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Graphene oxide amplifies the
phytotoxicity of arsenic in wheat
Xiangang Hu
1
, Jia Kang
1
, Kaicheng Lu
1
, Ruiren Zhou
2
,LiMu
3
& Qixing Zhou
1
1
Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of Education), Tianjin Key Laboratory of Environmental
Remediation and Pollution Control, College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China,
2
College of Life Science, Nankai University, Tianjin 300071, China,
3
Institute of Agro-environmental Protection, Ministry of
Agriculture, Tianjin 300191, China.
Graphene oxide (GO) is widely used in various fields and is considered to be relatively biocompatible.
Herein, ‘‘indirect’’ nanotoxicity is first defined as toxic amplification of toxicants or pollutants by
nanomaterials. This work revealed that GO greatly amplifies the phytotoxicity of arsenic (As), a widespread
contaminant, in wheat, for example, causing a decrease in biomass and root numbers and increasing
oxidative stress, which are thought to be regulated by its metabolisms. Compared with As or GO alone, GO
combined with As inhibited the metabolism of carbohydrates, enhanced amino acid and secondary
metabolism and disrupted fatty acid metabolism and the urea cycle. GO also triggered damage to cellular
structures and electrolyte leakage and enhanced the uptake of GO and As. Co-transport of GO-loading As
and transformation of As(V) to high-toxicity As(III) by GO were observed. The generation of
dimethylarsinate, produced from the detoxification of inorganic As, was inhibited by GO in plants. GO also
regulated phosphate transporter gene expression and arsenate reductase activity to influence the uptake and
transformation of As, respectively. Moreover, the above effects of GO were concentration dependent. Given
the widespread exposure to As in agriculture, the indirect nanotoxicity of GO should be carefully considered
in food safety.
G
raphene oxide (GO) nanosheets, a novel engineered nanomaterial, have been applied in various fields,
such as biology, chemistry, medicine, exploration and environmental protection
1,2
. Before using GO in
such applications, it is essential to scientifically understand ecological risk and health significance of this
material. The interactions of GO with biological molecules (DNA and proteins) as well as its cytotoxicity, uptake,
pharmacokinetics and hematopathology have mainly been examined in animal experiments
3,4
, while its phyto-
toxicity remains largely unexplored. Considering that plants are the primary producers in ecosystems, under-
standing the interactions between nanomaterials and plants is crucial for comprehending the impact of
nanomaterials on ecosystems and food safety. However, little progress has been made in this arena
5–7
.
The direct nanotoxicity of GO observed in living organisms suggests that GO is relatively biocompatible. For
instance, GO acts as a general enhancer of cellular growth by increasing cell attachment and proliferation
8
.
Further investigations based on histological examination of organ slices and hematological analyses revealed
that GO toxicity in the treated animals was insignificant
9
. The positive impacts of 400 mg/L of GO included
significant improvement of the plant health status indicated by decreased levels of H
2
O
2
and lipid and protein
oxidation
10
. In fact, exposure of living organisms to multi-toxicants or pollutants is generally concomitant in the
natural environment, rather than occurring in single exposure events. For instance, multiwalled carbon nano-
tubes and fullerenes increase the accumulation of pesticides in agricultural plants, with implications for food
safety
11
. Fullerenes also increase the toxicity and bioaccumulation of xenobiotic organic compounds in algae and
crustaceans
12
. Diuron sorbed onto carbon nanotubes enhances toxicity to Chlorella vulgaris
13
. Nano-Fe
2
O
3
/Al
2
O
3
significantly enhances the toxicity and accumulation of arsenic (As) (V) compounds in Ceriodaphnia dubia
14
.
These results all indicate toxic amplification of toxicants or pollutants by nanomaterials. Herein, this phenom-
enon is defined as ‘‘indirect’’ nanotoxicity.
In addition to the amplification of uptake indicated above, ‘‘indirect’’ nanotoxicity can involve multiple
phenomena, such as toxic amplification of coexistent toxicants via the regulation of transformation, metabolism,
genes, proteins and enzymes. As is a ubiquitous toxic element and shows the potential to pose a great risk to
animal and human health
15
. GO, which has a large hydrophilic-specific surface, can directly adsorb and indirectly
transform As species
16–18
. Wheat (Triticum aestivum L.) is one of the most important food crops globally and is a
recommended model organism in the guidelines for chemical testing proposed by the Organization for Economic
OPEN
SUBJECT AREAS:
PLANT STRESS
RESPONSES
METABOLOMICS
ECOTOXICOLOGY
ENVIRONMENTAL CHEMISTRY
Received
13 May 2014
Accepted
30 July 2014
Published
19 August 2014
Correspondence and
requests for materials
should be addressed to
Q.Z. (zhouqx@nankai.
edu.cn)
SCIENTIFIC REPORTS | 4 : 6122 | DOI: 10.1038/srep06122 1
Co-operation and Development (OECD)
19
. Widespread As contam-
ination in wheat is a global issue
20–22
. The influence of GO on the
uptake, transformation, metabolism and molecular toxicity of As in
wheat is a critical issue in food safety. However, there is little available
information related to this topic.
The primary goals of this study were to thoroughly assess the
indirect nanotoxicity of GO in relation to As in wheat. Specifically,
we determined: (i) the effects of GO on phytotoxicity of As, including
growth inhibition and oxidative stress; (ii) the metabolic regulation
of toxicity amplification; (iii) the influence of cell structure damage
and permeability on the uptake of GO and As; (iv) the regulation of
As uptake and transformation through the chemical interactions of
GO; and (v) the regulation of As uptake and transformation through
the biological interactions of GO, such as through gene expression
and enzymes catalysis.
Results
Characteristics of GO. The surface chemistry of GO is described in
Figure S1. X-ray photoelectron spectroscopy (XPS) images showed
that GO included 59.99% C1s, 36.57% O1s and 3.44% N1s. C1s was
shown to be a composite of C-C (46.79%), C-H (42.59%) and C-O-C
(10.62%), while O1s was a composite of C5O (75.24%) and O5C-O
(24.76%) and N1s was attributed to N-C (88.20%) and N5C
(11.80%). These oxygen- and nitrogen-containing groups result in
the hydrophilic surface of GO. The characteristics of GO are further
analyzed in Figure S2. A peak at 230 nm and a peak shoulder from
approximately 280 to 300 nm, which are the typical adsorption peaks
of GO, were observed. There was no obvious alteration of spectra
after 8 days in the GO (10 mg/L) solution, suggesting that GO is very
stable in the aqueous phase. This excellent stability represents a
benefit for the interactions between GO and organisms. Field
emission transmission electron microscopy images showed
irregular folds on GO nanosheets, which reflected the flexibility of
GO. Atomic force microscope imaging demonstrated that the
thickness of GO nanosheets was approximately 0.8–1 nm. The
diameter ranged from approximately 1–5 mm. Hence, as a wide
size distribution would increase the nondeterminacy of the
nanotoxicity test GO was treated using ultrasound (150 W,
30 min) prior to nanotoxicity testing, leading to a narrow size
distribution (465–486 nm, centered on 476 nm). The Raman
spectrum was used to characterize the structure of GO, as shown
in Figure S3. The typical D and G bands of graphene, located at
approximately 1355 and 1602 cm
21
, were detected; these bands
reflect the disordered structure and ordered sp
2
carbon system of
graphene, respectively
23,24
. Other features included the 2D band
and combined-mode D 1 G band located at approximately 2747
and 2939 cm
21
, respectively. The positions and intensity of the 2D
bands reflect the number of graphene layers
23,24
: the 2D band of
single-layer graphene centers around 2679 cm
21
, and the 2D/G
intensity ratio of single-layer graphene is .1.6
24
. In this work, the
2D peak was located at approximately 2747 cm
21
, and the 2D/G
intensity ratio was found to be 0.6 for the single-layer GO.
Compared to pristine graphene, the disagreement likely was due to
the oxygen-containing groups in GO
25
.
GO amplifies the toxicity of As(V). The effects of GO and As(V) on
wheat seed germination are presented in Figure 1. Compared with
the control, As(V) and GO did not significantly inhibit seed
germination, by having assessed using the fresh weight, the
germination rate, the root length, the root number and the shoot
length, as shown in Figure 1a. Concerning positive effects, As(V)
slightly increased the germination rate and the root number. The
shoot length significantly reduced with an increase in the GO
concentration in the AsGO groups. It appears that GO directly
affected the shoot length and As(V) enhanced the impact of GO
under the condition of As(V) and GO co-exposure. AsGO 0.1
markedly (P , 0.05) reduced the shoot length and this reduction
was more intensive with an increase in GO concentrations. AsGO 10
significantly reduced both the fresh weigh and the shoot length.
Chlorophyll biosynthesis, malondialdehyde (MDA) contents and
the activities of peroxidase (POD) and superoxide dismutase
(SOD) exhibited similar effects to growth inhibition, as shown in
Figure 1b. There were no significant differences between the
control and As(V) groups. GO directly affected the SOD and POD
activities, and the toxicity was significantly amplified by the co-
exposure of As(V) and GO. AsGO induced a significant reduction
of chlorophyll contents, especially that of chlorophyll b. The activity
of SOD increased with an increase in the concentration of GO in the
AsGO groups. The changes in MDA as an indicator of lipid
peroxidation were not significant among all groups. The above
results suggested that the relatively biocompatible GO markedly
enhanced the phytotoxicity of As.
Metabolism regulates the amplification of nanotoxicity. Com-
pared with proteins and genes, metabolic changes directly reflect
biological processes
26,27
. Herein, metabolomics analyses were
conducted to elucidate the mechanisms of indirect nanotoxicity of
GO in the presence of As. A total of 200 peaks in each sample were
analyzed, and 65 metabolites were identified, as shown in Tables S1
and S2. Compared with other results from metabolic analyses (e.g.,
using
1
H NMR technology), the applied extraction procedure and gas
chromatography-mass spectrometry/mass spectrometry with the
derivatization method were effective for analyzing the multiclass
metabolites of plant cells
28,29
. These metabolites correspond to the
main metabolic pathways involved in carbohydrate, amino acid and
fatty acid production, secondary metabolism and the urea cycle. The
metabolites were analyzed using t-tests in Multiple Experiment
Viewer 4, and the metabolites that were found to show significant
differences (P , 0.05) in the roots and shoots, which are listed in
Figure 2a and 2b, respectively. Based on hierarchical clustering
(HCL) analysis, the root metabolites were divided into two
clusters: the control and As/GO/AsGO. Furthermore, the As/GO/
AsGO cluster was divided into two sub-clusters: As and GO/AsGO.
Similarly, the shoot metabolites were divided into two clusters: the
control and As/GO/AsGO; and As/GO/AsGO was further divided
into two sub-clusters: As/GO and AsGO. Principal component
analysis (PCA) was also employed to illustrate the metabolic
specificity among different groups (Figure 2c). The samples were
divided into two groups: the control and As/GO/AsGO. The
discrete scores suggested that the metabolism displayed significant
differences among the As, GO and AsGO groups. The above results
demonstrated that AsGO markedly altered the metabolism in the
control, As and GO groups.
The results in Figure 1 suggest that the root number and chloro-
phyll b content were sensitive to AsGO toxicity in the roots and
shoots, respectively. To further assess metabolic regulation, using
both the root number and chlorophyll b as Y variables, an orthogonal
partial least squares discriminant analysis (OPLS-DA) model was
implemented (Figures S4–6). In the roots, 7 of 23 metabolites showed
a positive coefficient CS (coeffCS) value, indicating that these meta-
bolites made positive contributions to the observed root number.
Among these 7 metabolites, AsGO reduced the contents of xylose,
maltose, aspartic acid and aconitic acid with a VIP (variable import-
ance for the projection) of more than 0.7. Accordingly, the remaining
metabolites made negative contributions to the observed root num-
ber. AsGO increased the contents of 16 metabolites, and the meta-
bolites exhibiting a VIP of more than 1.4 included naphthalene,
monopalmitin, asparagine, galactose and cyclononasiloxane octade-
camethyl-. In the shoots, 9 of 57 metabolites with a positive coeffCS
made positive contributions to the anabolism of chlorophyll b.
Among these 9 metabolites, AsGO decreased the contents of galac-
topyranoside and galactose, which showed a positive coeffCS with a
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SCIENTIFIC REPORTS | 4 : 6122 | DOI: 10.1038/srep06122 2
VIP of more than 1.3. The remaining 48 metabolites made negative
contributions to the anabolism of chlorophyll b. AsGO enhanced the
contents of the 48 metabolites, and the metabolites with a VIP of
more than 1.3 included monopalmitin, galactofuranoside, pentaric
acid, propanoic acid, alkane, lysine, serine, threonine, phenylalanine,
leucine and isoleucine. The identified high-VIP metabolites are orga-
nized in Figure 3, which indicates the effects of AsGO on the main
metabolic pathways of cells. Compared with the control and the
As-only and GO-only treatments, AsGO inhibited carbohydrate
metabolism, enhanced amino acid and secondary metabolism and
disturbed fatty acid metabolism and the urea cycle. These results are
the first to reveal the mechanisms underlying indirect nanotoxicity
using metabolomics.
Promoted damage of the cell structure by uptake of GO and As. To
investigate the structural damage to the cell wall and plasma mem-
brane, the Fourier transform infrared spectroscopy (FTIR) spectra
and electrolyte leakage in root cells were studied. Compared with the
control/As-only/GO-only treatments, AsGO increased the abun-
dance of amino groups, hydroxyl groups, cellulose and polysaccha-
rides on the cell surface (Figure 4a), indicating structural damage to
the cell wall and plasma membrane. Electrolyte leakage was further
used to detect membrane permeability. As shown in Figure 4b, As
and GO did not induce obvious electrolyte leakage. However, AsGO
led to significant electrolyte leakage, which increased with the GO
concentration. Furthermore, transmission electron microscopy
(TEM) images confirmed the structural damage to the cell wall
Figure 1
|
Growth inhibition and oxidative effects in wheat exposed to As and GO. Number of replicates, n 5 3. In Figure 1a and b, * represents
significant differences (P , 0.05) compared with the control and As groups. The units for the enzyme activities of POD and SOD are U/mg/protein and
U/mg/protein, respectively. The units for chlorophyll a, chlorophyll b and MDA are mg/g. GO0.1–10, graphene oxide 0.1–10 mg/L; As, arsenic(V),
10 mg/L; POD, peroxidase; SOD, superoxide dismutase; MDA, malondialdehyde.
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SCIENTIFIC REPORTS | 4 : 6122 | DOI: 10.1038/srep06122 3
and plasma membrane (Figure 5). The plasma membrane was close
to the cell wall in the control, and slight plasmolysis occurred in the
As sample. However, remarkable plasmolysis was observed in the
GO sample and especially for the AsGO sample, as indicated by the
pink arrows in Figure 5a–d. The elliptical outlines of root cells
changed to irregular outlines in the GO and AsGO images. In the
shoot TEM images from the control, As and GO groups, elliptical
chloroplasts adhered to the plasma membrane and regular
thylakoids in the chloroplasts were visible. However, the elliptical
chloroplasts became circular and were distant from the plasma
membrane, and the structure of the thylakoid was damaged in
shoot TEM images from the AsGO group, as indicated by the
yellow and black arrows in Figure 5e–g. In addition, plasma
membrane folding was observed, as indicated with a blue circle in
an AsGO image. It is possible that GO increased the permeability of
plant cells and then enhanced the uptake of GO and As via passive
transport across the plasma membrane. Compared with the control
and As samples, remarkable GO deposition was observed in roots
exposed to GO and AsGO, as indicated by the green arrows.
Compared with GO (Figure 5g), AsGO induced a great deal of GO
deposition, which is indicated in the cytoplasm with blue arrows
(Figure 5h) for shoot samples.
There is no question of the intracellular uptake of As, but the
uptake of GO in plant cells remains unclear. To characterize the
uptake of GO, the compositions of the dark dots in plant cells
shown in Figure 5d (root) and h (shoot) were characterized using
the Raman spectra, as shown in Figure 6. The typical D and G
bands of GO located at 1,349 and 1,590 cm
21
were detected in the
dark dot compositions of the root samples. In contrast, ther e were
no obvious GO peaks outside of the dark dots. Similar results were
found in the shoot samples, although the signals were very weak.
These results demonstrated that GO entered the plant cel ls.
Furth ermore, TEM-EDX was used to semi-quantita tively analyze
the uptake of As (Figure 7). In the root sampl es, the co ntent of As
inside and outside of the dark dots was 0.5% and 0.1%, respect -
ively. For the shoot samples, the content of As inside and outside
of the dark dots was 0.4% and 0.0%, respectively. The above
results demonstrated that As loaded onto GO was taken up via
co-transport. In summary, ce ll structure damag e enhanced the
uptake of GO and As.
Figure 2
|
Metabolic analysis of plant cells exposed to GO and As(V). The relative contents of metabolites are represented by heat maps for root (a) and
shoot (b) samples. These metabolites show significant differences (P ,0.05) between the tested groups. Cluster analysis of the metabolites was carried out
using HCL. (c) Cluster analysis of the metabolites using PCA. GO, graphene oxide; HCL, hierarchical clustering; PCA, principal component analysis.
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SCIENTIFIC REPORTS | 4 : 6122 | DOI: 10.1038/srep06122 4
GO regulates As uptake and transformation through genes and
enzymes. The uptake and transformation of As(V) were further
quantified using the liquid chromatograph-inductively coupled
plasma mass spectrometer (HPLC-ICP-MS). The uptake rate of As
was high in the plants, as shown in Figure 8a. Compared with the
19.9% rate of As uptake recorded without GO exposure, the uptake
rate of As increased to 24.6% and 32.1%, then decreased to 16.1% in
the presence of GO concentrations of 0.1, 1 and 10 mg/L,
respectively. Therefore, the regulation of uptake of As by GO was
concentration dependent. Both of As(V) and As(III) were detected in
all groups, suggesting transformation of As(V) to As(III) in vivo.
Moreover, DMA was detected in the group exposed to As(V)
alone. GO enhanced the accumulation ratio of As(III) in the roots,
with root-to-shoot concentration ratio of 14.0, 10.9, 16.8 and 22.1
being observed in the As, AsGO 0.1, AsGO 1 and AsGO 10 groups,
respectively. The accumulation ratio for As(V) was 4.2, 9.1, 11.4 and
7.2 in As, AsGO 0.1, AsGO 1 and AsGO 10 groups, respectively,
showing concentration dependence. In summary, the generation of
DMA to detoxify inorganic As was inhibited by GO, and GO led to
the accumulation of high-toxicity As(III) in wheat roots.
To identify roles of direct chemical regulation in As uptake and
transformation by GO, the interaction of GO with As(V) in the
aqueous phase was investigated (Figure 8b). In the As-only group,
13.4% of As(V) was transformed to As(III). In the other groups, GO
did not significantly alter transformation of As(V). However, 73.5–
76.6% of the transformed As(III) was absorbed on GO, and the rate
of transformation was not significantly different in the different
groups. The ratio of absorbed As to total As increased with the GO
concentration, with values of 3.1, 8.9 and 12.1% being obtained in
GO 0.1, GO 1 and GO 10, respectively. These results showed that As,
especially As(III), was adsorbed onto GO, but GO played a limited
role in transformation of As through direct chemical reactions. To
further identify the functions of biological regulation in As uptake
and transformation by GO, the intracellular activities of key enzymes
and gene expression were quantified, as shown in Figure 8c. GO at
low and moderate concentrations (0.1 and 1 mg/L) enhanced the
expression of phosphate transporter genes that transport As through
the plasma membrane, but their expression decreased under a high
concentration of 10 mg/L GO. The activity of arsenate reductase,
which plays an important role in transformation of As(V) to
As(III), showed similar changes to the expression of phosphate
transporter genes. The activity of arsenate reductase was enhanced
and then inhibited by increasing GO concentrations. These results
demonstrated that GO regulated As uptake and transformation
through both chemical and biological processes and this regulation
was concentration dependent.
Discussion
In general, GO is thought to be relatively biocompatible compared
with metal nanoparticles and other carbon nanomaterials
3,30–32
.No
Figure 3
|
Effects of AsGO on the main metabolic pathways of plant cells. The metabolites labeled with arrows showed significant differences (P , 0.05)
between the tested groups. Compared with the control, As-only and GO-only groups, AsGO induced metabolic changes with a high VIP in roots and
shoots, which are indicated with blue and red arrows, respectively. The directions of the arrows indicate the up-regulation and down-regulation of
metabolites. The filled and dotted black arrows represent direct and indirect reactions, respectively. VIP, variable importance for the projection; GO,
graphene oxide; As, arsenic.
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SCIENTIFIC REPORTS | 4 : 6122 | DOI: 10.1038/srep06122 5
adverse effects of GO on the growth and biomass of higher plants,
such as cabbage, tomato, red spinach and lettuce, have been observed
at concentrations below 400 mg/L
5,10
. Treatment with GO dosages
ranging from 0.1 to 10 mg/L does not significantly disturb seed
germination in wheat, as demonstrated based on the growth, biomass
and oxidative stress of the plants. Although GO slightly reduced root
growth, the reduction was not remarkable. The amino groups on
biological cells could interact with GO, resulting in GO wrapping
or adsorbing around cells; the cells were then isolated from the cul-
ture medium, leading to a reduction in plant growth
33
. Treatment
with As(V) at a concentration of 10 mg/L also did not induce sig-
nificant adverse effects, but considerable adverse effects were
observed when wheat was exposed to As(V) and GO together, even
at a very low dose (0.1 mg/L) of GO. Compared with that in the
control, the application of 0.1 mg/L of GO with As triggered a reduc-
tion of shoot length and chlorophyll b contents and a increase of SOD
activity. Similarly, when the 24-h mortality of Ceriodaphnia dubia
was assessed, 2.4–3.2 mg/L As(V) led to an increase from 2.5–37.5%
without nanoparticles to 43–65% in the presence of 10–50 mg/L
nano-Al
2
O
3
and nano-Al
2
O
3
34
. Given that As contamination is wide-
spread in agricultural plants such as wheat, rice and corn
21,22
, the
impact of ‘‘indirect’’ nanotoxicity on food safety should be
considered.
The potential benefits of applying metabolomics to reveal molecu-
lar mechanisms of nanotoxicology and nanomedicine are only start-
Figure 4
|
Structural damage to the cell wall and plasma membrane.
Fourier transform infrared spectra (a) and electrolyte leakage (b) in root
cells. GO10 and GO1 are cells exposed to 10 and 1 mg/L GO, respectively.
As, arsenic (V) 10 mg/L; GO, graphene oxide. The chemical groups in the
Fourier transform infrared spectra refer to previous work
25–27
.
Figure 5
|
Transmission electron microscopy images of plant cells. Pink
arrows indicate the cell wall in (a)–(d). Green arrows indicate GO
deposition in the cytoplasm for (c) and (d). Yellow arrows indicate
chloroplasts in (e)–(h). Black arrows indicate thylakoids in (e)–(h). Blue
arrows indicate GO deposition in the cytoplasm for (g) and (h) images.
The blue circle indicates the plasma membrane in (h). Cw, cell wall; Pm,
plasma membrane; Chl, chloroplast; Thy, thylakoid. Scale bar, 5 mm.
Figure 6
|
Raman spectra of the dark dot compositions of the plant cells
shown in Figure 5d (root) and h (shoot) exposed to AsGO. GO, graphene
oxide. The chemical groups in the Raman spectra refer to previous
work
28,29
.
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SCIENTIFIC REPORTS | 4 : 6122 | DOI: 10.1038/srep06122 6
ing to be realised
3,35
. Compared with the control/As-only/GO-only
treatments, AsGO inhibited carbohydrate metabolism, except for
galactofuranoside. The inhibition of carbohydrate metabolism was
linked to the insufficiency of energy supplementation, which was
consistent with growth inhibition. Carbohydrate metabolism is also
considered to be an indicator of osmoprotection
36
. Thus, the results
depicted in Figure 4b indicate that AsGO increased cellular electro-
lyte leakage. There is extensive and unequivocal evidence that amino
acid metabolism and secondary metabolism in plants are associated
with the cellular response to the stress
37–39
. AsGO enhanced amino
acid and secondary metabolism, including that of leucine, glycine,
phenylalanine, naphthalene and octadecamethyl-cyclononasiloxane.
Compared with that in the other groups, AsGO significantly
enhanced the accumulation of a saturated fatty acid (alkane).
Increases in saturated fatty acids are linked to the reduction of mem-
brane fluidity
40
, which most likely led to the membrane structure
damage depicted in Figures 4 and 5. AsGO also disturbed the urea
cycle and induced the accumulation of amino acids, such as lysine,
threonine, asparagine and isoleucine. Specifically, the pathway of
nitrogen storage was altered. These metabolic responses may be
helpful for understanding the molecular mechanisms underlying
toxicity amplification from the co-exposure of As(V) and GO to
plants.
The mechanisms of the uptake of GO, and even carbon nanoma-
terials, into plant cells remain obscure at present. Fullerenes up to
100 nm in diameter enter the gaps between the cell wall and the
plasma membrane
41
, suggesting that nanomaterials can penetrate a
cell wall even when they are larger than the cell wall pores
41
.As
shown in Figure 5c and d, uptake of GO was observed in TEM images
and was confirmed by the Raman spectra presented in Figure 6.
Generally, the uptake of nanomaterials is increased by the enhance-
ment of permeability
41
. AsGO triggered structural damage to the cell
wall and plasma membrane and increased electrolyte leakage
(Figure 4), which generally occurred for very high concentrations
(more than 100 mg/L) of GO exposure to cells
31,32
. Moreover,
Figure 5c and d demonstrates that plasmolysis occurred. It has been
proposed that carbon nanotube and nano-ZnO materials enter the
roots of seedlings by increasing the permeability of plant cell walls
42
.
After the uptake of GO and As, the damage and structural alterations
observed in organelles were considerable, including destruction of
chloroplasts, which corresponded to a decrease of chlorophyll
biosynthesis.
Another important finding was that As accumulated on GO in
cells (Figure 7), demonstrating that GO increased the uptake of As
via co-transport. Figure 8a shows that 0.1 and 1 mg/L GO enhanced
the uptake of As, whereas 10 mg/L GO reduced As uptake.
Figure 7
|
Energy dispersive spectra of the dark dot compositions of the plant cells from Figure 5d (root) and h (shoot) exposed to AsGO.
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SCIENTIFIC REPORTS | 4 : 6122 | DOI: 10.1038/srep06122 7
Concerning direct chemical interactions, our results showed that As,
and especially As(III), could be absorbed on GO, and the absorption
increased with the GO concentration (Figure 8b). These findings
implied that the uptake of As involved pathways other than chemical
interactions alone. It has been reported that As is taken up by plants
via phosphate transporters
43
. Recently, the genotoxicity of graphene-
based materials has attracted much attention
32,41
. Figure 8c shows
that low concentrations of GO increased the expression of phosphate
transporter genes, while high concentrations of GO decreased their
expression. Therefore, GO influenced the uptake of As via at least
three pathways: permeability regulation, co-transport and trans-
porter gene regulation. DMA is a less toxic methylated form of inor-
ganic As
44
. GO inhibited the formation of DMA in plants and then
enhanced the phytotoxicity of As, as shown in Figures 1 and 8a.
As(III) is more toxic in terms of ecological and health risks than
As(V)
45
. Figure 8a shows that the ratio of As(III) to As(V) first
increased and then decreased with the GO concentration in both
the roots and shoots. Concerning direct chemical reactions, as indi-
cated in Figure 8b, GO increased the transformation of As(V) to
As(III), but there was no significant change associated with the GO
concentration. Arsenate reductase activity is considered to play a
critical role in the transformation of As(V) to As(III) in plants
44,46
.
GO regulated the transformation of As through arsenate reductase
activity (Figure 8c). Therefore, GO regulated the uptake and trans-
formation of As through chemical and biological processes together.
In conclusion, ‘‘indirect’’ nanotoxicity is defined as toxic amp-
lification of other toxicants or pollutants by nanomaterials. This
work comprehensively analyzed ‘‘indirect’’ nanotoxicity of the rela-
tively biocompatible GO, which amplified the phytotoxicity of wide-
spread As in wheat. The indirect nanotoxicity of GO involved at least
five components, as described in Figure S7: (i) enhancement of
growth inhibition and oxidative stress; (ii) regulation of key forms
of metabolism, such as carbohydrate, amino acid and secondary
metabolism; (iii) increasing damage to cell structures and permeab-
ility; (iv) regulation of As uptake and transformation via chemical
interactions, such as through As loading onto GO entering the cell;
and (v) regulation of As uptake and transformation via biological
interactions, such as transporter gene expression and enzyme cata-
lysis. Given the widespread exposure to As in agriculture, ‘‘indirect’’
nanotoxicity of GO should be considered in relation to food safety.
Methods
Characteristics of GO nanosheets. GO nanosheets were obtained from Nanjing
XFNANO Materials Tech Co., Ltd., China. XPS measurements were conducted using
the Axis Ultra XPS system (Axis Ultra DLD, Kratos) with a monochromatic Al Ka X-
ray source (1486.6 eV). The spectra were analyzed with Casa-XPS V2.3.13 software.
Samples were prepared and dispersed in absolute ethanol, and atomic force
microscopy (AFM) and field emission TEM measurements were then conducted on a
Veeco Nanoscope 4 or JEM-2010 FEF, respectively. UV spectra were obtained using a
TU-1901 spectrophotometer with UVWin5 software. The size distribution was
detected through wide-angle light scattering (BI-200SM, Brookhaven). Raman
spectrometry (Thermo Scientific, DXR) with a 514 nm laser was used to analyze GO
structure.
Seed germination experimen t. Wheat (Triticum aestivum L.) seeds were sterilized
for 20 min using 2% H
2
O
2
. Subsequently, the seeds were completely rinsed using pure
water (18.2 V/cm). Double-layer filter paper (diameter, 9 cm) was placed in a culture
dish. In a same volume of 6 mL, 10 mg/L As(V); 0.1–10 mg/L GO; 10 mg/L As(V)
plus 0.1–10 mg/L GO; and pure water were added to different culture dishes,
respectively. In each culture dish, 15 seeds with the same diameter and shape were
germinated. For uniformity, the seeds were arranged on the plates with consistent
growth spacing. The culture dishes were placed in a climate chamber at 3000 Lx
irradiation under a temperature of 24uC temperature 80% humidity.
Plant growth and oxidative stress. To avoid the influence of other factors, the culture
medium, temperature and spacing were kept uniform for all groups, such that these
factors did not have adverse effects on plant growth during germination. To reduce
differences among m anipulations, four dishes were prepared for each group.
Germination was performed for eight days. The fresh weight, leaf length, root length
and root number were recorded using a plant analyzer (EPSON Perfection V700
Photo, SilverFast STD4800). The content of chlorophyll a, the activities of SOD/POD
and content of MDA were analyzed using a TU-1901 spectrophotometer, as described
Figure 8
|
GO-regulated As uptake and transformation. GO-regulated As
uptake and transformation in roots and shoots (a). GO-regulated As
uptake and transformation in the aqueous phase (b). GO-regulated
activity of arsenate reductase and the expression of phosphate transporter
genes (c). GO, graphene oxide; AsGO0.1–10, 10 mg/L As(V) with GO
ranged from 0.1–10 mg/L. DMA, dimethylarsinate.
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 4 : 6122 | DOI: 10.1038/srep06122 8
previously
47
. The concentration of lipid peroxides was quantified in terms of the
MDA concentration. As monitored at 560 nm, SOD activity was assayed by
measuring the ability of the enzyme extract to inhibit the photochemical reduction of
nitrotetrazolium blue chloride. After the enzyme extract reacted with H
2
O
2
,POD
activity was measured based on the H
2
O
2
decomposition rate, using guaiacol as a
hydrogen donor, and CAT activity was estimated based on the decrease in absorbance
at 240 nm.
Metabolic analyses. To obtain representative material from large plant tissues, root
and shoot tips (length, 1 cm) were collected from more than 30 random plants using a
sharp knife. To avoid metabolic alterations during preparation, the root and shoot
tips (0.1 g) were snap-frozen in liquid nitrogen and then ground with 2 mL of a
solution of methanol5chloroform5water (volumetric ratio 5 2.55151) and 0.2 mg/
mL ribitol (50 mL) as an internal standard. Metabolites were intensively extracted
using ultrasound (200 W, 30 min) in an ice bath, then centrifuged for 10 min at
9,000 g at 4uC. The supernatant was subsequently collected, and the sediment was
extracted again using 1 mL of a methanol5chloroform solution (volumetric ratio 5
151), after which ultrasound and centrifugation were performed as described above.
This supernatant wa s mixed with the previously collected supernatant, and water
(500 mL) was added, followed by centrifugation at 9,000 g for 5 min. The lower phase
was dried through nitrogen blow-off. For the upper phase, methanol was removed via
nitrogen blow-off, and the remaining water was lyophilized. Methoxamine
hydrochloride (20 mg/mL, 50 mL) and N-methyl-N-
(trimethylsilyl)trifluoroacetamide (80 mL) were added as derivatives. Samples (1 mL)
were injected into the gas chromatography column in split mode (1525). Gas
chromatography (Agilent 6890N, Agilent Technologies, USA) linked with quadruple
mass spectrometry (Agilent 5973, Agilent Technologies, USA) was applied to analyze
metabolites. The detection and identification of metabolites were performed in a
previous study by our group
48
.
Electron microscopy and spectroscopy analysis. TEM was used to investigate the
cellular ultrastructure. Tips of roots and shoots were thoroughly washed using water,
prefixed in 2.5% glutaraldehyde, postfixed in 1% osmium tetroxide for 2 h,
dehydrated in a graded ethanol series and then embedded in epoxy resin (ETON 812).
Ultrathin sections (80 nm) were cut using an ultramicrotome with a diamond knife
(Leica EM FC7). TEM images were obtained on Hitachi HT7700 TEM. TEM coupled
EDX (JEM-2010FEF, phoenix 60 t) was conducted to detect elements in the roots and
shoots. The operation parameters were as follows: 200.00 kV, 45.00 azimuth, 51.2
ampT Sutw-sapphire detector type, with a resolution of 137.25. A Raman
spectrometer was used to analyze the GO compositions in the cells, which were
recorded on a Thermo Scientific DXR Raman microscope using 780 nm excitation
from a diode-pumped, solid state (DPSS) laser. FTIR was conducted to analyze the
chemical groups on the cellar surface, and the results were recorded on a Bruker
Tensor 27 infrared spectrometer with a resolution of 2 cm
21
at 4,000–400 cm
21
.A
measured amount of biomass was mixed with potassium bromide (mass ratios 5
2598). The mixture was ground into fine particles and compressed into a translucent
sample disk with a manual hydraulic press. The di sks were then fixed in the FTIR
apparatus for analysis.
Electrolyte leakage. Electrolyte leakage was measured to detect membrane
permeability and assessed as described in a previous report
49
. Root samples were
washed three times using pure water to remove surface-adhered electrolytes. The
roots were placed in closed vials containing 10 mL of pure water and incubated at
25uC on a shaker at 500 rmp for 24 h, after which the electrical conductivity of the
solution (EC
0
) was determined. The samples were then autoclaved at 120uC for
20 min, and the final electrical conductivity (EC
t
) was obtained after equilibration at
25uC. Electrolyte leakage was defined as follows: electrolyte leakage (%) 5 [EC
0
/EC
t
]
3 100.
Detection of As uptake and transformation. Samples of the roots and shoots with
a fresh weight of 0.1 g were lyophilized. The samples were then ground in 5 mL of
methanol and water (volume ratio, 251). As was extracted using ultrasound at
150 W for 30 min. Subsequently, the samples were centrifuged at 8,000 g for
15 min, and the supernatant was collected. The sediments were extracted and
centrifuged again, and the two supernatants were combined. After filtration
through a 0.22 mm membrane, 1 mL of the sample was injected into HPLC-ICP-
MS (Agilent 1260/7700X). As a sub-experiment, the direct interactions of GO with
As were studied in the aqueous phase. As(V) at 10 mg/L reacted with a GO at 0.1–
10 mg/L in the same incubator as was used for seed germination. The suspension
was filtered to separate GO (diameter, 465–486 nm, center at 476 nm) with an
ultrafilter (3KD, approximately 10 nm pore, Millipore). The filtrate was directed
analyzed using HPLC-ICP-MS. The adsorbed As was successively eluted using
0.5 mL of 1 M NaOH, 0.5 mL of 1 M HCl and 0.5 mL of water. The eluent was
analyzed through HPLC-ICP-MS. In all samples, As(V), As(III), dimethylarsinate
and monomethylarsinate were detected.
Arsenate reductase activity. Arsenate reductase activity was assayed using the
coupled enzymatic reaction described by Duan et al
46
. The assay was performed in
50 mM 3-(N-morpholino) propanesulfonic acid, 50 mM 2-(N-
morpholino)ethanesulfonic acid, pH 6.5, containing 1.5 mM nicotinamide adenine
dinucleotide phosphate, 1 unit of yeast glutaredoxins, 1 mM glutathione and 10 mM
sodium arsenate, in a total volume of 1.5 mL. All of the measurements were
performed at 30uC. The oxidation of nicotinamide adenine dinucleotide phosphate
was monitored by recording the decrease in absorbance using a TU-1901
spectrophotometer with UVWin5 software.
Expression of phosphate transporter genes. The analysis of phosphate transporter
gene expression was described in detail previously
50
. Briefly, based on known high-
affinity phosphate transporter sequences, degenerate polymerase chain reaction
(PCR) primers were designed against highly conserved regions identified through
sequence alignment. A combination of two upstream primers (59-TTYTTYCANG-
AYGCNTAYGAY-39 and 59-GTNCCNGGNTAYTGGTTYCANGT-39)anda
downstream primer (59-GGNCCRAARTTNGCRAARAA-39) were chosen for PCR.
For phosphate transporter sequences, first-strand cDNA synthesis was performed on
1 mg of total RNA as a template with 5 pmol of the antisense primer 59-
GGNCCRAARTTNGCRAARAA-39. For 18S RNA reactions, the antisense primer
59-CACTTCACCGGACCATTCAATCG-39 was employed for first-strand synthesis
using 0.5 mg of total RNA as a template.
Statistical analyses. All treatments included three replicates, and the mean 6 SD
(standard deviation) is presented with error bars. A t-test with Welch approximation
for unequal group variances with P-values based on the t-distribution was performed
with a cutoff of P , 0.05 in Multiple Experiment Viewer 4. PCA and OPLS-DA were
performed using SIMCA-P 11.5 software. A heat map was generated using MeV 4.8.1
software. The default distance metric for HCL was Pearson correlation, and the
linkage method selection was average linkage clustering.
1. Dreyer, D. R., Park, S., Bielawski, C. W. & Ruoff, R. S. The chemistry of graphene
oxide. Chem. Soc. Rev. 39, 228–40 (2010).
2. Novoselov, K. S. et al. A roadmap for graphene. Nature 490, 192–200 (2012).
3. Hu, X. & Zhou, Q. Health and ecosystem risks of graphene. Chem. Rev. 113,
3815–35 (2013).
4. Seabra, A. B., Paula, A. J., de Lima, R., Alves, O. L. & Duran, N. Nanotoxicity of
graphene and graphene oxide. Chem. Res. Toxicol. 27, 159–168 (2014).
5. Begurn, P., Ikhtiari, R. & Fugetsu, B. Graphene phytotoxicity in the seedling stage
of cabbage, tomato, red spinach, and lettuce. Carbon 49, 3907–3919 (2011).
6. Nair, R. et al. Effect of carbon nanomaterials on the germination and growth of
rice plants. J. Nanosci. Nanotechno. 12, 2212–2220 (2012).
7. Zhao, L. et al. Effect of surface coating and organic matter on the uptake of CeO
2
NPs by corn plants grown in soil: Insight into the uptake mechanism. J. Hazard.
Mater. 225, 131–138 (2012).
8. Ruiz, O. et al. Graphene oxide: A nonspecific enhancer of cellular growth. ACS
Nano 5, 8100–8107 (2011).
9. Yang, K. et al. In vivo biodistribution and toxicology of functionalized nano-
graphene oxide in mice after oral and intraperitoneal administration.
Biomaterials 34, 2787–2795 (2013).
10. Anjum, N. A. et al. Single-bilayer graphene oxide sheet impacts and underlying
potential mechanism assessment in germinating faba bean (Vicia faba L.). Sci.
Total Environ. 472, 834–841 (2014).
11. De La Torre-Roche, R. et al. Multiwalled carbon nanotubes and C
60
fullerenes
differentially impact the accumulation of weathered pesticides in four
Aagricultural plants. Environ. Sci. Technol. 47, 12539–12547 (2013).
12. Baun, A., Sorensen, S. N., Rasmussen, R. F., Hartmann, N. B. & Koch, C. B.
Toxicity and bioaccumulation of xenobiotic organic compounds in the presence
of aqueous suspensions of aggregates of nano-C
60
. Aquat. Toxicol. 86, 379–387
(2008).
13. Schwab, F. et al. Diuron sorbed to carbon nanotubes exhibits enhanced toxicity to
Chlorella vulgaris. Environ. Sci. Technol. 47, 7012–7019 (2013).
14. Hu, J. Wang, D. Forthaus, B. E. & Wang, J. Quantifying the effect of nanoparticles
on As(V) ecotoxicity exemplified by nano-Fe
2
O
3
(magnetic) and nano-Al
2
O
3
.
Environ. Toxicol. Chem. 31, 2870–2876 (2012).
15. Cohen, S. M., Arnold, L. L., Beck, B. D., Lewis, A. S. & Eldan, M. Evaluation of the
carcinogenicity of inorganic arsenic. Crit. Rev. Toxicol. 43, 711–752 (2013).
16. Zhang, J. et al. Graphene oxide as a matrix for enzyme immobilization. Langmuir
26, 6083–6085 (2010).
17. Chandra, V. et al. Water-dispersible magnetite-reduced graphene oxide
composites for arsenic removal. ACS Nano 4, 3979–3986 (2010).
18. Hu, X., Mu, L., Wen, J. & Zhou, Q. Covalently synthesized graphene oxide-
aptamer nanosheets for efficient visible-light photocatalysis of nucleic acids and
proteins of viruses. Carbon 50, 2772–2781 (2012).
19. Miralles, P., Johnson, E., Church, T. L. & Harris, A. T. Multiwalled carbon
nanotubes in alfalfa and wheat: Toxicology and uptake. J. R. Soc. Interface 9,
3514–3527 (2012).
20. Rico, C. M., Majumdar, S., Duarte-Gardea, M., Peralta-Videa, J. R. & Gardea-
Torresdey, J. L. Interaction of nanoparticles with edible plants and their possible
implications in the food chain. J. Agric. Food Chem. 59, 3485–3498 (2011).
21. Cao, Q., Hu, Q. H., Baisch, C., Khan, S. & Zhu, Y. G. Arsenate toxicity for wheat
and lettuce in six Chinese soils with different properties. Environ. Toxicol. Chem.
28, 1946–1950 (2010).
22. Bhattacharya, P., Samal, A. C., Majumdar, J. & Santra, S. C. Arsenic contamination
in rice, wheat, pulses, and vegetables: A study in an arsenic affected area of West
Bengal, India. Water Air Soil Pollut. 213, 3–13 (2010).
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 4 : 6122 | DOI: 10.1038/srep06122 9
23. Ferrari, A. C. et al. Raman spectrum of graphene and graphene layers. Phys. Rev.
Lett. 97, 187401 (2006).
24. Akhavan, O. & Ghaderi, E. Graphene nanomesh promises extremely efficient in
vivo photothermal therapy. Small 9, 3593–3601 (2013).
25. Kudin, K. N. et al. Raman spectra of graphite oxide and functionalized graphene
sheets. Nano Lett. 8, 36–41 (2008).
26. Kuehnbaum, N. L. & Britz-McKibbin, P. New advances in separation science for
metabolomics: Resolving chemical diversity in a post-genomic era. Chem. Rev.
113, 2437–2468 (2013).
27. Ellis, D. I. & Goodacre, R. Metabolomics-assisted synthetic biology. Curr. Opin.
Biotechnol. 23, 22–28 (2012).
28. Nagato, E. G. et al.
1
H NMR-based metabolomics investigation of Daphnia magna
responses to sub-lethal exposure to arsenic, copper and lithium. Chemosphere 93,
331–337 (2013).
29. Wu, H. et al. Proteomic and metabolomic responses of clam Ruditapes
philippinarum to arsenic exposure under different salinities. Aquat. Toxicol. 136 ,
91–100 (2013).
30. Bagri, A. et al. Structural evolution during the reduction of chemically derived
graphene oxide. Nat. Chem. 2, 581–587 (2010).
31. Zhang, Y. et al. Cytotoxicity effects of graphene and single-wall carbon nanotubes
in neural phaeochromo cytoma-derived PC12 cells. ACS Nano 4, 3181–3186
(2010).
32. Akhavan, O., Ghaderi, E. & Akhavan, A. Size-dependent genotoxicity of graphene
nanoplatelets in human stem cells. Biomaterials 33, 8017–8025 (2012).
33. Akhavan, O. Ghaderi, E. & Esfandiar, A. Wrapping bacteria by graphene
nanosheets for isolation from environment, reactivation by sonication, and
inactivation by near-infrared irradiation. J. Phys. Chem. B 115, 6279–6288 (2011).
34. Hu, J., Wang, D., Forthaus, B. E. & Wang, J. Quantifying the effect of nanoparticles
on As(V) ecotoxicity exemplified by nano-Fe
2
O
3
(magnetic) and nano-Al
2
O
3
.
Environ. Toxicol. Chem. 31, 2870–2876 (2012).
35. Duarte, I. F. Following dynamic biological processes through NMR-based
metabonomics: A new tool in nanomedicine? J. Control Release 153, 34–39
(2011).
36. Morsy, M. R., Jouve, L., Hausman, J. F., Hoffmann, L. & Stewart, J. M. Alteration of
oxidative and carbohy drate metabolism under abiotic stress in two rice (Oryza
sativa L.) genotypes contrasting in chilling tolerance. J. Plant Physiol. 164, 157–67
(2007).
37. Marti, G. et al. Metabolomics reveals herbivore-induced metabolites of resistance
and susceptibility in maize leaves and roots. Plant Cell Environ. 36, 621–639
(2013).
38. Hong, S.-Y. Roze, L. V. & Linz, J. E. Oxidative stress-related transcription factors
in the regulation of secondary metabolism. Toxins 5, 683–702 (2013).
39. Wang, C., Lu, J., Zhang, S., Wang, P., Hou, J. & Qian, J. Effects of Pb stress on
nutrient uptake and secondary metabolism in submerged macrophyte Vallisneria
natans. Ecotox. Environ. Safe. 74, 1297–1303 (2011).
40. Mortimer, M., Kasemets, K., Vodovnik, M., Marinsek-Logar, R. & Kahru, A.
Exposure to CuO nanoparticles changes the fatty acid composition of Protozoa
Tetrahymena thermophila. Environ. Sci. Technol. 45, 6617–6624 (2011).
41. Chen, R. et al. Differential uptake of carbon nanoparticles by plant and
mammalian cells. Small 6, 612–617 (2010).
42. Lin, D. H. & Xing, B. S. Root uptake and phytotoxicity of ZnO nanoparticles.
Environ. Sci. Technol. 42, 5580–5585 (2008).
43. Wang, J. R. et al. Mechanisms of arsenic hyperaccumulation in Pteris vittata.
Uptake kinetics, interactions with phosphate, and arsenic speciation. Plant
Physiol. 130, 1552–1561 (2002).
44. Molin, M. et al. Humans seem to produce arsenobetaine and dimethylarsinate
after a bolus dose of seafood. Environ. Res. 112, 28–39 (2012).
45. Watanabe, T. & Hirano, S. Metabolism of arsenic and its toxicological relevance.
Arch. Toxicol. 87, 969–979 (2013).
46. Duan, G. L., Zhu, Y. G., Tong, Y. P., Cai, C. & Kneer, R. Characterization of
arsenate reducatse in the extract of root and fronds of Chinese brake fern, an
arsenic hyperaccumuator. Plant Physiol. 138, 461–469 (2005).
47. Cheng, L.-J. & Cheng, T.-S. Oxidative effects and metabolic changes following
exposure of greater duckweed (Spirodela polyrhiza) to diethyl phthalate. Aquat.
Toxicol. 109, 166–175 (2012).
48. Hu, X. & Zhou, Q. Novel hydrated graphene ribbon unexpectedly promotes aged
seed germination and root differentiation. Sci. Rep. 4, 3782 (2014).
49. Nedjimi, B. & Daoud, Y. Effects of calcium chloride on growth, membrane
permeability and root hydraulic conductivity in two atriplex species grown at high
(sodium chloride) salinity. J. Plant Nutr. 32, 1818–1830 (2009).
50. Davies, T. G. E. et al. Expression analysis of putative high-affinity phosphate
transporters in Chinese winter wheats. Plant Cell Environ. 25, 1325–1339 (2002).
Acknowledgments
We thank Dr Yuming Chen, Mr Shaohu Ouyang, Dr Junjie Du and Ms Yuanyuan Gao for
their help in the characteristics of GO nanosheets and cells. This work was financially
supported by the National Natural Science Foundation of China (grant Nos: U1133006,
21037002 and 21307061), the Ministry of Education of China (grant No. IRT 13024),
Tianjin Natural Science Foundation (grant No. 14JCQNJC08900), the Doctoral Program of
Higher Education of China (grant No. 2013003112016), the Postdoctoral Science
Foundation of China (grant No. 2014M550138) and the Fundamental Research Funds for
the Central Univ ersities (grant no. 65121006).
Author contributions
H.X. and Z.Q. designed the research; H.X., M.L., Z.R., L.K. and K.J. performed the research;
H.X. and M.L. analyzed the data; H.X. wrote the paper; and Z.Q. revised the manuscript.
Additional information
Supplementary information accompanies this paper at http://www.nature.com/
scientificreports
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Hu, X. et al. Graphene oxide amplifies the phytotoxicity of arsenic
in wheat. Sci. Rep. 4, 6122; DOI:10.1038/srep06122 (2014).
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