Cadmium-induced responses in duckweed Lemna minor L

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: [email protected]

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


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


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


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


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


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.

References

Aebi H (1984) Catalase in vitro. Meth Enzymol 105:121–126

Aravind P, Prasad MNV (2005) Zinc mediated protection to the

conformation of carbonic anhydrase in cadmium exposed

Ceratophyllum demersum L. Plant Sci 169:245–254

Arbona V, Flors V, Jacas J, Garcıa-Agustın P, Gomez-Cadenas A

(2003) Enzymatic and non-enzymatic antioxidant responses of

carrizo citrange, a salt-sensitive citrus rootstock, to different

levels of salinity. Plant Cell Physiol 44:388–394

Arora A, Sairam RK, Srivastava GC (2002) Oxidative stress and

antioxidative system in plants. Curr Sci 82:1227–1238

Biswal UC, Biswal B (1988) Ultrastructural modifications and

biochemical changes during senescence of chloroplasts. Int

Rev Cytol 113:271–321

Blum H, Beier H, Gross HJ (1987) Improved silver staining of plant

proteins, RNA and DNA in polyacrylamide gels. Electrophoresis

8:93–99

Bradford MM (1976) A rapid and sensitive method for the

quantitation of microgram quantities of protein utilizing the

principle of protein-dye binding. Anal Biochem 72:248–254

Chance B, Maehly AC (1955) Assay of catalases and peroxidases.

Meth Enzymol 2:764–775

Chaoui A, Mazhoudi S, Ghorbal MH, El Ferjani E (1997) Cadmium

and zinc induction of lipid peroxidation and effects on antiox-

idant enzyme activities in bean (Phaseolus vulgaris L.). Plant Sci

127:139–147

Cho U-H, Seo N-H (2005) Oxidative stress in Arabidopsis thalianaexposed to cadmium is due to hydrogen peroxide accumulation.

Plant Sci 168:113–120

Chugh LK, Sawhney SK (1999) Photosynthetic activities of Pisumsativum seedlings grown in presence of cadmium. Plant Physiol

Biochem 37:297–303

Das P, Samantaray S, Rout GR (1997) Studies on cadmium toxicity in

plants: a review. Environ Pollut 98:29–36

Di Cagno R, Guidi L, De Gara L, Soldatini GF (2001) Combined

cadmium and ozone treatments affect photosynthesis and

ascorbate-dependent defences in sunflowers. New Phytol

151:627–636

Dixit V, Pandey V, Shyam R (2001) Differential antioxidative

responses to cadmium in roots and leaves of pea (Pisum sativumL. cv. Azad). J Exp Bot 52:1101–1109

Ensley HE, Barber JT, Polito MA, Olive AI (1994) Toxicity and

metabolism of 2, 4-dichlorophenol by the aquatic angiosperm

Lemna gibba. Environ Toxicol Chem 13:325–331

Feussner I, Wasternack C (2002) The lipoxygenase pathway. Annu

Rev Plant Biol 53:275–297

Feussner I, Kuhn H, Wasternack C (2001) Lipoxygenase-dependent

degradation of storage lipids. Trends Plant Sci 6:268–273

Foyer CH, Noctor G (2005) Oxidant and antioxidant signalling in

plants: a re-evaluation of the concept of oxidative stress in a

physiological context. Plant Cell Environ 28:1056–1071

Foyer CH, Lelandais M, Kunert KJ (1994) Photooxidative stress in

plants. Physiol Plant 92:696–717

Gomez JM, Jimenez A, Olmos E, Sevilla F (2004) Location and

effects of long-term NaCl stress on superoxide dismutase and

ascorbate peroxidase isoenzymes of pea (Pisum sativum cv.

Puget) chloroplasts. J Exp Bot 55:119–130

Heath RL, Packer L (1968) Photoperoxidation in isolated chloro-

plasts. I—Kinetics and stoichiometry of fatty acid peroxidation.

Arch Biochem Biophys 125:189–198

Hegedus A, Erdei S, Horvath G (2001) Comparative studies of H2O2

detoxifying enzymes in green and greening barley seedlings

under cadmium stress. Plant Sci 160:1085–1093

Ireland HE, Harding SJ, Bonwick GA, Jones M, Smith CJ, Williams

JHH (2004) Evaluation of heat shock protein 70 as a biomarker

of environmental stress in Fucus serratus and Lemna minor.

Biomarkers 9:139–155

Laemmli UK (1970) Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature 227:680–685

Landolt E (1986) The family of Lemnaceae—a monographic study.

In: Biosystematic investigations in the family of duckweeds

(Lemnaceae), vol 2. Veroffentlichungen des Geobotanischen

Institutes, Stiftung Rubel, ETH, No.71, Zurich

Lewis MA (1995) Use of freshwater plants for phytotoxicity testing: a

review. Environ Pollut 87:319–336

Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of

photosynthetic membranes. Meth Enzymol 148:350–382


123

Mendelssohn IA, McKee KL, Kong T (2001) A comparison of

physiological indicators of sublethal cadmium stress in wetland

plants. Environ Exp Bot 46:263–275

Mishra S, Srivastava S, Tripathi RD, Govindarajan R, Kuriakose SV,

Prasad MNV (2006) Phytochelatin synthesis and response of

antioxidants during cadmium stress in Bacopa monnieri. Plant

Physiol Biochem 44:25–37

Mittler R (2002) Oxidative stress, antioxidants and stress tolerance.

Trends Plant Sci 7:405–410

Mittler R, Zilinskas BA (1993) Detection of ascorbate peroxidase

activity in native gels by inhibition of the ascorbate dependent

reduction of nitroblue tetrazolium. Anal Biochem 212:540–546

Mohan BS, Hosetti BB (1997) Potential phytotoxicity of lead and

cadmium to Lemna minor grown in sewage stabilization ponds.

Environ Pollut 98:233–238

Mukherjee SP, Choudhari MA (1983) Implication of water stress-

induced changes in the levels of endogenous ascorbic acid and

hydrogen peroxide in Vigna seedlings. Physiol Plant 58:166–170

Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by

ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell

Physiol 22:867–880

Neumann D, Lichtenberger O, Gunther D, Tschiersch K, Nover L

(1994) Heat-shock proteins induce heavy-metal tolerance in

higher plants. Planta 194:360–367

Ouariti O, Gouia H, Ghorbal MH (1997) Responses of bean and

tomato plants to cadmium: growth, mineral nutrition, and nitrate

reduction. Plant Physiol Biochem 35:347–354

Ouzounidou G, Moustakas M, Eleftheriou EP (1997) Physiological

and ultrastructural effects of cadmium on wheat (Triticumaestivum L.) leaves. Arch Environ Contam Toxicol 32:154–160

Pandey N, Sharma CP (2002) Effect of heavy metals Co2+, Ni2+ and

Cd2+ on growth and metabolism of cabbage. Plant Sci 163:753–

758

Perfus-Barbeoch L, Leonhardt N, Vavasseur A, Forestier C (2002)

Heavy metal toxicity: cadmium permeates through calcium

channels and disturbs the plant water status. Plant J 32:539–548

Pirson A, Seidel F (1950) Zell- und stoffwechselphysiologische

Untersuchungen an der Wurzel von Lemna minor unter beson-

derer Berucksichtigung von Kalium- und Kalziummangel. Planta

38:431–473

Prasad SM, Dwivedi R, Zeeshan M, Singh R (2004) UV-B and

cadmium induced changes in pigments, photosynthetic electron

transport activity, antioxidant levels and antioxidative enzyme

activities of Riccia sp. Acta Physiol Plant 26:423–430

Prasad MNV, Malec P, Waloszek A, Bojko M, Strzałka K (2001)

Physiological responses of Lemna trisulca L. (duckweed) to

cadmium and copper bioaccumulation. Plant Sci 161:881–889

Reeves RD, Baker AJM (2000) Metal-accumulating plants. In: Raskin

I, Ensley BD (eds) Phytoremediation of toxic metals: using plants

to clean-up the environment. Wiley, New York, pp 193–230

Rodrıguez-Serrano M, Romero-Puertas MC, Zabalza A, Corpas FJ,

Gomez M, del Rıo LA, Sandalio LM (2006) Cadmium effect on

oxidative metabolism of pea (Pisum sativum L.) roots. Imaging

of reactive oxygen species and nitric oxide accumulation in vivo.

Plant Cell Environ 29:1532–1544

Sandalio LM, Dalurzo HC, Gomez M, Romero-Puertas MC, del Rio

LA (2001) Cadmium-induced changes in the growth and

oxidative metabolism of pea plants. J Exp Bot 52:2115–2126

Sanita di Toppi L, Gabbrielli R (1999) Response to cadmium in

higher plants. Environ Exp Bot 41:105–130

Schutzendubel A, Polle A (2002) Plant responses to abiotic stresses:

heavy metal-induced oxidative stress and protection by myco-

rrhization. J Exp Bot 53:1351–1365

Schutzendubel A, Schwanz P, Teichmann T, Gross K, Langenfeld-

Heyser R, Godbold DL, Polle A (2001) Cadmium-induced

changes in antioxidative systems, hydrogen peroxide content,

and differentiation in Scots pine roots. Plant Physiol 127:887–

898

Siedlecka A, Baszynski T (1993) Inhibition of electron flow around

photosystem I in chloroplasts of Cd-treated maize plants is due

to Cd-induced iron deficiency. Physiol Plant 87:199–202

Singh S, Eapen S, D’Souza SF (2006) Cadmium accumulation and its

influence on lipid peroxidation and antioxidative system in an

aquatic plant, Bacopa monnieri L. Chemosphere 62:233–246

Smeets K, Cuypers A, Lambrechts A, Semane B, Hoet P, Van Laere

A, Vangronsveld J (2005) Induction of oxidative stress and

antioxidative mechanisms in Phaseolus vulgaris after Cd

application. Plant Physiol Biochem 43:437–444

Smith S, Kwan KH (1989) Use of aquatic macrophytes as a bioassay

method to assess relative toxicity, uptake kinetics and accumu-

lated forms of trace metals. Hydrobiologia 188(189):345–351

Somashekaraiah BV, Padmaja K, Prasad ARK (1992) Phytotoxicity

of cadmium ions on germinating seedlings of mung bean

(Phaseolus vulgaris): involvement of lipid peroxides in chloro-

phyll degradation. Physiol Plant 85:85–89

Staples RC, Stahmann MA (1964) Changes in proteins and several

enyzmes in susceptible bean leaves after infection by the bean

rust fungus. Phytopathology 54:760–764

Stobart AK, Griffiths WT, Ameen-Bukhari I, Sherwood RP (1985)

The effect of Cd2+ on the biosynthesis of chlorophyll in leaves

of barley. Physiol Plant 63:293–298

Stoyanova DP, Tchakalova ES (1997) Cadmium-induced ultrastruc-

tural changes in chloroplasts of the leaves and stems parenchyma

in Myriophyllum spicatum L. Photosynthetica 34:241–248

Sudhakar C, Lakshmi A, Giridarakumar S (2001) Changes in the

antioxidant enzyme efficacy in two high yielding genotypes of

mulberry (Morus alba L.) under NaCl salinity. Plant Sci

161:613–619

Sun Q, Yec ZH, Wangb XR, Wong MH (2007) Cadmium hyperac-

cumulation leads to an increase of glutathione rather than

phytochelatins in the cadmium hyperaccumulator Sedum alfre-dii. J Plant Physiol 164:1489–1498

Vecchia FD, La Rocca N, Moro I, De Faveri S, Andreoli C, Rascio N

(2005) Morphogenetic, ultrastructural and physiological dam-

ages suffered by submerged leaves of Elodea canadensisexposed to cadmium. Plant Sci 168:329–338

Vitoria AP, Da Cunha M, Azevedo RA (2006) Ultrastructural changes

of radish leaf exposed to cadmium. Environ Exp Bot 58:47–52

Wang W, Vinocur B, Shoseyov O, Altman A (2004) Role of plant

heat-shock proteins and molecular chaperones in the abiotic

stress response. Trends Plant Sci 9:244–252

Woodbury W, Spencer AK, Stahmann MA (1971) An improved

procedure using ferricyanide for detecting catalase isoenzymes.

Anal Biochem 44:301–305


123

Cadmium-induced responses in duckweed Lemna minor L

Documents

Transcript of Cadmium-induced responses in duckweed Lemna minor L