Article pubs.acs.org/est
The Transcriptomic Response of Arabidopsis thaliana to Zinc Oxide: A Comparison of the Impact of Nanoparticle, Bulk, and Ionic Zinc Premysl Landa,† Sylva Prerostova,‡,§ Sarka Petrova,† Vojtech Knirsch,‡ Radomira Vankova,‡ and Tomas Vanek*,† †
Laboratory of Plant Biotechnologies, Institute of Experimental Botany AS CR, v.v.i., Rozvojova 263, 165 02 Prague 6 − Lysolaje, Czech Republic ‡ Laboratory of Hormonal Regulations in Plants, Institute of Experimental Botany AS CR, v.v.i., Rozvojova 263, 165 02 Prague 6 − Lysolaje, Czech Republic § Department of Experimental Plant Biology, Faculty of Science, Charles University in Prague, Vinicna 5, 128 44 Prague 2, Czech Republic S Supporting Information *
ABSTRACT: The impact of nanosize was evaluated by comparing of the transcriptomic response of Arabidopsis thaliana roots to ZnO nanoparticles (nZnO), bulk ZnO, and ionic Zn2+. Microarray analyses revealed 416 upand 961 down-regulated transcripts (expression difference >2-fold, p [FDR] < 0.01) after a seven-day treatment with nZnO (average particle size 20 nm, concentration 4 mg L−1). Exposure to bulk ZnO resulted in 816 up- and 2179 down-regulated transcripts. The most dramatic changes (1711 transcripts up- and 3242 down-regulated) were caused by the presence of ionic Zn2+ (applied as ZnSO4.7H20 at a concentration of 14.14 mg L−1, corresponding to the amount of Zn contained in 4 mg L−1 ZnO). Genes involved in stress response (e.g., to salt, osmotic stress or water deprivation) were the most relatively abundant group of gene transcripts up-regulated by all three Zn treatments while genes involved in cell organization and biogenesis (e.g., tubulins, arabinogalactan proteins) and DNA or RNA metabolism (e.g., histones) were the most relatively abundant groups of down-regulated transcripts. The similarity of the transcription profiles and the increasing number of changed transcripts correlating with the increased concentration of Zn2+ in cultivation medium indicated that released Zn2+ may substantially contribute to the toxic effect of nZnO because particle size has not demonstrated a decisive role.
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Administration,5 which allows its use in cosmetic formulations and the food industry. However, numerous studies have reported acute, chronic, and environmental toxicity of nZnO toward various organisms. Recently, studies that focused on nZnO toxicity toward animals and various animal cell lines,9,10 plants,11−14 aquatic organisms,15−17 ecotoxicological test organisms 18,19 and microorganisms 20,21 were reviewed. Although positive effects of nZnO on plants such as promoted seed germination, growth, and yield of peanuts were observed22 reports describing its negative effects dominate. Plants exposed to nZnO suffer by an inhibition of seed germination,23,24 root elongation23−27 and biomass accumulation.27,28 Moreover, nZnO altered roots morphology, 23,29 caused oxidative stress,27,30,31 decreased content of photosynthetic pigments31 and up-regulated transcription of abiotic and biotic stress
INTRODUCTION Nanotechnology has been rapidly developing in recent years, and nanoparticles are becoming more and more integrated into common commercial products. Nanotechnology Consumer Products Inventory listed 54 nanoenabled consumer products in 2005, while their number reached 1814 items in 2014. Most of these products (42%) fell into the health and fitness category including personal care, clothing, and cosmetics. Although silver nanoparticles are predominantly used in consumer products, majority of worldwide production includes TiO2, SiO2, and ZnO nanoparticles.1 Zinc oxide nanoparticles (nZnO) are used in sunscreens due to their ability to reflect and scatter UVA and UVB radiations and in paints because of their antimicrobial and photocatalytic properties.2,3 Moreover, they are potential material for sensing devices, food packaging, textiles, electronics, photovoltaics, catalysis, and rubber, oil and gas industry.4−6 Approximately 550 tons of nZnO is produced per year, which is still only a small quantity in compared with the 100 000 tons of total ZnO produced per year.4,7 Generally, nZnO (and ZnO itself) is considered nontoxic.8 ZnO is listed as safe (GRAS) material by the Food and Drug © XXXX American Chemical Society
Received: July 9, 2015 Revised: October 7, 2015 Accepted: November 11, 2015
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DOI: 10.1021/acs.est.5b03330 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
concentration of ZnSO4·7H2O was 14.14 mg L−1, which corresponded to the molar concentration of zinc in 4 mg L−1 of ZnO. The experiment duration was 7 days. Roots were frozen in liquid nitrogen and stored at −80 °C until they were processed for RNA isolation. The concentration of Zn2+ ions in the cultivation media was quantified using flame atomic absorption spectroscopy (SensAA, GBS, Australia) at a wavelength 279.5 nm. All samples were 10 min centrifuged at 14 000 rpm and then supernatant was collected and stored at 4 °C until the measurement. Calibration was based on a method of least-squares with R2 0.9975. Reporting limit was 0.5 μg L−1. Repeatability for Zn ranged from 0.16 to 2.71% RSD, and precision was better than 8% RSD. Recoveries were in the range of 83.5−116%. Microarray Analysis. The RNA was isolated from the roots of A. thaliana plants that were treated with nZnO, bulk ZnO, or ionic Zn2+ and control, untreated plants using the Plant RNA Isolation Mini Kit (Agilent Technologies, CA). RNA was then labeled using LowInput QuickAmp Labeling Kit (Agilent Technologies) by Cyanine 3 and Cyanine 5 using a dye swap design to avoid dye-based bias. Labeled cRNA was purified by RNeasy Plant Mini Kit (Qiagen, Germany), fragmented and hybridized on the Arabidopsis (V4) Gene Expression Microarray (Agilent Technologies) according to the manufacturer’s instructions. After a 17-h hybridization at 65 °C, slides were washed in GE Wash Buffers (Agilent Technologies), acetonitrile and Stabilization and Drying Solution (Agilent Technologies). Microarrays were scanned using a GenePix 4000B scanner controlled by GenePix Pro Microarray Analysis Software (Molecular Devices, CA). Experiments were repeated four times with root cRNA prepared independently from individual plants. The data acquired from the scanner were processed in R scripting environment using software package LIMMA according to Smyth and Speed (2003),47 Smyth (2004),48 and Smyth et al. (2005).49 The LOESS normalization method was used to balance the mean fluorescence intensities between the green and red channels in the frame of single arrays, and the Aquantile method was used to normalize signals among arrays. The background intensity was not subtracted from the overall spot intensities. The statistical analyses were performed without spots with zero weights. The false discovery rate (FDR) method was used for statistical evaluation. Genes showing ≥2-fold change in gene expression (p-value
