Environ Sci Pollut Res (2015) 22:17716–17723 DOI 10.1007/s11356-015-4976-7
RESEARCH ARTICLE
Response difference of transgenic and conventional rice (Oryza sativa) to nanoparticles (γFe2O3) Xin Gui 1 & Yingqing Deng 2 & Yukui Rui 1,2 & Binbin Gao 1 & Wenhe Luo 1 & Shili Chen 1 & Le Van Nhan 1,3 & Xuguang Li 1 & Shutong Liu 1 & Yaning Han 1 & Liming Liu 1 & Baoshan Xing 2
Received: 17 March 2015 / Accepted: 29 June 2015 / Published online: 9 July 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Nanoparticles (NPs) are an increasingly common contaminant in agro-environments, and their potential effect on genetically modified (GM) crops has been largely unexplored. GM crop exposure to NPs is likely to increase as both technologies develop. To better understand the implications of nanoparticles on GM plants in agriculture, we performed a glasshouse study to quantify the uptake of Fe2O3 NPs on transgenic and non-transgenic rice plants. We measured nutrient concentrations, biomass, enzyme activity, and the concentration of two phytohormones, abscisic acid (ABA) and indole-3-acetic acid (IAA), and malondialdehyde (MDA). Root phytohormone inhibition was positively correlated with Fe2O3 NP concentrations, indicating that Fe2O3 had a significant influence on the production of these hormones. The activities of antioxidant enzymes were significantly higher as a factor of low Fe2O3 NP treatment concentration and significantly lower at high NP concentrations, but only among transgenic plants. There was also a positive correlation between the treatment concentration of Fe2O3 and iron accumulation, and the magnitude of this effect was greatest among nontransgenic plants. The differences in root phytohormone production and antioxidant enzyme activity between transgenic and non-transgenic rice plants in vivo suggests that GM crops Responsible editor: Philippe Garrigues * Yukui Rui
[email protected] 1
College of Resources and Environmental Sciences, China Agricultural University, Yuanmingyuan West Road No.2, Haidian District, Beijing, China
2
Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA 01003, USA
3
Research Institute for Aquaculture No1, Tu Son Bac Ninh, Vietnam
may react to NP exposure differently than conventional crops. It is the first study of NPs that may have an impact on GM crops, and a realistic significance for food security and food safety. Keywords Nanoparticles . Transgenic rice . Phytohormones . Antioxidant system
Introduction Nanotechnology has enabled novel applications in a variety of fields, such as medicine, electronics, biomaterials, energy, and agricultural products (Das et al. 2008; Doane and Burda 2012; Haselman and Hauck 2010; Saito et al. 2011; Wu et al. 2012). For example, iron oxide nanoparticles (Fe2O3 NPs) have been used extensively in many fields including microelectronics, biomedical imaging, and the detection and visualization of various phases and interfaces in oil reservoirs (Basnet et al. 2013; Laurent et al. 2008; Mahmoudi et al. 2011). The increased prevalence of engineered nanoparticles (ENPs) has led to their release into the environment, which is potentially having an impact in living organisms (Shah et al. 2014; Zhao et al. 2014), especially plants (Ma et al. 2013; Liao et al. 2014; Martinez-Ballesta and Carvajal 2014). How these NPs may affect food safety and quality is of particular concern. The success of genetically modified (GM) crops is reshaping modern agriculture, with increased productivity and enhanced herbicide and pest resistance; however, there are many unknowns regarding how these crops respond to ENPs. GM crops might have different morphological, physiological, and biochemical alterations, compared with their conventional counterparts. Few studies have investigated the response of GM crops to NP contamination, and knowledge of the potential toxicity of ENPs to GM crops is critically needed
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as well as a better understanding of plant nano-interactions to promote safe use of ENPs in agriculture. Previous work on non-GM plants reported negative, positive, and/or no effect of different NPs on seed germination, root elongation, and other plant growth performance indicators. ENPs can influence nutrient uptake (Larue et al. 2012; Trujillo-Reyes et al. 2013), bioavailability (Lee et al. 2008; Marusenko et al. 2013), biotransformation (Zhang et al. 2012, 2013), antioxidant enzyme activity (Cui et al. 2014; Rico et al. 2013), and other physiological biomarkers in conventional plants. Fe2O3 NPs specifically have been shown to have key effects on plants. For example, superparamagnetic iron oxide NPs can significantly enhance the chlorophyll content in subapical leaves of soybean (Ghafariyan et al. 2013), and rice and soybean plants experienced lower toxicity from nano-sized iron oxide compared with micro-sized iron oxides (Alidoust and Isoda 2013, 2014). However, the effects of NPs are equivocal, as iron ions and NPs do not always affect physiological parameters, including chlorophyll content, catalase (CAT), and ascorbate peroxidase (APX) (Trujillo-Reyes et al. 2014. Some studies suggest that, compared with their non-transgenic counterparts, transgenic plants may confer more precise regulation of abiotic stress tolerance, such as to salinity, drought, and heavy metals. For example, transgenic tall fescue overexpressed both CuZn-superoxide dismutase and APX in response to metal treatment (Lee et al. 2007). Similarly, rice genotypes with expression of OsGSTLs have demonstrated greater tolerance to arsenic stress (Kumar et al. 2013). Rice is the staple food of more than half of the world’s population (Kennedy 2002). The uptake of nutrients (Mazumdar and Ahmed 2011), antioxidant property (phenol contents and radical scavenging ability) (Rico et al. 2013), and known toxicity of several NPs (ZnO, TiO2, Au, and carbon nanotubes) to conventional rice seedlings and cells has already been studied (Boonyanitipong et al. 2011; Koelmel et al. 2013; Shen et al. 2010). However, it is imperative to investigate the effects of NPs on transgenic rice specifically. To our knowledge, there are no published studies on the effects of Fe2O3 NPs on transgenic rice. In this study, we used 93–11 LRK1, which is a high-production transgenic rice cultivar with the LRK1 gene and its parent nontransgenic rice (93–11). In this greenhouse study, we exposed both transgenic and non-transgenic rice plants to various concentrations of Fe2O3 NPs. At the conclusion of the study, we harvested the plants and measured their uptake of Fe, biomass, content of chlorophyll, activity of peroxidase (POD) and superoxide dismutase (SOD), levels of two phytohormones, and the content of malondialdehyde (MDA).
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Materials and methods Characterization of Fe2O3 NPs Fe2O3 NPs with 99.9 % purity and particle sizes of less than 50 nm were purchased from Sigma Inc. The manufacturer descriptions of Fe2O3 NP’s crystalline are primarily γ, a specific surface area of 147 m 2 g−1, and a density of 5.25 g cm−3. Prior to use, Fe2O3 NPs were further characterized by scanning electron microscopy (SEM, S-4800, Japan) and dynamic light scattering (DLS). Fe2O3 NP suspensions were prepared at a concentration of 2 mg L−1 for measurement of hydrodynamic sizes and zeta potential (Nicomp 380 DLS Zeta potential/Particle system, Santa Barbara, CA, USA). Plant cultivation and exposure to Fe2O3 NPs Rice seeds (Oryza sativa L. ssp. indica) of transgenic LRK1 93–11 and non-transgenic 93–11 were obtained from College of Agriculture and Biotechnology, China Agriculture University. After being surface-sterilized in 5 % (v/v) H2O2 for 30 min, seeds were rinsed thoroughly with Milli-Q water (18 M cm) and placed in 10 cm × 1.5 cm Petri dishes for germination in a dark incubator at 25 °C. After 1 week, sets of four seedlings of uniform size were transferred to 2 L black plastic pots, with three replicate pots for each treatment. Each pot was fertilized with 1.8 L half-strength Kimura nutrient solution, which contained 0.18 mM (NH4)2SO4, 0.27 mM MgSO4·7H2O, 0.09 mM KNO3, 0.18 mM Ca(NO3)2·4H2O, and 0.09 mM KH 2 PO 4 and the micronutrients 20 μM NaEDTAFe· 3H2O, 6.7 μM MnCl2·4H2O, 9.4 μM H3BO3, 0.015 μM (NH 4 ) 6Mo 7 O 24 ·4H 2 O, 0.15 μM ZnSO 4 ·7H 2 O, and 0.16 μM CuSO4·5H2O. Each pot was given one of four nanoparticle treatments: 0, 2, 20, or 200 mg L−1 Fe2O3 NPs. The nanoparticle suspensions were sonicated in a water bath for 30 min prior to use and added to the pots with the Kimura nutrient solution. After 1 week, seedlings were harvested, washed with 1 mM HNO3, and rinsed three times with ice-cold 2 mM CaCl 2 solution and Milli-Q water. The roots were washed thoroughly to eliminate surface adsorbents that could compromise the accuracy of the subsequent analyses. TEM of root NP uptake Fresh roots from the control (0 mg L−1) and 200 mg L−1 NP treatments were observed using transmission electron microscopy (TEM) to analyze the uptake, translocation, and aggregation of Fe2O3 NPs. Samples for the TEM were prepared following the standard procedures (Zhang et al. 2012). Root apices were prefixed in 2.5 % glutaraldehyde, dehydrated in a
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graded acetone series, and embedded in Spurr’s resin. Ultrathin sections (90 nm) were obtained using a microtome with a diamond knife. To determine if Fe2O3 NPs had entered root cells, sections were observed using a JEM-1230 (JEOL, Japan) TEM operating at 80 kV.
ICP-MS analysis Roots and shoots were separated and washed thoroughly with deionized water and lyophilized with a freeze dryer (Alpha 1– 2 LD Plus, Christ, Germany). The dried tissues were digested with a mixture of concentrated plasma-pure HNO3 and H2O2 (v/v, 4:1) on a heating plate. The obtained residual solutions were then diluted with deionized water and analyzed by ICPMS (Thermo X7, USA). A standard reference (bush branches and leaves, GBW07602) was also digested and analyzed by ICP-MS to examine recovery. Indium (20 ng mL−1) was used as an internal standard to compensate for matrix suppression and signal drifting. Linearity was from 0.1 to 50 ng mL−1, and recovery from GBW07602 was 99 %. Spike recovery was 102 %, and detection limit was 0.01 ng mL−1.
Hormone measurements Frozen root and shoot samples (200–300 mg fresh weight) were ground and homogenized using a mortar and pestle with liquid nitrogen, then mixed with 5 mL cold 80 % methanol (v/ v) containing 100 mg L−1 ascorbic acid as an antioxidant. The mixture was stirred for 10 min and incubated for 48 h at 4 °C. The mixture was centrifuged at 3500 rpm for 15 min at 4 °C and the supernatant collected. Quantitative analyses of two common phytohormones, abscisic acid (ABA) and indole-3acetic acid (IAA), were performed by ELISA according to Gawronska et al. (2003).
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Results and discussion Fe2O3 NPs characterization The Fe2O3 nanoparticles used in this study had an average diameter of 10±3.2 nm (Fig. 1). After suspension in deionized water, they had a hydrodynamic radius of 154.3 nm and were stable, with a Zeta potential of −9.27 mV. Biomass of rice plants There was no significant difference in total biomass for transgenic or non-transgenic rice plants as a factor of Fe2O3 NP concentration treatment (Table 1). Biomass of plants was not correlated with treatment and that transgenic and nontransgenic plants did not differ significantly in biomass within treatments. There was also no significant difference in chlorophyll content among plants treated with all Fe2O3 NPs concentrations (data not shown). TEM imaging The distribution of Fe2O3 NPs in vivo was visualized by TEM (Fig. 2). Imaging showed that Fe2O3 NPs penetrated the cell membrane, emerged in cells, and accumulated on the epidermis of the root. Fe2O3 NPs were more frequently observed in the root cells from transgenic plants than non-transgenic plants. Further investigation should be done with elemental determination and metal speciation, but these results are in line with previous work suggesting that CeO2 NPs can be taken up by roots and transported to shoots in conventional and transgenic Bt cotton (Li et al. 2014). We found that SiO2 NPs were transported from roots to shoot via xylem sap in rice and CeO2 NPs also can be absorbed by this way (Le et al. 2014, 2015). Since xylem sap of rice contains Fe elements, we
Enzyme activities Roots and shoots were separated and homogenized with phosphate buffer solution (PBS, 50 mM, pH 7.8) in an ice bath, then centrifuged at 10,000 g and 4 °C for 10 min. The supernatants were kept for analyses of SOD and POD enzyme activities, and MDA contents using assay kits from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Statistical analysis The data across treatment groups were analyzed using oneway analysis of variance (ANOVA) and Turkey’s HSD test, which was performed using the statistical package SPSS Version 20.0. The data were expressed as means standard deviation (SD), a confidence interval of 95 % (p
