Toxicity and translocation of graphene oxide in Arabidopsis plants under stress conditions

Abstract

In the present study, we investigated the toxicity and translocation of graphene oxide (GO) in the μg L⁻¹ range in Arabidopsis plants under both normal and stress conditions. Exposure to GO for 4 weeks did not cause adverse effects on the development of Arabidopsis seedlings. In contrast, the combined exposure to GO and PEG 6000 (20%) or NaCl (200 mM) resulted in a more severe loss of morphology, decrease in fresh weight or root length, and increase in root-to-shoot ratio in Arabidopsis seedlings compared with exposure to stress alone. The combined exposure to GO and PEG 6000 (20%) or NaCl (200 mM) resulted in a greater increase in hydrogen peroxide content or membrane ion leakage, decrease in superoxide dismutase activity or catalase activity, and induction of reactive oxygen species production in Arabidopsis seedlings compared with exposure to stress alone. The combined exposure to GO and PEG 6000 (20%) or NaCl (200 mM) induced more severe alterations in expression patterns of genes required for development, abiotic stress, and membrane ion leakage in Arabidopsis seedlings. Moreover, under stress conditions, more GO was distributed in Arabidopsis seedlings and GO was translocated from roots to leaves. We hypothesize that, under stress conditions, GO may induce oxidative stress and membrane ion leakage, which may in turn induce GO translocation from the roots to the leaves. Our results will be useful for understanding toxicity and translocation of GO under different environmental conditions.

---

Introduction

The increased production and use of engineered nanomaterials (ENMs), such as metal nanoparticles (NPs), quantum dots (QDs), and carbon-based nanomaterials, for industry and medicine have led to great concerns about their possible adverse effects on both health and the environment.^1–3 As the global production and use of ENMs are growing rapidly, it is unavoidable that ENMs will enter the environment, especially aquatic and soil systems. Thus, plants will take up ENMs from these systems, since plants are always exposed to the environment.^4,5 Arabidopsis, a well-described and studied model plant, has a very rapid life cycle, and relevance for toxicity implications on edible food crops.⁶ So far, Arabidopsis has been widely used for toxicity assessment, toxicological studies, and translocation of ENMs such as metal oxide-NPs, QDs, and carbon nanotubes.^7–13

Graphene oxide (GO) is an important member of the graphene family, and has unique properties and 2-D structure. It has been shown that GO has great potential for both industrial and medical applications.^14–17 Meanwhile, a series of evidence has been raised that long-term exposure to GO or exposure to high concentrations of GO can cause adverse effects on organisms,^18–29 and result in the deposition of GO in cells or organisms.^21,25,27,30 Previous studies have further demonstrated that graphene and GO at high concentrations had adverse effects on plants.^31,32 In contrast, there are still no systematic studies on GO toxicity in plants under stress conditions.

Herein, we compared the phytotoxicity of GO under stress conditions with that under normal conditions in an in vivo assay system of Arabidopsis. Considering the fact that most of the ENMs released into the environment may be in the ng L⁻¹ or μg L⁻¹ range,^33–36 we assessed the toxicity of GO in the μg L⁻¹ range in Arabidopsis. Our data suggest that under stress conditions (drought stress or salt stress), exposure to GO induced a more severe adverse effect on Arabidopsis plants and altered the translocation patterns of GO in plants. Our results will be helpful for understanding the fate and toxicity of GO on environmental plants under stress conditions.

Results

Characterization of prepared GO

The detailed information for the characterization of the prepared GO, including Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy, has been described in our previous reports.²¹ The atomic force microscopy (AFM) result indicated that the thickness of the prepared GO was approximately 1.0 nm in topographic height, corresponding to approximately 1 layer (Fig. 1a). After sonication, the morphology of GO in Hoagland nutritive fluid was shown in Fig. 1b. The size distribution of GO suggests that most of the GO nanomaterials in Hoagland nutritive fluid were in the range of 40–50 nm (Fig. 1c). The zeta potentials of GO in Hoagland nutritive fluid, PEG 6000 (20%) in Hoagland nutritive fluid, and NaCl (200 mmol L⁻¹) in Hoagland nutritive fluid were −20.5 ± 2.4, −24.2 ± 3.2, and −23.7 ± 2.8 mV, respectively (Fig. 1d).

Fig. 1 Size distribution of GO in Hoagland nutritive fluid. (a) AFM assay of GO. (b) Image showing the GO distribution (after sonication) in Hoagland nutritive fluid. (c) Size distribution of GO in Hoagland nutritive fluid. (d) Zeta potential of GO.

Effects of GO at the examined concentrations on the development of Arabidopsis seedlings

We first investigated the possible effects of GO at the examined concentrations (10–1000 μg L⁻¹) on the development of Arabidopsis seedlings. The Arabidopsis seedlings exposed to 10–1000 μg L⁻¹ of GO showed similar morphology to that of Arabidopsis seedlings without GO exposure (Fig. 2a). The shoot development of Arabidopsis seedlings exposed to 10–1000 μg L⁻¹ of GO was not obviously affected (Fig. 2b). Moreover, exposure to 10–1000 μg L⁻¹ of GO did not significantly alter the fresh weight, root length, and root-to-shoot ratio of Arabidopsis seedlings (Fig. 2c–e). Thus, GO at the examined concentrations does not obviously influence the development of Arabidopsis seedlings.

Fig. 2 Combined effects of GO and stress on development of Arabidopsis seedlings. (a) Morphology of exposed Arabidopsis seedlings. (b) Shoot development of exposed Arabidopsis seedlings. (c) Fresh weight of exposed Arabidopsis seedlings. (d) Root length of exposed Arabidopsis seedlings. (e) Root-to-shoot ratio of exposed Arabidopsis seedlings. Bars represent means ± SEM. ^**P < 0.01 vs. 0 μg L⁻¹ GO.

Effects of GO at the examined concentrations on hydrogen peroxide (H₂O₂) production, lipid peroxidation, activity of H₂O₂ scavenging enzymes, and reactive oxygen species (ROS) production in Arabidopsis seedlings

Next, we investigated the physiological changes in GO exposed Arabidopsis seedlings. The Arabidopsis seedlings exposed to 10–1000 μg L⁻¹ of GO did not show obvious over-production of H₂O₂ compared with Arabidopsis seedlings without GO exposure (Fig. 3). Exposure to 10–1000 μg L⁻¹ of GO did not cause significant alterations in malondialdehyde (MDA) content in Arabidopsis seedlings compared with that in Arabidopsis seedlings without GO exposure (Fig. 3). The antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) are known to be involved in the detoxification of H₂O₂. Moreover, exposure to 10–1000 μg L⁻¹ of GO did not significantly alter the activities of both the SOD content and the CAT content in Arabidopsis seedlings compared with those in Arabidopsis seedlings without GO exposure (Fig. 3). We further employed the 3,3′-diaminobenzidine (DAB) staining method to detect the distribution of ROS production in Arabidopsis seedlings. We found that exposure to 10–1000 μg L⁻¹ of GO resulted in similar DAB staining results to those in Arabidopsis seedlings without GO exposure (Fig. S1†). These results imply that no detrimental effects of oxidative stress would develop in Arabidopsis seedlings exposed to GO at the examined concentrations.

Fig. 3 Combined effects of GO and stress on hydrogen peroxide generation, lipid peroxidation, and activities of antioxidant enzymes in Arabidopsis seedlings. The examined antioxidant enzymes are superoxide dismutase (SOD) and catalase (CAT). Bars represent means ± SEM. ^**P < 0.01 vs. 0 μg L⁻¹ GO.

Combined effects of GO and drought or salt stress on development of Arabidopsis seedlings

The above data suggest that GO at the examined concentrations did not induce adverse effects on Arabidopsis seedlings under normal conditions. We further investigated the effects of GO exposure on Arabidopsis seedlings under specific stress conditions. The stresses of drought and salt were selected for the assays. In Arabidopsis, we found that the combined exposure to PEG 6000 (20%) and 10 or 100 μg L⁻¹ of GO did not obviously influence the morphology of seedlings, shoot development, fresh weight, root length, and root-to-shoot ratio of seedlings compared with those in PEG 6000 (20%) exposed Arabidopsis seedlings (Fig. 2). Similarly, the combined exposure to NaCl (200 mmol L⁻¹) and 10 or 100 μg L⁻¹ of GO did not noticeably affect the morphology of seedlings, shoot development, fresh weight, root length, and root-to-shoot ratio of seedlings compared with those in NaCl (200 mmol L⁻¹) exposed Arabidopsis seedlings (Fig. 2). In contrast, interestingly, we found that the combined exposure to PEG 6000 (20%) and 1000 μg L⁻¹ of GO caused more severe adverse effects on the morphology of seedlings, shoot development, fresh weight, root length, and root-to-shoot ratio compared with those in PEG 6000 (20%) exposed Arabidopsis seedlings (Fig. 2). Similarly, the combined exposure to NaCl (200 mmol L⁻¹) and 1000 μg L⁻¹ of GO resulted in more severe adverse effects on the morphology of seedlings, shoot development, fresh weight, root length, and root-to-shoot ratio compared with those in NaCl (200 mmol L⁻¹) exposed Arabidopsis seedlings (Fig. 2). Thus, GO at the concentration of 1000 μg L⁻¹ may be able to induce more severe damage to Arabidopsis seedlings under drought or salt stress conditions.

Physiological basis for combined effects of GO with drought or salt stress on the development of Arabidopsis seedlings

We further examined the physiological basis for the observed combined effects of GO and drought or salt stress on the development of Arabidopsis seedlings. In Arabidopsis, we found that the combined exposure to PEG 6000 (20%) and 10 or 100 μg L⁻¹ of GO did not obviously alter the H₂O₂ content, MDA content, and activity of H₂O₂ scavenging enzymes, or induction of ROS production compared with those in PEG 6000 (20%) exposed Arabidopsis seedlings (Fig. 3 and S1†). Similarly, the combined exposure to NaCl (200 mmol L⁻¹) and 10 or 100 μg L⁻¹ of GO did not noticeably change the H₂O₂ content, MDA content, and activity of H₂O₂ scavenging enzymes, or induction of ROS production compared with those in NaCl (200 mmol L⁻¹) exposed Arabidopsis seedlings (Fig. 3 and S1†). Different from these, we found that the combined exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO induced greater production of H₂O₂ and ROS compared with that in PEG 6000 (20%) or NaCl (200 mmol L⁻¹) exposed Arabidopsis seedlings (Fig. 3 and S1†), implying that the combined exposure of PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO would induce a greater extent of oxidative stress compared with exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) alone in Arabidopsis seedlings. Moreover, the combined exposure of PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO resulted in a greater increase in MDA content compared with that in PEG 6000 (20%) or NaCl (200 mmol L⁻¹) exposed Arabidopsis seedlings (Fig. 3), suggesting that the combined exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO would induce more membrane damage, which is usually caused by oxidative stress in Arabidopsis seedlings. Furthermore, the combined exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO caused a greater decrease in both SOD content and CAT content compared with those in PEG 6000 (20%) or NaCl (200 mmol L⁻¹) exposed Arabidopsis seedlings (Fig. 3), suggesting that the combined exposure of PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO would more severely reduce the activities of antioxidant enzymes for detoxification of H₂O₂ than exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) alone in Arabidopsis seedlings.

Molecular basis for the combined effects of GO with drought or salt stress on the development of Arabidopsis seedlings

In Arabidopsis, the genes PIN2, PIN7, SHR, and SCR are required for root development.^37,38 The PIN genes encode key auxin efflux carriers which are crucial for the formation of instructive auxin distribution gradients and therefore the direction of root development.³⁷ The SCR/SHR pathway is required for specification and maintenance of the root stem cell niche.³⁸ Exposure to 1000 μg L⁻¹ of GO did not significantly alter the expression patterns of PIN2, PIN7, SHR, and SCR genes (Fig. 4a). Both PEG 6000 (20%) and NaCl (200 mmol L⁻¹) caused a decrease in expression levels of PIN2, PIN7, SHR, and SCR genes in Arabidopsis seedlings (Fig. 4a). The combined exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO caused a greater decrease in expression levels of PIN2, PIN7, SHR, and SCR genes in Arabidopsis seedlings than exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) alone (Fig. 4a). Therefore, GO exposure at a concentration of 1000 μg L⁻¹ may induce more severe damage to the molecular basis of the control of root development under drought or salt stress conditions.

Fig. 4 Expression pattern of genes required for root development (a) and abiotic stress control (b) in Arabidopsis seedlings. Bars represent means ± SEM. ^**P < 0.01 vs. 0 μg L⁻¹ GO.

In Arabidopsis, the genes ABI4, ABI5, AREB1, HKT1, SOS1, and RD29A are required for the control of drought and/or salt stress.^39–44 ABI4 is a master Apetala 2-type transcription factor targeted by multiple signaling pathways in plant cells.⁴¹ The abscisic acid response gene ABI5 encodes a basic leucine zipper transcriptional factor.³⁹ AREB1 encodes a bZIP factor. HKT1 encodes a Na⁺ transporter controlling retrieval of Na⁺ from the xylem. SOS1 encodes a Na⁺/H⁺ antiporter. RD29A encodes an ABA-responsive protein.⁴⁰ Exposure to 1000 μg L⁻¹ of GO did not significantly change the expression patterns of ABI4, ABI5, AREB1, HKT1, SOS1, or RD29A genes (Fig. 4b). Both PEG 6000 (20%) and NaCl (200 mmol L⁻¹) caused a decrease in the expression level of HKT1 and an increase in the expression levels of ABI4, ABI5, AREB1, SOS1, and RD29A in Arabidopsis seedlings (Fig. 4b). The combined exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO caused a greater decrease in the expression level of HKT1 and an increase in the expression levels of ABI4, ABI5, AREB1, SOS1, and RD29A in Arabidopsis seedlings than exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) alone (Fig. 4b). Therefore, GO exposure at the concentration of 1000 μg L⁻¹ may cause more severe damage to the molecular basis of the control of abiotic stress under the drought or salt stress conditions.

Effects of drought or salt stress on GO translocation in Arabidopsis seedlings

To further examine the possible mechanism for the combined effects of GO and drought or salt stress on Arabidopsis, we investigated the GO translocation in Arabidopsis seedlings by labeling GO with the molecular probe Rho-B. In Arabidopsis, exposure to 1000 μg L⁻¹ of GO/Rho-B or 1000 μg L⁻¹ of Rho-B did not obviously influence the development of seedlings (Fig. 5a). Exposure to 1000 μg L⁻¹ of Rho-B did not noticeably affect the development of seedlings exposed to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) (Fig. 5a). In contrast, exposure to 1000 μg L⁻¹ of GO/Rho-B caused the more severe developmental deficits (smaller plants and yellow leaves) in seedlings exposed to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) than exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) alone (Fig. 5a).

Fig. 5 Effects of drought or salt stress on GO/Rho-B translocation in Arabidopsis seedlings. (a) Combined effects of GO and stress on the development of seedlings. (b) Combined effects of GO and stress on GO/Rho-B translocation in root tips. (c) Combined effects of GO and stress on GO/Rho-B translocation in the mid-region of roots. (d) Combined effects of GO and stress on GO/Rho-B translocation in tissue slices of leaves. The exposure concentration for GO/Rho-B or Rho-B was 1000 μg L⁻¹. S, stoma.

In Arabidopsis, exposure to 1000 μg L⁻¹ of GO/Rho-B or 1000 μg L⁻¹ of Rho-B only caused moderate distribution of red fluorescence signals in roots, and very weak fluorescence signals in leaf tissues (Fig. 5b–d). The combined exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of Rho-B induced noticeable distribution of red fluorescence signals in root tissues, especially the root tips (Fig. 5b and c). Meanwhile, the combined exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of Rho-B caused obvious distribution of red fluorescence signals in leaf tissues, and some red fluorescence signals were also detected in the stoma of the leaves (Fig. 5d). In contrast, the combined exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO/Rho-B induced a greater distribution of red fluorescence signals in root tissues, especially the root tips, compared with those from the combined exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of Rho-B (Fig. 5b and c). Similarly, the combined exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO/Rho-B caused a greater distribution of red fluorescence signals in leaf tissues compared with those from the combined exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of Rho-B, and most of the stoma showed strong red fluorescence signals in leaves of Arabidopsis seedlings exposed to both GO/Rho-B and PEG 6000 (20%) or NaCl (200 mmol L⁻¹) (Fig. 5d). Therefore, these results imply that GO may be translocated from the roots to the leaves under drought or salt stress conditions. In addition, GO may further enhance the damage to plants exposed to PEG 6000 (20%) or NaCl (200 mmol L⁻¹).

Combined effects of GO and drought or salt stress on membrane ion leakage in Arabidopsis seedlings

Finally, we investigated the possible combined effects of GO and drought or salt stress on membrane ion leakage in Arabidopsis seedlings. In Arabidopsis, the genes KAT1, QUAC1, SLAH3, and TPC1 are required for the control of membrane ion leakage.^45–47 KAT1 encodes a K⁺ channel at the plasma membrane. QUAC1 gene encodes an R-type anion channel. SLAH3 encodes a slow anion channel. TPC1 encodes a Ca²⁺-activated channel. Exposure to 1000 μg L⁻¹ of GO did not significantly alter the expression patterns of the genes KAT1, QUAC1, SLAH3, and TPC1 (Fig. 6a). Both PEG 6000 (20%) and NaCl (200 mmol L⁻¹) caused an increase in expression levels of KAT1, QUAC1, SLAH3, and TPC1 in Arabidopsis seedlings (Fig. 6a). The combined exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO resulted in a greater increase in expression levels of KAT1, QUAC1, SLAH3, and TPC1 in Arabidopsis seedlings than exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) alone (Fig. 6a).

Fig. 6 Combined effects of GO and stress on ion leakage in Arabidopsis seedlings. (a) Combined effects of GO and stress on the expression patterns of genes required for the control of ion leakage. (b) Combined effects of GO and stress on ion leakage. Bars represent means ± SEM. ^**P < 0.01 vs. 0 μg L⁻¹ GO.

In Arabidopsis, exposure to 1000 μg L⁻¹ of GO did not obviously induce membrane ion leakage (Fig. 6b). Both PEG 6000 (20%) and NaCl (200 mmol L⁻¹) caused noticeable membrane ion leakage (Fig. 6b). Moreover, we found that the combined exposure of PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO resulted in more severe membrane ion leakage compared with exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) alone (Fig. 6b). Therefore, GO exposure at a concentration of 1000 μg L⁻¹ may induce more severe membrane ion leakage under drought or salt stress conditions.

Discussion

In the present study, we compared the effects of GO in the μg L⁻¹ range on plants under normal and stress conditions. The concentrations of 10–1000 μg L⁻¹ were selected, because this range reflects the possible concentrations of ENMs released into the environment.^33–36 Two widely studied and commonly occurring stresses, drought stress and salt stress, were selected for the combined exposure assay in this study. The selected concentration for PEG 6000 was 20%, and the selected concentration for NaCl was 200 mmol L⁻¹. The reason for selecting these concentrations is that exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) would induce moderate but adverse effects on Arabidopsis plants,^48–51 which would be helpful for our observations of more severe adverse effects on plants from the combined exposure to GO and PEG 6000 or NaCl. The selected Arabidopsis assay system is the most widely studied model plant. Arabidopsis is also an important environmental plant.

In this study, we observed that exposure to 10–1000 μg L⁻¹ of GO for 4 weeks did not induce obvious adverse effects on Arabidopsis seedlings under normal conditions. Exposure to 10–1000 μg L⁻¹ of GO did not induce noticeable alterations in morphology, fresh weight, root length, or root-to-shoot ratios of Arabidopsis seedlings (Fig. 2). Exposure to 10–1000 μg L⁻¹ of GO did not induce significant increases in H₂O₂ content or MDA content, or decreases in SOD content or CAT content, or the noticeable production of ROS in Arabidopsis seedlings (Fig. 3 and S1†). Exposure to 10–1000 μg L⁻¹ of GO did not induce obvious membrane ion leakage (Fig. 5b). Exposure to 1000 μg L⁻¹ of GO did not significantly influence the expression patterns of genes associated with the control of root development (Fig. 4a), abiotic stress (Fig. 4b), or membrane ion leakage (Fig. 6a) in Arabidopsis seedlings. Therefore, GO in the μg L⁻¹ range cannot cause adverse effects on Arabidopsis seedlings under normal conditions. A previous study has indicated that exposure to single walled carbon nanotubes (SWCNTs) at concentrations greater than 50 mg L⁻¹ induced toxic effects on Arabidopsis mesophyll cells, such as increased ROS generation, changes in protoplast morphology, and induction of necrosis or apoptosis of protoplast cells.⁵² The difference between our data on GO toxicity and the observations on SWCNTs toxicity⁵² may be due to the fact that exposure concentrations of GO in this study are relatively low. Moreover, in GO (1000 μg L⁻¹) exposed Arabidopsis seedlings, we did not observe the phenotype of cell surface trichome cluster as observed in Arabidopsis mesophyll cells exposed to 15 or 25 mg L⁻¹ of SWCNTs⁵² (data not shown). That is, formation of both beneficial effects and adverse effects may require exposure to carbon-based ENMs at certain concentrations.

Very different from the phenotypes under normal conditions, severe adverse effects were found in GO-exposed Arabidopsis seedlings under stress conditions. Under our experimental conditions, the combined exposure of PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO caused more severe adverse effects on morphology, fresh weight, root length, and root-to-shoot ratio of Arabidopsis seedlings compared with exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) alone (Fig. 2). These results are largely consistent with the observations in animals. In nematodes, it was reported that GO did not have adverse effects on animals under normal conditions.⁵³ However, under stress (juglone exposure) conditions, GO exposure caused more severe toxicity to animals.⁵³

For the physiological basis of stress-induced GO toxicity, firstly, we found that the combined exposure of PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO resulted in a more severe increase in H₂O₂ content (Fig. 3a) and induction of ROS production (Fig. S1†) in Arabidopsis seedlings compared with exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) alone, suggesting the possible crucial role of oxidative stress in the formation of stress-induced GO toxicity to plants. Previous studies have suggested that ENMs can induce oxidative stress in plants.^54,55 Secondly, we observed that the combined exposure of PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO induced a greater decrease in both the SOD content and the CAT content in Arabidopsis seedlings compared with exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) alone (Fig. 3c and d), implying a more severely damaged antioxidant system in GO exposed plants under stress conditions.

For the molecular basis of stress-induced GO toxicity, our results suggest that the molecular basis for development was altered. The combined exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO caused more severe alterations in the expression patterns of genes required for root development in Arabidopsis seedlings compared with exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) alone (Fig. 4a). Besides this, we further observed that the combined exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO induced more severe changes in the expression patterns of genes required for abiotic stress compared with exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) alone (Fig. 4b). These data also imply that specific signaling pathways, such as ABI4/5, AREB1, HKT1, SOS1, and RD29A, may participate in the control of stress-induced GO toxicity in Arabidopsis. The further elucidation of mechanisms of these signaling pathways in controlling the stress-induced GO toxicity will be helpful for our understanding of the molecular mechanism of nanotoxicity in plants.

For the cellular basis of stress-induced GO toxicity, with the aid of the Rho-B labeling technique, we found a more pronounced distribution of GO in both the roots and the leaves of Arabidopsis seedlings exposed to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) compared with that in GO exposed Arabidopsis seedlings (Fig. 5). More interestingly, most of the labeled signals were found in the root tips and the stoma of the leaves in Arabidopsis seedlings exposed to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) (Fig. 5), implying that the root tip and the stoma may be the favored sites for GO in plants. It is also possible that the root tip and stoma may have important functions in controlling the absorption and/or translocation of GO in plants. Moreover, we observed that relatively more GO/Rho-B was distributed in the roots and leaves than Rho-B in Arabidopsis seedlings exposed to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) (Fig. 5), which further suggests the toxic effects of GO on Arabidopsis seedlings under stress conditions. Previous studies have suggested that specific surface modifications such as PEG modification can reduce the toxicity of GO.^14,28 Our data further suggest that GO–PEG (1000 μg L⁻¹) did not obviously alter the effects of PEG 6000 (20%) or NaCl (200 mmol L⁻¹) on Arabidopsis seedlings, and the distribution or translocation pattern of GO–PEG/Rho-B was similar to that of Rho-B (data not shown). These data further suggest the potential toxicity of GO on Arabidopsis seedlings.

One of the possible explanations of the translocation pattern of GO in Arabidopsis seedlings under stress conditions is altered membrane ion leakage. We found that the combined exposure of PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO resulted in a more severe increase in membrane ion leakage in Arabidopsis seedlings compared with exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) alone (Fig. 6b). It was also observed that the combined exposure of PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO caused a greater increase in the MDA content in Arabidopsis seedlings compared with exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) alone (Fig. 3b). The combined exposure of PEG 6000 (20%) or NaCl (200 mmol L⁻¹) with 1000 μg L⁻¹ of GO caused a greater increase in the expression levels of genes associated with the control of membrane ion leakage in Arabidopsis seedlings compared with exposure to PEG 6000 (20%) or NaCl (200 mmol L⁻¹) alone (Fig. 6a), which provides further indirect evidence.

Conclusion

In the present study, we investigated the stress-induced toxicity and translocation of GO in the μg L⁻¹ range in the in vivo assay system of Arabidopsis. Our data suggest that, under normal conditions, GO did not cause obvious adverse effects on Arabidopsis seedlings. However, under drought stress or salt stress conditions, GO exposure induced more severe adverse effects on the development of Arabidopsis seedlings, and caused more oxidative stress compared with stress exposure alone. Under drought stress or salt stress conditions, GO exposure induced more severe alterations in the expression patterns of genes required for development, abiotic stress, and membrane ion leakage in Arabidopsis seedlings. Under drought stress or salt stress conditions, more GO was distributed in Arabidopsis seedlings and GO could be translocated from the roots to the leaves. Our results suggest that, under stress conditions, GO may induce severe oxidative stress and membrane ion leakage, which will further induce the translocation of GO from the roots to the leaves. Our results will be helpful for understanding the toxicity and translocation of GO and the possible underlying mechanisms in plants under different environmental conditions.

Experimental

Characterization of prepared GO

GO was prepared from natural graphite powder using the modified Hummer’s method.⁵⁶ Prepared GO was characterized by AFM (SPM-9600, Shimadzu, Japan). After sonication, morphology and size distribution of GO was examined by transmission electron microscopy (TEM). A few drops of GO suspension were deposited on the TEM grid, dried, and evacuated before analysis. The zeta potential was analyzed by a Nano Zetasizer (Nano ZS90, Malvern Instrument, UK) using a dynamic light scattering (DLS) technique. All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Plant growth and exposure

The A. thaliana ecotype Columbia (Col-0) was obtained from the Arabidopsis Biological Resource Center (ABRC, Columbia, OH, USA). The plant growth was performed as described previously.⁵⁷ The pH of the medium was maintained at 5.85. After sonication, GO was added to the media before autoclaving at the examined concentrations (10, 100, and 100 μg L⁻¹). Medium without GO was employed as the control. Seeds were surface sterilized and soaked in distilled water at 4 °C in the dark for 24 h. The seeds were cultured in plates with standard Murashige and Skoog (MS) medium containing GO at 22 °C in fully automated growth chambers (Conviron, Canada). The plates were positioned vertically to allow the roots to grow on the surface of the agar medium. Then, two week old seedlings on MS medium containing GO were transferred and cultured in Hoagland nutritive fluid containing GO. The seedlings were harvested freshly after two weeks, and the excess water was removed by soaking with absorbent paper. GO solutions were stable for at least 48 h. We replaced the Hoagland nutritive fluid containing GO every two days. Five replicates were performed.

For stress treatments, 2 week-old plants on MS medium were further exposed to 20% (w/v) polyethylene glycol (PEG) 6000 (ref. 49) or 200 mmol L⁻¹ NaCl (ref. 48) together with GO at the examined concentrations. PEG 6000 was used for the drought stress assay, and NaCl was used for the salt stress assay.^48,49

The seedlings were dried in an oven at 60–80 °C, and the dry weights of roots and shoots were determined, respectively, for analysis of the root-to-shoot ratio. Five replicates were performed.

Reverse-transcription and quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was isolated from four week-old seedlings using Tripure Isolation Reagent (Roche Co.) according to the manufacturer’s protocol. cDNA synthesis was performed in a 12.5 μL reaction volume containing 625 ng total RNA, 0.5 mmol L⁻¹ reverse-transcript primers, 50 mmol L⁻¹ Tris–HCl, 75 mmol L⁻¹ KCl, 3 mmol L⁻¹ MgCl₂, 10 mmol L⁻¹ dithiothreitol, 20 units ribonuclease inhibitor and 100 U reverse transcriptase (Takara, China). After cDNA synthesis, relative expression levels were determined by real-time PCR in an ABI 7500 real-time PCR system with Evagreen (Biotium, USA). All reactions were performed in triplicate with the same cDNA samples. PCR was processed with the following cycles: a 10 min activation and denaturation step at 95 °C, followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. Relative quantification of targeted genes in comparison to the reference actin gene was determined. The final results were expressed as the relative expression ratio between the targeted gene and the reference gene. The designed primers were shown in Table S1.†

MDA, H₂O₂, and antioxidant enzymes

The MDA assay was performed as described previously.^58,59 To assay MDA content, frozen plant samples were homogenized in 80% cold ethanol and centrifuged to pellet the debris. Different aliquots of the supernatant were mixed with 20% trichloroacetic acid and 0.5% thiobarbituric acid. The mixtures were allowed to react in a water bath at 90 °C for 1 h. The samples were cooled in an ice bath and centrifuged. Absorbance of the supernatant was read at 440, 534, and 600 nm against a blank. MDA content was expressed in terms of nmol g⁻¹ fresh weight (FW). Five replicates were performed.

The H₂O₂ assay was performed as described previously.⁶⁰ One hundred milligram of chilled sample was macerated in 4 mL cold acetone. The homogenate was filtered through Whatman filter paper. The filtrate (2 mL) was treated with 1 mL of titanium reagent (20% titanium tetrachloride in concentrated HCl, v/v) and 1 mL of concentrated ammonia solution to precipitate the titanium–hydroperoxide complex. After centrifugation at 5000g for 30 min, the precipitate was dissolved in 2 N H₂SO₄. The absorbance was read at 415 nm. The H₂O₂ content was calculated from a standard curve and expressed as μmol g⁻¹ FW. Five replicates were performed.

Before the assay of antioxidant enzymes, fresh plant samples (0.2 g) were homogenized in 1.5 mL of 50 mmol L⁻¹ potassium phosphate buffer (pH 7.8) containing 1 mmol L⁻¹ EDTA, 1 mmol L⁻¹ dithiotreitol, and 2% (w/v) polyvinyl pyrrolidone (PVP). The homogenate was centrifuged at 15 000g for 30 min at 4 °C, and the supernatant was used for the following enzyme assays. To analyze the total SOD activity, 1 mL of solution containing 50 mmol L⁻¹ potassium phosphate buffer (pH 7.8), 9.9 mmol L⁻¹ L-methionine, 0.025% Triton-X and 57 μmol L⁻¹ nitro blue tetrazolium (NBT) was added into glass tubes followed by addition of 20 μL of the sample. The reaction was started by adding 10 μL of riboflavin solution (4.4 mg 100 mL⁻¹) followed by placing the tubes in an aluminum foil-lined box with two 20 W fluorescent lamps for 7 min. After illumination, the absorbance of the solution was measured at 560 nm. The SOD activity was expressed as U per mg protein. CAT activity was determined by measuring by hydrogen peroxide consumption at 240 nm. CAT activity was assayed for 3 min in a CAT reaction solution composed of 50 mmol L⁻¹ potassium phosphate buffer (pH 7.0), 0.3% H₂O₂, and 20 μL of crude extract. The extinction coefficient for H₂O₂ was set at 23.148 mmol L⁻¹ cm⁻¹. Five replicates were performed.

In vivo detection of ROS

The method was performed as described previously.⁶¹ To detect the distribution of ROS production, we stained the seedlings with DAB. DAB is oxidized by hydrogen peroxide in the presence of some haem-containing proteins, such as peroxidases, to generate a dark brown precipitate. The samples were infiltrated in 1 mg mL⁻¹ DAB (pH 3.8) for 30 min, and then boiled in ethanol for 15 min to remove the chlorophyll from leaves. Images were captured using a stereo microscope equipped with a digital camera. Five replicates were performed.

Ion leakage measurement

The method was performed as described previously.⁶² The detached leaves were agitated in deionized water at 23 °C for 1 h. The conductivity of the examined solutions was measured using a meter (YSI model S5). The total ion strength of the examined solution was determined by boiling the solution in a water bath for 10 min and then cooling to 23 °C. Five replicates were performed.

Distribution of GO in Arabidopsis plants

To investigate the translocation and distribution of GO in Arabidopsis plants, Rho-B was loaded on GO by mixing Rho-B solution (1 mg mL⁻¹, 0.3 mL) with an aqueous suspension of GO (0.1 mg mL⁻¹, 5 mL), essentially as described previously.²⁸ Unbound Rho-B was removed by dialysis against distilled water over 72 h. The resulting GO/Rho-B was stored at 4 °C. The GO/Rho-B dispersed into the Hoagland nutritive fluid together with or without stress treatment to cultivate the two week-old Arabidopsis seedlings. After 48 h exposure, the exposed Arabidopsis seedlings were observed under a fluorescence stereomicroscope or a fluorescence microscope. Whole plants or tissue slices prepared with freezing microtome were used for observations of GO translocation. Before observation, the plants were thoroughly washed with water.

Statistical analysis

All data were presented as means ± standard error of the mean (S.E.M.). Graphs were generated using Microsoft Excel (Microsoft Corp., Redmond, WA). Statistical analysis was performed using SPSS 12.0 software (SPSS Inc., Chicage, USA). Differences between groups were determined using analysis of variance (ANOVA). Probability levels of 0.05 and 0.01 were considered statistically significant.

Acknowledgements

This work was supported by the grants from National Basic Research Program of China (no. 2011CB33404), National Natural Science Foundation of China (no. 81172698, 31000363), Jiangsu Province Ordinary University Graduate Research and Innovation Program (no. CXZZ13_0136), Southeast University Outstanding Doctoral Foundation, and Fundamental Research Funds for the Central Universities.

References

1. T. Xia, N. Li and A. E. Nel, Annu. Rev. Public Health, 2009, 30, 137–150.
2. Q. Rui, Y.-L. Zhao, Q.-L. Wu, M. Tang and D.-Y. Wang, Chemosphere, 2013, 93, 2289–2296.
3. A. Nouara, Q.-L. Wu, Y.-X. Li, M. Tang, H.-F. Wang, Y.-L. Zhao and D.-Y. Wang, Nanoscale, 2013, 5, 6088–6096.
4. D. Lin and B. Xing, Environ. Sci. Technol., 2008, 42, 5580–5585.
5. C. Larue, M. Pinault, B. Czarny, D. Georgin, D. Jaillard, N. Bendiab, M. Mayne-L’Hermite, F. Taran, V. Dive and M. Carriere, J. Hazard. Mater., 2012, 227–228, 155–163.
6. C. M. Rico, S. Majumdar, M. Duarte-Gardea, J. R. Peralta-Videa and J. L. Gardea-Torresdey, J. Agric. Food Chem., 2011, 59, 3485–3498.
7. J. Kurepa, T. Paunesku, S. Vogt, H. Arora, B. M. Rabatic, J. Li, M. B. Wanzer, G. E. Woloschak and J. A. Smalle, Nano Lett., 2010, 10, 2296–2302.
8. Y. Ze, C. Liu, L. Wang, M. Hong and F. Hong, Biol. Trace Elem. Res., 2011, 143, 1131–1141.
9. S. Wang, J. Kurepa and J. A. Smalle, Plant, Cell Environ., 2011, 34, 811–820.
10. D. L. Slomerg and M. H. Schoenfisch, Environ. Sci. Technol., 2012, 46, 10247–10254.
11. D. A. Navarro, M. A. Bisson and D. S. Aga, J. Hazard. Mater., 2012, 211–212, 427–435.
12. R. Kaveh, Y. Li, S. Ranjbar, R. Tehrani, C. L. Brueck and B. Van Aken, Environ. Sci. Technol., 2013, 47, 10637–10644.
13. J. Geisler-Lee, Q. Wang, Y. Yao, W. Zhang, M. Geisler, K. Li, Y. Huang, Y. Chen, A. Kolmakov and X. Ma, Nanotoxicology, 2013, 7, 323–337.
14. L. Feng and Z. Liu, Nanomedicine, 2011, 6, 317–324.
15. H. Yue, W. Wei, Z. Yue, B. Wang, N. Luo, Y. Gao, D. Ma, G. Ma and Z. Su, Biomaterials, 2012, 33, 4013–4021.
16. T. Kuila, A. K. Mishra, P. Khanra, N. H. Kim and J. H. Lee, Nanoscale, 2013, 5, 52–71.
17. K. Yang, Y. Li, X. Tan, R. Peng and Z. Liu, Small, 2013, 9, 1492–1503.
18. M. C. Duch, G. R. S. Budinger, Y. T. Liang, S. Soberanes, D. Urich, S. E. Chiarella, L. A. Campochiaro, A. Gonzalez, N. S. Chandel, M. C. Hersam and G. M. Mutlu, Nano Lett., 2011, 11, 5201–5207.
19. Y. Chang, S. Yang, J. Liu, E. Dong, Y. Wang, A. Cao, Y. Liu and H. Wang, Toxicol. Lett., 2011, 200, 201–210.
20. M. Lv, Y. Zhang, L. Liang, M. Wei, W. Hu, X. Li and Q. Huang, Nanoscale, 2012, 4, 3861–3866.
21. Q.-L. Wu, L. Yin, X. Li, M. Tang, T. Zhang and D.-Y. Wang, Nanoscale, 2013, 5, 9934–9943.
22. A. Bianco, Angew. Chem., Int. Ed., 2013, 52, 4986–4997.
23. G. Qu, S. Liu, S. Zhang, L. Wang, X. Wang, B. Sun, N. Yin, X. Gao, T. Xia, J. Chen and G. Jiang, ACS Nano, 2013, 7, 5732–5745.
24. X. Zhi, H. Fang, C. Bao, G. Shen, J. Zhang, K. Wang, S. Guo, T. Wan and D. Cui, Biomaterials, 2013, 34, 5254–5261.
25. Y.-P. Li, Q.-L. Wu, Y.-L. Zhao, Y.-F. Bai, P.-S. Chen, T. Xia and D.-Y. Wang, ACS Nano, 2014, 8, 2100–2110.
26. Q.-L. Wu, Y.-L. Zhao, Y.-P. Li and D.-Y. Wang, Nanoscale, 2014, 6, 11204–11212.
27. Q.-L. Wu, Y.-L. Zhao, G. Zhao and D.-Y. Wang, Nanomedicine: Nanotechnology, Biology and Medicine, 2014, 10, 1401–1410.
28. Q.-L. Wu, Y.-L. Zhao, J.-P. Fang and D.-Y. Wang, Nanoscale, 2014, 6, 5894–5906.
29. X. Hu and Q. Zhou, Chem. Rev., 2013, 113, 3815–3835.
30. J. Liu, S. Yang, H. Wang, Y. Chang, A. Cao and Y. Liu, Nanomedicine, 2012, 7, 1801–1812.
31. P. Begum and B. Fugetsu, J. Hazard. Mater., 2013, 260, 1032–1041.
32. X. Hu, J. Kang, K. Lu, R. Zhou, L. Mu and Q. Zhou, Sci. Rep., 2014, 4, 6122.
33. M. Hassellöv, J. W. Readman, J. F. Ranville and K. Tiede, Ecotoxicology, 2008, 17, 344–361.
34. K. Tiede, M. Hassellöv, E. Breitbarth, Q. Chaudhry and A. B. A. Boxall, J. Chromatogr. A, 2009, 1216, 503–509.
35. F. Gottschalk, T. Sonderer, R. W. Scholz and B. Nowack, Environ. Sci. Technol., 2009, 43, 9216–9222.
36. K. Doudrick, P. Herckes and P. Westerhoff, Environ. Sci. Technol., 2012, 46, 12246–12253.
37. I. Blilou, J. Xu, M. Wildwater, V. Willemsen, I. Paponov, J. Friml, R. Heidstra, M. Aida, K. Palme and B. Scheres, Nature, 2005, 433, 39–44.
38. J. R. Dinney and P. N. Benfey, Cell, 2008, 132, 553–557.
39. R. R. Finkelstein and T. J. Lynch, Plant Cell, 2000, 12, 599–609.
40. K. Nakashima, Y. Fujita, K. Katsura, K. Maruyama, Y. Natusaka, M. Seki, K. Shinozaki and K. Yamaguchi-Shinozaki, Plant Mol. Biol., 2006, 60, 51–68.
41. X. Sun, P. Feng, X. Xu, H. Guo, J. Ma, W. Chi, R. Lin, C. Lu and L. Zhang, Nat. Commun., 2011, 2, 477.
42. Y. Fujita, M. Fujita and K. Shinozaki, J. Plant Res., 2011, 124, 509–525.
43. D. Shkolnik-Inbar, G. Adler and D. Bar-Zvi, Plant J., 2013, 73, 993–1005.
44. K. Feki, F. J. Quintero, H. Khoudi, E. O. Leidi, K. Masmoudi, J. M. Pardo and F. Brini, Plant Cell Rep., 2014, 33, 277–288.
45. R. Hedrich, Physiol. Rev., 2012, 92, 1777–1811.
46. D. Imes, P. Mumm, J. Böhm, K. A. Al-Rasheid, I. Marten, D. Geiger and R. Hedrich, Plant J., 2013, 74, 372–382.
47. T. Maierhofer, C. Lind, S. Hüttl, S. Scherzer, M. Papenfuß, J. Simon, K. A. Al-Rasheid, P. Ache, H. Rennenberg, R. Hedrich, T. D. Müller and D. Geiger, Plant Cell, 2014, 26, 2554–2567.
48. T. Taji, M. Seki, M. Satou, T. Sakurai, M. Kobayashi, K. Ishiyama, Y. Narusaka, M. Narusaka, J. K. Zhu and K. Shinozaki, Plant Physiol., 2004, 135, 1697–1709.
49. S. M. Gupta, S. Singh, P. Pandey, A. Grover and Z. Ahmed, Plant Signaling Behav., 2013, 8, e25388.
50. X. Han, S. Tang, Y. An, D. C. Zheng, X. L. Xia and W. L. Yin, J. Exp. Bot., 2013, 64, 4589–4601.
51. Y. Shen, N. C. Silva, L. Audonnet, C. Servet, W. Wei and D. X. Zhou, Front. Plant Sci., 2014, 5, 290.
52. H. Yuan, S. Hu, P. Huang, H. Song, K. Wang, J. Ruan, R. He and D. Cui, Nanoscale Res. Lett., 2011, 6, 44.
53. W. Zhang, C. Wang, Z. Li, Z. Lu, Y. Li, J. Yin, Y. Zhou, X. Gao, Y. Fang, G. Nie and Y. Zhao, Adv. Mater., 2012, 24, 5391–5397.
54. R. Barrena, E. Casals, J. Colon, X. Font, A. Sanchez and V. Puntes, Chemosphere, 2009, 75, 850–857.
55. M. V. Khodakovskaya, K. de Silva, A. S. Biris, E. Dervishi and H. Villagarcia, ACS Nano, 2012, 6, 2128–2135.
56. N. I. Kovtyukhova, P. J. Olivier, B. R. Martin, T. E. Mallouk, S. A. Chizhik, E. V. Buzaneva and A. D. Gorchinskiy, Chem. Mater., 1999, 11, 771–778.
57. D.-Y. Wang, Y.-J. Xu, Q. Li, X.-M. Hao, F.-Z. Sun, K.-M. Cui and Y.-X. Zhu, Plant J., 2003, 33, 285–292.
58. D.-Y. Wang, Q. Li, K.-M. Cui and Y.-X. Zhu, J. Integr. Plant Biol., 2007, 49, 1627–1633.
59. D.-Y. Wang, Q. Li, K.-M. Cui and Y.-X. Zhu, J. Integr. Plant Biol., 2008, 50, 475–483.
60. A. K. Shaw and Z. Hossain, Chemosphere, 2013, 93, 906–915.
61. M. Iriti, L. Belli, C. Nali, G. Lorenzini, G. Gerosa and F. Faoro, Environ. Pollut., 2006, 141, 275–282.
62. Z. Y. Du, M. X. Chen, Q. F. Chen, S. Xiao and M. L. Chye, Plant, Cell Environ., 2013, 36, 300–314.

---

Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra10621k

‡ These authors contributed equally to this work.

---