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ORIGINAL PAPER
Cadmium-induced responses in duckweed Lemna minor L.
Mirta Tkalec Æ Tatjana Prebeg Æ Vibor Roje ÆBranka Pevalek-Kozlina Æ Nikola Ljubesic
Received: 30 November 2007 / Revised: 29 April 2008 / Accepted: 11 June 2008 / Published online: 1 July 2008
� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2008
Abstract Although duckweed Lemna minor L. is a known
accumulator of cadmium, detailed studies on its physio-
logical and/or defense responses to this metal are still
lacking. In this study, the effects of 10 lM CdCl2 on Lemna
minor were monitored after 6 and 12 days of treatment,
while growth was estimated every 2 days. Cadmium treat-
ment resulted in progressive accumulation of the metal in
the plants and led to a decrease in the growth rate to 54% of
the control value. The metal also considerably impaired
chloroplast ultrastructure and caused a significant reduction
in pigment content, i.e., at day 12, by 30 and 34% for
chlorophylls a and b, and by 25% for carotenoids. During
cadmium treatment, the contents of malondialdehyde and
endogenous H2O2 progressively increased (rising 77 and
46% above the controls by day 12), indicating that cadmium
induced considerable oxidative stress. On the other hand,
higher activities of pyrogallol peroxidase (PPX), ascorbate
peroxidase (APX) and catalase (CAT), as well as the
induction of a new APX isoform, in cadmium-treated
plants, clearly showed activation of an antioxidative
response. At day 6, only PPX activity was significantly
above the controls (15%), while, at day 12, PPX, APX and
CAT activities were increased (74, 78 and 63%). Cadmium
also led to accumulation of the heat shock protein 70
(HSP70) and induced an additional isoform of this protein.
The obtained results suggest that cadmium (10 lM) is
phytotoxic to Lemna minor, inducing oxidative stress, and
that antioxidative enzymes and HSP70 play important roles
in the defense against cadmium toxicity.
Keywords Lemna minor � Cadmium � Chloroplast �Oxidative stress � HSP70
Abbreviations
APX Ascorbate peroxidase
CAT Catalase
HSP70 Heat shock protein 70
MDA Malondialdehyde
PAGE Polyacrylamide gel electrophoresis
PPX Pyrogallol peroxidase
PVP Polyvinylpyrrolidone
ROS Reactive oxygen species
SDS Sodium dodecylsulfate
Introduction
Cadmium salts are particularly dangerous environmental
pollutants, due to their relatively high mobility in soils, large
water solubility and extreme toxicity, even at low doses (Das
et al. 1997). Anthropogenic activities such as metal industry,
mining, use of batteries, waste and sludge disposal and
application of pesticides and fertilizers result in increased
cadmium levels in terrestrial and aquatic ecosystems (Sanita
M. Tkalec and T. Prebeg contributed equally to this work
Communicated by G. Klobus.
M. Tkalec (&) � B. Pevalek-Kozlina
Department of Botany, Division of Biology, Faculty of Science,
University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, Croatia
e-mail: mtkalec@zg.biol.pmf.hr
T. Prebeg � N. Ljubesic
Division of Molecular Biology, Ruder Boskovic Institute,
Bijenicka 54, 10000 Zagreb, Croatia
V. Roje
Division for Marine and Environmental Research, Ruder
Boskovic Institute, Bijenicka 54, 10000 Zagreb, Croatia
123
Acta Physiol Plant (2008) 30:881–890
DOI 10.1007/s11738-008-0194-y
di Toppi and Gabbrielli 1999). For plant growth, cadmium is
a non-essential heavy metal which causes growth inhibition,
leaf chlorosis and even plant death (Mendelssohn et al. 2001;
Schutzendubel and Polle 2002). The metal was found to
disturb the uptake and distribution of essential elements
(Ouariti et al. 1997) and to imbalance water relations (Perfus-
Barbeoch et al. 2002). It also inhibits photosynthesis by
impairing chlorophyll synthesis (Stobart et al. 1985; Pandey
and Sharma 2002), the activity of photosystem II (Chugh and
Sawhney 1999), photosynthetic electron transport (Siedle-
cka and Baszynski 1993; Prasad et al. 2004), the dark
reactions (Di Cagno et al. 2001) and chloroplast organization
(Vecchia et al. 2005; Vitoria et al. 2006).
There is a growing body of evidence that the toxic
effects of cadmium can be attributed, at least to some
extent, to oxidative stress (Schutzendubel et al. 2001;
Rodrıguez-Serrano et al. 2006). Although cadmium is not a
redox-active metal, such as copper and iron, and cannot
catalyze Fenton-type reactions yielding reactive oxygen
species (ROS) directly (Schutzendubel and Polle 2002), it
can induce oxidative stress in an indirect manner, by dis-
turbing cellular equilibria between the generation and the
neutralization of ROS. Since ROS, as highly reactive
molecules, can damage cell structure and function, plants
possess effective scavenging mechanisms (Arora et al.
2002). Apart from low molecular weight metabolites like
ascorbic acid and reduced glutathione, important compo-
nents of the ROS-scavenging system are antioxidative
enzymes, such as peroxidases, catalase (CAT) and super-
oxide dismutase (Mittler 2002; Gomez et al. 2004). It was
found that cadmium treatment could modify their activi-
ties, although the results were contradictory and depended
on the type of the enzyme, cadmium concentration, the
plant species and the environmental conditions (Chaoui
et al. 1997; Sandalio et al. 2001; Smeets et al. 2005).
Exposure to cadmium was also found to trigger other
defense responses in plant cells, such as the induction of
heat shock proteins (HSPs), including HSP70 (Neumann
et al. 1994; Sanita di Toppi and Gabbrielli 1999; Ireland
et al. 2004). The HSP70 family is known to have essential
roles in preventing aggregation and assisting refolding of
non-native proteins under stress conditions and plays a
regulatory role in stress-associated gene expression as well
(Wang et al. 2004).
Lemna minor (Lemnaceae, duckweeds), is a monocot-
yledonous aquatic macrophyte consisting of floating plant
bodies (fronds) and submerged greenish roots. It mostly
reproduces vegetatively, forming daughter fronds in two
meristematic regions at the proximal end of the mother
fronds, thus creating colonies (Landolt 1986). Lemna minor
has already been shown to respond in a sensitive, repro-
ducible manner to a large number of environmental stresses
(Lewis 1995) and it also is a well-known accumulator of
heavy metals, including cadmium (Smith and Kwan 1989;
Mohan and Hosetti 1997). However, little is known about
the physiological mechanisms of cadmium toxicity in this
plant, especially when cadmium is applied in moderately
high but environmentally more relevant concentration. The
objectives of the present investigation were to study the
effects of 10 lM cadmium chloride, in Lemna minor
grown in axenic culture, on (1) growth and cadmium
accumulation, (2) chloroplast ultrastructure and content of
photosynthetic pigments, (3) lipid peroxidation and H2O2
content, (4) activities and isoenzyme patterns of antioxi-
dative enzymes and (5) induction of HSP70.
Materials and methods
Plant material and culture conditions
Stock cultures of Lemna minor L. (duckweed) were main-
tained under axenic conditions on modified Pirson–Seidel’s
nutrient solution, pH 4.55 (Pirson and Seidel 1950). Plants
were subcultured in 2-week intervals. Experimental cultures
were started by inoculation of a single colony with 2–3
fronds (for growth measurement), or 10–12 colonies (for
other analyses), into nutrient solution supplemented with
10 lM CdCl2. All cultures, stock and experimental, were
grown at 24 ± 2�C, under ‘white’ fluorescent light (Osram,
Germany) with a photon flux density of 40–
50 lmol m-2 s-1 and a day–night cycle of 16:8 h.
Plant growth
Growth was monitored for 12 days by counting the fronds
every 2 days, and expressed as relative frond numbers
according to Ensley et al. (1994).
Cadmium content
The plants were washed thoroughly with deionized water
and oven-dried to constant weight, at 80�C. The dry sam-
ples (70 mg) were digested with HNO3 using a microwave
sample preparation system (Anton Paar Multiwave 3000).
Cadmium content was determined by high resolution mass
spectrometry with inductively coupled plasma (HR-ICP-
MS), on a Thermo Finnigan Element 2 instrument. Exter-
nal calibration was performed using mixed standard
solutions (0, 1, 10 lg l-1).
Pigment analyses
Pigments were extracted after 6 and 12 days of cadmium
treatment with 80% (v/v) cold acetone and quantified
spectrophotometrically according to Lichtenthaler (1987).
882 Acta Physiol Plant (2008) 30:881–890
123
Electron microscopy
For ultrastructural analyses, small pieces of Lemna fronds
were fixed for 20 min in 1% glutaraldehyde in 0.05 M
cacodylate buffer (pH 7.2), and postfixed for 1 h in 1%
OsO4. After dehydration in a graded series of ethanol, the
tissue was embedded in Spurr’s resin. Ultrathin sections
were stained with uranyl acetate and lead citrate and
examined using a Zeiss EM10A electron microscope
operated at an accelerating voltage of 60 kV.
Lipid peroxidation and H2O2 content
The level of lipid peroxidation was measured according to
Heath and Packer (1968), after 6 and 12 days of cultivation,
as the amount of malondialdehyde (MDA), a product of
lipid peroxidation. The MDA content was calculated using
an extinction coefficient of 155 mM-1 cm-1 and expressed
on a fresh weight basis.
H2O2 was extracted by homogenizing 50 mg of plants
in 1 ml of ice-cold acetone (Mukherjee and Choudhari
1983). After centrifugation at 10,000g for 10 min, the
supernatant was mixed with titanium reagent (Fluka) and
ammonium solution to precipitate the titanium hydro-per-
oxide complex. The mixture was centrifuged at 10,000g,
for 10 min; the precipitate was dissolved in 2 M H2SO4
and the solution was clarified by centrifugation. The
absorbance of the supernatant was measured at 415 nm.
The amount of hydrogen peroxide was calculated using a
standard curve prepared with known concentrations of
H2O2.
Enzyme assays and isoenzyme analysis
Plant extracts were prepared after 6 and 12 days of culti-
vation and used for the determination of pyrogallol
peroxidase (PPX), ascorbate peroxidase (APX) and CAT
activities and isoenzyme patterns. Plants (50 mg) were
homogenized in 1 ml of cold 50 mM potassium phosphate
buffer (pH 7.0), containing 1 mM ethylenediaminetetra-
acetic acid, 5 mM ascorbate and 5% (w/v)
polyvinylpyrrolidone (PVP). The homogenate was centri-
fuged at 30,000g for 30 min at 4�C and the supernatant was
used for the following enzyme assays. PPX activity (EC
1.11.1.7) was determined by monitoring the increase in
absorbance at 430 nm due to the oxidation of pyrogallol
(e = 2.6 mM-1 cm-1), essentially as described by Nakano
and Asada (1981), and expressed as lmol of purpurogallin
(product of pyrogallol oxidation) per min and g fresh
weight. APX activity (EC 1.11.1.11) was determined by the
decrease in A290 (e = 2.8 mM-1 cm-1), as described by
Nakano and Asada (1981) and expressed as lmol of oxi-
dized ascorbate per min and g fresh weight. CAT activity
(EC 1.11.1.6) was assayed by measuring the decrease in
A240 (e = 36 mM-1 cm-1) according to Aebi (1984) and
expressed as lmol of decomposed H2O2 per min and g
fresh weight.
For isoenzyme analysis, polyacrylamide gel electro-
phoresis (PAGE) without sodium dodecylsulfate (SDS)
was performed on 8% (w/v) polyacrylamide gels (Laemmli
1970). The protein content in the enzyme extracts was
determined by a dye-binding technique (Bradford 1976)
using bovine serum albumin as a protein standard.
Approximately equal amounts of proteins, 35–45 lg per
well were loaded and electrophoresis was performed at
4�C. For APX, the gel was pre-run for 30 min before the
samples were loaded and ascorbate was added to the
electrode buffer. For PPX detection, the gels were, after
electrophoresis, stained according to Chance and Maehly
(1955). APX was stained by the procedure of Mittler and
Zilinskas (1993). For CAT detection, the gels were stained
according to Woodbury et al. (1971).
Protein analysis
Plants (50 mg) were homogenized in 1 ml of cold 0.1 M
Tris–HCl buffer (pH 8.0) (Staples and Stahmann 1964)
containing 5% (w/v) PVP and centrifuged at 30,000g for
30 min, at 4�C. Soluble protein content was determined
according to Bradford (1976). Protein extracts were mixed
with an equal volume of sample loading buffer (Laemmli
1970) and separated by 10% (w/v) SDS-PAGE. Two gels
were run simultaneously, one for protein staining with
silver nitrate (Blum et al. 1987) and the other for immu-
nochemical detection. Proteins were transferred to
nitrocellulose membranes in a Bio-Rad Mini Trans-Blot
cell. After overnight incubation with primary antibodies
(1:1000) against pea HSP70, raised in rabbits (antibody
provided by Professor H. Fulgosi), the protein blots were
incubated with alkaline phosphatase-conjugated anti-rabbit
IgG (1:2000) and probed for phosphatase activity using 5-
bromo-4-chloro-3-indolyl phosphate/nitrotetrazolium blue
chloride.
Statistics
Results were expressed as arithmetic means of 12 repli-
cates from at least three experiments ± standard errors,
except for measurements of growth and photosynthetic
pigments in which cases 10 and 6 replicates were used.
Significant differences between mean values were estab-
lished by one-way ANOVA followed by Duncan’s multiple
range test (STATISTICA 7.1). Significant (P B 0.05) dif-
ferences of the mean values presented in the Figures and
Tables are labeled with asterisks, only comparing samples
taken at the same time points.
Acta Physiol Plant (2008) 30:881–890 883
123
Results
Growth and cadmium accumulation
Throughout the experiment, all fronds in the control cul-
tures appeared green and vigorous, and no signs of
senescence were observed (Fig. 1a). In contrast, in the
cultures treated with 10 lM CdCl2, the fronds of the inoc-
ulum started to become chlorotic after 6–8 days. Chlorosis
firstly affected the edges of the fronds and, during further
cultivation, spread to their central parts. At the end of the
experiment (at day 12 after inoculation), most of these
fronds were completely yellow or sometimes even bleached
(Fig. 1b). During the experiment, the roots of these inocu-
lated fronds became brownish and very fragile, usually
dropping (Fig. 1c). The fronds which developed during
cultivation on cadmium-containing medium were green but
smaller than in the control colonies (Fig. 1b). Although
these fronds usually contained roots, they were consider-
ably shorter than those of the controls (Fig. 1c).
The growth of cadmium-exposed Lemna plants,
expressed as relative frond number, was significantly
impaired (P B 0.05). Decrease in the growth rate was
already observed at day 4 of cultivation, when the relative
frond number was 36% below that of the control. The
growth rate progressively decreased toward the end of the
experiment, dropping to 54% of the control value (Fig. 2).
Exposure of Lemna minor to cadmium led to its pro-
gressive accumulation in the plants. The high level found
after 6 days of cultivation (319.97 lg g-1 DW) almost
doubled after 12 days (Table 1).
Chloroplast ultrastructure
In the control fronds, chloroplasts showed well-developed
thylakoid systems. These plastids usually contained small
plastoglobules (mostly up to 90 nm in diameter) and 1–3
starch grains (Fig. 3a). In the fronds which were, at the
beginning of experiment, inoculated into cadmium-con-
taining medium, progressive degradation of chloroplasts
(accompanied by frond chlorosis) was observed. In the
early stages of chlorosis (when fronds appeared yellowish-
green), the thylakoid system in many plastids was reduced
(Fig. 3b, c). The thylakoids often became dilated and
wavy-shaped which, in some plastids, led to a markedly
disturbed organization of the thylakoid system (Fig. 3c).
Plastoglobules were often considerably enlarged (up to
350 nm in diameter). These plastids also commonly con-
tained starch grains, which frequently occupied large parts
of the plastid interior. Eventually, cadmium treatment
resulted in severely impaired chloroplast ultrastructure: in
the chloroplasts of bleached fronds, the thylakoid systems
were almost completely disintegrated and, in many cases,
Fig. 1 Lemna minor plants from a 12-day-old control culture (a) and
a culture treated with 10 lM CdCl2 (b, c). In b, the chlorotic fronds
(which were at the beginning of the experiment inoculated into
cadmium-containing medium; indicated by arrows) are seen along
with the green fronds (which developed during cultivation on
cadmium-containing medium). A chlorotic frond with shed root
(arrow) and newly developed green fronds with a short root are seen
in c
*
**
**
0
2
4
6
8
10
12
2 4 6 8 10 12
Time (d)
Rel
ativ
e fr
ond
num
ber
Fig. 2 Growth of Lemna minor during cultivation on medium
containing 10 lM CdCl2. Open square symbols indicate control
plants and dark filled square symbols indicate cadmium-treated
plants. Data represent arithmetic mean ± SE (n = 10). Columnsmarked by asterisks indicate a significant difference at P B 0.05
Table 1 Cadmium content in control and cadmium-treated Lemnaminor plants after 6 and 12 days of cultivation
Treatment Cadmium content (lg g-1 DW)
6 days 12 days
Control 0.61 ± 0.13 0.52 ± 0.18
10 lM CdCl2 319.97 ± 12.16 631.16 ± 29.04
Values represent the mean ± SE of six replicates
DW dry weight
884 Acta Physiol Plant (2008) 30:881–890
123
only single thylakoids remained (Fig. 3d). These plastids
commonly contained large plastoglobules (mostly up to
400 nm in diameter), while their stroma was usually
scarce. Starch grains were still present in many plastids. In
green fronds, which developed during the cultivation of
plants on cadmium-containing medium, the thylakoid
system was less developed than in the control fronds
(Fig. 3e). Plastoglobules were rather small (mostly up to
140 nm in diameter), while starch grains often filled large
portions of the plastid interior.
Photosynthetic pigments
Exposure to cadmium significantly decreased (P B 0.05)
the content of photosynthetic pigments in Lemna minor
plants: after 6 days, treated cultures contained 25% less
chlorophyll a and 33% less chlorophyll b than the controls.
At the same time, the carotenoid content was 20% lower
than in the controls. In plants exposed to cadmium for 12
days, the amounts of chlorophyll a and chlorophyll b had
decreased by 30 and 34%, respectively, while the carot-
enoid content was reduced by 25% (Table 2).
Oxidative stress
Exposure of Lemna minor to 10 lM cadmium significantly
enhanced (P B 0.05) the level of lipid peroxidation,
expressed as the content of MDA. MDA content increased
by 35% within the first 6 days of cadmium treatment,
while, at the end of the experiment, the increase was 77%,
as compared to the control plants (Fig. 4a). Enhancement
of lipid peroxidation in cadmium-treated fronds was
accompanied by increased H2O2 content. The latter
increased by 11%, after 6 days on cadmium-containing
medium, and by 46%, after 12 days of cultivation (Fig. 4b).
In plants exposed to cadmium, PPX activity also sig-
nificantly increased (P B 0.05). After 6 days of exposure,
the activity was 15% above the control value while, after
12 days, it was 74% larger than in the controls (Fig. 5a).
Gel electrophoresis revealed four PPX isoenzymes, two
slow-migrating (PPX 1 and 2) and two fast-migrating (PPX
3 and 4), both in the controls and the cadmium-treated
Fig. 3 Chloroplast ultrastructure in Lemna minor fronds from a
control culture (a) and from cultures treated with 10 lM CdCl2 (b–e).
a Control chloroplast with well-developed thylakoid system. b, cChloroplasts from fronds in the early stages of chlorosis showing
stages of progressive thylakoid system degradation. d Chloroplast
from a bleached frond containing remnants of thylakoid membranes.
e Chloroplast from a green frond showing a less developed thylakoid
system. Plastoglobules are indicated by arrowheads. s starch grain.
Bars in a, b, e 1 lm; c, d 0.5 lm
b
Acta Physiol Plant (2008) 30:881–890 885
123
plants. After 6 days of exposure to cadmium, the activities
of the PPX isoenzymes appeared to be at similar levels,
both in the controls and in the cadmium-treated plants.
However, after 12 days of cultivation, the activities of PPX
3 and 4 were more prominent in the cadmium-treated
plants (Fig. 5b).
APX activity increased slightly after 6 days of Lemna
exposure to cadmium, but this was not significant
(P [ 0.05) in comparison with the control. At day 12,
however, the activity in the control plants decreased and the
activity in cadmium-treated plants was significantly higher
(78%) than in the controls (Fig. 5a). Gel electrophoresis
revealed one slow-migrating (APX 1) and two fast-
migrating APX isoenzymes (APX 3 and 4) in the control
plants. In the cadmium-treated plants, in addition to APX 1,
3 and 4, a specific isoform, APX 2, was noticed at day 6.
After 12 days of cultivation the activities of APX 2, 3 and 4
in cadmium-treated plants were more pronounced (Fig. 5b).
Slightly increased CAT activity (10%) was noticed after
6 days of cadmium exposure, but the effect was not sig-
nificant (P [ 0.05) in comparison with the control. After
12 days of cultivation CAT activity decreased in the con-
trol plants, but, in the cadmium-treated plants, significantly
increased (63%) over the control (Fig. 5a). Both in the
controls and in the cadmium-treated plants, three very
slow-migrating CAT isoenzymes (CAT 1, 2 and 3) were
detected at days 6 and 12, with particularly prominent
activity found for CAT 3 (Fig. 5b).
HSP70 expression
During cadmium treatment, the total protein content in
Lemna fronds did not significantly change (Fig. 6a). How-
ever, SDS-PAGE showed increased levels of a number of
proteins, including bands at about 70 kDa (Fig. 6b).
Immunodetection with a specific HSP70 antibody revealed
one HSP70 isoform, both in the controls and in treated
plants. An additional HSP70 isoform was detected in cad-
mium-treated plants, after 12 days of exposure (Fig. 6c).
Discussion
Freshwater macrophytes including Lemnaceae are known
to accumulate, and to tolerate, high amounts of cadmium
(Prasad et al. 2001). In our experiments, Lemna minor
treated with 10 lM CdCl2 accumulated 631 lg g-1 DW
of cadmium (0.06% of dry weight) after 12 days of
cultivation on cadmium-containing medium, which is
above the level classified by Reeves and Baker (2000) as
‘hyperaccumulation’ (above 0.01% of dry weight). The
Table 2 Photosynthetic pigments in control and cadmium-treated Lemna minor plants after 6 and 12 days of cultivation
Treatment Chlorophyll a (mg g-1 FW) Chlorophyll b (mg g-1 FW) Carotenoids (mg g-1 FW)
6 days 12 days 6 days 12 days 6 days 12 days
Control 0.62 ± 0.03 0.59 ± 0.02 0.28 ± 0.02 0.27 ± 0.01 0.24 ± 0.01 0.23 ± 0.01
10 lM CdCl2 0.46 ± 0.03* 0.41 ± 0.020* 0.19 ± 0.01* 0.18 ± 0.01* 0.19 ± 0.01* 0.17 ± 0.01*
Values represent the mean ± SE of six replicates
Values followed by an asterisk (*) in the same column are significantly different at P B 0.05
FW fresh weight
*
*
0
5
10
15
20
MD
A c
onte
nt (n
mol
g-1F
W)
**
0
1
2
3
4
5
H2O
2 co
nten
t (µm
ol g
-1F
W)
C6 Cd6 C12 Cd12
a
b
Fig. 4 Effect of 10 lM CdCl2 on lipid peroxidation (a) and H2O2
content (b) in Lemna minor cultures Open square symbols indicate
control plants and dark filled square symbols indicate cadmium-
treated plants. Data represent arithmetic mean ± SE (n = 12).
Columns marked by asterisks indicate a significant difference at
P B 0.05. C6, C12, control plants after 6 and 12 days of cultivation,
respectively; Cd6, Cd12, cadmium-treated plants after 6 and 12 days of
cultivation, respectively
886 Acta Physiol Plant (2008) 30:881–890
123
amount of cadmium we measured comprised both extra-
cellular (apoplastic) and intracellular cadmium, as it is
commonly measured in studies on the overall capacity of
plants to accumulate heavy metals (Prasad et al. 2001;
Mishra et al. 2006; Sun et al. 2007). The level of cad-
mium accumulated by Lemna minor was apparently toxic
to the plants, as evidenced by progressive decrease in
their growth rate, dropping to 54% at day 12 of treatment.
High cadmium toxicity in Lemna minor was also dem-
onstrated by Smith and Kwan (1989) who found a
cadmium level of 1.7 lM as the half-maximal effective
concentration (EC50) for frond number reduction, after 10
days of treatment.
Ultrastructural analyses of the chloroplasts in Lemna
fronds showed that cadmium induced progressive degra-
dation of the thylakoid system, with thylakoids becoming
dilated and wavy-shaped. Simultaneously, plastoglobules
considerably enlarged. This was likely, at least in part, the
result of the deposition of lipid components from disinte-
grated thylakoids, as is thought to occur during thylakoid
breakdown which accompanies chloroplast senescence
(Biswal and Biswal 1988). Similar cadmium-induced dis-
turbances in chloroplast ultrastructure have also been
reported in other species (Ouzounidou et al. 1997; Stoya-
nova and Tchakalova 1997; Vecchia et al. 2005; Vitoria
et al. 2006). In addition to defective chloroplast ultra-
structure, cadmium, expectedly, also affected the levels of
photosynthetic pigments. It has been well documented in
many plant species that cadmium reduces chlorophyll
content (Chugh and Sawhney 1999; Sandalio et al. 2001),
by inhibition of its biosynthesis (Stobart et al. 1985) or by
inducing its degradation (Hegedus et al. 2001). The decline
of photosynthetic pigments found in Lemna cultures, along
with the observed degradation of the thylakoid system
*
*
0
100
200
300
PP
X
( µm
ol pr
oduc
t m
in-1
g-1F
W)
*
0
5
10
15
AP
X
( µm
ol ox
idis
ed a
scor
bate
min
-1g-1
FW
)
*
0
1
2
3
CA
T
( µm
ol H
2O2
min
-1 g
-1F
W)
Native-PAGE
PPX
APX
CAT
C6 Cd6 C12 Cd12
a bFig. 5 The effect of 10 lM
CdCl2 on antioxidative enzymes
in Lemna minor cultures. aActivities of PPX, APX and
CAT in crude extracts from
control and cadmium-treated
Lemna minor. Open squaresymbols indicate control plants
and dark filled square symbolsindicate cadmium-treated
plants. Data represent arithmetic
mean ± SE (n = 12). Columnsmarked by asterisks indicate a
significant difference at
P B 0.05. b Isoenzyme pattern
of PPX, APX and CAT in
control and cadmium-treated
Lemna minor. C6, C12, control
plants after 6 and 12 days of
cultivation, respectively; Cd6,
Cd12, cadmium-treated plants
after 6 and 12 days of
cultivation, respectively
Acta Physiol Plant (2008) 30:881–890 887
123
clearly demonstrated that cadmium considerably impaired
the structural and functional integrity of the chloroplasts.
In parallel with the reduction in growth of Lemna plants,
lipid peroxidation, expressed as MDA content, progres-
sively increased during cadmium exposure. This was, at
least to some extent, a result of higher H2O2 level, which
may either directly, or via its toxic derivatives (such as �OH
and 1O2), damage cellular lipids. An increase in MDA
content was also found in other species exposed to cad-
mium, and is considered a common symptom of cadmium
phytotoxicity (Sandalio et al. 2001; Singh et al. 2006). As
MDA and H2O2 are well-known indicators of oxidative
stress (Arbona et al. 2003; Cho and Seo 2005), the sup-
pression of growth observed in cadmium-treated Lemna
plants, could be, at least in part, attributed to oxidative
damage.
It is known that cadmium accumulation in plants can
modulate the activities of antioxidative enzymes, including
peroxidases and CAT (Das et al. 1997). Peroxidases
decompose H2O2 by oxidation of co-substrates such as
phenolic compounds, whereas CAT converts H2O2 into
H2O and O2 (Sudhakar et al. 2001). Cadmium was found to
increase the activities of these enzymes in Pisum sativum,
Phaseolus vulgaris and in the aquatic plant, Bacopa mon-
nieri (Dixit et al. 2001; Smeets et al. 2005; Singh et al.
2006), whereas the opposite effect, i.e., decreased activity,
was described for mung bean and Arabidopsis (Somashe-
karaiah et al. 1992; Cho and Seo 2005). In Lemna minor,
cadmium treatment resulted in considerably higher activi-
ties of PPX, APX and CAT at day 12 of the experiment,
indicating that these enzymes play important roles in
eliminating excessive H2O2 in cadmium-treated Lemna
plants. This is further corroborated by the fact that cad-
mium even induced a new isoform of APX, an enzyme
which is part of the ascorbic acid-glutathione cycle and one
of the most important elements in eliminating the toxic
H2O2 (Foyer et al. 1994).
After 6 days of Lemna exposure to cadmium, the
activities of PPX, APX and CAT were only slightly above
the control. This likely facilitated the accumulation of
H2O2 and, consequently, increased lipid peroxidation. It is
conceivable that, H2O2, which thus accumulated, might act
as a signal-transducing molecule (Foyer and Noctor 2005),
subsequently leading to considerably higher activities of
PPX, APX and CAT observed at day 12.
In the course of the experiment, the activities of anti-
oxidative enzymes and the levels of MDA and H2O2
changed not only in cadmium-treated plants, but also in the
control itself. In the latter case, the activities of PPX, APX
and CAT and the level of H2O2 were significantly lower at
day 12 than at day 6 of the experiment, while, at the same
time, the level of MDA increased. Since, during the
experiment, the control plants grew vigorously, their
chloroplasts showed normal ultrastructure, and the content
of photosynthetic pigments and total proteins remained at a
similar level, the above changes were likely the result of
normal fluctuations in the oxidative status of Lemna plants
during their growth and development in the culture media.
However, the decrease in H2O2 content was not expected to
be accompanied by an increased MDA level. This could be
a result of an increase in the content of other ROS but also
of the fact that lipid peroxidation may as well be a con-
sequence of increased activity of enzymes such as
lipoxygenases or a-dioxygenase (Feussner et al. 2001). It
should also be emphasized that lipid peroxidation does not
necessarily point to stress response but may also appear in
developmentally regulated processes (Feussner and
Wasternack 2002). Thus, the observed increase in MDA
levels could, at least in part, be the result of endogenous
0
2
4
6
8
10P
rote
in c
onte
nt (m
g g-1
FW
)
C6 Cd6 C12 Cd12
SDS-PAGE Immunoblot
a
b c
Fig. 6 The effect of 10 lM CdCl2 on HSP70 expression in Lemnaminor cultures. a Total protein content in crude extracts (n = 12). bTotal proteins separated by SDS-PAGE. c Immunoblot probed with
antibody against HSP70. C6, C12, control plants after 6 and 12 days
of cultivation, respectively; Cd6, Cd12, cadmium-treated plants after
6 and 12 days of cultivation, respectively; M molecular mass markers.
HSP70 is indicated by arrows. The arrowhead points to additional
HSP70 isoform
888 Acta Physiol Plant (2008) 30:881–890
123
lipid peroxidation which reflects regular metabolic pro-
cesses during plant growth and development.
In cadmium-treated Lemna minor, SDS-PAGE showed
increased level of a 70-kDa protein which was found to be
immunologically related to HSP70. It is known that the
accumulation of HSP70 correlates positively with
enhanced tolerance to abiotic stress, including cadmium
(Sanita di Toppi and Gabbrielli 1999; Wang et al. 2004).
However, the cellular mechanisms of HSP70 function
under stress conditions are not fully understood. In cell
cultures of Lycopersicon peruvianum exposed to cadmium,
a considerable amount of this protein was found to be
localized at the plasmalemma, mitochondrial membranes
and at the endoplasmatic reticulum, suggesting that HSP70
could be involved in the protection of membranes against
cadmium-induced damage (Neumann et al. 1994). In cad-
mium-treated Lemna minor, induction of HSP70 could, in
part, be related to oxidative stress. Namely, the HSP70
family is known to be involved in proteolytic degradation
of non-native proteins (Wang et al. 2004) and cadmium-
induced oxidation of proteins has been proposed to
increase susceptibility to proteolysis (Aravind and Prasad
2005).
In conclusion, the present work suggests that cadmium
applied in moderately high but environmentally realistic
concentration (10 lM) is phytotoxic to Lemna minor, as
indicated by decreased growth, significantly reduced levels
of photosynthetic pigments and considerably impaired
chloroplast ultrastructure. The cadmium-induced increase
in H2O2 content and lipid peroxidation indicates that oxi-
dative stress is an important mechanism of cadmium
toxicity. The increased activities of antioxidative enzymes
(PPX, APX and CAT) as well as HSP70 induction strongly
suggest their important roles in Lemna minor defense
against cadmium-induced stress.
Acknowledgments This work was supported by the Ministry of
Science, Education and Sports, Republic of Croatia (grants no. 119-
1191196-1202 and 098-0982913-2838). We are grateful to Professor
H. Fulgosi for his generous gift of antibody against pea HSP70. The
authors thank Dr Volker Magnus for helpful discussion and critical
reading of the manuscript.
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