Growth and Metabolic Responses of Rice (Oryza sativa L

Article pubs.acs.org/JAFC

Growth and Metabolic Responses of Rice (Oryza sativa L.) Cultivated in Phosphorus-Deficient Soil Amended with TiO2 Nanoparticles Zahra Zahra,† Naima Waseem,† Rubab Zahra,† Hwanhui Lee,‡ Mohsin Ali Badshah,§ Arshad Mehmood,∥ Hyung-Kyoon Choi,*,‡ and Muhammad Arshad*,† †

Institute of Environmental Sciences and Engineering, School of Civil and Environmental Engineering, National University of Sciences and Technology, Sector H-12, Islamabad 44000, Pakistan ‡ College of Pharmacy, Chung-Ang University, Seoul 06974, Republic of Korea § School of Mechanical Engineering, Chung-Ang University, Seoul 06974, Republic of Korea ∥ National Institute of Laser and Optronics, Nilore, Islamabad 45650, Pakistan S Supporting Information *

ABSTRACT: Plants have the natural ability to withstand stress conditions through metabolic adjustments. The present study aimed at investigating the effects of titanium dioxide nanoparticles (TiO2 NPs) application (0, 25, 50, 150, 250, 500, and 750 mg kg−1) in phosphorus-deficient soil in terms of growth responses, P contents, and metabolic alterations in rice. TiO2 NPs application increased shoot length up to 14.5%. Phosphorus contents in rice roots, shoots, and grains were increased by 2.6-, 2.4-, and 1.3-fold, respectively, at 750 mg kg−1 of TiO2 NPs. Gas chromatography-mass spectrometry (GC-MS)-based metabolomics revealed increased levels of amino acids, palmitic acid, and glycerol content in grains resulting from plants grown in 750 mg kg−1 TiO2 NPs-treated soil. Furthermore, no translocation of TiO2 NPs from the treated soil to rice grains was detected by inductively coupled plasma-optical emission spectrometry (ICP-OES), which suggests no risk of TiO2 NPs intake via grain consumption. The observed data indicates the strong relationship among NPs application, P contents, and metabolic alterations. KEYWORDS: titania, nanoparticles, phosphorus phytoavailability, metabolic profiling, rice

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INTRODUCTION Nutrient deficiencies in arid and semiarid regions remain a major obstacle for improving crop production and quality. Researchers are trying innovative ways to improve productivity in order to ensure food security for the ever-growing population of the world. Recently, addition of nanoparticles (NPs) has gained some attention for improving nutrient uptake by plants.1 Different effects of engineered NPs on various plant species have been reported in literature. For example, titanium dioxide nanoparticles (TiO2 NPs) increased spinach growth and biomass2,3 and improved photosynthesis in Arabidopsis thaliana,4 while they inhibited root growth in Vicia faba L.5 Ge et al. reported negative effects of metal oxide NPs on the bacterial communities in soil microcosms over 60 days following NPs treatment of soil.6 However, these effects may vary depending on soil properties and experimental conditions. Currently, there is a need to study the effects of engineered NPs and their potential role in nutrient management in agricultural soils. TiO2 is among the most extensively used engineered NPs. In recent years, global production of TiO2 was approximately 10 000 tons per year, with production levels increasing exponentially.7,8 Therefore, engineered NPs are the focus of modern research institutions and regulatory agencies in order to determine their potential effects in various applications.9−12 Recently, NPs have been considered as a factor for improving phytoavailable Pan essential macronutrient for plant growth and development. Approximately 30% of the world’s agricultural land is P-deficient. In soil, immobilization by Ca2+ © 2017 American Chemical Society

renders 88−99% of naturally occurring inorganic P unavailable to plants.13 A significant portion of the inorganic P originating from applied phosphate fertilizers is fixed in the soil forming insoluble complexes.14 Therefore, mobilization of naturally bound P is a significant challenge in the agricultural industry. As the Ti3+ possess more polarizing power (6.7 C/m2) as compared to Ca2+ (2.2 C/m2), so the application of TiO2 NPs in soil could provide more adsorption sites to the PO43− ions. The nano size of these NPs with high surface area could help in the direct increase in uptake of PO4 3− and enhance mobilization of P upon root exudation due to TiO2 NP application in the soil resulting into increased P uptake by plants.1 Hence, there is a need to study the effects of engineered NPs and their potential role in nutrient management in agricultural soils. Currently, there are few reports in the literature that describe P mobilization upon TiO2 NP application in the soil.1,15 Improved mobilization of P was also reported using ZnO NPs, resulting in higher P content in mung bean.15 Santner et al. reported increased P uptake in hydroponically grown plants using Al2O3 NP-bound P (10 mg L−1).16 The majority of these studies were short-term assays, and thus, information on the effects of NPs exposure on plant growth throughout its life cycle is still very limited. Received: Revised: Accepted: Published: 5598

April 21, 2017 June 21, 2017 June 26, 2017 June 26, 2017 DOI: 10.1021/acs.jafc.7b01843 J. Agric. Food Chem. 2017, 65, 5598−5606

Article

Journal of Agricultural and Food Chemistry

seeds were sterilized using 2.5% Ca(OCl)2 for 20 min to avoid fungal growth. Before cultivation in a prepared soil bed in the nursery, the seeds were washed thoroughly with distilled water and soaked for 6 h. Following 25 days of growth, seedlings were transplanted to the soil containing defined levels of TiO2 NPs (0, 25, 50, 150, 250, 500, and 750 mg kg−1). Plants were grown under ambient temperature and humidity throughout the experiment. Six replicates of each experimental treatment and the control were maintained. The plants were monitored until they reached maturity, after which they were harvested. During harvesting, all the plant parts were collected separately for detailed analysis. Assessment of Rice Physical Traits. Plant physical traits were assessed by measuring shoot−root length, shoot−root dry biomass, and total dry biomass of rice. Plants were harvested when they reached physical maturity. The harvested plants were shaken gently to remove loose soil followed by rinsing with distilled water several times. The shoot−root length of each plant was measured using a scale. For P content analysis, the shoots and roots were stored safely once they were oven-dried at 70 °C for 48 h. Grains from the control and TiO2 NP-treated plants were collected separately and stored at 4 °C for comprehensive global metabolic analysis. Analysis of P in Rice and Soil. Rhizosphere soil samples (200 g) from each treatment group were air-dried, ground using a mortar and pestle, sieved through a 2 mm mesh, and stored in plastic bags. Olsen’s P method was used to determine the phytoavailable P concentration in the soil.21 Analysis of the P content in shoots, roots, and grains of rice was performed following the wet digestion method.22 For this purpose, 5 mL of acid mixture (HNO3/HClO4 2:1) was added to 100 mg of powdered plant material. The mixture was digested on a hot plate until clear aliquots were obtained. Digested samples were then filtered through a Whatman #42 filter paper, and the vanadomolybdophosphoric acid colorimetric method was used to analyze P content.23 Comprehensive Global Metabolic Profiling of Rice. Representative grain samples of the control and TiO2 NPs-treated plants following harvest were used for global metabolic analysis. The samples were prepared according to a previously described method.24 In short, 30 mg of powdered grain (dehulled) sample was weighed in a 15 mL Eppendorf. For extraction, 3 mL of HPLC grade methanol/water mixture (4:1, v/v) was added to the samples. Capric acid (30 μL of 0.3 mg/mL) was added as an internal standard (IS) to each sample, and then samples were vortexed for 1 min. Following this, samples were incubated at 30 °C for 30 min, sonicated for 40 min, and centrifuged at 12 000 RCF for 10 min. The resulting supernatant was filtered using 0.45 μm PTFE Whatman syringe filters before drying using nitrogen gas. Methylation of extracts was performed using 90 μL of N,Obis(trimethylsilyl) trifluoroacetamide (BSTFA) with the addition of 1% of trimethylchlorosilane (TMCS) and 80 μL of pyridine, in which dried samples were resuspended by vortexing. Using a water bath, the extracts were incubated at 75 °C for 45 min and then transferred to GC vials containing inserts for further analysis. The extracted samples were analyzed using a GC (model 7890A, Agilent Technologies, Santa Clara, CA) equipped with a mass-selective detector (Agilent Technologies, model 5975C), an autosampler (Agilent Technologies, 7683B series,), an injection module, and the ChemStation software. The GC inlet temperature was set to 250 °C and 1.0 μL of sample was injected in duplicate. The carrier gas (Helium) flow rate was set at 1.0 mL/min. The initial oven temperature of 60 °C was held for 4 min before being ramped first to 170 °C at a rate of 8 °C/min, then to 259 °C at a rate of 4 °C/min, and finally to 300 °C at a rate of 8 °C/min. The total run time was 48.125 min. The data were obtained in full-scan mode. The full-scan data acquired from the GC system were processed with Genedata Expressionist MSX software, version 2013.0.39 (Basel, Switzerland). The spectral data obtained were subjected to nominal integration, chemical noise subtraction, m/z alignment, and baseline subtraction. For the comprehensive determination of metabolites, mass spectral data were processed using the GC-MSD software. The metabolites were identified using the National Institute of Standards and Technology (NIST) mass spectral search program. The standard for peak assignment was set to the following: (1) a match quality

According to various reports from the literature, altered nutrient contents of plants could also induce changes in metabolic profile. For example, application of mineral fertilization affected amino acid and protein contents in wheat.17 Similarly, P deficiency was responsible for different patterns of metabolites in soybean.18 However, none of these studies targeted application of NPs affecting the dynamics of P in NPs-treated cereal crop plants. Moreover, how the metabolic profile is influenced if P availability is improved by NPs soil application has not yet been explored. In the present study, rice was selected because it is an important cereal crop, and its consumption is considered to be one of the potential routes for dietary exposure to contaminants and NPs.19 Rice (Oryza sativa L.) is a staple food crop in many Asian countries, including Pakistan and Korea.20 Elevated levels of TiO2 NPs in soil might affect the nutritional quality and metabolic profiles of rice. A comprehensive metabolomics study of grains obtained from rice plants cultivated in soils with different TiO2 NPs concentrations is important for better understanding of plant physiological mechanisms and for a perspective on agricultural strategies for future cultivation. In this context, the first objective was to perform a physical trait assessment of rice in terms of plant growth and biomass. Second, P contents in shoots, roots, and grains in conjunction with the levels of phytoavailable P in the soil as altered by NPs application were analyzed. Finally, comprehensive global metabolic profile of grains from plants grown in TiO2 NPtreated soil was explored using gas chromatography-mass spectrometry (GC-MS).

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MATERIALS AND METHODS

Characteristics of TiO2 NPs. Commercially available TiO2 (GPR, BDH Chemicals Ltd., England) was purchased and further calcined at 500 °C for 6 h to obtain the pure nanosized crystal structure of TiO2. Scanning electron microscopy (SEM; JSM 6490 A, Jeol Ltd., Japan) and energy-dispersive X-ray spectroscopy (EDX; JED 2300, Jeol Ltd., Japan) analyses were performed to study the morphology and chemical composition of TiO2 NPs, respectively. The size, morphology, and purity of TiO2 NPs are shown in Supporting Information (Figure S1a−c). The average particle size of TiO2 NPs was 20 nm. The phase composition, crystal structure, and crystallite size for the TiO2 NPs were determined using an X-ray Diffractometer (Theta−Theta STOE, Germany). The X-ray was operated at 40 kV and 40 mA and step mode was used for an absolute scan, performed at 2θ angle over the range 20−80°. The XRD pattern was analyzed using the X’Pert High Score software package (PANalytical B.V. Almelo, The Netherlands). The crystallite size of TiO2 NPs was 43 nm calculated using the Scherer Formula. Spiking of Soil with TiO2 NPs and Rice Cultivation. Solutions containing TiO2 NPs in varying concentrations (0, 25, 50, 150, 250, 500, and 750 mg L−1) were prepared by suspending the solid material in distilled water using an ultrasonicator (JAC-1505, Jinwoo, Korea) for 60 min. The soil had clay loam texture, and its characteristics are shown in Table S1. The original concentration of P was 3.8 mg kg−1, which is considered deficient ( grains. In the case of rice grains, P contents at all TiO2 NP treatments (i.e., 25, 50, 150, 250, 500, and 750 mg kg−1) increased by 7.2%, 13.6%, 20.2%, 18.3%, 19.9%, and 29.2%, respectively. Whereas an increase of 17.2%, 50.4%, 78.8%, 88.4%, 104.9%, and 139.3% was observed in shoots and 5.3%, 9.9%, 55.6%, 76.5%, 138.9%, 166.8% in roots, respectively. In a recent study, increased P (34.0%) contents were reported in cucumber fruit resulting from plants exposed to 500 mg kg−1 TiO2 NPs.3 Improved P content in Lactuca sativa was observed in response to TiO2 NP treatments.1,15,27 In mung bean plants, P uptake was increased up to 10.8% following 10 mg L−1 of ZnO NPs application in the soil.15 Although an increase in P contents for different plant species has been reported in literature, there is no information on how the metabolic profile is affected. Soil application of NPs without an additional dose of P as fertilizer could potentially lead to metabolic activation of plant species, rendering improved uptake of P through rhizosphere exudation and modification. Effect of TiO2 NP Application on Phytoavailable P in Soil. Figure 2 illustrates the phytoavailable P in soil in response to TiO2 NP application after rice culture. Crop plants cultivated in P-deficient soil suffer from P deficiency, which ultimately affects crop yield. The mobility of naturally bound P is a key solution to improve crop productivity for sustainable agriculture. Recently, researchers have reported improved mobilization of naturally bound P using NPs, which can

preserve natural P resources for sustainable agriculture. In this study, an increase of 5.8% in soil phytoavailable P was observed following soil treatment with 750 mg kg−1 TiO2 NPs relative to that of the control. These results are in coherence with the improved P contents in rice grains, shoots, and roots, which consequently supported plant growth and biomass improvement. Improved phytoavailable P in soil increased the overall plant growth and development following NPs treatment.1,27 NPs application can result in a stressed environment, and forcing the plants to activate their metabolism in order to release primary metabolites in the rhizosphere1 can facilitate the conversion of bound P to mobile inorganic P, which improves P absorption by plant roots. The nano size of TiO2 can also promote the diffusion of inorganic P within the root system and its movement into upper parts of the plant, resulting in improved plant growth and development. Another reason could be that the TiO2 NPs could enter the chloroplasts, increasing oxygen evolution rate and consequently the photosynthetic activity of plants.28 This potential mechanism can trigger metabolic adjustments and variations in global metabolic profile. Comprehensive Metabolic Profiling of Rice Grains in Response to TiO2 NPs. Table 2 provides the details of 38 determined metabolites of rice grains in response to TiO2 NPs application. Metabolites were classified according to their respective category such as amino acids, sugars, alcohols, organic acids, and other compounds. In general, the fatty acid component of rice is mainly constituted of linoleic acid (50%), oleic acid (25−30%), and palmitic acid (16%), with lesser amounts of stearic acid,29,30 which varies according to the rice variety.31,32 Metabolic profile of rice grains (Table 2) from plants treated with 750 mg kg−1 TiO2 NPs indicated a significant increase (P < 0.05) in the levels of specific amino acids (proline, aspartic acid, and glutamic acid), whereas a decrease was observed in levels of certain fatty acids (linoleic and oleic acid) compared to the levels in control grains. Rico et al. reported a 36.6% decrease in total fatty acid content in rice following CeO2 NP treatments compared to the controls.33 In the present study, each amino acid level increased in response to NP treatments. Similarly, proline, glycine, and asparagine were also reported to increase in cucumber following nano copper application.34 The highest glycerol content was observed in grains treated with 750 mg kg−1 TiO2 NPs, which was 2.4-fold higher than that of control grains. Among the amino acids, a slight increase was observed in alanine, valine, asparagine, and tyrosine content in grains from plants subjected to both 500 and 750 mg kg−1 TiO2 NPs treatments compared to the control. Interestingly, the metabolic modification of P species was significantly increased in conjunction with the levels of amino acids (29% increase in glutamic acid, 33.6% increase in glycine, 39.7% increase in lysine, 56.8% increase in aspartic acid, 2.7fold increase in isoleucine and 2.8-fold increase in proline) and fatty acids (22.4% increase in palmitic acid) following 750 mg kg−1 TiO2 NP treatment compared to those of control grains (Table 2). Proline is considered as a multifunctional amino acid in plants that plays various roles in stress tolerance.34 Furthermore, it was determined that the accumulation of proline increases in large quantities during environmental stress.35,36 The degradation of glucose-6-phosphate or 2phosphoglyceric acid has been reported under stress. Bekele et al. suggested that this degradation might increase the phosphoric acid content.37 Concerning the organic acids,

Figure 2. Effect of TiO2 NPs treatment (0−750 mg kg−1) on the phytoavailable P content of the soil after rice cultivation. Dot represents the mean value and midline represents the median value of 6 replicates. 5601

DOI: 10.1021/acs.jafc.7b01843 J. Agric. Food Chem. 2017, 65, 5598−5606

Article

Journal of Agricultural and Food Chemistry Table 2. Comprehensive Metabolic Profile of Grains Obtained from Rice Cultivated in Soils Treated with Different Concentrations of TiO2 NPsa TiO2 NP treatments compound Amino Acids alanine valine isoleucine proline glycine aspartic acid lysine asparagine tyrosine glutamic acid glycerol-3-phosphate Sugars arabinose mannose sucrose glyceric acid beta-D-glucopyranose glycerol Fatty Acids palmitic acid linoleic acid stearic acid oleic acid myristic acid alcohols myo-inositol ribitol Organic Acids oxalic acid succinic acid adipic acid fumaric acid malic acid lactic acid pyruvic acid cinnamic acid isocitric acid nonanoic acid glycolic acid isobutanoic acid phosphoric acid Other Compounds oleamide

RT (min)

500 mg kg−1

control ± ± ± ± ± ± ± ± ± ± ±

10.21 12.49 13.92 14.00 14.15 17.69 24.97 20.15 25.31 19.30 21.79

88.43 21.20 4.28 12.64 48.76 73.29 3.70 69.83 12.34 215.10 60.31

19.68 26.16 39.22 14.57 24.33 13.58

1.93 ± 0.51a 50.97 ± 13.33b 7437.50 ± 134.69b 2.13 ± 0.64a 37.63 ± 11.00b 116.45 ± 6.13b

27.63 31.02 31.71 31.16 34.75

315.12 170.05 28.04 86.45 24.44

± ± ± ± ±

10.69a 2.72a 1.49b 2.25b 7.46b 12.69b 0.30b 13.83a 0.47a 1.21b 10.28b

8.97c 11.53a 0.68b 2.72a 1.70a

95.05 23.25 13.05 15.07 61.66 78.13 3.43 77.62 12.53 230.35 73.53

± ± ± ± ± ± ± ± ± ± ±

7.17a 3.27a 1.19a 1.38b 4.67ab 0.68b 0.28b 19.94a 0.54a 18.01b 9.71b

1.74 ± 0.18a 106.35 ± 4.49a 10112.45 ± 210.00a 2.24 ± 0.39a 72.52 ± 4.15a 124.77 ± 1.05b 350.21 186.94 40.16 95.58 26.69

± ± ± ± ±

17.08b 17.90a 0.97a 10.97a 2.80a

750 mg kg−1 124.45 29.93 11.63 35.33 65.12 114.91 5.17 94.14 14.92 278.31 102.29

± ± ± ± ± ± ± ± ± ± ±

22.77a 4.84a 1.90a 4.57a 5.03a 18.86a 0.93a 19.17a 3.64a 6.40a 11.75a

1.35 ± 0.45a 33.79 ± 4.91b 7105.04 ± 198.46b 2.85 ± 0.34a 22.51 ± 3.06b 283.48 ± 6.19a 385.57 135.43 27.63 44.24 22.81

± ± ± ± ±

12.57a 11.64b 1.27b 8.20b 1.20a

28.34 20.97 25.17

47.19 ± 1.76a 5.79 ± 1.20b

48.63 ± 3.93a 7.18 ± 0.92b

48.58 ± 2.24a 12.57 ± 2.41a

10.84 12.17 7.57 14.34 17.50 14.97 17.19 9.27 10.83 25.32 22.90 15.16 12.31 6.58 11.73

27.46 ± 2.65b

44.42 ± 2.43a

26.04 ± 1.39b

23.41 ± 4.38b

34.21 ± 3.24a

17.75 ± 2.61b

66.44 ± 4.12b 1.57 ± 0.52a 54.61 ± 6.32a 32.00 ± 1.49b 13.82 ± 1.11b 2.60 ± 0.07a 66.68 ± 0.84b 2.68 ± 0.25a 77.96 ± 4.37b 384.32 ± 19.65b 64.56 ± 4.38b

112.58 ± 10.05a 2.00 ± 0.16a 55.15 ± 5.14a 47.05 ± 3.26 a 20.17 ± 1.38a 2.89 ± 0.34a 84.55 ± 6.88ab 2.66 ± 0.09a 117.48 ± 7.27a 593.89 ± 39.12a 75.22 ± 2.02b

62.47 ± 3.41b 0.34 ± 0.05b 22.81 ± 5.43b 16.73 ± 2.02 c 13.14 ± 0.76 b 3.96 ± 1.00 a 96.10 ± 19.18a 2.13 ± 0.34a 68.22 ± 5.86b 377.37 ± 12.25b 96.70 ± 7.52a

35.08

5.67 ± 0.53a

7.13 ± 1.11a

7.65 ± 0.82a

a

Values in the table represent the relative intensities of each compound obtained by dividing the base peaks by IS value and multiplying it by 100. Means ± SD (N = 6) are shown for each treatment. Means within a row that share the same letter are not significantly different. Different letters indicate significant difference (P < 0.05). RT: retention time.

phosphate) along with the enhancement of nitrogen sources such as glutamic and aspartic acid. Coherent observations have been reported that P deficiency or enhancement could alter the concentration of various metabolites or seed components.38 Sugar compounds such as mannose, sucrose, and beta-Dglucopyranose were increased up to 108.7%, 36%, and 92.7%, respectively, following 500 mg kg−1 TiO2 NP treatment (Table

phosphoric acid was significantly increased by 49.8% following 750 mg kg−1 TiO2 NP treatment compared to that of control grains. This increase mirrored the improved P content of shoots, roots, and grains of TiO2 NP-treated plants relative to that of control plants. Therefore, the nutritional quality of rice grains in terms of P content was improved by the accumulation of P related metabolites (i.e., phosphoric acid and glycerol-35602

DOI: 10.1021/acs.jafc.7b01843 J. Agric. Food Chem. 2017, 65, 5598−5606

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Journal of Agricultural and Food Chemistry

importance of a variable to the entire model.42 A variable with VIP value >1 is considered important.43 In our study, seven compounds were determined as discriminating metabolites (P < 0.01) with VIP > 1 between TiO2 NPs treated groups and control (Figure S2 showed the VIP score of metabolites). These significantly modified metabolites included sucrose, glycerol, palmitic acid, glutamic acid, isobutanoic acid, glycerol-3-phosphate, and oleic acid. ANOVA showed that 23 metabolites were significantly (P < 0.05) altered by the TiO2 NPs relative to the control (Table 2). These results are in conformity with the VIP score. Twelve metabolites were significantly (P < 0.05) increased following 500 mg kg−1 TiO2 NP treatment compared to those in control and 750 mg kg−1 TiO2 NPs-treated grains. These significant differences following 500 mg kg−1 TiO2 NP treatment are likely the result of optimal or considerable amounts of TiO2 NPs applied to the soil. Although the higher concentration of 750 mg kg−1 TiO2 NPs promoted phytoavailable P content in the plant, the increased probability of TiO2 NPs agglomeration may not promote a significant improvement in most of the metabolite contents. Translocation of NPs in Rice Grains. FTIR spectra of both TiO2 NPs-treated and control groups of harvested rice grains are shown in Figure 4a. The P species identified in different wavenumber regions were assigned as P 2 O 7 4− (pyrophosphate) at 760 and 1180 cm−1, H2PO4− (primary orthophosphate) at 863 cm −1 , and PO 4 3− (inorganic orthophosphate) at 1012 cm−1.1,3 The P species in the tested groups (control and treated with 750 mg kg−1 TiO2 NPs) displayed clear variations in the spectra resulting from NPtreated and control rice samples. However, the PO43− species at 1012 cm−1 wavenumber was more abundant in TiO2 NPstreated samples compared to that of the control. These results are in agreement with those observed in previous reports.1,33,44 The metabolic variations in rice grains correspond to the increased variation in P species (as shown in Figure 4a), P contents, and plant physiological changes following TiO2 NPs treatment. Figure 4b displays Fe-SEM images representing the morphology of rice grains harvested from control (A) and TiO2 NPs-treated plants (B). The morphology of TiO2 NPs-treated rice grains differed compared to that of the control, in that fewer micro cavities were observed on the grain surface. EDX spectra of grain samples also indicated improvement in P for TiO2 NPs-treated grains compared to that of the control, which agreed with the results presented above. Ti was not detected by EDX in grains of the NP treated group. ICP-OES data did not show Ti presence in grain samples for both control and TiO2 NPs-treated groups (Table S2). Keeping in mind the above-mentioned results of microscopic and spectrometric techniques, it can be suggested that TiO2 NPs were not translocated to the rice grains (i.e., the edible part of the rice plant), confirming that, in this case, it is unlikely that TiO2 NPs might be introduced into the food chain via this dietary route. In a recent study on wheat plants, Fe3O4 NPs were also not translocated to the aerial parts of the plants, suggesting their potential use in agriculture sector.44 The absence of Ti in the grains indicated no threat to food chain contamination. On the basis of the previous reports1,15 and these findings, soil application of TiO2 NPs can be encouraged in order to mobilize fixed native P that has been an impediment in improving crop productivity.45 Changes in metabolic profile along with improved sequestration of phosphorus in P-deficient soil are an indicative of the fact that rice plants responded to the stress and improved P uptake through their metabolic

2), which consequently improved the carbon content of the grains. Furthermore, the levels of organic acids such as oxalic, succinic, adipic, pyruvic, and glycolic acid varied in both 500 mg kg−1 and 750 mg kg−1 TiO2 NP treatments as compared to those in control grains. A significant increase was observed in organic acid contents at 500 mg kg−1 TiO2 NP treatment, whereas overall organic acids were decreased in 750 mg kg−1 TiO2 NP treatment. In the rice plant’s life cycle, the grain defines a stage that is relatively inactive while preserving nutrients. Variations in the chemical constituents of grains are important in terms of quality control for the consumers and food/feed industries.39 For metabolic variations in grains as a target objective to improve nutrient contents following TiO2 NP application, it is important to consider which metabolite contents should be increased or decreased and to what extent they should be altered. However, these considerations are complex and ambiguous, and are affected by additional factors such as plant growth, grain yield, grain shelf life, and consumer habits (e.g., taste preferences, brown vs milled rice, etc.). In addition to the type of rice cultivar and the properties of the NPs used, different environmental factors including soil type, available plant nutrients, and climatic conditions also affect the final metabolic profile of rice grains. To visualize the differences between metabolic profiles of rice grains from plants grown under control conditions or treated with TiO2 NPs, the relatively quantified metabolites were subjected to PLS-DA analysis using MetaboAnalyst 3.0 software (http://www.metaboanalyst.ca).40,41 Data originating from the control and TiO2 NPs-treated groups were significantly different as shown in Figure 3. The combination of PLS

Figure 3. PLS-DA score plot of comprehensive metabolic profiles, composed of 38 metabolites, derived from GC-MS data of rice grains from plants grown under control conditions or treated with 500 and 750 mg kg−1 TiO2 NPs (N = 6).

components 1 and 2 accounted for 88.0% of the total variance. Metabolic changes in rice grains harvested from TiO2 NPtreated plants were clearly distinct from those in the control plants for PLS component 1, which showed 56.6% of the total variance. These findings suggest that TiO2 NPs induced strong metabolic variation in NPs-treated rice grains. Furthermore, parameters of variable importance in projection (VIP) were also assessed to determine the metabolites responsible for this variance or clear separation. VIP means the weighted sum of squares of the PLS-DA analysis, which represents the 5603

DOI: 10.1021/acs.jafc.7b01843 J. Agric. Food Chem. 2017, 65, 5598−5606

Article

Journal of Agricultural and Food Chemistry

Figure 4. (a) FTIR spectra of rice grain harvested from plants cultivated in soil treated with 0, 250, 500, and 750 mg kg−1 of TiO2 NPs. (b) Fe-SEM image (left three panels) and (c) EDX spectra (right panel) of rice grains harvested from (A) control and (B) plants treated with 750 mg kg−1 TiO2 NPs.

to 750 mg kg−1 TiO2 NPs treatment. The translocation of NPs from the soil to the rice grain was not observed, which suggests no or minimal risk of Ti entering into food chain through dietary consumption of TiO2 NPs-treated rice.

adjustments. However, thorough investigation for possible impacts in consumers of these grains with altered metabolic profile is desired. In summary, this is the first report to assess P contents over the life cycle of rice plants and global metabolic profiles of rice grains in response to soil application of TiO2 NPs. In paddy soil, TiO2 NPs application has significantly increased the growth and P contents in rice plant parts, i.e., roots, shoots, and grains. GC-MS-based metabolomics study revealed that TiO2 NPs treatment to rice plants until they reach maturity can significantly affect the metabolic profile of harvested grains. Substantial variation in various metabolites exhibited similar trends as P contents in TiO2 NPs-treated groups, regulating the metabolic pathways with multiple contributions. Several metabolites were significantly increased (proline, aspartic acid, glutamic acid, palmitic acid, glycerol, inositol, ribitol, phosphoric acid, and glycerol-3-phosphate), whereas overall organic acid, fatty acid, and sugar contents were decreased in response

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b01843. Table S1,S2 show the characteristics of soil used in this experiment and the ICP-OES results for the presence of titanium (Ti) in rice grains harvested from plants grown in soil amended with TiO2 NPs, respectively; Figure S1a−c present the SEM image, EDX spectra and XRD spectra of TiO2 NPs, respectively; and Figure S2 represents the VIP scores from PLS-DA analysis illustrating the discriminating metabolites made the group separation (PDF) 5604

DOI: 10.1021/acs.jafc.7b01843 J. Agric. Food Chem. 2017, 65, 5598−5606

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Journal of Agricultural and Food Chemistry

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in assessing environmental exposure, risk, and ecological implications of nanomaterials. Environ. Sci. Technol. 2009, 43, 6458−6462. (13) Vance, C. P.; Uhde-Stone, C.; Allan, D. L. Phosphorus acquisition and use: Critical adaptations by plants for securing a nonrenewable resource. New Phytol. 2003, 157, 423−447. (14) Vassilev, N.; Vassileva, M. Biotechnological solubilization of rock phosphate on media containing agro-industrial wastes. Appl. Microbiol. Biotechnol. 2003, 61, 435−440. (15) Raliya, R.; Tarafdar, J. C.; Biswas, P. Enhancing the mobilization of native phosphorus in the mung bean rhizosphere using ZnO nanoparticles synthesized by soil fungi. J. Agric. Food Chem. 2016, 64, 3111−3118. (16) Santner, J.; Smolders, E.; Wenzel, W. W.; Degryse, F. First observation of diffusion-limited plant root phosphorus uptake from nutrient solution. Plant Cell Environ. 2012, 35, 1558−1566. (17) Crista, F.; Radulov, I.; Crista, L.; Berbecea, A.; Lato, A. Influence of mineral fertilization on the amino acid content and raw protein of wheat grain. J. Food, Agric. Environ. 2012, 10, 47−50. (18) Tawaraya, K.; Horie, R.; Shinano, T.; Wagatsuma, T.; Saito, K.; Oikawa, A. Metabolite profiling of soybean root exudates under phosphorus deficiency. Soil Sci. Plant Nutr. 2014, 60, 679−694. (19) Seyfferth, A. L.; McCurdy, S.; Schaefer, M. V.; Fendorf, S. Arsenic concentrations in paddy soil and rice and health implications for major rice-growing regions of Cambodia. Environ. Sci. Technol. 2014, 48, 4699−4706. (20) Song, E. H.; Kim, H. J.; Jeong, J.; Chung, H. J.; Kim, H. Y.; Bang, E.; Hong, Y. S. A 1H HR-MAS NMR-based metabolomic study for metabolic characterization of rice grain from various Oryza sativa L. cultivars. J. Agric. Food Chem. 2016, 64, 3009−3016. (21) Olsen, S. R.; Cole, C. V.; Watanabe, F. S.; Dean, L. A. Estimation of available phosphorus in soil by extraction with sodium bicarbonate; United States Department of Agriculture: Washington, D.C., 1954; Vol. 939, pp 18−19. (22) Rashid, A. Mapping zinc fertility of soils using indicator plants and soils analyses. Ph.D. Dissertation, University of Hawaii, 1986. (23) Ryan, J.; Estefan, G.; Rashid, A. Soil and Plant Analysis: Laboratory Manual, 2nd ed.; International Center for Agricultural Research in the Dry Areas (ICARDA): Aleppo, Syria, 2001. (24) Zhou, J.; Ma, C.; Xu, H.; Yuan, K.; Lu, X.; Zhu, Z.; Wu, Y.; Xu, G. Metabolic profiling of transgenic rice with cryIAc and sck genes: An evaluation of unintended effects at metabolic level by using GC-FID and GC−MS. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2009, 877, 725−732. (25) Arshad, M.; Merlina, G.; Uzu, G.; Sobanska, S.; Sarret, G.; Dumat, C.; Silvestre, J.; Pinelli, E.; Kallerhoff, J. Phytoavailability of lead altered by two Pelargonium cultivars grown on contrasting leadspiked soils. J. Soils Sediments 2016, 16, 581−591. (26) Song, G.; Gao, Y.; Wu, H.; Hou, W.; Zhang, C.; Ma, H. Physiological effect of anatase TiO2 nanoparticles on Lemna minor. Environ. Toxicol. Chem. 2012, 31, 2147−2152. (27) Hanif, H. U.; Arshad, M.; Ali, M. A.; Ahmed, N.; Qazi, I. A. Phyto-availability of phosphorus to Lactuca sativa in response to soil applied TiO2 nanoparticles. Pak. J. Agric. Sci. 2015, 52, 177−182. (28) Hong, F.; Zhou, J.; Liu, C.; Yang, F.; Wu, C.; Zheng, L.; Yang, P. Effect of nano-TiO2 on photochemical reaction of chloroplasts of spinach. Biol. Trace Elem. Res. 2005, 105, 269−279. (29) Choudhury, N. H.; Juliano, B. O. Lipids in developing and mature rice grain. Phytochemistry 1980, 19, 1063−1069. (30) Kang, M. Y.; Rico, C. W.; Lee, S. C. Comparative analysis of the physicochemical properties of rice endosperm from cultivars with diverse amylose content. J. Crop Sci. Biotechnol. 2011, 14, 219−225. (31) Kim, J. B.; Ma, K. H.; Kwon, T. R.; Kang, H. K.; Lee, G. A.; Gwag, J. G.; Kim, T. S.; Park, Y. J. Amylose, tocopherol, free sugar and fatty acid content in selected mutant lines of Oryza sativa cv. Shindongjin. J. Crop Sci. Biotechnol. 2008, 11, 181−186. (32) Zhou, Z.; Blanchard, C.; Helliwell, S.; Robards, K. Fatty acid composition of three rice varieties following storage. J. Cereal Sci. 2003, 37, 327−335.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]; Tel.: +92-51-9085-4309. *E-mail: [email protected]; Tel.: +82 2 820 5605. ORCID

Muhammad Arshad: 0000-0002-2343-822X Funding

This work was supported by Higher Education Commission of Pakistan under Project No. 20-3060/NRPU/R&D/HEC/13 and by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (NRF2015RIA5A1008958). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED TiO2 NPs, titania nanoparticles; P, phosphorus; GC-MS, gas chromatography-mass spectrometry; ICP-OES, inductively coupled plasma optical emission spectrometry; FE-SEM, field emission scanning electron microscopy; EDX, energy-dispersive X-ray spectroscopy; FTIR, Fourier transform infrared spectroscopy

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REFERENCES

(1) Zahra, Z.; Arshad, M.; Rafique, R.; Mahmood, A.; Habib, A.; Qazi, I. A.; Khan, S. A. Metallic nanoparticle (TiO2 and Fe3O4) application modifies rhizosphere phosphorus availability and uptake by Lactuca sativa. J. Agric. Food Chem. 2015, 63, 6876−6882. (2) Yang, F.; Liu, C.; Gao, F.; Su, M.; Wu, X.; Zheng, L.; Hong, F.; Yang, P. The improvement of spinach growth by nano-anatase TiO2 treatment is related to nitrogen photoreduction. Biol. Trace Elem. Res. 2007, 119, 77−88. (3) Zheng, L.; Hong, F.; Lu, S.; Liu, C. Effect of nano-TiO2 on strength of naturally aged seeds and growth of spinach. Biol. Trace Elem. Res. 2005, 104, 83−91. (4) Ze, Y.; Liu, C.; Wang, L.; Hong, M.; Hong, F. The regulation of TiO2 nanoparticles on the expression of light-harvesting complex II and photosynthesis of chloroplasts of Arabidopsis thaliana. Biol. Trace Elem. Res. 2011, 143, 1131−1141. (5) Castiglione, M. R.; Giorgetti, L.; Bellani, L.; Muccifora, S.; Bottega, S.; Spanò, C. Root responses to different types of TiO2 nanoparticles and bulk counterpart in plant model system Vicia faba L. Environ. Exp. Bot. 2016, 130, 11−21. (6) Ge, Y.; Schimel, J. P.; Holden, P. A. Evidence for negative effects of TiO2 and ZnO nanoparticles on soil bacterial communities. Environ. Sci. Technol. 2011, 45, 1659−1664. (7) Robichaud, C. O.; Uyar, A. E.; Darby, M. R.; Zucker, L. G.; Wiesner, M. R. Estimates of upper bounds and trends in nano-TiO2 production as a basis for exposure assessment. Environ. Sci. Technol. 2009, 43, 4227−4233. (8) Hendren, C. O.; Mesnard, X.; Dröge, J.; Wiesner, M. R. Estimating production data for five engineered nanomaterials as a basis for exposure assessment. Environ. Sci. Technol. 2011, 45, 2562−2569. (9) Guzman, K. A. D.; Taylor, M. R.; Banfield, J. F. Environmental risks of nanotechnology: National nanotechnology initiative funding, 2000−2004. Environ. Sci. Technol. 2006, 40, 1401−1407. (10) Klaine, S. J.; Alvarez, P. J.; Batley, G. E.; Fernandes, T. F.; Handy, R. D.; Lyon, D. Y.; Mahendra, S.; McLaughlin, M. J.; Lead, J. R. Nanomaterials in the environment: behavior, fate, bioavailability, and effects. Environ. Toxicol. Chem. 2008, 27, 1825−1851. (11) Wiesner, M. R.; Lowry, G. V.; Alvarez, P.; Dionysiou, D.; Biswas, P. Assessing the risks of manufactured nanomaterials. Environ. Sci. Technol. 2006, 40, 4336−4345. (12) Wiesner, M. R.; Lowry, G. V.; Jones, K. L.; Hochella, M. F., Jr.; Di Giulio, R. T.; Casman, E.; Bernhardt, E. S. Decreasing uncertainties 5605

DOI: 10.1021/acs.jafc.7b01843 J. Agric. Food Chem. 2017, 65, 5598−5606

Article

Journal of Agricultural and Food Chemistry (33) Rico, C. M.; Morales, M. I.; Barrios, A. C.; McCreary, R.; Hong, J.; Lee, W. Y.; Nunez, J.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L. Effect of cerium oxide nanoparticles on the quality of rice (Oryza sativa L.) grains. J. Agric. Food Chem. 2013, 61, 11278−11285. (34) Zhao, L.; Huang, Y.; Zhou, H.; Adeleye, A. S.; Wang, H.; Ortiz, C.; Mazer, S. J.; Keller, A. A. GC-TOF-MS based metabolomics and ICP-MS based metallomics of cucumber (Cucumis sativus) fruits reveal alteration of metabolites profile and biological pathway disruption induced by nano copper. Environ. Sci.: Nano 2016, 3, 1114−1123. (35) Ashraf, M.; Foolad, M. Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ. Exp. Bot. 2007, 59, 206−216. (36) Hsu, S.; Hsu, Y.; Kao, C. The effect of polyethylene glycol on proline accumulation in rice leaves. Biol. Plant. 2003, 46, 73−78. (37) Bekele, E. A.; Annaratone, C. E. P.; Hertog, M. L. A. T. M.; Nicolai, B. M.; Geeraerd, A. H. Multi-response optimization of the extraction and derivatization protocol of selected polar metabolites from apple fruit tissue for GC-MS analysis. Anal. Chim. Acta 2014, 824, 42−56. (38) Singh, S. K.; Barnaby, J. Y.; Reddy, V. R.; Sicher, R. C. Varying response of the concentration and yield of soybean seed mineral elements, carbohydrates, organic acids, amino acids, protein, and oil to phosphorus starvation and CO2 enrichment. Front. Plant Sci. 2016, 7, 1−13. (39) Lou, Q.; Ma, C.; Wen, W.; Zhou, J.; Chen, L.; Feng, F.; Xu, X.; Lu, X.; Luo, L.; Mei, H.; Xu, G. Profiling and association mapping of grain metabolites in a subset of the core collection of Chinese rice germplasm (Oryza sativa L.). J. Agric. Food Chem. 2011, 59, 9257− 9264. (40) Xia, J.; Sinelnikov, I. V.; Han, B.; Wishart, D. S. MetaboAnalyst 3.0making metabolomics more meaningful. Nucleic Acids Res. 2015, 43, W251−W257. (41) Xia, J.; Mandal, R.; Sinelnikov, I. V.; Broadhurst, D.; Wishart, D. S. MetaboAnalyst 2.0a comprehensive server for metabolomic data analysis. Nucleic Acids Res. 2012, 40, W127−W133. (42) Garcia-Sevillano, M.; Garcia-Barrera, T.; Navarro, F.; Abril, N.; Pueyo, C.; Lopez-Barea, J.; Gomez-Ariza, J. Use of metallomics and metabolomics to assess metal pollution in Doñana National Park (SW Spain). Environ. Sci. Technol. 2014, 48, 7747−7755. (43) Xia, J.; Wishart, D. S. MSEA: a web-based tool to identify biologically meaningful patterns in quantitative metabolomic data. Nucleic Acids Res. 2010, 38, W71−W77. (44) Iannone, M. F.; Groppa, M. D.; de Sousa, M. E.; van Raap, M. B. F.; Benavides, M. P. Impact of magnetite iron oxide nanoparticles on wheat (Triticum aestivum L.) development: Evaluation of oxidative damage. Environ. Exp. Bot. 2016, 131, 77−88. (45) Hinsinger, P. Bioavailability of soil inorganic P in the rhizosphere as affected by root-induced chemical changes: a review. Plant Soil 2001, 237, 173−195.

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DOI: 10.1021/acs.jafc.7b01843 J. Agric. Food Chem. 2017, 65, 5598−5606