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Effect of Arsenic on Growth, Oxidative Stress,

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Article in Ecotoxicology and Environmental Safety December 2008
DOI: 10.1016/j.ecoenv.2008.09.022 Source: PubMed

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ARTICLE IN PRESS
Ecotoxicology and Environmental Safety 72 (2009) 11021110

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

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

Effect of arsenic on growth, oxidative stress, and antioxidant system

in rice seedlings$
Manju Shri, Smita Kumar, Debasis Chakrabarty, Prabodh Kumar Trivedi, Shekhar Mallick,
Prashant Misra, Devesh Shukla, Seema Mishra, Sudhakar Srivastava, Rudra D. Tripathi, Rakesh Tuli 
National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, India

a r t i c l e in f o

a b s t r a c t

Article history:
Received 13 February 2008
Received in revised form
13 August 2008
Accepted 26 September 2008
Available online 14 November 2008

The physiological, biochemical, and proteomic changes in germinating rice seedlings were investigated
under arsenic stress. A marked decrease in germination percentage, shoot, and root elongation as well as
plant biomass was observed with arsenic treatments, as compared to control, whereas accumulation of
arsenic and malondialdehyde (MDA) in seedlings were increased signicantly with increasing arsenic
concentration (both AsIII and AsV). The up-regulation of some antioxidant enzyme activities and the
isozymes of superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), peroxidase
(POD, EC 1.11.1.7), and glutathione reductase (GR, 1.6.4.2) substantiated that arsenic accumulation
generated oxidative stress, which was more pronounced in As(III) treatment. We also studied the
protective effect of reduced glutathione (GSH) and cysteine (Cys) to As(III)/As(V) stressed seedlings.
Both GSH and Cys imparted enhanced tolerance to seedlings against arsenic stress. Seedlings growth
improved while level of MDA declined signicantly when GSH and Cys were supplemented to
As(III)/As(V) treatments suggesting GSH and Cys-mediated protection against oxidative stress.
The arsenic content was highest in roots of seedlings grown in As(III) in the presence of GSH/Cys.
However, in case of As(V) plus GSH or Cys, the arsenic content in seedlings was highest in shoots.
The results are suggestive of differential metabolism of As(III) and As(V) in rice.
& 2008 Elsevier Inc. All rights reserved.

Keywords:
Arsenic
Cysteine
Glutathione
Oxidative stress
Rice
Seed germination

1. Introduction
Arsenic toxicity has been known for centuries, and has recently
received increased attention because of its chronic and epidemic
effects on human health (Abernathy et al., 1999). Arsenic can be
present in the terrestrial, marine, and freshwater environments in
various chemical forms. Organic arsenic species are less toxic than
inorganic species to aquatic plants, animals and humans, and this
has been presumed to be true for terrestrial plants also (Meharg
and Hartley-Whitaker, 2002). There is a general agreement that
arsenic contamination in the groundwater of south and southeast
Asia has resulted due to release of arsenic from solid phases under
anaerobic conditions (Polizzotto et al., 2008). Widespread use of
arsenic contaminated groundwater for irrigation in rice eld
elevates its concentration in surface soil and eventually into rice
plants and grains (Williams et al., 2007; Rahman et al., 2007).
Numerous studies have been carried out in relation to uptake and
translocation of arsenic from soil to plants (Tripathi et al., 2007).

This study does not involve human and experimental animal.

 Corresponding author. Fax: +91 522 2205836.

E-mail address: rakeshtuli@hotmail.com (R. Tuli).

0147-6513/$ - see front matter & 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2008.09.022

The predominantly accepted model for plant detoxication of

arsenic in plants is complexation of As(III) with glutathione (GSH)
and/or phytochelatins (PCs) through SH coordination. PCs have a
general structure [(g-GluCys)nGly) (n 211)] and are synthesized non-translationally by PC synthase using glutathione as a
substrate (Schmoger et al., 2000). For the detoxication purpose,
As(III) must be compartmentalized in vacuoles that may be
achieved by shuttling from the cytoplasm, probably as As-PC
complexes.
Plants normally take up arsenic predominantly in trivalent
(AsIII) and pentavalent (AsV) forms, which are known to interfere
with various metabolic pathways in cells like, interaction with
sulfhydryl groups and replacement of phosphate from ATP. Hence,
plants not tolerant to arsenic show toxic patterns such as decrease
in plant growth and crop yield (Carbonell-Barrachina et al., 1995,
1998; Knauer et al., 1999). Heavy metals including metalloid As
have been reported to stimulate the formation of free radicals and
reactive oxygen species leading to oxidative stress (Zhang et al.,
2003; Azevedo et al., 2005; Loureiro et al., 2006; H.P. Singh et al.,
2007; N. Singh et al., 2007). Requejo and Tena (2005) reported the
effect of arsenic exposure on maize (Zea mays L.) root proteome
and concluded that the induction of oxidative stress is the main
process underlying arsenic toxicity in plants. Plants respond to

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1103

oxidative stress by increasing the production of antioxidant

enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX) or peroxidase (POD). Of these, SOD is one among the
early induced enzymes, and is responsible for the detoxication of
the active superoxide radicals (Bowler et al., 1992). In plants
several isomeric forms of SOD catalyze the dismutation of
the superoxide radicals (Od
2 ) to hydrogen peroxide (H2O2). The
conversion of H2O2 into water in peroxisomes is carried out
by catalase (CAT), while that in cytosol and chloroplasts by
ascorbateglutathione cycle, which involves APX, ascorbate
(AsA), reduced glutathione (GSH), and glutathione reductase
(GR) (Noctor and Foyer, 1998). APX catalyzes the conversion of
H2O2 into water using AsA as substrate, while GSH and GR are
involved in the regeneration of AsA.
Several studies have been carried out on oxidative stress and
defense mechanism in plants under heavy metal stress (Hall,
2002; Ortega-Villasante et al., 2007; Lin et al., 2007; Liu et al.,
2007). However, the response of different antioxidant enzymes
and their isozymes in As stressed germinating rice seedlings has
not been examined. In this study the resultant effect of As(V) and
As(III) toxicity on seed germination, early seedling growth,
antioxidant enzyme activities and their isozymes in rice were
investigated. The role of GSH and Cys was evaluated in imparting
the protection to the seedlings against arsenic stress. Novel
ndings are presented on the effect of GSH and Cys in counteracting arsenic stress in plants.

activities. The preparation was applied to a column of sephadex G-25, equilibrated

with the same buffers and kept in an ice bath until the assays were completed.
Protein concentration of the enzyme extract was determined according to Bradford
(1976).
SOD activity was assayed by monitoring the inhibition of photochemical
reduction of nitro blue tetrazolium (NBT) according to the method of Beyer and
Fridovich (1987). Shoots and roots were homogenized in 1 mL cold 100 mM
K-phosphate buffer (pH 7.8) containing 0.1 mM ethylenediaminetetraacetic acid
(EDTA), 1% (w/v) polyvinyl-pyrrolidone and 0.5% (v/v) Triton X-100. One unit of
SOD activity was dened as the amount of enzyme required to cause 50%
inhibition of the reduction of NBT as monitored at 560 nm. For the determination
of APX, shoots and roots were homogenized in 100 mM Na-phosphate buffer
(pH 7.0) containing 5 mM AsA, 10% glycerol and 1 mM EDTA. APX activity was
determined in 1 mL reaction mixture containing 50 mM K-phosphate buffer (pH
7.0), 0.1 mM AsA (extinction coefcient, 2.8 mM1 cm1) and 0.3 mM H2O2. The
decrease in absorbance was recorded at 290 nm for 3 min (Chen and Asada, 1989).
The blank was made by adding of phosphate buffer instead of extracts in the
reaction mixture. For GR, shoots and roots were homogenized in 100 mM Naphosphate buffer (pH 7.0) containing 1 mM EDTA. GR activity was assayed by
following the reduction of 5,50 -dithio-bis (2-nitrobenzoic acid) (DTNB) at 412 nm
(extinction coefcient, 13.6 mM1 cm1) with some modications as described by
Smith et al. (1988) The assay mixture (1 mL) contained 100 mM K-phosphate
buffer (pH 7.5), 1 mM oxidized glutathione (GSSG), 1 mM EDTA, 0.08 mM DTNB,
0.1 mM NADPH and 100 mL of enzyme extract. GSSG was replaced by water in the
reaction mixture as blank. POD activity was measured by following the change of
absorption at 470 nm due to guaiacol oxidation (extinction coefcient, 26.6 mM1
cm1) following Putter (1974). The activity was assayed for 5 min in a reaction
mixture comprised of 50 mM K-phosphate buffer (pH 7.0), 20.1 mM guaiacol,
12.3 mM H2O2 and suitable amount of enzyme extract from shoots and roots.

2. Materials and methods

Native polyacrylamide gel electrophoresis (PAGE) was performed at 4 1C, 90 V,

following Laemmli (1970). For SOD, POD, APX, and GR, the enzyme solutions were
subjected to native PAGE with 10% polyacrylamide gel. Activity stain for each
enzyme was carried out as follows. APX activity was detected by the procedure
described by Mittler and Zilinskas (1993). The gel equilibrated with 50 mM
Na-phosphate buffer (pH 7.0) containing 2 mM ascorbate for 30 min was incubated
in a solution composed of 50 mM Na-phosphate buffer (pH 7.0), 4 mM ascorbate
and 2 mM H2O2 for 20 min. The gel was washed in the buffer for 1 min and
submerged in a solution of 50 mM Na-phosphate buffer (pH 7.8) containing 28 mM
N,N,N,N-tetramethyl ethylenediamine (TEMED) and 2.45 mM NBT for 1020 min
with gentle agitation in the presence of light. SOD activity was detected by the
procedure described by Beauchamp and Fridovich (1971). The gel equilibrated
with 50 mM K-phosphate buffer (pH 7.8) containing 2.8  105 M riboavin and
0.028 M TEMED for 30 min. The gel was washed in distilled water for 1 min and
submerged in the same solution (mentioned above) containing 2.45 mM NBT for
1020 min with gentle agitation in the presence of light. POD activity was revealed
with a modication of the method described by Prestamo and Manzano (1993),
the gel was covered with a solution containing 50 mL of 50 mM sodium acetate
buffer (pH 6.0), 3.4 mL of 0.5% H2O2 and 3.4 mL of 0.25% (w/v) o-dianisidine.
Finally, the gel was agitated until a brown color appeared (3060 min). GR activity
staining was adapted from Anderson et al. (1995) and was carried out in 0.4 mM
NADPH, 3.4 mM oxidized glutathione (GSSG), 1.2 mM 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT), 0.3 mM 2,6-dichlorophenol-indophenol
(DPIP) and 50 mM TrisHCl buffer (pH 7.7).

2.1. Plant material, growth conditions, and treatments

Mature seeds of Indica cultivar Lalat (Oryza sativa L.) were manually dehusked,
sterilized in 70% ethanol for 1 min, 0.1% mercuric chloride for 5 min, followed by
three washes in sterile distilled water. Sterilized seeds were cultured on 1/2 MS
medium containing As(III) (50 and 100 mM) and As(V) (100 and 500 mM). Sodium
arsenate (Na2HAsO4; ICN, USA) and arsenite (NaAsO2; J.T. Baker, UK) were used as
the source of arsenic. Twenty seeds were placed in each plate, covered by lid, and
incubated under a 16-h-light (110130 mE/m2/s) and 8-h-dark photoperiod in a
culture chamber at 26 1C. In some experiments, the medium was supplemented
with 0.1 mM GSH or 50 mg/L Cys along with 50 mM As(III) or 250 mM As(V) to see
the protective effect of GSH and Cys on germination and growth of rice seedling.
2.2. Seed germination and growth parameters
Geminated seeds were counted 10 d after initiation. Seeds were considered
germinated when both the plumule and radicle were extended from their junction.
Each treatment was replicated three times. After 10 d of growth, shoot length, root
length, root weight, and shoot weight were measured.
2.3. Measurement of lipid peroxidation
Shoot and root samples from 10-d-old rice seedlings were used for studying
lipid peroxidation. Lipid peroxidation was estimated as malondialdehyde (MDA)
produced using thiobarbituric acid (TBA) method as described by Heath and
Packer (1968). Briey, 1 g of sample was homogenized in 1 mL 0.5% trichloracetic
acid (TCA). The homogenate was centrifuged at 19,000  g for 20 min. The 0.5 mL
supernatant was mixed with 2.5 mL 20% TCA containing 0.5% TBA, heated in
boiling water bath for 30 min and then allowed to cool rapidly in an ice bath. The
supernatant was centrifuged at 10,000  g for 10 min and the resulting supernatant was used for determination of MDA. The concentration of MDA was
calculated from the absorbance at 532 nm (correction was made by subtracting
absorbance at 600 nm for turbidity) by using extinction coefcient of 155 mM1
cm1.

2.5. Native page and activity stain

2.6. Arsenic measurement

Shoots and roots from 10-d-old rice seedlings were dried in hot air oven at
40 1C till constant weight. Dried plants tissues (50 mg) were digested in HNO3 and
H2O2, 3:1 proportion through microwave digestion, in CEM-MDS 2000 Microwave
digester (Roychoudhury et al., 2002). The digested solutions were made up to 3 mL
from which 10 ml aliquot was quantitatively analyzed for arsenic through atomic
absorption spectrometry (PerkinElmer; AAnalyst 600) tted with graphite
furnace. Reference standard for calibration of the AAS was made using 1000 mg
mL1 (AA03N-5) standard supplied by Accustandard, USA.

2.4. Antioxidant enzyme activities

2.7. Statistical analysis

For determination of antioxidant enzyme activities, 0.5 g of shoots and roots

were homogenized in 1.5 mL of respective extraction buffer in a pre-chilled mortar
and pestle. The homogenate was ltered through four layers of cheesecloth and
centrifuged at 22,000  g for 20 min at 4 1C. The supernatant was centrifuged again
at 22,000  g for 20 min at 4 1C before determination of antioxidant enzyme

Each experiment was carried out under a completely randomized design with
three independent experiments with at least three replications. The data were
analyzed by two way analysis of variance (ANOVA) to conrm the variability and
validity of results, and Duncans multiple range test (DMRT) was performed to
determine signicant difference between treatments (Gomez and Gomez, 1984).

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M. Shri et al. / Ecotoxicology and Environmental Safety 72 (2009) 11021110

3. Results

a
b

90

c
b

60
bc
c

30
d d

a
Shoot

1.5

Root

b
b

bc

d
0.5

V)
5
s(
A

s(

V)
1

00

0
II I
)1
0
s(
A

s(

tr
on
C

00

0
ol

Arsenic accumulation by seedlings increased with increasing

concentrations of As(III) and As(V). The shoots accumulated more
arsenic in As(V) treatment than As(III). Surprisingly, the arsenic
accumulation was lower in both shoots and roots of 500 mM As(V)
exposed seedlings as compared to 100 mM As(V) (Fig. 2A). Root

Root

120

III
)5
0

3.2. Arsenic accumulation in root and shoot

Shoot

MDA ( molg-1 FW)

As a preliminary experiment, rice seeds were exposed to

different concentrations of As(V) and As(III) to determine their
effects on germination. Arsenic was highly toxic to germination of
rice seeds. The germination decreased signicantly with the
increase in arsenic concentration (Fig. 1A). The shoot and root
growth in terms of root length, shoot length, root and shoot fresh
weight was greatly inhibited at 50 mM As(III) as well as 500 mM
As(V) (Figs. 1B and C). In addition, no root formation was observed
at concentrations higher than 500 mM As(V) (Supplementary
Fig. 1A). The roots turned black in the presence of 100 mM As(III)
(Supplementary Fig. 1B). Root weight was affected more in
comparison to root length (Figs. 1B and C).

Arsenic (g mg-1 DW)

150

3.1. Effect of arsenic on growth parameters

Germination (%)

120

80

b
40

tissue accumulated 102.9 mg mg1 DW arsenic whereas in shoots

it was only 33.3 mg mg1 DW when germinated on 100 mM As(III).
In contrast to this, shoots accumulated more than 2-fold arsenic in
comparison to roots when grown on 100 mM As(V). This suggests
differential accumulation of arsenic species in different plant
tissues.

Length (cm)

12

Shoot

Root

ab

3.3. Effect on lipid peroxidation

ab

abc

ab
bc

0
Fresh weight (mg)

36

Shoot

Root

27

c
18

3.4. Effects of arsenic on antioxidant enzymes

V)
5

00

00
A

s(

V)
1
s(
A

s(

III
)1
0

I)5
0
A

s(
II

ol
tr
on

Lipid peroxidation in relation to arsenic stress in 10-d-old rice

seedlings was studied as a measure of oxidative stress. Lipid
peroxidation was estimated as MDA produced using thiobarbituric acid (TBA). The MDA content in shoots and roots was
increased with increasing concentration of As(III) and As(V),
indicating oxidative stress (Fig. 2B). MDA, a decomposition
product of polyunsaturated fatty acid hydroperoxides, is a
biomarker for lipid peroxidation, which is an effect of oxidative
damage. A signicant difference in MDA content was observed in
shoots as compared to roots of seedlings grown in 500 mM As(V).

Fig. 2. Arsenic (A) and MDA content (B) in shoots and roots of rice seedlings grown
on different As(III) and As(V) concentrations. AsIII and AsV followed by different
numbers represent concentration of As(III) and As(V) in mM. All values are the
mean of triplicates (7SD). ANOVA signicant at pp0.01. Different letters indicate
signicantly different values in a particular tissue (DMRT, pp0.05).

Fig. 1. Germination (A), shoot and root length (B), and fresh weight (C) of rice
seedlings grown on different As(III) and As(V) concentrations. AsIII and AsV
followed by different numbers represent concentration of As(III) and As(V) in mM.
All values are the mean of triplicates (7SD). ANOVA signicant at pp0.01.
Different letters indicate signicantly different values in a particular tissue (DMRT,
pp0.05).

The effect of arsenic on SOD, APX, POD, and GR activities and

isozymes in 10-d-old rice seeding was examined. In As(III) and
As(V) exposed plants, total SOD activity increased with increasing
arsenic concentration. The maximum activity was observed in
roots treated with 100 mM As(III) and 500 mM As(V). Shoots of
As(V) treated samples showed a higher increase in SOD activity as
compared to As(III) exposed shoots (Fig. 3A). Isozyme analysis
indicated that with both the treatments the observed increase of
SOD activity in roots was due to Cu/Zn-SOD (Fig. 4A). However, in
shoots only two isozymes of Mn-SOD were detected and highest

ARTICLE IN PRESS

0.1

V)
5
s(
A

bc

cd
c

0.02

00

00
V)
1
s(

s(
A

III
)1
0

50
A

s(

II I
)

tr
on
C

bc
e

0.04

ab

00

ab

ab

V)
5

ab

0.06

s(

Root

0.2

ab

Shoot

00

0.08

V)
1

Root

0.3

s(

Shoot

0.4

ol

POD ( mol min-1 mg-1 protein)

b c

bc

0.25

III
)1
0

s(

ol

d
4

Root
0.5

tr

50

Shoot

II I
)

s(

on

Root

1105

0.75

Shoot

GR ( mol min-1 mg-1 protein)

SOD (Unit mg-1 protein)

APX ( mol min-1 mg-1 protein)

M. Shri et al. / Ecotoxicology and Environmental Safety 72 (2009) 11021110

Fig. 3. Superoxide dismutase (A; SOD), ascorbate peroxidase (B; APX), guaiacol peroxidase (C; POD) and glutathione reductase (D; GR) activities in shoots and roots of rice
seedlings grown on different As(III) and As(V) concentrations. AsIII and AsV followed by different numbers represent concentration of As(III) and As(V) in mM. All values are
the mean of triplicates (7SD). ANOVA signicant at pp0.01. Different letters indicate signicantly different values in a particular tissue (DMRT, pp0.05).

activity was observed in 100 mM As(III) and 500 mM As(V)

exposures. In roots, total APX activity increased with increasing
arsenic concentrations, reaching a maximum at 100 mM As(III) and
500 mM As(V) (Fig. 4B). In shoot, maximum APX activity was
observed at 50 mM As(III), which decreased with increase in
arsenic concentration. Isozyme analysis showed that the increase
in total APX activity was due to an increase in the activity of the
APX-4 isozyme after treatment with both forms of arsenic.
Isozyme analysis also indicates new isoforms that were induced
in shoots as well as roots (Fig. 4B). The maximum POD activity
was observed in roots exposed to 100 mM As(III) and 500 mM As(V)
(Figs. 3C and 4C). However, no signicant changes were observed
in the POD activity in response to As(V) or As(III) in shoots
(Fig. 3C). Total GR activity increased with increasing As(III). GR
activity in As(V) exposed seedlings showed a differential pattern.
In shoots, the activity increased with increasing As(V) concentration whereas, in roots, activity declined at 500 mM As(V) in
comparison to 100 mM As(V) (Fig. 3D). Isoenzyme study suggested
that GR-5, 6, 7, and 8 specic activity was also induced with
increasing concentrations of As(III) in roots (Fig. 4D).

measured by root length, shoot length, and weight when cultured

either with GSH alone or supplemented with 50 mM As(III). On the
contrary, when supplemented with Cys (50 mg L1) along with
either As(III) or As(V) lesser growth inhibition of rice seedlings
was observed in comparison to As (III)/As(V) alone (Figs. 5B, C, and
6). The MDA content in shoots and roots were increased in As(III)
and As(V) treatments, indicating oxidative stress. However, when
the medium was supplemented with either GSH or Cys, there
were no signicant changes in MDA content in comparison to
control, indicating reduced oxidative stress (Fig. 5D).
Arsenic accumulation by rice seedlings indicates that there
was no variation between seedlings grown in 50 mM As(III) and in
the absence or presence of GSH. However, seedlings grown in
250 mM As(V) supplemented with GSH accumulated more arsenic
in their shoots as well as roots (Fig. 7). The plants grown in Cys
rich media along with 50 mM As(III), accumulated more arsenic in
roots. Whereas seedlings grown in either Cys rich or GSH
supplemented media along with 250 mM As(V) accumulated more
arsenic in shoots.

4. Discussion
3.5. Effects of GSH and Cys on arsenic toxicity
We investigated the role of supplementing GSH or Cys on
arsenic induced oxidative stress, germination, and seedling
growth. No signicant change in seed germination was observed
in different treatments in comparison to control (Fig. 5A).
Signicant inhibition of growth was observed under arsenic
(50 mM AsIII and 250 mM AsV) treatment (Figs. 5B and C). GSH
supplement along with 250 mM As(V) had a clear stimulating
effect on the growth of rice seedlings (Figs. 5B, C, and 6). However,
GSH had inhibitory effect on the growth of rice seedlings as

Arsenic (As) is a toxic metalloid, which enters the environment

mainly through anthropogenic activities (Abedin et al., 2002). Ascontaminated groundwater and food are a serious health risks
worldwide, especially in Bangladesh, China, and India (Karim
et al., 2008; Samanta et al., 2007; H.P. Singh et al., 2007; N. Singh
et al., 2007). Paddy soils and irrigation water are contaminated
with high concentrations of As in some areas in these countries
(Meharg and Hartley-Whitaker, 2002). Rice is the staple food for
populations in these countries. The buildup of As in paddy soils
and irrigation water adversely effects rice plant germination and

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M. Shri et al. / Ecotoxicology and Environmental Safety 72 (2009) 11021110

Cu/Zn SOD
Mn-SOD
Mn SOD

Mn-SOD

Mn SOD
Cu/Zn SOD
2 mM 5 mM
KCN H2O2

AsIII50 AsIII100 AsV100 AsV500

2 mM
KCN

5 mM
H2O2

AsIII50 AsIII100 AsV100 AsV500

1
2

3
2

4
3

5
6

5
6

7
C

AsIII50

AsIII100

AsV100

AsV500

AsIII50

AsIII100

AsV100

AsV500
1

1
2

3
C

AsIII50

AsV100

AsIII100

AsV500

AsIII50

AsIII100

AsV100

AsV500

1
2
3
4
1
5
6
7
2
3

AsIII50

AsIII100

AsV100

AsV500

Root

AsIII50

AsIII100

AsV100

AsV500

Shoot

Fig. 4. Superoxide dismutase (A; SOD), ascorbate peroxidase (B; APX), peroxidase (C; POD) and glutathione reductase (D; GR) in-gel assay of proteins from roots and shoots
of rice seedlings grown on different As(III) and As(V) concentrations. AsIII and AsV followed by different numbers represents concentration of As(III) and As(V) in mM.
Samples were electrophoresed on a 10% native polyacrylamide gel. 150 mg total protein were loaded per each well. Assays were carried out as described in methods. The
different isoforms are numbered from cathode to anode.

growth and leads to As accumulation in rice grains. Although,

there are several studies on oxidative stress and defense
mechanism in plants grown under heavy metal stress (Mascher
et al., 2002), not much information is yet available on rice. We

have studied biological effects of arsenic on seed germination,

early seedling growth of rice, antioxidant enzyme activities and
specially their isozymes and also evaluated the role of GSH and
Cys in protecting rice seedlings.

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M. Shri et al. / Ecotoxicology and Environmental Safety 72 (2009) 11021110

10

Shoot
Root

80

Length (cm)

Germination (%)

120

40

1107

de

e
a

a
a

Shoot
Root

abc

ab

abc

30

abc

a
bcd

20

cd

ab cd

ab
10

Shoot

1.5

ab

bcd
ab
ab

ab

00

MDA (mol g-1 FW)

Root

1.2
a

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b
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bc

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C
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+
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C
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0
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C
G
on
SH
tr
ol
+
A
C
sI
ys
II
50
A
+
sV
C
ys
25
0
+
C
ys

50

sV
A

sI
II

ro
l

0 0

on
t
C

40
Fresh weight (mg)

2
0

ab

ab

bc

cd

cd

ab

Fig. 5. Effect of As(III)/As(V) and GHS/Cys interaction on germination (A) root and shoot length (B), root and shoot fresh weight (C) and MDA content (D) of rice seedlings.
AsIII and AsV followed by different numbers represent concentration of As(III) and As(V) in mM. The medium was supplemented with 0.1 mM GSH and 50 mg L1 Cys along
with 50 mM As(III) or 250 mM As(V). All values are the mean of triplicates (7SD). ANOVA signicant at pp0.01. Different letters indicate signicantly different values in a
particular tissue (DMRT, pp0.05).

Seed germination is one of the most sensitive processes to

metal pollution because of lack of defense mechanisms and hence
is an important consideration while studying effects of heavy
metals on seedling growth (Liu et al., 2005). We observed
decrease in germination rate under As exposure in comparison
to control. Length and biomass of root and shoot were signicantly affected due to As stress. Reduced root length growth in
response to arsenic exposure has been reported by a number of
investigators in other plants (Hartley-Whitaker et al., 2001;
Kapustka et al., 1995; Sneller et al., 1999; Liu et al., 2005). The
inhibition was stronger in the root than in the shoot when
exposed to As, because the plant roots are the rst point of contact
for these toxic arsenic species in the nutrient media. Arsenic
accumulation by seedlings increased with increasing concentrations of As except at higher As(V) concentration where the root
growth was signicantly inhibited, thus reducing the transport of
arsenic. Further accumulation of As was higher in roots in
comparison to shoots upon exposure to As(III) while it was higher
in shoots in As(V) exposed plants except at 500 mM As(V). Higher
retention of As(III) might be attributed to its compartmentalization in root vacuoles. While in case of As(V) its reduction to As(III),
which is a prerequisite for compartmentalization, might takes
place in vascular tissues during transport to shoot (Pickering et al.,
2006). However, at higher exposure of As(V) membrane composition and functionality of transporters might be disrupted leading
to less transportation of As as well as other important nutrients.
This might be the reason for reduced growth of plant at 500 mM
As(V) despite less accumulation in comparison to 100 mM As(V).
Using proteomics approach, it has been reported that arsenic
exposure induces proteins related to oxidative stress in plants

(Requejo and Tena, 2005). Active oxygen radicals may induce

chain-like peroxidation of unsaturated fattty acids in the
membranes leading to formation of lipid peroxidation products
like MDA (Stoeva et al., 2005; H.P. Singh et al., 2007; N. Singh
et al., 2007; Mishra et al., 2008). Our results also indicate that
MDA content increased gradually with the increase in As stress.
However, at 500 mM As(V) exposure, the MDA content was lower
in roots in comparison to shoots. This might be attributed to
signicant increase in activity of POD, the enzyme responsible for
degradation of lipid peroxides, in roots at this concentration. On
the contrary, POD and GR showed opposite trends, which might
be due to differential protective mechanisms operating in shoots
and roots.
Comparing the activities of the four enzymatic antioxidants
SOD, APX, POD, and GR, it is evident that the uptake of As induced
a strong antioxidative response in rice seedlings. Activities of the
enzymes in different tissues were related to As concentrations but
in a differential manner. As(III) induced these enzymes more
prominently in roots whereas As(V) in the shoot tissue. It has been
reported that SOD in Zea mays was stimulated upon exposure to
both As(V) and As(III) (Requejo and Tena, 2005). Superoxide
radicals induced by As-stress in rice roots are converted to H2O2
by the action of SOD. The SODs exist in three isoforms, viz.,
Mn-SOD, Fe-SOD and Cu/Zn-SOD, which are localized in different
cell organelles depending upon their prosthetic groups (Bowler
et al., 1994). Rodrguez-Serrano et al. (2006) reported that Cd
treatment inhibited Cu/Zn SOD, but upregulated Mn-SOD and
Fe-SOD in pea roots. However, such studies in response to As
toxicity are lacking (H.P. Singh et al., 2007; N. Singh et al., 2007).
We observed that during arsenic stress Cu/Zn SOD was induced. It

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M. Shri et al. / Ecotoxicology and Environmental Safety 72 (2009) 11021110

Control +GSH

Control

Control +Cys

AsIII 50

AsIII 50 + GSH

AsIII 50+ Cys

AsV 250

AsV 250 + GSH

AsV 250 + Cys

Fig. 6. Effect of As(III)/As(V) and GHS/Cys interaction on growth of rice seedlings. Rice seeds were grown for 10 d on 1/2 MS plates containing 50 mM As(III) or 250 mM As(V)
alone or supplemented with 0.1 mM GSH or 50 mg L1 Cys along with 50 mM As(III) or 250 mM As(V).

Shoot
400

Root

a
b

300
200

C
+

25
0

sV

II
5
sI

ys

ys
C
+

C
0

+
ol

+
tr
on

sV

ys

H
S
G

G
25
0

0
A

sI
A

ed

SH

H
+

G
+

ol
tr
on

e d
S

25
0

sV

II
5

ol

sI

tr
A

on
C

e d

II
5

100

Arsenic (g mg-1 DW)

500

Fig. 7. Arsenic accumulation in shoots and roots of rice seedlings treated with
As(III) and As(V) alongwith GSH or Cys. AsIII and AsV followed by different
numbers represent concentration of As(III) and As(V) in mM. The medium was
supplemented with 0.1 mM GSH and 50 mg L1 Cys along with 50 mM As(III) or
250 mM As(V). All values are the mean of triplicates (7SD). ANOVA signicant at
pp0.01. Different letters indicate signicantly different values in a particular tissue
(DMRT, pp0.05).

has been reported (Lenartowicz, 1990) that treatment with As(III)

can produce extensive oxidation of intramitochondrial NAD(P)H
transhydrogenase. NAD(P)H shortage may result in accumulation of
oxidized glutathione and ROS. On the other hand, As(V) is reduced
rapidly to As(III) by arsenate reductase, using oxygen as a nal
electron acceptor (Tamaki and Frankenberger, 1992). During reduction, the generation of superoxide radicals is possible through the
reduction of oxygen (Mylona et al., 1998), which is supported by the
increase in SOD activity particularly Cu/Zn SOD during As(V) stress.

The accumulation of H2O2 is prevented in cells by the

ascorbateglutathione cycle where APX reduces it to H2O.
Signicant increase in APX activity was observed in As(III)
treatment in roots. One APX isoenzyme was detected markedly,
especially in roots of 100 mM As(III) treated seedlings. However, in
shoots maximum APX activity was observed at 50 mM As(III),
which decreased at higher concentration. At higher As(III)
concentration, roots experienced high ROS build up and detoxication by increased antioxidant systems before As(III) is
transported to shoots. According to POD isozyme chart, with the
increase of the concentration of As, POD-2 isozyme band
increased gradually, which coincided with the measured results
of POD activity in roots of As(III) treatment. It was also observed
that new isoforms of GR induced during As(III) stress. It is possible
that As(III) signicantly decreased the GSH content in rice roots,
due to its conversion to phytochelatins. The enhanced demand for
GSH in response to arsenic-induced oxidative stress might be met
by the increased GR activity and elevated GSH turn-over. The
results suggest that the antioxidative enzymes are highly
upregulated in As(III) stress than As(V).
Glutathione is an important antioxidant involved in cellular
defense against toxicants (Goldsbrough, 2000). Glutathione levels
in plant tissues are known to increase under metal stress (Sun
et al., 2007). Therefore, we studied the possible role of GSH and
Cys in protecting plants against toxicity by supplementing these
in As(III)/As(V) containing medium. GSH and Cys supplementation resulted in partial protection against arsenic stress. However,
GSH had negative effect on seedling growth, which may be due to
feedback regulation of glutathione biosynthesis within plants
(Richman and Meister, 1975). Further, GSH and Cys supplementation resulted in elimination of oxidative stress (MDA content) and
restored seedling growth As(V) exposed seedlings. GSH also
prevented the toxic effects of As(V).

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M. Shri et al. / Ecotoxicology and Environmental Safety 72 (2009) 11021110

The arsenic content in the roots and shoots of rice seedlings

grown on As(III)/As(V) along with GSH/Cys were measured. In
case of As(III)+GSH or Cys treated seedlings, the arsenic content
was higher in roots than shoots. However, in case of As(V)+GSH or
Cys treated seedlings, the arsenic content was higher in shoots
than roots. The results suggest that the metabolism of As(III) and
As(V) occurs involving different pathways in roots and shoots. The
nature of the transporters, antioxidant systems and sequestration
mechanisms has not been studied in details. Similar to our
ndings, differential role of antioxidant systems and phytochelatins have been proposed for As(III) and As(V) toxicity (Srivastava
et al., 2007). The main route of As(V) uptake in plants is through
the phosphate transporters as a phosphate analogue, whereas
As(III) is transported as neutral As(OH)3 through aquaglyceroporins. Recently, a silicon transporter from rice has been implicated in
transport of As(III) from root to shoot following the same pathway
as silicon ( Ma et al., 2008). Another important point is that the
arsenic content was much higher in As(V) exposed shoots
supplemented with GSH/Cys, though there was no signicant
effect on growth. This may possibly happen if As after movement
from root to shoot is sequestrated (most probably bound to PCs)
into vacuoles.

5. Conclusion
The study reports parameters related to oxidative stress and
the response of different antioxidant enzymes and their isozymes
in rice seedlings exposed to arsenic. The up-regulation of some
antioxidant enzyme activities and the isozymes of SOD, APX, POD,
and GR indicated that excess arsenic generates oxidative stress,
which is more pronounced in As(III) treatment. GSH and Cys gave
partial protection against arsenic stress. The results suggest
different pathways of metabolism of As(III) and As(V) in rice. In
some cases, the pathways between shoot and root appeared to be
different, that might involve differences in the transporters,
antioxidant systems, and the mechanism of sequestration. However, to clearly understand the metabolism of As(III) and As(V) in
germinating rice seeds during arsenic stress, more biochemical
and physiological analysis is needed.

Acknowledgments
This work was supported by the Council of Scientic and
Industrial Research, Govt. of India. One of the authors (RT)
acknowledges the DST, Govt. of India for providing nancial
assistance in the form of J.C. Bose Fellowship. SM and PM
acknowledge Council of Scientic and Industrial Research, Govt. of
India for Research Associate and Junior Research Fellowship.

Appendix A. Supplementary Materials

Supplementary data associated with this article can be found
in the online version at doi:10.1016/j.ecoenv.2008.09.022

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