Article
pubs.acs.org/est
Evidence of Translocation and Physiological Impacts of Foliar
Applied CeO2 Nanoparticles on Cucumber (Cucumis sativus) Plants
Jie Hong,† Jose R. Peralta-Videa,†,‡,§ Cyren Rico,‡,§ Shivendra Sahi,∥ Marian N. Viveros,†,⊥ Jane Bartonjo,∥
Lijuan Zhao,‡ and Jorge L. Gardea-Torresdey*,†,‡,§
†
Environmental Science and Engineering PhD Program, The University of Texas at El Paso, 500 West University Avenue, El Paso,
Texas 79968, United States
‡
Chemistry Department, The University of Texas at El Paso, 500 West University Avenue, El Paso, Texas 79968, United States
§
University of California Center for Environmental Implications of Nanotechnology (UC CEIN), The University of Texas at El Paso,
500 West University Avenue, El Paso, Texas 79968, United States
∥
Department of Biology, Western Kentucky University, Bowling Green, Kentucky 42101, United States
⊥
Biology Department, The University of Texas at El Paso, 500 West University Avenue, El Paso, Texas 79968, United States
S Supporting Information
*
ABSTRACT: Currently, most of the nanotoxicity studies in plants involve
exposure to the nanoparticles (NPs) through the roots. However, plants
interact with atmospheric NPs through the leaves, and our knowledge on their
response to this contact is limited. In this study, hydroponically grown
cucumber (Cucumis sativus) plants were aerially treated either with nano ceria
powder (nCeO2) at 0.98 and 2.94 g/m3 or suspensions at 20, 40, 80, 160, and
320 mg/L. Fifteen days after treatment, plants were analyzed for Ce uptake by
using ICP-OES and TEM. In addition, the activity of three stress enzymes was
measured. The ICP-OES results showed Ce in all tissues of the CeO2 NP
treated plants, suggesting uptake through the leaves and translocation to the
other plant parts. The TEM results showed the presence of Ce in roots, which
corroborates the ICP-OES results. The biochemical assays showed that
catalase activity increased in roots and ascorbate peroxidase activity decreased
in leaves. Our findings show that atmospheric NPs can be taken up and distributed within plant tissues, which could represent a
threat for environmental and human health.
■
In all the above-mentioned reports, plants were exposed to
the NPs through the roots. However, natural NPs from volcanic
or eolic sources are in the troposphere and interact with plants
through the leaves.13 NPs have also been used as foliar
antimicrobial products for agricultural purposes.14 However,
very few reports have mentioned the antimicrobial properties of
CeO2.15,16 Very likely, a portion of engineered NPs (ENPs)
released to the environment will be wind dispersed, reaching
the leaves of plants.17,18 Although there is no exact data about
the levels of CeO2 NPs in the air, their release to the
atmosphere from car exhaust is increasing. Johnson and Park19
estimated that in the United Kingdom there are 15.6−114.9
kg/year of CeO2 NPs released to the environment through
vehicles emissions. Thus, plants could be exposed to unusual
high concentrations of CeO2 NPs.
Reports indicate that particles and chemical elements enter
the leaves through the cuticle20−23 or stomata.24−26 Other
INTRODUCTION
Concerns about the release of excess NPs into the environment, with negative effects in ecosystem components, including
plants are increasing.1 Previous reports aimed to determine the
impacts of NPs in plants have shown varied results. While some
studies report benefits, others report decrements. For instance,
carbon nanotubes (CNT) have been found to increase root and
shoot lengths in Chickpea (Cicer arietinum),2 but they reduce
root elongation in tomato (Lycopersicon esculentum), cabbage
(Brassica oleracea), carrot (Daucus carota), and lettuce (Lactuca
sativa).3 Metal-based NPs have also shown different results. For
example, reports indicate that ZnO NPs enhance root length in
chick pea (C. arietinum)4 but reduce root elongation in some
desert plants.5 TiO2 NPs decrease biomass in wheat6 and
inhibit maize leaf growth;7 however, they increase root length
in cucumber.8 Other negative effects of metal-based NPs
include disturbance on cell division in Allium cepa by silver
NPs,9 genotoxic effects of ZnO NPs and CeO2 NPs on
soybean,10 excess Zn accumulation in maize exposed to ZnO
NPs,11 and reduction in squash (Cucurbita pepo) transpiration
by Ag and Cu NPs.12
© 2014 American Chemical Society
Received:
Revised:
Accepted:
Published:
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November 5, 2013
March 11, 2014
March 13, 2014
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Figure 1. Upper row: Hydroponic setup for cucumber plants treated with CeO2 NPs. Lower row: Nanoparticle application as powder (left) and
suspension (right). The plants were cultivated for 15 days before NP treatment and harvested 15 days after treatment. Each treatment had four
replicates.
corroborated by TEM analysis. Results from this research will
contribute to understand the interaction of ENPs with
terrestrial plants.
studies have shown that plants can also take up NPs through
leaves. For instance, Uzu et al.27 found submicrometric lead
particles inside stomata openings of lettuce leaves grown close
to an industrial plant. In Vicia faba (L.), water-suspended
hydrophilic polymeric NPs were found to penetrate leaves.24
Carbon coated magnetic NPs, sprayed or injected into pumpkin
(Cucurbita pepo) leaves were taken up and translocated to
different regions of the plants.28 Recently, Larue et al.29 found
that Lactuca sativa can take up Ag NPs through stomata, and,
after the penetration, some of these NPs can be transformed to
ionic Ag within the leaf. However, Birbaum et al.30 reported
uptake but no translocation of Ce in maize (Zea mays) plants
treated with CeO2 NPs, either as powder or in suspension.
Reactive oxygen species (ROS) produced in plants exposed
to abiotic or biotic stressors can damage cell structure.31
Antioxidant enzymes such as dehydroascorbate reductase
(DHAR), catalase (CAT), and ascorbate peroxidase (APX)
protect plants from ROS damage. Previous studies have shown
that NPs applied to the root system can modulate the activity of
stress enzymes. Zhao et al.32 have shown that CeO2 NPs
increased CAT and APX activity in maize plants. In cilantro
(Coriandrum sativum L.) plants, CAT and APX activity
increased in plants treated with CeO2 NPs at low concentration
but decreased at high NP concentrations.33 DHAR activity in
rice was significantly higher in plants treated with high
concentration of CeO2.34 To our knowledge, very few studies
have determined the physiological effects of foliar application of
ENPs.29
The aims of this study were to determine the Ce uptake and
translocation in cucumber plants exposed to CeO2 NPs
through the leaves and the effects of the CeO2 NPs on three
antioxidant enzymes. Hydroponically grown cucumber plants
were foliarly treated with CeO2 NPs as powder or aqueous
suspension. The uptake was determined by inductively coupled
plasma-optical emission spectroscopy (ICP-OES), and the
antioxidant enzyme activities were measured using UV−vis
spectroscopy. The uptake and translocation of Ce were
■
EXPERIMENTAL SECTION
Seed Germination and Plant Growth. All materials used
in the experiments, e.g. paper towels, tweezers, and water, were
autoclaved to reduce contamination in the germinating seeds.
Cucumber seeds were disinfected using a 4% NaClO solution,
stirred for 30 min, rinsed three times with distilled water, and
immersed in distilled water for 24 h. Then, seeds were placed
on the edge of wet germination paper towels, rolled, and added
with 10 drops of antimycotic/antibiotic solution (Sigma 5955).
The rolls were put into Mason jars with distilled water in the
bottom, set in the dark for four days, and, after that, exposed to
light for one day. After the sixth day, the young plants were
transferred to the hydroponic jars (magenta boxes) containing
300 mL of a modified Hoagland’s nutrient solution previously
described in the literature.35 All the jars had plastic lids with six
small holes. One cucumber seedling was introduced in each
hole, and the lid was covered with aluminum foil to avoid
contamination of the substrate with NPs (Figure 1). Aquarium
pumps were used to aerate the jars that were set in an
Environmental Growth Chamber (TC2Microcontroller, Chagrin falls, OH) with light intensity of 300 μmol m−2 s−1, 25/20
°C day/night temperature, and 65% relative humidity.
CeO2 Nanoparticles. The CeO2 NPs (Meliorum Technologies, NY) were obtained from the University of California
Center for Environmental Implications of Nanotechnology
(UC CEIN). Previous characterization at the UC CEIN
showed that CeO2 NPs are rods with primary size of 8 ± 1
nm, particle size of 231 ± 16 nm in DI water, surface area of
93.8 m2 g−1, and 95.14% purity.36
Powder Treatments. After two weeks of growth, the
young plants were transferred into three sealed chambers of
0.45 m × 0.45 m × 0.74 m, similar to those described in a
previous study.37 The plants were treated with 0 (control), 0.98
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g/m3 (low), and 2.94 g/m3 (high) NP concentrations. The
respective amount of CeO2 NPs was put on a tray in front of a
fan (120 V, 0.25 Amps, 60 Hz) inside the chamber. Two
blowing times were used, 15 and 45 min. Then, the cucumber
leaf samples were collected for analyses at 12, 24, 36, and 72 h.
The Ce concentration in roots, stems, and leaves of the
cucumber plants were measured by ICP-OES (Perkin-Elmer,
Optima 4300 DV, Shelton, CT), and samples were analyzed by
transmission electron microscope (H-7650 Transmission
electron microscope (TEM) (Hitachi High-Technologies,
Japan). The Ce content in the nutrient solution was also
measured to make sure there was no contamination. All the
samples were collected 7 days after the treatment.
Suspension Treatments. A second group of two week old
cucumber plants was treated with CeO2 NP suspensions.
Enough CeO2 NPs were suspended in distilled water to obtain
0, 40, 80, 160, and 320 mg/L and homogenized by sonication
for 45 min (Crest Ultrasonics, Trenton, NJ). Then, the
different CeO2 NP suspensions were sprayed on cucumber
leaves with a hand-held sprayer bottle. A total volume of 100
mL per treatment was split in three applications (every four h).
Six, 12, and 18 days after treatment, samples of leaves, stems,
and roots were collected and prepared for analysis.
Sample Preparation for ICP and TEM Analyses. For the
ICP-OES analyses, all the leaves in a treatment were severed by
half; a group of halves was processed for ICP-OES analysis
without washing, and the other halves were separated in three
groups. A group of halves was first rinsed with DI water, then
immersed for 20 min in DI water, and followed by another
rinse with DI water. Another group was rinsed with DI water,
immersed for 20 min in 0.01 M CaCl2, and rinsed again with DI
water. The third group of halves was rinsed with DI water,
immersed for 20 min in 0.01 M HNO3, and followed by
another rinsing with DI water. Afterward, the samples were
dried in an oven at 70 °C for 72 h, weighed, and microwaveassisted acid digested (CEM microwave accelerated reaction
system, MarsX, Mathews, NC) using a mixture of plasma pure
HNO3 and H2O2 (1:4). The digest were volume adjusted to 15
mL and analyzed for Ce concentration using the ICP-OES. For
quality control (QC) and quality assurance (QA), the NIST
SRM 1547 (Gaithersburg, MD) and spiked samples with 5, 10,
and 20 mg/L of CeO2 NPs were processed and read as samples.
Sample preparation for the TEM study is shown in the
Supporting Information (SI).
Enzyme Assays. All the enzyme activities were calculated
based on the changes of absorbance and Beer’s Law. Total
protein in the enzyme extracts was estimated based on the
Bradford38 method, and the specific activity of enzymes was
expressed as units per milligram protein. One unit of DHAR
was defined as 1 μmol of ascorbate formed per minute. One
unit of CAT was defined as the amount of enzyme required to
degrade 1 μmol of H2O2 per minute. One unit of APX was
defined as the amount of enzyme that oxidizes 1 μmol
ascorbate per minute.
Determination of Dehydroascorbate Reductase Activity.
DHAR enzyme activity was determined as per Doulis et al.39
The assay mixture contained 790 μL of 100 mM phosphate
buffer (pH 7.0), 100 μL of 12.5 mM reduced glutathione
(GSH), and 10 μL of extract solution. The reaction was
initiated with the addition of freshly prepared 100 μL of 2.5
mM dehydroascorbate (DAsA), and the absorbance was
recorded at 265 nm using a Perkin-Elmer Lambda 14 UV/vis
spectrometer (single-beam mode, Perkin-Elmer, Uberlinger,
Germany).
Determination of Catalase Specific Activity. The CAT
enzyme activity was determined following the procedure
previously described by Gallego et al. 40 Plants were
homogenized in 0.1 M KH2PO4 buffer at pH 7.0 and then
centrifuged at 4500 rpm during 5 min (Eppendorf AG bench
centrifuge 5417 R, Hamburg, Germany). The supernatant was
placed in a quartz cuvette with 73 mM H2O2 in phosphate
buffer, and the absorbance was recorded at 240 nm using a
Perkin-Elmer Lambda 14 UV/vis spectrometer.
Determination of Ascorbate Peroxidase Activity. The APX
enzyme activity was measured according to Nakano and
Asada.41 A ratio of 10% w/v of leaf sample was homogenized
on phosphate buffer (0.100 mg of leaves mixed with 900 μL of
0.1 M KH2PO4 at pH 7.4). The mixture was centrifuged for 25
min at 14000 rpm on a refrigerated centrifuge. The supernatant
was transferred to microtubes and assayed after centrifugation.
An aliquot of 886 μL of 0.1 M KH2PO4 buffer at pH 7.4, 10 μL
of the 17 mM H2O2, and 4 μL of 25 mM ascorbate were placed
in a quartz cuvette. Afterward, 100 μL of the sample was added
in order to obtain a final volume of 1 mL. The absorbance was
recorded at 290 nm in a Perkin-Elmer Lambda 14 UV/vis
spectrometer.
Statistics Analysis. The reported data are means of four
replicates ± standard error (SE). One-way ANOVA was used
to analyze the experiment variance (SPSS 19.0 package,
Chicago, IL). Tukey Honestly Significant Difference was used
to determine statistical differences between treatment means.
RESULTS AND DISCUSSION
Uptake and Accumulation of Ce from Powder
Treatments. As it has been previously shown in the literature,
■
Table 1. Cerium Concentration in Cucumber Leaves
Treated with CeO2 NPs at 2.94 g/m3 and Washed Three
Times Either with DI Water, CaCl2, or HNO3a
Ce (mg kg‑1)
nonwashed
washed
Ce removed
DI water
10 mM CaCl2
10 mM HNO3
745.5 ± 97.0
201.5 ± 56.1
73.0% ± 7.5%
1364.8 ± 450.6
259.1 ± 91.8
81.0% ± 1.0%
1244.6 ± 408.7
271.5 ± 67.9
78.2% ± 6.4%
a
The plants were hydroponically grown for 15 days in Hoagland
nutrient solution before treatment. Data are means ± SE (n = 4).
NPs can be adsorbed in the leaf surface without penetrating the
leaf tissue.29 Thus, for better assessment of the NPs penetration
into the leaf tissue, the leaves were previously washed before
preparation for Ce determination. Table 1 shows the efficiency
of washing processes to remove CeO2 NPs from the leaf
surface. As seen in this table, CaCl2 removed more Ce from the
leaf surface (81.3%) compared to HNO3 (76.5%) or water
(73.9%). The external surface of leaves of higher plants
comprises a cuticular layer covered by waxy deposits. The wax
contains a wide range of organic compounds that can trap the
NPs.42 The surface wax of the leaves can protect against
mechanical and pest damage, ultraviolet irradiation, and air
pollutants.43 In addition, the cucumber leaf is covered by many
glandular trichomes.44 These trichomes synthesize metabolites
like terpenoids, phenylpropenes, and fatty acids derivatives that
can bind the NPs electrostatically.45 These NPs can be
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Figure 2. A. Cerium concentrations in cucumber leaves after 12, 24,
36, and 72 h of exposure to 0.98 g CeO2 NPs/m3 and 2.94 g CeO2
NPs/m3. The samples were washed with 10 mM CaCl2. Data are
averages of four replicates ± SE. Different letters stand for statistical
differences at p ≤ 0.05. B. Cerium concentration in roots, stems, and
leaves of cucumber plants exposed to 2.94 CeO2 NPs/m3 for 15 and
45 min of fan blowing in a closed environment. Values are means ± SE
(n = 4). Capital letters represent statistical differences within each
blowing time period.
internalized into the leaf tissues and translocated within the
plant tissues.36
Figure 1 shows the experimental setting for the powder
treatments, and Figure 2 shows the Ce concentration in roots,
stems, and leaves of cucumber plants. Figure 2A shows the
concentration in leaves after washing with 10 mM CaCl2. As
expected, the Ce concentration in leaves treated with higher
concentration of NPs (2.94 g/m3) were significantly higher
than the concentration found in leaves treated with 0.98 g/m3
(Figure 2A). However, the uptake did not increase with time.
We hypothesize that the number of NPs on the surface
saturated the routes of entrance through the leaves avoiding
penetration.
Effect of Blowing Time. The effect of blowing time on
foliar uptake of CeO2 NPs is shown in Figure 2B. As seen in
this figure, the amount of Ce found in tissues of plants blown
for 15 min was significanlty higher (p ≤ 0.05) compared to the
amounts found in plants blown for 45 min. However, in both
blowing time periods, the Ce concentrations found in roots and
stems were very similar and significantly lower compared to the
Ce concentration found in leaves. It is possible that higher
blowing time removed the NPs from the surface of the leaves
and the blowing velocity was not enough to lift the particles
deposited on the floor. After the exposure, no Ce content was
detected in the hydroponic solution. Thus, this suggests that
Figure 3. Cerium concentration in A. leaves, B. roots and stems, and
C. flowers of cucumber plants treated with CeO2 NP suspensions.
Values are means ± SE (n = 4). * Stands for statistical differences at p
≤ 0.05.
the source for Ce in roots was due to translocation from the
leaves. According to Savvides et al.,46 stomata of cucumber have
∼21 μm length, ∼13 μm width, pore length of ∼12 μm, and
pore aperture of ∼1.23 μm. The size of the CeO2 NP powder
used is 8 ± 1 nm,36 which is small enough for the NPs to enter
into the stomata chamber.
Birbaum et al.30 reported that CeO2 NPs did not translocate
in maize plants; however, Wang et al.47 pointed out that
watermelon plant can take up aerosol NPs with diameter lower
than 100 nm through their leaves and transport them to the
root system through the phloem. All these results suggest that
foliar uptake of NPs are affected by different factors, including
size and type of nanoparticles, environmental conditions (light,
wind, and moisture), plant species, mode of application, and
others.47
Uptake of Ce by Cucumber Tissues from CeO2 NPs
Suspension. The concentration of Ce in leaves, roots, stems,
and flowers of cucumber plants sprayed with CeO2 NP
suspensions is shown in Figure 3. As seen in Figure 3A, Ce was
not detected in control leaves, and the concentration with 40
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Figure 4. A. TEM image of surface of cucumber leaf treated with 0.98 mg CeO2 NPs/m3. B. EDS of the spot highlight in A. C. TEM image of
cucumber root treated with 0.98 mg CeO2 NPs/m3. D. EDS of the spot 1 highlight in C.
mg L−1 (∼700 μg/g) was significantly lower than the other
treatments. Cerium was also detected in stems and roots of NP
treated plants (Figure 3B).
Results of this study have shown that CeO2 NPs may
penetrate the leaf surface when applied either as powder or
suspension. Corredor et al.28 proved that magnetic nanoparticles can get into the cells of pumpkin plants through the
stomata and the vascular system. It has also been reported that
water-suspended NPs can be taken up through stomata in Vicia
faba (L.).24 Eichert et al. observed that in leaves rewetted with
DI water, about 60% of all stomata were penetrated again by
NPs.24 In the present experiment, the spray treatment was
repeated three times in one day, which means the leaves were
dampened three times with NPs suspension. This increased the
chance of NPs to enter through the stomata. Even though the
Ce concentration in leaves was much higher in suspension
treatments, the Ce concentration in the roots and stems were
similar as in plants treated with NP powder. This suggests that
CeO2 NPs penetrate the leaf surface, but their translocation
through the leaf tissue is limited.
Ce concentrations in stems and flowers increased as the
external concentration of CeO2 NPs increased. Flower
structures appeared several days after treatment application.
This may indicate an effective uptake and translocation of the
CeO2 NPs through the cucumber leaves. As shown in Figure
3C, Ce in flowers increased as the concentration of NPs in
treatments increased. The Ce concentration at 160 mg/L
(∼9 μg/g) and above was statistically higher than at 40 mg/L.
In this study, we did not determine the speciation of Ce in
cucumber tissues; however, previous studies have shown that
most of the Ce in tissues of plants treated with CeO2 NPs
remains in the nanoparticulate form.47,48 This suggests that
atmospheric CeO2 NPs can be absorbed by plant leaves.
Further studies are needed in order to determine if these NPs
will reach the fruit, which will be significant for the risk
assessment of air pollution with CeO2 NPs.
TEM Studies of Cucumber Tissues from Plants
Treated with CeO2 Powder. Figure 4 shows TEM
micrographs and corresponding EDS spectra of cucumber
tissues from plants treated with 0.98 mg CeO2 NPs/m3. The
micrograph in Figure 4A shows a white spot in the epidermal
surface, and the corresponding EDS spectrum shows the
presence of Ce in the spot (Figure 4B). In addition, Figure 4C
shows the cross section of the cucumber root from a CeO2 NP
treated plant. Two spots (1 and 2) were selected from one of
the phloem areas, and the corresponding EDS spectrum shows
the presence of Ce in phloem (Figure 4D). Cerium dioxide
NPs are very stable in a variety of environments.43,44 Previous
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Figure 5. A. TEM image of control cucumber leaf sample showing a parenchymal cell (C-chloroplast; V-vacuole); B. TEM image of cucumber leaf
sample showing a parenchymal cell (C-chloroplast; M-mitochondria; V-vacuole) treated with 320 mg/L CeO2 NPs suspension; C. TEM image of
control cucumber root sample (I-interior of cell; B-between cells); D. TEM image of cucumber root sample treated with 320 ppm CeO2 NPs
suspension (I-interior of cell; B-between cells); E. Elemental analysis of dark precipitation found in the interior of cell shown in D.
studies have shown that a portion of the Ce taken up by the
roots of plants treated with CeO2 NPs is transported to the
aboveground tissues and remain as CeO2 NPs in vegetative and
reproductive organs.48,49 The presence of CeO2 NPs in
reproductive organs48 suggests phloem transportation. In our
work, the TEM image (Figure 4C) suggests that small CeO2
NPs penetrate the leaf surface through hydathodes (cuticle free
segments) and stomata,50 traverse cell walls of palisade
parenchyma, reach the leaf phloem, and distribute within the
plant body.
Figure 5A displays the electron micrograph of a control
cucumber leaf sample showing healthy chloroplasts filled with
starch granules, while Figure 5B shows a structurally normal
parenchymal cell from a cucumber leaf sample treated with 320
ppm CeO2 NPs suspension. Parenchyma cells are the only
photosynthetic cells in leaves and stems. Their primary cell
walls are thin, which allow light, water, gases, and metabolites
to pass through. In Figure 5B, the parenchymal cell shows an
increase of vacuole space, which has been shown to be an
ultrastructural sign of metal toxicity among various algae, such
as Laminaria saccharina,51 Chlamydomonas reinhardtii,52 and
Crypthecodinium cohnii.53 Meristematic cells contain many small
vacuoles that will form the large central vacuole when the cell is
matured.54 Epimashko et al.55 found that Mesembryanthemum
cystallinum L. can form two vacuoles in one cell under salt
stress. These two vacuoles can help the plant to separate the
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Figure 6. Antioxidant enzyme activities in cucumber plants foliarly treated with CeO2 NP powder at 0.98 and 2.94 g/m3 or 40, 80, 160, and
320 mg/L of CeO2 NP suspensions. A, B, and C correspond to APX, CAT, and DHAR activities, respectively, in plants treated with powder. D, E,
and F correspond to APX, CAT, and DHAR activities, respectively, in plants treated with CeO2 NP suspensions. Values are means ± SE (n = 4).
Means with the same letter are not significantly different (Tukey test, p ≤ 0.05).
interior of root cells and between root cells. The elemental
analysis spectrum identified the dark precipitation as Ce. The
dark precipitates were only observed in the treated plant tissues,
thus corroborating the uptake and translocation of the CeO2
NPs through the leaves.
Enzyme Activity Essays. Figure 6 shows the activity of
three antioxidant enzymes in cucumber leaves, stems, and roots
treated with either CeO2 NPs powder or suspension. As seen in
Figure 6A, in powder treatments the APX activity decreased in
stems and increased in roots as the external CeO2 NPs
increased. The activity of CAT significantly increased in leaves
and roots at the higher NP concentration, but no changes were
observed in stems (Figure 6B). On the other hand, DHAR did
contrasting vacuolar functions of salt storing and malate cycling.
There is no information on physiological or ultrastructural
changes due to CeO2 NPs. Although the mechanism of two
vacuoles coexisting in one cell is still poorly understood, this
abnormal phenomenon is hypothesized to be caused by CeO2
NPs, since this was not observed in the control. In Figure 5B,
dark precipitates are present inside the vacuole, near
mitochondria and in the tonoplast (circles). Also, there is
only one chloroplast shown in the cell. It has been proven that
the number of chloroplasts is reduced by metal toxicity.56,57
Figure 5C shows control root cells, while Figure 5D shows
cucumber root samples treated with 320 ppm CeO2 NPs
suspension. It clearly displays dark precipitates in both the
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not show changes in activity at any concentration of the CeO2
NPs powder (Figure 6C).
On the other hand, in plants treated with the CeO2
suspensions, the APX activity decreased in leaves at all NP
concentrations. However, in stems and roots, only the 40 mg/L
produced a significant increase in APX activity (Figure 6D).
The activity of CAT increased in leaves treated with 160 and
320 mg/L; it also increased in stems of plants treated with 80
and 160 mg/L and in roots of plants treated with 40 to 160
mg/L (Figure 6E). In addition, the activity of DHAR showed
an increase in leaves only in plants treated with 80 mg/L, but it
increased in stems and roots of plants treated with 80 to 320
mg/L (Figure 6F). The increase in DHAR in roots and stems
could lead to an increase in APX in these organs; however, the
result was contrary to the expected. Previous studies58,59 have
shown that metals can interfere with APX activity. As shown in
Figures 2 and 3, the Ce uptake from suspensions was higher
compared to powder applications. Thus, this could explain the
different response in enzyme activity in plants treated with NP
suspensions. Overall, CAT activity increased and APX activity
decreased in the treated samples compared to control.
The increase in CAT activity in all plants treated either with
powder or suspension at low concentrations suggests that the
CeO2 NPs in contact with leaves can modify this enzyme
activity in cucumber plants. On the other hand, the decrease of
CAT in stems and roots of plants treated with 320 mg/L
suggests that at this concentration CeO2 NPs caused toxicity.
Morales et al.33 reported that in cilantro, 62.5 and 125 mg/kg of
CeO2 NPs produced an increase in CAT activity, but at 250
and 500 mg/kg the NPs caused a decrease in CAT activity. In
addition, Zhao et al.32 found that in 10 day-old maize plants,
APX activity was higher at 400 mg CeO2 NPs/kg but lower at
800 mg/kg. In the present study, the APX activity decreased in
leaves of CeO2 NPs treated plants but increased in stems and
roots of plants treated with 40 mg/L. As previously reported,
APX utilizes ascorbic acid (AsA) as the electron donor to
remove H2O2 in the ASA-glutathione (GSH) cycle.60 In
chloroplasts, APX isoenzymes prevent oxidative stress,
maintaining the antioxidant system.61,62 In Figure 5B, we
observed the cell with only one chloroplast, and perhaps the
number of chloroplasts was reduced maybe because of CeO2
NPs toxicity. This could explain the decrease of APX activity in
leaves. As shown in Figure 6D, the activity of DHAR was higher
in stems and roots of plants treated with 80 to 320 mg/L of
CeO2 NPs. In rice plants, DHAR activity was also enhanced as
the CeO2 NP concentration in the treatments increased.34
In summary, this study has shown that cucumber plants can
take up Ce from CeO2 NPs applied to the leaves as powder or
suspension. In addition, the TEM studies have shown that the
Ce taken up by the leaves is translocated to other plant tissues.
The CeO2 NP treatments, even at concentrations of 40 mg/L
or 0.98 g/m3, can modify the enzyme activity in cucumber
plants. Although the speciation of Ce in tissues was not
determined, previous studies have shown that most of the Ce in
tissue of plants treated with CeO2 NPs remains in nanoparticulate form. This suggests that atmospheric CeO2 NPs
could be stored in the fruit of cucumber plants. Further studies
are needed in order determine the risk of atmospheric CeO2
NPs to human and environmental health.
■
ASSOCIATED CONTENT
S Supporting Information
*
Sample preparation for the TEM study. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*Phone: 915-747-5359. Fax: (915)747-5748. E-mail: jgardea@
utep.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This material is based upon work supported by the National
Science Foundation and the Environmental Protection Agency
under Cooperative Agreement Number DBI-0830117. Any
opinions, findings, and conclusions or recommendations
expressed in this material are those of the author(s) and do
not necessarily reflect the views of the National Science
Foundation or the Environmental Protection Agency. This
work has not been subjected to EPA review, and no official
endorsement should be inferred. The authors also acknowledge
the USDA grant number 2011-38422-30835 and the NSF
Grant # CHE-0840525. J. L. Gardea-Torresdey acknowledges
the Dudley family for the Endowed Research Professorship and
the Academy of Applied Science/US Army Research Office,
Research and Engineering Apprenticeship program (REAP) at
UTEP, grant # W11NF-10-2-0076, subgrant 13-7. The authors
also thank Dr. Peter Cooke (New Mexico State University, Las
Cruces, NM) for helping in the analysis.
■
■
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