Environ Sci Pollut Res (2013) 20:8672–8679
DOI 10.1007/s11356-013-1828-1
RESEARCH ARTICLE
Copper ultrastructural localization, subcellular distribution,
and phytotoxicity in Hydrilla verticillata (L.f.) Royle
Qinsong Xu & Han Qiu & Weiyue Chu & Yongyang Fu &
Sanjuan Cai & Haili Min & Sha Sha
Received: 6 January 2013 / Accepted: 14 May 2013 / Published online: 26 May 2013
# Springer-Verlag Berlin Heidelberg 2013
Abstract Laboratory experiments were conducted to inves-
tigate copper (Cu) subcellular distribution and toxicity in
Hydrilla verticillata. Fronds were subjected to different
concentrations (15, 75, and 150 μM) of Cu for 7 days. Cu
grains were found in cell walls, plasmodesmata, and within
the nuclei and chloroplasts using the autometallographic
technique. Subcellular fractionation of Cu-containing tis-
sues indicated that in leaves subjected to high Cu concen-
trations, 59–65 % of the element was located in the cell wall
fraction, followed by cell organelles (21–30 %) and the
soluble fraction (10–14 %). The levels of K, P, Zn, and
Mg declined under all Cu concentrations, but Ca, Mn, and
Fe contents reached their peak at 15 μM Cu and decreased
thereafter. Fv/Fm, F0, and Fm fell significantly in line with
the decrease in pigment content. Cu exposure also caused
significant damage to the chloroplasts, mitochondria, and
nuclei, including disintegration of the chloroplasts and
vacuolization of the mitochondria and nuclei, all of which
suggested that Cu hastened plant senescence. The Cu
maximum permissible concentration for H. verticillata was
10 μM, which was less than the existing general water
quality standard. This suggested that H. verticillata could
be used to assess Cu phytotoxicity.
Keywords Copper . Hydrilla verticillata . Subcellular
distribution . Localization . Ultrastructure . Physiology
Responsible editor: Elena Maestri
Q. Xu (*) : H. Qiu : W. Chu : Y. Fu : S. Cai : H. Min : S. Sha
Institute of Plant Cell and Molecular Biology, College of Life
Sciences, Nanjing Normal University, Nanjing 210023, People’s
Republic of China
e-mail: xuqinsong@163.com
Introduction
Heavy metals are one of the most persistent pollutants in
aquatic ecosystems because they are not biodegradable and
travel through the food chain via bioaccumulation. Copper
(Cu) is known as a nonspecific toxicant and is frequently
used as an algaecide, fungicide, bactericide, plant herbicide,
and mollusicide in the aquatic environment (de Oliveira-
Filho et al. 2004).
Cu, at low concentrations, is a structural and catalytic
component of many proteins and enzymes involved in a
variety of metabolic pathways in plants (Maksymiec 1997;
Mal et al. 2002; Andrade et al. 2004). However, at supra-
optimal concentrations, Cu exhibits strong toxicity, inhibits
plant growth, and induces plant senescence or even death
(Srivastava et al. 2006; Lequeux et al. 2010). The phytotox-
icity of Cu is generally ascribed to its possible reaction with
glutathione and the thiol group of proteins, as well as its
potential to induce the production of reactive oxygen spe-
cies via the metal-catalyzed Haber–Weiss reaction, causing
subsequent lipid peroxidation, protein degradation, and
DNA damage (Maksymiec and Krupa 2006; Monferrán et
al. 2009; Kanoun-Boulé et al. 2009; Yildiz et al. 2009).
Hydrilla verticillata (L.f.) Royle, a rooted, submerged
aquatic plant species, is widely distributed in freshwater
ponds, lakes, ditches, and streams throughout the world.
H. verticillata has been suggested to be used in the
phytoremediation of metal-contaminated aquatic bodies
due to its strong adaptability, fast growth, high biomass,
and high metal (such as Hg, Cu, Cd, As, and Zn) accumu-
lation (Gupta and Chandra 1996; Xu et al. 2004; Srivastava
et al. 2006, 2009; Xue et al. 2010; Xue and Yan 2011).
Previous research has shown that the accumulation of heavy
metals in H. verticillata is often accompanied by a number
Environ Sci Pollut Res (2013) 20:8672–8679
of physiological and cellular changes (Gupta and Chandra
1996; Xu et al. 2004, 2006; Srivastava et al. 2006), but the
mechanisms behind its toxicity still remain unclear. The
aims of this study were (1) to investigate the ultrastructural
localization of Cu by autometallography; (2) to evaluate the
pattern of Cu subcellular distribution using tissue fraction-
ation; and (3) to investigate the effects of Cu on photosyn-
thetic activity, mineral nutrient content, and the ultrastruc-
ture of H. verticillata. The objectives of this study were to
elucidate possible phytotoxic effects of Cu on H. verticillata
and to evaluate the use of this submerged macrophyte as a
bioindicator for the assessment of Cu pollution in aquatic
ecosystems.
Materials and methods
Plant material and treatment conditions
H. verticillata (L.f.) Royle plants were collected from Lake
Tai, China, and were grown for 6 months in a culture pool
for hydrophytes, one fourth filled with soil. Before Cu
exposure, plant shoots (top 10–15 cm of the plant) were
acclimatized in glass aquarium for at least 1 week under
laboratory conditions (115-μmolm−2 s−1 light with a 14-h
photoperiod at 25/18 °C day/night temperature) in 10 %
Hoagland’s solution. The plants were grown in 10 %
Hoagland’s solution with the addition of copper sulfate (0,
15, 75, and 150 μM) under the above mentioned laboratory
conditions for 7 days. The Cu concentrations were based on
earlier studies (Xu et al. 2006). All the solutions were
renewed every 2 days. After 7 days, the plants were washed
with deionized water, blotted, and used for further analysis.
The experiments were carried out in triplicate and each
replicate contained 20 plants of equal size.
Tissue fractionation
The subcellular distribution of Cu within the leaves was
determined according to the procedure used by Xiong et
al. (2009). Leaves were homogenized in chilled extraction
buffer containing 50 mM HEPES (pH 7.5), 500 mM su-
crose, 1 mM dithiothreitol, 5 mM ascorbate, and 1 %
polyvinylpolypyrrolidone. The homogenate was centrifuged
at 500×g for 5 min in order to isolate the cell wall fraction
(F1). The supernatant was then centrifuged at 20,000×g for
45 min to sediment out the cell organelles (F2) from the
supernatant solution, referred to as the soluble fraction (F3).
All steps were performed at 4 °C. After digestion (cell wall
and organelle-containing fractions) with concentrated
HNO3–HClO4 (4:1, v/v), the Cu concentrations in the dif-
ferent fractions were quantified by a GBC 932 Plus Atomic
Absorption Spectrophotometer (AVANTA, Australia) using
8673
an air–acetylene flame. The detection limit for Cu was
0.0011 ppm.
Mineral analysis
The leaves were washed thoroughly with 10 mM EDTA at
4 °C for 30 min and then with double distilled water in order
to remove the metals adsorbed to the surface. They were
digested with HNO3–HClO4 (10:1, v/v) at 160 °C until the
digest solution became clear. The digested residue was
dissolved in 0.7 ml HCl and diluted with pure water to
10 ml. The concentrations of P, K, Ca, Mn, Zn, Fe, and
Mg in the extracts were determined using inductively
coupled plasma atomic emission spectroscopy (Leeman
Labs, Prodigy, USA).
Photosynthetic pigment, EC50, and MPC estimation
Leaf samples (∼0.4g fresh sample) were taken and the chloro-
phyll and carotenoid contents were extracted in 80 % chilled
acetone. After centrifugation at 10,000×g for 10 min at 4 °C,
chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids
(Car) were calculated according to the equations 12.25×A663 −
2.79×A647, 21.50×A647 −5.10×A663, and (1,000×A470 −1.82×
Chl a−85.02×Chl b)/198 based on Lichtenthaler’s coefficients
(Lichtenthaler 1987), respectively. According to Mohan and
Hosetti (2006), the EC50 is the concentration of a toxicant
which produces 50 % reduction of the chlorophyll content in
experimental plants as compared to the controls. The maxi-
mum permissible concentration (MPC) was calculated by con-
sidering 10 % of EC50 as a conventional safety factor.
Determination of chlorophyll a fluorescence parameters
The fluorescence parameters were determined using a Handy-
PEA instrument (Hansatech Instruments, UK; Rau et al.
2007). Fully expanded, intact leaves from the control (n=6)
and exposed plants (n=6) were used to monitor chlorophyll a
fluorescence. H. verticillata leaves were dark-adapted for at
least 20 min at room temperature so that all the reaction
centers were open. The following parameters were measured:
F0 (initial/minimal fluorescence), Fm (maximum fluores-
cence), and Fv/Fm (maximum quantum yield of PSII).
Ultrastructural localization of Cu
The ultrastructural localization of Cu in leaf cells was evalu-
ated according to Pihl (1967) and Xu et al. (2004). The
samples were fixed in 2.5 % glutaraldehyde in 0.1 M phos-
phate buffer (pH 7.4) and saturated with H2S for 1 h. After the
samples had been washed in two changes of 0.1 M Tris–
maleate buffer (pH 7.4, containing 7.5 % sucrose), they were
dehydrated through a graded ethanol series and embedded in
8674
Epon 812 resin. Ultrathin sections of 70-nm thickness were
cut using an Ultracut E ultra-microtome (Leica, Germany),
with a diamond knife, and were incubated in the dark in 10 ml
distilled water solution containing 30 % dextran, 0.015 M
quinol, 0.022 M citric acid, 0.293 M sucrose, and 0.09 ml
10 % AgNO3. After the physical developer had been drained,
the sections were washed with distilled water, stained in 2 %
uranyl acetate, and examined using a Hitachi H-600 transmis-
sion electron microscope (TEM).
Ultrastructural observation
Samples taken from the leaves of the control and Cu-treated
plants were initially fixed for 2 h at 4 °C in 2.5 % (v/v)
glutaraldehyde in 0.1 M phosphate buffer solution (pH 7.3)
and then post-fixed in 1 % (w/v) aqueous osmium tetroxide
for 2 h. The samples were dehydrated through an ethanol
gradient and finally embedded in Epon 812 resin. Ultrathin
sections of 70-nm thickness were cut using an Ultracut E
ultramicrotome (Leica), with a diamond knife, and then
stained in 2 % (w/v) uranyl acetate and lead citrate before
they were examined using a Hitachi H-600 transmission
electron microscope.
Data analysis
The results are expressed as the mean values±SD of at least
three individual experiments. All the data were subjected to
an analysis of variance (ANOVA) using the method given
by Schefler (1969) in order to determine the level of signif-
icance within a concentration. The correlation coefficients
were expressed using r values.
Results
Subcellular distribution of Cu
Compared to the control, Cu contents in the different sub-
cellular fractions significantly rose in a dose-dependent
manner as the Cu concentration was increased in the nutrient
solution (rF1 =0.91, p<0.05; rF2 =0.91, p<0.05; rF3 =0.83,
p>0.05; Table 1). Most Cu (711.9–1353.1 mg kg−1 fresh
weight, FW) was stored in the cell walls of leaves from
Cu-treated plantlets. A smaller quantity of Cu (229.8–
548.6 mg kg−1 FW) accumulated in the organelles, and even
less Cu (155.6–210.9 mg kg−1 FW) accumulated in the
soluble fraction.
Concentrations of selected mineral nutrients
The concentrations of nutrients in plants exposed to increased
Cu changed markedly compared to the control (Table 2). A
Environ Sci Pollut Res (2013) 20:8672–8679
significant reduction in K, Zn, P, and Mg concentrations in
Cu-treated leaves was observed in a dose-dependent manner
(rk =−0.64, p>0.05; rZn =−0.96, p<0.01; rP =−0.88, p<0.05;
rMg =−0.90, p<0.05), whereas the levels of Ca, Mn, and Fe
peaked at 15 μM Cu; increases over the corresponding values
in the control plants were 43, 151, and 112 %, respectively. As
the concentration of Cu continued to rise, their concentrations
began to decline, but Fe content remained higher than the
control, even at the highest Cu concentration.
Photosynthetic pigments
After 7 days of growth, a significant loss in Chl a (r=−0.99,
p<0.01) and Car (r=−0.90, p<0.05) contents were ob-
served as the levels of Cu in the nutrition medium increased
(Fig. 1). The maximum decreases of Chl a and Car com-
pared to the control plants were 77 and 98 %, respectively, at
150 μM Cu.
In contrast, Chl b was less sensitive than Chl a and Car
with regard to Cu (Fig. 1). Its content increased by 12 and
8 % at 15 and 75 μM Cu, respectively, compared to the
control plants and declined by 36 % at 150 μM Cu.
Change in chlorophyll a fluorescence parameters
Chlorophyll fluorescence parameters were significantly af-
fected by exposure to Cu (Table 3). The values for Fv/Fm
(r=−0.90, p<0.05), F0, and Fm decreased progressively as
the Cu concentration increased. This meant that severe
damage to the PSII reaction center of H. verticillata leaf
occurred as the Cu concentration reached toxic levels.
Localization of Cu in leaves as shown
by autometallographic staining
Under the control conditions, the cytoplasm and cell organ-
elles were normally free of any deposits (Fig. 2a). In con-
trast, dark grains were clearly visible, in particular compart-
ments in the cells of Cu-treated leaves: cell walls contained
grains of Cu and, occasionally, groups of plasmodesmata
containing densely packed particles were found (Fig. 2b). A
large number of electron-dense grains were also found in the
nuclei and chloroplasts (Fig. 2c–e).
Ultrastructure
In the leaf cells of the control H. verticillata plants, the
chloroplasts appeared to be oblong with an orderly arrange-
ment of grana and stroma thylakoids. The nuclei were intact
and there was a distinct boundary between the nucleolus and
the caryoplasm. Furthermore, the chromatin was well distrib-
uted within the caryoplasm. The mitochondria appeared to be
round, with distinct tubular cristae (Fig. 3a). In contrast, the
Environ Sci Pollut Res (2013) 20:8672–8679 8675

Table 1 Cu distribution in the cell wall, cell organelles, and soluble fraction in leaf cells of H. verticillata grown in the presence of 15, 75, and
150 μM Cu for 7 days

Cu concentration (μM) Cu in subcellular fractions (mg kg−1 FW)

Cell wall (% of total) Cell organelles (% of total) Soluble fraction (% of total) Total Cu content

0 89.1a±0.2 (38) 84.2a±1.5 (36) 59.9a±0.2 (26) 233.2a±2.0 (100)

15 711.9b±1.0 (65) 229.8b±1.9 (21) 155.6b±0.3 (14) 1,097.3b±2.9 (100)
75 1,003.6c±2.5 (59) 510.3c±1.2 (30) 178.7c±1.9 (11) 1,692.6c±5.6 (100)
150 1,353.1d±10.9 (64) 548.6d±1.7 (26) 2,10.9d±1.3 (10) 2,112.5d±13.9 (100)

Values are the mean±SD (n=3). Different letters in the same column indicate significant difference at the 5 % level after one-way ANOVA

ultrastructures of the chloroplasts, mitochondria, and nuclei
were markedly different in the Cu-treated plants, although the
effects were limited at 15 μM Cu (data not shown). In the leaf
cells of fronds treated with 75 μM of Cu, the chloroplasts
seemed to be oval, the thyladoids were disorderly arranged,
and breakages had occurred in the outer membrane (Fig. 3b).
In the nuclei, chromatin had agglomerated into lumpish matter
with a fairly high electron density (Fig. 3b, c). Some of the
nuclear envelopes were broken, but the nucleoli were still
intact (Fig. 3c). In some of the mitochondria, the number of
cristae declined and became vacuolar (Fig. 3d). When plants
were treated with 150 μM Cu, the chloroplasts disintegrated
(Fig. 3e), the nuclear envelope disappeared, and vacuolar
nuclei were seen (Fig. 3f).
Discussion
H. verticillata can take up mineral elements directly from
water through their leaves since it is completely submerged
and has a very thin surface cuticle (Jackson 1998). As an
essential trace element, Cu is readily taken up by plants
using many transporters (Dučić and Polle 2005).The results
of this study confirmed that H. verticillata leaves, over
7 days, effectively accumulated Cu in a dose-dependent
manner at culture medium concentrations up to 150 μM.
The bioaccumulation of Cu in H. verticillata leaves also
induced considerable disturbance in plant physiology and
metabolism that could be observed through changes in sev-
eral biomarkers.
This study found that, following the increase in external
Cu, more than half of the accumulated Cu was bound to the
cell wall of the leaves (Table 1), which suggested that the
cell wall played an important role in Cu accumulation, an
observation consistent with those of earlier studies by
MacFarlane and Burchett (2000) and Xue et al. (2010) and
also matched the in situ tissue distribution of Cu shown by
autometallography (Fig. 2b–e). Although only a small
amount of Cu was stored in the cell organelles and the
soluble fraction (Table 1), this study showed that a number
of metabolic processes (e.g., photosynthesis) were disturbed
(Fig. 1), which confirmed the results of earlier investigations
(Xu et al. 2006) wherein it was reported that Cu treatment
resulted in a significant loss in the synthesis of protein.
Although Cu can interfere with a number of physiological
processes, the primary target of its toxicity is probably the cell
membrane (Maksymiec and Krupa 2006). This investigation
showed that the presence of Cu in the growth medium mark-
edly modified the concentrations of K, P, Ca, Mn, Zn, Fe, and
Mg in the leaves of H. verticillata (Table 2). Decreases in P, K,
Zn, and Mg concentrations have also been observed in Cucumis
sativus (Rouphael et al. 2008), Arabidopsis thaliana (Lequeux
et al. 2010), and Glaucium flavum (Cambrollé et al. 2011),
which probably occurred due to a nonspecific effect of Cu on
ion absorption (Rouphael et al. 2008) caused by alterations to
membrane permeability (Srivastava et al. 2006) or metal-

Table 2 Total phosphorus,

potassium, zinc, magnesium, Element content Cu concentration (μM)
calcium, manganese, and iron (mg kg−1 FW)
concentrations in leaves of H. 0 15 75 150
verticillata in response to treat-
ment with a range of Cu con- P 990.1a±28.2 632.4b±16.4 469.8c±9.6 320.2d±6.8
centrations (15, 75, and 150 μM) K 3,580.4a±76.1 431.7b±18.4 263.9c±8.8 181.8d±6.9
for 7 days (in milligrams per Zn 81.7a±1.4 69.1b±1.8 62.2b±1.3 46.4c±1.2
kilogram FW)
Mg 379.3a±5.8 273.8b±7.6 164.1c±3.8 132.7d±2.4
Data are the mean±SD (n=3). Ca 2,103.8c±47.2 2,999.7a±54.9 2,600.3b±87.9 1,784.9d±67.1
Within a row, values followed by Mn 133.2b±2.4 334.7a±9.6 115.3c±3.6 91.5d±1.8
different letters are significant at Fe 191.4d±3.2 405.9a±9.8 376.6b±10.1 325.3c±5.6
p<0.05 after one-way ANOVA
8676 Environ Sci Pollut Res (2013) 20:8672–8679

Fig. 1 Cu influence on the

chlorophyll, Chl a and Chl b,
and Car contents in leaves of H.
verticillata after 7 days of
exposure to 15, 75, and 150 μM
Cu. Data are the mean±SD
(n=3). Values designated over
the bars in different letters are
significantly different at p<0.05
after one-way ANOVA

induced disorder in cellular metabolism. The presence of Fe–
EDTA in the nutrient solution could explain the high accumu-
lation of Fe (Table 2) in H. verticillata exposed to Cu because
Cu ions can displace Fe from Fe–EDTA complexes, making Fe
ions more available for plant uptake (Lequeux et al. 2010).
Chlorophyll degradation is a common response in aquatic
plants to Cu stress (Prasad et al. 2001; Srivastava et al. 2006;
Monferrán et al. 2009; Kanoun-Boulé et al. 2009). This study
found that there was a significant reduction in the levels of Chl
a and Car in H. verticillata after exposure to Cu (Fig. 1). The
drop in chlorophyll is thought to be due to (a) copper-induced
modification of chlorophyll degradation, together with struc-
tural and functional damage (Prasad et al. 2001; Monferrán et
al. 2009); (b) impairment in the supply of Mg, which is
required for the synthesis of chlorophylls (Table 2); (c) Zn
deficiency (Table 2) resulting in the inhibition of enzymes,
such as carbonic anhydrase (Table 2); or (d) changes in
chloroplast ultrastructure (Fig. 3b, e, f) induced by elevated
Cu. For fronds treated with Cu, as shown in Fig. 1, the Chl b
content rose slightly at lower Cu concentrations (up to 75 μM)
and only decreased in the highest concentration. Similarly, the
relative concentrations of pigments in Lemna trisulca rose
significantly in Cu concentration up to 10 μM, followed by
a decrease (Prasad et al. 2001). The results also revealed that
Chl a and Car were more sensitive than Chl b to Cu damage,
and it has been suggested that the slower degradation of Chl b
was due to the involvement of photo-oxidative mechanisms in
copper-induced damage (Prasad et al. 2001; Srivastava et al.
2006; Kanoun-Boulé et al. 2009). The dose-dependent rela-
tionship between chlorophyll and heavy metals has been used
to determine the 50 % effective concentration (EC50) and the
MPC of a given toxicant (Mohan and Hosetti 2006). In
this investigation, the EC50 and MPC of Cu, with regard to
H. verticillata, were 100 μM (7.5 mg l−1) and 10 μM
(0.75 mg l−1), respectively, over 7 days of exposure. Since
the MPC in this study was less than the existing general water
quality standard of the World Health Organization (2 mg l−1),
the Cu concentrations of wastewaters should be limited to a
value of 10–15 μM if the normal growth of aquatic plants,
such as H. verticillata, is to be maintained.
It is widely recognized that Cu toxicity has a considerable
effect on photosynthetic processes (Prasad et al. 2001;
Tanyolaç et al. 2007; Cambrollé et al. 2011). Chlorophyll
fluorescence induction measurements have shown that

Table 3 Effects of Cu on leaf maximum quantum yield (Fv/Fm), initial/minimal fluorescence (F0), and maximum fluorescence (Fm) following
exposure to different Cu concentrations (15, 75, and 150 μM) for 7 days

Parameters Cu concentration (μM)

0 15 75 150

F0 387.2a±16.4 150.2b±6.4 26.8c±1.1 23.2.0c±1.1

Fm 1,198.8a±36.2 399.8b±16.5 32.7c±2.9 24.5d±1.7
Fv/Fm 0.7a±0.04 0.6a±0.07 0.2b±0.03 0.1b±0.03

Data are the mean±SD (n=6). Within a row, values followed by different letters are significant at p<0.05 after one-way ANOVA
Environ Sci Pollut Res (2013) 20:8672–8679 8677

Fig. 2 Transmission electron

microscopy micrographs of H.
verticillata leaf sections stained
with sulfide–silver for
localization of Cu. Cu was seen
as black deposits in this
method. a Few grains were in
control leaf cells (bar, 1 μm).
b, c Leaf cells treated with
15 μM Cu showing Cu deposits
in the cell wall and
plasmodesmata (arrow; bar,
1 μm) (b) and the chloroplast
and nucleus (bar, 1 μm) (c).
d Cu particles were in the
nucleus and chloroplast in leaf
cells treated with 75 μM (bar,
2 μm; arrow showing the
breakage occurred in the
nuclear envelope). e Leaf cells
treated with 150 μM Cu
showing Cu grains in the
nucleus (bar, 2 μm). CW cell
wall, CP chloroplast, N nucleus

alterations occur in the efficiency of the photosynthetic
apparatus (Tanyolaç et al. 2007). For example, variations
in the Fv/Fm ratio are seen as reflecting changes in the
photochemical efficiency of PSII. In this study, the signifi-
cant decrease in Fv/Fm (Table 3) in Cu-treated fronds indi-
cated an impairment of the primary photochemical efficien-
cy of the photosynthetic apparatus. Moreover, the finding
that most of the Cu grains distributed in the thylakoid
membranes (Fig. 2c, d) was also correlated with the de-
creased capacity of PSII as well (Table 3). In addition, the
change in the ultrastructure of the thylakoid membrane
would affect the electron transport rate (Monnet et al.
2001). The loss of chlorophyll pigments (Fig. 1) and the
damage to chloroplasts (Fig. 3b, d, e) induced by Cu also
supported the overall disturbances in photosynthesis evident
in chlorophyll a fluorescence.

Fig. 3 Effects of Cu (15, 75,

and 150 μM) after 7 days of
exposure on the ultrastructure
of leaf cells in H. verticillata.
a The chloroplasts,
mitochondria, and nuclei in
control leaf cells (bar, 1 μm).
b Global chloroplast and
breakage occurred on the outer
membrane in 75 μM Cu-treated
leaf cells (bar, 2.0 μm).
c Nuclei in 75 μM Cu-treated
leaf cells (bar, 2.0 μm).
d Vacuolar mitochondria in
75 μM
Cu-treated leaf cells (bar,
1.0 μm; arrow points at the
breakage which occurred in the
nuclear envelope).
e Disintegrated chloroplasts in
150 μM Cu-treated leaf cells
(bar, 1.0 μm). f Leaf cells
treated with 150 μM Cu
showing the nucleus (bar,
2.0 μm). CW cell wall, CP
chloroplast, Mi mitochondrion,
N nucleus
8678
In order to show the ultrastructural distribution of Cu in the
leaves with greater accuracy, a histochemical method, called
autometallography, was used to analyze the in situ tissue distri-
bution of Cu. In environmental toxicology, due to its high level
of sensitivity, this approach has been used to elucidate the sites
of metal accumulation and transport in plant tissues (Heumann
2002; Xu et al. 2004). In this study, after fixation in glutaralde-
hyde saturated with H2S and physical development in a modi-
fied sulfide–silver developer, it was found that abundant
electron-dense grains were distributed throughout the cell wall
and in the plasmodesmata (Fig. 2b), intracellularly in the nuclei
and chloroplasts (Fig. 2c–e), and, to a limited extent, in the
cytoplasm (Fig. 2c), but they did not appear in the control
treatment (Fig. 2a). The current results were in considerable
agreement with the work of MacFarlane and Burchett (2000),
who reported that SEM X-ray microanalysis showed that the
greatest accumulation of Cu occurred in the leaf cell walls of
Avicennia marina. The electron energy loss spectroscopy spec-
tra of cell wall-bound Cu from the root cells of Armeria
maritima also showed that Cu was strongly bound, which
agreed with the model spectra of protein/copper complexes
(Neumann et al. 1995). It is well known that cell walls are
mainly composed of polyose (including cellulose, hemicellu-
lose, and pectin) and protein, which provide negative charge
sites on their surfaces. This means that they can strongly bind Cu
ions. Cu has been shown to accumulate in plastids and nuclei by
binding to proteins (Neumann et al. 1995). The patterns for Cu
ultrastructural localization showed a similar Cu tissue distribu-
tion to the traditional tissue fractionation method.
In this experiment, TEM observation revealed that expo-
sure to Cu dramatically affected ultrastructural integrity and
ultrastructural damage became more serious as the Cu con-
centration increased, e.g., increased chloroplast swelling
(Fig. 3b, d), chloroplast disintegration (Fig. 3e), chromatin
condensation in the nucleus, disruption of the nuclear mem-
brane (Fig. 3b, c), and vacuolization of the mitochondria
(Fig. 3d) and nuclei (Fig. 3f). All these pronounced changes
could be attributed to the serious oxidative stress caused by Cu
(Srivastava et al. 2006; Xu et al. 2006). The changes observed
in the organelles were in line with the ultrastructural localiza-
tion characteristics of Cu and might indicate that the destruc-
tion was a direct consequence of Cu accumulation. The direct
action of high concentrations of Cu on these organelles con-
firmed its toxicity to plants (Prasad et al. 2001). Undoubtedly,
the damage to the ultrastructure of the nuclei, chloroplasts, and
mitochondria in Cu-treated leaf cells (Fig. 3b–f) would inev-
itably induce important metabolic changes.
Conclusions
The present study investigated the ultrastructural localiza-
tion and subcellular distribution of Cu and the physiological
Environ Sci Pollut Res (2013) 20:8672–8679
and ultrastructural responses of H. verticillata to varying
doses of Cu (up to 150 μM). The experimental results
indicated that (1) the patterns of Cu ultrastructural localiza-
tion were confirmed by the traditional tissue fractionation
method; (2) the observed ultrastructural damage to the chlo-
roplasts, mitochondria, and nuclei suggested that these three
organelles were acutely prone to harm by high levels of Cu
and were indicative of a general disruption in cellular func-
tion; (3) Cu stress disturbed the nutrient balance and photo-
synthetic capacity and ultrastructural damage was related to
changes in these two physiological parameters; and (4) H.
verticillata showed potential as a phyto-assay for Cu toxic-
ity in ecotoxicological studies.
Acknowledgments This work was supported by the National
Natural Science Foundation of China (30800055) and a project funded
by the Priority Academic Program Development of Jiangsu Higher
Education Institutions (PAPD).We are very grateful to Mrs. Tong-Shun
Jin, Mr. Yao-Ming Zhou, Mr. Kai-he Du, and Mr. Da-Wei Shi for
technical assistance.
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