Article pubs.acs.org/est
CuO Nanoparticle Interaction with Arabidopsis thaliana: Toxicity, Parent-Progeny Transfer, and Gene Expression Zhenyu Wang,†,‡ Lina Xu,† Jian Zhao,*,†,‡ Xiangke Wang,§ Jason C. White,∥ and Baoshan Xing*,⊥ †
Institute of Costal Environmental Pollution Control, and Ministry of Education Key Laboratory of Marine Environment and Ecology, Ocean University of China, Qingdao 266100, China ‡ Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China § School of Environment and Chemical Engineering, North China Electric Power University, Beijing 102206, China ∥ Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station, New Haven, Connecticut 06504, United States ⊥ Stockbridge School of Agriculture, University of Massachusetts, Amherst, Massachusetts 01003, United States S Supporting Information *
ABSTRACT: CuO nanoparticles (NPs) (20, 50 mg L−1) inhibited seedling growth of different Arabidopsis thaliana ecotypes (Col-0, Bay-0, and Ws-2), as well as the germination of their pollens and harvested seeds. For most of growth parameters (e.g., biomass, relative growth rate, root morphology change), Col-0 was the more sensitive ecotype to CuO NPs compared to Bay-0 and Ws-2. Equivalent Cu2+ ions and CuO bulk particles had no effect on Arabidopsis growth. After CuO NPs (50 mg L−1) exposure, Cu was detected in the roots, leaves, flowers and harvested seeds of Arabidopsis, and its contents were significantly higher than that in CuO bulk particles (50 mg L−1) and Cu2+ ions (0.15 mg L−1) treatments. Based on X-ray absorption near-edge spectroscopy analysis (XANES), Cu in the harvested seeds was confirmed as being mainly in the form of CuO (88.8%), which is the first observation on the presence of CuO NPs in the plant progeny. Moreover, after CuO NPs exposure, two differentially expressed genes (C-1 and C-3) that regulated root growth and reactive oxygen species generation were identified, which correlated well with the physiological root inhibition and oxidative stress data. This current study provides direct evidence for the negative effects of CuO NPs on Arabidopsis, including accumulation and parent-progeny transfer of the particles, which may have significant implications with regard to the risk of NPs to food safety and security.
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INTRODUCTION CuO nanoparticles (NPs) are widely used in commercial applications, including as catalysts, gas sensor, heat transfer fluids, semiconductors and photovoltaic cells.1,2 CuO NPs have raised health and environmental concerns because of this widespread application and their relatively high toxicity to biota.3,4 Plants are primary produces in ecosystems and may serve as a potential route for the transfer of NPs into food chain.5 Adverse effects of different NPs on terrestrial plants have been reported, although many of these nanotoxicity studies have focused on basic physiological response such as seed germination,6 root elongation7 and NPs transport/ accumulation.8 It is reported that CeO29 and TiO2 NPs10 were translocated from root to the fruit of tomato and cucumber, respectively. CuO NPs could also be taken up by the root of maize and then distributed in the shoots through xylembased transport.1 However, to our knowledge, current data regarding the transfer of CuO NPs from roots to fruits or © XXXX American Chemical Society
harvested seeds are not available. In addition, CuO NPs may be dissolved or biotransformed during uptake and transport, which increases the complexity of parent-progeny transport of CuO NPs in plant. Upon exposure to CuO NPs and other metalbased NPs, adverse responses of plants have been widely reported.11 Kim et al. reported that nano zerovalent iron (nZVI) enhanced root elongation by inducing cell wall loosening in Arabidopsis thaliana.12 The swelling of root tips by TiO2 NPs was observed as a result of isotropic growth of epidermal cells.13 In addition to morphological alteration of roots, CuO NPs also caused ROS generation and accumulation in rice.14 Atha et al. observed that DNA damage of radish and ryegrass upon CuO NPs exposure due to the oxidative damage Received: February 28, 2016 Revised: May 15, 2016 Accepted: May 18, 2016
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DOI: 10.1021/acs.est.6b01017 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Environmental Science & Technology of base nucleotides.15 Gene expression analyses could help capture the molecular basis of the aforementioned adverse responses. Kohan-Baghkheirati and Geisler-Lee reported that both Ag NPs and Ag+ induced the up-regulated expression of reactive oxygen species (ROS) associated genes.16 Arabidopsis is a model plant with ecotypes originating from a wide range of geographical areas, characterized by a variety of climates and selective pressures, which could bring the differences in morphological development, physiological reaction and adaptive traits.17 Therefore, different ecotypes of Arabidopsis have different tolerance to environmental stresses. For example, it is reported that Ws ecotype had higher tolerance to heavy metal (Cu(II)) than other ecotypes such as Col-0.18 Therefore, three representative Arabidopsis ecotypes (Col-0, Bay-0 and Ws-2) were used to investigate phytotoxicity and genotoxicity of CuO NPs in comparison to equivalent bulk and ion exposures. For the three ecotypes, the toxicity of CuO NPs to different stages of Arabidopsis, as well as the distribution of CuO in plants, flowers, and harvested seeds, was studied. The differentially expressed genes induced by CuO NPs and the related physiological responses were further analyzed. The findings in this work will help to gain insight into the distribution and parent-progeny transfer of NPs in plants and will provide critical data to inform risk assessment efforts.
dicotyledonous nutrient solution (Supporting Information (SI) Table S1). After an additional 3-day culturing, the seedlings were transferred to distilled water amended with CuO NPs (0, 20, and 50 mg L−1), CuO BPs (50 mg L−1) or Cu2+ ions solution (0.15 mg L−1). The inhibition test was conducted in distilled water instead of nutrient solution to minimize CuO NPs aggregation. Growth inhibition was determined at 0, 24, 48, and 96 h, respectively (8 plants/pot, 3 pots/treatment). During the exposure, seedlings were grown under 24 °C/20 °C; 16 h/8 h light/dark cycle; 80%−85% of humidity and 400− 450 μmol m−2s−1 of light intensity. The CuO NPs/BPs suspensions and Cu2+ ions solutions were aerated continuously and replaced every 24 h. After exposure, shoot and root tissues of Arabidopsis were sampled, and the fresh weight was measured. The relative growth rate was determined as (RGR, mg g−1 h−1) = [ln(M2)-ln(M1)]/(T2-T1), where M1 and M2 are the plant fresh mass at time T1 and T2, respectively.20 The roots were scanned and the morphological parameters were analyzed with the WinRHIZO Pro 2005b (Regent instruments Inc., Canada). The morphology of roots in each treatment was also examined by scanning electron microscopy (SEM, S-4800, Hitachi, Japan). The roots were fixed in 2.5% glutaraldehyde (GA) and 2% paraformaldehyde (PA) in 0.1 M PBS (pH 7.4), postfixed in 1% osmium tetroxide, dehydrated, critical-point dried, sputter-coated, and then observed with SEM. Viability of Pollens and Harvested Seeds. Arabidopsis seedlings were cultured in the dicotyledonous nutrient solution until the squaring stage (around 30 days). The plants were then moved to distilled water amended with CuO NPs (0, 20, and 50 mg L−1), CuO BPs (50 mg L−1) or Cu2+ ions solution (0.15 mg L−1). At the full-bloom stage (around 35 d), 50 flowers were randomly collected from each treatment, and the germination of the collected pollen particles was determined following the procedure of Fan et al.21 To investigate the harvested seeds, the squaring stage plants were exposed to CuO NPs (0, 20, and 50 mg L−1), CuO BPs (50 mg L−1) or Cu2+ ions (0.15 mg L−1) until harvest (around 42 d). The harvested seeds were collected, and germinated following the same procedure as described above. After germination and culture for 8 days, the seedling root lengths of harvested seeds were determined. Cu Content in Plants and Speciation in Harvested Seeds. During the above CuO NPs exposure experiments, the roots, shoots, flowers and harvested seeds were sampled. The roots were prewashed thoroughly with Na2-EDTA solution three times, and then all the samples were oven-dried at 70 °C for 24 h. The dried samples were digested with 6 mL of 68% HNO3.1 Cu contents in these samples were determined by FAAS. X-ray absorption near-edge spectroscopy (XANES) was used to determine the speciation of Cu in harvested seeds. The XANES data at the Cu K-edge were obtained on the beamline 14W1 at the Shanghai Synchrotron Radiation Facility (SSRF, Shanghai, China). The storage-ring current during the data acquisition was in the range of 130−210 mA at 3.5 GeV. A Cu foil internal reference was used for energy calibration with the first inflection point set at 8979 eV. The XANES spectra of Cu reference samples, including commercial CuO NPs, Cu2O, Cu2S, CuS, CuSO4, Cu2(OH)PO4, Cu-citrate, and Cu-acetate in their solid form, were collected using X-ray transmission mode. The spectra of the harvested seeds were collected using a 19 elements solid-state detector (SSD). The XANES data
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MATERIAL AND METHODS CuO NPs Characterization. CuO NPs and bulk particles (BPs) were purchased from Beijing Nachen S&T Ltd. Surface areas of CuO NPs and BPs were measured by the N2−BET method (Autosorb-1, Quantachrome Instruments Ltd., USA). Particle suspensions (50 mg L−1) were prepared by adding CuO NPs or BPs powder into distilled water, and the solutions were sonicated (100 W, 40 kHz) for 30 min to increase particle dispersion. One drop of the suspension was evaporated on a nickel grid to observe particle morphology using transmission electron microscopy (TEM) (H-7650, Hitachi, Japan). Zeta potentials of CuO NPs and BPs suspensions were estimated using a Nanosizer (Nano Series ZS90, Malvern, Britain). The dissolution kinetics of CuO NPs was examined at different time intervals (1−168 h). The CuO NPs suspensions were centrifuged twice at 10 000 rpm for 30 min to separate CuO NPs from suspension. Cu2+ ions concentration in the supernatant was determined by flame atomic absorption spectrophotometry (FAAS, SOLAAR M6, Thermo, USA). Seeds and Germination. Seeds of Arabidopsis ecotypes (Col-0, Bay-0 and Ws-2) were provided by Prof. Jihong Xing at Agricultural University of Hebei, China. For germination, seeds were surface-sterilized with 75% alcohol for 30 s, washed three times with sterilized H2O, and then sterilized by 10% NaClO solution for 10 min. To minimize residual NaClO, the seeds were further washed three times with sterilized H2O.19 The sterile seeds were soaked in CuO NPs (0, 20, and 50 mg L−1) or BPs (50 mg L−1) suspensions or in Cu2+ ions solution (0.15 mg L−1) for 48 h; all seeds were then placed in the agarcontaining Murashige and Skoog (MS) medium. The ion level of 0.15 mg L−1 was selected as the test Cu2+ concentration based on the dissolution kinetics of CuO NPs and BPs in distilled water as described above. After 5 days, the germination of seeds (20 seeds/dish, 3 dishes/treatment) was examined and counted following the approach by Wang et al.1 Growth Inhibition of Seedlings. The seeds were sterilized as described above, and then sown on MS agar plates. After 10day germination, the seedlings with four leaves were moved to B
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in the chloroplast. Therefore, three different inhibitors which specifically block the electron transport in photosystem were used to locate the sites of ROS generation. The inhibitors employed were 3-(3,4-dichloro-phenyl)-1,1-dimethylure (DCMU), 2,5-dibromo-3-methyl-6-isopropyl benzoquinone (DBMIB), and 1,1-dimethyl-4,4-bipyridinium (Paraquat), which blocks the electron transport from quinone A (QA) to quinone B (QB),28 from plastoquinone (PQ) to cytochrome bf (cty bf),29 and from iron−sulfur centers (Fe−S) to Ferredoxin (Fd),30 respectively. The cultured seedlings at the four-leaf stage (as described above) were exposed to 50 mg L−1 CuO NPs, 25 μM DCMU, 75 μM DBMIB, 50 μM Paraquat, 50 mg L−1 CuO NPs with 25 μM DCMU, 50 mg L−1 CuO NPs with 75 μM DBMIB, and 50 mg L−1 CuO NPs with 50 μM Paraquat, respectively. The three inhibitors did not show any negative effects on the growth of Arabidopsis seedlings in our preliminary experiments. After exposure for 96 h, the chloroplasts of leaves were extracted by differential centrifugation.31 The generation of ROS in the extracted chloroplast was determined according to the approach by Naydov et al.32 Briefly, after exposure to CuO NPs (both in the presence and in the absence of inhibitors) for 96 h, the intracellular generation of ROS in chloroplast was examined with 2′, 7′dichlorofluorescein diacetate (H2DCF-DA, Beyotime, China). The fluorescence intensities were determined using fluorescence microscopy (IX70, Olympus, Japan) with an excitation wavelength at 485 nm and emission wavelength at 522 nm. Statistical Analysis. All the experiments were run in triplicates unless stated otherwise. Data were analyzed by oneway ANOVA with a LSD test using SPSS Statistics. “p < 0.05” was used for the statistical significance.
processing and linear combination (LC) were done using the software program ATHENA.22 Differential Display Reverse Transcription Polymerase Chain Reaction (DDRT-PCR). DDRT-PCR was used to explore changes in gene expression of CuO NPs-treated Arabidopsis. DDRT-PCR is a reliable and highly sensitive technique to identify differently expressed genes in organisms.23 Seedlings at the four-leaf stage were exposed to distilled water, 50 mg L−1 CuO NPs or 0.15 mg L−1 Cu2+ ions for 0, 24, 48, and 96 h. The seedlings were then washed with deionized water, immediately frozen in liquid N2 and grinded to fine powders in a prechilled mortar. Total RNA was extracted from the seedling samples using RNeasy plant mini kit (Cat. No 74904, Germany). The RNA extract was purified using DNAfree DNase Treatment (Promega No. M6101, Germany). The analytical procedure of DDRT-PCR was based on the method described by Wang et al.24 mRNA in the purified RNA samples was also reverse-transcribed into cDNA. After the amplification of cDNA, the PCR products were separated on a 6% denaturing polyacrylamide gel electrophoresis and visualized by silver stain.25 Differences in band intensities were caused by differential expression. Reverse Northern blotting and semiquantitative PCR are commonly employed to improve candidate selection and to avoid possible false positives obtained from DDRT-PCR.26 The differentially expressed gene bands from DDRT-PCR were recycled and reamplified, and then hybridized with DIG labeled cDNA probe (Roche Applied Science, Germany). The positive gel-purified DNA genes were cloned and sequenced by Sangon Biotech (Shanghai) Co. These sequences (accuracy >98.5%) were identified on the basis of sequence homology using a basic local alignment search tool (BLAST) program with Arabidopsis genome sequences from the Arabidopsis information resource (TAIR). Semiquantitative PCR was used to investigate the expression levels of these identified genes. The sequences of reference and target genes are shown in SI Table S2. The cDNA (1 μL) was used for PCR amplification in a solution with a final volume of 25 μL (12.5 μL SuperReal PreMix, 1 μL forward primer, 1 μL reverse primer and 9.5 μL double distilled water). PCR amplification was performed for 32 cycles using the following parameters: 30 s denaturation at 94 °C, 30 s annealing at 51 °C, 50 s elongation at 72 °C, and a final 10 min elongation at 72 °C. These PCR products were then electrophoresed on a 1% agarose gel. The expression level of each differentially expressed gene was calculated by the software Gel-Pro analyzer 4 with the tubulin gene as the control. Quantitative Real-Time PCR Analysis. The first gene (Auxin signal F-box protein) obtained from the above DDRTPCR was further investigated by quantitative RT-PCR. This gene could regulate the growth of root hair and lateral root. PCR reaction contained 1 μL cDNA template, 5 μL SYBR Green (FP205, TIANGEN, China), 0.4 μL primer (SI Table S3) and 3.6 μL RNase free H2O. Samples were run concurrently with a standard curve obtained from the PCR products (15 min 95 °C, 40 cycles of 95 °C for 10 s and 60 °C for 30 s). A melting curve analysis was used to confirm specific replicon formation. Relative expression of target gene =2−ΔΔCt, where ΔΔCt = (Ctgene, treatment − Ct actin, treatment) − (Ctgene, control − Ctactin, control), Ct is the threshold cycle.27 Oxidative Stress. Another identified gene that was differentially expressed is Fe superoxide dismutase (Fe-SOD) and relates to the generation of reactive oxygen species (ROS)
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RESULTS AND DISCUSSION Effect of CuO NPs on Seed Germination and Seedling Growth. The characterization of the CuO NPs and BPs is shown in SI Figure S1 and Table S4. The surface areas of CuO NPs and BPs were 13.3 and 0.52 m2 g−1, respectively. The particle size of CuO NPs was 20−40 nm as determined from TEM imaging (20 particles in 5 different TEM images). The zeta potential of CuO NPs in the suspension was −9.76 mV. The equilibrium concentration of Cu2+ ions released from the CuO NPs (50 mg L−1) and BPs (50 mg L−1) suspension were 0.10 and 0.012 mg L−1 (SI Figure S2), respectively. In order to investigate the contribution of Cu2+ ions to phytotoxicity in the subsequent experiments, 0.15 mg L−1 Cu2+ ions were therefore included as a treatment. Seed germination and plant growth experiments were first conducted. For all the three ecotypes of Arabidopsis (Col-0, Bay-0 and Ws-2), CuO NPs did not inhibit their seed germination (p < 0.05) (SI Figure S3). This observation may be due to the selective permeability of seed coat, which has evolved to protect the embryo from harmful external factors. Both Lin and Xing,6 and Wang et al.1 reported that plant seeds insensitive to metal oxide NPs. For seedling growth, after exposure for 96 h, CuO NPs at 20 mg L−1 and 50 mg L−1 significantly decreased the root and shoot fresh weight of all the three ecotypes as compared to the unexposed control, Cu2+ ions (0.15 mg L−1) and CuO BPs (50 mg L−1) treatments (p < 0.05) (SI Figure S4−S6). In addition, the observed growth inhibition of CuO NPs on seedlings was exposure time-dependent. CuO NPs (50 mg L−1) caused significantly different (p < 0.05) inhibition ratios in the root growth of the Col-0 and Bay-0 ecotypes after 96 h exposure C
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increasing cell width and decreasing cell elongation, as well as more cortical cell columns; this has been observed previously in plants exposed to rare earth oxide NPs (e.g., CeO2, La2O3, Gd2O3, and Yb2O3).34 Further observation was performed by SEM imaging. Lateral roots and root hairs of Col-0 seedlings were significantly decreased (SI Figure S7B), and swelling in the root elongation zone was obvious (SI Figure S7C) after exposure to 50 mg L−1 CuO NPs for 96 h. Similar changes in root morphology were observed in Arabidopsis when exposed to TiO2 NPs13 and water-soluble fullerenes.19 Aberrant root morphology has been linked to isotropic growth of epidermal cells, auxin disruption, aberrant microtubule arrangement and cell division,19 which could cause the observed swelling of roots in the present study. However, the enhancement of root elongation was observed during exposure to nZVI due to OH radical-induced polysaccharides degradation and cell wall loosening.12 The OH radicals were generated via Fenton reaction on the surface of nZVI. In our study, the root elongation enhancement was not observed because Fenton reaction could not occur on the surface of CuO NPs. Effect of CuO NPs on the Viability of Pollen and Harvested Seeds. To investigate the transgenerational effect of CuO NPs in Arabidopsis, the viability of pollen and harvested seeds was examined. As shown in Figure 2A, the germination of pollen grains from 50 mg L−1 CuO NPs-treated Arabidopsis was significantly reduced (p < 0.05) in comparison with the pollen collected from unexposed plants. The inhibition ratios for Col0, Bay-0 and Ws-2 were 10.0%, 10.0%, and 18.0%, respectively. The inhibition of pollen germination may result from (1) the damage of pollen plasmalemma after CuO NPs exposure (as observed in SI Figure S8), which is a crucial factor inhibiting pollen germination; and (2) the accumulation of CuO NPs or dissolved Cu2+ in flowers or pollens, which may lead to disrupted biochemistry or physiology. Similarly, germination ratios of harvested seeds were significantly reduced after exposing CuO NPs (both 20 and 50 mg L−1) to Arabidopsis plants for all the three ecotypes (Figure 2B). Cu2+ ions (0.15 mg L−1) or CuO BPs (50 mg L−1) had no effect on the germination of harvested seed. The root elongation of Col-0 was reduced by 16.5%, 28.0%, 63.5%, and 12.5% for the seedlings that were harvested from Arabidopsis plants that were pretreated with Cu2+ ions, CuO NPs, and CuO BPs, respectively (Figure 2C). Notably, CuO NP-induced root toxicity was concentration-dependent (Figure 2D). The negative effects of CuO NPs on both pollen and harvested seeds suggest potential impacts of NPs exposure on food yield and quality. Uptake of Cu in Plant. Figure 3 shows the distribution of Cu in different tissues (roots, leaves and flowers) of exposed Arabidopsis after 96 h. With exposure to 50 mg L−1 CuO NPs, there was a significant increase Cu content in the plants (p < 0.05) in comparison to the other treatments. The Cu contents in the roots were obviously higher than that in leaves, flowers, or harvested seeds for all the three ecotypes (p < 0.05). For all tested tissues, the Cu content after CuO NPs exposure (50 mg L−1) was significantly higher than that exposed to Cu2+ ions (0.15 mg L−1), suggesting that CuO NPs were likely taken up by plants and distributed in roots, leaves, and flowers. Interestingly, Cu was detected in the harvested seeds after CuO NPs treatment, and the content was higher than the Cu2+ ions treatment for all the ecotypes (Figure 4A). The speciation of Cu in the harvested seeds of Col-0 (the lowest Cu content of the three ecotypes) was further analyzed using XANES (Figure
(Figure 1). In the shoots, significant differences (p < 0.05) occurred only after 96 h exposure, indicating that roots were
Figure 1. Relative growth rate (RGR) of Arabidopsis after exposure to CuO NPs (6−96 h). (A) RGR of roots in the presence of 50 mg L−1 CuO NPs for 96 h; (B) RGR of shoots in the presence of 50 mg L−1 CuO NPs for 96 h. RGR (mg g−1h−1) = [ln(M2)-ln(M1)]/(T2-T1), where M1 and M2 are the plant fresh mass at time T1 and T2, respectively. The values were given as mean ± SD (standard deviation). For a given ecotype, significant difference between 6 h exposure and other exposures (12, 24, 48, or 96 h) was marked with“*” (p < 0.05, t test, n = 3). For a given exposure time, significant difference among different Arabidopsis ecotypes was marked with different letters (p < 0.05, LSD, n = 3).
more sensitive to CuO NPs than the shoots. The root and shoot RGRs for Col-0 (3.12, 9.35 mg g−1 h−1) were significantly lower (p < 0.05) than those of Bay-0 (10.8, 11.5 mg g−1 h−1) and Ws-2 (10.5, 12.5 mg g−1 h−1) after 96 h exposure. This may result from different adaptive traits of these three ecotypes.17 Detailed information (e.g., phenotypes, place of origin, tolerance) of these three ecotypes (Col-0, Bay-0, and Ws-2) is presented in SI Table S5. Ws-2 being a medium phenotype in all the three ecotypes (SI Table S5) showed the highest resistance. Murphy and Taiz investigated the toxicity of Cu to 10 Arabidopsis ecotypes, and Ws ecotype had the highest tolerance and showed the higher level of expression of metallothionein genes in all the test ecotypes,18 which is in agreement with our findings that Ws-2 was more tolerant to the CuO NPs. Vashisht et al. also found that Ws-2 ecotype had significant tolerance to flooding stress than Col-0 and Bay-0.33 Due to the anticipated higher sensitivity of root, tissue morphology changes were investigated further. Root length, surface area and volume, and the number of root tips of Arabidopsis seedlings were significantly reduced (p < 0.05) after exposure to 50 mg L−1 CuO NPs for 96 h (SI Table S6). Interestingly, the average root diameter of Col-0 ecotype was significantly increased (p < 0.05) compared with the unexposed, Cu2+ ions and CuO BPs treatments (SI Table S6). The increase in root diameter could be explained by D
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Figure 3. Cu contents in roots (A), leaves (B), and flowers (C) of different Arabidopsis ecotypes after treated with distilled water (control), 0.15 mg L−1 Cu2+ ions (0.15 Cu2+), 50 mg L−1 CuO NPs (50 NPs) and 50 mg L−1 CuO BPs (50 BPs). The values were given as mean ± SD (standard deviation). Significant difference among different treatments compared control was marked with “*”. For a given treatment, different letters represent significant differences among different ecotypes (p < 0.05, LSD, n = 3).
Figure 2. Germination of pollen (A), seeds (B) and root length of seeds (C) obtained from CuO NPs-treated Arabidopsis. The five treatments were distilled water (control), 0.15 mg L−1 Cu2+ ions (0.15 Cu2+), 20 mg L−1 CuO NPs (20 NPs), 50 mg L−1 CuO NPs (50 NPs), and 50 mg L−1 CuO BPs (50 BPs), respectively. The values were given as mean ± SD (standard deviation). For a given ecotype, significant difference among different treatments was marked with letters (p < 0.05, LSD, n = 3); (D) Photo of Arabidopsis harvested seedlings obtained from 0.15 mg L−1 Cu2+ ions, 50 mg L−1 CuO BPs, and 0−50 mg L−1 CuO NPs exposure.
the Cu contents in roots, leaves, flowers and harvested seeds were statistically equivalent to the untreated controls and were significantly less than the NPs treatments (Figure 3,4A), demonstrating that CuO BPs cannot be readily accumulated and distributed in the plant. Several others have reported on the particle-size dependent uptake of metal oxide nanoparticles. For example, Larue et al. described the size-dependent uptake of TiO2 NPs in wheat, where particles smaller than 36 nm were translocated from the roots to the leaves, but particles between 40 and 140 nm were retained in the roots. Those above 140 nm were actually excluded from the plants.35 Similarly, Hawthorne et al. observed that the accumulation of CeO2 NPs in zucchini roots was 4-fold greater than that of the equivalent BPs.36 Differential Gene Expression Following CuO NPs Exposure. NPs (e.g., Ag) are known to trigger changes in gene expression in plants;37 thus, we employed a DDRT-PCR technique to search for the genes whose expression was affected by CuO NPs. Eighteen differentially expressed genes in
4B). LC fits of the spectrum with the standard spectra indicated that 88.8% of total Cu in the seeds was in the form of CuO, suggesting that CuO NPs were indeed accumulated in the progeny of exposed Arabidopsis. In accordance with our previous study of CuO uptake by maize,1 the NPs may pass through the root apex of Arabidopsis and moved to the stele, followed by transport to the shoots via xylem. Small amounts of Cu2(OH)PO4 (2.0%), Cu-acetate (3.2%) and Cu2O (6.0%) were observed in the harvested seeds. This finding may result from uptake of free Cu2+ ions that were released from CuO NPs in medium or from ions that dissolved from internalized CuO NPs. In addition, upon exposure to 50 mg L−1 CuO BPs, E
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Figure 4. (A) Cu contents in harvested seeds of different Arabidopsis ecotypes after treated with distilled water (control), 0.15 mg L−1 Cu2+ ions (0.15 Cu2+), 50 mg L−1 CuO NPs (50 NPs) or 50 mg L−1 CuO BPs (50 BPs). The values were given as mean ± SD (standard deviation). Significant difference among different treatments compared control was marked with “*”. For a given treatment, different letters represent significant differences among different Arabidopsis ecotypes (p < 0.05, LSD, n = 3). (B) Cu K-edge XANES spectra of the harvested seeds of Arabidopsis (Col-0) after treated with 50 mg L−1 CuO NPs. In panel B, the black dashed line is the fitting curve.
Arabidopsis after CuO NPs or Cu2+ ions exposure were found (SI Figure S9). Using reverse Northern dot-blot analysis, six positive genes (truly differently expressed genes) were obtained from the 18 differentially expressed genes. In these six positive genes, three genes belonged to Col-0, and the others belonged to Bay-0. No positive genes were found in Ws-2. The six positive differentially expressed genes were further selected for homology analysis using the BLAST program. As shown in SI Table S7, the three clones of Bay-0 (B-1, B-2, and B-3) showed no significant homology with the known expressed sequence tags, whereas the other three clones that belonged to Col-0 (C-1, C-2, and C-3) had significant homology. C-1 was 93% homologous to auxin signaling Fbox protein; C-2 and C-3 showed 73% and 94% homology to DNA mismatch repair protein MSH5 and Fe-SOD, respectively. The time-dependent expression of the three genes of Col-0 (C-1, C-2, C-3) was further analyzed by semiquantitative PCR (Figure 5). C-1 (auxin signaling F-box protein) was downregulated after exposure to both CuO NPs (24, 48, and 96 h) and Cu2+ ions (48 and 96 h). F-box protein plays a negative feedback regulation of auxin signaling;38 thus, the down-
Figure 5. Semiquantitative PCR analysis of differential expression genes (C-1, C-2, C-3) of Col-0. These genes were obtained by DDRTPCR and had significant homology as shown in SI Table S7. The values were given as mean ± SD (standard deviation). Significant difference among different treatments was marked with letters (p < 0.05, LSD, n = 3).
regulation of this gene suggests a decrease of F-box protein function and of auxin signaling transduction after exposure. C-2 (DNA mismatch repair protein MSH5) and C-3 (Fe-SOD) were up-regulated after treated with CuO NPs and Cu2+ ions for 48 and 96 h. MSH5 takes part in the recombination in the process of meiosis,39 indicating that CuO NPs and Cu2+ ions could promote the DNA repair at the stage of meiosis. C-3 (FeSOD) takes part in the regulation of oxidative stress;40 this gene was up-regulated, probably due to the enhanced oxidative stress of Col-0 after exposed to CuO NPs or Cu2+ ions. The detailed interaction between the function of the selected genes (C-1, C-3) and the stress responses of CuO NPs (e.g., growth F
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Oxidative Response. The increased expression of C-3 suggested activation of Fe-SOD, which is a metalloenzyme SOD and primarily exists in the chloroplasts of higher plants.40 We thus investigated the generation of ROS in chloroplasts after CuO NPs exposure. As shown in Figure 6B, ROS generation was significantly increased (1.58 times) (p < 0.05) when treated with CuO NPs (50 mg L−1), a finding that correlates well with the up-regulation of C-3. In addition, three chloroplast electron transport chain inhibitors (DCMU, DBMIB and Paraquat) were employed to locate the specific sites of ROS generation. The relative ROS levels were 158%, 148%, 165%, and 130% for CuO NPs, DCMU, DBMIB, and Paraquat treatments, as compared to the untreated controls (Figure 6B). Moreover, the ROS levels in the two CuO NPsinhibitor treatments (CuO + DCMU, CuO + DBMIB) were significantly higher than the individual CuO NPs or inhibitor treatments, whereas there was no significant difference for the “CuO + Paraquat” exposure. It has been reported that DCMU can block electron transport from quinone A (QA) to quinone B (QB);28 DBMIB can block electron transport from plastoquinone (PQ), to cytochrome bf (cty bf);29 and Paraquat can block electron transport from iron−sulfur centers (Fe−S) to Ferredoxin (Fd).30 The results in Figure 6B thus suggested that CuO NPs blocked the electron transport from QA to QB, and from PQ to cytochrome, but had no interaction with the electron transport from Fe−S to Ferredoxin (SI Figure S10). This blockage of electron transport could result in excessive ROS accumulation and oxidative stress, thus causing oxidative damage of biological molecules (e.g., DNA and protein), disruption of cellular metabolism, and ultimately, growth inhibition of Arabidopsis.
and oxidative stress) are further discussed in the next section. C-2 (DNA mismatch repair protein MSH5) was not studied further due to its low homology. Differentially Expressed Genes: Growth and Oxidative Response. Growth Response. Auxin signaling regulates the development of lateral roots and root hairs according to the expression of AXR2/IAA7, AXR3/IAA17, and SLR1/IAA14.41 The expression of these three genes after CuO NPs exposure were thus examined (Figure 6A). The expression of AXR2/
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ENVIRONMENTAL IMPLICATION The current study investigated the toxicity of CuO NPs to Arabidopsis, as well as their impacts on parent-progeny transfer and the regulation of gene expression upon exposure. The growth inhibition and Cu uptake of Arabidopsi in the Cu2+ ions or CuO BPs treatment were much lower than CuO NPs treatment, indicating a nanospecific series of effects. Importantly, Cu in the harvested seeds was mainly present (88.8%) in the form of CuO NPs according to the XANES analysis. The observed negative physiological effects and the parent-progeny transfer of CuO NPs in Arabidopsis may also occur in other plants, especially in agricultural crop, which presents the potential risk on food yield, quality, and safety. Although CuO was the dominant form in the harvested seeds, Cu2O (6.0%) was also present in the harvested seeds after CuO NPs exposure, indicating the transformation of CuO NPs during uptake and/or transport of CuO NPs in Arabidopsis. However, it is unknown that at what location of Arabidopsis Cu2O was formed. The role of Cu2O formation in CuO NPs toxicity and gene expression of Arabidopsis deserves further study. In our previous studies, Cu2O was also detected in maize root1 and prokaryotic alga Microcystis aeruginosa44 after CuO NPs exposure using high resolution transmission electron microscopy (HRTEM). It is suggested that the transformation of metal-based NPs could be further investigated through characterizing NPs products (e.g., size, crystalline phase, metal species) by combining XANES and HRTEM. To our knowledge, this is the first observation for CuO NPs being present in the plant progeny, and the information provided here will be helpful for better understanding the fate, transfer and molecular basis of nanotoxicity in terrestrial plants.
Figure 6. Relative expression amounts of AXR2/IAA7, AXR3/IAA17, and SLR1/IAA14 (A) after exposure to 50 mg L−1 CuO NPs or 0.15 mg L−1 Cu2+ ions for 48 and 96 h, and assessment of ROS level (B) in Col-0 chloroplast after exposure to CuO NPs (NPs), DCMU, DBMIB, Paraquat, NPs+DCMU, NPs+DBMIB, NPs+Paraquat. The values were given as mean ± SD (standard deviation). In panel (A), significant difference among all the treatments was marked with latters for a given gene. In panel (B), significant difference among different treatments was marked with letters (p < 0.05, LSD, n = 3).
IAA7, AXR3/IAA17 and SLR1/IAA14 genes was increased after exposure to CuO NPs (50 mg L−1) for 48 and 96 h. After CuO NPs exposure for 96 h, the expression of AXR2/IAA7, AXR3/IAA17 and SLR1/IAA14 were 2.80, 1.48, and 6.95 times higher than control, respectively, and 2.56, 1.45, and 2.70 times higher than Cu2+ ions, respectively. This obvious activation of AXR2/IAA7 and AXR3/IAA17 can improve the stability of encoded proteins, and can induce the auxin-related developmental defect.42 SLR1/IAA14 is known to interrupt the cell cycle in the pericycle in Arabidopsis lateral root initiation.43 Therefore, the increased expression of these three genes could explain the observed inhibition of Arabidopsis lateral roots and root hairs upon CuO NPs treatment, which was supported by our observed root morphology results (SI Figure S7). This impact on lateral roots and root hairs caused by CuO NPs could affect water and nutrient absorption, and result in plant growth inhibition. G
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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.est.6b01017. Ten figures and seven tables (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*(B.X.) Phone: +1 413 545 5212; e-mail:
[email protected]. *(J.Z.) E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by Natural Science Foundation of China (41325013, 41120134004, 41403086), Natural Science Foundation of Shandong (ZR2014DM018), USDA-AFRI (2011-67006-30181), and USDA-NIFA Hatch program (MAS 00475).
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