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The mutual effects of graphene oxide nanosheets and cadmium on the growth, cadmium uptake and accumulation in rice
Plant Physiology and Biochemistry 147 (2020) 289–294
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Short communication
The mutual effects of graphene oxide nanosheets and cadmium on the growth, cadmium uptake and accumulation in rice
T
Jie Lia,b, Fan Wua,b, Qing Fanga,b, Zheng Wua,b, Qingyun Duana,b, Xuede Lia,b, Wenling Yea,b,∗ a Hefei Scientific Observing and Experimental Station of Agro-Environment, Ministry of Agriculture, Anhui Agricultural University, 130 Changjiang West Road, Hefei, 230036, Anhui, PR China b Anhui Province Key Laboratory of Farmland Ecological Conservation and Pollution Prevention, School of Resources and Environment, Anhui Agricultural University, 130 Changjiang West Road, Hefei, 230036, Anhui, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Graphene oxide Cadmium uptake Rice Germination Growth
The broad application and unique properties of graphene oxide (GO) nanosheets make them interact with other pollutants and subsequently alter their behaviors and toxicities. However, investigation on the effects of GO nanosheets on plant uptake of co-occurring heavy metals is scarce. We evaluated the mutual effects of cadmium (Cd) at 1 mg/L and different concentrated GO nanosheets (0, 1 and 10 mg/L) on the rice seed germination, further seedling growth, Cd uptake and accumulation in rice roots and shoots in a hydroponic system. The effects of GO were concentration dependent. GO alone at 1 mg/L showed no apparent effects, while GO alone at 10 mg/ L accelerated the rice seed germination and root growth due to the improved water uptake. Cd alone showed adverse effects on the rice seed germination, which was alleviated by the presence of GO at 1 or 10 mg/L. GO at 10 mg/L also increased the membrane permeability, thus enhancing Cd uptake by rice roots and shoots. These results indicate that GO can change the effects of Cd on the rice seed germination and Cd uptake as well as accumulation in the roots and shoots of rice seedlings, which is helpful for understanding the fate and ecotoxicological impacts of both GO and Cd.
1. Introduction Cadmium (Cd), as a non-nutritive and non-biodegradable element, can enter the ecosphere due to the various agricultural and industrial activities (Nagajyoti et al., 2010; Cui et al., 2013), which could further exert multiple adverse effects on the agriculture, including the inhibition of seed germination (Rizwan et al., 2016a), root elongation (Rizwan et al., 2016b) and overall growth as well as disruption of photosynthesis (Volland et al., 2014). Chronic exposure to Cd would cause various health problems such as renal dysfunction, cancer, osteoporosis and cardiovascular disease (Zhao et al., 2011). Additionally, the ecotoxicological effects of Cd to food crops is influenced by the cooccurring other contaminants in the environment, such as engineered nanoparticles (NPs). Understanding the processes which may potentially impact the toxicity of Cd or aggravate the Cd accumulation in corps is therefore of great importance. Graphene oxide (GO) nanosheets are graphene analogs possessing ample oxygen-containing functional groups, such as epoxy, hydroxyl, and carboxyl groups, decorating on the planes and edges of the
graphene layer. Therefore, they carry numerous isolated hydrophobic sp2 clusters within the hydrophilic sp3 C–O matrix (Li et al., 2017). Due to the unique physicochemical properties, GO nanosheets have exhibited great promise for potential applications in various fields, including hybrid materials, batteries, sensors, environmental pollution management, hydrogen storage, nanoelectronics, etc. (Zhao et al., 2014) The market for GO products is projected to approach $675 million by 2020, which could lead to a large amount of graphene-based waste (Ahmed and Rodrigues, 2013). With the continued increase in the demand and consumption of GO, GO will be inevitably discharged into the ecosphere either intentionally or unintentionally with adverse effects on human health and ecological security (Goodwin et al., 2018; Fadeel et al., 2018). Hence, clarifying the potential toxicity of GO and the corresponding response of plants is very important and is a critical issue for agricultural research. To date, several studies have been conducted to assess the potential ecotoxicological effects of GO, including the impacts on the seed germination, seedling growth, plant physiological and biochemical response and accumulation in plants (Chen et al., 2017; Li et al., 2018; Hu et al., 2015; He et al., 2018; Zhao et al., 2018),
∗ Corresponding author. Hefei Scientific Observing and Experimental Station of Agro-Environment, Ministry of Agriculture, Anhui Agricultural University, 130 Changjiang West Road, Hefei, 230036, Anhui, PR China. E-mail address: [email protected] (W. Ye).
https://doi.org/10.1016/j.plaphy.2019.12.034 Received 12 November 2019; Received in revised form 25 December 2019; Accepted 26 December 2019 Available online 27 December 2019 0981-9428/ © 2019 Elsevier Masson SAS. All rights reserved.
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2.3. Effects of GO nanosheets and Cd on the rice seed germination and further growth
and the results vary. The plentiful oxygen-containing functionalities on the GO surface can form stable surface complexes with heavy metal ions (e.g., Pb, Cd and As), thus further impacting the environmental fate and ecotoxicological effects of both GO nanosheets and heavy metal ions (Boukhvalov and Katsnelson, 2008; Ren et al., 2018). On the one hand, for example, GO nanosheets were good adsorbents for Cd uptake from aquatic environment, showing a maximum adsorption amount of 106.3 mg/g, which is more than 9 times higher than that of granular activated carbon (10.1 mg/g) (Zhao et al., 2011). Consequently, GO nanosheets could perform as carriers for heavy metals to enter the plant tissues or retain heavy metals in the plant rhizosphere (Hu et al., 2014, 2018). These complex reciprocal interactions between GO and heavy metals suggest that the impact of GO on the seed germination and further seedling growth as well as the plant uptake of heavy metals could be different in the ternary GO-metal ions-plant systems from binary GO-plants or metal ion-plants systems. Therefore, illuminating the mutual effects of co-occurring GO and heavy metals is helpful for understanding about their environmental toxicities on plants, while researches clarifying these combined effects are less and inconsistent. The potential effects of GO on plants under environmental stress could be complicated. Therefore, the environmental response and toxicity of GO on plants in the presence of heavy metal ions cannot be directly extrapolated from the available findings. However, available information in connection with this topic is scarce and is crucial for food safety. Considering the high probability of Cd and GO copresence in the ecological environment and their potential complicated reciprocities, the exploration on the response of plants to the co-occurring Cd and GO is therefore meaningful. The Cd concentration used in this study was 1 mg/L based on our previous study (Ye et al., 2019). Two relatively low concentrations (1 and 10 mg/L) of GO nanosheets were selected to probe their influence on the physiological actions of plants and plant Cd uptake at environmentally relevant levels (Hu et al., 2014, 2018). We assumed that the copresence of GO and Cd could affect the seed germination and seedling growth of rice as well as influence the Cd toxicity in rice. Thus, the main targets herein were to study the mutual effects of Cd and GO on the seed germination, seedling growth as well as Cd uptake by rice and to investigate the potential mechanisms resulting in the altered changes.
The surface of rice seeds with similar size and shape were sterilized by using 0.5% NaOCl for 15 min and then thoroughly washed by deionized water. Double-layer filter paper in a diameter of 9 cm was placed in each Petri dish followed by adding a mixed solution with different concentrated GO nanosheets and Cd. Two concentrations of GO nanosheets (1 and 10 mg/L) and Cd at one level of 1 mg/L were predesigned in this study. Each treatment had four replicates. 30 seeds were arranged in each Petri dish with consistent growth spacing and germinated in a climate chamber at 3000 Lx irradiation under 28 °C and 80% humidity. After 10 days, the germination rate, dry root and shoot weight of rice seedlings were measured. 2.4. Effect of GO nanosheets on the Cd uptake by rice seedlings The rice seeds were germinated in 0.5 mM CaCl2 Nylon yarn net for 7 days. After germination, each rice seedling was transferred into 200 mL plastic cask filled with Hoagland solution as described before (Ye et al., 2017) and were cultivated in these solutions at pH 5.5 for 30 days under plant growth lamp providing 250 μmol m2 s−1 photosynthetic photon flux density (16 h light–8 h dark photoperiod, temperature 30 °C/25 °C and 70% humidity). The growth media were renewed every three days. Afterward, rice seedlings were cultivated in Hoagland solution containing different concentrations of GO and Cd for an additional 7 days under the abovementioned conditions. Seedlings grown in just Hoagland solution were set as controls. Each treatment had three replicates. 2.5. Detection of total Cd in the roots and shoots of rice seedlings Root samples were immersed in 10 mmol/L ethylenediaminetetraacetic acid solution for 5 min to remove surface-adsorbed Cd and then washed by pure water for three times. The total Cd in the roots and shoots of rice seedlings was determined by HNO3/HClO4 (87/13, v/v) (Ye et al., 2011). This final solution was then analyzed through an inductively coupled plasma mass spectrometry (ICP-MS, PerkinElmer mod. DRCII, Waltham, MA). 2.6. Electrolyte leakage analysis
2. Materials and methods Electrolyte leakage was measured to detect the membrane permeability. The roots of rice seedlings were washed by using pure water for three times to remove surface-adhered electrolytes. Then, the root samples were put into closed vials containing 30 mL of pure water and incubated at 25 °C on a shaker at 500 rpm for 1 h followed by measuring the electrical conductivity of the solution (EC0). The samples were then put in the boiling water for 10 min, and the final electrical conductivity (ECf) at 25 °C was measured after equilibrium. The electrolyte leakage was calculated as follows: electrolyte leakage (%) = (EC0/ECf) × 100.
2.1. Chemicals and materials GO nanosheets were obtained by using the improved Hummers' method (Li et al., 2017) from the flake graphite with an average particle diameter of 20 mm purchased from Qingdao Tianhe Graphite Co. Ltd., China. Rice (Oryza sativa L. cv. Liangyou 8106) seeds were provided commercially by the Win-All Hi-Tech Seed Co. Ltd. Cd(NO3)2 (analytically pure) was purchased from Aladdin reagent (Shanghai) Co. Ltd.
2.2. Characterization of GO nanosheets
3. Results and discussion
GO nanosheets were identified by using the transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy (Zhao et al., 2011). The XPS measurement was performed on an ESCALab 220IXL system. The TEM image was obtained with a JEM-2010. Raman spectra were conducted on a Renishew in Via Raman spectrometer (Renishaw plc, UK). The laser excitation was provided by a conventional model laser operated at a wavelength of 514 nm. Fourier-transformed infrared (FTIR) spectroscopy was achieved on a Nicolet Magana-IR750 spectrometer using the KBr pellet method.
3.1. Characteristics of GO nanosheets A visibly homogenous dispersion of GO nanosheets was obtained by using the concentrated H2SO4 and KMnO4 to oxidize the graphite layers (Li et al., 2017). The C1s XPS spectrum (Fig. 1A) reveals that the GO nanosheets were significantly oxidized by the oxidants with different functional groups, including the nonoxygenated ring C centered at 284.5 eV (70.5%), the C atom in C–O bond centered at 286.2 eV (19.1%), the carbonyl C centered at 287.8 eV (10.2%) and the carboxylate carbon (O–C]O) centered at 289.0 eV (0.2%) (Zhao et al., 2011). The dispersion with a brown-yellow color of GO nanosheets 290
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Fig. 1. C 1s XPS spectrum (A), TEM image (B) and Raman pattern (C) of GO nanosheets.
hydrophilic and may not favorable for the release and transport of water. For example, germination rates of rice seeds were remarkably decreased with the addition of large amounts of GO (100, 500 and 1500 mg/L) (Yin et al., 2018). Thus, an appropriate amount of GO may function as a promising nontoxic additive to promote the germination of rice seeds. On the contrary, nanoparticles such as Au, Ag, ZnO, CeO2 or CNTs would penetrate the seeds to stimulate the germination (Lahiani et al., 2013; Khodakovskaya et al., 2013; Gardea-Torresdey et al., 2003; Bandyopadhyay et al., 2015; Zhao et al., 2012), and their accumulation in plants could cause food safety problems. Fig. 2 reveals that the germination of rice seeds was inhibited by the addition of 1 mg/L Cd (57.0%, P = 0.041) in comparison with the blank experiment (62.7%). Yin et al. (2018) observed that the germination rate of rice seeds (Oryza sativa L. cv. Changyou 99-1) was remarkably inhibited by Cd at high concentration of 20 mg/L but was not influenced by Cd at a relatively low concentration of 5 mg/L. Different types of rice seeds may be the reason for this discrepancy. In the copresence of Cd and GO (Cd at 1 mg/L and GO at 1 mg/L, abbr. Cd1GO1, Cd at 1 mg/L and GO at 10 mg/L, abbr. Cd1GO10), the germination ratios of rice seeds treated with Cd1GO1 and Cd1GO10 were 68.0% (P = 0.038) and 76.0% (P = 0.010), respectively, disclosing that the germination ratios of rice seeds were promoted by the coexistence of Cd and GO in comparison with the single Cd treatment (57.0%). This experimental phenomenon also reveals that the addition of GO nanosheets alleviated the inhibitive effects of Cd on the germination of rice seeds. Compared with the single GO treatment, there is no significant difference in the germination of rice seeds treated with Cd1GO1 (P = 0.535) and Cd1GO10 (P = 0.641), revealing that the presence of Cd cannot influence the acceleration effect of GO exerted on the rice seed germination. One reasonable explanation was that Cd was adsorbed by GO and left in the water, which was evidenced by the previous study (Zhao et al., 2011). Low concentration of Cd (1 mg/L) cannot consume all GO (1 mg/L or 10 mg/L) present in the nutrient solutions due to the abundant oxygen-containing functional groups, thus no noticeable effect of Cd exerted on GO was observed.
remained stable over several months of aging time, which promotes the formation of complexes with metal ions. The TEM image reveals heterogeneous GO nanosheets with sharp and irregular edges (Fig. 1B). It is reported that the thickness of one-layered GO nanosheet is ~0.8–1.0 nm (Zhang et al., 2010). The Raman spectrum of GO nanosheets is presented in Fig. 1C. The peak intensity ratio (ID/IG) of D band (1350 cm−1) and G band (1580 cm−1) is widely applied to indicate the local defects/disorders in GO nanosheets. In this study, the ID/IG was 0.85, disclosing low GO defects/disorders. 3.2. Effects of GO nanosheets and Cd on the seed germination of rice The seed germination results are shown in Fig. 2. In comparison with the blank treatment (62.7%), the germination ratios of rice seeds treated with GO at 1 and 10 mg/L reached 64.0% (P = 0.846) and 79.0% (P = 0.019) after 10 days, respectively, revealing that the existence of GO nanosheets stimulated the germination of rice seeds. Moisture is crucial for the rice seed germination. In the initial germination stage, the hydrotropic substances in the rice seeds began to collect water, and when the embryo started to develop, the rice seeds continued to collect water. The oxygen-containing functional groups in GO nanosheets could uptake water, and the hydrophobic sp2 domains could transport water to the rice seeds, thus accelerating the germination of rice seedlings. He et al. (2018) also found that GO at a low concentration remarkably stimulated the germination of spinach and chive in soil due to the hydrophilic nature and water-transporting properties of GO. They also found no GO either on the surface or inside the cells of spinach and chive, disclosing that GO was not phytotoxic. Note that, an excessive amount of GO nanosheets, which carry large amounts of oxygen-containing functionalities, was probably overly
3.3. Effects of GO nanosheets and Cd on the growth of rice seedlings The roots and shoots of all rice seedlings cultivated in Cd alone (1 mg/L), GO alone (1 and 10 mg/L) and the co-presence of Cd1GO1 and Cd1GO10 remained healthy, and no visible adverse effects on rice seedling growth were observed. Dry root and shoot weight further acted as growth indicators to evaluate the effects of Cd and GO on the growth of rice seedlings and the results are shown in Fig. 3. Obviously, Cd alone at 1 mg/L had no significant effects on the dry root or shoot weight of rice seedlings in comparison with the controls. Rossi et al. also found that the dry biomass of root and shoot systems in soybean seedlings was not influenced by 1 mg/L of Cd (Rossi et al., 2018). Similarly, GO alone at 1 mg/L had no obvious impacts on the dry root or shoot weight, while 10 mg/L of GO remarkably increased the dry root weight of rice seedlings (Fig. 2A). Thus, GO at the concentration of 10 mg/L can promote the root growth of rice seedlings, which is in accordance with
Fig. 2. The germination ratios of rice seeds after 10-day exposure to various concentrations of Cd and/or GO. GO, 1 or 10 mg/L; Cd, 1 mg/L. 291
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Fig. 3. The dry root (A) and shoot (B) weights of rice seedlings after 10-day exposure to various concentrations of Cd and/or GO. GO, 1 or 10 mg/L; Cd, 1 mg/L.
Fig. 4. The Cd uptake in the root (A) and shoot (B) systems of rice seedlings after exposure to various concentrations of Cd and/or GO. GO, 1 or 10 mg/L; Cd, 1 mg/L.
Fig. 5. Fourier transform infrared spectra (A) and electrolyte leakage in root cells (B). GO, 1 or 10 mg/L; Cd, 1 mg/L.
nanoparticles (50 μg/L) remarkably facilitated the root elongation and biomass of hydroponic rice plants (Yang et al., 2019). Generally, mechanisms for nanomaterials to improve the plant growth include: (i) low dose toxicant-induced repair and overcompensation (Yang et al., 2019; Wang et al., 2013); (ii) reduction in the formation or action of ethylene in plants (Negi et al., 2008); (iii) improving the water uptake (He et al., 2018); and so on. In this study, the introduction of GO would induce new pores or enlargement of seed coat pores, thus enhancing water uptake and improving root growth. For instance, the dry mass of roots especially shoots increased upon the introduction of GO at 10 mg/L compared with the single Cd treatment. Although the difference between single Cd treatment and Cd1GO10 group is not remarkable, the low Cd concentration could be the reason. The positive response of GO at an appropriate concentration towards
the acceleration effects of carbon nanotubes and C60 at 50 μg/mL (Nair et al., 2012), and AgNO3 as well as Ag nanoparticles at 50 μg/L on the root length of rice seedlings (Yang et al., 2019). Nair et al. (2012) found that graphene at the concentration of 50 μg/mL inhabited the root and shoot growth of rice seedlings. Another work reported by Hu et al. found that the growth of wheat seedlings was not inhabited by GO at 0.1 and 10 mg/L (Hu et al., 2014). Recently, this group (Hu et al., 2018) observed that GO at a concentration of 1 mg/L slightly accelerated the growth of L. minor. Thus, the effect of GO on the growth of plants was dependent on its concentration and the kinds of plants, which is consistent with that of Ag nanoparticles (Yang et al., 2019; Thuesombat et al., 2014). Previous studies reported that high-dose Ag nanoparticles (above 10 mg/L) remarkably inhibited the growth of Rice (Oryza sativa L. cv. KDML 105) (Thuesombat et al., 2014), while low concentrated Ag 292
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5. Funding information
seed germination and further growth of the seedlings is superior to that of metallic engineered nanoparticles, such as CeO2 or ZnO NPs (Wang et al., 2018). Unfortunately, this promotion effect was lessened by the presence of Cd, as evidenced by the no discernible effect of Cd1GO1 and Cd1GO10 on the dry weight.
We acknowledge the financial support from the National Natural Science Foundation of China (21806001), the Natural Project of Anhui Provincial Department of Education (KJ2018A0126), the Provincial Foundation for Excellent Young Talents of Colleges and Universities of Anhui Province (No.gxyq2018004) and the Open Fund of Anhui Province Key Laboratory of Farmland Ecological Conservation and Pollution Prevention, FECPP201904. CAS Key Laboratory of Photovoltaic and Energy Conservation Materials, Chinese Academy of Sciences is also acknowledged.
3.4. Effect of GO nanosheets on the Cd uptake by rice seedlings The intracellular Cd uptake in rice seedlings is unquestionable, but the impact of GO nanosheets on the intracellular Cd uptake remains unclear. Fig. 4 reveals that the Cd uptake amount without GO exposure was 366.7 and 18.3 mg/kg in the root and shoot system, respectively. Compared with the Cd alone treatment in both root and shoot systems, significantly higher Cd uptake was observed by the Cd1GO10 groups, while no significant change in Cd uptake by the Cd1GO1 groups. Moreover, the exposure of Cd1GO10 (34.6 mg/kg) induced obviously larger Cd uptake with respect to that of Cd1GO1 (25.0 mg/kg) in the shoot system. Thus, GO at the concentration of 10 mg/L promoted Cd uptake and accumulation in both root and shoot systems, while GO at 1 mg/L led to no noticeable effect on Cd uptake. The regulation of Cd uptake and accumulation by GO nanosheets in the roots and shoots of rice seedlings was therefore concentration dependent. Some similar effects have also been observed by Hu et al. (2014) for the mutual impacts of GO and As on the As uptake in wheat, and by Hu et al. (2018) for the effect of GO on Cu uptake in Lemna minor L. Previously, several studies have investigated the impact of NPs including GO nanosheets on the heavy metal uptake by plants (Hu et al., 2014, 2018; Rossi et al., 2017, 2018; Wang et al., 2018; Rizwan et al., 2019). The underlying mechanisms reported with relation to the alteration of heavy metal uptake onto plants by the presence of NPs include: (i) the adsorption of heavy metals onto NPs so that NPs served as heavy metal carriers; (ii) the chemical alternation in the plant rhizosphere, including enhanced excretion of root exudates; and (iii) the changes in the anatomical structure of plant roots. In this study, the FTIR spectra and electrolyte leakage in the rice roots were conducted to probe the structural damage in the root cell wall and plasma membrane. Compared with the blank treatment, the FTIR spectra of rice seedlings treated with Cd at 1 mg/L, GO at 1 or 10 mg/L, Cd1GO1 and Cd1GO10 have no noticeable changes (Fig. 5A), disclosing no distinct structural damage to the root cell wall. Fig. 5B reveals that compared with the control, GO alone did not induce apparent electrolyte leakage, while Cd alone and CdGO group lead to remarkable increase in the electrolyte leakage. Moreover, the electrolyte leakage caused by the CdGO group was much higher than that by Cd alone. It is possible that the co-presence of Cd and GO nanosheets promoted the permeability of rice root cells and therefore facilitated Cd uptake through passive transport across the plasma membrane. Hence, we can conclude that the increased membrane permeability enhanced the Cd uptake.
Contribution Wenling Ye and Jie Li conceived and designed the study. Fan Wu, Qing Fang, Zheng Wu, and Qingyun Duan performed the experiments. Jie Li wrote the paper. Xuede Li reviewed and edited the manuscript. All authors read and approved the manuscript. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Ahmed, F., Rodrigues, D.F., 2013. Investigation of acute effects of graphene oxide on wastewater microbial community: a case study. J. Hazard Mater. 256–257, 33–39. Bandyopadhyay, S., Plascencia-Villa, G., Mukherjee, A., Rico, C.M., José-Yacamán, M., Peralta-Videa, J.R., Gardea-Torresdey, J.L., 2015. Comparative phytotoxicity of ZnO NPs, bulk ZnO, and ionic zinc onto the alfalfa plants symbiotically associated with Sinorhizobium meliloti in soil. Sci. Total Environ. 515–516, 60–69. Boukhvalov, D.W., Katsnelson, M.I., 2008. Modeling of graphite oxide. J. Am. Chem. Soc. 130, 10697–10701. Chen, L., Wang, C., Li, H., Qu, X., Yang, S., Chang, X., 2017. Bioaccumulation and toxicity of 13C-skeleton labeled graphene oxide in wheat. Environ. Sci. Technol. 51, 10146–10153. Cui, H., Zhou, J., Zhao, Q., Si, Y., Mao, J., Fang, G., Liang, J., 2013. Fractions of Cu, Cd, and enzyme activities in a contaminated soil as affected by applications of micro- and nanohydroxyapatite. J. Soils Sediments 13, 742–752. Fadeel, B., Bussy, C., Merino, S., Vázquez, E., Flahaut, E., Mouchet, F., Evariste, L., Gauthier, L., Koivisto, A.J., Vogel, U., Martín, C., Delogu, L.G., Buerki-Thurnherr, T., Wick, P., Beloin-Saint-Pierre, D., Hischier, R., Pelin, M., Candotto Carniel, F., Tretiach, M., Cesca, F., Benfenati, F., Scaini, D., Ballerini, L., Kostarelos, K., Prato, M., Bianco, A., 2018. Safety assessment of graphene-based materials: focus on human health and the environment. ACS Nano 12, 10582–10620. Gardea-Torresdey, J.L., Gomez, E., Peralta-Videa, J.R., Parsons, J.G., Troiani, H., JoseYacaman, M., 2003. Alfalfa sprouts: a natural source for the synthesis of silver nanoparticles. Langmuir 19, 1357–1361. Goodwin, D.G., Adeleye, A.S., Sung, L., Ho, K.T., Burgess, R.M., Petersen, E.J., 2018. Detection and quantification of graphene-family nanomaterials in the environment. Environ. Sci. Technol. 52, 4491–4513. He, Y., Hu, R., Zhong, Y., Zhao, X., Chen, Q., Zhu, H., 2018. Graphene oxide as a water transporter promoting germination of plants in soil. Nano Res. 11, 1928–1937. Hu, X., Kang, J., Lu, K., Zhou, R., Mu, L., Zhou, Q., 2014. Graphene oxide amplifies the phytotoxicity of arsenic in wheat. Sci. Rep. 4, 6122. Hu, X., Ouyang, S., Mu, L., An, J., Zhou, Q., 2015. Effects of graphene oxide and oxidized carbon nanotubes on the cellular division, microstructure, uptake, oxidative stress, and metabolic profiles. Environ. Sci. Technol. 49, 10825–10833. Hu, C., Liu, L., Li, X., Xu, Y., Ge, Z., Zhao, Y., 2018. Effect of graphene oxide on copper stress in Lemna minor L.: evaluating growth, biochemical responses, and nutrient uptake. J. Hazard Mater. 341, 168–176. Khodakovskaya, M.V., Kim, B.-S., Kim, J.N., Alimohammadi, M., Dervishi, E., Mustafa, T., Cernigla, C.E., 2013. Carbon nanotubes as plant growth regulators: effects on tomato growth, reproductive system, and soil microbial community. Small 9, 115–123. Lahiani, M.H., Dervishi, E., Chen, J., Nima, Z., Gaume, A., Biris, A.S., Khodakovskaya, M.V., 2013. Impact of carbon nanotube exposure to seeds of valuable crops. ACS Appl. Mater. Interfaces 5, 7965–7973. Li, J., Wu, Q., Wang, X., Chai, Z., Shi, W., Hou, J., Hayat, T., Alsaedi, A., Wang, X., 2017. Heteroaggregation behavior of graphene oxide on Zr-based metal–organic frameworks in aqueous solutions: a combined experimental and theoretical study. J. Mater. Chem. 5, 20398–20406. Li, F., Sun, C., Li, X., Yu, X., Luo, C., Shen, Y., Qu, S., 2018. The effect of graphene oxide on adventitious root formation and growth in apple. Plant Physiol. Biochem. 129, 122–129. Nagajyoti, P.C., Lee, K.D., Sreekanth, T.V.M., 2010. Heavy metals, occurrence and
4. Conclusions The physical and biochemical response of rice seeds and seedlings as well as the Cd uptake were probed by the co-presence of GO nanosheets and Cd in a hydroponic system. GO nanosheets could uptake and transport water due to their hydrophilic nature, thus promoting the rice seed germination and further seedling growth. The inhibitive effects of Cd on the seed germination was alleviated by the co-accurring GO nanosheets. Besides, the presence of GO nanosheets at 10 mg/L improved Cd uptake in both root and shoot system due to the enhancement in the membrane permeability. These findings can provide new insights for understanding the reciprocal effects of heavy metals and engineered or new emerging nanomaterials in plants. Additionally, further studies should be carried out in a soil matrix considering its complex environmental conditions, such as the microbial communities, rhizosphere and coating, which may influence the heavy metal toxicity and accumulation. 293
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Short communication
The mutual effects of graphene oxide nanosheets and cadmium on the growth, cadmium uptake and accumulation in rice
T
Jie Lia,b, Fan Wua,b, Qing Fanga,b, Zheng Wua,b, Qingyun Duana,b, Xuede Lia,b, Wenling Yea,b,∗ a Hefei Scientific Observing and Experimental Station of Agro-Environment, Ministry of Agriculture, Anhui Agricultural University, 130 Changjiang West Road, Hefei, 230036, Anhui, PR China b Anhui Province Key Laboratory of Farmland Ecological Conservation and Pollution Prevention, School of Resources and Environment, Anhui Agricultural University, 130 Changjiang West Road, Hefei, 230036, Anhui, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Graphene oxide Cadmium uptake Rice Germination Growth
The broad application and unique properties of graphene oxide (GO) nanosheets make them interact with other pollutants and subsequently alter their behaviors and toxicities. However, investigation on the effects of GO nanosheets on plant uptake of co-occurring heavy metals is scarce. We evaluated the mutual effects of cadmium (Cd) at 1 mg/L and different concentrated GO nanosheets (0, 1 and 10 mg/L) on the rice seed germination, further seedling growth, Cd uptake and accumulation in rice roots and shoots in a hydroponic system. The effects of GO were concentration dependent. GO alone at 1 mg/L showed no apparent effects, while GO alone at 10 mg/ L accelerated the rice seed germination and root growth due to the improved water uptake. Cd alone showed adverse effects on the rice seed germination, which was alleviated by the presence of GO at 1 or 10 mg/L. GO at 10 mg/L also increased the membrane permeability, thus enhancing Cd uptake by rice roots and shoots. These results indicate that GO can change the effects of Cd on the rice seed germination and Cd uptake as well as accumulation in the roots and shoots of rice seedlings, which is helpful for understanding the fate and ecotoxicological impacts of both GO and Cd.
1. Introduction Cadmium (Cd), as a non-nutritive and non-biodegradable element, can enter the ecosphere due to the various agricultural and industrial activities (Nagajyoti et al., 2010; Cui et al., 2013), which could further exert multiple adverse effects on the agriculture, including the inhibition of seed germination (Rizwan et al., 2016a), root elongation (Rizwan et al., 2016b) and overall growth as well as disruption of photosynthesis (Volland et al., 2014). Chronic exposure to Cd would cause various health problems such as renal dysfunction, cancer, osteoporosis and cardiovascular disease (Zhao et al., 2011). Additionally, the ecotoxicological effects of Cd to food crops is influenced by the cooccurring other contaminants in the environment, such as engineered nanoparticles (NPs). Understanding the processes which may potentially impact the toxicity of Cd or aggravate the Cd accumulation in corps is therefore of great importance. Graphene oxide (GO) nanosheets are graphene analogs possessing ample oxygen-containing functional groups, such as epoxy, hydroxyl, and carboxyl groups, decorating on the planes and edges of the
graphene layer. Therefore, they carry numerous isolated hydrophobic sp2 clusters within the hydrophilic sp3 C–O matrix (Li et al., 2017). Due to the unique physicochemical properties, GO nanosheets have exhibited great promise for potential applications in various fields, including hybrid materials, batteries, sensors, environmental pollution management, hydrogen storage, nanoelectronics, etc. (Zhao et al., 2014) The market for GO products is projected to approach $675 million by 2020, which could lead to a large amount of graphene-based waste (Ahmed and Rodrigues, 2013). With the continued increase in the demand and consumption of GO, GO will be inevitably discharged into the ecosphere either intentionally or unintentionally with adverse effects on human health and ecological security (Goodwin et al., 2018; Fadeel et al., 2018). Hence, clarifying the potential toxicity of GO and the corresponding response of plants is very important and is a critical issue for agricultural research. To date, several studies have been conducted to assess the potential ecotoxicological effects of GO, including the impacts on the seed germination, seedling growth, plant physiological and biochemical response and accumulation in plants (Chen et al., 2017; Li et al., 2018; Hu et al., 2015; He et al., 2018; Zhao et al., 2018),
∗ Corresponding author. Hefei Scientific Observing and Experimental Station of Agro-Environment, Ministry of Agriculture, Anhui Agricultural University, 130 Changjiang West Road, Hefei, 230036, Anhui, PR China. E-mail address: [email protected] (W. Ye).
https://doi.org/10.1016/j.plaphy.2019.12.034 Received 12 November 2019; Received in revised form 25 December 2019; Accepted 26 December 2019 Available online 27 December 2019 0981-9428/ © 2019 Elsevier Masson SAS. All rights reserved.
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2.3. Effects of GO nanosheets and Cd on the rice seed germination and further growth
and the results vary. The plentiful oxygen-containing functionalities on the GO surface can form stable surface complexes with heavy metal ions (e.g., Pb, Cd and As), thus further impacting the environmental fate and ecotoxicological effects of both GO nanosheets and heavy metal ions (Boukhvalov and Katsnelson, 2008; Ren et al., 2018). On the one hand, for example, GO nanosheets were good adsorbents for Cd uptake from aquatic environment, showing a maximum adsorption amount of 106.3 mg/g, which is more than 9 times higher than that of granular activated carbon (10.1 mg/g) (Zhao et al., 2011). Consequently, GO nanosheets could perform as carriers for heavy metals to enter the plant tissues or retain heavy metals in the plant rhizosphere (Hu et al., 2014, 2018). These complex reciprocal interactions between GO and heavy metals suggest that the impact of GO on the seed germination and further seedling growth as well as the plant uptake of heavy metals could be different in the ternary GO-metal ions-plant systems from binary GO-plants or metal ion-plants systems. Therefore, illuminating the mutual effects of co-occurring GO and heavy metals is helpful for understanding about their environmental toxicities on plants, while researches clarifying these combined effects are less and inconsistent. The potential effects of GO on plants under environmental stress could be complicated. Therefore, the environmental response and toxicity of GO on plants in the presence of heavy metal ions cannot be directly extrapolated from the available findings. However, available information in connection with this topic is scarce and is crucial for food safety. Considering the high probability of Cd and GO copresence in the ecological environment and their potential complicated reciprocities, the exploration on the response of plants to the co-occurring Cd and GO is therefore meaningful. The Cd concentration used in this study was 1 mg/L based on our previous study (Ye et al., 2019). Two relatively low concentrations (1 and 10 mg/L) of GO nanosheets were selected to probe their influence on the physiological actions of plants and plant Cd uptake at environmentally relevant levels (Hu et al., 2014, 2018). We assumed that the copresence of GO and Cd could affect the seed germination and seedling growth of rice as well as influence the Cd toxicity in rice. Thus, the main targets herein were to study the mutual effects of Cd and GO on the seed germination, seedling growth as well as Cd uptake by rice and to investigate the potential mechanisms resulting in the altered changes.
The surface of rice seeds with similar size and shape were sterilized by using 0.5% NaOCl for 15 min and then thoroughly washed by deionized water. Double-layer filter paper in a diameter of 9 cm was placed in each Petri dish followed by adding a mixed solution with different concentrated GO nanosheets and Cd. Two concentrations of GO nanosheets (1 and 10 mg/L) and Cd at one level of 1 mg/L were predesigned in this study. Each treatment had four replicates. 30 seeds were arranged in each Petri dish with consistent growth spacing and germinated in a climate chamber at 3000 Lx irradiation under 28 °C and 80% humidity. After 10 days, the germination rate, dry root and shoot weight of rice seedlings were measured. 2.4. Effect of GO nanosheets on the Cd uptake by rice seedlings The rice seeds were germinated in 0.5 mM CaCl2 Nylon yarn net for 7 days. After germination, each rice seedling was transferred into 200 mL plastic cask filled with Hoagland solution as described before (Ye et al., 2017) and were cultivated in these solutions at pH 5.5 for 30 days under plant growth lamp providing 250 μmol m2 s−1 photosynthetic photon flux density (16 h light–8 h dark photoperiod, temperature 30 °C/25 °C and 70% humidity). The growth media were renewed every three days. Afterward, rice seedlings were cultivated in Hoagland solution containing different concentrations of GO and Cd for an additional 7 days under the abovementioned conditions. Seedlings grown in just Hoagland solution were set as controls. Each treatment had three replicates. 2.5. Detection of total Cd in the roots and shoots of rice seedlings Root samples were immersed in 10 mmol/L ethylenediaminetetraacetic acid solution for 5 min to remove surface-adsorbed Cd and then washed by pure water for three times. The total Cd in the roots and shoots of rice seedlings was determined by HNO3/HClO4 (87/13, v/v) (Ye et al., 2011). This final solution was then analyzed through an inductively coupled plasma mass spectrometry (ICP-MS, PerkinElmer mod. DRCII, Waltham, MA). 2.6. Electrolyte leakage analysis
2. Materials and methods Electrolyte leakage was measured to detect the membrane permeability. The roots of rice seedlings were washed by using pure water for three times to remove surface-adhered electrolytes. Then, the root samples were put into closed vials containing 30 mL of pure water and incubated at 25 °C on a shaker at 500 rpm for 1 h followed by measuring the electrical conductivity of the solution (EC0). The samples were then put in the boiling water for 10 min, and the final electrical conductivity (ECf) at 25 °C was measured after equilibrium. The electrolyte leakage was calculated as follows: electrolyte leakage (%) = (EC0/ECf) × 100.
2.1. Chemicals and materials GO nanosheets were obtained by using the improved Hummers' method (Li et al., 2017) from the flake graphite with an average particle diameter of 20 mm purchased from Qingdao Tianhe Graphite Co. Ltd., China. Rice (Oryza sativa L. cv. Liangyou 8106) seeds were provided commercially by the Win-All Hi-Tech Seed Co. Ltd. Cd(NO3)2 (analytically pure) was purchased from Aladdin reagent (Shanghai) Co. Ltd.
2.2. Characterization of GO nanosheets
3. Results and discussion
GO nanosheets were identified by using the transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy (Zhao et al., 2011). The XPS measurement was performed on an ESCALab 220IXL system. The TEM image was obtained with a JEM-2010. Raman spectra were conducted on a Renishew in Via Raman spectrometer (Renishaw plc, UK). The laser excitation was provided by a conventional model laser operated at a wavelength of 514 nm. Fourier-transformed infrared (FTIR) spectroscopy was achieved on a Nicolet Magana-IR750 spectrometer using the KBr pellet method.
3.1. Characteristics of GO nanosheets A visibly homogenous dispersion of GO nanosheets was obtained by using the concentrated H2SO4 and KMnO4 to oxidize the graphite layers (Li et al., 2017). The C1s XPS spectrum (Fig. 1A) reveals that the GO nanosheets were significantly oxidized by the oxidants with different functional groups, including the nonoxygenated ring C centered at 284.5 eV (70.5%), the C atom in C–O bond centered at 286.2 eV (19.1%), the carbonyl C centered at 287.8 eV (10.2%) and the carboxylate carbon (O–C]O) centered at 289.0 eV (0.2%) (Zhao et al., 2011). The dispersion with a brown-yellow color of GO nanosheets 290
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Fig. 1. C 1s XPS spectrum (A), TEM image (B) and Raman pattern (C) of GO nanosheets.
hydrophilic and may not favorable for the release and transport of water. For example, germination rates of rice seeds were remarkably decreased with the addition of large amounts of GO (100, 500 and 1500 mg/L) (Yin et al., 2018). Thus, an appropriate amount of GO may function as a promising nontoxic additive to promote the germination of rice seeds. On the contrary, nanoparticles such as Au, Ag, ZnO, CeO2 or CNTs would penetrate the seeds to stimulate the germination (Lahiani et al., 2013; Khodakovskaya et al., 2013; Gardea-Torresdey et al., 2003; Bandyopadhyay et al., 2015; Zhao et al., 2012), and their accumulation in plants could cause food safety problems. Fig. 2 reveals that the germination of rice seeds was inhibited by the addition of 1 mg/L Cd (57.0%, P = 0.041) in comparison with the blank experiment (62.7%). Yin et al. (2018) observed that the germination rate of rice seeds (Oryza sativa L. cv. Changyou 99-1) was remarkably inhibited by Cd at high concentration of 20 mg/L but was not influenced by Cd at a relatively low concentration of 5 mg/L. Different types of rice seeds may be the reason for this discrepancy. In the copresence of Cd and GO (Cd at 1 mg/L and GO at 1 mg/L, abbr. Cd1GO1, Cd at 1 mg/L and GO at 10 mg/L, abbr. Cd1GO10), the germination ratios of rice seeds treated with Cd1GO1 and Cd1GO10 were 68.0% (P = 0.038) and 76.0% (P = 0.010), respectively, disclosing that the germination ratios of rice seeds were promoted by the coexistence of Cd and GO in comparison with the single Cd treatment (57.0%). This experimental phenomenon also reveals that the addition of GO nanosheets alleviated the inhibitive effects of Cd on the germination of rice seeds. Compared with the single GO treatment, there is no significant difference in the germination of rice seeds treated with Cd1GO1 (P = 0.535) and Cd1GO10 (P = 0.641), revealing that the presence of Cd cannot influence the acceleration effect of GO exerted on the rice seed germination. One reasonable explanation was that Cd was adsorbed by GO and left in the water, which was evidenced by the previous study (Zhao et al., 2011). Low concentration of Cd (1 mg/L) cannot consume all GO (1 mg/L or 10 mg/L) present in the nutrient solutions due to the abundant oxygen-containing functional groups, thus no noticeable effect of Cd exerted on GO was observed.
remained stable over several months of aging time, which promotes the formation of complexes with metal ions. The TEM image reveals heterogeneous GO nanosheets with sharp and irregular edges (Fig. 1B). It is reported that the thickness of one-layered GO nanosheet is ~0.8–1.0 nm (Zhang et al., 2010). The Raman spectrum of GO nanosheets is presented in Fig. 1C. The peak intensity ratio (ID/IG) of D band (1350 cm−1) and G band (1580 cm−1) is widely applied to indicate the local defects/disorders in GO nanosheets. In this study, the ID/IG was 0.85, disclosing low GO defects/disorders. 3.2. Effects of GO nanosheets and Cd on the seed germination of rice The seed germination results are shown in Fig. 2. In comparison with the blank treatment (62.7%), the germination ratios of rice seeds treated with GO at 1 and 10 mg/L reached 64.0% (P = 0.846) and 79.0% (P = 0.019) after 10 days, respectively, revealing that the existence of GO nanosheets stimulated the germination of rice seeds. Moisture is crucial for the rice seed germination. In the initial germination stage, the hydrotropic substances in the rice seeds began to collect water, and when the embryo started to develop, the rice seeds continued to collect water. The oxygen-containing functional groups in GO nanosheets could uptake water, and the hydrophobic sp2 domains could transport water to the rice seeds, thus accelerating the germination of rice seedlings. He et al. (2018) also found that GO at a low concentration remarkably stimulated the germination of spinach and chive in soil due to the hydrophilic nature and water-transporting properties of GO. They also found no GO either on the surface or inside the cells of spinach and chive, disclosing that GO was not phytotoxic. Note that, an excessive amount of GO nanosheets, which carry large amounts of oxygen-containing functionalities, was probably overly
3.3. Effects of GO nanosheets and Cd on the growth of rice seedlings The roots and shoots of all rice seedlings cultivated in Cd alone (1 mg/L), GO alone (1 and 10 mg/L) and the co-presence of Cd1GO1 and Cd1GO10 remained healthy, and no visible adverse effects on rice seedling growth were observed. Dry root and shoot weight further acted as growth indicators to evaluate the effects of Cd and GO on the growth of rice seedlings and the results are shown in Fig. 3. Obviously, Cd alone at 1 mg/L had no significant effects on the dry root or shoot weight of rice seedlings in comparison with the controls. Rossi et al. also found that the dry biomass of root and shoot systems in soybean seedlings was not influenced by 1 mg/L of Cd (Rossi et al., 2018). Similarly, GO alone at 1 mg/L had no obvious impacts on the dry root or shoot weight, while 10 mg/L of GO remarkably increased the dry root weight of rice seedlings (Fig. 2A). Thus, GO at the concentration of 10 mg/L can promote the root growth of rice seedlings, which is in accordance with
Fig. 2. The germination ratios of rice seeds after 10-day exposure to various concentrations of Cd and/or GO. GO, 1 or 10 mg/L; Cd, 1 mg/L. 291
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Fig. 3. The dry root (A) and shoot (B) weights of rice seedlings after 10-day exposure to various concentrations of Cd and/or GO. GO, 1 or 10 mg/L; Cd, 1 mg/L.
Fig. 4. The Cd uptake in the root (A) and shoot (B) systems of rice seedlings after exposure to various concentrations of Cd and/or GO. GO, 1 or 10 mg/L; Cd, 1 mg/L.
Fig. 5. Fourier transform infrared spectra (A) and electrolyte leakage in root cells (B). GO, 1 or 10 mg/L; Cd, 1 mg/L.
nanoparticles (50 μg/L) remarkably facilitated the root elongation and biomass of hydroponic rice plants (Yang et al., 2019). Generally, mechanisms for nanomaterials to improve the plant growth include: (i) low dose toxicant-induced repair and overcompensation (Yang et al., 2019; Wang et al., 2013); (ii) reduction in the formation or action of ethylene in plants (Negi et al., 2008); (iii) improving the water uptake (He et al., 2018); and so on. In this study, the introduction of GO would induce new pores or enlargement of seed coat pores, thus enhancing water uptake and improving root growth. For instance, the dry mass of roots especially shoots increased upon the introduction of GO at 10 mg/L compared with the single Cd treatment. Although the difference between single Cd treatment and Cd1GO10 group is not remarkable, the low Cd concentration could be the reason. The positive response of GO at an appropriate concentration towards
the acceleration effects of carbon nanotubes and C60 at 50 μg/mL (Nair et al., 2012), and AgNO3 as well as Ag nanoparticles at 50 μg/L on the root length of rice seedlings (Yang et al., 2019). Nair et al. (2012) found that graphene at the concentration of 50 μg/mL inhabited the root and shoot growth of rice seedlings. Another work reported by Hu et al. found that the growth of wheat seedlings was not inhabited by GO at 0.1 and 10 mg/L (Hu et al., 2014). Recently, this group (Hu et al., 2018) observed that GO at a concentration of 1 mg/L slightly accelerated the growth of L. minor. Thus, the effect of GO on the growth of plants was dependent on its concentration and the kinds of plants, which is consistent with that of Ag nanoparticles (Yang et al., 2019; Thuesombat et al., 2014). Previous studies reported that high-dose Ag nanoparticles (above 10 mg/L) remarkably inhibited the growth of Rice (Oryza sativa L. cv. KDML 105) (Thuesombat et al., 2014), while low concentrated Ag 292
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5. Funding information
seed germination and further growth of the seedlings is superior to that of metallic engineered nanoparticles, such as CeO2 or ZnO NPs (Wang et al., 2018). Unfortunately, this promotion effect was lessened by the presence of Cd, as evidenced by the no discernible effect of Cd1GO1 and Cd1GO10 on the dry weight.
We acknowledge the financial support from the National Natural Science Foundation of China (21806001), the Natural Project of Anhui Provincial Department of Education (KJ2018A0126), the Provincial Foundation for Excellent Young Talents of Colleges and Universities of Anhui Province (No.gxyq2018004) and the Open Fund of Anhui Province Key Laboratory of Farmland Ecological Conservation and Pollution Prevention, FECPP201904. CAS Key Laboratory of Photovoltaic and Energy Conservation Materials, Chinese Academy of Sciences is also acknowledged.
3.4. Effect of GO nanosheets on the Cd uptake by rice seedlings The intracellular Cd uptake in rice seedlings is unquestionable, but the impact of GO nanosheets on the intracellular Cd uptake remains unclear. Fig. 4 reveals that the Cd uptake amount without GO exposure was 366.7 and 18.3 mg/kg in the root and shoot system, respectively. Compared with the Cd alone treatment in both root and shoot systems, significantly higher Cd uptake was observed by the Cd1GO10 groups, while no significant change in Cd uptake by the Cd1GO1 groups. Moreover, the exposure of Cd1GO10 (34.6 mg/kg) induced obviously larger Cd uptake with respect to that of Cd1GO1 (25.0 mg/kg) in the shoot system. Thus, GO at the concentration of 10 mg/L promoted Cd uptake and accumulation in both root and shoot systems, while GO at 1 mg/L led to no noticeable effect on Cd uptake. The regulation of Cd uptake and accumulation by GO nanosheets in the roots and shoots of rice seedlings was therefore concentration dependent. Some similar effects have also been observed by Hu et al. (2014) for the mutual impacts of GO and As on the As uptake in wheat, and by Hu et al. (2018) for the effect of GO on Cu uptake in Lemna minor L. Previously, several studies have investigated the impact of NPs including GO nanosheets on the heavy metal uptake by plants (Hu et al., 2014, 2018; Rossi et al., 2017, 2018; Wang et al., 2018; Rizwan et al., 2019). The underlying mechanisms reported with relation to the alteration of heavy metal uptake onto plants by the presence of NPs include: (i) the adsorption of heavy metals onto NPs so that NPs served as heavy metal carriers; (ii) the chemical alternation in the plant rhizosphere, including enhanced excretion of root exudates; and (iii) the changes in the anatomical structure of plant roots. In this study, the FTIR spectra and electrolyte leakage in the rice roots were conducted to probe the structural damage in the root cell wall and plasma membrane. Compared with the blank treatment, the FTIR spectra of rice seedlings treated with Cd at 1 mg/L, GO at 1 or 10 mg/L, Cd1GO1 and Cd1GO10 have no noticeable changes (Fig. 5A), disclosing no distinct structural damage to the root cell wall. Fig. 5B reveals that compared with the control, GO alone did not induce apparent electrolyte leakage, while Cd alone and CdGO group lead to remarkable increase in the electrolyte leakage. Moreover, the electrolyte leakage caused by the CdGO group was much higher than that by Cd alone. It is possible that the co-presence of Cd and GO nanosheets promoted the permeability of rice root cells and therefore facilitated Cd uptake through passive transport across the plasma membrane. Hence, we can conclude that the increased membrane permeability enhanced the Cd uptake.
Contribution Wenling Ye and Jie Li conceived and designed the study. Fan Wu, Qing Fang, Zheng Wu, and Qingyun Duan performed the experiments. Jie Li wrote the paper. Xuede Li reviewed and edited the manuscript. All authors read and approved the manuscript. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Ahmed, F., Rodrigues, D.F., 2013. Investigation of acute effects of graphene oxide on wastewater microbial community: a case study. J. Hazard Mater. 256–257, 33–39. Bandyopadhyay, S., Plascencia-Villa, G., Mukherjee, A., Rico, C.M., José-Yacamán, M., Peralta-Videa, J.R., Gardea-Torresdey, J.L., 2015. Comparative phytotoxicity of ZnO NPs, bulk ZnO, and ionic zinc onto the alfalfa plants symbiotically associated with Sinorhizobium meliloti in soil. Sci. Total Environ. 515–516, 60–69. Boukhvalov, D.W., Katsnelson, M.I., 2008. Modeling of graphite oxide. J. Am. Chem. Soc. 130, 10697–10701. Chen, L., Wang, C., Li, H., Qu, X., Yang, S., Chang, X., 2017. Bioaccumulation and toxicity of 13C-skeleton labeled graphene oxide in wheat. Environ. Sci. Technol. 51, 10146–10153. Cui, H., Zhou, J., Zhao, Q., Si, Y., Mao, J., Fang, G., Liang, J., 2013. Fractions of Cu, Cd, and enzyme activities in a contaminated soil as affected by applications of micro- and nanohydroxyapatite. J. Soils Sediments 13, 742–752. Fadeel, B., Bussy, C., Merino, S., Vázquez, E., Flahaut, E., Mouchet, F., Evariste, L., Gauthier, L., Koivisto, A.J., Vogel, U., Martín, C., Delogu, L.G., Buerki-Thurnherr, T., Wick, P., Beloin-Saint-Pierre, D., Hischier, R., Pelin, M., Candotto Carniel, F., Tretiach, M., Cesca, F., Benfenati, F., Scaini, D., Ballerini, L., Kostarelos, K., Prato, M., Bianco, A., 2018. Safety assessment of graphene-based materials: focus on human health and the environment. ACS Nano 12, 10582–10620. Gardea-Torresdey, J.L., Gomez, E., Peralta-Videa, J.R., Parsons, J.G., Troiani, H., JoseYacaman, M., 2003. Alfalfa sprouts: a natural source for the synthesis of silver nanoparticles. Langmuir 19, 1357–1361. Goodwin, D.G., Adeleye, A.S., Sung, L., Ho, K.T., Burgess, R.M., Petersen, E.J., 2018. Detection and quantification of graphene-family nanomaterials in the environment. Environ. Sci. Technol. 52, 4491–4513. He, Y., Hu, R., Zhong, Y., Zhao, X., Chen, Q., Zhu, H., 2018. Graphene oxide as a water transporter promoting germination of plants in soil. Nano Res. 11, 1928–1937. Hu, X., Kang, J., Lu, K., Zhou, R., Mu, L., Zhou, Q., 2014. Graphene oxide amplifies the phytotoxicity of arsenic in wheat. Sci. Rep. 4, 6122. Hu, X., Ouyang, S., Mu, L., An, J., Zhou, Q., 2015. Effects of graphene oxide and oxidized carbon nanotubes on the cellular division, microstructure, uptake, oxidative stress, and metabolic profiles. Environ. Sci. Technol. 49, 10825–10833. Hu, C., Liu, L., Li, X., Xu, Y., Ge, Z., Zhao, Y., 2018. Effect of graphene oxide on copper stress in Lemna minor L.: evaluating growth, biochemical responses, and nutrient uptake. J. Hazard Mater. 341, 168–176. Khodakovskaya, M.V., Kim, B.-S., Kim, J.N., Alimohammadi, M., Dervishi, E., Mustafa, T., Cernigla, C.E., 2013. Carbon nanotubes as plant growth regulators: effects on tomato growth, reproductive system, and soil microbial community. Small 9, 115–123. Lahiani, M.H., Dervishi, E., Chen, J., Nima, Z., Gaume, A., Biris, A.S., Khodakovskaya, M.V., 2013. Impact of carbon nanotube exposure to seeds of valuable crops. ACS Appl. Mater. Interfaces 5, 7965–7973. Li, J., Wu, Q., Wang, X., Chai, Z., Shi, W., Hou, J., Hayat, T., Alsaedi, A., Wang, X., 2017. Heteroaggregation behavior of graphene oxide on Zr-based metal–organic frameworks in aqueous solutions: a combined experimental and theoretical study. J. Mater. Chem. 5, 20398–20406. Li, F., Sun, C., Li, X., Yu, X., Luo, C., Shen, Y., Qu, S., 2018. The effect of graphene oxide on adventitious root formation and growth in apple. Plant Physiol. Biochem. 129, 122–129. Nagajyoti, P.C., Lee, K.D., Sreekanth, T.V.M., 2010. Heavy metals, occurrence and
4. Conclusions The physical and biochemical response of rice seeds and seedlings as well as the Cd uptake were probed by the co-presence of GO nanosheets and Cd in a hydroponic system. GO nanosheets could uptake and transport water due to their hydrophilic nature, thus promoting the rice seed germination and further seedling growth. The inhibitive effects of Cd on the seed germination was alleviated by the co-accurring GO nanosheets. Besides, the presence of GO nanosheets at 10 mg/L improved Cd uptake in both root and shoot system due to the enhancement in the membrane permeability. These findings can provide new insights for understanding the reciprocal effects of heavy metals and engineered or new emerging nanomaterials in plants. Additionally, further studies should be carried out in a soil matrix considering its complex environmental conditions, such as the microbial communities, rhizosphere and coating, which may influence the heavy metal toxicity and accumulation. 293
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