Article pubs.acs.org/est
Internalization and Phytotoxic Effects of CuO Nanoparticles in Arabidopsis thaliana as Revealed by Fatty Acid Profiles Jin Yuan, Anfei He, Shidi Huang, Jing Hua, and G. Daniel Sheng* State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China S Supporting Information *
ABSTRACT: Internalization and phytotoxic effects of CuO nanoparticles (nCuO) in plants were studied at the cellular level. Arabidopsis thaliana was hydroponically challenged by nCuO (100 mg/L), as compared to Cu2+ ions (1.2 mg/L), to account for nCuO dissolution for 96 h and 28 days to monitor Cu accumulation in the plant as well as the fatty acid (FA) profiles of the plant cell membrane. Under the same growing conditions, the nCuO exposure resulted in more Cu accumulation than did the Cu2+ exposure. Multiple microscopic techniques confirmed the internalization and sequestration of nCuO in root cell vacuoles, where transformation of Cu(II) to Cu(I)Cl occurred. Short and long exposures (96 h versus 28 days) to both nCuO and Cu2+ elevated FA saturation degrees in plant cells through oxidative stress, as verified by in situ detection of superoxide radicals, with conversions mostly from C18:3, C16:3, and C18:2 to C16:0. Only the long exposure to nCuO significantly brought about an additional elevation of FA saturation degree in root cells. These results demonstrated that the acute effects of plant exposure to nCuO were mainly produced from the stress of Cu2+ ions released from nCuO dissolution, while the chronic effects in roots were significantly developed by the nCuO particle stress. The findings in this work are novel and may offer significant implications in better understanding nanoparticle-induced phytotoxicity and potential risks in ecosystems.
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induced DNA lesions,11 and macromolecular damages.17 Because copper may become a toxicant to nontarget organisms at elevated levels, knowledge of the interactions between nCuO and plants is warranted for both the environmental risk assessment and the safety of nanotechnology in agroecosystems. The current paradigm to account for the phytotoxic effects has mostly been attributed to the oxidative stress from nCuOinduced generation of reactive oxygen species (ROS).10,11,13,17,22,23 However, as a redox-active transition metal, Cu can generate ROS via the Fenton or Haber−Weiss reactions, both in vitro and in vivo.24 Understanding the mode of action with nCuO is thus complicated by the extra- or intracellular Cu2+ released by nCuO dissolution, and no consensus has yet been reached. The phytotoxicities of nCuO have been ascribed to the nanoparticle-specific,10,12,15,16,18 Cu2+-specific,13,19,20,25 or combined nanoparticle−Cu2+ effects.11,14 These discrepancies arise from not only the extremely intricate interactions between nanoparticles and organisms but also the experimental artifacts. Examples of the latter are the use of nCuO samples with heterologous properties, the
INTRODUCTION Among myriad practices of nanotechnology, the use of nanopesticides and nanobiocides for plant protection results intentionally in the release of nanoparticles into terrestrial and aquatic ecosystems.1−4 Owing to their potent biocidal activity toward a wide spectrum of microorganisms,5−7 nanosized copper oxides (nCuO) have been integrated into, with growing production,8 products of nanobiocides available in the markets.9 The nCuO−plant interactions result in the nanoparticle internalization and transformation in plants. For example, the presences of nCuO were reported in the root cells of cucumbers (Cucumis sativus),10 radishes (Raphanus sativus),11 and bulrushes (Schoenoplectus tabernaemontani).12 nCuO were suggested to cross cell walls and cell membranes to distribute themselves around nucleus and organelles and to form agglomerates, either with themselves or with other cellular materials within the root cells. The transformations of nCuO were reported to form Cu(I)−sulfur complexes in the shoots of wheat (Triticum aestivum),13,14 Cu2S and Cu2O in the roots of maize (Zea mays L.)15 and Cu−alginate, Cu−cysteine, and Cu−oxalate in the whole aromatic madder (Elsholtzia splendens).16 As a consequence, the multifarious detrimental effects of nCuO on plants have been documented, with such symptoms as growth inhibitions,13−20 photosynthesis interferences,20 decreased chlorophyll content,13,16,18,19 metabolic disturbances,21 modified antioxidant enzyme activity,10,13,17 © XXXX American Chemical Society
Received: May 25, 2016 Revised: July 14, 2016 Accepted: September 2, 2016
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DOI: 10.1021/acs.est.6b02613 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
Table S1). Details of characterization and all the data can be found in Table S2 and Figures S1−S3. In brief, the negatively charged (∼-13 mV) nCuO (100 mg/L) readily aggregated to form larger particles and quickly sedimented in 20% Hoagland solution, probably due to the ionic strength (5.5 mM). The concentration of released Cu2+ at nCuO dissolution equilibrium was about 1.05 mg/L (Figure S3). Hydroponic Plant Exposure. A hydroponic system, modified from Tocquin et al.46 and Norén et al.,47 was adopted for the A. thaliana culture and exposure (Figure S4). A. thaliana (ecotype Columbia [Col]) were germinated and grown in a culture room (22 ± 2 °C) under fluorescent lighting (approximately 200 μmol/m2/s photons flux) with a 14 h light−10 h dark cycle. Detailed culture conditions were described in the Supporting Information. The overall germination rate exceeded 85%. A. thaliana (4 weeks old) were exposed in triplicates to the fresh 20% Hoagland solution containing nominal 100 mg/L nCuO (nCuO treatment) or nominal 1.2 mg/L Cu 2+ (Cu(NO3)2·3H2O) (Cu2+ treatment). Choice of this Cu2+ concentration was based on the measured Cu2+ concentration from nCuO dissolution (Figure S3). Addition of NO3− resulted only in a 1.3% difference in NO3− concentration between the nCuO and Cu2+ treatments. About 70 plantlets were used in each replicate (container). Another replicate of plants growing with no added copper was used as the control. During exposure, the hydroponic media were supplemented as necessary but not renewed for consistency consideration. Their pHs (initial value of 6.3; Table S2) were monitored and allowed to fluctuate naturally (Figure S5). The nCuO suspensions were stirred by a glass rod for 10 min every 8 h to compensate for the rapid sedimentation of nCuO (Figure S2B) and protection of roots. For all treatments, nine plantlets were collected from each container at 2, 6, 12, 24, 36, 48, and 96 h in the 96 h exposure and at 1, 3, 6, 10, 16, 21, and 28 days in the 28 day exposure. Wilted leaves were removed (if any); roots were rinsed with 10 mM Na4EDTA twice under ultrasonic condition (2 min) to remove the adsorbed nCuO−Cu2+ on root surfaces48 and then washed using copious deionized water. Control plants were only collected in triplicates before (0 h or 0 days) and after (96 h or 28 days) exposures. The intact plantlets and separated tissues were lyophilized prior to Cu content determination (see details in the Supporting Information) and FA analysis. Aliquots of exposure media were also collected, centrifuged, filtrated (0.22 μm filter for the Cu2+ treatment), acidified, and analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES; Agilent 720 ES) for Cu2+ concentrations when A. thaliana were sampled. Besides, before and after the 28 day exposure, six additional fresh plantlets in each treatment were harvested for the in situ detection of superoxide radicals (O2•−) using a nitroblue tetrazolium (NBT) histochemical staining method modified from Ramel et al.49 and Qian et al.50 The O2•− visualized as blue precipitates of the NBT−formazan were then quantified according to a previously described assay.51 Details of O2•− detection and quantification can be found in the Supporting Information. FA Analysis. The overall FA in A. thaliana tissues, i.e., roots, rosettes−leaves, stems, fruits (immature seeds), seeds, and wilted leaves, were simultaneously extracted and methyl esterified using the HCl−methanol procedure of Sayanova et al.52 with a slight modification. The detailed procedure for methyl esterification and analysis conditions of fatty acid
selection of plants with species differences, and inconsistent exposure conditions.26,27 The most-tangible shortcomings are the parsimonious experimental designs (e.g., one sample point from exposures to nCuO and Cu2+ for Cu accumulations and phytotoxicity end points instead of evaluation through the course of exposure with more sample points), as well as insensitive indicators of phytotoxicity at the physiological level instead of those that are more-sensitive at cellular level. Cell membrane is the first “living” target of many pollution challenges, including nanoparticles.28 The impacts of nanoparticles on membrane components (e.g., phospholipids, proteins, and saccharides) can thus reflect the initial toxic effects of, and plant responses to, the nanoparticles. The resulting variations in membrane components may thus be a range of suitable indicators at the cellular level to reveal unambiguously the mode of action of nCuO on plants. The fatty acid (FA) profile in membrane phospholipids has significant implications in both phytotoxicity and plant accumulation of nanoparticles. There have been only a few studies in the literature that reported the alteration of the FA profile (for example, in tetrahymena (T. thermophila) by nCuO and nano-TiO2 as well as in rice (Oryza sativa) by nanoCeO2),29−31 although such alteration in plants from heavymetal toxicity, including copper stress, is a well-established phenomenon.32−35 The FA are sensitive to various abiotic stresses, especially ROS, as unsaturated FA (UFA) are highly susceptible to oxidative attack.32,36,37 This results in an increase in FA saturation degree, transiting membranes from the fluid phase to the rigid phase38 and hence damaging such membrane functions as endocytosis and root-to-shoot translocation across the membrane.15,26,39−44 To the best of our knowledge, no literature is currently available with respect to the impact of nCuO and most of the current nanoparticles on the plant FA profiles. In this study, Arabidopsis (Arabidopsis thaliana), a fully sequenced model plant,45 was exposed to nCuO and Cu2+ ions in a hydroponic system for 96 h and 28 days to evaluate both the acute and chronic effects of nCuO on the plant FA profiles. The internalization and transformation of nCuO in A. thaliana cells were examined by transmission electron microscopy (TEM), energy-dispersive spectroscopy (EDS), and highresolution TEM (HRTEM) with the fast Fourier transform (FFT) technique. The seven-sample-point evaluation of the FA saturation degree with the Cu uptake kinetics through the course of exposures revealed that the chronic effects of nCuO on A. thaliana FA profiles in root and rosette−leaf samples were developed under the respective stresses of nanoparticles and Cu2+ ions released from nCuO dissolution, while the acute effects were predominantly produced from the released Cu2+ stress. This work is among the first done at the cellular level using fatty acid profiles to study the nanoparticle−plant interactions, and the results may offer significant implications in better understanding the phytotoxicity and potential risks of current nanoparticles in ecosystems.
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MATERIALS AND METHODS nCuO Characterization. Prior to exposure, nCuO particles (Sigma-Aldrich, catalog no. 544868; advertised particle size of
