Ecotoxicology and Environmental Safety 171 (2019) 771–780

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Effects of copper on the growth, antioxidant enzymes and photosynthesis of T

spinach seedlings
Qin Gonga,b,1, Ling Wanga,1, Tongwei Daia, Jingyi Zhoua, Qun Kanga, Hongbin Chena, Kun Lia,
⁎
Zhaohua Lia,c,
a
Faculty of Resources and Environmental Science, Hubei University, Wuhan 430062, China
b
Xinjiang Vocation College of Agriculture, Changji, Xinjiang 831100, China
c
Hubei Rural Safe Drinking Water Engineering Technology Research Center, Wuhan, Hubei 430062, China

A R T I C LE I N FO A B S T R A C T

Keywords: Examination of plants with strong Cu tolerance and an understanding of their Cu-tolerance mechanisms are of
Copper concentrations considerable significance for the remediation of Cu-contaminated soil. Although spinach may be a plant with
Spinach seedlings strong Cu tolerance, the threshold of Cu tolerance in this plant and its physiological response mechanisms to Cu
Plant growth are still unclear. In this study, we examined that the effects of different Cu concentrations on the growth
Antioxidant enzyme
parameters, antioxidant enzyme activities, and photosynthesis of spinach seedlings. The results showed that
Photosynthesis
when treated with a low Cu concentration (100 mg L−1 CuSO4), the biomass of spinach seedlings increased,
whereas the MDA content, the activities of antioxidant enzymes, Pn, gs and Tr were not significantly different
from those in the control (P > 0.05), and Y(II), qP reached their maximum values, indicating that a low Cu
concentration (100 mg L−1 CuSO4) had minimal negative effects on the life activities of spinach seedlings. In
contrast, when treated with high Cu concentrations (800–1000 mg L−1 CuSO4), the total biomass of spinach
seedlings was markedly decreased, the MDA contents increased, antioxidant enzyme activities initially increased
and then decreased to varying degrees, the contents of chlorophyll, Pn, Tr, Fv/Fm, qP, NPQ, and Y(II) were all
decreased. However the growth of spinach did not terminate, implying that the lethal threshold concentration of
Cu for spinach is greater than 1000 mg L−1 CuSO4 used in this study. In summary, spinach exhibits a high
tolerance to Cu and can be considered as an alternative plant for the remediation of Cu-contaminated soils.

1. Introduction term pollution, which is not easily discerned and non-biodegradable.

As a consequence of a variety of anthropogenic activities, such as
the rampant discharge of urban and industrial waste, combustion of
fossil fuels, and excessive application of chemical fertilizers, large
amounts of exogenous heavy metals are transported to the soil en-
vironment, where excessive accumulation result in contents of heavy
metals that exceed their standard levels in soil (Chandrasekhar and Ray,
2017). Soil is an important medium for plant growth, and when the
concentrations of heavy metals in soil exceed the physiological load of
plants, the normal growth and survival of plants will be detrimentally
affected. In soil, heavy metal elements can lead to wide ranging, long-
Moreover, heavy metals in soil can progressively accumulate in living
organisms through the food chain, poisoning certain organisms in the
food chain, and eventually accumulating in the human body to levels
that may endanger health. Therefore, heavy metal pollution in soil has
currently emerged as a global environmental crisis. Concentrations of
heavy metals in soil that exceed their standard levels have also received
widespread attention, and there is an urgent need to assess what
measure need to be taken to remedy heavy metal-contaminated soil
(Chen et al., 2015; Możdżeń et al., 2016).
In china, about 20 million hectares of farmland was contaminated
by heavy metals every (Cai et al., 2019). According to the national

Abbreviations: FW, Fresh weight; DW, Dry weight; MDA, Malondialdehyde; TCA, Trichloroacetic acid; SOD, Superoxide dismutase; POD, Peroxidase; CAT, Catalase;
APX, Ascorbate peroxidase; ROS, Reactive oxygen species; Chl a/b, Chlorophyll a/b; Car, Carotenoid; Pn, Net photosynthetic rate; gs,, Stomatal conductance; Ci,
Internal CO2 concentration; Tr, Transpiration rate; Fv/Fm, Maximum quantum yield of PSII photochemistry; qP, Photochemical quenching; NPQ, Nonphotochemical
quenching; YⅡ, PSII actual photosynthetic efficiency
⁎
Corresponding author at: Faculty of Resources and Environmental Science, Hubei University, Wuhan 430062, China.
E-mail address: Zli@hubu.edu.cn (Z. Li).
1
These authors contribute equally to this work.

https://doi.org/10.1016/j.ecoenv.2019.01.016
Received 14 August 2018; Received in revised form 4 January 2019; Accepted 6 January 2019
0147-6513/ © 2019 Elsevier Inc. All rights reserved.
Q. Gong et al.
communique of the soil pollution survey conducted by the Ministry of
Environmental Protection of China and the Ministry of Land and Re-
sources in 2014 (MEP, 2014), the total out-standard rate of soil pollu-
tion reached 16.1%, among which the over-standard rates of cultivated
lands were the highest. In addition, the out-standard rate of Copper
(Cu) was 2.1% which ranked the fourth. Zhang et al. reported that the
level of copper pollution in soil reached 102.5 mg kg-1 among 31 pro-
vincial capitals in China (Zhang et al., 2018), Other research found that
the Cu concentration in mining area was as high as 739 mg kg-1(Sun
et al., 2018). During Cu smelting process, heavy metals in Cu ores
would be emitted into the surrounding environment, degrading en-
vironmental quality, exceeding levels of heavy metal contents in the
vegetables, and consequently posing high risks to human health (Cai
et al., 2019).
Copper (Cu) is a heavy metal element with dual characteristics.
Although it is a trace element that is necessary for plant growth, ex-
cessive Cu can cause toxicity to plants (Rombel-Bryzek et al., 2017). In
recent years, due to the unreasonable use of Cu-containing pesticides
and Cu-containing manure in agricultural production, as well as waste
incineration and sewage irrigation, the soil content of Cu in many re-
gions has exceeded its standard level. Plants cultivated in soil con-
taining excess Cu will suffer from growth retardation, decreased yield,
increased accumulation of reactive oxygen species, enhanced activity of
oxygen scavenging systems, altered cell membrane structure, enhanced
cell membrane permeability, imbalance of endogenous hormones, and
changes in nutrient absorption and distribution (Chandrasekhar and
Ray, 2017; Chen et al., 2015).
To date, research on the physiological response mechanism of plants
to Cu stress has yielded certain results, which not only provides a basis
for remediation of contaminated soil but also lays a theoretical foun-
dation for further study on the mechanism of Cu toxicity to plants as
well as plant tolerance to Cu. Previous research in this field has tended
to focus on crops such as wheat (Triticum aestivum L.) (Atabayeva et al.,
2017) and sorghum (Sorghum bicolor L.) (Roy et al., 2017), fruit vege-
tables such as tomato (Solanum lycopersicon L.) (Chamseddine et al.,
2009) and cabbage (Brassica oleracea L. var. capitata L.) (Ali et al.,
2015), as well as Cu-accumulating plants such as Elsholtzia haichowensis
(Zhang et al., 2008, 2010) and India mustard (Brassica juncea L.) (Feigl
et al., 2013, 2015), whereas other plants are seldom studied. There are
substantial differences in the Cu tolerance range among different plants.
The examination of plants with strong Cu tolerance and an under-
standing of their Cu-tolerance mechanism are of considerable sig-
nificance for the remediation of Cu-contaminated soil.
Spinach (Spinacia oleracea L.), a herb belonging to the
Caryophyllales order, contains large amounts of vitamins, carotenoids,
organic acids and alkaline minerals, as well as antioxidants (Aehle
et al., 2004; Lisiewska et al., 2011; Becker et al., 2014), and is char-
acterized by large leaf area, high relative growth rate, and high rate of
heavy metal absorption (Alia et al., 2015). Given these characteristics,
spinach and other plants belonging to the Caryophyllales order are
often used in scientific research to observe their growth in response to
heavy metal contamination and toxicity (Alexander et al., 2006; Naz
et al., 2013, 2016). Studies have also shown that when spinach is grown
in sewage-irrigated soil (Cu2+ content of 7.93 mg L-1), the Cu2+ content
in spinach exceeds its standard level, whereas spinach growth and yield
are significantly increased (Naz et al., 2016); similarly, when irrigated
with sewage spiked with CuO nanoparticles at a lower concentration,
spinach shows promoted growth (Singh and Kumar, 2016). It is thus
speculated that spinach may have a certain level of Cu tolerance,
however, its Cu tolerance threshold and physiological response me-
chanisms are still unclear. How much Cu concentration can spinach
tolerate? How does the physiological mechanism of spinach change
under high copper concentration? Should this tolerance mechanism be
used to remedy copper contaminated soils? Therefore, in this study we
conducted a pot experiment to investigate the effects of different Cu
concentrations on the growth state and physiological mechanisms of
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Ecotoxicology and Environmental Safety 171 (2019) 771–780
spinach seedlings, with the aim of determining the Cu tolerance
threshold and Cu tolerance mechanisms of spinach.
2. Materials and methods
2.1. Culture of test materials
The experiment was conducted in November 2017 in solar green-
house of the Faculty of Resources and Environmental Science of Hubei
University. The test variety was Japanese big-leaf spinach, which was
provided by the seed breeding station of Wangzhendian, Qingxian
County, Hebei Province. Seeds with full granules were selected, disin-
fected with 0.1% NaClO for 10 min, rinsed with deionized water, and
evenly placed in a filter paper-containing petri dish (9 cm in diameter),
which was placed in a 25 °C incubator to allow seed germination in the
dark for 48 h, Seeds showing white buds were subsequently selected
and planted in peat soil until the seedlings had grown to a height of
approximately 10 cm. Seedlings showing the same growth character-
istics were selected and transplanted into 53 cm × 23 cm × 18 cm
(length × width × height) plastic pots. Each pot contained 10 seed-
lings and was supported by a tray from underneath. The potting matrix
comprised peat soil and perlite mixed at a 7:3 ratio, of which 5.5 kg
(3.85 kg DW) was used to fill each pot. The seedlings were cultivated
for 20 days to allow a vigorous root system to establish, followed by Cu
treatment. During cultivation, the soil was irrigated with a complete
Hoagland nutrient solution according to soil moisture levels.
2.2. Test treatment
Eleven treatments of Cu concentrations were set: 0 (CK), 100, 200,
300, 400, 500, 600, 700, 800, 900, and 1000 mg L-1 Cu supplied as
CuSO4 (analytical reagent), each of which was replicated three times.
The 0 mg L-1 concentration was used as the control treatment in which
only Hoagland nutrient solution was added. The Hoagland nutrient
solution, including 47.9 mg L-1 CuSO4, was adjusted to a pH of
6.5 ± 0.3 by using 0.1 mmol L-1 NaOH or HCl. The CuSO4 solutions at
each treatment concentration, prepared using the Hoagland nutrient
solution, were used to irrigate the soil at 2–3 day intervals, with a vo-
lume of 400 mL per pot per irrigation, and the total volume of CuSO4
solutions used were 1200 mL per pot. Solution overflowing into the tray
was added back to the soil. Water loss due to evaporation was com-
pensated for by adding deionized water until a weight of 6.1 ± 0.03 kg
was obtained. Cultivation was conducted at a day/night temperature of
22/15 °C under a relative humidity of 70–75%, with 14 h of illumina-
tion (light intensity of 8000 Lx) and 10 h of darkness. After 8 days of
treatment, plant samples were collected for the measurement of in-
dicators.
2.3. Measurement indicators and methods
2.3.1. Determination of biomass
The seedlings of each treatment group were carefully taken out from
the pots, initially rinsed with tap water, and then rinsed three times
with deionized water. After drying with blotting paper, the leaves,
stems, and roots were separated, immediately followed by a rapid
measurement of fresh weight (FW). Thereafter, the samples were he-
ated at 105 °C for 10 min, and then dried in an oven at 80 °C until a
constant weight was obtained, which was recorded as the dry weight
(DW).
2.3.2. Determination of root system parameters
In each treatment group, three of the root systems showing average
growth vigor were selected for scanning to assess root morphology. We
used an Epson Expression 10,000 XL image scanning system combined
with Win RHIZO root image software to analyze total root length, total
root surface area, total number of branches, and total number of root
Q. Gong et al.
tips (Graaff et al., 2013).
2.3.3. Determination of malondialdehyde and proline contents
Malondialdehyde (MDA) was determined using the thiobarbituric
acid method (Tewari et al., 2002). Fresh leaf samples (0.5 g) were
collected and ground with 5 mL of 0.5% TCA to yield a homogenized
slurry, which was then centrifugated at 15,000 r/min for 10 min. To the
resulting supernatant, 2 mL of 0.67% TBA were added followed by
mixing via shaking and heating at 90 °C for 20 min, after which the
mixture was subjected to rapid cooling and centrifugation at 1000r/min
for 5 min, followed by measurement of supernatant absorbances at
532 nm and 600 nm.
Proline content was determined by acidic ninhydrin colorimetry.
(Bates et al., 1973). Fresh leaf samples (0.5 g) were weighed and mixed
with 5 mL of 3% sulfosalicylic acid and extracted in a boiling water bath
for 10 min, after which the mixture was allowed to cool. The resulting
supernatant was isolated by suction filtration, followed by the addition
of 2 mL glacial acetic acid and 2 mL acidic ninhydrin and heated in a
boiling water bath for 30 min, after which the resulting mixture was
allowed to cool to room temperature. Thereafter, 4 mL toluene was
added, and the mixture was thoroughly shaken and allowed to stand
until stratified, followed by isolation of the upper layer for absorbance
measurement at 520 nm.
2.3.4. Determination of antioxidant enzyme activities
Superoxide dismutase (SOD) assay (Beauchamp and Fridovich,
1971). The reaction mixture (3.9 mL) contained 50 mmol L-1 phosphate
buffer (pH 7.8), 0.1 mmol L-1 EDTA, 13.37 mmol L-1 methionine, 750
μmol L-1 nitroblue tetrazolium (NBT) and 20 μmol L-1 riboflavin. The
reaction was started by adding enzyme extracts under illumination
(10 min, 5000 lx). The absorbance of the sample was determined at
560 nm.
Peroxidase (POD) assay (Zheng and Huystee, 1992). The reaction
mixture (2.9 mL) contained 10 mmol L-1 phosphate buffer (pH 7.0),
50 μL 0.02 mol L-1 guaiacol, 0.1 mL of enzyme extracts, and 10 μL
0.04 mol L-1 H2O2. The POD activity was determined by monitoring the
increase in absorbance at 470 nm.
Catalase (CAT) assay (Lu et al., 2018). The reaction mixture
(3.3 mL) contained 100 mmol L-1 phosphate buffer (pH 7.4), 0.1 mL of
enzyme extracts, 0.5 mL 65 μmol L-1 H2O2 and 0.5 mL 32.7 mol L-1
ammonium molybdate. The absorbance of the sample was determined
at 405 nm.
Ascorbate peroxidase (APX) assay (Nakano and Asada, 1981). The
Fig. 1. Effect of CuSO4 concentrations on the fresh weight (a) and dry weight (b) of spinach seedlings. Each column represents the average value of triplicate
measurements, and error bars represents the standard error. Different letters on the columns denote significant differences (P < 0.05).
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Ecotoxicology and Environmental Safety 171 (2019) 771–780
reaction mixture (1.7 mL) consisted of 50 mmol L-1 Na-phosphate buffer
(pH 7.8) containing 0.1 mmol L-1 EDTA, 5 mmol L-1 ascorbate,
20 mmol L-1 H2O2 and 100 μL of enzyme extract. The decrease in ab-
sorbance at 290 nm was monitored.
2.3.5. Determination of photosynthetic pigments, photosynthetic
parameters, and chlorophyll fluorescence parameters
The contents of Chlorophyll a (Chl a), Chlorophyll b (Chl b) and
carotenoids were determined according to the methods described by
Hiscox and Israeltem (Hiscox and Israeltem, 1979), and the content per
gram of leaves was calculated according to Arnon's formula (Arnon,
1949) as follow:
Ca = 13.95A 665 − 6.88A 649; Cb = 24.96A 64 9 − 7.32A 665; CT = Ca + Cb
C x·c = (1000A 470 − 2.05 Ca − 114.8 Cb)/245.
The net photosynthetic rate (Pn), intercellular CO2 concentration
(Ci), stomatal conductance (gs), and transpiration rate (Tr) of functional
leaves were determined using a Li-6400 portable photosynthesis meter
(LI-COR, USA). Chlorophyll fluorescence parameters were determined
using a PAM-2100 chlorophyll fluorometer (Walz, Germany), including
minimum fluorescence (F0), maximum fluorescence after dark adapta-
tion (Fm), photochemical quenching (qP), nonphotochemical quenching
(NPQ), and actual photosynthetic efficiency of PSII [Y(II)]. The ratio of
Fv/Fm was calculated according to the formula (Fm-F0)/Fm, and the
remaining parameters could be measured directly.
2.4. Data analysis
A normal distribution test was used to assess the data of each in-
dicator, and those data not conforming to a normal distribution were
converted to a normal distribution. One-way analysis of variance
(ANOVA, LSD test) was performed to evaluate the significance of the
inter-treatment differences. Pearson's correlation analysis was per-
formed to determine correlations among the indicators. All statistical
analyses were conducted using SPSS 17.0 statistical software. Graphic
representations were generated using Origin Pro 9.0 software.
3. Results
3.1. Effect of Cu concentrations on growth parameters of spinach seedlings
The total biomass of spinach seedlings reached a maximum value in
response to treatment with 100 mg L-1 of CuSO4, and the total FW and
Q. Gong et al.
Fig. 2. Effect of CuSO4 concentrations on the root morphology of spinach seedlings. The treatments CuSO4 concentrations ( mg L-1) are as follows: I: 0 (CK), II: 100,
III: 200, IV: 300, V: 400, VI: 500, VII: 600, VIII: 700, IX: 800, X: 900, XI: 1000.
total DW increased by 23.45% and 41.88%, respectively, compared
with those in the control treatment (Fig. 1). Thereafter the total biomass
of spinach continued to decrease with an increase in Cu concentrations.
When treated with Cu at high concentrations (800–1000 mg L-1 CuSO4),
the average value of total FW and total DW were reduced by 38.11%
and 44.28%, respectively, compared with those in the control group.
Changes in the FW and DW of leaves, stems, and roots of spinach
seedlings with increasing Cu concentrations showed essentially the
same trend as that of total biomass, that is, an initial increase and
subsequent decrease after reaching a maximum value at 100 mg L-1
CuSO4.
The root parameters of spinach seedlings showed change trends
similar to that of biomass. When treated with 100 mg L-1 CuSO4, the
total root length, total root surface area and total root tips number of
the seedlings were not significantly different, compared with those in
the control group (P > 0.05). As the treatment concentrations in-
creased, each root system parameter was decreased in a gradient
manner (Supplementary material Table 1). As shown by the change in
the root morphology of spinach seedlings (Fig. 2), when treated with
100 mg L-1 CuSO4, the number of fibrous roots of the seedlings was
slightly reduced, although the roots became stouter. With an increase of
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Ecotoxicology and Environmental Safety 171 (2019) 771–780
Cu treatment concentrations, although the number of fibrous roots and
the length of the roots of seedlings continued to decline, the roots of
seedlings remained viable when treated with relatively high Cu con-
centrations (> 700 mg L-1 CuSO4).
3.2. Changes in spinach leaves MDA and proline contents in response to
treatment with different concentrations of Cu
The MDA content in the leaves of spinach seedlings was no sig-
nificant difference from that in the control at 100 mg L-1 CuSO4
(P > 0.05) (Fig. 3a). Increases in MDA contents were highest in
seedlings treated with CuSO4 at concentrations greater than 800 mg L-1,
and were significantly higher than those in the control and other
treatments (P < 0.05). The contents of proline in spinach leaves in-
creased significantly with an increase in CuSO4 concentrations
(P < 0.05) (Fig. 3b). When the CuSO4 concentrations were in the range
between 100 and 600 mg L-1, there was no significant difference in
proline content among the treatments (P > 0.05), however, at con-
centrations greater than 800 mg L-1, the proline contents in each
treatment were significantly higher than those in other treatments
(P < 0.05), which increased by 1.23, 1.39 and 1.53 times at
Q. Gong et al.
Fig. 3. Effect of CuSO4 concentrations on the contents of malondialdehyde (a) and proline (b) in the leaves of spinach seedlings.
concentrations of 800, 900, and 1000 mg L-1, respectively， compared
with that in the control group.
3.3. Changes in the antioxidant enzyme activities of spinach leaves in
response to increasing Cu concentrations
Differences were observed in the antioxidant enzyme activities in
leaves of spinach in the response to treatment with different CuSO4
concentrations (Fig. 4). At a CuSO4 concentration of 100 mg L-1, there
was no significant difference in the activities of SOD, POD, CAT, and
APX compared with that in the control group (P > 0.05). However, at
treatment concentrations higher than 500 mg L-1, the activities of these
four enzymes were significantly increased, among which the activities
of SOD and APX reached their maximum values in response to treat-
ment with 700 mg L-1 CuSO4, which were 2.07 and 5 times higher, re-
spectively, than those in the control group Similarly, at CuSO4 con-
centrations of 800 and 900 mg L-1, CAT and POD activities reached their
maximum values, which were 4.76 and 4.75 times higher, respectively,
than those in the control group. At the highest Cu concentration
(1000 mg L-1 CuSO4), the activities of all four enzymes declined to
varying degrees.
3.4. Effects of different concentrations of Cu on spinach leaves chlorophyll
contents and photosynthetic characteristics
Table 1 shows that with an increase in CuSO4 concentrations, there
was a downward trend in the contents of Chlorophyll a, Chlorophyll b
and total chlorophyll in spinach seedlings. At the highest Cu con-
centration (1000 mg L-1 CuSO4), each of these three pigment contents
declined to a minimum value, with reductions of 42.24%, 64.07%, and
51.49% relative to those in the control group, respectively. In contrast,
carotenoids content showed an initial increase and subsequent decrease
with an increase in CuSO4 concentrations. Whereas in response to
treatment with 100 mg L-1 CuSO4, there was no significant change in
the carotenoid content of spinach seedlings (P > 0.05). At a CuSO4
concentration of 400 mg L-1, the content of carotenoids reached a
maximum value, with an increase of 52.6% relative to that in the
control group. However, at the highest Cu concentration (1000 mg L-1
CuSO4), the content had declined to a minimum value, although this
was still higher than that in the control group.
In response to treatment with the different concentrations of CuSO4,
we detected no obvious changes in the Pn and Tr of spinach seedling
leaves, compared with the control group (P > 0.05) (Table 1). At this
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Ecotoxicology and Environmental Safety 171 (2019) 771–780
concentration (100 mg L-1 CuSO4), gs was also increased by 16.7%
compared with that in the control group, whereas Ci was decreased,
although not to a significant extent compared with that in the control
group (P > 0.05). Subsequently, with an increase in the concentrations
of CuSO4, Pn and Tr values decreased, Ci increased, and gs intially in-
creased and then decreased. When treated with high Cu concentrations
(800–1000 mg L-1 CuSO4), Pn, gs, and Tr were decreased by an average
of 65.6%, 58.8%, and 44.5%, respectively, compared with those in the
control group. In addition, the values of Fv/Fm and NPQ in the leaves of
spinach seedlings were decreased with increasing treatment con-
centrations (Fig. 5). At 100 mg L-1 CuSO4, Y(II) and qP reached max-
imum values, whereas in response to higher treatment concentrations,
the values decline. In response to high concentrations Cu treatments
(800–1000 mg L-1 CuSO4), the values of Fv/Fm, Y(II), qP, and NPQ were
decreased by 21.2%, 51.6%, 34.4% and 44.4%, respectively, compared
with those in the control group.
3.5. Correlation analysis
In this study, we analyzed the effects of different CuSO4 con-
centrations on spinach indicators of biomass, antioxidant enzymes,
chlorophyll content, photosynthetic parameters, and chlorophyll
fluorescence parameters. As indicated by correlation analysis of these
indicators (Table 2), under the Cu treatment conditions, there were
significant positive between the total FW of spinach seedlings and total
DW, Pn, Tr, Fv/Fm, Y(II), qP, NPQ and total chlorophyll content, all of
which had associated correlation coefficients greater than 0.7
(P < 0.01). In contrast, total FW was found to be significantly negative
correlation with CAT activity and Ci (P < 0.01). The MDA contents in
spinach leaves showed a significant positive correlation with proline
content, and POD, CAT activities (P < 0.01), but showed a significant
negative correlation with the photosynthetic indicators Pn, Fv/Fm, Y
(II), qP and NPQ (P < 0.01).
4. Discussion
4.1. Physiological response of spinach seedlings to Cu treatment at low
concentration
Although Cu is one of the essential nutrients for plant growth and
development, there are significant differences in the demand for Cu
among different plants. The results of the present study showed that the
biomass of spinach seedlings treated with a low concentration of Cu
Q. Gong et al.
Fig. 4. Effect of CuSO4 concentrations on the activities of SOD (a), POD (b), CAT (c) and APX (d) in the leaves of spinach seedlings.
(100 mg L-1 CuSO4) was significantly higher than that in the control
group and in the other treatment groups (200–1000 mg L-1 CuSO4)
(P < 0.05). In addition, the root length, root surface area, and total
root tip number of spinach seedlings subjected to the 100 mg L-1 CuSO4
treatment showed no significant changes compared with those of the
control group (P > 0.05), indicating that the roots growth of spinach
seedling was little affected by this low Cu concentration. It can also be
speculated that Cu concentration at this time is beneficial to spinach
growth. Similar studies have also found that the application of CuO
nanoparticles (concentrations ≤ 100 mg L-1) had no obvious influence
on the growth of the roots and above-ground parts of spinach (Singh
and Kumar, 2016).
In plants, internal physiological regulation and phenotypic changes
constitute a unified mechanism. Therefore, it is particularly important
to analyze the effects of different Cu concentrations on the growth of
spinach seedlings from the perspective of plant physiology. MDA is an
important product of membrane lipid peroxidation, which can bind to
amino acids, proteins, nucleic acids and other substances to form in-
soluble compounds (lipofuscin), causing intracellular metabolic dis-
orders (Rombel-Bryzek et al., 2017; Lu et al., 2010). In this study, we
found that when spinach seedlings were treated with CuSO4 at a con-
centration of 100 mg L-1, the MDA contents showed no significant dif-
ference (P > 0.05), compared with control seedlings, indicating that
damage to membrane lipids via peroxidation is negligible in spinach
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Ecotoxicology and Environmental Safety 171 (2019) 771–780
seedlings treated with low Cu concentration. In response to Cu stress,
the suite of antioxidant enzymes act synergistically to protect plants
against physiological damages caused by Cu stress such as cell mem-
brane damage and the inactivation of enzyme activities (Sgherri et al.,
2007; Zhang et al., 2008). The results showed that the activities of
antioxidant enzymes (SOD, POD, CAT, APX) in spinach seedlings
treated with 100 mg L-1 CuSO4 were not significantly different from
those in the control group (P > 0.05), indicating that 100 mg L-1
CuSO4 could have minimal detrimental effects on the life activities of
spinach seedlings. Therefore, by slightly modifying the activities of
antioxidant enzymes, plants can resist the damage to growth attribu-
table to Cu, indicating that the tolerance mechanism of spinach plays a
role, and its regulatory role is very little to resist the damage of Cu
treatment.
Excessive Cu can have varying degrees of adverse effects on pho-
tosynthesis. Plants maintain photosynthetic efficiency and reduce
photooxidative damage as much as possible by physiological regula-
tion. (Chamseddine et al., 2009; Moustakas et al., 2011). The results of
the present study showed that the Pn, gs, and Tr values of spinach leaves
at the low Cu concentration (100 mg L-1 CuSO4) were not significantly
different from those in the control group (P > 0.05), which may be
attributable to the fact that the spinach seedlings use transpirational
pull to absorb water and supplement transpirational water loss, thereby
diluting the higher Cu concentrations in the leaves and reducing their
Q. Gong et al. Ecotoxicology and Environmental Safety 171 (2019) 771–780

own damage. This is consistent with the findings of Wang et al. (2013),

1.709 ± 0.099ab

1.652 ± 0.069ab
2.437 ± 0.461bc

1.433 ± 0.287a
1.313 ± 0.131a
1.312 ± 0.013a
1.653 ± 0.28ab
3.093 ± 0.159c
3.063 ± 0.224c
2.691 ± 0.712c
(mmol m-2s-1)

2.545 ± 0.67c
who reported that after a 9-day treatment of the "Wutaicai" (Chinese
cabbage) with 10 μmol L-1 CuSO4, there were increases in the values of
Pn, gs, Tr, and biomass. Furthermore, we also observed that when plants
were treated with 100 mg L-1 CuSO4, the Y(II) and qP values increased
Tr

to their maximum values, indicating that the photochemical conversion

efficiency and photochemical activity of PSⅡ had improved, and that
285.763 ± 33.148abcd

303.779 ± 10.784bcde
300.522 ± 3.458bcde
303.559 ± 9.379bcde
284.087 ± 1.163abc

318.381 ± 0.743cde
there had been an increase in plant photosynthetic activity (Kumar and
276.998 ± 3.582ab

321.839 ± 17.58de
251.16 ± 33.515a

324.95 ± 6.219e
Prasad, 2015), ultimately enhancing the photosynthesis of spinach

383.72 ± 5.679f
seedlings. Similar results have also been obtained in previous studies.
(μmol m-1)

For example, when treated with 1 mmol L-1 Cu2+ for 15 days, pi-
geonpea (Cajanus cajan L.) showed the highest qP value and enhanced
Ci

photosynthetic activity, as well as increased total biomass (Sharma

et al., 2017). Furthermore, we found that the carotenoid contents of
spinach seedlings subjected to the 100 mg L-1 CuSO4 treatment showed
0.114 ± 0.028ab
0.133 ± 0.015ab

0.134 ± 0.009ab
0.112 ± 0.037ab
0.398 ± 0.426b

0.064 ± 0.003a
0.064 ± 0.004a
0.058 ± 0.003a

0.045 ± 0.002a
0.045 ± 0.006a

no significant difference compared with the control group (P > 0.05),

(mol m-2s-1l)

0.05 ± 0.01a

implying that the Cu damage to chloroplasts in spinach seedlings could

be reduced via the synthesis of carotenoids ( Pätsikkä et al., 2002). In
summary, the minimal damage to photosynthesis in spinach seedlings
gs

when treated with low Cu concentration (100 mg L-1CuSO4) is another

important reason for the significant increase in biomass, implying that
10.152 ± 2.311a
10.869 ± 0.337a
10.312 ± 1.895a
9.269 ± 0.309ab

5.129 ± 0.083de
6.429 ± 0.415cd
7.854 ± 0.792bc

4.072 ± 0.344e
3.618 ± 0.693e
3.495 ± 0.331e
3.368 ± 1.194e

the tolerance mechanism plays a role in the spinach.

(μmol m-2s-1)

4.2. Analysis of the mechanism of underlying the tolerance of spinach

seedlings to high Cu concentrations
Pn

Although excessive Cu inhibits plant growth, different plants have

Carotenoids content

differing levels of Cu tolerance. Studies have shown that Elsholtzia

0.951 ± 0.399ab

0.951 ± 0.006ab
1.264 ± 0.060bc

1.252 ± 0.074bc

1.251 ± 0.006bc

haichowensis, a plant with strong Cu tolerance can withstand 600 mg kg-

0.911 ± 0.144a

1.304 ± 0.063c
1.390 ± 0.131c

1.361 ± 0.027c
1.381 ± 0.012c
1.371 ± 0.005c
Effect of CuSO4 concentrations on chlorophyll contents and photosynthetic parameters in the leaves of spinach seedlings.

1
of Cu (Jiang et al., 2004), and Moso bamboo (Phyllostachys pubescens),
（mg g-1）

a plant used for soil remediation, can withstand 300 mg kg-1 of Cu

(Chen et al., 2015). In this study, although the total biomass of spinach
seedlings decreased significantly when seedlings were treated with high
Cu concentrations (800–1000 mg L-1 CuSO4), the plants remained in a
1.819 ± 0.263abc

1.748 ± 0.332abc

viable state. Whereas the Cu tolerance level of spinach seedlings was

1.951 ± 0.133bc

2.027 ± 0.072bc
1.371 ± 0.104a
1.353 ± 0.506a
1.58 ± 0.118ab

2.108 ± 0.006c
2.187 ± 0.026c
1.931 ± 0.01bc
Chl a/b value

2.15 ± 0.061c

not as high as that of the aforementioned Cu-tolerant plants, it is still

significantly higher than that of other common plants. For example,
when treated with Cu at only 2.5 μmol L-1 concentration (equivalent to
0.399 mg L-1 CuSO4) or 5 μmol L-1 (equivalent to 0.798 mg L-1 CuSO4)
for 14 days, the roots and the above-ground parts of Arabidopsis thaliana
show significantly decreased growth (Lequeux et al., 2010). Similarly,
Total chlorophyll content

when treated with Cu at 100 μmol L-1 concentration (equivalent to

15.96 mg L-1 CuSO4) for15 days, the shoot and roots lengths of maize
10.155 ± 0.164cde
10.837 ± 0.158bc

(Zea mays L.) are both decreased (Aly and Mohamed, 2012).
9.884 ± 0.021de
9.915 ± 0.065de
11.255 ± 0.214c
10.85 ± 0.349bc
12.408 ± 1.16b
14.76 ± 0.881a

9.677 ± 0.549e

7.929 ± 0.028f

High Cu concentrations induce the production of large amounts of

7.16 ± 0.055f

ROS in plants, which leads to the peroxidation of membrane lipids, and

(mg g-1)

subsequent increases in cell membrane permeability and inactivation of

enzyme activities, thereby compromising cell viability (Arora et al.,
2012; Hussain et al., 2014). The results of the present study showed that
MDA content increased significantly in spinach seedlings when treated
3.372 ± 0.016bcd
3.278 ± 0.101bcd
3.223 ± 0.012bcd
2.551 ± 0.012cd
3.683 ± 0.227bc
3.601 ± 0.664bc

with high Cu concentrations (800–1000 mg L-1 CuSO4), indicating that

2.247 ± 0.036d
4.374 ± 0.274b
3.893 ± 0.479b
6.254 ± 0.638a
5.587 ± 1.472a
Chl b content

the spinach seedlings were experiencing raised levels of stress. Simi-

(mg g-1)

larly, the proline content in spinach also increased significantly, prob-

ably due to the fact that proline is an osmolyte in plants (Vijendra et al.,
2016), and that spinach reduced cell osmotic potential to maintain
growth by increasing the content of proline. Antioxidant enzymes
6.638 ± 0.036cd
6.82 ± 0.315bcd
6.88 ± 0.065bcd
6.957 ± 0.132bc

6.931 ± 0.175bc

protect cells during heavy metal stress and can scavenge ROS sub-
6.512 ± 0.014d
7.155 ± 0.068b

4.913 ± 0.021g
8.506 ± 0.243a

6.075 ± 0.289e

5.378 ± 0.017f
Chl a content

stances. In the present study, we observed that the activities of SOD,

POD, CAT, and APX in spinach seedlings initially increased to the
(mg g-1)

maximum values, indicating that the plants reduced the damage in-
duced by high Cu concentrations by increasing antioxidant enzyme
activities (Sgherri et al., 2007; Zhang et al., 2008). However, when the
Treatment

CuSO4 concentrations exceed the physiologically controllable range of

(mg L-1)

spinach (about approximately 800 mg L-1 CuSO4), the activities of an-

Table 1

1000
100
200
300
400
500
600
700
800
900

tioxidant enzymes were decreased, the possible reason was high metal
CK

concentrations declined the antioxidant activity due to metal binding to

777
Q. Gong et al.
Fig. 5. Effect of CuSO4 concentrations on chlorophyll fluorescence parameters Fv/Fm (a), Y(II) (b), NPQ (c) and qP (d) in the leaves of spinach seedlings.
the active centres of enzymes (Sidhu et al., 2017).
High Cu concentrations also affect factors relating to plant photo-
synthesis, including carbon dioxide diffusion, PSII efficiency, electron
transport, ROS formation, RuBP content (dependent on the supply of
ATP and NADPH) (Qureshi et al., 2010; Allen et al., 2011), and pho-
torespiration (Saibo et al., 2009). The results showed that the contents
of Chlorophyll a, Chlorophyll b and the total chlorophyll in the leaves of
spinach seedlings were decreased with an increase in Cu treatment
concentrations. At the highest Cu concentration applied (1000 mg L-1
CuSO4), the average decrease in chlorophyll content exceeded that in
the control group by 50%, indicating that the stress of high Cu con-
centration had affected chlorophyll synthesis( Pätsikkä et al., 2002;
Możdżeń et al., 2016). Furthermore, Cu2+ binds to -SH or replaces
Fe2+, Zn2+ or Mg2+ in chloroplast proteins, thereby altering the ionic
composition at the center of the protein, such that it becomes inactive
or unstable and undergoes degradation, which results in a decrease of
chlorophyll contents (Maksymiec, 1998; Demidchik et al., 2001). In
addition, at high Cu concentrations (800–1000 mg L-1 CuSO4), the
average values of Pn and Tr were decreased by 65.6%and 44.5%, re-
spectively, compared with those in the control group. On the basis of
these observations, it can be inferred that high Cu concentrations in-
duce a regulatory resistance in spinach to inhibit photosynthesis (Wang
et al., 2015), resulting in a decrease in spinach biomass. In addition, the
Fv/Fm, qP, NPQ, and Y(II) values of spinach leaves decreased to the
778
Ecotoxicology and Environmental Safety 171 (2019) 771–780
largest extent when seedlings were treated with high Cu concentrations
(800–1000 mg L-1 CuSO4), among which, the values of Fv/Fm, qP, and
Y(II) showed significant differences from those in the control group
(P < 0.05). These observations indicate that high Cu concentrations
interferes with electron transport in the photochemical reactions of
PSII, thereby inhibiting the electron transport of plastoquinones (Yruela
et al., 1996; Kumar and Prasad, 2015), and thus suppressing the dis-
sipation of excess light energy in the form of heat. This in turn ag-
gravates photodamage and decreases photoprotective ability, thereby
leading to a decrease in PSII activity (Pätsikkä et al., 2002; Baker,
2008), and eventually, to a decrease in the photosynthesis of spinach
seedlings. Although we observed that the growth of spinach seedlings
was substantially inhibited in response to treatment with a high Cu
concentration (1000 mg L-1 CuSO4), this high Cu concentration did not
terminate the growth of spinach, implying that the concentration is
below the lethal threshold of Cu for spinach. Further research on the
tolerance threshold of spinach for Cu may provide a scientific basis for
the remediation of Cu-contaminated soil.
5. Conclusion
Treatment with a low-concentration of Cu treatments (100 mg L-1
CuSO4) had minimal adverse effects on the life activities of spinach
seedlings. In contrast, at high Cu concentrations (800–1000 mg L-1
Q. Gong et al. Ecotoxicology and Environmental Safety 171 (2019) 771–780

CuSO4), there was an increase in the stress-induced damage to spinach

Carotenoids
seedlings, antioxidant enzyme activities were substantially disturbed,

-0.624*
-0.339
-0.437

-0.136
0.451 photosynthetic mechanisms were impaired, and photosynthetic rate
0.389

0.075
0.253
0.211
0.166

0.431
0.238
0.349
0.204
0.408
0.192
0.11
and biomass were both decreased. However this high Cu concentrations

1
did not terminate the growth of spinach, indicating that spinach has a
Total chlorophyll

higher Cu tolerance threshold, and can accordingly be considered as a

candidate plant for effective phyoremediation of Cu-contaminated soils.
-0.822**

-0.745**

-0.786**
0.789**

0.811**

0.936**
0.785**
0.941**
0.789**
-0.665*

-0.612*
0.731*

-0.589

-0.377 Acknowledgements

0.453

0.495

1
This work was supported by Grants from the key projects of Hubei
-0.803**
-0.746**

-0.795**
-0.769**

-0.878**
0.978**
0.929**

0.933**

0.811**
0.850**
0.996**
0.918**
-0.682*

0.721*

Natural Fund (innovation group) in China (2016CFA016), the National

-0.562
NPQ

Science and Technology for the Benefit of the People in China

1
(S013GMD100042) and China Postdoctoral Science Foundation
-0.789**
-0.834**

-0.838**
-0.786**

-0.862**
0.901**
0.816**

0.953**

0.966**
0.921**
-0.726*

(2017M622386). We would like to thank Editage [www. editage. cn]

0.687*

0.654*
-0.556

for English language editing.

qP

1
-0.782**

-0.807**
-0.805**

-0.855**

Appendix A. Supplementary material

0.964**
0.912**

0.943**

0.804**
0.852**
-0.722*
-0.718*

-0.625*

0.729*
Y(Ⅱ)

Supplementary data associated with this article can be found in the

-0.802**
-0.893**

-0.839**
-0.786**

-0.855**

online version at doi:10.1016/j.ecoenv.2019.01.016.

0.869**
0.743**

0.915**
-0.730*
Fv/Fm

-0.543

0.589

0.587
1

References
0.775**

0.735**
-0.714*
-0.603*

-0.648*
-0.626*

-0.677*
0.674*

-0.513

-0.391

0.534

Aehle, E., Reynaudle Grandic, S., Ralainirina, R., Baltorarosset, S., Mesnard, F., Prouillet,
Tr

C., Maziere, J.C., Fliniaux, M.A., 2004. Development and evaluation of an enriched
natural antioxidant preparation obtained from aqueous spinach (Spinacia oleracea)
-0.876**
-0.770**

-0.844**
0.826**

-0.659*

extracts by an adsorption procedure. Food Chem. 86 (4), 579–585. https://doi.org/

0.722*

0.689*
0.527

0.553
0.352

10.1016/j.foodchem.2003:10.006.
Ci

Alexander, P.D., Alloway, B.J., Dourado, A.M., 2006. Genotypic variations in the accu-
mulation of Cd, Cu, Pb and Zn exhibited by six commonly grown vegetables. Environ.
0.821**
-0.706*

-0.621*
-0.708*
-0.699*
0.684*

-0.586

-0.519

Pollut. 144, 736–745. https://doi.org/10.1016/j.envpol.2006.03.001.

0.523

Ali, S., Shahbaz, M., Shahzad, A.N., Khan, H.A., Anees, M., Haider, M.S., Fatima, A., 2015.
gs

Impact of copper toxicity on stone-head cabbage (Brassica oleracea var. capitata) in

hydroponics. PeerJ 3 (4), e1119. https://doi.org/10.7717/peerj.1119.
-0.851**
-0.822**
-0.824**
-0.914**
-0.887**
0.921**
0.780**

-0.677*

Alia, N., Sardar, K., Said, M., Salma, K., Sadia, A., Sadaf, S., Toqeer, A., Miklas, S., 2015.
Toxicity and bioaccumulation of heavy metals in Spinach (Spinacia oleracea) grown
Correlation analysis among various indicators of spinach seedlings under different CuSO4 concentrations.
Pn

in a controlled environment. Int. J. Environ. Res. Public Health 12, 7400–7416.

https://doi.org/10.3390/ijerph120707400.
0.860**

0.829**
0.653*
-0.529
-0.473

Allen, J.F., de Paula, W.B., Puthiyaveetil, S., Nield, J., 2011. A structural phylogenetic
0.361
0.263
APX

map for chloroplast photosynthesis. Trends Plant Sci. 16, 645–655. https://doi.org/
1

10.1016/j.tplants.2011.10.004.
Aly, A.A., Mohamed, A.A., 2012. The impact of copper ion on growth, thiol compounds
-0.772**

0.778**

0.862**
0.856**
-0.619*

and lipid peroxidation in two maize cultivars (Zea mays L.) grown in vitro. Aust. J.
0.647*
CAT

Crop Sci. 6, 541–549.

1

Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in beta

vulgaris. Plant Physiol. 24 (1), 1–15. https://doi.org/10.1104/PP.24.1.1.
0.843**
0.841**
0.892**
-0.829
-0.637

Arora, P., Bhardwaj, R., Kanwar, M.K., 2012. Effect of 24-epibrassinolide on growth,
POD

protein content and antioxidative defense system of Brassica juncea L. subjected to

1

cobalt ion toxicity. Acta Physiol. Plant. 34 (5), 2007–2017. https://doi.org/10.1007/

s11738-012-1002-2.
0.656*
-0.693

0.592
-0.57

Atabayeva, S., Nurmahanova, A., Asrandina, S., Alybayeva, R., Meldebekova, A.,
SOD

Ablaikhanova, N., 2017. Effect of copper on physiological and biochemical peculia-

1

rities of wheat (Triticum aestivum L.) varieties. Pak. J. Bot. 49 (6), 2189–2196.
Baker, N.R., 2008. Chlorophyll fluorescence: a probe of photosynthesis in vivo. Annu.
Proline

0.894**
-0.808
-0.587

Rev. Plant Biol. 59 (1), 89. https://doi.org/10.1146/annurev.arplant59.032607.

** Significant correlation at the 0.01 level (both sides);
* Significant correlation at the 0.05 level (both sides).

092759.
1

Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for
-0.862
-0.674

water-stress studies. Plant Soil 39, 205–207.

MDA

Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay
1

applicable to acrylamide gels. Anal. Biochem. 44, 276–287.

Becker, C., Klaering, H.P., Kroh, L.W., Krumbein, A., 2014. Cool-cultivated red leaf lettuce
Total DW

accumulates cyanidin-3-O-(6''-O-malonyl)-glucoside and caffeoylmalic acid. Food

0.930**

Chem. 146, 404–411. https://doi.org/10.1016/j.foodchem.2013.09.061.

Cai, L.M., Wang, Q.S., Luo, J., Chen, L.G., Zhu, R.L., Wang, S., Tang, C.H., 2019. Heavy
1

metal contamination and health risk assessment for children near a large Cu-smelter
in central China. Sci. Total Environ. 650, 725–733. https://doi.org/10.1016/j.
Total FW

scitotenv.2018.09.081.
Chamseddine, M., Wided, B.A., Guy, H., Marie-Edith, C., Fatma, J., 2009. Cadmium and
copper induction of oxidative stress and antioxidative response in tomato (Solanum
1

lycopersicon) leaves. Plant Growth Regul. 57 (1), 89.

Chandrasekhar, C., Ray, J.G., 2017. Copper accumulation, localization and antioxidant
Total chlorophyll

response in Eclipta alba L. in relation to quantitative variation of the metal in soil.

Acta Physiol. Plant. 39. https://doi.org/10.1007/s11738-017-2508-4.
Carotenoids

Chen, J., Shafi, M., Li, S., Wang, Y., Wu, J., Ye, Z.Q., Peng, D., Yan, W.B., Liu, D., 2015.
Total DW
Total FW

Proline

Copper induced oxidative stresses, antioxidant responses and phytoremediation po-

Fv/Fm
Table 2

MDA

Y(Ⅱ)

NPQ
POD

tential of Moso bamboo (Phyllostachys pubescens). Sci. Rep. 5, 13554. https://doi.

SOD

APX
CAT

qP
Pn
　

Tr
Ci

org/10.1038/srep13554.
gs

779
Q. Gong et al.
Demidchik, V., Sokolik, A., Yurin, V., 2001. Characteristics of non-specific permeability
and H+-ATPase inhibition induced in the plasma membrane of Nitella flexilis by
excessive Cu2+. Planta 212, 583–590.
Feigl, G., Kumar, D., Lehotai, N., Molnar, A., Racz, E., Ordog, A., Erdei, L., Kolbert, Z.,
Laskay, G., 2015. Comparing the effects of copper in the leaves of Brassica juncea (L.
Czern) and Brassica napus (L.) seedlings: growth inhibition, oxidative stress and
photosynthetic damage. Acta Biol. Hung. 66.
Feigl, G., Kumar, D., Lehotai, N., Tugyi, N., Molnar, A., Ordog, A., Szepesi, A., Gemes, K.,
Laskay, G., Erdei, L., Kolbert, Z., 2013. Physiological and morphological responses of
the root system of Indian mustard (Brassica juncea L. Czern.) and rapeseed (Brassica
napus L.) to copper stress. Ecotoxicol. Environ. Saf. 94, 179–189. https://doi.org/10.
1016/j.ecoenv.2013.04.029.
Graaff, M.A.D., Six, J., Jastrow, J.D., Schadt, C.W., Wullschleger, S.D., 2013. Variation in
root architecture among switchgrass cultivars impacts root decomposition rates. Soil
Biol. Biochem. 58, 198–206. https://doi.org/10.1016/j.soilbio.2012.11.015.
Hiscox, J.D., Israeltem, G.F., 1979. A method for extraction of chlorophyll from leaf tissue
without maceration. Rev. Can. Bot. 57, 1332–1334. https://doi.org/10.1139/b79-
163.
Hussain, I., Rasheed, R., Wahid, A., 2014. Seasonal variations in some water relations and
biochemical attributes of two genetically diverse maize cultivars. Braz. J. Bot. 37,
417–428. https://doi.org/10.1007//s40415-014-0095-9.
Jiang, L.Y., Yang, X.E., He, Z.L., 2004. Growth response and phytoextraction of copper at
different levels in soils by Elsholtzia splendens. Chemosphere 55, 1179–1187.
https://doi.org/10.1016/j.chemosphere.2004.01.026.
Kumar, A., Prasad, M.N.V., 2015. Lead-induced toxicity and interference in chlorophyll
fluorescence in Talinum triangulare grown hydroponically. Photosynthetica 53,
66–71. https://doi.org/10.1007/s11099-015-0091-8.
Lequeux, H., Hermans, C., Lutts, S., Verbruggen, N., 2010. Response to copper excess in
Arabidopsis thaliana: impact on the root system architecture, hormone distribution,
lignin accumulation and mineral profile. Plant Physiol. Biochem. 48, 673–682.
https://doi.org/10.1016/j.plaphy.2010.05.005.
Lisiewska, Z., Kmiecik, W., Gębczyński, P., Sobczyńska, L., 2011. Amino acid profile of
raw and as-eaten products of spinach (Spinacia oleracea L.). Food Chem. 126,
460–465. https://doi.org/10.1016/j.food chem.2010.11.015.
Lu, Q., Zhang, T., Zhang, W., Su, C., Yang, Y., Hu, D., Xu, Q., 2018. Alleviation of cad-
mium toxicity in Lemna minor by exogenous salicylic acid. Ecotoxicol. Environ. Saf.
147, 500–508. https://doi.org/10.1016/j.ecoenv.2017.09.015.
Lu, Y., Li, X.R., He, M.Z., Wang, Z.N., Tan, H.J., 2010. Nickel effects on growth and
antioxidative enzymes activities in desert plant Zygophyllum xanthoxylon (Bunge)
Maxim. Sci. Cold Arid Reg. 02, 436–444. https://doi.org/10.3724/SPJ.1226.2010.
00436.
Maksymiec, W., 1998. Effect of copper on cellular processes in higher plants.
Photosynthetica 34, 321–342. https://doi.org/10.1023/A:1006818815528.
Ministry of Environmental Protection, 2014. A National Survey of Soil Pollution Bulletin.
Moustakas, M., Sperdouli, I., Kouna, T., Antonopoulou, C.I., Therios, I., 2011. Exogenous
proline induces soluble sugar accumulation and alleviates drought stress effects on
photosystem II functioning of Arabidopsis thaliana leaves. Plant Growth Regul. 65,
315. https://doi.org/10.1007/s10725-011-9604-z.
Możdżeń, K., Wanic, T., Rut, G., Łaciak, T., Rzepka, A., 2016. Toxic effects of high copper
content on physiological processes in Pinus sylvestris L. Photosynthetica 55,
193–200. https://doi.org/10.1007/s11099-016-0229-3.
Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specific per-
oxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880.
Naz, A., Khan, S., Qasim, M., Khalid, S., Muhammad, S., Tariq, M., 2013. Metals toxicity
and its bioaccumulation in purslane seedlings grown in controlled environment. Nat.
Sci. 05, 573–579. https://doi.org/10.4236/ns.2013.55073.
Naz, S., Anjum, M.A., Akhtar, S., 2016. Monitoring of growth, yield, biomass and heavy
metals accumulation in spinach grown under different irrigation sources. Int. J.
Agric. Biol. 18, 689–697. https://doi.org/10.17957/ijab/15.0129.
Pätsikkä, E., Kairavuo, M., Sersen, F., Aro, E.M., Tyystjã¤Rvi, E., 2002. Excess copper
780
Ecotoxicology and Environmental Safety 171 (2019) 771–780
predisposes photosystem II to photoinhibition in vivo by outcompeting iron and
causing decrease in leaf chlorophyll. Plant Physiol. 129, 1359–1367. https://doi.org/
10.1104/pp.004788.
Qureshi, M.I., D'Amici, G.M., Fagioni, M., Rinalducci, S., Zolla, L., 2010. Iron stabilizes
thylakoid protein-pigment complexes in Indian mustard during Cd-phytoremediation
as revealed by BN-SDS-PAGE and ESI-MS/MS. J. Plant Physiol. 167, 761–770.
https://doi.org/10.1016/j.jplph.2010.01.017.
Rombel-Bryzek, A., Rajfur, M., Zhuk, O., 2017. The impact of copper ions on oxidative
stress in garden cress Lepidium sativum. Ecol. Chem. Eng. S. 24. https://doi.org/10.
1515/eces-2017-0041.
Roy, S.K., Cho, S.W., Kwon, S.J., Kamal, A.H.M., Lee, D.G., Sarker, K., Lee, M.S., Xin, Z.,
Woo, S.H., 2017. Proteome characterization of copper stress responses in the roots of
sorghum. Biometals 30, 765–785. https://doi.org/10.1007/s10534-017-0045-7.
Saibo, N.J.M., Lourenço, T., Oliveira, M.M., 2009. Transcription factors and regulation of
photosynthetic and related metabolism under environmental stresses. Ann. Bot. 103,
609–623. https://doi.org/10.1093/aob/mcn227.
Sgherri, C., Quartacci, M.F., Navariizzo, F., 2007. Early production of activated oxygen
species in root apoplast of wheat following copper excess. J. Plant Physiol. 164,
1152–1160. https://doi.org/10.1016/j.jplph.2006.05.020.
Sharma, P., Sirhindi, G., Singh, A.K., Kaur, H., Mushtaq, R., 2017. Consequences of copper
treatment on pigeon pea ph otosynthesis, osmolytes and antioxidants defense.
Physiol. Mol. Biol. Plants 23, 809–816. https://doi.org/10.1007/s12298-017-0461-8.
Sidhu, G.P.S., Singh, H.P., Batish, D.R., Kohli, R.K., 2017. Tolerance and hyper-
accumulation of cadmium by a wild, unpalatable herb Coronopus didymus (L.) Sm.
(Brassicaceae). Ecotoxicol. Environ. Safe 135, 209–215. https://doi.org/10.1016/j.
ecoenv.2016.10.001.
Singh, D., Kumar, A., 2016. Impact of irrigation using water containing CuO and ZnO
nanoparticles on Spinach oleracea grown in soil media. Bull. Environ. Contam.
Toxicol. 97, 548–553. https://doi.org/10.1007/s00128-016-1872-x.
Sun, Z., Xie, X., Wang, P., Hu, Y., Cheng, H., 2018. Heavy metal pollution caused by
small-scale metal ore mining activities: a case study from a polymetallic mine in
South China (http://dx.doi:). Sci. Total Environ. 639, 217–227. https://doi.org/10.
1016/j.scitotenv. 2018.05. 176.
Tewari, R.K., Kumar, P., Sharma, P.N., Bisht, S.S., 2002. Modulation of oxidative stress
responsive enzymes by excess cobalt. Plant Sci. 162, 381–388. https://doi.org/10.
1016/S0168-9452(01)00578-7.
Vijendra, P.D., Huchappa, K.M., Lingappa, R., Basappa, G., Jayanna, S.G., Kumar, V.,
2016. Physiological and biochemical changes in moth bean (Vigna aconitifolia L.)
under cadmium stress. J. Bot. 2016 (2016-3-30), 1–13. https://doi.org/10.1155/
2016/6403938.
Wang, L., Yang, X.Y., Ren, Z.H., Hu, X.Y., Wang, X.F., 2015. Alleviation of photosynthetic
inhibition in copper-stressed tomatoes through rebalance of ion content by exogenous
nitric oxide. Turk. J. Bot. 39, 10–22. https://doi.org/10.3906/bot-1312-17.
Wang, X.H., Song, Y.P., Ren, J., Liu, T.K., Hou, X.L., Li, Y., 2013. Response of biomass and
photosynthesis in non-heading Chinese cabbage to excess copper. J. Anim. Plant Sci.
23, 1659–1665.
Yruela, I., Pueyo, J.J., Alonso, P.J., Picorel, R., 1996. Photoinhibition of photosystem II
from higher plants. Effect of copper inhibition. J. Biol. Chem. 271, 27408–27415.
Zhang, H., Yan, X., Wang, G., Shen, Z., 2008. Excess copper induces accumulation of
hydrogen peroxide and increases lipid peroxidation and total activity of copper-zinc
superoxide dismutase in roots of Elsholtzia haichowensis. Planta 227, 465–475.
Zhang, H.F., Zhang, Y.X., Wang, G., Shen, Z., 2010. Excess copper induces production of
hydrogen peroxide in the leaf of Elsholtzia haichowensis through apoplastic and
symplastic CuZn-superoxide dismutase. J. Hazard Mater. 178, 834–843. https://doi.
org/10.1016/j.jhazmat.2010.02.014.
Zhang, X., Zha, T., Guo, X., Meng, G., Zhou, J., 2018. Spatial distribution of metal pol-
lution of soils of Chinese provincial capital cities. Sci. Total Environ. 643, 1502–1513.
https://doi.org/10.1016/j.scitotenv.2018.06.177.
Zheng, X., Huystee, R.B.V., 1992. Peroxidase-regulated elongation of segments from
peanut hypocotyls. Plant Sci. 81, 47–56.