with the growth rate on the basis of phenotypic observations; for instance, yellow and dried leaves should increase along with leaf fall after 60 days in tomato plants with stressors. This is because the growth rate tends to increase more in the presence of stressors than in their absence, which finally results in rapid aging and weak immunologic response. Meanwhile, the secondary enzymes of CAT and APX showed no significant change with respect to stressors because of the competitive reaction to achieve H2O2 elimination, as is the case with many types of antioxidants, including CAT, APX, peroxidases, glutathione peroxidase, glutathione, ascorbic acid, and anthocyanin (Landi et al., 2015). 3.3.3. Influence of nanoTiO2 on the photochemistry in plant leaves Photosynthesis is a crucial process that produces chemical energy by absorbing photons and occurs in chloroplasts. However, translocated nanoTiO2 has been reported to trigger interruption or

Fig. 6. Schematic illustration of superoxide (O2_-) production by sensitized nanoTiO2 in leaf cells.

improvement of photosynthesis (Tan et al., 2018a; Gao et al., 2013; Conway et al., 2015). We recognized noteworthy changes in photochemical parameters, indicating enhancement of photosynthetic efficiency in the presence of nanoTiO2. For this measurement, tomato plants with a similar level of chlorophyll, those grown under LL and harvested at 30 days, were used to observe any critical effects caused by nanoTiO2, rather than by chlorophyll, as Chl. a and b play are essential for photosynthesis, and distinct concentrations could alter our understanding of the effect of nanoTiO2 on the photosynthetic process. As shown in Fig. 7a, we noticed a weak increase of A in tomato plants with nanoTiO2, 6.98 mmol m
2 s
1 for LL-Con and 8.55 mmol m
2 s
1 for LL-nanoTiO2. Moreover, other physiological parameters showed a positive correlation. For instance, an elevation of ~41% was recorded relative to the control for ФPSII and ФCO2 (Fig. 7b and c, respectively), which represent increases in absorbed light in PS II that is useable for the photochemistry and high efficiency of chemosynthesis with CO2. Also, gsw and E (Fig. 7d and e, respectively) showed noticeable increases of ~120% compared with the control, improving photosynthetic capacity by fostering CO2 and H2O mobility in stomata as well as continuous water and nutrient transportation from the roots. The results also showed a weak increase in intercellular-to-ambient CO2 ratio (Ci-to-Ca) at LLnanoTiO2 (Fig. 7f), e.g., 0.64 for control and 0.71 for nanoTiO2, although there was no significant difference in consideration of the SD despite a much higher gsw for nanoTiO2 translocated leaves, indicating effective CO2 consumption in leaf cells. Improved photosynthetic ability with nanoparticle uptake has been reported in various papers. For example, Giraldo et al. (2014) explained increased photochemical efficiency with strongly augmented electron-transfer efficiency and photo-absorption induced by single-walled carbon nanotubes, and Rossi et al. (2016) and Sun et al. (2016) suspected that induced synthesis of chlorophyll was responsible for enhanced photochemistry. We also assume that nanoTiO2 uptake plays an important role in photochemical activities. NanoTiO2 in plant cells helps collect more light, particularly in specific light regions, as discussed in Suppl. Fig. 6. Absorption increased by approximately 19 times in the presence of nanoTiO2. However, a minor reduction also occurred in the absorption region for chlorophyll, an effect that was probably caused by translocated nanoTiO2 in the thylakoid membrane and attached nanoTiO2 on the surface of chloroplasts blocking the light absorption on chlorophyll (Laurue et al., 2012). Additionally, faster electron transfer is suspected to be a crucial factor for achieving higher photosynthetic efficiency because photosynthesis occurs as a sequential reaction, e.g., the excited electron in PSII can only move

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Fig. 7. Influence of nanoTiO2 on photochemical and physiological activity of tomato plants. (a) Accumulation rate (A, mmol m
2 s
1), (b) quantum yield of photosystem II (ФPSII), (c) quantum yield of CO2 (ФCO2, mmolCO2 mmol
1photon), (d) stomatal conductance (gsw, mmol m
2 s
1), (e) transpiration efficiency (E, molH2O m
2 s
1), (f) intercellular-to-ambient CO2 concentration ratio (Ci-to-Ca), and (g) electron transport rate (ERT, mmol m
2 s
1).

to other proteins and processes when the primary plastoquinone acceptor of QA is empty. 4. Conclusions In this study, we monitored physicochemical alterations triggered by high irradiance and nanoTiO2 on tomato plants. Confirmation of nanoTiO2 accumulation in plant cells using various instruments, such as TEM, EDS, XRD, and ICP-MS, was performed with leaves to provide direct evidence of nanoTiO2 accumulation in leaf cells as well as translocation from the root. Exposure to strong irradiance and nanoTiO2 resulted in numerous phenotypic and physiological reactions in tomato plants, including increased flower and fruit production, increase of anthocyanin and carotenoids, and high enzyme activity. Among the most notable observations was high fruit production, probably driven by a stress-induced reaction to accelerate propagation. However, despite nanoTiO2 treatment, no significant increase in titanium concentration was found in the fruits. Similarly, concentrations of elements such as magnesium, manganese, copper, and zinc also showed no remarkable concentration difference compared with controls. In contrast, accelerated growth and rapid aging, i.e., yellow leaves, leaf drop, and withered leaves, were evident in the presence of stressors. Increased stress also resulted in physiological changes. A statistically significant decrease in chlorophyll concentration in chloroplasts owing to aging and direct and indirect damage caused by

strong light, and nanoTiO2 was observed. Although concentrations of both carotenoids and anthocyanin increased, there were differences in accordance with the function of the pigment. For example, carotenoid concentration relied strongly on the intensity of irradiance, while anthocyanin was influenced by the presence of nanoTiO2 as it can eliminate produced ROS from the nanoTiO2. In addition, enzyme activity of SOD increased under HL and nanoTiO2 exposure, and the increase in roots was significantly higher than that in leaves because of direct physical damage by particles as well as enhanced physiological activity based on high levels of water evaporation caused by strong irradiance and increased photosynthetic efficiency from accumulated nanoTiO2. Although leaves with stressors showed a weak increase in enzyme activity, exposure of nanoTiO2 led to enhanced SOD activity owing to expanded absorbable light and also to increased redox reaction on the surface of nanoTiO2 because plant pigments can act as sensitizers. To address the possible effects of nanoTiO2 on photochemistry, photosynthetic efficiency in leaf tissues was measured with an infrared gas analyzer. The results showed enhancement of photochemistry parameters, possibly because of improved physiological and photochemical activities as well as expansion of the absorbable light to the ultraviolet region owing to dispersed nanoTiO2 in leaves. Meanwhile, a rapid decrease in photosynthetic efficiency is expected in the plants, with stressors due to reduction of chlorophyll and an increase of damaged leaves as the culture period increased, although fruit productivity was significantly enhanced in presence of stressors.

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J.A. Ko, Y.S. Hwang / Environmental Pollution 255 (2019) 113141

The results from this study may indicate that nanoTiO2 has potential application in agricultural production. However, the safety issues regarding the use of nanoTiO2 must be carefully evaluated since evidence mounts that nanoTiO2 in food may be harmful (Bettini et al., 2017), although we obtained no critical evidence of nanoTiO2 accumulation in tomato fruits. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) [grant numbers: NRF-2017R1C1B2009920]. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2019.113141. References Amde, M., Liu, J.F., Tan, Z.Q., Bekana, D., 2017. Transformation and bioavailability of metal oxide nanoparticles in aquatic and terrestrial environments. A review. Environ. Pollut. 230, 250e267. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276e287.
ra, C., Gaultier, E., Dupuy, J., Naud, N., Bettini, S., Boutet-Robinet, E., Cartier, C., Come
, S., Grysan, P., Reguer, S., Thieriet, N., Re
fre
giers, M., Thiaudie re, D., Tache re, M., Audinot, J.-N., Pierre, F.H., Guzylack-Piriou, L., Cravedi, J.-P., Carrie Houdeau, E., 2017. Food-grade TiO2 impairs intestinal and systemic immune homeostasis, initiates preneoplastic lesions and promotes aberrant crypt development in the rat colon. Sci. Rep. 7, 40373. Bian, S.W., 2011. Aggregation and Dissolution of 4 nm ZnO nanoparticles in aqueous environments: influence of pH, ionic strength, size, and adsorption of humic acid. Langmuir 27, 6059e6068. Boss, P.K., Bastow, R.M., Mylne, J.S., Dean, C., 2004. Multiple pathways in the decision to flower: enabling, promoting, and resetting. Plant Cell 16, 18e31. Supplement. Calogero, G., Yum, J.-H., Sinopoli, A., Di Marco, G., Gr€ atzel, M., Nazeeruddin, M.K., 2012. Anthocyanins and betalains as light-harvesting pigments for dyesensitized solar cells. Sol. Energy 86, 1563e1575. Conway, J.R., Beaulieu, A.L., Beaulieu, N.L., Mazer, S.J., Keller, A.A., 2015. Environmental stresses increase photosynthetic disruption by metal oxide nanomaterials in a soil-grown plant. ACS Nano 9, 11737e11749. Daly, G.L., Lei, Y.D., Teixeira, C., Muir, D.C.G., Wania, F., 2007. Pesticides in western canadian mountain air and soil. Sci. Total Environ. 41, 6020e6025. Du, W., Tan, W., Peralta-Videa, J.R., Gardea-Torresdey, J.L., Ji, R., Yin, Y., Guo, H., 2017. Interaction of metal oxide nanoparticles with higher terrestrial plants: physiological and biochemical aspects. Plant Physiol. Biochem. (Paris) 110, 210e225. Gao, J., Xu, G., Qian, H., Liu, P., Zhao, P., Hu, Y., 2013. Effects of nano-TiO2 on photosynthetic characteristics of Ulmus elongata seedlings. Environ. Pollut. 176, 63e70. Giraldo, J.P., Landry, M.P., Faltermeier, S.M., McNicholas, T.P., Iverson, N.M., Boghossian, A.A., Reuel, N.F., Hilmer, A.J., Sen, F., Brew, J.A., Strano, M.S., 2014. Plant nanobionics approach to augment photosynthesis and biochemical sensing. Nat. Mater. 13, 400e408. Gottschalk, F., Sonderer, T., Scholz, R.W., Nowack, B., 2009. Modeled environmental concentrations of engineered nanomaterials (TiO2, ZnO, Ag, CNT, Fullerenes) for different regions. Sci. Total Environ. 43, 9216e9222. Havaux, M., 2014. Carotenoid oxidation products as stress signals in plants. Plant J. 79, 597e606. Hong, F., Zhou, J., Liu, C., Yang, F., Wu, C., Zheng, L., Yang, P., 2005. Effect of nano-TiO2 on photochemical reaction of chloroplasts of spinach. Biol. Trace Elem. Res. 105, 269e279. Joyard, J., Ferro, M., Masselon, C., Seigneurin-Berny, D., Salvi, D., Garin, J., Rolland, N., 2009. Chloroplast proteomics and the compartmentation of plastidial isoprenoid biosynthetic pathways. Mol. Plant 2, 1154e1180. Keller, A.A., Lazareva, A., 2014. Predicted releases of engineered nanomaterials: from global to regional to local. Environ. Sci. Technol. Lett. 1, 65e70. Kirchhoff, H., 2013. Architectural switches in plant thylakoid membranes. Photosynth. Res. 116, 481e487. Kohli, A., Sreenivasulu, N., Lakshmanan, P., Kumar, P.P., 2013. The phytohormone crosstalk paradigm takes center stage in understanding how plants respond to abiotic stresses. Plant Cell Rep. 32, 945e957.  kov
Kol
ar, J., Sen a, J., 2008. Reduction of mineral nutrient availability accelerates flowering of Arabidopsis thaliana. J. Plant Physiol. 165, 1601e1609. Landi, M., Tattani, M., Gould, K.S., 2015. Multiple functional roles of anthocyanins in plant-environment interactions. Environ. Exp. Bot. 119, 4e17.

Laurue, C., Laurette, J., Herlin-Boime, N., Khodja, H., Fayard, B., Flank, A.M., Brisset, F., Carriere, M., 2012. Accumulation, translocation and impact of TiO2 nanoparticles in wheat (Triticum aestivum spp.): influence of diameter and crystal phase. Sci. Total Environ. 431, 197e208. Li, J., Naeem, M.S., Wang, X., Liu, L., Chen, C., Ma, N., Zhang, C., 2015. Nano-TiO2 is not phytotoxic as revealed by the oilseed rape growth and photosynthetic apparatus ultra-structural response. PLoS One 10, e0143885. Lichtenthaler, H.K., Buschmann, C., 2001. Chlorophylls and carotenoids: measurement and characterization by UV-VIS spectroscopy. In: Wrolstad, R.E., et al. (Eds.), Current Protocols in Food Analytical Chemistry. John Wiley & Sons, Inc., New Jersey, USA. F4.3.1-F4.3.8. Liu, S., Lim, M., Fabris, R., Chow, C., Drikas, M., Amal, R., 2008. TiO2 photocatalysis of natural organic matter in surface water: impact on trihalomethane and haloacetic acid formation potential. Environ. Sci. Technol. 42, 6218e6223. Ludin, N.A., Mahmoud, A.M.A.-A., Mohamad, A.B., Kadhum, A.A.H., Sopian, K., Karim, N.S.A., 2014. Review on the development of natural dye photosensitizer for dye-sensitized solar cells. Renew. Sustain. Energy Rev. 31, 386e396. Ma, C., Liu, H., Chen, G., Zhao, Q., Eitzer, B., Wang, Z., Cai, W., Newman, L.A., White, J.C., Dhankher, O.P., Xing, B., 2017. Effects of titanium oxide nanoparticles on tetracycline accumulation and toxicity in Oryza sativa (L.). Environ. Sci.: Nano 4, 1827e1839.
n, J., 2004. Salicylic acid regulates flowering time Martínez, C., Pons, E., Prats, G., Leo and links defense responses and reproductive development. Plant J. 37, 209e217. Mittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7, 405e410. Murray, J.R., Hackett, W.P., 1991. Dihydroflavonol reductase activity in relation to differential anthocyanin accumulation in juvenile and mature phase Hedera helix L. Plant Physiol. 97, 343e351. Neill, S.O., Gould, K.S., Kilmartin, P.A., Mitchell, K.A., Markham, K.R., 2002. Antioxidant activities of red versus green leaves in Elatostema rugosum. Plant Cell Environ. 25, 539e547. Ren, X., Zeng, G., Tang, L., Wang, J., Wan, J., Liu, Y., Yu, J., Yi, H., Ye, S., Deng, R., 2018. Sorption, transport and biodegradation-an insight in to bioavailability of persistent organic pollutants in soil. Sci. Total Environ. 610e611, 1154e1163. Rossi, L., Zhang, W., Lombardini, L., Ma, X., 2016. The impact of cerium oxide nanoparticles on the salt stress responses of Brassica napus L. Environ. Pollut. 219, 28e36. Schneider, J., Matsuoka, M., Takeuchi, M., Zhang, J., Horiuchi, Y., Anpo, M., Bahnemann, D.W., 2014. Understanding TiO2 photocatalysis: mechanisms and materials. Chem. Riv. 114, 9919e9986. Smijs, T.G., Pavel, S., 2011. Titanium dioxide and zinc oxide nanoparticles in sunscreens: focus on their safety and effectiveness. Nanotechnol. Sci. Appl. 4, 95e112. Sun, D., Hussain, H.I., Yi, Z., Rookes, J.E., Kong, L., Cahill, D.M., 2016. Mesoporous silica nanoparticles enhance seedling growth and photosynthesis in wheat and lupin. Chemosphere 152, 81e91. Takeno, K., 2016. Stress-induced flowering: the third category of flowering response. J. Exp. Bot. 67, 4925e4934. Tan, W., Du, W., Darrouzet-Nardi, A.J., Hernandez-Viezcas, J.A., Ye, Y., PeraltaVidea, J.R., Gardea-Torresdey, J.L., 2018a. Effects of the exposure of TiO2 nanoparticles on basil (Ocimum basilicum) for two generations. Sci. Total Environ. 636, 240e248. Tan, W., Peralta-Videa, J.R., Gardea-Torredey, J.L., 2018b. Interaction of titanium dioxide nanoparticles with soil components and plants: current knowledge and future research needs e a critical review. Environ. Sci.: Nano 5, 257e278. Tanaka, R., Tanaka, A., 2011. Chlorophyll cycle regulates the construction and destruction of the light-harvesting complexes. Biochim. Biophys. Acta 1807, 968e976. Wada, K.C., Kondo, H., Takeno, K., 2010. Obligatory short-day plant, Perilla frutescens var. crispa can flower in response to low-intensity light stress under long-day conditions. Physiol. Plant. 138, 339e345. Xia, X.J., Zhou, Y.H., Shi, K., Zhou, J., Foyer, C.H., Yu, J.Q., 2015. Interplay between reactive oxygen species and hormones in the control of plant development and stress tolerance. J. Exp. Bot. 66, 2839e2856. Xiao, J., Chen, W., Wang, F., Du, J., 2013. Polymer/TiO2 hybrid nanoparticles with highly effective UV screening but eliminated photocatalytic activity. Macromolecules 46, 375e383. Yamamoto, Y., 2016. Quality control of photosystem II: the mechanisms for avoidance and tolerance of light and heat stresses are closely linked to membrane fluidity of the thylakoids. Front. Plant Sci. 7, 1136. Yi, Y., Yang, Z., Zhang, S., 2011. Ecological risk assessment of heavy metals in sediment and human health risk assessment of heavy metals in fishes in the middle and lower reaches of the Yangtze River basin. Environ. Pollut. 159, 2575e2585. Young, A.J., Frank, H.A., 1996. Energy transfer reactions involving carotenoids: quenching of chlorophyll fluorescence. J. Photochem. Photobiol., B 36, 3e15. Zhang, F.Q., Wang, Y.S., Lou, Z.P., Dong, J.D., 2007. Effect of heavy metal stress on antioxidative enzymes and lipid peroxidation in leaves and roots of two mangrove plant seedlings (Kandelia candel and Bruguiera gymnorrhiza). Chemosphere 67, 44e50.