Graphene Oxide-Induced pH Alteration, Iron Overload, and Subsequent Oxidative Damage in

Rice ( Oryza sativa L.): A New Mechanism of Nanomaterial Phytotoxicity

Original authors: Peng Zhang, Zhiling Guo, Wenhe Luo, Fazel Abdolahpur Monikh, Changjian Xie,
Eugenia Valsami-Jones, Iseult Lynch, Zhiyong Zhang.

Student analysis: Jhosep Castillo. Departamento de Ciencias de la Vida y de la Agricultura,

Universidad de las Fuerzas Armadas – ESPE, Salgolquí, Ecuador

Abstract

Graphene is a nanomaterial that has great potential to be used in various sectors, which leads to
an increase in its release into the environment. The graphene-based nanomaterials phytotoxicity
mechanism and their association with their physicochemical properties have not yet been fully
studied. The present study compared the effects of graphene oxide (GO) and reduced graphene
oxide (rGO) on the growth of rice plants under hydroponic conditions for 3 weeks. GO reduced
the shoots length and biomass, and caused oxidative damage, while rGO didn´t show any overt
effect, suggesting that surface O2 content is a critical factor affecting the biological impacts of
GBM. GO treatments caused excessive Fe accumulation in shoots, which was found to be the
main reason for oxidative damage to leaves. GO-induced acidification of the nutrient solution was
the main reason for Fe overload in plants. In addition to antioxidant regulators, plants activated
other pathways to defend themselves against Fe toxicity, through the negative regulation of
metabolites associated with Fe transport (mainly coumarins and flavonoids). Exudates from plant
roots facilitated the reduction of toxic GO to non-toxic rGO, acting as another route for plant
adaptation to GO-induced phytotoxicity.

Keywords: graphene-based nanomaterials, phytotoxicity, graphene oxide, reduced graphene

oxide

are also reported even at relevant environmental

1. Introduction
concentrations. On the other
Graphene is a two-dimensional carbon-
hand, positive effects of GBM exposure on plant
based nanomaterial made up of a single layer
growth have been reported, Chen et al., (2018)
of carbon atoms. It is one of the most promising
stated that exposure with G-NH2 (GO with an
nanomaterials in various fields due to its
amine) improved wheat plant growt ; root and
unique properties (thermal, electronic, and
stem length increased by 19.27% and 19.61%,
mechanical) (Zhang et al., 2020). Despite the
respectively, at 2000 µg / mL and no
benefits they bring to society and economy,
accumulation of G-NH2 was observed in the
graphene-based nanomaterials (GBM) should
roots.
be also considered for its potential to present
environmental and health risks due to the Common forms of graphene, including
increasing release of these nanomaterials into graphene (G), graphene oxide (GO), and reduced
the ecosystem (Wang et al., 2019). graphene oxide (rGO) differ in their surface
oxygen content; GO is the most oxidized form and
There are several comprehensive studies
includes carboxylic, hydroxyl, epoxy, and
about GBM toxicity in cells and
carbonyl groups; that give it a high dispersion in
microorganisms, while its impact on higher
water and an opportunity of greater
plants is unknown. Some studies have
functionalization for more applications.
reported that GBM induce reduction of
biomass, shoot and root lengthening; This study investigates the impact of GO and
Physiological alterations and oxidative stress rGO in rice plants growth under hydroponic
conditions for 3 weeks; for this, oxidative
stress, disturbance of the absorption of macro
and micro elements, metabolic alteration in
plants and transformation of GO and rGO were
evaluated in rice plants.
2. Materials and methods
• Nanomaterials and seeds
GO and rGO morphology, lateral size,
thickness and chemical structure were
characterized. The rice seeds were obtained
from the Chinese Academy of Agricultural
Sciences.
• Plant cultivation and exhibition
GO and rGO were added to a freshly
prepared Hoagland nourishing solution to
obtain suspensions with concentrations of 5,
50, 100 and 250 mg/L, followed by an
ultrasonic treatment for 10 min. The rice plants
were exposed to the suspensions for 3 weeks.
• Biomass production, seedling length and
nutrient content
After 3 weeks the roots and shoots were
dried and its fresh weight and dry weight were
measured. Nutrient content (Fe, Cu, Mn, Zn, K,
Ca, Mg, P) in roots and shoots was quantified
by inductively coupled plasma optical emission
spectroscopy (ICP-OES, Thermo, USA).
• Response to stress induced by GO and
rGO
The activities of superoxide dismutase
(SOD), peroxidase (POD) and
malondialdehyde content (MDA) were
analyzed using test kits purchased from
Nanjing Jiancheng Institute of Bioengineering.
The reactive oxygen species (ROS)
accumulation in the roots and leaves was
evaluated by a DCFH-DA staining method and
observation under a fluorescence microscope.
• Characterization of GO and rGO after
interaction with plants
The GO and rGO adhered to the surface of
the roots (called GO-W and rGO-W) and the
residual solutions of the beakers (GO-R) after
the removal of the plants were collected for
analysis. The above described was analyzed with
Raman (Horiba Scientific, Japan), FTIR (Bruker
Tensor 27 spectrometer, Germany), UV-vis
spectroscopy (Purkinje General, Beijing) and XPS
(ESCALAB 250Xi, Thermo Scientific, USA).
• Collection of xylem sap and analysis of Fe
concentration
A cut was made above the root shoot interface
and a silicone tube was fitted. The xylem sap was
collected 24 h later and the Fe concentration was
analyzed by ICP-OES (Thermo, USA).
• Analysis of the metabolomics of rice leaves.
The prepared fresh rice leaf samples were
analyzed by tandem liquid MS-chromatography
(LC-MS / MS)
3. Results and Discussion.
• GO and rGO characterization
For GO and rGO morphology it was observed
that the mean lateral size of the GO layers was
0.089 µm and for rGO it was 0.078 µm; which
indicates that they are not significantly different. It
was also observed that the GO and rGO layers
had a thicknesses of 0.78 µm and 0.44 µm
respectively. It was determined that the amount of
oxygen-containing groups in GO was 61% and for
rGO was 23%. In previous studies of GBM effects
in higher plants, it has been observed that the
physicochemical properties of graphene-based
nanomaterials are very diverse and in certain
studies they are not even described as observed
in Table 1 of the supplementary data. This study
provides data on lateral size, thickness, oxygen
content, which are critical characteristics that
determine GBM biological effects; for example
Asli & Neumann, (2009) reported that there is a
physical blockage that is closely related to the
size of the GBM. If the diameter of the
nanoparticles is greater than the diameter of the
pores of the root cell wall, the particles will
accumulate on the root surfaces and form surface
layers, thus decreasing the hydraulic conductivity
and absorption of nutrients.
• GO and rGO showed different effects on GO induced the alteration of the levels of
the rice seedlings growth. several elements, including P and Fe in the
shoots and Fe and Zn in the roots; but the most
The effects of GO and rGO on rice plants
significant alteration was observed in the Fe level
growth were different as seen in Figure 1. GO
in the shoot treated with 250 mg/L of GO since it
reduced shoot length by 11% at 100 mg/L and
increased 3.6 times more than the control (Fig. 2).
by 40% at 250 mg/L; GO also reduced the
Rice plants experience toxicity when Fe exceeds
fresh biomass of the roots and shoots, and the
400 mg/kg due to an iron overload. Fe
dry weight of the shoots to 250 mg/L. rGO did
translocates and accumulates in leaves
not show any impact on elongation and
producing a notable amount of ROS, which
biomass. These effects on growth are common
correlates with the excessive accumulation of
since Chen et al., (2017) reported that GO
ROS and altered antioxidant enzymatic activities
negatively affected biomass accumulation and
in the leaves of plants, this suggests a possible
stem elongation in wheat seedlings. The
mechanism of phytotoxicity since GO induced
present study does not provide the GBM effect
overload Fe and the consequent oxidative stress
on seed germination, since there are several
in the leaves.
studies that indicate adverse effects, for
example Nair et al., (2012) reported that in rice • Fe overload contributes more than GO to
seeds treated with 50 mg/L of graphene , GO-induced phytotoxicity.
germination started 3 days after the control
It follows that Fe overload is the main cause of
group. It is suggested that a bioaccumulation
toxicity rather than GO itself. To test this
study of GBM can be carried out as well as they
hypothesis, an excessive amount of Fe (4 mM
are essential for the biosafety assessment.
FeSO4) was added to a 50 mg/L GO suspension
Chen et al., (2019) applied stable isotope
and the rice seedlings growth was examined. The
labeling with 13C on the carbon skeleton of GO
dry weight of the leaves was reduced by 27% (Fig.
to investigate the bioaccumulation and toxicity
3A) and the content of MDA in the leaves
of GO in pea; GO was found to accumulate in
increased by 46% (Fig. 3B) and the level of Fe
the root and rGO to translocate from the root to
increased significantly in the leaves (Fig. 3C )
the leaves, inhibiting photosynthesis. GO
compared to GO alone. These results suggest
accumulation is due to the large size of its
that Fe overload contributed more than GO to
layers, which makes translocation difficult.
oxidative stress and subsequent toxicity in rice
• Responses to oxidative stress induced by plants.
GO and rGO.
• Mechanisms involved in Fe overload in
Antioxidant enzymes activities including leaves.
SOD and POD in shoots after GO treatment
Fe generally exists in Fe2+ form, which is
significantly increased at 100 mg/L and 250
bioavailable for absorption by the plant. To
mg/L, while evidence showed that ROS and
prevent excessive Fe accumulation, rice roots
MDA excessively accumulated in GO exposed
can release oxygen and oxidase, to oxidize Fe2+
plants. In particular, the most manifested
into Fe3+, which precipitates to form a coating on
accumulation of MDA and ROS in response to
the roots called "Fe plaque" which prevents
GO treatment was found in shoots rather than
excessive Fe2+ absorption, as well (Mahender,
roots. No significant alterations were observed
2019). It is indicated that a pH decrease can
in the rGO treatments. GBMs cause oxidative
significantly promote the reduction of insoluble
stress, which has been recognized as one of
Fe3+ to Fe2+ which eventually leads to Fe toxicity.
the most important mechanisms in limiting
It was found that GO acidified the nutrient solution
effects of plants growth (Wang et al., 2019).
while rGO did not significantly change the pH (Fig.
• Alteration of macro and micro elements 4A). A low pH itself is not the main driver of toxicity
absorption in rice plants. because of the rice plant tolerance to acidic
conditions (Zhao, 2007). In contrast, a pH
decrease induced by GO can increase Fe
mobilization and cause a Fe overload observed
in shoots. Fig. 4B further demonstrated that the
flux of Fe content in the xylem sap increased
significantly with GO treatment.
• The plant defense against Fe overload
through the reduced production of
metabolites associated with Fe transport.
Depressed production of iron-mobilizing
coumarin-type compounds and iron-chelating
flavonoid-type compounds were found in
leaves of GO-treated plants, which may act as
an important component of iron depletion. For
example, psoralen, a linear furanocoumarin,
decreased by 36% and naringenin (a
flavanone) concentration, decreased by 48%.
This decreases suggest that metabolic
regulatory mechanisms were evoked in the
leaves to sustain iron homeostasis in response
to GO-induced Fe overload.
• GO biotransformation as a pathway to
alleviate GO-induced phytotoxicity.
Nanomaterials can be transformed by
interaction with plants, this determines their
subsequent behavior, fate and toxicity in
plants. CeO2 nano-material has been reported
to transform in many plant species, it depletes
and releases Ce3+, which is considered to be
responsible for CeO2 ENMs toxicity in Lactuca
plants (Zhang, 2015). The transformation of
GBM in bacteria, plants and in water under
sunlight has also been reported (Hou, 2015). In
this study, GO was found to be adsorbed on
the root surface; similarly, a layer of exudate
could be observed between GO and the cells
of the epidermis of the root, which could act as
a barrier to prevent GO from entering the roots.
It was also reported that the O/C
(oxygen/carbon) ratio of GO decreased
significantly after interaction with plants; there
was a greater reduction of GO-W (washed from
the root surface) than in GO-R (residual
solutions from beakers) suggesting that direct
contact with the roots of the plants accelerated
the transformation of the "toxic" GO in a
relatively non-toxic "rGO" that could act as a
pathway to alleviate GO toxicity.
4. Conclusions
This study shows a new GBM phytotoxicity
mechanism, in which the alteration of the pH
induced by GO of the nutrient solution produces
Fe overload and oxidative damage in the leaves.
GO and rGO impact on plants growth suggests
that phytotoxicity is related to their surface oxygen
content, since GO (with more oxygen) had more
negative effects than rGO on rice plants.
This study was carried out under hydroponic
conditions, so it is suggested to carry out studies
with similar conditions in a more realistic soil
environment and with a longer time exposure to
observe its effects.
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 Supplementary data

Table 1. Studies on the effects of GBM on higher plants and physicochemical characteristics
Species Nanomaterial Lateral size Thickness Oxygen Effect on plant growth
content
Rice GO y rGO 0.089 µm y 0.78 µm y 61% y Reduction shoot biomass and
0.078 µm 0.44 µm 23% length
Wheat GO 30 µm 0.9 µm 31.9% Decrease in dry weight (≥ 40
mg / L); inhibition of root
elongation (≥ 400 mg / L)
Apple GO 0.05-0.2 µm 0.8-1.2 µm - Inhibition of root elongation
Wheat GO y G-NH2 - - - Decreased length of
seedlings, biomass, tissue

Adapted from: (Zhang, 2020)

Figure 1. Seedling lengths (A and B), fresh weights (C and D) and dry weights (E and F) of rice
seedlings after exposure to different concentrations of GO and rGO for 3 weeks.
Element content (mg/kg)

Figure 2. Macronutrient and micronutrient content in sprouts

 Figure 3. Dry weight (A), MDA content (B) and Fe content (C) in rice plants after exposure to GO (50
mg / L) and a mixture of GO (50 mg / L) and FeSO 4 (4 mM) for 3 weeks.

Figure 4. PH values of GO and rGO suspensions in nutrient solution (A), the concentration of free Fe in
xylem bags collected (B).